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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116163 10.31557/APJCP.2023.24.4.1389 Research Article Comparative Study of DNA Ploidy and BRAF Immunohistochemistry between Colonic Adenocarcinoma and Inflammatory Colonic Lesions Khalil Heba Khalil Mohamed 1 Hammam Olfat Ali 1 Anis Shady Elia 2 El-Hady Elashry Mohamed Abd 3 El-Yasergy Dina Fawzy 2* 1 Department of Pathology, Theodor Bilharz Research Institute, Giza, Egypt. 2 Department of Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt. 3 Department of Surgery, Theodor Bilharz Research Institute, Giza, Egypt. * For Correspondence: dandoon3000@hotmail.com 2023 24 4 13891400 2 1 2023 17 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objectives: To evaluate DNA ploidy and S-phase fraction (SPF) in non-Lynch colonic adenocarcinoma, ulcerative colitis (UC), Crohn disease (CD) which are known as risk factors, and colitis. We correlated ploidy and SPF with tumor grading, staging and BRAF expression. Methods: All studied adenocarcinomas have intact mismatch repair genes as proved by immunohistochemistry. All were assessed for ploidy by automated image-based DNA cytometry and histograms were drawn. Immunostaining by anti-BRAF V600E was performed. Diagnostic laparoscopy (DL) was done as a preliminary step for staging GI cancers. Results: there is significant difference in DNA ploidy between groups; 77.5% and 17.5% of aneuploid cases are adenocarcinoma and UC. Groups are compared in terms of 2C, 4C, above 4C DNA content and SPF and significant difference is principally found between adenocarcinoma group and others. In adenocarcinomas, DNA ploidy is significantly correlated with tumor staging and grading. Regarding BRAF expression, there is significant difference between groups; all adenocarcinomas, 83.33% of UC were positive, while all cases of colitis, bilharzial colitis, CD were negative. There is significant relation between BRAF and SPF among all diploid cases including adenocarcinoma, and among non-neoplastic diploid cases. There is direct significant relation between BRAF intensity and adenocarcinoma staging. There is no significant difference between BRAF and ploidy among UC cases, although 75% of aneuploid UC are positive. DL helps in GI cancer staging. Routine laparoscopy before laparotomy, especially in cancers which have equivocal operability helps to avoid unnecessary laparotomies. Conclusion: Based on significant difference in ploidy between adenocarcinoma and UC and between SPF and ploidy, assessment of ploidy by DNA cytometry for UC and other colitis could therefore predict impending malignant transformation before development of colonic dysplasia. Also measuring SPF in adenocarcinoma helps to select patients who could greatly benefit from chemotherapy. DL has vital role in staging GI cancers. Key Words DNA ploidy Histogram BRAF colon adenocarcinoma ==== Body pmcIntroduction Colorectal cancer (CRC) is one of most common malignancy worldwide. It develops from sequential activation of oncogenes and inactivation of tumor suppressor genes. Mutation in v-Raf murine sarcoma viral oncogene homolog B (BRAF) occurs in approximately 10%–15% of CRC (Barras, 2015). BRAF oncogene encodes BRAF protein, stimulates mitogen-activated protein kinase (MAPK) pathway. Change from valine to glutamic acid at codon 600 (V600E mutation) represents 80% of all BRAF mutations (Ritterhouse and Barletta, 2015). BRAF mutation is as well an independent predictor of poor prognosis in CRC (Barras, 2015). BRAF mutations in CRC can be detected by allele-specific polymerase chain reaction (PCR) or Sanger sequencing, but both are expensive and time-consuming. Nowadays, IHC against BRAF V600E mutant protein becomes feasible as an alternative diagnostic tool for BRAF V600E mutation detection in CRC (Bledsoe et al., 2014). DNA ploidy is a measurement of DNA content. In the histogram, the x-axis shows DNA-ploidy values (2c, 4c, and so on), where 2c are diploid cells where the nuclear DNA ploidy are in the G0/G1 phases of the cell cycle. The y-axis illustrates the number of nuclei corresponding to each interval in the histogram. A diploid histogram has a major peak corresponding to the DNA content of the 46 chromosomes (point 2c; DNA index, DI of 1), with a small proportion of cells captured at stages in which DNA is being, or had been, replicated, thus, possess up to double the normal amount of DNA (up to 4c on the x-axis). A tetraploid histogram shows an abnormal ‘tetraploid’ peak at the 4c indicating increased number of cells containing 4 sets of 23; DI 1.99 which represents either cells at G2 phase, or viable cells with tetraploid genome, or both. An aneuploid peak indicates that some cells possess an abnormal DNA content that is exceeds that of diploid cells, but less than if the whole set chromosome was duplicated. Aneuploidy is an inevitable result of chromosomal instability and can be detected quantitively using automated image-based DNA cytometry. The DI is the degree of aneuploidy and vary according to the extent of chromosomal losses or gains. S phase fraction (SPF) is the percentage of cells synthesizing DNA for chromosomal replication, lies along a continuum between diploid (2c) and tetraploid (4c) before completion of mitosis (Danielsen et al., 2016). DNA ploidy and SPF are significant independent factors for prognosis of CRC. Cases with DNA diploid tumors have a higher survival rate than those patient with aneuploid tumors. Aneuploid tumors have a higher median SPF than diploid tumors (Zhao et al., 2021). Ulcerative colitis is associated with a significantly increased risk for CRC development that is directly related to both extent and disease duration. To detect patients at high risk, colonoscopic surveillance with mucosal biopsy to detect dysplasia is widely used nowadays. Total reliance on microscopic changes as the only marker for malignant transformation has limitations owing to the subjective nature of the dysplasia assessment and interobserver and intraobserver variability due to the influence of inflammation. In addition, sampling error may occur because of the patchy nature of dysplasia. Detection of DNA aneuploidy in mucosal biopsies may be more reliable marker for subsequent malignant transformation of the colorectal mucosa (Beaugerie and Itzkowitz, 2015). Aneuploidy as well occurs as an early genetic alteration preceding morphologic changes of neoplasia, it is not confined to dysplastic epithelium only, DNA aneuploidy in flat mucosa may constitute an additional marker in the identification of patients at increased cancer risk who could benefit from a closer surveillance (Schimmelpenning et al., 2000). One plausible reason for colon cancer cells being chemotherapy unresponsiveness is the large cell number in the G0 phase (Jedema et al., 2003). In this study, we compared DNA ploidy and SPF in colonic adenocarcinoma, UC, CD, non-bilharzial colitis, bilharzial colitis and control group. We correlated DNA ploidy and SPF with tumor grading, staging and BRAF expression. Assessment of DNA ploidy could therefore be a main element in future surveillance programs for screening early malignant transformation. Evaluation of SPF of tumor cells could select the patients who have the greatest benefit from chemotherapy. Diagnostic laparoscopy (DL) has several advantages over other diagnostic modalities. This is reflected on its effectiveness in visualizing abdominal and pelvic cavities and detecting minimal ascites and small peritoneal deposits that may be missed by other imaging studies. It also detects adenopathies and small hepatic metastases. Moreover, it makes it possible to evaluate the local infiltration of the structures surrounding a given lesion and to decide whether it should be resected. An important goal of this technique is to reduce the number of negative laparotomies and reduce the surgical burden in patients who appear to have resectable abdominal masses. This results in reduced perioperative morbidity and mortality, shorter hospital stays, and earlier referral to chemoradiotherapy (Yeola et al., 2018). Materials and Methods Ninety patients were enrolled in this study. They were admitted to the Gastroentrology and Surgery Departments at the Theodor Bilharz Research Institute (TBRI) Hospital. The study protocol was approved by the Ethics committee of Theodor Bilharz Research Institute (TBRI) according to the institutional committee for the protection of human subjects and adopted by the 18th world medical assembly, Helsinki, Finland. The patients were grouped into: Group (1): control, 6 cases Group (2): non-bilharzial colitis, 8 cases. Group (3): chronic bilharzial colitis, 10 cases. Group (4): UC, 12 cases, 6 with dysplasia, 6 without dysplasia Group (5): CD, 10 cases. Group (6): CRC, 44 cases, all were non-Lynch adenocarcinoma (have intact mismatch repair genes; MMR). Immunostaining for MMR genes were performed (Figure 1, 2). The studied cases include 6 colonic biopsies with unremarkable pathological lesions as a control group. The patients of groups 2-5 underwent colonoscopy and biopsy from apparent gross lesions. Tissue samples of group 6 were obtained through partial colectomy specimens. All tissue specimens were delivered to the Pathology Department of TBRI for histopathological assessment. Operation protocol The peritoneal cavity was insufflated to 15 mm Hg and two trochars (10m, 5m) were introduced. Any suspicious lesion was biopsied and sent for frozen section, and if ascites was present, the fluid was collected and sent for cytology, if negative, we proceed for colectomy according to site of tumor. Histopathological study Tissues were fixed in 10% buffered formalin. A paraffin-embedded tissue biopsy is routinely processed and stained by Hematoxylin and Eosin stains. Histopathological diagnosis was done using the standard criteria for diagnosis of inflammatory bowel disease (IBD), WHO classification (Nagtegaal et al., 2020) and modified Dukes’ classification (Bresalier, 2010) of colo-rectal tumors. In the 10 patients of chronic bilharzial colitis, schistosomal infestation was diagnosed by identifying the bilharzial ova in tissue samples or detecting schistosomal antibodies in serum using ELISA technique. Immunohistochemical evaluation of BRAF Tissue sections from all different studied cases were immunohistochemically stained for BRAF V600E (ROCHE USA Tuscon) using the Dako autostainer (48 link, Dako, Denmark): Preparation of Slides - Three unstained Paraffin sections 5µ thickness for each case were cut by the microtome. Sections were mounted on the glass slides. - All slides were deparaffinized according to the setup of the laboratory where the autostainer is being installed. - After deparaffinization, we rinsed slides thoroughly under running water and placed them in a bath of autostainer wash buffer to soak for at least 5 minutes before staining. The reagent vials were labelled, the time was set on the computer to the time at the current autostainer location, and a protocol template was created using the programming as presented in DAB programming subsection. We loaded the reagents and slides onto the autostainer, then started the run. - After the run had ended, the slides were removed from the autostainer, placed in a bath and rinsed thoroughly under running water, counterstained with Hematoxylin, and finally we coverslip the slides using DPX. Interpretation of BRAF immunohistochemistry The intensity of anti-BRAF V600E in cells was graded on a 0–3 scale. Strong cytoplasmic staining was scored as 3, medium cytoplasmic staining as 2, weak cytoplasmic staining as 1 and the absence of staining was scored as 0. The staining is considered positive if diffuse (>50% of tumor cells) with intensity ≥1 (Dvorak et al., 2019) Evaluation of DNA Content Using Image Analysis System In analysis of DNA ploidy, the DNA content is measured per nucleus for all nuclei of a tissue specimen to yield a histogram illustrating the DNA content in each sample. This is an objective measure of genomic instability which discriminates between nuclei of normal DNA content and nuclei of abnormal DNA content. Principle of the Procedure The Feulgen stain specifically and quantitatively stains the nuclear DNA blue while the cytoplasm appears transparent. The quantity of this blue colored compound formed is directly proportional to the DNA content within the nucleus of the cell. In a population of normal and abnormal cells there is an obvious difference between the nuclei in the staining intensity. Cells in the S phase appear darker than their normal counterparts because of their increased amount of DNA content (Schutte et al., 1989). DNA Image Analysis Automated image analysis assessment of nuclear DNA of hepatocytes was performed using the computer-controlled analysis system (Image Analysis System, Zeiss Germany), this essentially consists of a computer-controlled microscope (Zeiss Axioscope microscope), video camera, one monitor and a computer unit. Image analysis technique was performed using the software program Axio vision 4.8. which allowed the colored compound that develops in the stained nuclei by Feulgen to be directly proportional to DNA content within the nucleus and can be measured as quantifiable integrated optical density (IOD) (Ranaldi et al., 1992). A number of nuclei ranging from 150-200 cells were submitted for DNA analysis in each case at 400x lens magnification. Only single monolayer nuclei without overlapping were analyzed. Reference cells are necessary for DNA scaling of densometric measurements. Imprint from normal mice liver were used as standard control. Reference cells were stained exactly as the cells under analysis in the same staining bath with slides of the sample. On analyzing their DNA content, reference histograms were elaborated and considered reference control histograms for sections under study (Schutte et al., 1989). The elaborated DNA histograms were classified into Diploid or aneuploid histograms based on DNA index (DI) of the main peak. Diploid Exhibiting a significant peak in the diploid range (DI=1±10%) were further sub classified according to the percentage of proliferating cells in the S phase fraction into: - Diploid histogram with mild increase in S phase, when the percentage of proliferating cells at S phase equal 10-20% of total number of analyzed cells. - Diploid histogram with moderate increase in S phase, when the percentage of proliferating cells at S phase equal 21-30 of total number of analyzed cells. - Diploid histogram with marked increase in S phase, when the percentage of proliferating cells at S phase exceeds 30 % of total number of analyzed cells (Eskelino et al., 1995; Yosef et al., 1996) Aneuploid histogram displaying a mass peak or multiple peaks outside the diploid or tetraploid range. Terms used in DNA analysis Ploidy: Analysis of DNA content Diploid: DNA content of the normal cell or 2C. Aneuploid: Abnormal DNA content. S Phase: The synthesis phase during which, there is rapid replication of the DNA content of the cell, which varied from 2C to 4C. DNA Index (DI): DI= Modal aneuploid Go/Gl DNA content /Modal diploid GO/Gl DNA content. The DI is commonly used to compare the DNA content of abnormal with that of normal cells G0: Resting phase of the cell cycle. G l: pre-synthetic phase. M phase: period of mitosis (Aziz et al., 1991; Borgmama et al., 1991). Results Ninety patients were enrolled in this study, they range in age from 30-86 years with mean age 58 years. Regarding the DNA content, there was significant difference between the studied groups. 77.5% of the aneuploid cases are adenocarcinoma, 17.5% are ulcerative colitis and 5% are Crohn’s disease. Regarding the diploid histogram, all the control cases (6/6), colitis (8/8), and bilharzial colitis (10/10) are diploid. Five out of the 12 cases of UC and 8 out of the 10 cases of CD are diploid. The variation between the different groups is statistically significant (P < 0.01). In analysis the relation between histogram of adenocarcinoma cases and Duke staging, there was significant difference between DNA content of adenocarcinoma cases and Duke stage (P= 0.01). 51.6% and 38.7% of the adenocarcinoma with aneuploid histogram are Duke B and C respectively. 37.5% and 50% of the adenocarcinoma with diploid histogram are Duke A and B respectively. Regarding the relation between histogram of adenocarcinoma cases and histological grading, there was significant difference between DNA content of adenocarcinoma cases and histological grade (P<0.01). 71% and 19.4% of the adenocarcinoma with aneuploid histogram are grade II and III respectively. 62.5% and 37.5% of the adenocarcinoma with diploid histogram are grade I and II respectively. Regard to the 2C DNA content, each of the control, colitis, bilharzial colitis, CD is significantly different from the adenocarcinoma group and dysplastic UC group. Non dysplastic UC is as well significantly different from the adenocarcinoma group. However no significant difference between the dysplastic UC group and adenocarcinoma group. Regarding the SPF, there is significant difference between the dysplastic UC and each of control, colitis and malignancy group. Regarding the above 4C DNA content, each of the control, colitis, bilharzial colitis, CD, non-dysplastic UC and dysplastic UC is significantly different from the adenocarcinoma group (Table 1). Regard to the 2C DNA content, Duke A is significantly different from that of B and C. In the SPF, there is significant difference between Duke B and C. No significant difference is found between Duke A, B or C in the above 4C DNA content (Table 2). Regard to the percentage of cells expressing BRAF and intensity of expression, there was significant difference between the groups (P<0.01). All adenocarcinoma cases show >50% BRAF expression and 72.7% show marked intensity. 83.33% of UC are positive for B raf showing >50% BRAF expression with moderate and marked intensity. BRAF was negative in all cases of colitis, bilharzial colitis and CD (Table 3). There was significant relation between percentage of BRAF expression and SPF, (P=0.02) and also significant relation between intensity and SPF among the diploid cases (P=0.002). 90% of diploid cases with mild increase in S phase are negative for BRAF. On the other hand, 44.4% of diploid cases with marked increase in SPF show >50% BRAF expression and 33.3% show marked BRAF expression (Table 4). There was significant relation between percentage of BRAF expression and SPF, (P=0.001) and also significant relation between intensity and SPF among the non-neoplastic diploid cases (P=0.01) (Table 5). All non-neoplastic diploid cases with mild increase in SPF are negative for B raf and 33.3% show mild intensity. On the other hand, 28.6% of non-neoplastic diploid cases with marked increase in S phase show >50% BRAF expression, and 57.1% and 21.4% show moderate and marked BRAF BRAF intensity respectively (Table 5). All adenocarcinoma cases show >50% BRAF expression. There was significant relation between intensity of BRAF expression and Duke staging. All adenocarcinoma cases in Duke C stage and 70% of Duke B show marked BRAF intensity. On the other hand, 33.3% and 66.7% of Duke A show mild and moderate expression respectively. This difference was statistically significant (P<0.01) (Table 6). Among the UC cases, there was no significant relation was detected between percentage of BRAF expression and DNA ploidy (P=1), BRAF although 75% of aneuploid cases show >50% BRAF expression. Also, no significant relation between intensity of BRAF expression and DNA ploidy (P=0.1), however, all cases of aneuploid cases show moderate and marked expression (Table 7). Among the IBD cases, there was no significant relation was detected between percentage of BRAF expression and DNA ploidy (P=0.3), however, 60% of aneuploid cases show >50% BRAF expression. Also, no significant relation between intensity of BRAF expression and DNA ploidy (P=0.3), however, all cases of aneuploid cases show moderate and marked expression (Table 8). Data was analysed using the Statistical Package for the Social Science Version 22 (IBM Corp., Armonk, NY, USA). Chi square, fisher exact test and analysis of variance (ANOVA) were used for comparing the qualitative variables. P value above 0.05 was considered significant. Histogram 1 A histogram of control mucosa (control case), the majority of the nuclei (70%) are diploid at 2C with S phase (20.73%), tetraploid at 4c (5.61) and DI peak 0.99 Histogram 2. A histogram of bilharzial colitis case, the nuclei (27.78%) are diploid at 2C with S phase (58.97%), tetraploid at 4c (13.25) and DI peak 0.94 Histogram 3 A histogram of non-bilharzial colitis case, the nuclei (43.22%) are diploid at 2C with S phase (47.46%), tetraploid at 4c (8.90), the nuclei above 5C (0.42%) and DI peak 1.11 Histogram 4 A histogram of UC case, the nuclei (26.60%) are diploid at 2C with S phase (58.87%), tetraploid at 4c (12.77), the nuclei above 5C (1.77%) and DI peak 1.28 Histogram 5 A histogram of Crohn’s case, the nuclei (46.88%) are diploid at 2C with S phase (46.88%), tetraploid at 4c (6.25), and DI peak 0.94 Histogram 6 A histogram of adenocarcinoma case, the majority of the nuclei are aneuploid (27.63 %) above 4C, S phase (25.99%), tetraploid at 4c (29.61), diploid at 2c (13.82%) and DI peak 1.17 Table 1 Nuclear DNA Content, in Colonic Lesions Group 2C (Mean ±SD) 4C (Mean ±SD) SPF (Mean ±SD) above 4C (Mean ±SD) Control (6) 69.70±13.72@$ 5.01±0.89@ 21.32±8.83$ 00.00±0.00@ Colitis (8) 63.75±6.43@$ 11.59±3.36@ 24.56±7.04$ 00.00±0.00@ Bilhcolitis (10) 57.40±7.88@$ 8.20±1.75@ 36.30±7.87 00.0 ±0.00@ Non-dysplastic UC (6) 50.79±8.87@ 12.63±6.17 39.37±10.19 00.0 ±0.00@ DysUC (6) 29.50±10.68 12.35±4.41 49.80±14.70**#@ 00.58±0.93@ CD (10) 62.06±8.65@$ 7.75±4.04@ 35.12±19.20 0.36 ±00.58@ CRC (44) 28.41±15.25 21.89±11.09 31.91±10.74$ 12.83±7.79 ** P<0.05, Significant difference from control group; # P<0.01 Significant difference from colitis group; @ P<0.01 Significant difference from adenocarcinoma group; $ P<0.01 Significant difference from Dysplastic UC group. Figure 1 Colorectal Adenocarcinoma Hematoxylin & Eosin (Original Magnification, x200) Figure 2 Colorectal Adenocarcinoma. Cases Showing Intact MMR Genes Immunostaining (Original Magnification, (a) 100x, (b) 200x, (c, d) 400x) Table 2 Nuclear DNA Content, in Adenocarcinoma Group 2C (Mean ±SD) 4C (Mean ±SD) SPF (Mean ±SD) Above 4C (Mean ±SD) Duke A (6) 46.72±9.10 21.06±10.15 29.38±9.01 13.30±13.11 Duke B (20) 29.27±14.27* 17.13±6.51 37.99±10.13 11.76±8.34 Duke C (18) 21.35±12.83* 27.46±13.17# 26.01±8.39# 13.86±4.76 * P<0.01, Significant difference from A duck group; #P<0.01 Significant difference from B duck group. Table 3 Relation between BRAF Immunohistochemical Expression among the Studied Groups Pathological Diagnosis Control Colitis Bilhcolitis UC Crohn Malignant BRAF percentage 0 6 100.00% 0 0.00% 0 0.00% 0 0.00% 0 0.00% 0 0.00% 1-25% 0 0.00% 5 62.50% 0 0.00% 0 0.00% 1 10% 0 0.00% 25-50% 0 0.00% 3 37.50% 10 100% 2 16.67% 9 90% 0 0% >50% 0 0.00% 0 0.00% 0 0.00% 10 83.33% 0 0.00% 44 100% BRAF intensity 0 6 100.00% 0 0.00% 0 0.00% 0 0.00% 0 0.00% 0 0.00% mild 0 0.00% 7 87.50% 1 10% 0 0.00% 4 40% 2 4.50% moderate 0 0.00% 1 12.5 9 90% 6 50% 6 60% 10 22.70% marked 0 0.00% 0 0.00% 0 0.00% 6 50% 0 0.00% 32 72.70% Total 6 100% 8 100% 10 100% 12 100% 10 100% 44 100% Figure 3 Control Case Immunostaining Showing Negativity for BRAF (Original Magnification, x200) Figure 4 Colonic Adenocarcinoma Cases Showing Immunostaining Positivity for BRAF, (a, b) marked intensity, (c) moderate, (d) weak intensity (Original Magnification, 200x) Table 4 Relation between BRAF Expression and SPF of the Diploid Cases Including Adenocarcinoma Cases S phase Fraction (SPF) Diploid with mild increase Diploid with moderate increase Diploid with marked increase P value BRAF percentage 0 5 50% 0 0.00% 1 5.60% 0.002 1-25% 1 10% 4 23.50% 1 5.60% 25-50% 3 30% 10 58.80% 8 44.40% >50% 1 10% 3 17.70% 8 44.40% BRAF intensity 0 5 50% 0 0.00% 1 5.60% Mild 3 30% 6 35% 3 16.70% Moderate 2 20% 9 52.90% 8 44.40% 0.002 Marked 0 0.00% 2 11.80% 6 33.30% Total 10 100% 17 100% 18 100% Table 5 Relation between BRAF Immunohistochemical Expression and SPF of the Non-neoplastic Diploid Cases S phase Fraction (SPF) Diploid with mild increase Diploid with moderate increase Diploid with marked increase P value BRAF percentage 0 5 55.60% 0 0.00% 1 7.10% 0.001 1-25% 1 11.10% 4 28.60% 1 7.10% 25-50 3 33.30% 10 71.40% 8 57.10% >50 0 0.00% 0 0.00% 4 28.60% BRAF Intensity 0.00 5 55.60% 0 0.00% 1 7.10% 0.001 Mild 3 33.30% 6 42.90% 2 14.20% Moderate 1 11.10% 8 57.10% 8 57.10% Marked 0 0.00% 0 0.00% 3 21.40% Total 9 100% 14 100% 14 100% Table 6 Relation between BRAF Immunohistochemical Expression and Duke Staging of Adenocarcinoma Duke Staging Duke A Duke B Duke C BRAF percentage 1-25% 0 0.00% 0 0.00% 0 0.00% 25-50% 0 0.00% 0 0.00% 0 0.00% >50% 6 100% 20 100% 18 100.00% BRAF intensity Mild 2 33.30% 0 0.00% 0 0.00% Moderate 4 66.70% 6 30% 0 0.00% Marked 0 0.00% 14 70% 18 100.00% Total 6 100% 20 100% 18 100% Table 7 Relation between BRAF Immunohistochemical Expression and DNA Ploidy in UC Cases UC Diploid Aneuploid BRAF % 0 0 0% 0 0.00% 1-25% 0 0% 0 % 25-50% 1 25% 2 25% >50% 3 75% 6 75% BRAF intensity Mild 0 0% 0 0% Moderate 2 50% 5 62.50% Marked 2 50% 3 37.50% Total 4 100% 8 100% Table 8 Relation between Immunohistochemical BRAF Expression and DNA Ploidy in IBD Cases IBD Diploid Aneuploid BRAF % 0 0 0% 0 0% 1-25% 1 8.30% 0 0% 25-50% 8 66.70% 4 40% >50% 3 25% 6 60% BRAF intensity Mild 4 33.30% 0 0.00% Moderate 6 50% 6 60% Marked 2 16.70% 4 40% Total 12 100.00% 10 100.00% Table 9 Cases in which DL Changed the Staging of the Disease Diagnosis Preoperative CT staging DL findings Post DL staging 2 cases Sigmoid cancer II Liver metastasis IV 1 case Rectal cancer II Liver metastasis and mimimal ascites IV 2 cases Colonic cancer II Liver metastasis IV Discussion Colorectal cancer (CRC) is one of most common malignancy worldwide. Aneuploidy is an inevitable result of chromosomal instability and can be detected quantitively using automated image-based DNA cytometry (Danielsen et al., 2016). DNA ploidy and SPF are significant independent factors for prognosis of colorectal carcinoma (Zhao et al., 2021). Aneuploidy is an early genetic alteration preceding morphologic changes of dysplasia. Thus, measuring DNA ploidy in UC could be used to screen early malignant transformation. (Schimmelpenning et al., 2000). In our study, there was significant difference in the DNA content between the studied groups. 70% of the adenocarcinoma cases (31/44) are aneuploid and 18% (8/44) are diploid. Also, Chapman et al., (1995); Jotti et al., (1995); Kouri, 1993; Flyger et al., (1999) and Hong-yi et al., (2001) found aneuploidy in 70%, 70%, 62%, 89%, 62% and 58% of colon adenocarcinoma, respectively. According to Salud et al., (1999); Karelia et al., (2001) and Çobanoğlu et al., (2009), 58.9% (44/107), 41% (32/79) and 58% of colorectal adenocarcinoma are aneuploid. According to our results, all cases of non-bilharzial colitis (8/8), bilharzial colitis (10/10) are diploid. No other studies were found testing the DNA ploidy in these two groups. We didn’t detect abnormal DNA content in any of the normal appearing colonic mucosa (control, 0/6). Also, Miyazaki et al., 1999, found no aneuploid peaks in normal appearing colonic mucosa. In our study, 58% (7/12) of UC show aneuploidy and 42% are diploid and there was significant difference in DNA content (2c) between non-dysplastic UC (mean =50.79) and adenocarcinoma (mean=28.41). According to Meling et al., (1991), DNA aneuploidy was found in 60% of the mucosa samples of UC (dysplastic and non- dysplastic). According to Fozard et al., 1986, 42% of UC are aneuploid with higher rate of DNA aneuploidy in dysplastic tissues (21%) compared with non-dysplastic tissues (15%). Schimmelpenning et al., (2000) detected aneuploidy in 25% of UC cases and all developed adenocarcinoma within seven years follow up. In our work, 80% (8/10) of CD are diploid and CD show less SPF (35±19)% than dysplastic UC (49.80±14.70) and non-dysplastic UC (39.37±10.19). Keeping with our study, Porschen et al., (1992), noted diploid histogram in 90% of CD with higher SPF in UC (17.8±7.7) % than in CD (13.1±4.6) %. In our study, the mean SPF of adenocarcinoma cases is 31.91±10.74. According to Karelia et al., (2001), 30 % of colorectal cancers have a high (>10%) S-phase fraction. Bazan et al., (2002) detected high SPF (>18.3%) among colonic adenocarcinomas. In our work, a significant difference was detected between DNA content of adenocarcinoma cases and histological grade and Bazan et al., (2002) documented this significant relation. Opposite to our results, Suzuki, (1988); Chapman et al., (1995); Salud et al., (1999) and Hong-yi et al., (2001) found no significant relation between DNA content of adenocarcinoma cases and histological grade. As regarding the relationship between DNA ploidy and pathologic staging, there was significant difference between DNA content of adenocarcinoma cases and Duke stage (P= 0.01). Aneuploid tumors were identified among those with a more advanced pathologic stage in studies done by Tribukait et al., (1983); Jones et al., (1988); Halvorsen and Johannesen, (1990); Kouri et al., (1990); Salud et al., (1999); Bazan et al., (2002) and Çobanoğlu et al., (2009). In contrast to us, Armitage et al., (1985); Suzuki et al., (1988); Armitage et al., (1991); Chapman et al., (1995) found no significant relation between DNA ploidy and tumor stage. In our work, Duke B is significantly lower from Duke C adenocarcinoma in SPF. In agreement to our finding, Pinto et al., (1997); Bazan et al., (2002) also found this significant positive relation. Chen et al., (2002) found no significant relation between SPF of adenocarcinoma and tumor staging. In our work, all adenocarcinoma cases show >50% BRAF expression and 72.7% show marked intensity. Similarly, González-Colunga et al., (2020) found intense and diffuse reaction against BRAF V600E in all cases of CRC with known BRAF mutation indicating that BRAF V600E IHC is sensitive, making it feasible as an alternative method for molecular evaluation. According to our study, BRAF positivity was found in 83% (10/12) of UC cases. Yang et al., (2018) detected positivity in 51% of UC cases. In our study, BRAF was negative in all cases of non-bilharzial and bilharzial colitis. No corresponding studies were found. In our work, all cases of CD are negative for BRAF. Marcuzzi et al., (2013) studied genetic changes in CD and detected no BRAF expression. It is not from the susceptibility mutated genes. We found a significant increase relation between Duke stage of adenocarcinoma and intensity of BRAF expression. Similarly, Luu and Timothy (2018); Ardekani et al., (2012); Barras (2015), Clarke and Kopetz, (2015) documented this significant relation. In the current study, DL altered the staging of 5 of the cancer patients (12.5%) by detecting liver deposits or minimal malignant ascites not identified by radiological investigations. This is consistent with a prospective study done on 40 patients by El Zanati et al., (2020), proved the efficacy of DL that also altered the staging in 24.3% of the cases in their study. Author Contribution Statement They share in putting the idea of the research, collecting the studied cases after revising their data and examining the slides. They supervised the results and their statistical analysis. The corresponding author wrote the paper and other authors read it and discussed all points. Acknowledgements Recommendation of The Study Further extended studies are recommended to prove the efficacy of DNA ploidy assessment to detect early malignant transformation in non-neoplastic colitis even development of any dysplastic morphologic change. Also, correlation between measuring SPF and chemotherapeutic response of the tumor needs more research. Ethics approval The research was approved by the Ethics committee of Theodor Bilharz Research Institute (TBRI) according to the institutional committee for the protection of human subjects and adopted by the 18th world medical assembly, Helsinki, Finland. The study haven’t been submitted to any scientific body. ==== Refs References Ardekani GS Jafarnejad SM Tan L Saeedi A Li G The Prognostic Value of BRAF Mutation in Colorectal Cancer and Melanoma: A Systematic Review and Meta-Analysis PLoS One 2012 7 e47054 23056577 Armitage NC Robins RA Evans DF The influence of tumourcell DNA abnormalities on survival in colorectal cancer Br J Surg 1985 72 828 30 4041716 Armitage NC Ballantyne KC Sheffield JP A prospective evaluation ofthe effect of tumor cell DNA content on recurrence in colorectal cancer Cancer 1991 67 2599 604 2015560 Aziz DC Peter JB Wax S DNA ploidy and cell cycle analysis tools for assessment of cancer Prognosis J Clin Pathol 1991 5 422 38 Barras D BRAF Mutation in Colorectal Cancer: An Update Biomark Cancer 2015 7 9 12 26396549 Bazan V Migliavacca M Zanna I DNA Ploidy and S-phase fraction, but not p53 or NM23-H1 expression, predict outcome in colorectal cancer patients Result of a 5-year prospective study J Cancer Res Clin 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116168 10.31557/APJCP.2023.24.4.1435 Research Article Relationship between Symptom Burden and HRQOL among Kuwaiti Women Recently Diagnosed with Breast Cancer: A Cross-Sectional Study Safar Hanan 1* Mazanec Susan R. 2 1 College of Nursing, the Public Authority for Applied, Education and Training, Kuwait. 2 Frances Payne Bolton School of Nursing, Case Western Reserve University, Cleveland, OH, USA. * For Correspondence: hanansafar2023@gmail.com 2023 24 4 14351441 14 2 2023 8 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Breast cancer is a life-threatening chronic condition associated with distress and psychological symptoms. Breast cancer also leads to ongoing ambiguity around the symptom burden of the disease and its treatment over the long-term, which impacts health-related quality of life (HRQOL). The factors influenced HRQOL of Kuwaiti women with breast cancer is unclear and not well understood. Purpose: The purpose of the study was to explore the relationships between the symptom burden and HRQOL of Kuwaiti women diagnosis with breast cancer within their first year. Methods: This cross-sectional correlational study surveyed 100 Kuwaiti women diagnosed with breast cancer within the previous year at the Kuwait Cancer Control Center Hospital (KCCC). The study collected data using a combination of five questionnaires: The Memorial Symptom Assessment-Short Form Scale (MSAS-SF), the Medical Outcomes Study Social Support Survey (MOS-SSS), the Functional Assessment of Cancer Therapy-General (FACT-G), and demographic/clinical questionnaire. Results: The 100 Kuwaiti women in the study experienced moderate symptom burden (M = 2.35, SD = 0.28), which is significantly negatively associated with HRQOL. The most prevalent symptoms the women reported were pain, difficulty sleeping, lack of energy, and hair loss. Symptom burden was significantly negatively associated with HRQOL. Conclusion: The findings of this study suggest the need for more training for clinicians to diagnose and treat common symptoms. Improved screening tools and psychosocial interventions also need to be developed. Future research should focus on longitudinal data and qualitative methods to gain a more comprehensive understanding of Kuwaiti women’s experiences with breast cancer. Key Words Female quality of life breast neoplasms Kuwait symptom assessment ==== Body pmcIntroduction Worldwide, breast cancer is the most frequently diagnosed malignancy, accounting for over a million cases every year, and it is the leading cause of death among women worldwide (Arnold et al., 2022). In Kuwait, breast cancer is the ninth leading cause of death, and in 2014, the Kuwait registry reported an incidence rate of 58.5 cases per 100,000 (World Health Rankings, 2017). Being diagnosed with breast cancer could be a life-altering experience associated with feelings of uncertainty in illness. Undergoing the process of diagnosis and treatment can be physically and emotionally taxing for women with breast cancer and those close to her. After being diagnosed with breast cancer, a woman may realize her life has a different meaning and she will not be the same (Leão et al., 2022). As the diagnosis of breast cancer and its treatment could be unpleasant and experience (Traboulssi et al., 2022). Women with breast cancer experience uncertainty in illness (UII) (Hong et al., 2022), due to the inability to define the feels and value of their disease and related incidents and the potential risks associated with a breast cancer diagnosis and consequently try to manage it through coping strategies (Greco, 2022). Breast cancer leads to uncertainty in illness, indicating a challenging familiarity that can include health-related quality of life (HRQOL) and the capability to cope with the illness (Sharif, 2017; Sharif et al., 2017). Contemporary scientific improvement for the diagnosis and treatment of breast cancer has raised patients’ survival (Rah et al., 2019); hence, improving HRQOL during the disease has become a more pressing concern(Ho et al., 2018). There are insufficient data and limited studies regarding the uncertainty in illness and HRQOL among women with breast cancer in the Middle East (ME) and Arabia Gulf countries. Hence, this study aims to identify the associations of symptom burden and HRQOL in Kuwaiti women with breast cancer, thereby raising awareness among Kuwaiti women with breast cancer regarding factors influencing their HRQOL. The study also aims to enable healthcare providers to set strategies that promote patients’ wellbeing. The purpose of the proposed study is to explore the relationship between symptom burden, uncertainty in illness, perceived social support, and HRQOL among Kuwaiti women with breast cancer within the first 12 months of their diagnosis. This is a descriptive correlational, cross-sectional study, Materials and Methods Design and participants This correlational cross-sectional study was conducted on 100 Kuwaiti women recently diagnosed with breast cancer referral to the medical and surgical oncology outpatient clinic during follow-up appointments at the Kuwait Cancer Control Center Hospital (KCCC). Participants were either under active treatment or receiving checkups. A private room was utilized to ensure participant confidentiality and privacy. This hospital was chosen because it is the largest cancer hospital in Kuwait. Data was collected over a four-month period (10/2019 – 02/2020). Approval for the study was sought from the University Institutional Review Board (IRB) of Case Western Reserve University (CWRU), the Kuwaiti Ministry of Health (MOH), and the KCCC hospital ethics board. Clinical staff at the KCCC hospital were informed about the study purpose, needs, and inclusion and exclusion criteria. Staff at the KCCC clinic served only in the coordination role and were not involved in the data collection. Only the researcher obtained informed consent and collect data. Data collection Following approval from the Institutional Review Board (IRB) at Case Western Reserve University (CWRU), the Kuwaiti Ministry of Health (MOH), and the ethics board at the Kuwait Cancer Control Center Hospital (KCCC), recruitment of participants began. A convenience sampling method was accomplished using the electronic records from the outpatient clinic database after gotten permission from the ministry of health and hospital administration. Recruited participants included women within the first 12 months of their diagnosis of breast cancer from all Kuwaiti cities referred to KCCC hospital, with no restriction on breast cancer treatment type. Recruited participants were in stable clinical condition (i.e., not acutely ill, not requiring hospitalization, or actively dying), and able to independently provide informed consent to participate in the study without the assistance of family members. Eligibly criteria All research participants met the following inclusion criteria:18 years or older, Female, A citizen of Kuwait, Within one year of their clinical diagnosis of stage (1–III) breast cancer, An outpatient (pre-treatment, chemotherapy/radiation treatment, and post-treatment follow-up), Speak Arabic and able to provide signed consent. Participants were excluded from this study if they were: Acutely ill, hospitalized, or actively dying, diagnosed with stage 4 breast cancer, Diagnosed with any other type of cancer previously except non-melanoma skin cancer and Cognitively impaired (i.e., disoriented to place, person, or time) and Unable to give consent independently. Determination of the sample size was carried out using G*Power 3.1 software. Using a multiple regression model with one independent variables (symptom burden), and one dependent variable (HRQOL) and an alpha of 0.05 and power of 0.80, G*Power calculated a total required sample of 92. Measurements Empirically, three instruments were used as measures in the study. The first questionnaire assessing demographic and medical characteristics (age (in years), marital status, educational level (degree), and current employment status. Health history includes: family cancer history (the type of cancer, when the family member’s cancer was diagnosed, with a primary interested in breast cancer among family), cancer stage, time since diagnosis and current treatment, and other medical conditions.), and then the Memorial Symptom Assessment- Short Form Scale (MSAS-SF), and the Functional Assessment of Cancer Therapy-General (FACT-G). The Memorial Symptom Assessment Scale Short Form (MSAS-SF), a shortened version of the original MSAS, measures each of 28 symptoms concerning distress or frequency alone (Chang et al., 2000). The instrument scoring is designed to include three subscales: The Global Distress Index (GDI), the Physical Symptom subscale (PHYS), and the Psychological Symptom subscale (PSYCH). The Global Distress Index (GDI) consists of 10 items that evaluate overall symptom distress. The FACT-G has 27 questions, measure the respondents’ health state over the last seven days in four subscales: physical well-being (PWB; seven items, score range 0 to 28), social/family well-being (SWB; seven items, score range 0 to 28), emotional well-being (EWB; six items, score range 0 to 24), and functional well-being (FWB; seven items, score range 0 to 28), and some items being reverse-scored (Cella et al., 1993). Data Analysis Descriptive statistics conducted include frequencies for all study variables. Means and standard deviations reported for continuous variables, frequencies and percentages for categorical variables, and skewness and kurtosis for the shape of the distribution. Descriptive and inferential statistics including Pearson’s correlation coefficient and multiple linear regressions were conducted to answer the research questions. Relationship between symptom burden and HRQOL was assessed using Pearson’s correlation coefficient test. Results Demographic Characteristics There was no refusal of participation and no one was excluded. The study consists of a sample of 100 participants. The mean age of the participants was 50.8 year. Further, the result shows that the maximum number of the participants were from the age group 58 and above and an equal number of participants n (%), 21 (21%) came from the age group 38-48. Moreover, only 34 (34%) of the participants were married. More than 50 (50%) of the participants were employed and the majority of the participants had either a diploma 32 (32%) or an undergraduate degree 39 (39%) (Table 1). Results of the study indicated that out of the 100 participants n (%) 72 (72%) had a family history of cancer. It can also be noticed that 53 (53%) of the participants had stage III breast cancer. At the time of the survey, 91 (91%) of the participants were receiving some kind of therapy. More than half of the participants were receiving chemotherapy 61 (61%). Almost all the participants had some type of surgery 99 (99%). Moreover, the average time since diagnosis was six months (SD = 3.08. Ninety-eight percent (n= 98) of the participants had co-morbid conditions, most commonly diabetes mellitus, dyslipidemia, lung disease, and heart disease. On average, the total number of co-morbidities was three (SD = 1.79). The average of ECOG score was calculated to be 1.54 (SD = 0.93) Table 2. Main results The overall score for (FACT-G) was (M = 62.25, SD = 8.96), which is a range of moderate. The average value of (SWB was (M = 18.67, SD = 3.92) followed by the average value of (FWB) (M = 16.27, SD = 3.32) and (EWB) (M = 14.34, SD = 2.88). The lowest average was observed for the subscale Physical Well-Being (M = 12.97, SD = 3.08) Table 3. The average overall symptom burden score was found to be (M = 2.35, SD = 0.28), which is near to the mid-range score considered to be in the moderate range. The score of subscale MSAS-PSYCH (M = 2.61, SD = 0.38) was higher than the score of the subscale MSAS-PHYS (M = 2.51, SD = 0.28). This means that Kuwaiti women with breast cancer complain of psychological symptom burden more than physical symptom burden, although the mean scores were very close (Table 4). The results showed that the most distressing physical symptom was pain, followed by difficulty sleeping, lack of energy, a change in the way food tastes, hair loss, lack of appetite, difficulty concentrating, weight loss, dizziness, and difficulty swallowing. The most distressing psychological symptom was feeling irritable, worrying and feeling nervous, whereas the least distressing symptom was feeling sad. The most frequent symptoms were pain, difficulty sleeping, lack of energy, hair loss, a change in the way food tastes, lack of appetite, weight loss, difficulty concentrating, numbness/tingling in hands/feet, and dizziness (Table 5). A Pearson correlation was performed to check the associations between symptom burden overall, physical, and psychological (MSAS-SF, MSAS-PHYS, and MSAS-PSYCH), and HRQOL (FACT-G). The results of the study showed a statistically significant negative correlation between symptom burden and HRQOL with a correlation coefficient (r= - 0.354; p= .000). It shows that as the value of overall symptom burden increases, HRQOL decreases. Further, the variables of physical symptom burden and psychological symptom burden also show a significant negative correlation with HRQOL, with the values of correlation coefficient (r= - 0.209; p=0.037) and (r= - 0.528; p= 0.000) respectively. The negative correlation of physical and psychological symptom burden with HRQOL indicates that as the value of physical and psychological symptom burden increases HRQOL decreases (Table 6). Table 1 Demographic Characteristics Participants (N=100) Mean (SD) Age (24-72) 50.8 (11.9) Age Group: N=100 from 18 to less than 28 2 (2.0%) from 28 to less than 37 14 (14.0%) from 38 to less than 48 21(21.0%) from 48 to less than 58 21 (21.0%) from 58 and above 42 (42.0%) Marital Status: Married 34 (34.0%) Divorced 40 (40.0%) Widowed 14 (14.0%) Separated 12 (12.0%) Current Employment Status: Employed 54 (54.0%) Retired 29 (29.0%) Not Employed 14 (14.0%) Other 3 (3.00%) Education Level: Elementary school degree 1 (1.00%) Intermediate school degree 8 (8.00%) High school degree 13 (13.0%) Diploma degree 32 (32.0%) Undergraduate degree 39 (39.0%) Graduate degree 7 (7.00%) Table 2 Medical Characteristics of Participants N=100 Family history of cancer 72 (72.0%) Cancer stage: I 2 (2.00%) II 45 (45.0%) III 53 (53.0%) Receiving therapy currently 91 (91.0%) Type of treatments currently received: Chemotherapy 56 (60.9%) Hormonal 11 (12.0%) Combination 24 (26.1%) Surgery 99 (99.0%) Type of surgery: Lymphadenectomy and Mastectomy 27 (27.0%) Lumpectomy Only 13 (13.0%) Lumpectomy and Lymphadenectomy 1 (1.00%) Mastectomy Only 58 (58.0%) Time since diagnosis (until enrolment) Mean (SD) 6.00 (3.08) N=100 Less than 4 months 27 (27.0%) 4 to 8 months 38 (38.0%) More than 8 months 35 (35.0%) Time since complete treatment (until enrolment): 3.18 (2.50) Less than 4 months 60 (60.6%) 4 to 8 months 33 (33.3%) More than 8 months 6 (6.06%) Told by a doctor has co-morbid conditions 98 (98.0%) Total number of co-morbid conditions Mean (SD) 2.55 (1.79) Types of co-morbidities: N=100 High blood pressure 2 (2.00%) Diabetes mellitus 37 (37.0%) Dyslipidemia 22 (22.0%) Heart disease 10 (10.0%) Lung disease 13 (13.0%) Stroke 8 (8.00%) Anemia 6 (6.00%) Kidney disease 1 (1.00%) Liver disease 1 (1.00%) ECOG[2] Score Mean (SD 1.54 (0.93) ECOG Status: N=100 Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature 10 (10.0%) Ambulatory, capable of all self-care, unable to carry out any work activities more than 50% of waking hour 45 (45.0%) Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 28 (28.0%) Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair 17 (16.70%) [2], Eastern Cooperative Oncology Group (ECOG) Table 3 Assessment of Quality of Life of Participants Using Functional Assessment of Cancer Therapy-Breast Cancer (FACT-G) Scale/ subscale Mean Median SD 95% CI Range Alpha Functional Assessment of Cancer Treatment-General (FACT-G) 62.25 62.5 8.96 [60.50-64.03] 40-87 0.821 Physical well-being 12.97 13.0 3.08 [12.40-13.60] 6-21 0.745 Social/family well-being 18.67 18.5 3.92 [17.70-19.50] 10-28 0.721 Emotional well-being 14.34 15.0 2.88 [13.80-14.90] 9-20 0.694 Functional well-being 16.27 16.0 3.32 [15.60-17.00] 8-26 0.782 Breast cancer-specific concern 19.5 19.0 3.43 [18.90-20.00] 13-30 0.586 Alpha, Cronbach’s alpha Table 4 Assessment of Symptom Burden among Participates Scale/ subscale Mean Median SD 95% CI Range Alpha MSAS-SF 2.35 2.32 0.28 [2.30-2.40] 1.75-3.05 0.875 MSAS-PHYS 2.5 2.47 0.28 [2.45-2.56] 1.67-3.13 0.871 MSAS-PSYCH 2.61 2.58 0.38 [2.53-2.68] 1.67-3.57 0.884 Note: Alpha, Cronbach’s alpha; The Memorial Symptom Assessment-Short Form Scale (MSAS-SF); The Memorial Symptom Assessment-physical symptom distress (MSAS-PHYS), The Memorial Symptom Assessment-psychologic symptom subscale(MSAS-PSYCH) Table 5 Most Distressing Physical and Psychological Symptoms Reported on the MSAS-SF Scale items Mean Median SD 95% CI Range Physical Symptoms Pain 3.10 3.2 0.32 [3.03-3.16] 2.4-4 Difficulty sleeping 3.02 3.2 3.02 [2.91-3.14] 1.6-4 Lack of energy 2.98 3.2 0.42 [2.90-3.07] 1.6-4 Change in the way food tastes 2.94 3.2 0.47 [2.85-3.04] 1.6-4 Hair loss 2.89 3.2 0.67 [2.76-3.03] 1.6-4 Lack of appetite 2.89 3.2 0.48 [2.79-2.98] 1.6-4 Difficulty concentrating 2.87 3.2 0.46 [2.78-2.96] 1.6-4 Weight loss 2.81 3.2 0.62 [2.68-2.93] 1.6-4 Dizziness 2.79 2.4 0.48 [2.69-2.89] 1.6-4 Difficulty swallowing 2.74 2.4 0.56 [2.63-2.86] 0.8-4 Psychological Symptoms Felling irritable 2.53 3.2 0.67 [2.40-2.66] 4-Jan Worry 2.53 3.2 2.53 [2.41-2.65] 4-Jan Felling nervous 2.52 3.2 0.42 [2.38-2.66] 4-Jan Felling sad 2.16 3.2 0.66 [2.06-2.26] 4-Jan Table 6 Associations between Symptom Burden Overall and HRQOL among Breast Cancer Patients Variable Pearson's r (FACT-G) P-value MSAS-SF -0.354** 0.001 MSAS-PHYS -0.209* 0.037 MSAS-PSYCH -0.528** 0.001 Note. *p<0.1, **p<0.05; The Memorial Symptom Assessment-Short Form Scale (MSAS-SF),The Memorial Symptom Assessment-physical symptom distress (MSAS-PHYS), The Memorial Symptom Assessment-psychologic symptom subscale(MSAS-PSYCH) Discussion The purpose of this quantitative, cross-sectional correlational study was to explore the associations between symptom burden and HRQOL among 100 Kuwaiti women with breast cancer. Total symptom burden in this study showed a moderate mean score. These findings are consistent with other studies reporting findings on the same measure in Middle Eastern patients with breast cancer (Huijer and Abboud, 2012) and other type of cancer (Huijer et al., 2012). However, descriptive statistics of psychological and physical symptom burden in the present study showed a higher mean score than other studies reporting findings on the same measures in patients with cancer (Huijer and Abboud, 2012; Huijer et al., 2012). This could be due to the affective nature of breast cancer as a disease as well as the side effects of treatment (Hamer et al., 2017). Of these four studies only two were focused on women with breast cancer undergoing active chemotherapy treatment (Huijer and Abboud, 2012), while the other two studies were on Lebanese men and women with different types of cancer, including breast cancer. The most frequent symptoms reported in this study on patients with breast cancer were pain, difficulty sleeping, lack of energy, hair loss, changes in the way food tastes, lack of appetite, and weight loss. This study finding was consistent with the other two studies about patients with cancer (Huijer and Abboud, 2012; Huijer et al., 2012), in which patients reported that pain, difficulty sleeping, and lack of energy were the most frequent symptoms. Mishel and colleagues stated that fatigue, sleep disturbance, and disrupted mood are the most prevalence reported by 313 breast cancer survivors’ even years post treatment (Hall et al., 2014). Also, these results are similar to a study conducted in Kuwait, where women with breast cancer undergoing chemotherapy treatment reported high rates of fatigue, pain, and difficulty sleeping (Alawadi and Ohaeri, 2009). However, this study used a different tool to assess women’s symptoms under active chemotherapy treatment, the European Organization for Research and Treatment of Cancer and Breast Cancer-Specific Quality of Life Questionnaire (EORTC QLQ–C30 and QLQ-BR23), rather than the assessment tools used in the current study. The current study’s findings were inconsistent with the study conducted by Abu-Saad Huijer et al., (2015), in which lack of energy was the most frequent symptom, followed by feeling nervous and feeling sad. The most distressing physical symptoms reported in the current study were pain, difficulty sleeping, and lack of energy, changes in the way food tastes, hair loss, and lack of appetite. Lack of energy and pain are consistent with physical symptoms that Lebanese women with breast cancer reported as highly distressing (Huijer and Abboud, 2012) However, inconsistent with the current study’s findings, Abu-Saad Huijer et al., (2012) reported pain, difficulty swallowing, worry, feeling like “I don’t look like myself,” and feeling nervous as the most distressing symptoms. These results indicate that Lebanese patients with cancer experienced more distress associated with psychological symptoms than with physical symptoms. In the present study, pain was the most frequent and the most distressing physical symptom, which is supported by the findings of Dhingra et al., (2015) on patients with cancer. The most distressing psychological symptom reported in this study was feeling irritable, followed by worrying, feeling nervous, and feeling sad respectively. These findings are consistent with other studies examining symptom management in patients with various types of cancer in Lebanon (Huijer and Abboud, 2012; Huijer et al., 2012). The current study illustrated that psychological symptom burden was more strongly correlated with HRQOL than physical symptom burden, a conclusion that is supported by a previous study on women with breast cancer (Huijer and Abboud, 2012), where the most prevalent psychological symptoms women described were feeling nervous and feeling sad (Huijer and Abboud, 2012). Consistent with (Rogers et al., 2017), patients with breast cancer experience depression, and/or anxiety, and fatigue months to years post diagnosis beside these symptoms were correlated with low HRQOL. This finding indicates that healthcare providers treating women for breast cancer should address the need for psychological education for patients on the nature of breast cancer as a disease, the implications of treatment, side effects, and psychological concerns. Thus, medical practitioners and researchers can work together to explore approaches for coping with breast cancer which can boost the psychosocial welfare of women with a breast cancer diagnosis. The results indicate a moderate level of HRQOL among the Kuwaiti women in this sample. The mean score (62.25) is lower than other studies of Middle Eastern women with breast cancer measuring QOL with the FACT-G. Reported FACT-G mean scores were 68, 69.63, and 84.74 in studies conducted in Iraq, Iran, and Lebanon, respectively (Zamanian et al., 2015; Al-Naggar et al., 2016; Akel et al., 2017). The findings in the present study are consistent with the findings of Khater et al., (2019), whose participants reported a FACT-G score of (62), and where more than half (56.8%) of the Egyptian women in the study were in stage III breast cancer and (83.8%) and under active chemotherapy treatment (Khater et al., 2019). According to a study by Al-Alawadi and Ohaeri (2009), Kuwaiti women with breast cancer under active chemotherapy treatment had globally poor HRQOL (45.3), as measured by the EORTC QLQ-C30 tool (Alawadi and Ohaeri, 2009). The results of these studies may be explained by the fact that they only capture a snapshot of a single moment in a women’s life, rather than exploring stability or change in the HRQOL throughout the cycle of diagnosis, treatment, and/or remission. Overall symptom burden, physical symptom burden, and psychological symptom burden each had an inverse, significant relationship with HRQOL. As physical and psychological symptom burden increased, HRQOL decreased. This study supports previous research showing an inverse relationship between symptom burden and HRQOL. These findings are consistent with Abu-Saad Huijer and Abboud’s (2012) study that found a significant, inverse correlation between symptom burden and HRQOL among Lebanese women with breast cancer. Similarly, Nho et al., (2018) found symptom clusters had a negative impact on HRQOL among Korean women with breast cancer. Women with breast cancer often have multiple physical and psychological symptoms, such that making a comprehensive symptom assessment a requisite toward good symptom control and an important component of QOL assessment (Alawadi and Ohaeri, 2009; Hamer et al., 2017; Hashemi et al., 2019; Lee and Park, 2020). This study can help fill the gap in the literature on Kuwaiti women in particular, and the connections between HRQOL, symptom burden in relation to a breast cancer diagnosis. This study will serve as a foundation for additional research on symptom burden, and HRQOL practice among Kuwaiti women with breast cancer which will advance the nursing body of knowledge by researchers using the uncertainty in illness theory and scales. In particular, the sections below make a number of recommendations for nursing clinical practice, nursing education, and nursing research. Included patients in present study were in stage 1 to 3 that the symptoms can be different from breast cancer patients in stage 4 so the variety and severity of symptoms can be impact on quality of life in breast cancer patients in different stages (Zaker et al., 2021). About study limitations first, the study used a cross-sectional and correlational design that examined the association between the variables rather than considering cause and effect. Since a cross-sectional design was used, the degree of uncertainty in illness due to life circumstances prior to the diagnosis of breast cancer cannot be determined in this study’s design. The absence of a baseline assessment limits the study from being able to determine the complete extent of the effects that the diagnosis, as well as the illness event itself, have had on women’s level of uncertainty in illness. Second, the selection of a convenience sampling method may have caused a sampling bias that might jeopardize the external validity of the study. For example, this study did not represent Kuwaiti women with breast cancer who get their treatment abroad (which is optional for Kuwaiti patients), and, thus, the generalizability of the study findings is limited among Kuwaiti women as a whole. Also, this sample was limited to women who had been diagnosed within a year. This limited sample leaves out women who have been diagnosed over longer periods and who are in different stages of treatment and/or recovery. Additionally, the participants of this study were all recruited from one particular hospital (KCCC) in Kuwait. Thus, these findings cannot be generalized to all Kuwaiti patients. Third, self-reporting questionnaires were used to collect data, which might have resulted in response bias, social desirability bias, and inaccuracies that could have affected the findings. A qualitative study could integrate data on a greater variety of factors that influence participants’ uncertainty in illness. About strengths of study since there have been no studies since 2009 conducted with Kuwaiti women with breast cancer, this study represents an important update and expansion of the literature on the subject. Data were collected over four months with a positive response rate of 100 participants. The actual sample size was adequate to detect existing associations between and among the major study variables. Additionally, there were no missing data, which could reduce the study’s statistical power and the result is well representativeness of the samples. The study outcomes were assessed and analyzed through Arabic versions of validated and reliable Arabic questionnaires. Finally, the flexibility of a cross-sectional study with data collection taking place at one particular point in time allowed for a fairly quick response, which allowed for timely analysis and interpretation of the study’s findings. In conclusions, the results of this study suggested that Kuwaiti women reported a moderate level of symptom burden, which is significantly associated with their HRQOL. This study helps to raise awareness of Kuwaiti women’s experiences and highlights the need to develop a more holistic model of care for women with breast cancer and survivors that incorporates their physical and psychological needs. Author Contribution Statement All authors contributed equally in this study. Acknowledgements This study obtained from PhD thesis conducted in Frances Payne Bolton School of Nursing CASE Western Reserve University. We would like to extend our sincere thanks to the staff in Kuwait Cancer Control Center Hospital (KCCC). Ethics approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee-of Iran and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study. Data Availability Statement The data that support the findings of this study are available from the corresponding author, upon reasonable request. Conflict of Interest The authors declare no competing interests. ==== Refs References Akel R El Darsa H Anouti B Anxiety, depression and quality of life in breast cancer patients in the levant Asian Pac J Cancer Prev 2017 18 2809 29072421 Al-Naggar RA Osman MT Al-Baghdadi N Study of quality of life and characteristic factors in women with breast cancer undergoing different types of therapy J Appl Pharm Sci 2016 6 147 52 Alawadi SA Ohaeri JU Health–related quality of life of Kuwaiti women with breast cancer: A comparative study using the EORTC Quality of Life Questionnaire BMC Cancer 2009 9 1 11 19118499 Arnold M Morgan E Rumgay H Current and future burden of breast cancer: Global statistics for 2020 and 2040 Breast J 2022 66 15 23 Cella DF Tulsky DS Gray G The Functional Assessment of Cancer Therapy scale: development and validation of the general measure J Clin Oncol 1993 11 570 9 8445433 Chang VT Hwang SS Feuerman M The memorial symptom assessment scale short form (MSAS-SF) validity and reliability Cancer 2000 89 1162 71 10964347 Dhingra LK Lam K Cheung W Variation in symptom distress in underserved C hinese A merican cancer patients Cancer 2015 121 3352 59 26059972 Greco C The uncertain presence: experiences of living with metastatic breast cancer Med Anthropol 2022 41 129 40 34187222 Hall DL Mishel MH Germino BB Living with cancer-related uncertainty: associations with fatigue, insomnia, and affect in younger breast cancer survivors Supportive Care Cancer 2014 22 2489 95 Hamer J McDonald R Zhang L Quality of life (QOL) and symptom burden (SB) in patients with breast cancer Supportive Care Cancer 2017 25 409 19 Hashemi S-M Balouchi A Al-Mawali A Health-related quality of life of breast cancer patients in the Eastern Mediterranean region: a systematic review and meta-analysis Breast Cancer Res Treat 2019 174 585 96 30632022 Ho PJ Gernaat SA Hartman M Health-related quality of life in Asian patients with breast cancer: a systematic review BMJ Open 2018 8 e020512 Hong S Lee J Lee J The Mediating Effect of Uncertainty in Illness on Cancer Coping in Patients With Primary Malignant Brain Tumors Cancer Nurs 2022 10 1097 Huijer HA-S Abboud S Health-related quality of life among breast cancer patients in Lebanon Eur J Oncol Nurs 2012 16 491 97 22257429 Huijer HA-S Abboud S Doumit M Symptom prevalence and management of cancer patients in Lebanon J Pain Symptom Manage 2012 44 386 99 22727948 Khater AI Noaman MK Hafiz MNA Health-related quality of life among Egyptian female breast cancer patients at the National Cancer Institute, Cairo University Asian Pac J Cancer Prev 2019 20 3113 31653162 Leão DCM Pereira ER Silva RMC Spiritual and emotional experience with a diagnosis of breast cancer: A scoping review Cancer Nurs 2022 45 224 35 33654011 Lee I Park C The mediating effect of social support on uncertainty in illness and quality of life of female cancer survivors: A cross-sectional study Health Qual Life Out 2020 18 143 Nho JH Kim SR Park MH Symptom clusters and quality of life in breast cancer survivors after cancer treatment in a tertiary hospital in Korea Eur J Cancer Care 2018 27 E12919 Rah B Ali S Dar MI Breast cancer: management and survivorship Cancer Surviv 2019 2019 16 37 Rogers LQ Courneya KS Anton PM Effects of a multicomponent physical activity behavior change intervention on fatigue, anxiety, and depressive symptomatology in breast cancer survivors: randomized trial Psychooncology 2017 26 1901 6 27530961 Sharif SP Locus of control, quality of life, anxiety, and depression among Malaysian breast cancer patients: The mediating role of uncertainty Eur J Oncol Nurs 2017 27 28 35 28279393 Saeed Pahlevan S Ashraf Sadat A Harjit Kaur P Uncertainty and quality of life of Malaysian women with breast cancer: Mediating role of coping styles and mood states Appl Nurs Res 2017 38 88 94 29241526 Zaker MR Safaripour A Sabegh SRZ Supportive Intervention Challenges for Patients with Breast Cancer: A Systematic Review Asian Pac Environ Cancer 2021 4 19 24 Zamanian H Eftekhar-Ardebili H Eftekhar-Ardebili M Religious coping and quality of life in women with breast cancer Asian Pac J Cancer Prev 2015 16 7721 5 26625787

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116169 10.31557/APJCP.2023.24.4.1443 Research Article The High Presence of HCMV pp71 Proteins, Correlate with P63 Expression in Pancreatic Cancer Tumor Tissue Abdul-Razzaq Lana Nazar 1 Jassim Marwa Mohammed Ali 2 Mahmood Majid Mohammed 3* 1 Ministry of Education, Educational Rusafa Directorate 1, Baghdad, Iraq. 2 College of Dentistry, Al-Muthanna University, Al-Muthanna, Iraq. 3 Department of Biology, College of Science, Mustansiriyah University, Baghdad, Iraq. * For Correspondence: majidmahmood93@yahoo.com 2023 24 4 14431447 16 3 2023 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: The purpose of this retrospective investigation was (1) to screen the existence of HCMV in pancreatic cancer tissues in relation to the histopathological grading system of such tumor tissues. (2) To evaluate the expression of the (P63) tumor suppressor gene in these tissues. (3) To find out the impact of the coexistence of (HCMV) along with the p63 on the occurring histopathological alterations. Methods: The current retrospective cohort study included 35 paraffinized pancreatic tissues from the archives of major hospitals and numerous private histopathological laboratories from 2015 to 2020. (Twenty-five pancreatic carcinomatous tissues and 10 biopsies from seemingly normal pancreatic tissues were examined). Tissue slices from the desired tissue blocks were subjected to the immunohistochemistry (IHC) technique to detect Human Cytomegalovirus pp71 and tumor suppressor P63 proteins with aid of monoclonal primary antibodies. Results: The HCMV pp71 proteins were found in 92% (23 out of 25) of pancreatic tumor tissues, while it was in two (20%) of healthy pancreatic tissues. in comparison, the p63 proteins were found in 76% (19 out of 25) of tumor tissues and in four (40%) of their correlative healthy tissues. Conclusion: The increased expression of HCMV in malignant pancreatic tissue may indicate its primary or secondary role in the emergence of this type of cancer, whereupon HCMV inactivation may be useful in the treatment of this type of cancer. On the other hand, p63’s high levels of expression in malignant pancreatic tumors reflect either an oppressive function or an unfortunate mutation that prevents it from functioning. Key Words Pancreatic cancer P63- Human cytomegalovirus immunohistochemistry ==== Body pmcIntroduction Pancreatic carcinomas (PC) is a cancerous tumor that develops from the cells of the pancreas, a glandular organ. Pancreatic malignancies can be classified as endocrine or non-endocrine. Based on histological features, pancreatic malignancies include Ductal adenocarcinoma, Cystadenocarcinoma, and others (Pappalardo et al., 2021). The PC incidence and fatality rates have been rising year after year across the world. In (2015), 367,411 new cases were reported worldwide, with 359,335 fatalities (Hu et al., 2021). Pancreatic carcinomas are responsible for roughly 4% of all cancer fatalities. Furthermore, it is a very aggressive malignancy, with 80% of patients having locally progressed or metastasized at the time of diagnosis (Puckett et al., 2023). According to The GLOBOCAN (Global Cancer Observatory), pancreatic cancer accounts for 2.7 percent of all cancer cases recorded in Iraq (Castanon et al., 2019). Human cytomegalovirus (HCMV) is a herpes virus that is found all over the world. In immunocompromised persons, such as organ transplant recipients and AIDS patients, human cytomegalovirus causes severe and frequently fatal infections. It reactivates in healthy viral carriers on a regular basis, but the host immune response normally controls this (Kiros et al., 2021). Monocytes may serve as a reservoir for latent HCMV, which can be reactivated by cellular differentiation or inflammation. HCMV infection and cancer have been studied for decades. HCMV infection may cause numerous human malignancies, according on seroepidemiologic and tumor tissue viral DNA, mRNA, and/or antigens (De Groof et al., 2021; Elder et al., 2019). Some researchers employ the term “oncomodulation” to explain HCMV’s role in malignancies, even though human CMV is not an oncogenic virus. The fact that HCMV is frequently found in tumor tissues could be explained by the fact that it promotes tumor growth while not being an oncogenic virus (Kwon et al., 2021). Oncomodulation occurs when HCMV infects tumor cells and alters their aggressiveness (Touma et al., 2021). Virus regulatory proteins and noncoding RNA affect tumor cell features such as proliferation, survival, invasion, angiogenic factor production, and immunogenicity, causing human cytomegalovirus-induced oncomodulation. Consequently, HCMV infection may induce tumor cells to change phenotype and become more malignant, as well as tumor progression (Adamson et al., 2020). The TP63 gene, located on chromosome 3q28, encodes tumor protein 63 (p63), a transcription factor in the p53 gene family. Cell cycle-regulating genes and basal layer keratins are only two examples of the many genes whose expression is controlled by p63 throughout the development of the ectoderm and its offspring. The diagnostic value of sustained elevated p63 expression in the selected cell and tissue types is based on their use as biomarkers. It is a widespread practice in diagnostic pathology to employ p63 immunohistochemistry (IHC) to detect cancer-relevant cell types, such as basal cells in the prostate and breast glands (Nasiri et al., 2021; Steurer et al., 2021) . Homologs of the tumor suppressor gene p53, called p63, produce proteins with or without a transactivating domain (TAp63) at their amino termini (Np63). Furthermore, various isoforms emerge from alternative splicing. When overproduced, TAp63 isoforms can cause apoptosis, whereas Np63 isoforms inhibit apoptosis. The relevance of isoform profiles in different cancers has been explored by several researchers (Bartas et al., 2019). The prognostic significance of p63 expression and its level of expression has been shown to vary widely amongst tumor types. Head and neck squamous cell carcinomas have been associated with increased p63 staining, and p63 may be a marker for metaplastic breast carcinoma. Loss of p63 expression, on the other hand, has been linked to tumor development and reduced survival in lung and urothelial malignancies. P63 was expressed nearly exclusively in endometrioid carcinomas, and its lack was linked to myometrial infiltration (Sinha et al., 2015; Zaha, 2014). Materials and Methods The study aims to explore whether or not HCMV expression is linked to oncomodulatory or transformative functions in pancreatic cancer, as well as to extend more to probable involvement of the p63 tumor suppressor protein in pancreatic carcinogenesis in Iraqi patients. Material and methods: Thirty-five formalin-fixed paraffin-embedded archival tissues representing years from 2018 to 2021, collected from private shistopathology laboratories, were used in this investigation (25 samples from carcinomatous pancreatic tissues and 10 biopsies from seemingly normal pancreatic tissues taken from cancer-negative samples). Tissue sections about 4-5 mm thick were put on a positively charged slide for an immunohistochemical (IHC) technique to look for HCMV pp71 and p63 expression proteins. This was done using Anti- pp71 HCMV and Anti-p63 antibodies from the Abcam company in the UK, according to the manufacturer’s instructions. The samples were then looked at under a microscope at 10 and 40 times magnification (Areej et al., 2013). Statistical analysis The data were analyzed using the SPSS software tool, version 24. Pearson chi-square were employed to accept or reject statistical hypotheses. As well as, Spearman’s rho to find out the relationships between markers. P value (P ≤ 0.05) was regarded as statistically significant (Wang, 2003). Results Results of Immunohistochemical Detection of Human Cytomegalovirus pp71 among Studied Tissues Table (1) reveal the HCMV-IHC expression and scoring signals in PC and control tissues. Out of 25 PC sections, 23(92%) showed positive HCMV-IHC reactions as in the following grade distribution; well-differentiated (4.35%), moderately differentiated (13.04) poorly differentiated (17.39%), and undifferentiated (65. 2%). In comparison 2(20%) out of the ten healthy sections showed positive reactions. There were significant differences (P=0.05) between PC sections and healthy ones on one side and (P<0.05) between the different grades of PC sections themselves on the other side. Results of the Expression of p63 -IHC and Signal Scoring among Study Groups As shown in table (2), p63 proteins were identified in 76% (19 of 25) of malignant tissues and 40% (4 of 8) of their corresponding healthy tissues. In the pancreatic cancers group, it was expressed in well-differentiated tissues in a ratio of (5.26%), in moderately differentiated (10.53%), in poorly differentiated (26.32%) and in undifferentiated grade, it was expressed in a ratio of (57.89%). Statistically, Significant differences were found among the four grades (well, moderate, poorly, and undifferentiated) at (P<0.05) level. While there was a Non-Significant difference (P>0.05) between cancerous and control tissues. Correlations between HCMV and p63 Signal Scores in Pancreatic Carcinomas Table (3) shows the relationship between the expression of HCMV and p63 signal scores in pancreatic carcinomatous tissues. The correlation was positive (0.009 **) and highly significant at P < 0.05 level by using Spearman’s rho correlations. Table 1 Human Cytomegalovirus-pp71 IHC Expression in Relation to Histopathological Pancreas Cancer Grading Studied Groups Diagnosis HCMV-pp71 Score Pearson Chi-Square Positive Negative (P-value) Pancreatic Carcinomas Well N 1 2 + 1 P=0.03 differentiated % 4.35% 100% ++ 0 Sign. (P<0.05) +++ 0 Moderately N 3 0 + 0 differentiated % 13.04% 0.00% ++ 2 +++ 1 Poorly N 4 0 + 0 differentiated % 17.39% 0.00% ++ 2 +++ 2 Undifferentiated N 15 0 + 1 % 65.22% 0.00% ++ 3 +++ 11 Total N 23 2 % 92% 8% Control N 2 8 % 20% 80% P=0.05 Total N 10 Sign. (P=0.05) % 100% A significant difference (P≤0.05) by using Pearson’s Chi-Square test. Table 2 Signal Scores Expression of p63-IHC in Relation to Pancreatic Cancer Grading Studied Groups Diagnosis P63-IHC signal scores Score Pearson Chi-Square Positive Negative (P-value) Pancreatic Carcinomas Well N 1 2 + 0 P=0.047 differentiated % 5.26% 33.33% ++ 1 Sign. (P<0.05) +++ 0 Moderately N 2 0 + 0 differentiated % 10.53% 0.00% ++ 1 +++ 1 Poorly N 5 3 + 1 differentiated % 26.32% 50% ++ 2 +++ 2 Undifferentiated N 11 1 + 0 % 57.89% 16.67% ++ 2 +++ 9 Total N 19 6 % 76% 24% Control N 4 6 % 40% 60% P=0.13 Total N 10 Non-Sign. (P>0.05) % 100% Table 3 Spearman's rho Statistical Testing to Evaluate HCMV and P63 Scoring Relationships in Pancreatic Carcinoma Spearman's rho Correlation (PC) scores HCMV scores P63 scores HCMV scores r. 0.009 * P63 scores P-value 0.049 *, Correlation is significant at the P < 0.01 level (Highly Significant). Discussion Pathologic evidence suggests that HCMV infections may be involved in the aetiology of numerous human cancers (Geisler et al., 2019). Infecting the majority of human organs, including the blood, uterine cervix, brain, breast, colon, eyes, kidneys, liver, lungs, and gingival sulcus fluid, the virus has a broad tropism in many healthy individuals (Jassim and Ali, 2019; Scrivano et al., 2011). However, there is a scarcity of evidence on the involvement of CMV (reactivation and/or disease-causing) in individuals with solid tumors (Schlick et al., 2015). In addition to triggering cell cycle arrest, HCMV infection changed the steady-state concentrations of a number of proteins involved in cell cycle control. HCMV generated higher levels of both cyclins E and B, as well as their respective kinase activity (Branch et al., 2021). In contrast, the production of cyclin A appeared to be suppressed, and the levels of the protein and its associated kinase activity increased only at the late stages of infection. Additionally, it was shown that the disease causes the accumulation of high quantities of the proteins p53 and retinoblastoma (Groves et al., 2021). This study’s findings are consistent with those of Ali et al. (2019), who found a high prevalence of cytomegalovirus infections in the sinonasal and nasopharyngeal malignant tumors of a cohort of Iraqi patients, and suggest that cytomegalovirus may play an oncomodulatory role in the development of these tumors (Jassim et al., 2020). CMV has been implicated in modifying cellular immune responses in cancer in both human and preclinical investigations (Erkes et al., 2017; Meng et al., 2018). The majority and most strong immunohistochemical expressions of CMV-pp71 proteins were clearly observed in the undifferentiated grades of the pancreatic tumors investigated (Table1). It is possible that CMV infections play starting and/or cofactor roles in pancreatic carcinomas, combining with other carcinogenic agents. Many different types of cancer showed positive p63 expression, including squamous cell carcinomas (96–100%), thymic tumors (100%), urothelial carcinomas (81–100%), basal type tumors (e.g., basal cell carcinomas, 100%), and various salivary gland neoplasias (81–100%). Furthermore, malignancies derived from p63-negative tissues showed positive p63 immunostaining in 422 pancreatic tumors, although this was unrelated to an aggressive character (Steurer et al., 2021). Ito et al., (2001) demonstrated that p63 overexpression has been linked to the development of pancreatic adenocarcinoma. Another research on undifferentiated pancreatic cancer (UC) discovered moderate-to-strong pan-p63 expression in anaplastic (pleomorphic giant cell) UC (n = 4), sarcomatoid UC (n = 2), UC with osteoclast-like giant cells (n = 3), and ductal carcinomas with partial squamous differentiation (n = 3). Pan-p63 expression was observed to be weak and localized in monomorphic UC (n = 3) and in the majority of neuroendocrine carcinomas (6/7 cases) (Liszka, 2020). Cancer metastasis can be restrained by TAp63, which triggers cell death and senescence. Previous research has established a correlation between p63 deficiency and increased tumor growth and invasiveness (Flores et al., 2005). Recent research has elaborated on this finding, demonstrating that p53 mutation with TAp63 deletion or inactivation increases tumorigenicity through TGF-induced pathways and DNA repair gene alterations (Adorno et al., 2009). In this context, a study by Jassim et al., (2022) on malignant lung tumors found abnormal expressions of some other tumor suppressor proteins such as BRCA1, BCL2, and the RB. Likewise, (Jassim et al., 2021) on tumor suppressor protein “p53” in breast cancerous tumors. The findings about how TAp63 works and what happens when it is lost or turned off have also been proven in pancreatic cancer (Jahedi et al., 2019). Some mutant forms of p53 that are produced from tumors are able to downregulate p63 and p73 by making direct contact with the p53 core domain. This interaction between “p63 and p53” is critical in the development of malignancies like pancreatic carcinoma (Gaiddon et al., 2001). In conclusion, The elevated expression of HCMV infection in cancerous pancreatic tissue could reflect the molecular role of this virus in the disease. The infection may have a primary and/or cooperative function in the development of this kind of cancer, meaning that HCMV inactivation may have a role in cancer treatment. The high expression rates of p63 in malignant pancreatic tumors, on the other hand, reflect an oppressive role or an unlucky mutation that renders it nonfunctional. Author Contribution Statement All authors contributed equally in this study. 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116156 10.31557/APJCP.2023.24.4.1331 Research Article Aberrant Expression of FGFRL1 in Esophageal Cancer and Its Regulation by miR-107 Sharma Priyanka 12 Kaushik Vaishali 1 Saraya Anoop 3 Sharma Rinu 1* 1 University School of Biotechnology, Guru Gobind Singh Indraprastha University, Dwarka, New Delhi, India. 2 Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 3 Department of Gastroenterology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India. * For Correspondence: rinusharma@gmail.com 2023 24 4 13311341 13 12 2022 7 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of cancer. However, little is known about the expression and function of FGFRL1 in esophageal cancer (EC). Methods: We systematically evaluated the expression of FGFRL1 in TCGA and GETex datasets followed by expression analysis in EC cell lines and clinical specimens using immunofluorescence (IF) and immunohistochemistry (IHC) respectively. Results: GEPIA analysis on TCGA and GETex datasets identified significant upregulation of FGFRL1 in EC patients (n=182) compared to normal controls (n=286, p<0.05). IHC analysis showed significantly higher FGFRL1 expression in EC tissues as compared to the distant matched non-malignant tissues (p<0.001). Immunoflourescence in EC cells suggested increased expression of FGFRL1 from WDSCC (KYSE30) to MDSCC (KYSE140) and finally to PDSCC (KYSE410). In-silico tools predicted miR-107 as most significant miRNA regulating FGFRL1 expression. qRT-PCR revealed miR-107 expression to be significantly and inversely correlated with FGFRL1 expression in 73% (22/30) EC tissues (p=0.015) and over-expression of miR-107 resulted in significantly decreased expression of FGFRL1 at mRNA (fold change=0.11, p=0.0016) as well as protein level in miR-107 versus NC treated cells. Luciferase reporter assay using FGFRL1-3’UTR further confirmed it to be a direct target of miR-107. Conclusion: Our results herein document clinical as well as functional relevance of FGFRL1 in EC and its regulation by miR-107. Key Words Esophageal squamous cell carcinoma Fibroblast growth factor receptor miRNA mediated regulation TCGA ==== Body pmcIntroduction Fibroblast growth factor receptors (FGFRs) are high affinity cell surface tyrosine kinase receptors, exerting their roles in embryogenesis, tissue homeostasis, and implicated in development of cancer. Mutations and aberrant expression of FGFR 1-4 promote the initiation and progression of bladder and prostate cancer (Turner and Grose, 2010). Aberrant activation of FGFRs promote tumorigenesis by increasing cell proliferation, migration, survival and differentiation. Fibroblast growth factor receptor like 1 (FGFRL1) is the poorly understood member of FGFR family and shows up to 40% amino acid sequence similarity to other FGFRs (Wiedemann et al., 2000; Rieckmann et al., 2009) but lack kinase domain (Olsen et al., 2007; Wesche et al., 2011; Yang et al., 2017; Bonifacino et al., 2003; di Martino et al., 2013 ). The FGFRL1 gene is expressed in all vertebrates albeit at lower levels than that of conventional FGFRs. In human, FGFRL1 mRNA is expressed at a high level in pancreas, thyroid and adrenal gland, kidney, skeletal muscle and heart. Whereas, the expression is negative in lung, stomach, esophagus, and smooth muscle (di Martino et al., 2013; Tsuchiya et al., 2011). In contrast to other FGFRs, FGFRL1 has been shown to act as a decoy receptor to FGF ligands and antagonizes FGFR signalling in early development (Steinberg et al., 2010) and inhibits cell proliferation. Antagonistic effect of FGFRL1 on FGFR signalling as a decoy receptor has been reinforced by its lack of kinase domain and interaction with Spred1, a negative regulator of FGF (Trueb et al., 2003; Steinberg et al., 2010; Zhuang et al., 2011). However, this negative regulation theory has been challenged by the presence of genes that are regulated by FGFRL1. Emerging evidences suggest that cytoplasmic domain of FGFRL1 contains an SH2 binding motif that interacts with the tyrosine phosphatase SHP1 and overexpression of Fgfrl1 results in activation of ERK1/2 signaling (Silva et al., 2013). Chen et al., (2020) also demonstrated FGFRL1 mediated regulation of ENO1-PI3K/Akt pathway via combining to ENO1 in SCLC cells (Chen et al., 2020). These observations highlighted the oncogenic role of FGFRL1 independent of FGFR signalling. MicroRNAs play a crucial role in regulating the expression of various genes and modulate tumor cellular processes. FGF pathway activity during development or regeneration can be regulated by miRNAs and loss of miRNA regulation of FGF signaling can result in disease progression or cancer. Several miRNAs including miR-338, miR-17, miR-424 and miR-503, miR-710, miR-34a have been shown to affect cell differentiation by directly regulating FGF or FGFR expression (Liu et al., 2014; Carraro et al., 2009; Kim et al.,2013 ; Uchiyama et al., 2010; Fu et al., 2014; Liu et al., 2014; Cheng et al., 2014 )Decreased expression of FGF/FGFR regulating miRNAs in cancers has been reported thereby leading to upregulation of FGFR mediated signalling and cancer progression (Cheng et al., 2014; Yang et al., 2014 ; Yin et al., 2013). However, limited information is available for miRNA mediated regulation of FGFRL1. miR-210 has been shown to directly regulate FGFRL1 in Esophageal squamous cell carcinoma (ESCC). FGFRL1 promotes proliferation of ESCC cell by inhibiting the cell cycle arrest at the G1/G0 and G2/M phase (Tsuchiya et al., 2011). Later, similar results were observed in osteosarcoma (Liu et al., 2018) bladder cancer (Yang et al., 2017) and hepatocellular carcinoma (Yang et al., 2016) as well. In present study, we have demonstrated the clinical significance of FGFRL1 in EC and identified miR-107 as regulator of FGFRL1 expression. Materials and Methods Data mining from TCGA and GTEx datasets The online database Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/index.html) was used to analyse the expression of FGFRL1 in different cancers including EC as compared to the normal controls. GEPIA is an interactive web that includes 9,736 tumors and 8,587 normal samples from TCGA and the GTEx projects, which analyse the RNA sequencing expression (Silva et al., 2013). GEPIA was used to generate box plots, based on gene expression using Anova on log2 (TPM+1) and |Log2FC| cut-off of 1 in 33 different types of cancers. Differential expression of FGFRL1 in EC was determined on ESCA (TCGA n=182) v/s 286 normal controls (GTEx n=273 + TCGA n=13). p<0.05 was considered as statistically significant. Patients and clinicopathological data collection This study was approved by the institutional human ethics committee prior to its commencement. Tumor and matched distant non-malignant esophageal tissue biopsy specimens (obtained from a region atleast 8 cm away from the tumor) were collected from patients who underwent endoscopy in Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India with the prior consent of patients. The diagnosis was based on clinical examination and histopathological analysis of the tissue specimens. The tumors were histopathologically graded as preneoplastic (30) and neoplastic (21). Seven endoscopic biopsies collected from a region atleast 8 cm away from the tumor were histopathologically confirmed to be non-malignant esophageal tissue. The samples were collected and immediately snap frozen in liquid nitrogen and stored at −80◦C till further use. One part of the sample was collected in 10% formalin and embedded in paraffin was used for hematoxylin/eosin staining and immunohistochemical analysis. The clinicopathological data were recorded in a predesigned performa that included site of lesion, histopathological differentiation, age, gender, nature of diet, tea, alcohol and tobacco consumption, and family history. Immunohistochemistry Paraffin-embedded sections (5 μm) of histologically confirmed human esophageal normal (n = 7), preneoplastic tissues (n=30) and ESCC (n = 21) tissues were obtained on poly-L-lysine coated slides. Briefly, the tissue sections were deparaffinized and rehydrated. Tris–EDTA buffer (10 mM Tris-base, 1 mM EDTA, pH 9.0) was used for carrying out antigen retrieval followed by incubation with 0.3% v/v hydrogen peroxide in methanol for 30 min and blocking in 1% normal horse serum. Slides were incubated overnight with rabbit polyclonal anti-FGFRL1 antibody (CloudClone) at 4 °C followed by 30 min incubation with HRP conjugated anti-rabbit IgG (ImmPRESS anti-rabbit Ig (peroxidase) Polymer Detection kit, Vector Laboratories Inc, USA) at RT. The colour was developed using diaminobenzidine as the chromogen. Haematoxylin was used for nuclear staining. Esophageal tissue sections not treated with anti-FGFRL1 antibody were used as negative controls (NC). For FGFRL1 protein expression, sections were counted as positive if epithelial cells showed immunopositivity in the membrane/nucleus/cytoplasm when observed independently by three of us (VK, RS and PD). The slides were scored based on the percentage of immunostained cells as ≤ 10% = 0; 11–20% = 1; 21–40%=2;41–60%=3;61–80%=4and>81%=5. Slides were also scored on the basis of staining intensity as faint = 1; moderate = 2 and strong = 3. Finally, a total score was found by adding the scores of percentage positivity and intensity. Based on sensitivity and specificity, calculated by ROC curve analysis, a total score cut-off value of 4 was defined as FGFRL1 immunopositivity. Cell culture Human ESCC cell line KYSE-410 was purchased from the European Collection of Authenticated Cell Cultures (ECACC), supplied by Sigma- Aldrich (Bangalore, India). KYSE-30 and -140 were a kind gift from Dr. Y.Shimada (Japan). The cell culture was maintained in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS (HiMedia Laboratories Pvt. Ltd., Mumbai, India), 100 U/ml of penicillin and 100 μg/ml of streptomycin (HiMedia Laboratories). The HEK-293T cell line was a kind gift by Dr Nimisha Sharma (GGSIPU, New Delhi). It was maintained in DMEM (HiMedia Laboratories) supplemented with 10% FBS, 100 U/ml of penicillin and 100 μg/ml of streptomycin. The cell lines were maintained in a humidified incubator with 5% CO2 and 95% humidity at 37 °C. Immunofluorescence microscopy To determine the localization of FGFRL1 protein in EC cells, confocal and immunofluorescence microscopy were performed. Briefly, the cells were fixed in methanol at –20 °C for 20 min and permeabilized with 0.5% Triton X-100 in 1X-PBS for 10 min. To prevent non-specific binding, the cells were blocked with 3% BSA and 0.05% Triton X-100 in PBS for 1 h. Then the cells were incubated with rabbit polyclonal anti-FGFRL1 antibody (1:50 dilution) overnight at 4 °C, followed by incubation with Alexa Fluor 488-conjugated goat anti- rabbit secondary antibody (Invitrogen) at room temperature for 30 min (1:1000 dilution). The cells were observed under an inverted fluorescence microscope (Nikon), after nuclei counterstaining with propidium iodide (PI) for immunofluorescence microscopy. In-silico tools to predict miRNAs regulating FGFRL1 Three miRNA prediction tools viz. miRanda, miRdb and miRNet was used for prediction of potential miRNAs targeting FGFRL1. In order to minimize false positives, only miRNAs predicted by all the three software were selected. The most putative miRNA was further screened out on the basis of number of target sites, type of sites (canonical 8-mers were preferred over canonical 7-mer which were preferred than marginal 6-mers) and their miSVR score as calculated using miRanda. Transient transfection of miR-107 mimic using Lipofectamine 3000 KYSE-410 cells were seeded in 96-well plates (1.2x104 cells/well), 24-well plate (5x104 cells/well) or 6-well plate (3x105 cells/well). hsa-miR-107 mimic (100 nM) (Ambion, Inc., Foster City, CA, USA) was transfected in the cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and Opti-MEM medium (Invitrogen) according to the manufacturer’s instructions. Negative control #1 (Ambion) was used as NC. RNA extraction and quantitative RT-PCR Total RNA was extracted using RNeasy Mini Kit (Qiagen) followed by first-strand cDNA synthesis using universal cDNA synthesis kit (Exiqon A/S, Vedbaek, Denmark) according to the manufacturer’s protocol. qRT-PCR analysis of miR-107 was performed using SYBR-Green Master Mix (Exiqon A/S) and predesigned miR-107 specific LNATM PCR primer sets (Exiqon A/S) as described previously (Song et al., 2017). The first strand cDNA for FGFRL1 was synthesized using oligo dT (Fermentas, Canada) and MMLV reverse transcriptase (Fermentas, Burlington, ON, Canada). qRT-PCR of FGFRL1 was carried out using gene-specific primers (Table 1) and KAPA SYBR FAST real-time PCR kit (Kapa Biosystems, Inc., Wilmington, MA, USA) following the manufacturer’s protocol. Its expression was analyzed at 48 and 72 h post miR-107 transfection using qRT-PCR. The expression of FGFRL1 mRNA in the mimic-treated cells was normalized to that of the cells treated with NC. A small RNA, 5S rRNA (Table 1) was used as the endogenous control for data normalization. The 2-ΔΔCT method was used to calculate the fold-change. Western blot analysis Protein was isolated using RIPA buffer (Invitrogen) supplemented with a protease inhibitor cocktail (Invitrogen) using sonication. 70 μg of protein were separated by 12.5% SDS-PAGE and transferred to PVDF membranes (Microdevices, Inc., New Delhi, India). The membranes were blocked with 5% non-fat milk in PBS overnight at 4 °C followed by incubation for 1 h with primary antibodies of FGFRL1 (CloudClone) and GAPDH (Santa Cruz Biotechnology, CA, USA) at dilution of 1:200. The membranes were washed and incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (DAKO A/S Copenhagen, Denmark) for 1 h. After washing, bound secondary antibody was detected using enhanced chemi-luminescence (ECL) system (Pierce Biotechnology Rockford, IL, USA). Western blot results were analyzed quantitatively using ImageJ. FGFRL1 3’UTR reporter construct FGFRL1 has ten miR-107 MRE (miRNA recognition elements) in 3’UTR of FGFRL1 (MFE of -28.2 Kcal/mol). A 989 bp region of FGFRL1-3’UTR (NM_001004358.1) containing all the ten predicted binding sites for miR-107 was amplified and cloned in pMIR-REPORT luciferase vector (Promega) between the Mlu1 and HindIII restriction sites, downstream to the luciferase reporter gene using primers as described in Table 1. Luciferase reporter assay HEK-293T cells were seeded into 24-well plates and cultured until 80% confluent. The cells were then co-transfected with either miR-107 mimic or NC at a 100 nM final concentration and with 100 ng of pMIR-REPORT construct containing FGFRL1 3’UTR along with 10 ng Renilla luciferase vector using Lipofectamine 3000 transfection reagent according to the manufacturer’s recommendations (Invitrogen). Relative firefly luciferase activity, which was normalized with Renilla luciferase, was measured using a Dual-luciferase reporter gene assay system (Promega) and the results were plotted as the percentage of change over the respective control. Statistical analysis All experiments were repeated at least three times and the Student’s t-test was used to analyze the statistical significance between two groups while one-way ANOVA was used to determine the significance of differences among multiple groups. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis were performed using GraphPad Prism software version 6.00 (GraphPad Software, Inc., CA, USA) and SPSS software 16.0 (SPSS, Inc., Chicago, IL, USA). To assess the diagnostic accuracy, receiver-operating characteristic (ROC) curves were generated and area under the ROC curve (AUC) was determined. The correlation of miR-107 and target gene, FGFRL1 was analyzed using non-parametric Spearman’s rho test. Results Data mining from TCGA and GTEx datasets In-silico analysis of FGFRL1 expression in TCGA and GTEx datasets using GOPIA revealed a signficantly decreased expression of FGFRL1 mRNA in 182 EC patients as compared to 286 normal controls (p<0.05, Figure 1A and 1B). cBioportal showed alterations in FGFRL1 gene in 11 patients (mutation = 5, amplification = 3, Deletion=3) out of 185 patients in TCGA dataset and in 1 patient out of 88 in ICGC dataset (Figure 1C). 9 patients out of these 12 patients had disease free survival data and alteration in FGFRL1 was significantly associated with poor DFS with median DFS of 8.7 months for patients with alteration as compared to the unaltered group (median =21.4 months) (Figure 1D). Though, it had no significant association with overall survival (p=0.141) (Data not shown). Increased expression of FGFRL1 protein in ESCC tissues To determine the clinical significance of FGFRL1 protein in ESCC, its expression and localization was investigated in 21 ESCC tissues, 30 preneoplastic tissues and 7 histologically confirmed distant matched non-malignant esophageal tissues using immunohistochemistry. Increased expression of FGFRL1 protein was observed in 20/21 (95%) ESCC tissues and 13/30 (43%) preneoplastic tissue. FGFRL1 expression was observed in the cytoplasm of EC tissues. A high percentage positivity and increased intensity of FGFRL1 staining was observed in ESCC tissues as compared to distant matched non-malignant tissues which either did not show any detectable staining for FGFRL1 protein or a very faint staining in lesser proportion of cells in the tissues was observed (Figure 2A). Student t-test showed a significant increase in FGFRL1 protein expression in ESCC tissues as compared to distant non-malignant tissues (p<0.001, Table 2). ROC analysis revealed an AUC of 0.786 with sensitivity of 90.47% and specificity of 48.64% (Figure 2B). However, no significant correlation was observed between FGFRL1 protein expression in tissues and clinicopathological parameters (Table 2). FGFRL1 protein localizes in the cytoplasm and the membrane of ESCC cells Expression and localisation of FGFRL1 was analysed in EC cell lines (KYSE30, KYSE140 and KYSE410) varying in histopathological grades using immunofluorescence assay. Specific staining was observed in cytoplasm and membrane as a positive staining (green) for FGFRL1 protein expression. Nucleus was stained (red) using PI. Upon merging the two images no nuclear localisation (yellow) was observed i.e. the expression is negative for nucleus. NC (lacking primary antibody) showed no signal from the antibody (i.e. no FGFRL1 expression). Also, the expression seemed to be increasing as we progressed from WDSCC (KYSE 30) to MDSCC (KYSE 140) and finally to PDSCC (KYSE 410) (Figure 3A-C). In-silico prediction of miRNAs regulating FGFRL1 Putative miRNAs targeting FGFRL1 were predicted using miRanda, miRdb and miRNet as shown in Figure 4A and Table 3. miRdb identified 71 miRNAs while miRanda and miRNet resulted in 10 and 20 potential miRNAs respectively. Three miRNAs viz. miR-107, miR-103 and miR-210 were identified by all the three tools. Notably, FGFRL1 is a validated and well-established target of miR-210 which corroborates with our analysis (Eswarakumar et al., 2005; Sharma et al., 2013; Steinberg., 2010). miR-107 had 10 MREs in FGFRL1 3’UTR out of which 9 were canonical 7 or 8 mers suggesting it to be a strong target. Therefore, in order to avoid false positives, we selected miR-107 for further validation using a series of systematic invitro assays. Correlation between miR-107 and its in-silico predicted target, FGFRL1 miR-107 expression was found to be significantly and inversely correlated with FGFRL1 expression in 72.5% (11/40) esophageal tissues (r=-0.332; p=0.036) suggesting FGFRL1 to be the downstream target of miR-107 (Figure 4B). Transfection efficiency Real-time PCR analysis revealed an increase in miR-107 expression by 400-fold at 72 h post miR-107 mimic transfection in the KYSE-410 cells as compared to the cells treated with NC indicating that miR-107 was successfully transfected (Figure 4c). miR-107 overexpression results in decreased expression of FGFRL1 at the mRNA level Interestingly, enforced expression of miR-107 in ESCC cell line KYSE-410 significantly decreased expression of FGFRL1 mRNA by 70.05% at 48 h post transfection and 91.69% at 72 h post transfection (p<0.001) (Figure 4D). Therefore, we further validated if it is the direct target of miR-107. Enforced expression of miR-107 results in decreased expression of FGFRL1 protein To verify that miR-107 acts as a FGFRL1 suppressor, KYSE-410 cells were transfected with 100 nM miR-107 and the NC. The relative expression of FGFRL1 protein was calculated as band intensity of FGFRL1/band intensity of GAPDH. Densitometry analysis showed that at 48 h and 72h post-transfection, overexpression of miR-107 decreased the FGFRL1 protein level by 20% and 57.27% as compared to the NC (Figure 4E). miR-107 targets FGFRL1-3’UTR directly To examine whether FGFRL1 is a direct target of miR- 107, we looked for the presence of miR-107 target sites in its 3’UTR. Interestingly, miRanda showed presence of ten miR-107 MRE in 3’UTR of FGFRL1 (Figure 5A). It is noteworthy, that out of these ten sites, two were 8-mer canonical sites, 7 were canonical 7-mers and one was marginal site, suggesting it to be a strong target of miR-107. The two 8-mer sites were present at positions 467 and 753 in 3’UTR of FGFRL1 and showed maximum miSVR score of - 0.2823 and -0.3879 respectively. A 989 bp region of FGFRL1-3’UTR (NM_001004358.1) containing all the ten predicted binding sites for miR-107 was cloned in miR-Report vector by our group. Luciferase reporter assay showed that co-transfection of miR-107 and pMIR-FGFRL1- 3’UTR construct in HEK293T cells significantly (p=0.001) decreased luciferase activity by 67.62% at 24 h post miR-107 transfection as compared to the NC (Figure 5B). Table 1 Primer Sequences Used in This Study Primers for qRT-PCR FGFRL1 Forward Primer 5' ACACAGCCCTCCAAGATGAG 3' FGFRL1 Reverse Primer 5' GCAGGTTCTTCAGGCTCAGT 3' 5S rRNA Forward Primer Forward: 5’- GTCTACGGCCATACCACCCTG-3’ 5S rRNA Reverse Primer Reverse: 5’-AAAGCCTACAGCACCCGGTAT-3’ Primers for cloning FGFRL1-ECOR1-Forward Primer 5’ GCCGAATTCGTTATGGCCATGACGCCGAGCCCCCTGTT 3’ FGFRL1- Xho1-Reverse Primer 5’ CGCCTCGAGCTAGCACTGATAGTGGATGTGCTGGTGGA 3’ Figure 1 Panel A) Expression profile of FGFRL1 in 33 different cancers including EC from TCGA dataset v/s normal controls obtained from GTEx database. Analysis was performed using GoPIA tool. B) Box plot showing expression of FGFRL1 in EC versus normal controls. C) Diagram showing association of FGFRL1 expression with mutation status in EC patients from TCGA and ICGC datasets. D) Altered FGFRL1 expression was significantly associated with poor disease-free survival Figure 2 A) Representative esophageal tissue section immunostained for FGFRL1 in (a) NC showing no detectable FGFRL1 (no primary antibody) (b) histologically normal tissue showing no immunoreactivity (c) preneoplastic tissue showing cytoplasmic immunoreactivity (d&e) moderately differentiated tissue showing intense cytoplasmic and membranal staining (f) poorly differentiated section showing even darker staining. B) ROC analysis for FGFRL1 protein expression in ESCC tissues as compared to preneoplastic tissues and normal controls Table 2 Relationship of FGFRL1 Protein Expression with Clinicopathological Parameters of ESCC Patients Clinicopathological Parameters Total Cases FGFRL1 protein positivity n (%) p-value Distant matched non-malignant 7 1 (14) ESCC 21 20 (95) <0.001 Age (years) <40 10 7 (70) >0.05 ≥40 49 23 (92) Gender Male 30 19 (30) >0.05 Female 29 11 (29) Histopathology grading MDSCC/PDSCC 21 19 (21) >0.05 PRENEO 30 18 (30) Figure 3 A) Subcellular distribution of FGFRL1 in WDSCC (KYSE30): The top panel shows fluorescence emitted from the antibody (green; absent), cell nuclei (red) stained with PI and the nuclear presence of the antibody (yellow; absent) as demonstrated by superimposition of the two images. Similarly, the lower panel show the fluorescence emitted from the antibody(green), cell nuclei (red) stained with PI and the nuclear presence of the antibody (yellow; absent) as demonstrated by superimposition of the two images (Magnification: 10X). B) Subcellular distribution of FGFRL1 in MDSCC cells (KYSE140): The top panel shows fluorescence emitted from the antibody (green; absent), cell nuclei (red) stained with PI and the nuclear presence of the antibody (yellow; absent) as demonstrated by superimposition of the two images. Similarly, the lower panel show the fluorescence emitted from the antibody(green), cell nuclei (red) stained with PI and the nuclear presence of the antibody (yellow; absent) as demonstrated by superimposition of the two images (Magnification: 10X). C) Subcellular distribution of FGFRL1 in PDSCC cells (KYSE410): The top panel shows fluorescence emitted from the antibody (green; absent), cell nuclei (red) stained with PI and the nuclear presence of the antibody (yellow; absent) as demonstrated by superimposition of the two images. Similarly, the lower panel show the fluorescence emitted from the antibody(green), cell nuclei (red) stained with PI and the nuclear presence of the antibody (yellow; absent) as demonstrated by superimposition of the two images (Magnification: 10X) Table 3 List of miRNAs Predicted by insilico Analysis as Potential Regulators of FGFRL1 Expression miRanda miRDB (71) miRNet miR-103 miR-107 miR-371-5p miR-210 miR-495 miR-150 miR-34c-5p miR-34a miR-449a miR-449-b miR-889-3p, miR-7-2-3p, miR-7157-5p, miR-7157-3p, miR-7-1-3p, miR-7111-5p, miR-103a-3p, miR-107, miR-6891-3p, miR-6889-3p, miR-6870-5p, miR-6818-5p, miR-6798-5p, miR-6787-5p, miR-6754-5p, miR-6749-3p, miR-6737-3p, miR-6736-3p, miR-663a, miR-661, miR-656-3p, miR-6529-3p, miR-6504-3p, miR-618, miR-6072, miR-593-3p, miR-5698, miR-5688, miR-567, miR-520b-5p, miR-519a-2-5p, miR-5008-3p, miR-495-3p, miR-4800-3p, miR-4793-3p, miR-4723-5p, miR-4692, miR-4686, miR-4666b, miR-4520-5p, miR-4514, miR-4455, miR-4442, miR-4441, miR-4310, miR-4288, miR-4270, miR-3922-3p, miR-371a-5p, miR-3190-5p, miR-3175, miR-3158-5p, miR-3130-3p, miR-302c-5p, miR-302a-5p, miR-2114-5p, miR-210-3p, miR-198, miR-1973, miR-1908-5p, miR-1227-5p, miR-10524-5p, miR-10401-3p, miR-10399-5p, miR-10396b-5p, miR-10226, miR-3657, miR-4673, miR-4669, miR-6867-5p, miR-4645-5p miR-3130-3p, miR-21-5p, miR-6867-5p, miR-4658, miR-7157-5p, miR-335-5p, miR-107, let-7b-5p, miR-574-5p, miR-6765-5p, miR-103a-3p, miR-3659, miR-1304-5p, miR-1827, miR-6790-5p, miR-6818-5p, miR-140-5p, miR-210-3p, miR-4793-3p Figure 4 Insilico analysis for predicting putative miRNAs targeting FGFRL1: Prediction of miRNAs regulating FGFRL1 using different tools (Miranda, miRDB, mirnet). B) Non-parametric Spearman’s rho test showing negative correlation between miR-107 and FGFRL1 in clinical cohort of EC patients identified by qRT-PCR C) Histogram showing fold-change in miR-107 expression at 72 h post-transfection in KYSE-410 cells. D) Overexpression of miR-107 resulted in significantly decreased expression of FGFRL1 mRNA at 48h and 72 h post transfection. E) Effect of miR-107 overexpression on FGFRL1 protein levels in the KYSE-410 cells. The relative expression of Cdc42 protein was calculated as band intensity of FGFRL1/band intensity of GAPDH Figure 5 FGFRL1 is a Direct Target of miR-107: A) Figure showing potential binding sites of miR-107 in FGFRL1 3’UTR along with miSVR score and position. miRanda revealed presence of 10 MRE sites for miR-107 in FGFRL1 3’UTR. Canonical 8-mer sites for miR-107 present in FGFRL1 3’UTR are highlighted in red boxes. B) Relative luciferase activity in cells co-transfected with miR-107 mimic and FGFRL1-3′UTR-miR-Report construct in HEK293T cell line as compared to NC at 24 h post transfection; UTR, untranslated region; NC, Negative Control Discussion Esophageal Cancer (EC) is the seventh most common cancer in the world (Ferlay et al., 2019). Since its 5 year survival rate is very low, poor outcomes are common even after the symptoms have been accurately correlated (Enzinger et al., 2003; Pennathur et al., 2008) Although treatment options for EC have improved in recent years, the overall prognosis for patients remains very poor (Pennathur et al., 2008). Therefore, in order to identify biomarker and therapeutic targets in EC, investigations into the molecular mechanisms of EC progression are urgent. FGF/FGFR pathway activation is implicated in the development and progression of a variety of cancers (Jang et al., 2001; Chin et al., 2006; Behrens et al., 2008) FGFRL1 is the fifth and latest addition in the FGFR family of receptor tyrosine kinases. Unlike the other four members of the FGFR family, it lacks the classical kinase domain but has still been shown to activate downstream signal transduction via interaction with key signalling proteins (Gerber et al., 2012; Tai et al., 2018). Although, FGFRL1 has been shown to be aberrantly expressed in SCLC, ovarian cancer (OC), bladder cancer and osteosarcoma, its expression in EC is yet to be investigated (Yang et al., 2017 ; Chen et al., 2020; Liu et al., 2018; Tai st al., 2018). High FGFRL1 levels correlated with poor prognosis in OC and hypoxia induced FGFRL1 expression promoted tumor progression by crosstalk with Hedgehog signalling (Tai et al., 2018). This appears to be consistent with our findings of increased FGFRL1 expression in ESCCs as compared to the distant matched non-malignant tissues.Analysis of data obtained from TCGA and GTeX portals also reveals significant upregulation of FGFRL1 in EC tissues compared to normal controls thus supporting our findings. Deregulation of FGFRs has been attributed to amplification, point mutation, or translocation and amplification being the most common deregulation form in multiple cancer types (Eswarakumar et al., 2005; Helsten et al., 2016 ; Grose et al., 2005). Although FGFR alteration are well studied in several cancers, limited information is available in EC. Recently, Song et al., (2017) investigated the frequency and the prognostic impact of FGFR1 amplification in ESCC patients. They demonstrated that high FGFR1 amplification presented as a delayed adverse prognostic factor in resected stage I-II ESCC patients. FGFRL1 being a relatively new member of the FGFR family has not yet been explored in this regard. cBioPortal showed 6% alterations in the FGFRL1 gene in esophageal tumors and its expression was associated with poor disease-free survival in patients with FGFRL1 alterations as compared to unaltered group, thus further establishing the clinical relevance of FGFRL1 in EC. Moreover, the prevalence of FGFR amplification is the primary determining factor for the efficacy and application of FGFR inhibitors in management of EC patients. This necessitates further well-orchestrated investigation in this area. In regards of potential limitations, our current study is retrospective in nature, and our results must be validated in future prospective studies with follow up samples to validate association of FGFRL1 with prognosis in EC patients. Aberrant expression of miRNAs contributes significantly to development and progression of cancers by subsequent changes in expression of their target genes. miRNAs regulating FGFRs have been shown to be downregulated in several cancers thereby leading to activation of FGF/FGFR signalling (Wang et al., 2013; Schelch et al., 2018) This is emerging to be another mechanism which might lead to FGFR overexpression in cancers. Herein, using bioinformatics tools viz. mirnet, miRanda, and miRdb, we identified miR-107 to be one of the most potential miRNA that may regulate FGFRL1. Enforced expression of miR-107 in EC cells significantly decreased FGFRL1 mRNA and protein expression and its co-transfection significantly decreased the luciferase activity thus confirming it to be a direct target of miR-107. Moreover, FGFRL1 expression was found to be significantly and negatively correlated with the expression of miR-107 in EC tissues. These results collectively demonstrate miR-107 mediated regulation of FGFRL1 in EC. Our lab has previously reported miR-107 to be a tumor suppressor miRNA. Its overexpression suppressed ESCC cell proliferation and migration and induced G1/S arrest (Sharma et al., 2017; Liu et al., 2018). FGFRL1 has previously been linked with cell proliferation while its downregultion has been shown to cause cell cycle arrest in various cancers (Tsuchiya et al., 2011). Our in-silico analysis identified miR-210 as other potential miRNA targeting FGFRL1. Previous studies corroborate with our finding (Zuo et al., 2015; Huang et al., 2009). Hypoxia induced miR-210 downregulated the expression of FGFRL1 in laryngocarcinoma thereby promoting cells in G1/G0 phase and decreasing in S and G2/M phases (Zuo et al., 2015). Moreover, FGFRL1 could partially rescue the inhibitory effect of miR-210 on tumor growth in tumor xenografts (Huang et al., 2009). miR-210 directly targeted the 3’UTR site of FGFRL1 and inhibited EC cell proliferation while FGFRL1 accelerated cancer cell proliferation by preventing G0/G1 cell cycle arrest (Tsuchiya et al., 2011). Taken together these studies suggest that FGFRL1 positively regulates cancer cell proliferation including EC and inhibits cell cycle arrest. In conclusion, using a systematic invitro assays and EC patient samples we established the clinical relevance of FGFRL1 in EC and identified miR-107 mediated regulation as one of the possible mechanisms contributing to its enhanced expression in EC. Author Contribution Statement Concept and design: Rinu Sharma; Acquisition, analysis, or interpretation of data: Priyanka Sharma,Vaishali Kaushik, and Rinu Sharma, Drafting of the manuscript: Priyanka Sharma, Vaishali Kaushik, Anoop Saraya and Rinu Sharma; Statistical analysis: Priyanka Sharma, Vaishali Kaushik, and Rinu Sharma; Administrative, technical, or material support: Anoop Saraya and Rinu Sharma; Supervision: Rinu Sharma Priyanka Sharma, Vaishali Kaushik, Anoop Saraya, and Rinu Sharma take responsibility for the integrity of the work. Acknowledgements The authors acknowledge and thank CSIR, ICMR, and Guru Gobind Singh Indraprastha University for funding the study. Funding This work was supported by FRGS (GGSIPU/DRC/FRGS/2018/19(1115)) and GGSIPU/DRC/FRGS/2021/594/17]., ICMR (2019-0940/GEN/ADHOC/BMS) and CSIR (27(0342)/19/EMR-II). Ethical Approval This study was approved by the institutional ethics committee prior to its commencement and informed consent was obtained from each patient. The research was carried out according to the principles set out in the Declaration of Helsinki 1964 and all subsequent revisions. Reference Number [GGSIPU/ IEC/ 1.2/2010] ,[ IEC/NP-320/2010] The study was approved by University School Research Committee in addition to Institutional Ethical Committee (GGSIPU and AIIMS , Delhi). This study was a part of an approved thesis of Ph.D. student Dr. Priyanka Sharma. Data And Materials Availability Statement Not applicable Conflict of Interest The authors have no conflicts of interest to disclose. ==== Refs References Behrens C Lin HY Lee JJ Immunohistochemical expression of basic fibroblast growth factor and fibroblast growth factor receptors 1 and 2 in the pathogenesis of lung cancer Clin Cancer Res 2008 14 6014 22 18829480 Bonifacino JS Traub LM Signals for sorting of transmembrane proteins to endosomes and lysosomes Annu Rev Biochem 2003 72 395 447 12651740 Carraro G El-Hashash A Guidolin D miR-17 family of microRNAs controls FGF10-mediated embryonic lung epithelial branching morphogenesis through MAPK14 and STAT3 regulation of E-Cadherin distribution Dev Biol 2009 333 238 50 19559694 Chen R Li D Zheng M FGFRL1 affects chemoresistance of small-cell lung cancer by modulating the PI3K/Akt pathway via ENO1 J Cell Mol Med 2020 24 2123 34 31957179 Cheng Z Ma R Tan W Zhang L MiR-152 suppresses the proliferation and invasion of NSCLC cells by 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116155 10.31557/APJCP.2023.24.4.1321 Research Article The Feasibility and Safety of Induction Chemotherapy Followed by Definitive Chemoradiation in Patients with Locally Advanced Cervical Cancer: A Single-Arm Phase II Clinical Trial Ahmadloo Niloofar 1 Heidarpourfard Marzieh 1* Najib Fatemeh Sadat 2 Shiravani Zahra 34 Omidvari Shapour 5 Mosalaei Ahmad 1 Mohammadianpanah Mohammad 6 Ansari Mansour 4 Nasrolahi Hamid 1 Khanjani Nezhat 1 Kadkhodaei Behnam 1 Hamedi Seyed Hassan 1 1 Department of Radiation Oncology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. 2 Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. 3 Division of Oncology and Gynecology, Department of Obstetrics and Oncology, Shiraz University of Medical Sciences, Shiraz, Iran. 4 Maternal-Fetal Medicine Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. 5 Breast Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. 6 Colorectal Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. * For Correspondence: m.heidarpourfard@gmail.com 2023 24 4 13211330 17 12 2022 8 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: The present study aimed at investigating the feasibility and safety of induction chemotherapy followed by definitive chemoradiation (dCRT) in patients with locally advanced cervical cancer. Materials and Methods: In this single-arm clinical trial, patients with cervical cancer (stages IB3-IVA) received a median four cycles of induction chemotherapy (paclitaxel and carboplatin, every three weeks) followed by dCRT (which consisted of the whole pelvis at the dose of 45-50 Gy along with weekly cisplatin (40 mg/m2) followed by intracavitary brachytherapy at the total dose of 80-90 Gy). Primary end point was local control at three months, which was assessed by gynecologic examination and pelvic MRI. The secondary outcome of the study was treatment-related toxicity. Results: Seventy-four patients with the mean age of 51.6 ± 9.5 years were included. The most frequent (51.4%) disease stage was IIB. Complete and partial clinical responses were observed in 60.8% and 14.9% of patients, respectively. The frequency of progressive disease and stable disease were 14.9% and 9.5%, respectively. Grade II and III neutropenia (during neoadjuvant chemotherapy were 13.5% and 2.7%, respectively; these figures during chemoradiation were 29.7% and 13.5%, respectively. A treatment interruption was observed for 60.8% (45 cases) of patients during chemoradiation and 31.1% during induction chemotherapy. Discussion and conclusion: Induction chemotherapy followed by chemoradiation is feasible in patients with locally advanced cervical cancer; however, the toxicity should be managed properly to avoid delayed e treatment. More than three quarters of the patients achieved complete or partial clinical response within a three-month follow-up. Key Words Locally advanced cervical cancer induction chemotherapy definitive chemoradiation ==== Body pmcIntroduction Cervical cancer is one of the most common cancers of the female reproductive system, and is the leading cause of cancer-related deaths in women, especially in developing countries. The frequency of cervical cancer is varying in different parts of the world. While cervical cancer is the second most common cancer in developing countries, this cancer ranks the tenth place in developed countries (Jemal et al., 2011). Overall, the survival of patients with cervical cancer is dismal and even in the early-stage of disease, only 80% of patients will survive up to five years. At higher stages, the 5-year survival drops to 50-60 % when it is confined to the pelvis (Balasubramaniam et al., 2021). There are three main treatment modalities for cervical cancer, which include surgery, radiotherapy, and chemotherapy. The choice of surgical treatment in patients with cervical cancer and its type depend on the cancer stage specified by the International Federation of Gynecology and Obstetrics (FIGO) staging system. The standard treatment for patients with bulky cervical cancer in stages IB2–IVA has been chemoradiation over the past decade. The results of five published clinical trials (three of them exclusively addressed locally advanced patients) revealed a significant improvement in the overall survival and disease-free survival as a result of using chemoradiation treatment. This finding changed the direction of treating this group of patients worldwide (Keys et al., 1999; Morris et al., 1999; Peters et al., 2000; Rose et al., 1999; Whitney et al., 1999). Recently, more intense treatments have been of interest. A relatively large randomized phase III clinical trial by Dueñas-González et al. investigated the concurrent chemoradiation with the gemcitabine/cisplatin regimen. This approach was followed by surgery, after the completion of which two additional cycles of this regimen was prescribed as an adjuvant treatment (Duenas-Gonzalez et al., 2011). Although their study showed a 9% improvement in the three-year progression-free survival, this approach is still not considered as a standard approach due to its considerable toxicity. Currently, some studies have been conducted regarding the usefulness of adding chemotherapy before (ongoing INTERLACE trial) or after (OUTBACK) chemoradiation. However, none of the proposed approaches have been associated with positive results (Marth et al., 2017). The rationale for using the neoadjuvant chemotherapy approach in patients with cervical cancer has been to reduce the size of the primary tumor, increase the probability of complete resection, eradicate micrometastases, potentially increase blood supply to tumors, and reduce hypoxic cells. The findings of a meta-analysis showed that neoadjuvant chemotherapy followed by surgery, as compared to radiotherapy, was associated with a 35% reduction in the risk of death (p=0.00004 and HR=0.65), and improved patient survival by 14% (from 52% to 64%) (Neoadjuvant Chemotherapy for Locally Advanced Cervical Cancer Meta-analysis Collaboration, 2003). In that study, 872 patients with locally advanced cervical cancer enrolled in five different clinical trials were evaluated. In this study, 441 patients with FIGO stage IB2-III cervical cancer were subjected to a secondary meta-analysis, and neoadjuvant chemotherapy based on cisplatin followed by radical surgery was compared to conventional radiotherapy. The most important problem of the studies included in this meta-analysis was their insufficient radiotherapy dose such that brachytherapy was not performed in 27% of the patients, and the prescribed dose to point A was less than 60 gray (Gy) in 11% of the patients. Moreover, in all the evaluated studies, the control group included patients treated with radiotherapy alone, which is currently not the standard treatment for patients with advanced cervical cancer; presently, concurrent chemoradiation is the standard treatment for this group of patients. Therefore, the present study was designed to investigate the treatment response to induction chemotherapy followed by chemoradiation in patients with locally advanced cervical cancer. Materials and Methods This single arm non-randomized phase II clinical trial included patients with cervical cancer admitted to the radio-oncology department of Namazi Hospital in Shiraz, Iran in 2020. According to a 2016 study by Narayan and colleagues (Narayan et al., 2016), considering alpha equal to 5% and the formula n=(Z_(1-α⁄2)^2 pq)/d^2 (p = 84%, d = 0.10 , Z= 1.96), the sample size was calculated to be 74 patients by considering the 10% drop of the initial 81 patients. Non-random and consecutive sampling was done among patients with cervical cancer that met the inclusion criteria of the present study. Inclusion criteria consisted of all women providing the signed written consent with newly diagnosed cervical cancer, the age of more than or equal to 18 to 75 years, the functional status of less than two based on the Eastern Cooperative Oncology Group (ECOG), the confirmed diagnosis of squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma by histology, the early clinical stage IB3, IIA with the size of larger than 4 cm, IIB-IVA in gynecologic examinations based on FIGO staging system, the adequate bone marrow function (neutrophil count of more than 1,500, hemoglobin level of higher than g 10/dL, and platelet count of more than 100 x 103), the appropriate liver function (liver enzyme level of less than 5 times normal, bilirubin level of less than 1.5 times normal, and alkaline phosphatase level of less than 5 times normal), and adequate renal function (mL/ min60< glomerular filtration rate (GFR)). The exclusion criteria included pregnancy, previous pelvic radiotherapy, presence of grade two or higher neuropathy, history of hypersensitivity reactions to paclitaxel or cisplatin, serious medical illness (severe heart, liver, or renal failure), presence of other concurrent malignancies, distant metastasis, hearing impairment, and presence of contraindications for magnetic resonance imaging (MRI) with injectable contrast. Moreover, the exclusion criteria during the study encompassed the neoadjuvant treatment discontinuation, lack of response and disease progression, failure to refer for gynecologic examinations, and failure to refer for brachytherapy. The study protocol was approved by the Research Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.MED.REC.1400.173). The study was then registered in the Iranian Registry of Clinical Trials (IRCT) (IRCT20210808052110N1). First, the eligible patients’ demographic and clinical information such as age, sex, medical and surgical history, and physical examination along with the clinical stage based on the 2018 FIGO staging system were recorded using the data collection form. Patients’ height and weight were measured before treatment. Moreover, body mass index (BMI), body surface area (BSA), and glomerular filtration rate (GFR) were calculated for each patient. All patients were examined by the gynecologic oncology fellowship who checked the position, characteristics, and stage of the tumor. The diagnosis of squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma of the cervix was confirmed by pathological evaluation on the sample obtained from the biopsy of the cervix. Then, the patients were visited by the gynecologic oncology fellowship surgeon, and if the cancer was considered locally advanced, they were referred to the radio-oncology clinic to receive definitive chemoradiation (dCRT) treatment. Before starting the treatment, all patients underwent a gynecologic examination, pelvic MRI with and without injection, computerized tomography (CT) scan of abdomen and pelvis, chest CT scan, or chest X-ray (CXR). Moreover, the clinical staging of the tumor was performed using the 2018 FIGO staging system. The patients’ functional status was evaluated based on the ECOG performance status scale. Moreover, laboratory parameters such as complete blood count (CBC), liver function test (LFT), renal function test (RFT), and electrolytes were measured for all patients prior to the initiation of the treatment. After obtaining written informed consent, the induction chemotherapy was prescribed every three weeks for four cycles with the carboplatin (area under the curve: 5) and paclitaxel (175 per square meter) regimen before chemoradiation and brachytherapy. Before each cycle of chemotherapy, patients were visited and examined every time. In addition to controlling the treatment complications, CBC, LFT, and RFT were checked as well. After completing the induction chemotherapy cycles and controlling CBC, LFT, and RFT, the patients underwent chemoradiation of the whole pelvis. To do so, at first, patients were subjected to CT scan with SOMATOM Definition AS Siemens scanner (Siemens Healthcare GmbH, Erlangen, Germany), and the obtained images were transferred to the treatment planning system through a DICOM network (INFINITT). Then, treatment volumes including gross tumor volume (GTV), clinical target volume (CTV), and planning target volume (PTV) were drawn on the prepared images. The information obtained from the gynecologic examination and MRI before radiotherapy was used to determine the GTV. Next, tumor CTV or CTV1 was defined as covering the entire uterus, cervix, and gross tumor (excluding muscles, small intestine, and bones). In order to draw PTV1, a margin of 15 mm was added to CTV1. Subsequently, CTV2 was added to cover the parameters and one-third to the upper half of the vagina. Moreover, a margin of 10 mm was added to CTV2 to draw PTV2. Finally, to cover the pelvic lymph nodes, CTV3 was drawn by covering internal iliac, external iliac, common iliac, and pre-sacral lymph nodes, and PTV3 was obtained by adding 7 mm margin to CTV3. The radiotherapy complications including diarrhea, proctitis, non-infectious cystitis, and hematological disorders were graded (grades 1 to 5) based on the radiotherapy oncology group (RTOG) criteria and the 5th version of the common terminology criteria for adverse events (CTCAE). Severe complications equal to RTOG grades three/four were defined as the patient required hospitalization during induction chemotherapy or chemoradiation. Just after completion of chemoradiation, the volume-based 3D planning for each tandem and ovoid insertion of HDR brachytherapy were used to keep the whole treatment time less than nine weeks. Although most patients in our study suffered from locally advanced tumors, all patients underwent brachytherapy using tandem and ovoid because of lack of access to interstitial brachytherapy. Evaluation of treatment response was performed by the gynecologic examination, Pap smear, and pelvic MRI three months after the completion of all treatments. If there was a suspicious result in any of the examinations (feeling a cervical mass in the gynecologic examination, abnormal Pap smear results, or abnormal signal of cervix in pelvic MRI), consultation with gynecological oncology fellowship was done to obtain a biopsy. The primary and secondary outcomes of the study were the response rate in the three-month follow-up and complications, respectively. Response evaluation criteria in solid tumours (RECIST) criteria were used to evaluate the primary outcome. According to the mentioned criteria, the patients were divided into four categories: complete response (i.e., the absence of all malignant lesions with less than 10 mm lymph node size), partial response (i.e., at least 30% reduction in the size of the malignant lesion), progressive disease (i.e., an increase of at least 20% in the size of the malignant lesion), and stable disease (i.e., not having enough characteristics to be included in the progressive and relative disease criteria). Patients were then followed up during induction chemotherapy every three weeks (before each cycle of chemotherapy) and weekly during chemoradiation in terms of acute toxicity and acceptance of the treatment protocol. Finally, the effectiveness and safety of the treatment protocol were evaluated. The collected data were analyzed using Statistical Package for Social Sciences (SPSS) v. 24. Statistical indicators such as frequency, percentage, mean, and standard deviation were used to describe the data. Graphs and tables were drawn to graphically represent and tabulate the data. Inferential statistics including Chi-square and Fisher’s Exact Test were used to compare qualitative data. In order to compare patients’ age and duration of treatment regarding the treatment response in the three-month follow-up, normal distribution of data was checked using the Kolmogorov-Smirnov test, which showed the normal distribution of this variable in the studied population. Then, one-way analysis of variance (ANOVA) was used. Considering the significance of the ANOVA test results regarding the duration of treatment, Tukey’s post hoc test was used to compare the two groups. The significance level for the tests was considered 0.05. Results In the present study, 74 patients with cervical cancer with the mean age of 51.6 ± 9.5 years (median 52.0 and range 30-70 years) were subjected to induction chemotherapy, chemoradiation, and brachytherapy during the study period, and their response rate was addressed by evaluating pelvic MRI images after treatment, Pap smear results, and gynecologic examinations three months after treatment. The patients’ entrance into the study is shown in Figure 1. Majority of the patients were in stage IIB based on the 2018 FIGO staging system (51.4%, 38 cases). Moreover, most patients had squamous cell carcinoma (85.1%, 63 cases). Regarding the external radiotherapy, 59 patients (79.7%) only underwent whole pelvic treatment without para-aortic region (Table 1). In terms of treatment response, the results of the three-month follow-up of patients with cervical cancer undergoing induction chemotherapy followed by chemoradiation and brachytherapy based on RECIST criteria revealed that 60.8% of patients (45 cases) had a complete response to treatment while 14.9% of them (11 cases) had partial response. The frequency of progressive and stable diseases were 14.9% (11 cases) and 9.5% (7 cases), respectively. The most severe hematological complication were grade two and three neutropenia (during induction chemotherapy: 13.5% and 2.7%, respectively; during chemoradiation: 29.7% and 13.5%, respectively). In total, the occurrence of complications was associated with the temporary treatment discontinuation in 60.8% of patients (45 cases). Temporary treatment discontinuation was reported in 31.1% of patients (23 cases) under induction chemotherapy and in 56.8% of patients (42 cases) under chemoradiation (Table 2 and Figure 2). The mean age of patients with complete response, partial response, progressive disease, and stable disease were 51.6 ± 10.3 years, 50.9 ± 10.6 years, 52.6 ± 7.5 years, and 51.2 ± 6.6 years, respectively (p=0.980). Complete pathological response was reported in 66.7% of stage IB3 patients, 75% of stage IIA2 patients, 73.7% of stage IIB patients, 25% of stage IIIA patients, 50% of stage IIIB patients, 50% of stage IIIC1 patients, and 40% of stage IIIC2 patients (p=0.179). Complete pathological response in patients with and without delay in chemotherapy were reported to be 47.8% and 66.7%, respectively (p=0.369). Complete pathological response was similar in patients with and without delay in chemoradiation (59.4% vs. 61.9%, respectively, p=0.910). Complete pathological response in patients with squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma were reported to be 60.3% (38 cases), 66.7% (4 cases), and 60% (5 cases), respectively (p=0.444). Examining the response rate in patients with cervical cancer undergoing induction chemotherapy followed by chemoradiation and brachytherapy indicated no significant association in terms of treatment delay regardless of its type (p=0.857). Complete pathological response was similar in patients with and without treatment delay (57.8% vs. 65.5%, respectively, p=0.857). The comparison of the mean duration of treatment according to treatment response in patients with cervical cancer undergoing induction chemotherapy followed by chemoradiation and brachytherapy is presented in Figure 3. The mean duration of treatment in patients with complete response, partial response, progressive disease, and stable disease were 151.1 ± 6.9 days, 159.6 ± 9.1 days, 157.4 ± 9.8 days, and 154.2 ± 6.5 days, respectively. The result of ANOVA test showed a significant difference between the compared groups (p=0.008). Furthermore, the result of Tukey’s post hoc test showed that only the mean duration of treatment in patients with complete response was significantly less than the mean duration of treatment in patients with partial response (p=0.014), and no association was observed between the other subgroups compared. Figure 1 Flowchart of Patients Entering the Study Table 1. Patients’ Demographic Characteristics Frequency Percentage FIGO stage IB3 3 1.4 IIA2 4 4.5 IIB 38 51.4 IIIA 4 4.5 IIIB 6 8.1 IIIC1 4 4.5 IIIC2 15 20.3 Histology SCC 63 85.1 Adenocarcinoma 6 8.1 Adenosquamous carcinoma 5 6.8 Treatment field Whole pelvis 59 79.7 Whole pelvis and para-aorta 15 20.3 Figure 2 Distribution of the Temporary Treatment Discontinuation due to the Occurrence of Complications in the Treatment of Patients with Cervical Cancer Undergoing Induction Chemotherapy Followed by Chemoradiation and Brachytherapy Table 2 Treatment Complications Characteristics 0 N (%) 1 N (%) 2 N (%) 3 N (%) 4 N (%) Dermatitis 12 (16.2) 56 (75.7) 3 (4.1) 3 (4.1) 0 GI toxicity 7 (9.5) 41 (55.4) 22 (29.7) 2 (2.7) 2 (2.7) GU toxicity 9 (12.5) 47 (63.5) 17 (23) 1 (1.4) 0 During induction chemotherapy Neutropenia 4 (4.5) 10 (13.5) 2 (2.7) 0 Thrombocytopenia 2 (2.7) 2 (2.7) 3 (4) 0 Anemia 40 (54) 12 (16.2) 4 (4.5) 0 During chemoradiation Neutropenia 4 (4.5) 22 (29.7) 10 (13.5) Thrombocytopenia 0 4 (4.5) 4 (4.5) 0 Anemia 32 (43.2) 12 (16.2) 0 0 Figure 3 Comparison of the duration of Treatment in Patients with Cervical Cancer Undergoing Induction Chemotherapy Followed by Chemoradiation and Brachytherapy According to the Pathological Response Discussion Cervical malignancies are among the most common malignancies of women. Timely treatment is very significant in their management such that according to the available scientific literature, the interval between the start and the end of dCRT and brachytherapy should be kept less than eight weeks (Hong et al., 2017). However, the start of treatment may be delayed in some clinical scenarios, especially in developing countries such as Iran due to their limited access to radiotherapy facilities (Mayadev et al., 2022). In such situations, it is suggested to use chemotherapy as an auxiliary approach with the aim of reducing the load of the accelerator device and create an opportunity to plan the treatment while inducing a significant oncologic effects (Ahmed et al., 2021; Ouabdelmoumen et al., 2018). Such an approach may be associated with a significant reduction in tumor size, a reduction in the risk of distant metastasis, and an increase in the probability of clinical response. Of course, it should be noted that the use of multi-drug regimens may be associated with bone marrow or non-blood complications, which often cause delays in the patients’ treatment (Benson et al., 2019; da Costa et al., 2019; de Azevedo et al., 2017; McCormack et al., 2013; Narayan et al., 2016; Singh et al., 2013). Unfortunately, until now, there has not been a randomized phase III clinical trial investigating different approaches in the non-surgical treatment of cervical malignancy, and the findings are mainly obtained from single-group and phase II or cohort trials. The present single arm non-randomized clinical trial aimed at investigating the treatment response to induction chemotherapy followed by chemoradiation in patients with locally advanced cervical cancer, during which the patients underwent four cycles of induction chemotherapy every three weeks with carboplatin and paclitaxel regimen before dCRT. In terms of treatment response, the results showed that 60.8% of patients (45 cases) had complete response to treatment while 14.9% of them (11 cases) had partial response. The frequency of progressive and stable diseases were 14.9% (11 cases) and 9.5% (7 cases), respectively. Moreover, the most common non-hematological complications were grade one dermatitis (75.7%, 56 cases), grade one gastrointestinal complications (41 cases, 55.4%), and grade one genitourinary complications (63.5%, 47 cases). The study of hematological complications in patients with cervical cancer undergoing induction chemotherapy followed by chemoradiation and brachytherapy showed that the most severe hematological complication were grade two and three neutropenia (during induction chemotherapy: 13.5% and 2.7%, respectively; during chemoradiation: 29.7% and 13.5%, respectively). Of course, no cases of fever and neutropenia were observed in the examined patients. Grade three and two thrombocytopenia were observed during induction chemotherapy in 4% of patients (3 cases) and 2.7% (2 cases) of patients, respectively. The frequency of grade three and two thrombocytopenia during chemoradiation were 4.5% (4 cases) and 4.5% (4 cases), respectively. Grade three anemia was reported only in 4.5% of patients (4 cases) during induction chemotherapy, and none was observed during chemoradiation. Furthermore, the occurrence of complications during chemoradiation led to the removal of one simultaneous cycle of chemotherapy in 18 patients (24.3%). Overall, the occurrence of complications was associated with the temporary treatment discontinuation in 45 patients (60.8%). Temporary treatment discontinuation was reported in 31.1% of patients (23 cases) under induction chemotherapy and in 56.8% of patients (42 cases) under chemoradiation. None of the studied variables including age, treatment delay, tumor histology, and disease stage had a significant association with the pathological response. In the present study, treatment response was observed in three quarters of patients after treatment with induction chemotherapy and dCRT, and most of the patients indicated a complete response. Lack of a response indicating a stable or progressive disease was also observed in a quarter of patients. In Tian et al.’s study (2021), 120 women with stage IB2 and IIB-IVA cervical cancer were retrospectively examined after induction chemotherapy and dCRT. The complete/partial response rates were 81.7% and 99.2% after the completion of induction chemotherapy and chemoradiation, respectively (Tian et al., 2021). In a phase II clinical trial conducted by Benson et al., (2019), 27 patients with advanced cervical malignancies underwent induction chemotherapy followed by chemoradiation; the results of their follow-up examinations revealed the complete response observed in 76% and 87.5% of patients after induction chemotherapy and chemoradiation, respectively. The treatment regimen used in the study by Tian et al., (2021) for induction treatment was paclitaxel with cisplatin or loplatin every three weeks for two to four cycles while the regimen in Benson et al., (2019)’s study, it consisted of six cycles of weekly chemotherapy with paclitaxel and carboplatin regimen (Benson et al., 2019; Tian et al., 2021). Both of the aforementioned studies were different from the present study in terms of the duration of treatment and regimen. It should also be noted that the follow-up approach and the tools used for determining the tumor response rate to induction treatment in these two studies were different from the approach used in the present study. In addition, Tian et al. did not describe the protocol they used for evaluating the treatment response in their study. Furthermore, Benson et al., (2019)’s study used abdominal and pelvic CT scans along with clinical examinations and Pap smear results to follow up patients and evaluate the response to induction treatment. Therefore, if the findings of clinical examinations and Pap smear are based on inaccurate tools such as CT scan , the results of such studies cannot be considered as a reliable criterion for data comparison (Benson et al., 2019; Tian et al., 2021). However, in addition to the gynecologic examination and Pap smear results, the results of MRI were used in the present study three months after the treatment to evaluate the treatment response. In the randomized phase II clinical trial conducted by da Costa et al., (2019), patients with cervical cancer in stage IIB-IVA were randomly assigned to one of the induction chemotherapies and chemoradiation group (n=55) or chemoradiation alone group (n=52). After the completion of the treatment, the complete response rate was significantly higher in the chemoradiation alone group as compared to the induction chemotherapy group (80.3% vs. 56.3%, P = 0.008), which was associated with better survival in the chemoradiation alone group. However, approximately 60% and 15% of the patients in the current study had a complete and partial treatment response, respectively. It should be considered that the regimen used for chemotherapy by da Costa et al., (2019) included three cycles of gemcitabine and cisplatin, which was associated with significant treatment discontinuation in the patients receiving induction treatment (20% vs. 6.8 %). Therefore, in their study, one of the most important reasons for the weaker results of induction chemotherapy as compared to chemoradiation alone can be the higher rate of treatment discontinuation in patients undergoing induction chemotherapy as well as the delay in starting chemoradiation due to the prescription of induction chemotherapy. The mentioned delay in starting definitive treatment can have adverse effects on the survival of patients with cervical malignancy such that the prescription of three cycles of treatment every three weeks in the best case and in the absence of treatment toxicity is associated with a 9-week delay in the initiation of chemoradiation. The meta-analysis conducted in this respect showed that an interval of 14 days or less in induction chemotherapy cycles was associated with clinical benefits in patients with cervical malignancy although caution should be exercised in interpreting the results of this study (Neoadjuvant Chemotherapy for Locally Advanced Cervical Cancer Meta-analysis Collaboration, 2003). The phase II study conducted by de Azevedo et al., (2017) regarding the effectiveness of induction chemotherapy and then chemoradiation in the treatment of stage Ib2-IVa cervical cancer showed that the use of induction chemotherapy regimen including cisplatin and gemcitabine was associated with an 81% response rate, which is relatively similar to the findings of the present study. In the present study, treatment response, most of which were complete responses, was observed in three quarters of patients after treatment with induction chemotherapy and dCRT. The regimen used by de Azevedo et al., (2017) was gemcitabine and cisplatin, which was also similar to the regimen used by da Costa et al., (2019) although de Azevedo et al., (2017)’s study was different from da Costa et al., (2019)’s study in terms of the dose of cisplatin (35mg/m2) and the number of treatment cycles (two cycles). Considering the results of the present study and those of de Azevedo et al., (2017)’s study, it seems that prescribing two cycles of gemcitabine and cisplatin chemotherapy every three weeks or four cycles of paclitaxel and carboplatin chemotherapy every three weeks were associated with similar clinical responses (da Costa et al., 2019; de Azevedo et al., 2017). In the present study, the highest complete pathologic response rates were recorded in patients with stage IIA2 (75%) and IIB (73.7%). To compare our findings with the findings of other studies addressing standard treatment (chemoradiation and then brachytherapy), the results of the study by Pereira et al., (2017) can be taken into consideration. They examined the data of 75 patients with stage IB2-IIIB cervical cancer under chemoradiation and revealed that the complete clinical response rate in this group of patients was 33.3%. It is worth noting that the complete clinical response rate was 80% in the subgroup of patients who used cisplatin as a sensitizing drug to radiotherapy, while the complete clinical response rate was 31% in other patients who used other chemotherapy drugs for this purpose. In our study, the highest complete clinical response rates were observed in patients with stage IB2 (100%,), IIA (50%), and IIB (22%). In another study, Duenas-Gonzalez et al., (2002) investigated the response to chemoradiation and then brachytherapy in 41 patients with stage IB2-IIIB cervical cancer and reported a clinical response of 87% in those patients. Moreover, while interpreting the results reported by Pereira et al., (2017) and Duenas-Gonzalez et al., (2011) it should be taken into account that the evaluation of the treatment response in these two studies was performed only by considering the findings of the clinical examinations with or without the reported cytology evaluation, which is less sensitive than the pelvic MRI (Nawapun et al., 2021). In another study by Lee et al., (2017), 225 patients with FIGO stage Ib2-IVa cervical cancer (based on pre-treatment MRI) were subjected to pelvic MRI examination between cisplatin-based chemoradiotherapy and brachytherapy. Their results showed response rates of 49.7% (Lee et al., 2017). But the results regarding the response rate are not presented according to the clinical stage; rather, the evaluation of the response between chemoradiotherapy and brachytherapy instead of the end of the treatment, is different from the present study. In a study by Schernberg et al., (2018), 260 patients with cervical cancer were evaluated in terms of tumor size reduction in MRI before brachytherapy. A volume reduction of more than 90% was reported in 54.6% of patients. The highest rate of response was reported in stage IIB patients (50%) and subsequently in stage IB2 patients (31%). In the study by Schernberg et al., (2018) MRI was performed between the completion of chemoradiotherapy and brachytherapy. In another study by Beriwal et al., (2018), the complete metabolic response was investigated in 155 patients with stage IB1-IV4 cervical cancer after chemoradiotherapy and brachytherapy. In their study, the metabolic response was evaluated by FDG-PET/CT scan and pelvic MRI 10-16 weeks after the completion of treatment. Complete metabolic response was reported in 72% of patients, partial response in 18.7% of patients, and progressive disease in 9% of patients. The results were not presented by stage, but most of the patients were in stage IIB. In the present study, a complete response to treatment was reported in 60.8% of patients and a partial response in 14.9% of them, which was similar to the results of the study by Beriwal et al., (2012) It seems that using the neoadjuvant chemotherapy approach followed by standard treatment or starting treatment with the standard approach without neoadjuvant chemotherapy (definitive chemoradiotherapy and then brachytherapy) has the same short-term results; therefore, radio-oncology specialists can consider obtaining the clinical conditions of the patient (for example, the need to observe a quick response in a patient with urinary tract obstruction) and the radiotherapy center (for example, a long waiting list in the field of high load of patients) should use each of these two approaches. In a study by Javadinia et al., (2020) author suggested that the 6-, 12-, 18-, and 24-month overall survival of patients with cervical cancer was 98%, 86%, 75%, and 50%, respectively. Investigating the hematological complications in patients with cervical cancer undergoing induction chemotherapy followed by chemoradiation and brachytherapy showed that grade two and three neutropenia during induction chemotherapy occurred in 13.5% of patients (10 cases) and 2.7% of patients (3 cases), respectively, while grade two and three neutropenia during chemoradiation occurred in 29.7% of patients (22 cases) and 13.5% of patients (10 cases), respectively. Of course, no cases of fever and neutropenia were reported in the examined patients. Grade three and two thrombocytopenia were observed in 4% of patients (3 cases) and 2.7% of patients (2 cases) during induction chemotherapy. The frequency of grade three and two thrombocytopenia during chemoradiation were 4.5% (4 cases) and 4.5% (4 cases), respectively. Grade three anemia was reported only in 4.5% of patients (4 cases) during induction chemotherapy, and no cases were reported during chemoradiation. Moreover, the occurrence of complications during chemoradiation led to the removal of one cycle of concurrent chemotherapy in 24.3% of patients (18 cases). Among the non-hematological complications, the most common complications were grade one dermatitis, grade one gastrointestinal complications, and grade one genitourinary complications. Overall, the occurrence of complications was associated with the temporary treatment discontinuation in 60.8% of patients (45 cases). Temporary treatment discontinuation was reported in 31.1% of patients (23 cases) under induction chemotherapy and in 56.8% of patients (42 cases) under chemoradiation. In Tian et al., (2021)’s study, the use of induction chemotherapy before dCRT was associated with grade three, four, or higher gastrointestinal toxicity, as well as leukopenia, neutropenia, and thrombocytopenia. The treatment regimen used in Tian et al., (2021)’s study for induction treatment was paclitaxel with cisplatin and or loplatin every three weeks for a minimum of two to a maximum of four cycles and weekly cisplatin chemotherapy regimen. Unfortunately, Tian et al.did not provide a detailed description of the number of treatment cycles. In the randomized phase II clinical trial conducted by da Costa et al., (2019), induction chemotherapy and dCRT were compared to chemoradiation alone in patients with cervical cancer in stage IIB-IVA. Their results showed that grade three neutropenia, grade three nausea/vomiting, and neuropathy were significantly higher in patients undergoing induction treatment. In their study, induction chemotherapy included three cycles of gemcitabine (1,000 mg/m2 on day 1 and day 8) and cisplatin (50 mg/m2 on day 1), and complications in 20% of patients were associated with the treatment discontinuation. The phase II study conducted by de Azevedo et al., (2017) showed that using an induction chemotherapy regimen including cisplatin and gemcitabine was associated with significant complications, and hematological and gastrointestinal toxicity were the most common toxicities. Grade three/four toxicity was observed during induction chemotherapy and chemoradiation in 20% and 44% of patients, respectively. In the study of Narayan et al., (2016), the use of chemotherapy before the standard chemoradiation treatment with one of Platin/5-FU or Taxol/Platin/5-FU regimes were associated with a significant treatment toxicity such that grade three/four hematological toxicities were higher in the induction chemotherapy group as compared to chemoradiation alone. However, the occurrence of non-hematological toxicity was not significantly different between the two groups. The use of TPF triple chemotherapy regimen was associated with higher complications. Baruah et al., (2022) reported similar results in patients with cervical cancer receiving chemoradiation. Besides, the psychological effects of suffering from cancer and its treatments should be taken into consideration (Moezian et al., 2022; Salek et al., 2021; Shirzadeh et al., 2016; Shomoossi et al., 2013). Despite the fact that the use of induction chemotherapy is associated with good therapeutic effects on the treatment of patients with cervical malignancy, caution should be exercised regarding the type of regimen selected as well as the number and dose of drugs used so that the treatment approach adopted does not decrease the patient’s tolerance leading to a delay in definitive chemoradiation. The present study suffers from some limitations as well, which includes the dissatisfaction of some patients that met the criteria for entering the study to complete the treatment protocol as well as their unwillingness to perform brachytherapy after the completion of induction chemotherapy and chemoradiation, which reduced the sample size. In order to manage this limitation, not only the objectives and benefits of the study were explained to the patients but also the time of data collection and sampling was expanded. Furthermore, since conducting the study as a single arm trial precluded the possibility of comparing the obtained results with the findings of the peer group, the generalizability of the results of the present study was at risk. Moreover, a significant number of the studied patients were suffering from large cervical masses so that the use of tandem ovoid as the only brachytherapy tool cannot be a breakthrough in delivering the appropriate dose and creating optimal local control. In addition, the use of 45 Gy dose in the treatment of para-aortic lymph nodes due to lack of access to IMRT was also associated with underdosing of these lesions. It is proposed to conduct, in future, two-group randomized controlled clinical trials with a larger sample size by controlling the confounding factors to confirm the findings of this study. Moreover, it is essential to follow up the patients in terms of their overall survival and disease-free survival for 12 to 60 months and compare the results with other patients treated in the same center. In addition, considering that the use of induction chemotherapy may be associated with a delay in the prescription of definitive treatment (chemoradiation and brachytherapy) in patients with cervical malignancy, comparing the findings obtained from patients treated with induction and adjuvant chemotherapy approaches seems to be illuminative. Furthermore, since targeted treatments and immunotherapy are an integral part in the treatment of patients with malignancy nowadays, it is necessary to examine the role of simultaneous administration of this group of treatments during neoadjuvant/adjuvant chemotherapy or in combination with chemoradiation in future studies and clinical trials. Additionally, other brachytherapy approaches such as interstitial brachytherapy and new external radiotherapy techniques such as IMRT should also be attended to in future studies to address the limitations of the present study. The presentation of the results of this study in scientific conferences and its publication in reliable scientific and research journals can effectively help other researchers in designing upcoming studies. In conclusion, the results of the present study revealed the appropriate clinical response of patients with cervical cancer malignancy to the approach of induction chemotherapy followed by chemoradiation such that more than three quarters of patients had a complete/partial response in the three-month follow-up. Of course, it should be noted that the use of induction chemotherapy was associated with the treatment discontinuation in a significant number of patients. Although the treatment discontinuation during induction chemotherapy was less frequent than the treatment discontinuation during chemoradiation, it is worth considering that the main treatment of patients with locally advanced cervical cancer is the chemoradiation, and that the administration of induction chemotherapy reduces the patients’ tolerance of chemoradiation. Author Contribution Statement Study concept and design: N.A. and F.S.N.; acquisition of data: Z. Sh. and Sh. O.; analysis and interpretation of data: M.H. and A.M.; drafting the manuscript: M.M, M. A., and H.N.; critical revision of the manuscript for important intellectual content: N. Kh., B. K., and S.H.H.; statistical analysis: biostatical expert. Acknowledgements This research was funded by Shiraz University of Medical Sciences (grant number 16754 to N.A.). Authors would like to thank all patients who participated in the project. Ethical approval statement The study protocol was approved by the Research Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.MED.REC.1400.173), and a written informed consent form was obtained from the patients or their legal guardian. Clinical trial registration number The study was registered in the National Clinical Trials System (IRCT20210808052110N1). Data availability statement All data generated and analyzed during this study can be accessed through direct communication with the corresponding author upon the agreement of all research team members. Conflict of interest statement The authors report no conflicts of interest. ==== Refs References Ahmed I Krishnamurthy S Bhise R Definitive Chemoradiation with Concurrent Weekly Cisplatin in the Treatment of Esophageal Squamous Cell Carcinoma: A Simplistic Approach Asian Pac J Cancer Care 2021 6 181 7 Balasubramaniam G Gaidhani RH Khan A Survival rate of cervical cancer from a study conducted in India Indian J Med Sci 2021 73 203 11 Baruah C Paul T Sarma B Hematological Toxicity in Carcinoma Cervix Patients Undergoing Concurrent Chemo Radiation Therapy or Radiation Therapy alone in a Tertiary Hospital of North East India: A Prospective Study Asian Pac J Cancer Care 2022 7 651 55 Benson R Pathy S Kumar L Locally advanced cervical cancer - neoadjuvant chemotherapy followed by concurrent chemoradiation and targeted therapy as maintenance: A phase II study J Cancer Res Ther 2019 15 1359 64 31898673 Beriwal S Kannan N Sukumvanich P Complete metabolic response after definitive radiation therapy for cervical cancer: Patterns and factors predicting for recurrence Gynecol Oncol 2012 127 303 6 22902917 da Costa SCS Bonadio RC Gabrielli FCG Neoadjuvant Chemotherapy With Cisplatin and Gemcitabine Followed by Chemoradiation Versus Chemoradiation for Locally Advanced Cervical Cancer: A Randomized Phase II Trial J Clin Oncol 2019 37 3124 31 31449470 de Azevedo C Thuler LCS de Mello MJG Phase II trial of neoadjuvant chemotherapy followed by chemoradiation in locally advanced cervical cancer Gynecol Oncol 2017 146 560 65 28709705 Duenas-Gonzalez A Lopez-Graniel C Gonzalez-Enciso A Concomitant chemoradiation versus neoadjuvant chemotherapy in locally advanced cervical carcinoma: results from two consecutive phase II studies Ann Oncol 2002 13 1212 9 12181244 Duenas-Gonzalez A Zarba JJ Patel F Phase III, open-label, randomized study comparing concurrent gemcitabine plus cisplatin and radiation followed by adjuvant gemcitabine and cisplatin versus concurrent cisplatin and radiation in patients with stage IIB to IVA carcinoma of the cervix J Clin Oncol 2011 29 1678 85 21444871 Hong JC Foote J Broadwater G Data-Derived Treatment Duration Goal for Cervical Cancer: Should 8 Weeks Remain the Target in the Era of Concurrent Chemoradiation? 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116142 10.31557/APJCP.2023.24.4.1209 Research Article Potential Anti-Inflammatory and Growth Inhibitory Effect of Cyrtopodion scabrum Extract on Colon Cancer; An in vivo Study Babaei Zahra 12 Namavari Ghazal 3 Khademi Fatemeh 12 Koohpeyma Farhad 4 Rashidi Mojtaba 5 Shafiee S. M. 2 Zal Fatemeh 2 Seghatoleslam Atefeh 26* 1 Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran. 2 Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. 3 School of Medicine, Iran University of Medical Sciences, Tehran, Iran. 4 Shiraz Endocrinology and Metabolism Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. 5 Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 6 Autophagy research Center, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. * For Correspondence: seghatolea@sums.ac.ir 2023 24 4 12091216 24 10 2022 7 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: The use of complementary and/or alternative medicine to increase the efficacy and decrease the side effects of current cancer treatment is highly required. In this in-vivo study, we aimed to investigate the anti-tumor activity and probable side effects of a natural treatment, Cyrtopodion scabrum extract (CsE), in a model of tumor bearing mice. Methods: We established 28 female CT26-tumor bearing balb/c-mice model. We divided them randomly into four groups (n=7): Negative control received distilled water (DW) and the three treatment groups were administered with 5-FU and two different doses (300 and 600 mg/kg) of the gecko aqueous extract, respectively. The changes in the tumor volumes and weights during and after treatment, along with the blood cell counts; spleen and thymus indices were assessed in the treatment groups. We have also measured the serum TNF-α, VEGF, AST, ALT and GSH, as well as the physical activities of the experimental mice. Results: We found that the means of tumor weights and volumes in both CsE and 5-FU treated groups were significantly lower than the untreated group (p<0.05). Serum TNF-α and VEGF levels in both CsE treated groups were remarkably lower than 5-FU and untreated groups (p<0.05). The 5-FU treatment caused a remarkably decrease in serum GSH, RBC count, WBC count, thymus index, and spleen index , while CsE treatment maintained these quantities, with no significant changes, compared to the control group. AST and ALT were not significantly changed in none of the treated groups compared to control. Conclusion: Altogether, data suggest C. scabrum, as an effective and safe anti-cancer natural source, which could be used as an alternative/complementary medicine for the treatment of patients who suffer from colon cancer. Key Words Cyrtopodion scabrum natural anti-tumor medicine anti-inflammation side effect 5-FU ==== Body pmcIntroduction Cancer is one of the major public health problems in the world (Torre et al., 2016). The statistics show an estimated 19.3 million new cases and 10 million cancer deaths worldwide in 2020 and a global cancer burden of 28.4 million cases in 2040, a 47% rise from 2020 (Sung et al., 2021). Colorectal cancer (CRC), the third most common malignancy, is predicted to increase by 60% to more than 2.2 million new cases and 1.1 million deaths by 2030 (Arnold et al., 2017). The incidence of this type of cancer is the fourth and third among Iranian men and women, respectively (Halimi et al., 2020; Mansori et al., 2018). Chemotherapy, one of the main powerful treatments for cancer, has been shown to increase the survival rate by 10% (Blauwhoff-Buskermolen et al., 2016). It not only kills cancer cells, but also affects normal cells intensively. Various side effects including cardiocytotoxicity, nephrotoxicity, myelosuppression, neurotoxicity, hepatotoxicity, and gastrointestinal toxicity have been reported to be associated with chemotherapy and strongly impair the quality of life of cancer patients (Liu et al., 2021). Therefore, finding less toxic and safer drugs against cancer is urgent, and many scientists have turned the spotlight to the use of natural agents such as herbal extracts or animal products. For almost thousands of years, the traditional use of natural products including zoo therapy and herbal therapy has represented a source of effective drugs (Fakher et al., 2019; Gohari et al., 2018; Seghatoleslam et al., 2014; Tavakoli et al., 2015). In Traditional Chinese Medicine (TCM), the whole-body extract of a kind of Chinese gecko (swinhonis Gūenther) has been used as a drug to treat various diseases such as cancer for hundreds of years (Duanet al., 2018; Jeong et al., 2012; Lee et al., 2019; Yang and Wang, 2020). In our previous studies, we also showed that a kind of gecko, named Cyrtopodion scabrum (so-called rough-tailed home gecko), has anti-proliferative effects on the breast, colorectal (Amiri et al., 2015), stomach, and liver cancer cells (Rashidi et al., 2017). C. scabrum is distributed around the Indus Valley to the eastern borders of the Caspian Sea, including Iran, Turkey, Pakistan, Iraq, etc. (Rastegar-Pouyani et al., 2010). Our recent studies revealed that the C. scabrum extract (CsE) had antitumor properties and growth inhibitory effects of 30-78% on different human cancer cells with a significant impact on SW742, MKN45 and HepG2, and no significant effect on the normal cells. We also found that treatment with CsE induced apoptosis in cancer cells and the observed anti-cancer effect of CsE might occur through TP53 up-regulation, but P53-independent transcriptional activity. According to our last interesting findings, C. scabrum homogenate and extract significantly attenuated the 5-fluorouracil (5-FU)-induced liver dysfunction in rats through strengthening antioxidant defense system, resulting in liver function improvement (Amiri et al., 2015; Diba et al., 2021; Rashidi and Seghatoleslam, 2021). We designed the present study to investigate the potential in-vivo anticancer properties and side effects of CsE, which are still unclear. We also compared the effects of CsE with 5-FU, as the main backbone of chemotherapy for colorectal cancer patients which have not been reported previously. We also aimed to identify a new inexpensive anticancer drug with natural sources which might be an efficient noninvasive therapy compared with current treatment strategies. Materials and Methods Cell culture The CT26 cell line was purchased from National Center for Cell Sciences, Pasteur Institute of Iran (Tehran). The cells were cultured as monolayers in RPMI-1640 medium (Bioidea, Iran) supplemented with 10% FBS (Shellmax, China), 1% Penicillin Streptomycin (Shellmax, China) and maintained in a humidified atmosphere of 5% CO2 at 37°C. Establishment of CT26-tumor bearing mice 28 female BALB/c mice (5-7-week-old and weight of 16-18 g) were purchased from Pasteur Institute of Iran (Tehran). The mice were housed under standard conditions with light: dark cycle 12:12 h, humidity 23-32%, temperature 22-25°C, and allowed access to food and water ad libitum. All the procedures were approved by Institutional Animal Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (Ethical code: IR.SUMS.REC. 1395.S772). The CT26 model was established by subcutaneous injection of 3×106 viable cells to the armpit region of BALB/c mice, as previously reported (Zhang et al., 2013). Tumor growth was monitored daily during 2 weeks for tumor development. When the tumor volume reached approximately 90-100 mm3, the mice were considered ready for starting the treatments. Preparation of the Cyrtopodion scabrum extracts (CsE) C. scabrum was provided by Razi Research Institute of Vaccine and Serum, Shiraz Branch, Iran. The identification of the specimens had been performed by a taxonomist (F.Torki, the head of the department at FTEHCR). The extract was prepared according to the protocol described previously by Amiri et al., (2015). Briefly, the animals were weighted, cleaned, and crushed by liquid nitrogen; then, homogenization, water extraction, and ethanol precipitation were done to obtain aqueous crude extract (CsE). It was then dialyzed, freeze dried by freeze dryer Alpha 1-2/ LD plus (Christ, Germany), and kept at -20°C for later use. Experimental design The established tumor-bearing mice (n=28, 7-9 weeks, 20±3 gr) were randomly divided into four experimental groups as follows: Group I, the negative control group, was given 0.2 ml DW orally by gavages. Groups II and III received CsE (300 and 600 mg/kg BW- according to the pilot study) orally by gavages in a daily schedule (0.2 ml of DW consisting of required amount of CsE). Group IV was given daily intraperitoneal injections of 5-FU (2 mg/kg BW) (Cipla, Iran). Animal body weight and the tumor size were measured on the first day, and the changes were monitored daily until the end of the treatment. The duration of treatment was determined based on the tolerability of the growing tumors by the animals in the control group, their ability to move, body weight loss, infirmity and weakness, in the mice of 5-FU group. Thus, in this experiment, the treatments were terminated on the day 12, according to the above-mentioned items. At the end of the treatment, the mice were anesthetized using CO2. Blood sampling was performed from the heart, and the sera were collected and stored at − 20°C until use for biochemical and immunological measurements. The final tumor size and volume were also measured. The mice were sacrificed, and the whole-body skin of the tumor-bearing mice was removed for complete detection (Figure 1A). Anti-tumor assay The length and width of the tumors were measured daily using a caliper (Heng Liang, China) and the tumors’ volumes were calculated using the standard formula: V=ab2/2 where a, is the length, and b is the width of the tumors. Moreover, final body and tumor weights were measured after dissection and the tumor volumes were also evaluated after dissection by immersion method. The anti-tumor activity of the treatments was evaluated using the following formula: Tumor inhibitory rate (%) = (Average tumor weight of treatment groups / Average tumor weight of control group) ×100 Immune function analysis To evaluate the impact of the treatments on the immune organs, we calculated the thymus and spleen indexes, as follows: Organ index = average weight of organ (mg) / body weight (g) The number of WBCs was also analyzed using neubauer counting chamber, after dilution of the blood samples with Marcano solution (2% acetic acid + 1% methylene blue + 97% DW). Serum TNF-α, as an important pro-inflammatory cytokine, was also measured using mouse TNF-α (IBL, Germany) ELISA kit, following the manufacturer’s instructions. Hematologic and non-hematologic toxicity study On day 13, the blood samples were collected via cardiac puncture under CO2 anesthesia. To study hematologic toxicity of CsE, we analyzed the whole blood for red blood cell (RBC) count using neubauer-counting chamber. Also, to study the probable side effects of CsE on the liver, we analyzed the sera for aspartate transaminase (AST) and alanine transaminase (ALT) activity using colorimetric assay kits (MAN, Iran). Moreover, the body weight was also measured to evaluate the effect of CsE treatments on the body weight of the experimental mice. Determination of antioxidant state The level of GSH, as a main antioxidant parameter in the body, was evaluated in the sera of experimental mice, using Ellman’s method with minor modification (Rostampour et al., 2017). The process was based on the formation of GS-TNB complex from DTNB (5, 5’ dithiobis 2-nitrobenzoic acid) which developed yellow color because of the DTNB reduction. The GSH level was measured using spectrophotometer (SHIMADZU, Japan) at 412 nm. The total level of GSH was calculated using a standard curve. Analysis of VEGF angiogenic marker As a preliminary study to explore the anticancer mechanism of CsE, serum level of VEGF, as a main angiogenic marker, was analyzed using mouse VEGF ELISA kit (IBL, Germany), following the manufacturer’s instructions. Statistical analysis All data were expressed as mean ± SD from duplicate experiments and analyzed using SPSS software (Version 16.0; Chicago, USA). Mann-Whitney U test was also applied to analyze the significant groups with one factor. P<0.05 was considered statistically significant. Results In this study, we evaluated the in-vivo antitumor efficacy of CsE in CT26-tumor-bearing mice and compared it to 5-FU chemotherapy treatment. We also studied its effects on hemato-immunological indices and liver function as well. Effects of CsE on tumor growth and body weight in CT26-tumor bearing mice Daily changes in the tumor volume were measured and summarized in Figure 1. These results revealed unchanged tumor growth and volume in the groups treated with 5-FU and two doses of CsE compared to the control group, suggesting a significant antitumor effect of both CsE and 5-FU. On day 12, the mice were sacrificed, and the final body weight, tumor volume, and tumor weight were measured after dissection. The results are summarized in Table1. Based on the results, the tumor volume and weight were decreased dramatically in the treated groups compared to the control group (p<0.05), with no significant differences between CsE and 5-FU treated groups. The tumor growth suppression was approximately 63.36 and 65.99% for the mice treated with two doses of CsE and 78.11% for 5-FU treated mice, compared to the control group. The dissected tumors were also photographed, and the tumor size and volume in the different groups were compared (Figure 2 A,B). As demonstrated, the sizes of the dissected tumors in the treatment groups diminished significantly, and hopefully they were about to be cured in some cases. In the 5-FU group, the tumor growth was suppressed compared to the control group, but two of the mice died due to the side effects of the chemotherapy. We also observed that although 5-FU, as a chemotherapeutic agent, dramatically suppressed the tumor weight, it simultaneously reduced the body weight of mice compared to the control group (p=0.017), which indicated that 5-FU had a strong toxicity on the body. Meanwhile, the body weights of CsE-treated mice were significantly higher than those in the 5-FU group, with no significant differences with the control mice. These results suggest that CsE had no toxicity to the organism (Table 1). The effect of CsE on immune function As summarized in Table 2, WBC count in the experimental groups revealed no significant changes in the CsE treated groups, but a significant (P=0.004) decrease was observed in the 5-FU-treated group when compared to the control group. There was no significant difference between the thymus and spleen index of the treatment groups of CsE compared to the untreated control group, but these indexes decreased significantly (P<0.05) in the 5-FU-treated group. The serum level of TNF-α was significantly lower in the CsE treated groups compared to the control and 5-FU groups (p<0.05), but the difference between the control and 5-FU groups was not significant (Figure 3). The Effect of CsE on organ function We further studied the safety and potential toxicity of CsE on the hematologic (RBC) and nonhematologic (serum ALT and AST) parameters, in response to the CsE and 5-FU treatment. As shown in Table 3, CsE treatment did not make any significant change in the RBC count compared to the control group, while treatment with 5-FU significantly (p=0.010) reduced the RBC count compared to the control group. Thus, unlike 5-FU, treatment with CsE was not associated with hematological toxicity. However, no significant changes, which indicate liver toxicity, were observed in the serum AST and ALT levels in the treatment groups compared to the control one. The Effect of CsE on GSH level Serum GSH level, as a main antioxidant parameter, was also measured in the CT26-tumor- bearing mice. As shown in Table 3, while the serum GSH level in the 5-FU group significantly decreased (P<0.05), CsE treatment did not shown reduction, compared to the control group. The Effect of CsE on VEGF angiogenic marker Serum VEGF levels, as the main regulator which induces tumor angiogenesis, are represented in Figure 4. According to the results, CsE treatment significantly decreased the VEGF levels compared to the control and 5-FU groups (p<0.05), while the difference between the control and 5-FU groups was not significant. Tables 1 The Effects of CsE on Body Weight, Tumor Weight, Calculated Volume and Inhibitory Rate at the End of the Treatments Groups Body weight (g) Tumor weight (g) Tumor volume (%) Inhibitory Rate (%) Control 18.80±3.1 5.52±2.67 5.18±2.50 - CsE 300 20.00±3.4 1.87±1.27a 1.24±1.01a 65.99 CsE 600 23.60±4.5 2.02±1.98a 1.90±1.40a 63.36 5-FU 12.83±2.9a 1.2±0.90a 1.31±0.84a 78.11 The values (n=7) are presented as mean±SD; a, indicates significant at P < 0.05 vs Control group Figure 1 The Effect of CsE on Tumor Growth during 12-day Treatments in the CT26-Tumor-Bearing Mice. The lengths and widths of the tumors were measured individually every day and presented as an average for each group in tumor growth curve Table 2 The Effects of CsE on the Immune Function in the Tumor-Bearing Mice Groups WBC count (×103 /ml) Spleen index (mg/g) Thymus index (mg/g) Control 7.22±1.5 5.16±2.5 16.45±4.5 CsE 300 5.35±0.82 4.98±0.94 14.01±3.7 CsE 600 5.57±1.2 4.60±0.68 14.85±2.5 5-FU 0.72±0.16a 1.60±0.94 a 8.9±4.7a The values (n=7) are presented as mean±SD; a, indicates significant at P < 0.05 vs Control group Figure 2 (A) Photograph of the whole body of the tumor-bearing mice, on day 13, after removing the skin. (B) Photograph of the dissected tumors from the mice of each group on day 13 Table 3 The Potential Toxicity of CsE in the Tumor-Bearing Mice Groups RBC count (×109/ml) Serum AST Level (U/L) Serum ALT Level (U/L) Serum GSH Level (mmol/ml) Control 0.31±.12 181.22±25.3 62.7±7.7 5.70±1.7 CsE 300 0.40±.38 164.00±24.1 55.83±10.7 5.90±2.1 CsE 600 0.32±.13 166.00±29.1 60.80±3.5 5.90±.98 5-FU 0.15±.10a 198.17±42.5 67.78±10.9 2.44±.88a The values (n=7) are presented as mean±SD; a, indicates significant at P < 0.05 vs Control group Figure 3 The Effect of CsE on the Serum TNF-α Level in the Tumor-Bearing Mice. a, indicates significance at P < 0.05 vs. Control group; b, indicates significant difference vs. 5-FU group Figure 4 The effect of CsE on the serum VEGF level in the tumor-bearing mice. a, indicates significance at P < 0.05 vs. Control group; b, indicates significant difference vs. 5-FU group Discussion The selective anticancer properties and growth inhibitory effects of C. scabrum, a genus of the rough-tailed geckonid lizard of Iran, was approved for the first time in our previous studies (Amiri et al., 2015; Rashidi et al., 2017). The present study was designed to have a better understanding of its anti-tumor properties, potential toxicity, and possible mechanism for tumor suppression, in-vivo. For this purpose, we selected a colon cancer cell line, CT26, to establish BALB/c tumor-bearing mice. CT26 tumor-bearing mice have been used extensively in the literature, as a model of colorectal cancer (Tuo et al., 2020; Wei et al., 2018). The results obtained from this study indicated that the tumors of groups subjected to different concentrations (300 and 600 mg/kg BW) of CsE as well as 5-FU-treated group shrank significantly, compared to control group. These results are consistent with those of Liu et al.’s study on anti-tumor activities of one kind of Gecko powder on the tumor model of the mouse S180 sarcoma (Liu et al., 2008). While the unwanted side effects of 5-FU treatment are evident, we extended our investigation on CsE treatment to explore whether it may have negative side effects on the treated groups as well. Therefore, we evaluated some immune and hematopoietic parameters in the treatment groups. Immune system and hematopoietic system are two important sensitive systems that are the target of harmful effects of drugs on living organisms. It is well known that the thymus and spleen, as the main immune organs, play important roles in the immunity of the host. Therefore, a higher organ index indicated a stronger immune capability (Yin et al., 2007; Zhu et al., 2016). The toxic effect of drugs on hematopoietic system can be evaluated through measurement of blood parameters such as the total WBC and RBC count. The results obtained from this part of the study indicated that 5-FU, as a conventional chemotherapy agent, had adverse effects by the significant reduction in WBCs and RBCs in the peripheral blood and a decline in the spleen and thymus indexes as well. In close agreement with our results, Li et al., (2018) and Gelen and Şengül, (2018). Also reported immune suppression and cytopenia followed by 5-FU-treatment. Unlike the results of 5-FU-treated group, CsE exerted its antitumor effects without any immune suppression or cytopenia, indicating its prominence in immune safety. Moreover, the GSH marker, as a representative of antioxidant state, significantly reduced in the 5-FU-treated group compared to the CsE-treated and control groups and there was no difference in the GSH level in the CsE treatment groups compared to the control group. Taken together, these observations strongly indicate that CsE, as a natural source-based remedy, is a safer antitumor agent compared to 5-FU chemotherapy drug. In order to further confirm the safety of CsE, we measured the activity of hepatic enzymes, ALT and AST, to assess the potential liver function or liver damage (Moreno, 2009). Fortunately, CsE consumption revealed no hepatotoxicity in the animals. Moreover, the mice treated with CsE demonstrated good physical activity and normal weight gain, while in the 5-FU group, two mice died and the rest had become very weak; in some cases, they were even unable to eat normally; these results also approved the safety of CsE, as a natural antitumor treatment, compared to 5-FU. To uncover the mechanism of tumor suppression and the possible role of gecko extract, as an anti-angiogenic and/or anti-inflammatory factor, we measured serum TNF-α and VEGF of the treatment groups. The results obtained from our study showed that the level of these factors decreased significantly following CsE, but not 5-FU treatment, which suggests that the antitumor mechanisms of CsE is different from that of 5-FU. In close agreement with our results, there was the reduced level of TNF-α and VEGF following some Gecko species extract treatments in some in-vivo and in-vitro experiments (Song et al., 2012; Tang et al., 2015). Therefore, based on the results of our study, CsE treatment can lead to effective cancer treatment due to the reduction of pro-inflammatory cytokine TNF-α together with VEGF. However, there is a complicated interplay between inflammation and carcinogenesis. Most recent reports revealed a direct causal link between inflammation and carcinogenesis. During inflammation, cytokines are released by immune and stromal cells of the tumor by cell-to-cell signaling. Elevated expression of TNF-α, as a pro-inflammatory cytokine, is the characteristic of many malignant tumors and is associated with poor prognosis (Wu and Zhou, 2009). Inflammation could lead to not only the induction of neoplastic transformation or promotion of tumor growth, but also recurrence after therapy. Inflammatory conditions also increase the production of growth factors such as VEGF, which increase angiogenesis and results in tumor growth and proliferation (Avraamides et al., 2008; Li et al., 2007). Limitations Further biochemical and Molecular studies are required for investigating the exact antitumor mechanisms of CsE treatment as well as exploring the active component of the extract. In addition, more extensive research should be conducted to verify and optimize CsE for using in Clinical trial. In conclusion, to the best of our knowledge, this is the first in-vivo experimental study to examine the anti-tumor effects of C. scabrum compared to 5-FU, an important chemotherapy agent. The most remarkable result emerging from our data is that C. scabrum is a valuable source of anti-cancer materials with no unwanted side effects, which possibly exerting its anti-tumor effects through anti-inflammatory and anti-angiogenic mechanisms. Author Contribution Statement A.S. designed the study and Z.B. and S.M.S. participated in the design of the work. Z.B., G.N., F.Kh. and F.Ko conducted the experiments and analyzed the data. M.R., F.Z. and S.M.S. provided conceptual and technical guidance for all aspects of the study. A.S. and Z.B. wrote the manuscript with input from all the authors. All the authors contributed to the manuscript revisions. Acknowledgements The authors would all like to thank Dr. M.Namavari from Razi Research Institute of Vaccine and Serum, Shiraz Branch for providing C.scabrum, and Dr. Narges mashkour for critical reviewing the manuscript. We also acknowledge the English editing done by Dr. N.Shokrpour at the RCC department of vice chancellor for research at Shiraz University of Medical Sciences. This article was extracted from the MSc thesis written by Z. Babaei. Funding The present study was financially supported by research grant no. 95-01-01-11796, from Shiraz University of Medical Sciences, Shiraz, Iran. Ethical approval All the procedures were approved by Institutional Animal Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (Ethical code: IR.SUMS.REC. 1395.S772). Availability of data data are available from the corresponding author on reasonable request. The present study follows the guidance of Critical Appraisal Tool (AIMRDA) developed for the Peer-Review of Studies Assessing the Anticancer Activity of Natural Products (Ahmad et al., 2022). 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116149 10.31557/APJCP.2023.24.4.1265 Research Article Anti-cancer Effect and Active Phytochemicals of Houttuynia cordata Thunb. against Human Breast Cancer Cells Inthi Pitsinee 1 Pandith Hataichanok 23 Kongtawelert Prachya 1 Banjerdpongchai Ratana 1* 1 Department of Biochemistry, Faculty of Medicine, Chiang Mai University, 110 Inthawaroros Road, Sri Phoom, Muang, Chiang Mai 50200, Thailand. 2 Department of Biology, Faculty of Science, Chiang Mai University, 239 Huaykaew Road, Suthep, Muang, Chiang Mai 50200, Thailand. 3 Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. * For Correspondence: ratana.b@cmu.ac.th 2023 24 4 12651274 30 11 2022 7 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Houttuynia cordata Thunb (HCT) is a medicinal herb used in Southeast Asia. Aim of this work: This study aimed at investigating the cytotoxicity of this plant extract and fractions towards human breast cancer MDA-MB-231 and MCF-7 cells. HCT’s phytoactive compounds are determined. Materials and methods: Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mode of cell death was measured by staining with annexin V-FITC and propidium iodide (PI) employing flow cytometry technique. The oxidative stress was measured by using 2’,7’-dihydrodichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE+) fluorescent probes and using a fluorescence microplate reader. HCT phytochemicals were characterized by high performance liquid chromatography (HPLC). Results: The proliferation of MDA-MB-231 and MCF-7 cells was dramatically decreased by the crude extract and individual fraction of HCT. Ethyl acetate was the solvent fraction with the highest toxicity against MCF-7 cells, followed by dichloromethane, crude, and hexane fractions, respectively, whereas in MDA-MB231 cells, dichloromethane, crude, hexane, and ethyl acetate fractions each had the strongest impact, respectively. The methanol fraction had no effect on either cell line up to 200 μg/ml. The extract and fractions were less harmful to the NIH3T3 normal murine fibroblast cell line. The mode of both cell death was apoptosis evidenced by the increase of cell population stained with annexin V-FITC and PI. The fluorescence probes of both DCFH-DA and DHE in MDA-MB-231 cell line were enhanced. Phenolic acids included chlorogenic acid (CA), gallic acid (GA), transcoumaric acid (TCA), vanillic acid (VA), and syringic acid (SA), as well as flavonoids like quercetin and rutin, were identified as the active phytochemicals in the crude and fractions by using HPLC method. Conclusion: MDA-MB-231cells underwent apoptosis via oxidative stress when induced with HCT hexane fraction. Phenolic acids and flavonoids were identified in HCT’s extract and fractions. Key Words Houttuynia cordata Thunb anti-cancer human breast cancer active phytochemicals ==== Body pmcIntroduction According to a molecular docking method that determines the binding affinities between the phytochemicals and the growth factor receptors in cancer cells, Houttuynia cordata Thunb. (HCT), has anti-cancer ligands. According to Das et al., in 2022, the top-hit phytochemicals from HCT can be employed to dock with the overexpressed growth factor receptors. The ligand-binding pocket of the HER2 and VEGFR2’ kinase domains is docked against a total of 100 physiologically active phytochemicals from HCT (Das et al., 2022). Finding high affinity phytochemicals that have a perfect fit for the ATP ligand-binding pocket requires competitive docking. When docked to HER2 and VEGFR2 using both hydrogen and hydrophobic contacts of the kinase domains of HER2 and VEGFR2, β-sitosterol and quercetin have the highest binding affinity among the top-hit phytochemicals from HCT (Das et al., 2022). We also previously reported the anti-cancer effect of HCT extract against human breast cancer cell proliferation, migration, invasion and apoptosis (Subhawa et al., 2020). In castrate-resistant (PCai1) and androgen-sensitive prostate cancer cells (LNCaP), Subhawa et al., in 2021, showed that HCT extract and its ethyl acetate fraction (EA) induce apoptosis through activation of caspases, down-regulation of androgen receptor, and inactivation of AKT/ERK/MAPK signaling. One of HCT’s active ingredients, rutin, exerts effects on LNCaP cells that are comparable to those of HCT/EA. Moreover, in LNCaP cells, HCT/EA block the STAT3/Snail/Twist and epithelial-mesenchymal transition phenotypes. Treatment with 1% HCT orally dramatically slows the growth of PCai1 xenograft tumors. HCT inhibits the development of prostate cancer and CRPC both in vitro and in vivo (Subhawa et al., 2021). In 2021, Liu et al., conducted research on the effects of heat treatment on various HCT components and the inhibitory effects on gastric cancer cell proliferation. Alamar blue assay is used to evaluate the effect of various extracts (heated aerial stem, heated subterranean stem, heated leaves, and non-heated classified as NAS, NSS, and NL) on cancer cell viability. It’s interesting to note that heat treatment intensifies the effects of cellular growth inhibition and triggers apoptosis via the mitochondrial pathway (Liu et al., 2021). In order to treat cervical cancer cells biologically, Chen et al., in 2021, produced the copper nanocomplex utilizing HCT (Hc-CuONPs) herbal extract. With a maximum peak at 350 nm, the UV-visible spectroscopy research demonstrated the existence of the nanocomplex, whilst the Fourier transform infrared (FT-IR) spectroscopy absorption peaks exhibited the existence of distinct function groups. Studies using X-ray diffraction and transmission electron microscopy (TEM) revealed that the Hc-CuONPs have a face-centered cubic structure with a size of 40–45 nm. Moreover, the Hc-CuONPs is hazardous to cervical HeLa cancer cells (IC50=5 μg/ml). Acridine orange/ethidium bromide (AO/EtBr), propidium iodide (PI), and 4’,6-diamidino-2-phenylindole (DAPI) staining, are used to evaluate apoptotic cell death in the nanocomplex-treated HeLa cells. The analysis results showed that the Hc-CuONPs inhibit HeLa cell proliferation and promote apoptosis in an in vitro model through PI3K/AKT signaling pathways (Chen et al., 2021a). New clinical data show that HCT is highly effective in treating radiation-induced lung injury (RILI). A deadly and dose-limiting consequence of radiation for thoracic malignancies was the subject of two studies, one by Liu et al., in 2023, and the other by Lai et al., in 2022. There are two types of the conditions: irreversible advanced-stage fibrosis and early reversible pneumonitis. Reliable biomarkers are still needed for RILI clinical diagnosis and early prediction. Inflammation plays a major role in the onset and development of RILI, and certain associated biomarkers are identified during this process. 75 differentially expressed genes (DEGs) are filtered out of two expression profiles that were retrieved from the Gene Expression Omnibus (GEO) database. MMP-9, IL-1, CCR1, and S100A9 were found to be four inflammation-related hub genes in the progression of RILI by combining Gene Oncology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) network analysis, viz., created using STRING. In RILI mice models, the hub genes’ levels of expression are confirmed, with S100A9 showing the greatest level of overexpression. S100A9 expression in lung tissues and its amount in bronchoalveolar lavage fluid (BALF) are positively linked with the level of inflammation in RILI (Liu et al., 2023). 12 potent compounds against RILI were found by Lai et al., in 2022, after they searched for the main active components from HCT that corresponded to the targets in the Traditional Chinese Medicine System Pharmacology (TCMSP) Database. The GeneCards database is used to retrieve the targets of RILI. By Venn diagram analysis, common targets between active chemicals and disorders are discovered. The network interaction between the medications, active chemicals, targets, and disease is built and visualized using Cytoscape. The effectiveness of the primary active compounds’ binding affinities to the central targets is confirmed by molecular docking research. Quercetin, kaempferol, hyperoside, and rutin are the main active chemical constituents. The PPI network’s hub nodes for the cancer-signaling pathway include TP53, VEGFA, JUN, TNF, and IL-6. The GO categories are divided into three functional areas: 32 cellular components from the active chemicals in the HCT, 112 biological processes, and 9 molecular functions. The target genes are found in multiple important cancer-related signaling pathways, including the TNF-, PI3K-, AKT-, and HIF-1 signaling pathways, according to the KEGG pathway enrichment study (Lai et al., 2022). The chemopreventive effect of HCT and its bioactive component 2-undecanone against benzo(a)pyrene (B[a]P)-initiated lung carcinogenesis and the underlying mechanism were reported by Lou et al., in 2019, using a B[a]P-stimulated lung cancer animal model in A/J mice and a normal lung cell model (BEAS.2B) in vitro. Without causing systemic toxicity in vivo, HCT and 2-undecanone effectively reduce B[a]P-induced lung carcinogenesis. Furthermore, BEAS.2B cells are further noticeably protected by HCT and 2-undecanone, which successfully reduce B[a]P-induced intracellular ROS overproduction, DNA damage, and inflammation by preventing interleukin-1 (IL-1) and phosphorylated H2A.X overexpression. By strikingly activating the Nrf2 pathway, HCT and 2-undecanone, as confirmed by an Nrf2 siRNA molecule, cause the production of the antioxidant enzymes: heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO-1). The Nrf2-HO-1/NQO-1 signaling pathway is thus effectively activated by HCT and 2-undecanone to reduce intracellular ROS production, thereby reducing DNA damage and inflammation brought on by B[a]P activation (Lou et al., 2019). There are, however, still few studies in comparing the cytotoxicity of HCT extract and different components. As a result, the objectives of this study were to determine the cytotoxic effect of crude extract in comparison to hexane, ethyl acetate, dichloromethane and methanol fraction on human breast cancer MDA-MB-231 and MCF-7 cells, as well as normal murine fibroblast NIT3T3 cell line, mode of cell death, and its preliminary molecular mechanism, including whether or not ROS were generated. The phytochemical components were identified by using HPLC. Materials and Methods Chemicals Rutin, quercetin, 2’,7’dihydro dichlorofluorescein diacetate (DCFH-DA), and dihydroethidium (DHE+) were purchased from Sigma-Aldrich in addition to MTT (3-(4,5-dimethyl)-2,5-diphenyl tetrazolium bromide, gallic acid, chlorogenic acid, vanillic acid, syringic acid, and transcoumaric acid (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) was bought from Invitrogen (USA). A Roche Annexin V-FITC kit was purchased (Indianapolis, Indiana, USA). The fluorescence probes Fluo3-AM and Rhod2-AM were purchased from Molecular Probes (Eugene, Oregon, USA). Analytical quality hydrogen peroxide, ferrous sulfate, methanol, ethyl acetate, hexane, dichloromethane, and ethanol were purchased from RCI Labscan Limited (Bangkok, Thailand). Plant materials and extraction The complete Houttuynia cordata Thunb. plants were procured in Thailand’s Lop Buri province in May 2018. Plant species was identified and authenticated by Dr. Narin Printarakul, a lecturer and senior plant taxonomist, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand. The voucher specimen number is CMUB003997001, deposited in CMU Herbarium, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand. After the plant’s authenticity was confirmed, HCT that had been air dried and finely ground was percolated five times with ten liters of ethanol over the course of five days at room temperature. To create a crude ethanolic extract, the extracts were mixed and dried under decreased pressure. The ethanolic extract was redissolved in a 3:7 mixture of methanol and water, and then partitioned three times with different solvents in a ratio of 1:1: hexane (H), dichloromethane (CH2Cl2), and ethyl acetate (EtOAc). The solvent was eliminated from each fraction using a rotary evaporator. Every fraction was kept at 4oC until used. Cell lines and cell culture The American Type Culture Collection provided the human breast cancer MDA-MB-231, MCF-7 and murine embryonic fibroblast NIH3T3 cells (ATCC, Manassas, VA, USA). Cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% inactivated fetal bovine serum, 25 mM NaHCO3, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were incubated at 37°C in an environment with 5% CO2. Cytotoxicity test The crude extract, various fractions, including hexane, dichloromethane, ethyl acetate, and methanol fractions, were applied to MCF-7, MDA-MB-231, and NIH3T3 cells for 24 hours at various concentrations, i.e., two-fold dilution, such as 50, 100, 150, 200, 300, and 400 μg/ml. Cell viability was assessed using a mitochondrial enzyme reaction that changes MTT into formazan crystals. MTT dye solution was added and incubated at 37oC for four hours after the extract or each fraction had been treated for 24 hours. A microplate reader was used to measure the absorbance at 540 nm and the reference wavelength at 630 nm after the formazan crystal was dissolved in dimethyl sulfoxide (DMSO) (BioTek, Winooski, VT, USA) (Banjerdpongchai et al., 2010). As in the prior study, the percentage of cell viability was calculated. Using the formula below. To further investigate the dosage response effect, the inhibitory concentrations at 20 percent (IC20) and 50 percent (IC50) were computed. Calculating the percentage of cell viability was performed using the following formula (Banjerdpongchai et al., 2010). %Cell viability=mean absorbance in treated wellsmean absorbance in control wells×100 Mode of cell death determination by flow cytometry In a 24-well culture plate, 5x105 MDA-MB-231 and MCF-7 cell lines were incubated at IC0, IC20, and IC50 doses. The cells were centrifuged after being washed twice with phosphate-buffered saline (PBS) and incubated at 37°C in a 5% CO2 environment for 24 hours. The cells were then reconstituted and stained for 15 minutes in the dark with 100 μl of binding buffer containing 5 μl of annexin V-fluorescein isothiocyanate (FITC) and PI (Roche Diagnostics, Mannheim, Germany). The cells’ fluorescence intensity was assessed. Using the flow cytometer’s Cell Quest software, the cells’ dot plot character was examined (Beckman, Dickinson and Company, NJ, USA). Annexin V-FITC and Annexin V-FITC combined with PI staining cells were identified as early and late apoptotic cells. A bar graph was created and the proportion of total apoptotic cells was computed (Banjerdpongchai et al., 2016). Determination of reactive oxygen species generation At IC0, IC20, and IC50 concentrations, particular hexane fractions were applied to cells (1x105 cells/ml in a 96-well culture plate). Each unique set of treated cells received a final concentration of either DHE+ (25 μM) or DCFH-DA (20 mM) before an hour of incubation. The fraction was applied to the cells for 4 hours at 37°C and 5% CO2. After that, the DCF fluorescence was recorded. As a positive control, hydrogen peroxide (0.3% for 30 minutes; H2O2) was used. A fluorescence microplate reader was employed with wavelengths of 485 nm and 530 nm as the excitation and emission wavelengths, respectively (Prommaban et al., 2012). When employing FeSO4 as a positive control, the excitation and emission wavelengths for ethidium (E+) were 535 nm and 635 nm, respectively (Rachakhom et al., 2019). Characterization of phytochemicals by high performance liquid chromatography (HPLC) analysis All fractions and the ethanolic extract were dissolved in methanol at a concentration of 1 mg/ml. In methanol at a concentration of 100 μg/ml, all typical chemicals, including gallic acid, chlorogenic acid, vanillic acid, syringic acid, transcoumaric acid, rutin, and quercetin, were dissolved. Agilent 1260 Infinity DAD detector (Santa Clara, CA, USA) with a binary pumping system was used for HPLC-UV/DAD analysis. Zorbax Eclipse plus C18 column (4.6 mm 100 mm, particle size 3.5 m) was injected with samples (10 ml) (Kumar et al., 2014). The standard mixture’s gradient program designated for 0.5% formic acid in water (solvent A) and methanol (solvent B). For 4 minutes, the solvent gradient was 90:10 (A:B), for 11 minutes, 80:20, for 50 minutes linearly 10:90, and for 5 minutes constantly. Prior to injecting the second sample, an equilibrium phase of 90:10 (A: B) lasted for 5 minutes. The flow rate was 0.6 ml/min, the detecting wavelength was 272 nm, and the injection volume was 5 ml. The extract was subjected to HPLC under the conditions of 0.5% formic acid in water (solvent A) and methanol (solvent B) as the mobile phase. Before injecting the next sample, the initial A:B ratio was 90:10 for the first 4 min, changing to 70:30 for 16 min, 50:50 for 40 min, 30:70 for 10 min, constant for 10 min, and 90:10 for the final 10 min. The detection wavelength was 272 nm, the injection volume was 5 ml, and the flow rate was 0.6 ml/min. Formic acid (solvent A) and methanol (solvent B) were the solvents used in the HPLC procedure for the hexane fraction. 50:50, 10:90 for the opening 15 minutes, for A:B. Before injecting the next sample, there was a 5-min 90:10 (A: B) equilibrium phase that followed. Volume of 5 ml was used for the injection, with a flow rate of 0.6 ml/min and a 272 nm detecting wavelength. Using methanol as the solvent B and 0.5% formic acid in water as the solvent A, HPLC was used to separate the dichloromethane fraction. Initially, the 4:1 ratio of A:B was 90:10, followed linearly by 30 minutes of 80:20 and 20 minutes at 10:90. The following session was a 5-minute 90:10 equilibrium period. The flow rate was 600 μl/ml, the detecting wavelength was 272 nm, and the injection volume was 5 ml. Statistical analysis The results are presented as mean + S.D. Three independent experiments carried out in triplicate were used to determine the statistical difference between the control and treated groups using one-way ANOVA (Kruskal Wallis analysis) with a p-value cutoff of 0.05. Data were examined using Student’s t-test to compare between two groups. Results Cytotoxic effect of H. cordata extract and fractions against human breast cancer MDA-MB-231, MCF-7 cells and normal murine fibroblast NIH3T3 cells In a dose-dependent way, the crude extract decreased the percentage of cell viability in both MCF-7 and MDA-MB-231 cells. However, it had no harmful effects on NIH3T3 cells that were in their normal state (Figure 1A). The crude extract was hazardous to MDA-MB-231 and MCF-7 cells at concentrations below 200 μg/ml, but not to NIH3T3 cells at concentrations over 200 μg/ml. Hexane fraction dramatically decreased the viability of both breast cancer cells in a concentration-dependent manner, but it had no harmful effects on normal NIH3T3 cells (Figure 1B). At doses under 200 μg/ml for both types of breast cancer MCF-7, MDA-MB-321; and normal healthy fibroblast NIH3T3 cells the dichloromethane fraction treatment showed cytotoxicity. The particular fraction was therefore not included in subsequent research (Figure 1C). The IC50 concentration for the ethyl acetate fraction in MDA-MB-231 cells was greater than 200 μg/ml, whereas it was less than 200 μg/ml in the MCF-7 cell line. This demonstrated that MDA-MB-231 cells have lower sensitivity to ethyl acetate than MCF-7 cells. Additionally, NIH3T3 cells that were given the ethyl acetate fraction treatment displayed no harm (Figure 1D). Methanol fraction did not exhibit cytotoxicity towards MCF-7, MDA-MB-231 and normal NIH3T3 cells at maximal concentration of 200 μg/ml (Figure 1E). Thus, methanol fraction was also excluded in further experiment. We selected hexane fraction for further study since it was intriguing to determine the mode and mechanism of such cell death effect, and the relevant mechanism in breast cancer cells (used as representatives). When this particular hexane fraction was applied to MCF-7 and MDA-MB-231 cells, the mode of cell death was identified. In order to determine the manner of cell death using the annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining and flow cytometry technique, the IC20 and IC50 values of H fraction on MDA-MB-231 cells were calculated to be 189 and 289 μg/ml, respectively. The dosages were 194 and 337 μg/ml, respectively, for the MCF-7 cells. MDA-MB-231 cells appeared to be more sensitive to the particular H fraction than MCF-7 cells based on these inhibitory concentrations (IC) or values. Apoptosis induction by H. cordata specific hexane fraction in MDA-MB-231 and MCF-7 cell lines Apoptosis resistance is one of the characteristics of cancer cells (Hanahan and Weinberg, 2011). Therefore, we investigated whether or not a particular hexane fraction may cause the cell lines MDA-MB-231 and MCF-7 to undergo apoptosis. Using data from dot plots and the CellQuest flow cytometer software (Becton, Dickinson and Company, NY, USA), it was discovered that both breast cancer cell lines died in an apoptotic-regulated manner (Figure 2A and 2B). Higher concentrations of a particular hexane fraction significantly increased the proportion of the entire population of apoptotic cells in MDA-MB-231 cells (p < 0.01). Additionally, there was a significant difference between the effects caused by IC20 and IC50 (p < 0.05). (Figure 2C). Although not dose dependent, the percentage of MCF-7 apoptotic cells significantly increased at both IC20 and IC50 (p < 0.01) (Figure 2C). Specific Human breast cancer MDA-MB-231 cells generated ROS as a result of the H. cordata fraction treatment The fluorescent intensity of DCFH-DA in MDA-MB-231 cells was considerably raised by a specific percentage of HCT dosage at IC50 (p < 0.05). The formation of ROS, also known as hydrogen peroxide or peroxide radicals, was much higher between the IC20 and IC50 ranges. (p < 0.01) (Figure 3A). At IC50, there was a substantial increase in the fluorescence intensity of dihydroethidium fluorescence (p < 0.01) (Figure 3B). Between IC20 and IC50, there were also noticeably variable dihydroethidium intensities (p < 0.05). Radical superoxide anion is represented by dihydroethidium. Active phytochemicals in the extract and fractions of H. cordata determined by HPLC approach A medicinal herb, H. cordata Thunb., is grown across Asia (Chun et al., 2014). The following common compounds are among the active phytochemicals found in the plant. Several phenolic acids, including gallic acid (GA), chlorogenic acid (CA), vanillic acid (VA), syringic acid (SA), transcoumaric acid (TCA), and sinapinic acids, were shown to be present in commercial fermented H. cordata products (Senawong et al., 2014). The phenolic acids and flavonoids in the H. cordata extract and fractions were detected in our investigation using the unique HPLC standard system. We employed the standard chemicals displayed in Figure 4A and their retention times are reported in Table 1 for our HPLC analytical apparatus. Figures 4B–4E show the HPLC chromatograms for the crude extract of natural H. cordata and each distinct fraction. In the HPLC analysis of crude extract, flavonoids like rutin and quercetin and phenolic acids, such as syringic acid, vanillic acid, chlorogenic acid, transcoumaric acid and gallic acid, were detected, respectively, ranging in quantity from high to low (Figure 4B). Flavonoids like quercetin were the most prevalent flavonoid component in the hexane fraction (Figure 4C). The primary substances in the dichloromethane fraction were flavonoids, specifically quercetin and rutin, as well as phenolic acids like syringic acid and vanillic acid (Figure 4D). The phenolic acids, such as transcoumaric acid, chlorogenic acid, gallic acid, syringic acid and vanillic acid, were the most prevalent substances in the ethyl acetate fraction, followed by flavonoids such as rutin and quercetin, respectively (Figure 4E). Table 1 The Standard Components in the Mixture together with Their Retention Time (RT) Determined by HPLC technique Standard compounds Retention times (minutes) Gallic acid (GA) 10.83 Chlorogenic acid (CA) 23.17 Vanillic acid (VA) 25.77 Syringic acid (SA) 26.81 Transcoumaric acid (TCA) 30.49 Rutin (RU) 33.87 Quercetin (QC) 40.07 Figure 1 H. cordata Crude Extract and Different Fractions' Cytotoxic Effects on the Normal NIH3T3 Murine Embryonic Fibroblast Cell Line as Well as the Breast Cancer MDA-MB-231 and MCF-7 Cell Lines. After 24 hours of exposure to crude extract (A), hexane (B), dichloromethane (C), ethyl acetate (D), and methanol (E) fractions at varied concentrations, the percentage of cell viability was calculated. The data are shown as the mean ± SD of three independent trials done in triplicate. The statistical significance of values is indicated by the symbols *p < 0.05, **p < 0.01, ***p < 0.001 Figure 2 Dot plots showing the induction of apoptosis by the H. cordata hexane fraction in MDA-MB-231 (A) and MCF-7 (B) cells after 24 hours of treatment at IC0, IC20, and IC50, respectively. The positive control utilized was Navelbine (5 μM). Total apoptotic cell bar graphs (C) from three separate studies are displayed as mean ± SD (black bar represents MDA-MB-231 and white bar represents MCF-7 cells). The statistical significance of values is indicated by the symbols *p < 0.05, **p < 0.01, ***p < 0.001 Figure 3 MDA-MB-231 Cells Treated with the Hexane Fraction of H. cordata Produce Reactive Oxygen Species (ROS). Fluorescence probes for ROS generation include 2',7'-dichlorodihydrofluoresceine diacetate (DCFH-DA) for measuring peroxide radicals or hydrogen peroxide (H2O2) and dihydroethidium (DHE+) for determining superoxide anion radicals (O2•−). The fluorescence intensity was determined using a fluorescence microplate reader. The results of three separate tests are shown as mean ± SD. The statistical significance of values is indicated by the symbols *p < 0.05, **p < 0.01, ***p < 0.001 vs control. The comparison between groups are marked with, # p < 0.05, ## p < 0.01. NC, negative control; PC, positive control, H2O2 for DCFH-DA and FeSO4 for DHE Figure 4 The Following HPLC Chromatograms are Shown: standard chemicals (A) in standard mixes (retention periods 0–50 min), crude extract (B), hexane fraction (C), dichloromethane fraction (D), and ethyl acetate fraction (E). Gallic acid (GA), cholrogenic acid (CA), vanillic acid (VA), syringic acid (SA), transcoumaric acid (TCA), rutin (RU) and quercetin (QC) are the conventional benchmarks. Each tiny panel was not in the same scale Discussion H. cordata considerably boosts the mRNA and protein expression of hypoxia-inducible factor (HIF-1A) and Forkhead box (FOXO3) in hepatocellular carcinoma HepG2 cells. It also promotes the expression of MEF2A and induces apoptosis. MEF2A operates as a proapoptotic factor through increased caspase-3 and caspase-7, as well as the production of proteins from the Bcl-2 family (viz., Bax, Bcl-xL, and Bcl-2) (Kim et al., 2017). Additionally, primary colorectal cancer cells are subjected to cytotoxicity by HCT ethanol extract. It causes a reduction in the mitochondrial membrane potential (ΔΨ(m)) and an increase in the generation of reactive oxygen species (ROS). The elevated amounts of cytochrome c, Apaf-1, and caspase-3 and -9 are the mitochondrial dependent apoptotic signaling pathway. Caspase-9 and Caspase-3 are activated by a decrease in ΔΨ(m) and a rise in the Bax/Bcl-2 ratio. It showed that a mitochondria-dependent signaling mechanism in human primary colorectal cancer cells causes HCT to trigger apoptotic cell death (Lai et al., 2010). Additionally, the intrinsic pathway is used to trigger apoptosis in the human colon cancer HT-29 cell line (Tang et al., 2009). The antiproliferative properties and phenolic acid content of water and ethanolic extracts of the powdered formulation of H. cordata Thunb. fermented broth and P. emblica fruit mixture were also investigated (Kumnerdkhonkaen et al., 2018). Aqueous extract is less cytotoxic than ethanolic extract. According to the susceptibility of cancer cells to the extract, the ethanolic extract is toxic to human breast MCF-7 cells, human colon cancer HCT116 and HT29 cells, human T-cell leukemia Jurkat cell line, and non-cancer Vero cell lines in that order. The Jurkat cells respond to both extracts the greatest. Apoptosis induction, however, is the underlying mechanism for antiproliferative action. The most vulnerable cells to extract-induced apoptosis are HT29 cells. It has been proven that ethanolic extract works better than water extract at apoptosis induction. Additionally, cell cycle arrest is also shown (Kumnerdkhonkaen et al., 2018). The powder mix extracts of ethanol and water contain seven recognized phenolic acids, including gallic, p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic, and sinapinic acids, with p-coumaric acid having the greatest concentration followed by ferulic acid, according to HPLC examination (Kumnerdkhonkaen et al., 2018). However, in our study we could further identify two flavonoids in the extract and fraction, which were rutin and quercetin. There are several conventional methods for treating cancer, such as chemotherapy or radiotherapy, or even targeted therapy. These, however, frequently have a number of negative consequences and several drawbacks in practical practice. Additionally, concerns regarding drug resistance are growing. Natural chemicals originating from plants are of great interest in the ongoing search for safer and more efficient treatments. Secondary metabolites known as plant phenolics have grown in significance as possible anti-cancer agents. Phenolics have a significant potential as cytotoxic anti-cancer drugs since they target several components of cancer, increase apoptosis, and decrease proliferation (including angiogenesis, growth, differentiation and metastasis). The subclass of plant phenolics known as phenolic acids, which is further classified into benzoic and cinnamic acids, has been linked to powerful anti-cancer properties in a variety of in vitro and in vivo scenarios. Additionally, the therapeutic effects of phenolic acids are supported by their functions as epigenetic regulators and promoters of unfavorable reactions or resistance to conventional anticancer therapy. It was stated that one difficult component of the effectiveness of natural chemicals employed in cancer treatment is the encapsulation of phyto-substances into nanocarrier systems (Abotaleb et al., 2020). Investigated in the human lung cancer A549 cell line are the anti-human lung cancer activity and growth suppression mechanisms of ethanol HCT extract. The G0/G1 and Sub-G1 cell (apoptosis) populations are increased by HCT extract, which also promotes apoptosis with characteristic cell shape. According to the Comet assay, the extract enhances DNA fragmentation and DNA condensation. Additionally, it triggers the activation of caspase-3 and -8. Fas/CD95 protein levels are raised in A549 cells treated with HCT extract. The G0/G1 phase and apoptotic related protein levels, cyclin D1, cyclin A, CDK4 and CDK2 are decreased, and p27, caspase-8 and caspase-3 are upregulated in A549 cells after HCT treatment. Hence, A549 cells died as a result of HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptosis (Chen et al., 2013). As previously established, Houttuynia cordata Thunb. (HCT) is a plant with multiple biological features and uses as both food and medicinal. Investigated are the impact of food preparation methods or styles on the anticancer activity of HCT. The plant extracts (designated as heated aerial stem, heated subterranean stem, heated leaves, and not heated as NAS, NSS and NL, respectively) were found to have an inhibitory effect on the viability of four cancer cell lines. Four human tumor cell lines that have received heat treatment from HCT demonstrated anticancer activity. It is intriguing to expect that heat treatment might boost anticancer activity. Heat treatment promotes apoptosis through intrinsic signaling pathways and boosts anticancer activity of aerial stem by 2–14 times in the human gastric cancer SCG–7901 cell line. It is intriguing to note that the caspase-9 specific inhibitor prevented the arial stem - treated cells to die or less viable, demonstrating that apoptosis was being induced by the mitochondrial pathway. HCT’s anticancer action is increased by heat treatment, which can be used as a dietary strategy to prevent stomach cancer (Liu et al., 2021). The absence of some specific types of standard chemicals employed in this technique is the restriction of our HPLC analysis. Due to the use of insufficiently representative reference chemicals, several HPLC peaks could not be distinguished. Gallic acid (GA), chlorogenic acid (CA), vanillic acid (VA), syringic acid (SA), and transcoumaric acid (TCA) were our reference components in the mixture and overlapped with the results of the prior studies (Senawong et al., 2014; Kumnerdkhonkaen et al., 2018). But instead, we have employed two flavonoids, quercetin (QC) and rutin (RU), which are also present in some fractions, as the HPLC standard compounds. However, we discovered and proved that HCT extract and its various fractions were made up primarily of phenolic acids and a small number of flavonoids from the results of the thin layer chromatograms (data not shown). According to research on flavonoids and apoptosis induction, flavonoids like quercetin’s with in the flavanol classification group are distinguished by their unsaturated C rings at C2-C3, which are discovered oxidized at C4 and hydroxylated at C3 (Panche et al., 2016). The most prevalent flavonoid, 3,5,7,3′,4′-pentahydroxyflavone, or quercetin, is a phytoestrogen with structural similarities to natural estrogen 17-estradiol (Ranganathan et al., 2015). Numerous research back up quercetin’s capacity to fight cancer. Quercetin is demonstrated as high propensity to enhance Bax and capsase-3, decrease Bcl-2 and necroptosis by overexpression of RIPK1 and RIPK3 in MCF-7 cells (Ranganathan et al., 2015; Duo et al., 2012; Chou et al., 2010). But in the invasive ductal breast cancer BT-474 cell line, quercetin increases extrinsic apoptosis without having any effect on Bcl-2 or Bax, and as a result, does not start any intrinsic apoptotic pathway (Seo et al., 2016). In HepG2 liver cancer cells, the substance lowers ERK1/2 and AKT phosphorylation and blocks the NF-kappaB pathway (Fantini et al., 2015; Granado-Serrano et al., 2010). Our findings showed that HCT-specific hexane fractions could reduce breast cancer cell viability and trigger cell death by generating superoxide anion and hydroxide radicals. According to the current findings, oxidative stress, or excess ROS generation, caused human invasive breast cancer MDA-MB-231 cells to undergo apoptosis. However, each flavonoid or phenolic acid should be examined separately on breast cancer cells in order to demonstrate that they were the culprits behind the controlled apoptotic cell death. To confirm the effects of the active phytochemicals, the compound should be utilized to test in human primary breast cancer MDA-MB-231 cells in vitro and in vivo and evaluate the antiproliferative effect and mode / mechanism(s) of cell death at molecular levels. The current study has shown, in summary, that a specific extract and fraction of H. cordata have a strong potential to suppress the growth of human invasive breast cancer cells by inducing apoptosis and oxidative stress through the production of hydroxide and superoxide anion radicals. This herb is a good source of flavonoids, particularly quercetin and phenolic acids. The growth of human breast cancer cells may have been inhibited by phenolic acids and flavonoids. Further investigation into the use of H. cordata as a supplementary medicine for cancer treatment and prevention in both animal models and clinical trials is necessary in light of the study’s findings. Author Contribution Statement All authors have contributed to the current work. PI performed laboratory investigations. RB contributed substantially to the conception and design of the study, and funding acquisition. PI, HP and RB carried out the acquisition, analysis and interpretation of data. PI and RB drafted the manuscript, and RB performed the critical revision and correction of the manuscript. PK supervised the study. All the authors approved the final version submitted for publication and take responsibility for the statements made in the published article. Acknowledgements Authors would like to thank all the Molecular Biology Research lab team, Faculty of Medicine, Chiang Mai University; Dr. Narin Printarakul at Department of Biology, Faculty of Science, Chiang Mai University; and Dr. Hataichanok Pandith’s lab team at Department of Biology, Faculty of Science and Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Chiang Mai University. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests Neither of the authors discloses any potential or actual conflict of interest. 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116150 10.31557/APJCP.2023.24.4.1275 Research Article Analysis and Validation of the Prognosis ability of the M7G-Related miRNAs in Lung Adenocarcinoma Qiu Xiaowen 12 Chen Yongting 3 Zhu Xingzhuang 2 Gong Zheng 2 Yu Fengyuan 2 Zhang Pengfei 2 Song Yipeng 2* Li Hongwei 2* 1 Department of Oncology,Binzhou Medical University, Binzhou,China. 2 Department of Radiotherapy,The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China. 3 The People’s Hospital of Zhaoyuan City,Zhaoyuan, China. * For Correspondence: syp1972@sina.com, 873662676@qq.com. Xiaowen Qiu and Yongting Chen have equal contribution in this study. 2023 24 4 12751287 5 12 2022 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: N7-methylguanosine (m7G) modification plays a crucial role in the development and progression of lung cancers. MicroRNAs (miRNAs) are closely involved in programmed cell death and the mechanism of tumor growth. The m7G-associated miRNAs genes in lung adenocarcinoma (LUAD), and their prognosis prediction ability of LUAD, however, had not been investigated. Methods: The RNA transcriptomes, clinical indices, and immune scores of LUAD patients were searched and downloaded from The Cancer Genome Atlas (TCGA) and the ESTIMATE database. The miRNAs targeting METTL1 and WDR4 were extracted from the TargetScan database. Differentially expressed m7G-related miRNAs were identified and their prediction power of LUAD prognosis was systematically investigated. Results: Among 40 the differentially expressed m7G-related miRNAs in LUAD, five (hsa-miR-31-5p, hsa-miR-5571-3p, hsa-miR-4697-3p, hsa-miR-6858-5p, and hsa-miR-873-3p) demonstrate significant predictive value for prognosis. The risk score constructed by these five miRNAs was an independent prognostic factor (univariate Cox regression results: hazard ratio (HR) = 1.6619, 95% confidential interval (CI) = 1.2103-2.2819, p = 0.0017; multivariate Cox regression results: HR = 1.6004, 95% CI = 1.1633-2.2017, p = 0.0039). The survival curves showed that patients with high-risk scores had a poor prognosis. Calibration curves indicated good predictability in a nomogram constructed combining the miRNA and the clinical indices of age, sex, chemotherapy, radiotherapy, stage, and risk score. GO and KEGG analysis of the overlapping genes showed that the prognostic miRNAs were closely associated with the neuropeptide signaling pathway. Besides, the immune infiltration analysis showed that the expression of the AMPD1 gene was strongly associated with immune cells and immunology functions in LUAD. Conclusion: This study identified DE m7G-related miRNAs and demonstrated their prediction ability in the prognosis of LUAD patients. The risk signature based on these miRNAs demonstrates high accuracy in predicting the prognosis of LUAD patients. Key Words m7G miRNA nomogram prognosis immune ==== Body pmcIntroduction Lung adenocarcinoma (LUAD), the most common subtype of non-small cell lung cancer (NSCLC) Sung et al., (2021), has the highest incidence and mortality rate in many countries. Therefore, it is an urgent desire to establish a prognostic panel with accurately stratifying risk and high specificity and sensitivity that could assist the selection of treatment strategy, promote the survival rate, and improve the long-term prognosis of LUAD patients. N7-methylguanosine (m7G) modifies the 5’ caps of tRNAs and rRNAs under the catalyzing of the Methyltransferase complex composed of the Methyltransferase-like 1 (METTL1) and the WD repeat domain 4 (WDR4) in the tRNA variable loop in humans (Alexandrov et al., 2005; Boulias et al., 2019; Lin et al., 2018; Pandolfini et al., 2019). Ma et al., (2021) found in human lung cancer samples that the gene expressions of METTL1 and WDR4 were significantly upregulated and were associated with poor patient prognosis. Chen et al., (2022) study demonstrated the mechanism of the methyltransferase complex of METTL1/WDR4 in promoting the disease progression of HNSCC by impacting the immune microenvironment and regulating the PI3K/AKT/mTOR signaling pathway. Zhao et al., (2021) research shows that METTL1 could promote post-ischemic angiogenesis by increasing the protein production of VEGFA. All these studies indicate that the METTL1/WDR4 complex might also be associated with the mechanism and progression of LUAD. MicroRNAs (miRNAs) are a category of non-coding RNAs that are universally involved in cell proliferation, differentiation, and apoptosis in tumors (Baltimore et al., 2008; Karrich et al., 2013), their abnormal gene expression has been detected in types of human cancers(El Bezawy et al., 2017; He et al., 2017; Hummel et al., 2014; Wang et al., 2018; Xu et al., 2017). Although miRNAs lack protein-coding capacity, recent studies have revealed their ability in regulating the expression of tumor suppressor genes and oncogenes via binding to tumor-related genomic regions or sensitive areas of the genome (Wu et al., 2021). Previous studies have identified several miRNAs as potential prognostic indicators for LUAD, such as mir-582 and mir-584, the expressions of which were associated with poor prognosis in patients (Siriwardhana et al., 2019). Although miRNAs, as a whole, their associations with the LUAD have been well studied, the impact of m7G-related miRNAs, a subtype of them, had scarcely been studied on LUAD. The tumor microenvironment (TME) is a blend of tumor cells, immune cells, and other endogenous molecules impacting tumor development such as inflammatory mediators (Hanahan et al., 2012). In the TME, TIICs are critical players in tumor progression and immunotherapeutic response (Binnewies et al., 2018). Close association with the prognosis of lung cancer has been observed in many types of immune cells, such as M1/M2 macrophages, CD8+/CD4+ T cells, mature and immature dendritic cells (DC), and the regulatory T cells (Tregs) (Catacchio et al., 2018). Even some indices of systemic inflammation, such as the high neutrophil to lymphocyte ratio, have also been reported to be associated with the overall survival (OS) and progression free survival (PFS) in cancer patients (Gu et al., 2015). This suggests that the study of the TME, especially immune cells and immunology functions, is critical for the development of therapeutical treatments for cancer patients. To systematically evaluate the prognostic value of m7G-related miRNAs in LUAD, we designed this study. First, relevant data were extracted from the TCGA, TargetScan, and ESTIMATE databases. Second, through a series of analyses, the DE miRNAs associated with LUAD prognosis were identified and a risk score model was constructed. Third, a prognostic signature for individualized assessment of patient prognosis was established in the combination of the differentially expressed (DE) m7G-related miRNAs and clinical indices, which showed improved prediction accuracy. Finally, DE mRNAs of the LUAD patients were calculated after the grouping of the patients according to their immune scores and risk scores, and their associated biological pathways and immune cell infiltration were further analyzed. The flow chart of the present study is illustrated in Figure 1. Materials and Methods Data Collection The miRNA and mRNA expression data and clinical characteristics of patients with LUAD were downloaded from The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/tcga). LUAD patients were included only when their clinical information was complete, including survival time, survival status, age, sex, chemotherapy, radiotherapy, and the stages of cancer. The upstream miRNAs of m7G-related genes were extracted from the TargetScan database (http://www.targetscan.org/). Next, the immune scores of the LUAD patients were obtained from the ESTIMATE database (https://bioinformatics.mdanderson.org /estimate/). Differential expression analysis of m7G associated miRNAs The differentially expressed (DE) m7G-related miRNAs between LUAD and matched healthy tissues were calculated using the R packages of “limma” and “edgeR”. Heatmaps were drawn using the R package “heatmap”. A statistically significant differential expression was determined when the false discovery rate (FDR) < 0.05 and |log2fold changes (FC)| > 1. Calculation of risk scores of DE miRNAs and evaluation of their association with prognosis The relationship between m7G-related DE miRNA and the overall survival (OS) of the patients was investigated using the TCGA-LUAD cohort. Univariate and multivariate Cox regression analyses were performed using the R package “survival” and “forestplot, with a p-value <0.05 as the cutoff criterium. Kaplan-Meier curves were utilized for survival analyses, with log-rank tests and univariate Cox proportional hazard regression models used to generate p-values as well as hazard ratios (HRs) with 95% confidence intervals (CIs). The Lasso regression analysis was conducted using the R package “glmnet”. The DE miRNAs with prognostic relevance resulted in these analyses were selected for further assessment. First, a risk score was defined based on the expression of these prognostic miRNAs for each patient following centralization and standardization with the R “scale” function. The risk scores were calculated using the following formula: risk score =∑5iXi×Yi where X and Y are the coefficients and miRNA expression levels, respectively. Second, we then validated the accuracy of the risk scores using principal component analysis (PCA). Patients from the TCGA-LUAD cohort were divided into low- and high-risk groups by the median risk score, and their Oss were compared using the Kaplan-Meier approach. Their 1, 3, and 5-year OS were compared in ROC curves generated by the R packages “survival”, “ROCR”, and “timeROC”. Besides, dot and line plots were used to verify the accuracy of the prognostic model. The prognostic relevance of m7G-associated miRNAs was further assessed by Cox regression analyses. K-M survival analyses were conducted and miRNAs with high prognostic potential were chosen for next stage evaluation. Construction and validation of a nomogram Demographics and clinical data, including patients’ age, sex, chemotherapy, radiotherapy, cancer stage, and risk score, were extracted from the TCGA database. Univariate and multivariate Cox regression models were used to analyze the prognostic value of these variables. Then, a prognostic nomogram was created by Cox regression analysis and was subsequently validated by the calibration plots drawn by the R package “rms”. Calculation of the DE mRNAs associated with immune score and risk score The immune scores of LUAD patients were obtained from the ESTIMATION database. Based on the risk scores and immune scores of the patients, their mRNA transcriptome data downloaded from the TCGA database were divided into high-risk and low-risk groups. DE genes between the two groups were calculated using the R package “limma” and “edgeR”. A statistically significant differential expression was determined when the false discovery rate (FDR) < 0.05 and |log2fold changes (FC)| > 1. Functional enrichment analysis of the overlapping genes The overlapping genes of two groups of DE genes obtained by risk score and immune score were identified and plotted by the R package “VennDiagram”. Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the R language. Functional analysis of the immune correlation DE genes associated with immune score were analyzed by the ssGESA algorithm, and the correlation coefficients between the immune cells (imc) and the immunology function (imf) scores were calculated. We then analyzed the immune cells and immunology functions in the high- and low- risk groups. Univariate regression analyses were performed for the intersecting genes of statistical significance and the correlations coefficients were calculated among the immune genes, immune cells, and immunology functions. Results Differentially expressed m7G-related miRNAs in LUAD As a result, we identified a total of 443 LUAD patients who meet our criteria in the TCGA database, the clinical characteristics of which were shown in Table 1. Based on the data sets we collected from the TCGA database and the TargetScan databases, we analyzed the expression of m7G-miRNA-associated miRNAs in lung adenocarcinoma. The gene expression differences of miRNAs between LUAD patients and healthy subjects were shown in the volcano plot and heatmap (Figure 2). As a result, there are a total of 40 DE miRNAs, including 34 upregulated and 6 downregulated ones (Figure 2a), and their differential expressions in each sample are shown in the heat map (Figure 2b). Construction of A multi-miRNA Prediction Model for the Prognostic of LUAD The prognostic value of these 40 DE miRNAs in LUAD was further evaluated by univariate and multifactorial Cox regression analysis. The univariate Cox regression analysis showed that hsa-miR-31-5p (p=0.0002), hsa-miR-5571-3p (p=0.0298), hsa-miR-4697-3p (p=0.0394), hsa-miR-6858-5p (p=0.0299), and hsa-miR-873-3p (p = 0.0001) have a predictive value for LUAD prognosis (Figure 3a). The multifactorial Cox regression analysis showed that hsa-miR-31-5p (p<0.001), hsa-miR-5571-3p (p=0.0027), hsa-miR-4697-3p (p=0.0417), hsa-miR-6858-5p (p=0.0105), and hsa-miR-873-3p (p=0.0026) might be independent prognostic indicators for LUDA (Figure 3b). Using the lasso regression analysis, five miRNAs with the best prognosis ability, including hsa-miR-31-5p, hsa-miR-5571-3p, hsa-miR-4697-3p, hsa-miR-6858-5p, and hsa-miR-873-3p, were identified as prognostic markers according to the minimum criteria and coefficients (Figure 3c-d). The median risk score in the multifactorial Cox regression analysis was used as the cut-off point to classify LUAD cancer patients into low-risk and high-risk groups for further assessments. To validate this grouping, we first performed a principal components analysis (PAC), which showed good discrimination between the high- and low-risk groups (Figure 3e). Then Kaplan-Meier curves were plotted to compare the survival times between the low-risk patients and high-risk patients. The results showed that the average OS time was significantly longer in low-risk patients than that in high-risk patients (p<0.001, Figure 3g). The area under the ROC curve for time-dependent survival at 1, 3, and 5 years was 0.717, 0642, and 0.582 for the two groups of patients, respectively, indicating the good performance of this risk model in predicting the survival of LUAD patients (Figure 3f). We also plotted the distribution of the risk scores and survival status for each patient (Figure 3h-i). It was observed that along with the increase in patients’ risk scores, the number of patient deaths had a discriminable increase. Prognostic Analysis and Clinical Correlation of the Independent Risk Factors The calculated risk scores of the patients, as well as other clinical features collected from the TCGA database data, including age, sex, chemotherapy, radiotherapy, and cancer stage, were analyzed by the univariate Cox analysis (Table 1). Among them, chemotherapy (p = 0.0452; HR = 1.3827), radiotherapy (p < 0.0001; HR = 2.0463), cancer stage (p < 0.0001; HR = 2.4182), and risk score (p = 0.0017; HR = 1.6619) were used as independent risk factors (Figure 4a). The results of the multivariate Cox analysis showed that radiotherapy (p = 0.0022; HR = 1.7399), stage (p < 0.001; HR = 2.2199), and risk score (p = 0.0039; HR = 1.6004) could be independent risk factors affecting the prognosis of LUAD (Figure 4b). The area under the ROC curve for time-dependent survival of risk score, age, sex, chemotherapy, radiotherapy, and stage were 0.718, 0503, 0.578, 0.524, 0.537, and 0.616, respectively, indicating high prediction accuracy of the risk score and cancer stage (Figure 4c). Next, a prediction model was constructed by the clinical features and the risk scores (*: p < 0.05, **: p < 0.01, *** p < 0.001) (Figure 4e), based on which the total points of each patient were calculated, and their 1-, 3-, and 5-year survival rates were predicted. According to this model, a higher point of a patient predicts a worse prognosis. We also used a calibration curve to test the accuracy of the nomogram. As shown in the calibration curves, the predicted1-, 3-, and 5-years survival curves are very close to the observed survival curves, indicating a high prediction accuracy of the nomogram has (Figure 4d). Prognostic significance of the miRNAs and associated DE genes We further screened two miRNAs with survival significance from the five miRNAs used to construct the risk score models and drew their survival curves at p <0.05. As shown in Figure 5, a poor prognosis was observed in the has-miR-315p (p=0.008) (Figure 5a) high expression group and the has-miR-55713p (p=0.048) (Figure 5b) low expression group, suggesting that the high expression of has-miR-315p could promote the occurrence and development of tumor, while the high expression of has-miR-55713p might inhibit the caner exacerbation. Following the grouping of the risk scores, the DE mRNA genes were calculated between the high-risk group and the low-risk group from the TCGA transcriptome data sets. As shown in the volcanic map, there were 1066 DE genes, including 764 up-regulated genes and 302 down-regulated genes (Figure 5c). Similarly, the DE mRNA was also calculated after the patients were grouped by their immune scores obtained from the ESTIMATION database. A total of 1727 DE genes were obtained, of which 864 genes were up-regulated and 863 genes were down-regulated (Figure 5d). Functional Enrichment Analysis of the DE mRNA Genes To further explore the biological functions associated with these DE mRNA genes, we first obtained 540 DE genes that were associated with the grouping of both the risk score and the immune score, the overlapping of which were displayed in a Venn diagram (Figure 6a). Next, we utilized the GO and KEGG analyses to investigate the related molecular mechanisms of these genes. As a result, in the GO groups of biological processes (BP), cellular components (CC), and molecular function (MF), the “neuropeptide receptor activity”, “antimicrobial humoral response”, and “neuropeptide signaling pathway” were the most significantly terms, respectively (Figure 6b-c). The significantly enriched KEGG pathways were listed in Table 2, which were mainly associated with the pathways of neuroactive ligand-receptor interaction, taste transduction, and protein digestion. Immune Infiltration analysis of the DE genes To further study the immune cells and immune function of associated with these DE genes in LUAD, we drew a heatmap of immune scores calculated using the ssGSEA algorithm (Figure 7a). The heatmap showed that the composition of 16 types of immune cells (imc) and 13 immunology functions (imf) in each sample had significant differential expression. Among the biological cells, T helper cells were highly expressed in each sample, while NK cells were lowly expressed. In immunology functions, HLA, and MHC class I were highly expressed in almost all samples. As shown in Figure 7b, the proportions of differentially infiltrated immune cells were moderately-to-highly-correlated. For instance, the correlation coefficient between the pDCs and the TIL was 0.82 and the correlation coefficient between the T helper cells and the macrophages was 0.8. In the imf correlation matrix, the correlation between T cell co-inhibition and the checkpoint activity reached 0.93 (Figure 7c). Next, we analyzed the differences in immune cells and immunology functions in the high and low risk groups. For the imc, when compared to high-risk groups, low-risk groups had significantly higher aDCs, iDCs, Mast cells (p < 0.001), B cells, Neutrophils (p < 0.01), and TIL (p < 0.05), while had significantly lower infiltration level of NK cells (p < 0.01) (Figure 7d). For the imf, the low-risk group was significantly higher than the high-risk group in HLA (p < 0.001) and type I IFN response (p < 0.01), while significantly lower in MHC class I (p < 0.01) (Figure 7e). Significant DE genes were obtained by univariate Cox regression analysis of all overlapping DE genes (Table 3). Finally, the immune correlation analysis of these genes showed that AMPD1 was positively correlated with most immune cells and immunology functions (Figure 7f), indicating that the role of AMPD1 in LUAD immunity deserves further study. Taken together, these results suggest that differential gene expression plays an important role in the immune regulation of LUAD. Figure 1 The Flow Chart of the Overall Study Design Figure 2 Expression of M7G-related DE miRNAs. (a) Volcano plot of the DE miRNAs. Red dots represent upregulated m7G-associated miRNAs, green dots represent downregulated ones, and black dots represent miRNAs with no significant differential expression. (b)The heatmap of top 20 DE miRNAs between the LUAD group and the healthy group Table 1 Summary of Basic Clinical Features Covariates Group Patient number (%) p-value1 p-value2 Age ≤65 216 (48.8%) 0.654 0.326 ˃65 227 (51.2%) Sex Male 200 (45.1%) 0.845 0.769 Female 243 (54.9%) Chemotherapy Yes 181 (40.9%) 0.045 0.714 No 262 (59.1%) Radiation Yes 105 (23.7%) 1.76E-05 0.009 No 338 (76.3%) Stage Stage I-II 3539 (79.7%) 2.14E-07 1.01E-05 Stage III-IV 90 (20.3%) 1 p-value of univariate Cox regression; 2 p-value of multivariate Cox regression Table 2 KEGG Enrichment Analysis miRNA ID Description Count p.adjusted hsa04080 Neuroactive ligand-receptor interaction 32 1.57E-10 hsa04742 Taste transduction 10 0.001002972 hsa04974 Protein digestion and absorption 10 0.003294531 hsa05033 Nicotine addiction 6 0.007720087 hsa00140 Steroid hormone biosynthesis 7 0.009797174 hsa04915 Estrogen signaling pathway 10 0.017160914 hsa04913 Ovarian steroidogenesis 6 0.017160914 hsa04950 Maturity onset diabetes of the young 4 0.046678348 hsa04927 Cortisol synthesis and secretion 6 0.048272193 Figure 3 Construction and Validation of the Prognosis Prediction Model. (a-b) Univariate and Multivariate analysis of five miRNAs associated with LUAD prognosis. (c-d) The process of lasso regression analysis to select predictive variables. (e) PCA and (f)ROC curve verified the accuracy of the predictive power of the risk score model. (g)Survival curve compares the survival rate of the high-risk group and low-risk group. (h) Survival status for patients. (i) Distribution of patients’ risk scores Table 3 Univariate Cox Regression of Overlapping Genes Gene HR HR-95Low HR-95Up p-value HIST1H2AL 1.008655118 1.004280181 1.013049114 0.000102011 EBF3 1.000362325 1.000179036 1.000545648 0.000106714 HIST1H2AJ 1.006645254 1.003219751 1.010082453 0.000139917 ANXA13 1.000250209 1.000121232 1.000121232 0.000143259 DCAF12L2 1.00449623 1.002106005 1.006892155 0.000223595 FAM9A 1.003356779 1.001554714 1.005162087 0.000258448 HIST1H2BI 1.005418447 1.002484183 1.008361301 0.000290327 HIST1H4B 1.004285452 1.001956247 1.006620071 0.000306649 SYT10 1.003409143 1.00153566 1.005286131 0.00035801 BARHL2 1.003325081 1.001479382 1.005174181 0.000410029 HIST1H2BL 1.006749345 1.002907984 1.010605419 0.000563306 RBMY1E 1.116062011 1.047945539 1.188606054 0.000631982 HIST1H3I 1.008520839 1.00361963 1.013445983 0.000641138 ASIC2 1.000816023 1.00034647 1.001285796 0.000657398 HIST1H4C 1.001254315 1.000531476 1.001977677 0.000669059 HIST1H2AB 1.006352989 1.002676392 1.010043067 0.000695741 GDPD2 1.000434487 1.000180611 1.000688428 0.000794776 LCN15 1.000440633 1.000179027 1.000702307 0.000961541 SLC10A2 1.000224638 1.000089788 1.000359506 0.001094193 NPY5R 1.002951746 1.001168353 1.004738315 0.001170847 HIST1H4D 1.003464179 1.001226871 1.005706486 0.002392559 COL9A1 1.002087607 1.000707756 1.00346936 0.003013852 CRABP1 1.000073924 1.000024559 1.000123291 0.00333467 AMPD1 0.995589173 0.992598941 0.998588412 0.003971938 TGIF2LY 1.02684375 1.008439511 1.04558387 0.004095319 KRTAP29-1 1.005106966 1.001617293 1.008608796 0.004096565 UGT2B28 1.002495079 1.000766639 1.004226505 0.0046493 SLC3A1 1.000421841 1.000127915 1.000715853 0.00490643 SLC22A10 0.906487055 0.845723518 0.971616329 0.005548349 NKAIN1 1.00068796 1.000198611 1.00117755 0.005856298 RAB3B 1.000100968 1.000028359 1.000173582 0.006419937 EPHA5 1.000175252 1.000044985 1.000305536 0.008367732 TM4SF20 1.000083007 1.000020439 1.000145579 0.009315098 HIST1H4L 1.020764987 1.004927419 1.036852153 0.009993429 FABP1 1.000620756 1.000132007 1.001109744 0.012792349 SCGB1A1 0.999975762 0.999956361 0.999995164 0.01434692 CYP17A1 0.991524759 0.984741181 0.998355067 0.015099843 PEX5L 1.000195205 1.000036249 1.000354186 0.01608583 HMX2 1.004416694 1.000799395 1.008047067 0.016663167 SCGB3A1 0.999992487 0.99998633 0.999998645 0.016785424 CLVS2 1.001220833 1.000219674 1.002222995 0.016835501 HIST1H3J 1.009735816 1.001709563 1.01782638 0.017338146 NPY 1.000015359 1.000002579 1.000028139 0.018499329 HOXD13 1.00082481 1.000121615 1.0015285 0.021500875 TRIM58 1.00085058 1.000124858 1.001576829 0.021600885 HIST1H2AH 1.005753838 1.000609254 1.010924873 0.028325951 CT47B1 1.00699033 1.000564052 1.013457882 0.032957884 KRT33A 1.000628673 1.000027531 1.001230176 0.040388504 GLRA2 1.003543926 1.000139818 1.00695962 0.04128971 Gene HR HR-95Low HR-95Up p-value C8orf86 1.023568218 1.00086135 1.046790243 0.041832284 FAM216B 0.999679134 0.999365642 0.999992724 0.044916679 CYP2B6 1.000085411 1.000001506 1.000169323 0.046026185 PRAC1 1.002561706 1.000025576 1.005104268 0.047731097 EN1 1.000799915 1.000006595 1.001593865 0.048125021 Figure 4 Construction and Validation of a Nomogram. (a-b) Univariate and multivariate Cox regression analysis of the clinical features and the risk scores. (c) The ROC curve of the independent risk factors. (d) Prediction accuracy of the nomogram. The observed and predicted 1-, 3-, and 5-year survival were in good agreement. Y-axis: actual survival; X-axis: nomogram-predicted survival. (e) In the upper part, six lines are drawn for each patient, representing the points of the six predictors in the nomogram, the sum of which is annotated on the ‘Points’ axis. At the bottom, three lines are drawn to display the predicted 1-, 3-, and 5-year overall survival of LUAD Figure 5 DE Genes in LUAD Associated with the Grouping by the Risk Score and the Immune Score. (a) Survival curves of differential hsa−miR−31−5p expression. (b)Survival curves of differential hsa-miR-557-3p expression. Volcano plots of the DE genes when patients are grouped by the risk score (c) and the immune score (d). Red dots represent upregulated genes, green dots represent down-regulated genes, and black dots represent genes with nonsignificant differences Figure 6 Enrichment Analysis of the 540 DE Genes. (a), Venn diagram displays the intersection of the two DE gene sets; (b), Bar chart of the GO enrichment analysis; (c), Dot plot of the GO enrichment analysis Figure 7 Analysis of Immune Cells and Immunology Functions Associated with DE Genes. (a) Heatmap of the scores of immune cells and immunology functions. (b-c) Matrix of the correlation coefficients between the immune cells and immunology functions. (d) Immune cell analysis of the high- and low-risk groups. (e) Immunology function analysis of the high- and low-risk groups. (f) Correlation analysis of immune genes with immune cells and immunology functions. The red color represents positive correlations, the blue color represents negative correlations, and the white indicates relationships without a statistical difference Discussion The 7-methylguanosine (M7G) is one common tRNA methylation modification that occurs at nucleotide position 46 in tRNAs (Guy et al., 2014), it is catalyzed by the methyltransferase complexes formed by METTL1 and WDR4 (Lin et al., 2018). METTL1 is overexpressed in cancers, and studies showed that it was associated with drug resistance to chemotherapies and poor prognosis in the patients (Wang et al., 2021a; Wang et al., 2021b). Ma et al., (2021) found that METTL1 and WDR4 were involved in the development of lung cancers by promoting the m7G tRNA modification and regulating the protein production of cell cycle genes. Shi et al., (2019) found that mir-107 has also been shown to be involved in lung cancer growth and metastasis. However, no study had been reported on the association of m7G-related miRNAs with LUAD and their application in the prognosis prediction of LUAD patients. A number of studies have proved the prognostic value of miRNA-based signatures for patients with various types of cancers (Lv et al., 2020; Tang et al., 2019). X Xu et al., (2019) identified three miRNAs in patients with hypopharyngeal squamous cell carcinoma, the DE expression of which was significantly correlated with the OS and disease-specific survival (DSS) of the patients. In patients with clear cell renal cell carcinoma, Xie et al., (2018) identified four miRNAs the DE expression of which could be used to predict the survival of patients. Qian et al., (2019) constructed two prediction models based on miRNAs identified in colorectal cancer patients, the prediction from which were in good agreement with the patients’ OS and recurrence-free survival (RFS). Our study identified 40 DE miRNAs in LUAD patients that were associated with m7G, including 34 upregulated and 6 downregulated. Then, we further identified five m7G-related miRNAs (hsa-miR-31-5p, hsa-miR-5571-3p, hsa-miR-4697-3p, hsa-miR-6858-5p, and hsa-miR-873-3p) which show a significant impact on the patients’ prognosis. The prediction performance of this 5-miRNA signature was then evaluated by the K-M survival analysis and ROC analysis in the patients after they were stratified into 2 groups according to the risk score. To establish a more reliable, individualized clinical prediction model, we construct a nomogram by incorporating these five miRNAs with six other independent clinical indices. independent clinical indices. The calibration plots showed that the prediction of the nomogram was in good agreement with the observed patient outcome, and the ROC curve analysis also suggested that this nomogram had higher prognostic efficiency. Furthermore, the K-M survival analysis indicated that has-miR-315p and has-miR-55713p have research significance for the overall survival of LUAD patients. All these results, taken together, strongly suggest that m7G-related miRNAs have a significant prognostic ability in LUAD. Almost all the five prognostic miRNAs we identified in this study have been previously reported to be associated with cancers. Mi et al., (2020) study showed that upregulated expression of miR-31-5p might promote exacerbation of colon adenocarcinoma by targeting TNS1 thus predicting a poor OS. Lu et al., (2019) study showed that miR-31-5p promoted the progression of oral cancer by enhancing the proliferation of oral epithelial cells. In our study, we found that high expression of miR-31-5p worsen the prognosis of LUAD patients, suggesting miR-31-5p’s involvement in the progression of LUAD. DF Li et al., (2015) found that miR-4697-3p plays an important role in maintaining physiological balance, and its destruction led to the development of gastric cancer. Wang et al., (2021b) study showed that miR-6858-5p plays an essential in melatonin’s inhibiting the malignant biological behaviors of Glioma cells. Some researchers found that mir-873-3p might promote the development of gastric cancer, pancreatic cancer, estrogen-receptor-positive breast cancer, and some other cancers (Deng et al., 2021; Fang et al., 2021; Zhang et al., 2021). The miR-5571-3p we identified was a novel gene that had not been previously studied, our study showed that high expression of miR-5571-3p plays a protective role in LUAD patients and has a good prediction ability of prognosis. It is suggested that miR-5571-3p might inhibit tumor development by regulating m7Gmethylation, which indicates the necessity of future investigations on it as a novel potential therapeutical target of LUAD. These m7G-related miRNAs could be used as predictors of LUAD. Among them, the expression of miR-31-5p and miR-5571-3p affects the prognosis of patients, which is worthy of our further studies on their molecular mechanism. Furthermore, we analyzed overlapping genes associated with the immune scores and the risk scores using the KEGG and GO enrichment analysis to detect the potential molecular mechanisms. In the KEGG analysis, the neuropeptide receptor activity, antimicrobial humoral response, and neuropeptide signaling pathway were the most significantly enriched pathways. In the groups of biological processes, cellular components, and molecular function, the neuropeptide receptor activity, serine-type endopeptidase inhibitor activity, and neuropeptide signaling pathway were the most significantly enriched gene sets, respectively. The biological functions of neuropeptide receptors have been reported in previous studies. Lee et al., (2020) found that the neuropeptide bombesin receptor stimulates the proliferation of lung cancer cells through the activation of HER3. Some studies have also revealed that immune-related lncRNAs, such as SCHLAP1, could mediate the neuropeptide signaling pathway and the cytokine - cytokine receptor interactions in low-grade gliomas (Li et al., 2019). This indicates that the neuropeptide signaling pathway plays a critical role in the development and progression of cancer, thus suggesting requirement of future studies on the impacting mechanism of the neuropeptide signaling pathway in LUAD. The number, location, and phenotype of tumor-infiltrating lymphocytes (TILs) play essential roles in the development and progression of tumors (Chen et al., 2017). Tumor immunotherapy is currently the main motive power for personalized precision medicine, on which great efforts are currently being made in treating advanced or metastatic cancers utilizing the functions of the immune system (Ganesh et al., 2019; Szekely et al., 2018; Wang et al., 2017). The tumors’ responses to immunotherapy are closely related to the infiltration of immune cells, including the T helper (Th) cells which are associated with the activation of T lymphocytes (Antony et al., 2005; Hung et al., 1998; Pardoll et al., 1998). Thus, we investigated the role of immune cell infiltration and immunology functions in the immune microenvironment in LUAD. Among the immune cells, T helper cells are highly expressed in each sample, showing that Th may play an important role LUAD. Among the immunology functions, human leukocyte antigen (HLA) and MHC class I were highly expressed in almost every sample. Previous studies have shown that MHC class I is essential for immunology functions in coordinating the killing effect of NK cells and CD4T cells (Shklovskaya et al., 2021). Thereafter, we analyzed the differences in immune cells and immunology functions between the high- and low-risk groups. Our results showed that compared to high-risk groups, low-risk groups had significantly higher infiltration of Dendritic cells, Mast cells, B cells, Neutrophils, and TIL, and significantly lower infiltration of NK cells. The Univariate Cox regression analysis showed that the expression of AMPD1 was positively correlated with most of the immune cells and the immunology functions. Studies on the AMPD1 in LUAD, however, are very limited, thus further studies are required to unveil the impacting mechanism of the AMPD1 gene in LUAD. The above results revealed the possible involvement of immune cell infiltration and immunology functions in LUAD and provide a reference for future studies on the development of novel immunotherapy targeting LUAD. Besides the novelty and significant findings, we acknowledge certain limitations in the present study. This study integrates the associations between m7G, miRNA, and LUAD prognosis, which has implications for innovation and provides accuracy for clinical prognosis prediction. However, this study only analyzed the data of LUAD patients collected from the TCGA database, the model constructed in this study had not been validated by real world data collected from a purposely designed clinical trial due to a limitation of the research area of our laboratory. Besides, the expression and mechanism of these miRNAs had not been validated using wet-lab experiment, which requires further investigations. In conclusion, our study is the first in the area to explore the application of m7G-related miRNAs in predicting the prognosis of LUAD. We identified DE m7G-related miRNAs associated with LUAD, five from which with significant prognostic value were extracted to construct an independent prognostic signature for LUAD. Furthermore, we constructed a nomogram by integrating these five m7G-related miRNAs with six other independent clinical indices, which demonstrated a more accurate and reliable predict ability of the prognosis of LUAD patients. In a summary, our study unveiled the prognostic value of m7G-related miRNAs in LUAD, and it should serve as reference for the identification of more m7G-related genes in the future. Author Contribution Statement Methodology and Writing – original draft: XiaowenQiu; Data curation:Yongting Chen; Supervision,Writing – review & editing: Yipeng Song and Hongwei Li; Formal analysis:Xingzhuang Zhu,Zheng Gong,Fengyuan Yu and Pengfei Zhang; Final approval of manuscript: All authors. Acknowledgements Ethics statement There were no cell, tissue, or animal studies. No ethical requirements are involved. Consent for publication All authors agree to publish the paper. Availability of data and materials The datasets in this study are available from TCGA (https://portal.gdc.cancer.gov) TargetScan (http://www.targetscan.org/) and ESTIMATE database (https://bioinformatics.mdanderson.org/estimate/disease.htmlhttps://bioinformatics.mdanderson.org/estimate/disease.html). 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PMC010xxxxxx/PMC10352731.txt

==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116138 10.31557/APJCP.2023.24.4.1173 Research Article Disparities in Compliance with Colorectal Cancer Screening: Evidence from Two US National Surveys Ahmed Nasar U 1* Chowdhury Muhammad Abdul Baker 2 Rodriguez Anny 1 Azim Syeda Ishra 3 Taskin Tanjila 1 Ahmed Shyfuddin 1 1 Department of Epidemiology, Robert Stempel College of Public Health & Social Work, Florida International University, Miami, Florida, USA. 2 Department of Neurosurgery, University of Florida College of Medicine, Gainesville, Florida, USA. 3 School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia. * For Correspondence: ahmedn@fiu.edu 2023 24 4 11731180 20 9 2022 6 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: Colorectal cancer (CRC) is the most preventable cancer if adherence to its screening guidelines through compliance with physician recommendations are met. Lack of access to care is the most significant barrier which was decreased by the Affordable Care Act (ACA), that may influence healthcare behaviors/practices. The aim of this study was to determine the factors affecting compliance with recommendations for CRC screening between two US National Health Interview Surveys (NHIS) in 2010 and 2015. Methods: We used individual data of adults aged ≥50 years from the Cancer Module of NHIS that repeats every-5-years. Multiple logistic regression analyses were employed to identify the compliance associated factors and their changes after five years. Results: We included final data of 1,553 and 2259 and individual from 2010 and 2015, respectively. Overall, compliance to physician recommendations for colorectal cancer was 85.70% in 2010 and 81.54%. Men compiled more in 2010 than women which was reversed in 2015. The multivariable-adjusted odds of compliance were increased with age; lower for female [Odds ratio (OR)= 0.45 Confidence Interval (CI 95% 0.27, 0.75), having a family history of CRC [OR=3.05 CI:1.02, 9.05], having insurance [OR 3.58 CI:1.4, 9.12], and Odds increased with the number of doctor visit in 2010. However, in 2015 the odds were substantially increased with the increasing age, reversed odds for female [OR= 3.49 CI: 1.67, 7.29)], increased for non-Hispanic Blacks [OR= 4.87 CI: 2.05, 11.55] and lower for Asian [OR=0.33 CI:0.15, 0.74], higher for family history of colorectal cancer [OR=3.31 CI:1.92, 5.69]. Although insurance coverage and the number of doctor visits were significant predictors of compliance in 2010, those became non-significant in 2015. Conclusions: Compliance disparities by gender and access to healthcare either reduced in strength or reversed between 2010 and 2015. The non-Hispanic Black significantly higher in compliance than other race-ethnicities in 2015. Key Words Colorectal cancer colonoscopy compliance racial and gender disparities affordable care act ==== Body pmcIntroduction Colorectal cancer (CRC) is the 2nd leading cause of cancer mortality accounting for 53,200 deaths of US men and women in 2020 with 147,950 new cases (Howlader et al., 2017; Seigel, 2020). The 5-year survival rate of CRC is 90% if detected at the early localized stage. In contrast, if diagnosed at a late stage, the survival rate goes down to only about 11.7% (Siegel et al., 2012). The US Preventive Services Task Force (USPSTF) recommended that adults aged 50-75 years should screen for CRC to detect and prevent CRC at an early stage. However, 60.6% of the US adults 50-75 years or above were up to date with CRC screening (Joseph et al., 2020). Surprisingly, only 37% of CRC is detected at an early localized stage, and 21% are diagnosed at a distant stage (Seigel, 2020). Improving strategies to increase screening adherence could decrease morbidity and mortality from colorectal cancer. Colonoscopy can view the entire colon. It can detect presence of any polyps which can be removed during the same procedure which established it the gold standard for prevention (Seigel, 2020). The national polyp study has shown that colonoscopies were effective in reducing CRC incidence up to 90% as compared with the historical controls (Winawer et al., 2000). Endoscopic screening has also been found to be more effective in reducing mortality than fecal occult blood test (FOBT) alone (Crespi et al., 2001). The national guidelines have recommended that persons aged ≥50 years should be screened for colorectal cancer using the gold standard-- colonoscopy once every ten years or sigmoidoscopy once in every five years (Smith et al., 2006). Despite a consensus among experts regarding the effectiveness of colorectal screening (Crespi et al., 2001; Pignone et al., 2002; Walsh et al., 2003), screening rates remain low (Ioannou et al., 2003; Janz et al., 2003; Seeff et al., 2002). The benefits of CRC screening are not fully realized due to a lack of adoption and compliance with the screening guidelines by US populations. In 2018, the Centers for Disease Control and Prevention (CDC) reported that around 40% of eligible adults did not receive the Gold Standard test- colonoscopy in the past decade (Clarke et al., 2020). Receiving physician recommendations for CRC screening is associated with sociodemographic characteristics and healthcare access in the US population (Ahmed et al., 2013). Insurance coverage has been found to play a critical role in both healthcare access and utilization (Buchmueller et al., 2005). Healthcare access may be the central conduit to involved physicians (Ahmed et al., 2013). Physicians play a crucial role in identifying and recommending eligible persons for CRC screening (Ahmed et al., 2013; Janz et al., 2003). However, not all those who received the recommendation complied. It is well recognized that the concept of health is quite complex, however, understanding how social determinants actively playing a role in compliance and impacting the inequities within their pathways is imperative (Shokouh et al., 2017). With no true gold standard, one proposed method is to include multiple indicators along with the life-course approach for social determinants as well as socio-economic factors (Shokouh et al., 2017). Thus, non-compliance related factors need to be further explored and identified. The purpose of this study is to identify the predictors of compliance with physician recommendation (prescription) of colonoscopy for CRC screening among the screen-eligible US population and to examine the changes among these factors between in the year 2010 and after five years in 2015. Materials and Methods Data source and study population We used publicly available data from the National Health Interview Survey (NHIS) for the years 2010 and 2015. The National Center for Health Statistics (NCHS) of the CDC house the data and made the survey data file available for research via internet and other electronic media. The cancer module of this survey is done only once every 5 years within the NHIS. The NHIS is a cross-sectional household survey where the sample design is multistage probability sampling. In the sample, basic information on the health and demographics of all household members was collected by an in-person interview. The NHIS cancer module is collected as a supplement which is given to individuals over the age of 40. Of the 35,153 eligible adults, a total of 27,157 were interviewed in 2010, resulting in a final sample adult response rate of 60.8%. Of the 42,270 eligible adults, a total of 33,673 were interviewed in 2015, yielding a final sample adult response rate of 55.2%. There was a high percentage of missing data in cancer module potentially due to the reason that the cancer module was in the near end of the survey, which may have contributed to respondent fatigue, or they became reluctant to answer questions. However, missing data issues had been addressed by the NHIS technical team using non-response, design effect, and post-stratification adjustments. The details on the design of NHIS can be found elsewhere. (Centers for Disease Control and Prevention, 2022) For this analysis, the inclusion criteria (Fig 1) were as follows: a) Age 50 years or older; b) No history of colon or rectal cancer; c) Visited doctor in the last 12 months; and d) Received recommendation from a physician to perform colonoscopy within last twelve months. The exclusion criteria were a) The participants less than 50 years old; b) Sigmoidoscopy screening done within 5 years before the survey, c) Colonoscopy screening done within 10 years before the survey, d) Those diagnosed with colon or rectal cancer, or e) The participant did not visit a doctor within the last 12 months. Variables and Measures Compliance with colorectal screening was measured by whether a person who is 50 years or older had completed either a colonoscopy or sigmoidoscopy after receiving a recommendation from a physician. We created a dichotomous outcome variable (complied-yes or no). Participants who had the screening done in the last year were measured as complied. Based on the literature, we classified independent variables into three thematic groups: i) Sociodemographic (age, sex, race/ethnicity, education, and marital status); ii) Health care access (insurance coverage and doctor’s visit in the past 12 months); and iii) Health risk and health status (family history of any cancer, citizenship, income, and region). Statistical analysis We performed a descriptive analysis of demographic and other variables by calculating the proportion of compliance for colorectal cancer by adjusting the sampling weight of the survey. Pearson Chi-square test was used to assess the relationship between the outcome variable (compliance) and the co-variates. We estimated crude odds ratios to assess patients’ compliance with physician recommendations for colorectal cancer screening using bivariate logistic regression models. Then we built adjusted models using the stepwise forward-backward variable selection method with purposeful criteria of p ≤ 0.20 as inclusion and p ≤ 0.25 as exclusion criterion for each sample. Thus, in the final model 12 variables: age, sex, race/ethnicity, education, marital status, family history of CRC, citizenship, income, insurance coverage, usual source of care, number of doctor visits in the past 12 months, and region of residence were included. Data were analyzed separately for each survey year. The data sets contained the national weights to address the design and post-stratification effect of oversampling of a certain race, region, interviewer effect, and non-response. The complex multistage cluster sampling adjustment was done using the Taylor Series Linearization technique for the NHIS nationally representative sample. All estimates were generated using SAS version 9.4, which allows deriving correct standard errors for estimates of complex surveys. Results The nationally representative sample consisted of 1,553 participants in NHIS 2010 and 2,259 in NHIS 2015, aged 50 years and older, who were never diagnosed with colorectal cancer (Figure 1). Demographics were similar for both 2010 and 2015, with three-fourth of the sample being non-Hispanic White, one-third of the sample were college graduates, and notable about one-fifth of the participants had a family history of cancer. The descriptive statistics, bivariate and multivariable analyses Table 1 for the year 2010 and Table 2 for the year 2015 are shown, respectively. Gender and racial disparities In multiple logistic regression analyses, after controlling for all other variables, females were less likely (OR: 0.45, 95% CI: 0.27, 0.75) to adhere to a recommendation for CRC screening as compared with males in 2010. However, in 2015, the odds of compliance with the screening recommendation among females were 3.49 (95% CI: 1.67, 7.29) compared to males. In 2015, after adjusting for the predictor variables, CRC screening recommendations compliance differed by race/ethnicity. Non-Hispanic Blacks are almost five-fold odds (OR: 4.87, CI: 2.05, 11.55) of adhering to the recommendation in 2015 compared to non-Hispanic Whites. A decrease in compliance with recommendations is observed among Asians (OR: 0.37, CI: 0.16, 0.84). Comparison between 2010 and 2015 The models for each of the two survey years showed differences in several factors that explained compliance with a physician recommendation for CRC screening. In both 2010 and 2015, factors significantly associated with compliance for screening were family history of colorectal cancer and the age of participants. Those who had a family history of colorectal cancer were at least three times odds of compliance with a physician recommendation in each of the two survey years: (in 2010, OR 3.05 vs. 3.32 in 2015). The odds of participants’ age on compliance to physician recommendations were almost doubled from 2010 to 2015. In 2010, 55-59 years old participants were 79% more likely, and 60-64 years old were 144% more likely to adhere to the physician recommendation compared to 50-54 years old. However, in 2015, there is a significant increase of odds 5.4 times and 4.6 times to adhere to the physician recommendation among 55-59 years and 60-64 years, respectively. A higher frequency of physician visits was significantly positively associated with compliance with physician recommended screening in 2010; however, this association was not observed in 2015. In 2010, those who had 2-3 visits were 3.21 times odds of compliance, and those who had more than four visits were 3.88 odds of compliance with physician recommendation in comparison to with a single physician visit during a year. However, in 2015, there was no significant association with the physician visit frequency with compliance after adjusting for other factors. Disparities were also observed in participants with a college education and higher income (more than $65,000) in 2015. Participants with a college education were 5.07 times as likely to adhere to physician recommendations for CRC screening than participants with no high school education. Similarly, participants with an income of more than $65,000 were 4.35 times as likely to adhere to the physician recommendation. In contrast, in 2010, no significant disparities in income or education were observed. In 2010, those who were insured were 3.58 times as likely to comply with physician recommendations for CRC screening compared to those who were not insured. However, in 2015, no significant association of health insurance has been found with CRC screening compliance. Table 1 Sociodemographic Characteristics of Complied to Physician Recommendation for Colorectal Cancer Screening among Eligible United States Population: National Health Interview Survey, 2010 Characteristics Total n=1553 Sample (%) Complied (%) n=1331 Crude OR (95%CI) Multivariate OR (95%CI) Age, years 50-54 324 (25.42) 79.01 Ref Ref 55 -59 266 (17.24) 85.71 1.82* (1.13, 2.91) 1.79* (1.02, 3.22) 60- 64 297 (18.8) 86.2 1.96** (1.26, 3.03) 2.44* (1.04, 5.7) 65-74 417 (25.09) 86.81 1.64* (1.10, 2.42) 1.25 (0.67, 2.35) ≥75 249 (13.45) 91.97 3.24*** (1.9, 5.52) -- Sex Female 858 (50.97) 84.62 Ref Ref Male 695 (49.03) 87.05 1.24 (0.9, 1.72) 2.25*** (1.36, 3.73) Race/ Ethnicity Non-Hispanic White 1048 (78.72) 84.35 Ref Ref Hispanic 168 (7.73) 87.5 1.29 (0.71, 2.33) 1.07 (0.42, 2.74) Non-Hispanic Black 259 (9.91) 91.12 2.26*** (1.4, 3.65) 2.49 (0.96, 6.47) Non-Hispanic Asian 68 (3.09) 80.88 0.86 (0.47, 1.56) 2.22 (0.28, 17.56) Education Non-High school 240 (12.01) 90.42 Ref Ref High school 402 (24.52) 82.84 0.63 (0.38, 1.07) 0.68 (0.06, 8.45) Some college 440 (28.82) 86.14 0.87 (0.51, 1.49) 0.60 (0.05, 7.76) College graduate 463 (34.65) 85.1 0.74 (0.46, 1.2) 0.74 (0.06, 9.67) Marital status Married/living together 848 (70.28) 86.44 Ref Ref Widowed/Separated 293 (11.64) 88.4 0.97 (0.62, 1.51) 1.07 (0.41, 2.79) Divorced 304 (13.21) 81.91 0.61*** (0.43, 0.87) 0.54 (0.3, 0.98) Never married 106 (4.87) 83.02 0.81 (0.44, 1.52) 0.61 (0.25, 1.53) Family History No 778 (84.53) 83.8 Ref Ref Yes 148 (14.71) 91.22 1.55 (0.8, 3.0) 3.05* (1.02, 9.05) Income > $20,000 184 (25.9) 80.43 Ref Ref $20,000-$64999 316 (47.26) 83.86 1.14 (0.74, 1.74) 1.11 (0.63, 1.95) >$65000 164 (26.84) 82.93 1.2 (0.69, 2.1) 1.19 (0.49, 2.88) Insurance coverage Not Insured 43 (2.53) 67.44 Ref Ref Insured 1506 (97.47) 86.19 2.05*** (0.82, 5.1) 3.58 ** (1.4, 9.12) Usual source of care No 20 (1.12) 75 Ref Ref Yes 1533 (98.88) 85.84 1.42 (0.44, 4.6) 1.01 (0.42, 2.43) Doctor visit in 12 mo 1 143 (10.1) 79.02 Ref Ref 2-3 413 (26.93) 82.57 1.35 (0.76, 2.39) 3.21*** (1.97, 5.24) ≥4 997 (62.97) 87.96 2.09*** (1.25, 3.48) 3.88*** (2.15, 7) *P<0.05; **P<0.01; ***P<0.001 Table 2 Sociodemographic Characteristics of Complied to Physician Recommendation for Colorectal Cancer Screening Among Eligible United States Population: NHS 2015 Characteristics Total n=2259 Sample (%) Complied (%) n=1842 Crude OR (95%CI) Multivariate OR (95%CI) Age, years 50-54 448 (23.76) 73.44 Ref Ref 55 -59 396 (19.61) 79.8 1.59* (1.04, 2.44) 5.4** (1.79, 16.28) 60- 64 387 (17.23) 80.88 1.8** (1.18, 2.74) 4.6 *** (2.25, 9.41) 65-74 751 (28.81) 85.89 2.09*** (1.4, 3.1) 8.82*** (3.75, 20.72) ≥75 277 (10.59) 86.28 2.68*** (1.65, 4.35) 6.77** (1.64, 27.87) Sex Female 1199 (50.04) 82.9 Ref Ref Male 1060 (49.96) 80 0.83 (0.63, 1.1) 0.29*** (0.14, 0.59) Race-Ethnicity Non-Hispanic White 1608 (75.94) 80.97 Ref Ref Hispanic 202 (7.46) 81.68 0.88 (0.53, 1.47) 0.4 (0.11, 1.48) Non-Hispanic Black 354 (12.4) 85.31 1.76** (1.22, 2.53) 4.87*** (2.05, 11.55) Non-Hispanic Asian 70 (3.43) 78.57 0.95 (0.46, 1.96) 0.37* (0.16, 0.84) Education Non-High school 293 (10.81) 79.52 Ref Ref High school 573 (25.13) 81.15 0.78 (0.49, 1.21) 2.01(0.72, 5.62) Some college 699 (29.54) 79.97 0.75 (0.49, 1.15) 5.07** (1.81, 14.18) College graduate 689 (34.52) 84.33 1.03 (0.65, 1.63) 1.23 (0.45, 3.37) Marital status Married/living together 1168 (66.96) 84.5 Ref Ref Widowed/Separated 390 (12.16) 81.03 0.69 (0.46, 1.04) 0.15** (0.06, 0.41) Divorced 483 (14.88) 77.85 0.78 (0.55, 1.09) 0.45* (0.24, 0.85) Never married 213 (6) 74.65 0.72 (0.46, 1.12) 0.61 (0.21, 1.82) Family History No 1117 (81.47) 81.56 Ref Ref Yes 255 (18.53) 91.37 3.19*** (1.94, .26) 3.32*** (1.92, 5.75) Income > $20,000 195 (19.84) 80.51 Ref Ref $20,000-$64999 460 (46.19) 79.35 1.08 (0.59, 1.95) 1.97(0.88, 4.38) >$65000 252 (33.97) 80.56 1.23 (0.69, 2.22) 4.35*** (1.87, 10.12) Insurance coverage Not Insured 40 (1.9) 65 Ref Ref Insured 2213 (98.1) 81.83 2.05 (0.82, 5.1) 0.9 (0.27, 2.95) Usual source of care No 53 (2.14) 66.04 Ref Ref Doctor visit in 12 mo 1 276 (10.86) 74.64 Ref Ref 2-3 601 (27.63) 81.53 1.34 (0.88, 2.06) 0.79 (0.32, 1.99) ≥4 1382 (61.51) 82.92 1.71** (1.18, 2.47) 2.61 (0.97, 7.07) *P<0.05; **P<0.01; ***P<0.001 Figure 1 Study Flow Chart Discussion In the present study, we found a substantial change among factors between 2010 and 2015 in compliance with colorectal cancer recommendations. Our findings of an increase in compliance are strongly associated with increasing age in both 2010 and 2015. This association may be attributed to receiving more attention from family members as well as frequent doctor visits by older individuals. Several other studies also found higher screening adherence among the older age group (Beydoun et al., 2008). Some research documented that older patient with three or more comorbidities had early and repeat-colonoscopy (Parsons, 2014). In our study, the younger group is less compliant with physician recommendations for CRC screening. Increasing physician recommendations for the younger eligible population plays a crucial role in reaching the National Colorectal Cancer Roundtable’s goal for CRC screening of 80% by 2024. Age plays a substantial role in human health and well-being. Almost a quarter of the total global burden of disease is attributed to disorders in those 60 years and older (Hudson et al., 2012). Studies have shown that healthcare utilization increases with those older groups compared with their younger counterparts (Buchmueller et al., 2005). It is known that aging adults have varying health conditions; however, the vast majority have at least one chronic condition that requires medical care (Goodwin et al., 2011). The establishment of Medicare in 1966 allowed for increased healthcare access among the older, eligible adults. With the implementation of the Affordable Care Act (ACA) in 2010, the additional provisions created in conjunction with Medicare led to a significant increase in the number of diagnosed early-stage CRC among older adults (Prince et al., 2015). There are gender differences in screening, detection, treatment, and survival of colorectal cancer. Our study has found that the odds of adherence to CRC screening have substantially increased among females compared with males between 2010 and 2015. There was an almost three folds increase in compliance among females as compared with that in males in five years. One possible explanation is that the ACA might have provided availability of affordable insurance plans and, by nature of females’ health-seeking behavior, took advantage of the opportunity and thus improved their ability to get access to health care, including preventive services (Lissenden et al., 2017) and utilized it. The signs and symptoms of CRC present differently for women. Women present with right-sided colon cancer at a higher rate than men and are often diagnosed at a later stage due to the location (Gunja et al., 2017). When compared to left-sided rectal cancer presents as polyps, right-sided colon cancer often presents as flat tumors, making it difficult to distinguish (Gunja et al., 2017) and diagnosis for women. Therefore, it was known as a non-female disease, despite women over age 65 and older have higher mortality and lower 5-year survival rates when compared with men (Gunja et al., 2017). Due to the advancement of diagnostic procedures, the perception of CRC being solely a man’s disease changed, and CRC is recognized as women’s disease too. Mounted nationwide awareness campaigns and promotions resulted in a substantial expansion of CRC screening recommendations to women (Eom et al., 2020). This may lead to being another explanation for our findings showing the much higher rate of compliance for women. Other factors for the decline in compliance among men could be the perceived threat of loss of masculinity or sexuality, fear of cancer diagnosis, or discomfort of CRC screening procedure (Eom et al., 2020; Kim et al., 2015). A study in the UK also found significantly higher screening adherence among females compared with males (Gwede et al., 2015). Addressing sex differences in CRC screening is challenging. A higher rate of recommendation for CRC screening played a significant role for men to adhere to CRC screening (Beydoun et al., 2008), but lack of awareness regarding screening could be a reason for lower screening among this study population. It is essential to have an increased level of conversation between health care providers and patients to increase CRC screening and compliance among males (Friedemann-Sánchez et al., 2007). A study conducted in Australia found that a notification letter before the fecal immunochemical test (FIT) increases the CRC screening by 12% among males compared with those who were not contacted (A White et al., 2018). Our study findings confirm previous studies on the association between family history of colorectal cancer and adherence to CRC screening recommendations (Wilkins, 2011). The research documented that a family history of colorectal cancer positively influenced CRC screening (B White et al., 2015). A systematic review identified that an individual with a positive family history of CRC has 1.4 to 3.3 times more likely to perform CRC screening than those with no family history (Holden et al., 2010). It has been found that first-degree relatives are 70% more likely to perform a colonoscopy compared to non-first-degree relatives. More studies supported higher CRC screening among first-degree relatives compared with non-first-degree relatives (Henrikson et al., 2015; Shapiro et al., 2012; Townsend et al., 2013). The reasons behind higher screening adherence among those individuals with family history could be due to a combination of self-awareness of the susceptibility, familiarity of this disease, family encouragement, and physician recommendation (Perencevich et al., 2013). One of our key findings that insurance coverage became non-significance in predicting CRC compliance in 2015, whereas it was significant in 2010. It has been reported that insurance plays a significant role in physician recommendations (Perencevich et al., 2013). Insurance coverage, including copayments, may have been a key factor for compliance prior to 2010. The Medicaid expansion under the ACA made impacts on the affordability of care, access to care, utilization of services, especially for the underserved population (Dillon et al., 2018). This expansion includes preventive services, including CRC (Lissenden et al., 2017). This may have in part reduced the variability and perhaps one reason for not observing any significant association of insurance coverage with compliance CRC screening in 2015. The ACA and other forms of health insurance are referred to as a financial mechanism for paying for healthcare, while access refers to the receiving of care (Buchmueller et al., 2016). The ACA has been found to be associated with increased healthcare access, use of preventative and outpatient services (Tangka et al., 2019). The initial implementation of the ACA in 2010 might have been playing some role in decreasing the insurance barrier yielding an impact on access to care and healthcare delivery, which in turn, perhaps influenced compliance with the CRC screening recommendation in 2015. We also observed the reduction of health disparities in the complying recommendation to CRC screening by race. We found increased compliance with CRC recommendations among non-Hispanic Black in 2015 than that in 2010. It has been reported that ACA decreased racial and ethnic disparities (Brawarsky et al., 2004). After the ACA Medicaid expansion, the insurance coverage gap significantly decreased for non-Hispanic Black Medicaid colorectal cancer patients (Antonisse et al., 2018). We found that Blacks have a higher rate of compliance than the White population, which may be of interest. Several programs or initiatives targeted to reduce health disparities, such as community health centers, Colorectal Cancer Control Program, and the National Colorectal Cancer Roundtable Initiative, could facilitate the reduction of racial disparities and increase access to CRC screenings (Antonisse et al., 2018; Buchmueller et al., 2016; Tangka et al., 2019). This study has a few limitations the main one self-reported information such as income and completion of CRC screening. The income missing information has been adjusted with a standard statistical method by the National Health Statistics Technical Team. Colonoscopy and sigmoidoscopy procedures require substantial preparatory involvements by the patients and these tests are quite invasive in nature. It is unlikely not to recall correctly that tests were done or not within a year of timeframe. Moreover, a satisfactory agreement was found between self-reported information and medical records of CRC screenings (Hoover et al., 2019). Despite all, one must exercise caution in making any causal inference based on the cross-sectional nature of the data. In Conclusion, the disparities in compliance with CRC recommendations by education, and income which widened in 2015. Interestingly, gender and healthcare access variables showed reduced strength or reversed comparative disparities with compliance in 2015. Author Contribution Statement N. Ahmed conceptualized the study, designed the analytic approach, drafted, led and supervised the study. M.A.B. Chowdhury and S.I. Azim designed the analytic approach, managed and analyzed the data, and interpreted the results. N. Ahmed, M.A.B. Chowdhury, and S. I. Azim drafted the article. A. Rodriguez, T. Taskin, and S. Ahmed reviewed, edited, and updated the manuscript. All authors reviewed and approved the final version of the manuscript for submission. Acknowledgements Availability of data and material All data presented here in the manuscript is freely available at cdc.gov Data availability Data is freely available at https://www.cdc.gov/nchs/nhis/index.htm Ethics approval This study was exempt from the ethical review approval as we used publicly available de-identified data. Disclosures/Conflict of Interest There are no potential conflicts (financial, professional, or personal) to disclose by any of the authors. ==== Refs References Ahmed NU Pelletier V Winter K Factors explaining racial/ethnic disparities in rates of physician recommendation for colorectal cancer screening Am J Public Health 2013 103 91 9 Antonisse L Garfield R Rudowitz R The effects of Medicaid expansion under the ACA: updated findings from a literature review 2018 Published March Beydoun HA Beydoun MA Predictors of colorectal cancer screening behaviors among average-risk older adults in the United States Cancer Causes Control 2008 19 339 59 18085415 Brawarsky P Brooks DR Mucci LA Effect of physician recommendation and patient adherence on rates of colorectal cancer testing Cancer Detect Prev 2004 28 260 8 15350629 Buchmueller TC Grumbach K Kronick R Book review: The effect of health insurance on medical care utilization and implications for insurance expansion: A review of the literature Med Care Res Rev 2005 62 3 30 15643027 Buchmueller TC Levinson ZM Levy HG Effect of the Affordable Care Act on racial and ethnic disparities in health insurance coverage Am J Public Health 2016 106 1416 21 27196653 Clarke TC Thompson TD Sabatino SA QuickStats: Reason for the Most Recent Colonoscopy, Among Adults Aged 50–75 Years Who Had a Test in the Past 10 Years-National Health Interview Survey 2020 United States Crespi M Stigliano V Assisi D Current trends in screening and secondary prevention of colorectal cancer Hepatogastroenterology 2001 48 1635 40 11813590 Dillon M Flander L Buchanan DD Family history–based colorectal cancer screening in Australia: A modelling study of the costs, benefits, and harms of different participation scenarios PLoS Med 2018 15 e1002630 30114221 Eom KY Jarlenski M Schoen RE Sex differences in the impact of Affordable Care Act Medicaid expansion on colorectal cancer screening Prev Med 2020 138 106171 32592796 Friedemann-Sánchez G Griffin JM Partin MR Gender differences in colorectal cancer screening barriers and information needs 1 Health Expect 2007 10 148 60 17524008 Goodwin JS Singh A Reddy N Overuse of screening colonoscopy in the Medicare population Arch Intern Med 2011 171 1335 43 21555653 Gunja MZ Collins SR Doty MM How the Affordable Care Act has helped women gain insurance and improved their ability to get health care 2017 The Commonwealth Fund Gwede CK Koskan AM Quinn GP Patients’ perceptions of colorectal cancer screening tests and preparatory education in federally qualified health centers J Cancer Educ 2015 30 294 300 25249181 Henrikson NB Webber EM Goddard KA Family history and the natural history of colorectal cancer: systematic review Genet Med 2015 17 702 12 25590981 Holden DJ Harris R Porterfield DS Enhancing the use and quality of colorectal cancer screening Evid Rep Technol Assess 2010 2010 1 195 Hoover S Subramanian S Tangka F Developing a Web-Based Cost Assessment Tool for Colorectal Cancer Screening Programs Prev Chronic Dis 2019 16 E54 31050637 Hudson SV Ferrante JM Ohman-Strickland P Physician recommendation and patient adherence for colorectal cancer screening J Am Board Fam Med 2012 25 782 91 23136316 Ioannou GN Chapko MK Dominitz JA Predictors of colorectal cancer screening participation in the United States Am J Gastroenterol 2003 98 2082 91 14499792 Janz NK Wren PA Schottenfeld D Colorectal cancer screening attitudes and behavior: a population-based study Prev Med 2003 37 627 34 14636796 Joseph DA King JB Dowling NF Vital signs: colorectal cancer screening test use—United States, 2018 Morb Mortal Wkly Rep 2020 69 253 Kim SE Paik HY Yoon H Sex-and gender-specific disparities in colorectal cancer risk World J Gastroenterol 2015 21 5167 25954090 Lissenden B Yao NA Affordable Care Act changes to Medicare led to increased diagnoses of early-stage colorectal cancer among seniors Health Aff 2017 36 101 7 Perencevich M Ojha RP Steyerberg EW Racial and ethnic variations in the effects of family history of colorectal cancer on screening compliance Gastroenterology 2013 145 775 81 23796457 Pignone M Rich M Teutsch SM Screening for colorectal cancer in adults at average risk: a summary of the evidence for the U S Preventive Services Task Force Ann Intern Med 2002 137 132 41 12118972 Prince MJ Wu F Guo Y The burden of disease in older people and implications for health policy and practice Lancet 2015 385 549 62 25468153 Seeff LC Shapiro JA Nadel MR Are we doing enough to screen for colorectal cancer? 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116134 10.31557/APJCP.2023.24.4.1137 Research Article Genetic Polymorphisms of Gene Methionine Synthase Reductase (MTRR) and Risk of Urinary Bladder Cancer Gautam Kirti Amresh 1* Raghav Alok 2 Sankhwar S N 3 Singh Rajender 4 Tripathi Prashant 5 1 Department of Basic & Applied Sciences, School of Engineering & Sciences, GD Goenka University, Gurugram, Haryana, India. 2 Multidisciplinary Research Unit, GSVM Medical College, Kanpur, India. 3 Department of Urology, King George’s Medical University, Lucknow, India. 4 Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow, India. 5 Department of Biochemistry, GSVM Medical College, Kanpur, India. * For Correspondence: emails2kirti@gmail.com 2023 24 4 11371141 23 7 2023 12 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Methionine synthase reductase (MTRR) gene involved in the signaling for production of enzyme called methionine synthase reductase that use for the synthesis of methionine, which further used in DNA replication and repair. Genetic variation in MTRR gene may alter the susceptibility of developing urinary bladder cancer. The present study undertaken to identify the contribution of genetic polymorphisms in the MTRR gene on the selected polymorphic sites including c.66A>G and c.524C>T towards urinary bladder cancer risk. Direct-DNA sequencing method was applied for the observation of genotyping distribution of MTRR c.66A>G and c.524C>T polymorphisms in 232 histopathological confirmed cases of transitional cell carcinoma (TCC) of urinary bladder cancer and 250 age-, sex- and ethnicity-matched cancer free controls. With significant difference (p = 0.05) of genotype analysis further corresponding Odds ratio (OR) and 95% confidence interval (CI) were calculated. Multivariable logistic regression analysis was applied for adjusting significant confounder variables. Haploview software (version 4.2) was used to perform pairwise Linkage Disequilibrium (LD) analysis. Age (p = 0.01), Habit of smoking (p = 0.05), tobacco consumption (p = 0.001) and diet (p = 0.02) were significantly differed between cases and controls. Both the MTRR substitution showed higher risk of developing urinary bladder cancer (p = <0.001), although this effect alters in multivariable logistic regression analysis in a protective association for both the substitution. No LD observed between the c.66A>G and c.524C>T substitutions. In conclusion, MTRR c.66A>G and c.524C>T substitutions showed a joint effect with the other associated risk factors. Further studies with a greater number of subjects of different ethnicity and polymorphisms are recommended for the better understanding urinary bladder cancer etiology and to screen the population who are at higher risk of developing urinary bladder cancer. Key Words Urinary bladder cancer MTRR Methionine synthase reductase genetic polymorphisms ==== Body pmcIntroduction Urinary bladder cancer is the 10th most common cancer in the world (Bray et al., 2018). According to the GLOBOCAN data, in 2018, estimated incidence of urinary bladder cancer was 5,55,000 (Bray et al., 2018). The most common urothelial carcinoma or transitional cell carcinoma comprises about 90% of all primary tumors of the urinary bladder (Longe., 2005). Smoking tobacco is accounting for approximately 50-60% of urinary bladder cancer incidence each year as a result relative risk of urinary bladder cancer mortality is second only to lung cancer (Freedman et al., 2011). Epidemiological studies observed a relationship between low folate consumption or folate deficiency with an increased risk of developing urinary bladder cancer. The major genes involved in folate (one-carbon) metabolism involved methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), cystathionine β-synthase (CBS), and thymidylate synthase (TS). The important role of one-carbon metabolism is in the synthesis of purines, pyrimidines and S-adenosylmethionine (SAM) (Sharp and Little, 2004), these key components genes and associated proteins are involved in DNA replication, DNA repair and methylation of DNA, RNA and protein (Hannibal and Blom, 2017). Methionine synthase reductase (MTRR) gene is located on short arm of chromosome 5 (5p15.2-15.3). This gene involved in the cell-signaling for the production of enzyme called methionine synthase reductase that use for the synthesis of methionine by regenerating methionine synthase (MS) enzyme that converts homocysteine (Hcy) to methionine in a cobalamin-dependent (folate) manner. MTRR plays an important for providing methyl group in folate metabolism. Genetic variations in MTRR gene directly affects functions of the expressed proteins which ultimately affects the process of DNA synthesis, repair and methylation (Wang et al., 2008). Genetic polymorphism of MTRR c.66 A>G results in amino acid substitution of isoleucine by a methionine at codon 22 position (Wilson et al., 1999). This polymorphism marginally reduces the protein’s biological activity (Dong et al., 2012). The variant genotype GG is associated with a decrease level of Hcy in plasma (Gaughan et al., 2001). Other investigated MTRR polymorphism showed a C>T substitution at position 524 results in a Ser→Leu change at codon 175 has been reported, and to date no relationship with the risk of cancer has been identified (Ravel et al., 2009). Association data for the genetic polymorphisms of MTRR c.66A>G and c.524C>T with the risk of urinary bladder cancer have still inconsistent. Therefore, this prompts us to execute this case-control study with the aim to identify the contribution of genetic polymorphisms in the MTRR gene on the selected polymorphic sites including c.66A>G and c.524C>T towards urinary bladder cancer risk. Materials and Methods Study Design and Subjects characteristics This case-control study was conducted in the Department of Urology, King George’s Medical University, Lucknow and Division of Endocrinology, Central Drug Research Institute, Lucknow. A total of 498 subjects were recruited based on inclusion and exclusion criteria. Study comprises 232 histopathological confirmed cases of transitional cell carcinoma (TCC) of urinary bladder cancer and 250 age-, sex- and ethnicity-matched cancer free controls. Subjects with any chronic disease or any kind other cancer were excluded from the study. Demographical, personal habits and clinical data of the recruited subjects were collected using questionnaire and arranged in excel sheet for further data analysis. After collecting subject’s details 3 ml peripheral blood sample was aspirated and transferred into the EDTA vial and stored at -20˚C for the DNA isolation and genotype analysis. This study was approved by the Ethics Committee of King George’s Medical University, Lucknow (Ref. No. XLIIECM/B-P31). Detailed description about the study was given to subjects and written consent form was taken for their recruitment. Genetic Polymorphisms analysis Genomic DNA was isolated from the stored peripheral blood samples. For the DNA isolation phenol-chloroform isoamyl alcohol (PCI) extraction method was adopted. DNA quality was quantified by measuring absorbance at 260nm using Nano-Drop spectrophotometer (Nano Drop Technologies, Inc., Wilmington, DE, USA). The quality of DNA was assessed by agarose gel-electrophoresis. Subjects with good quality of DNA extracted were included for the further study. Primer used for the amplification of c.66A>G and c.524C>T sites are design using Primer3 online tool and custom synthesized by Eurofins (Bengaluru, India). Primer used for c.66 A>G was forward: GCAAAGGCCATCGCAGAAGACAT and reverse: GTGAAGATCTGCAGAAAATCCATGTA with product size of 296 bp and for c.524 C>T was forward: TTGTGGTTGAGCCGTGGATTG reverse: GAGAGTGGGGGTACCGAAC with product size 375 bp. The amplicons were directly sequenced using dideoxy chain terminator cycle sequencing protocol (Big Dye V3.1, Applied Biosystems, Foster City, Ca, USA) on ABI 3730 DNA analyzer. In Brief, the sequencing PCR reaction consisted of Big-Dye 3.8 μl, PCR product 0.5-1.0 μl, forward or reverse primer 0.1-0.5 μl and variable volume of Milli-Q to make the reaction volume to 5 μl. The sequencing PCR was conducted under PCR conditions consisting of initial denaturation at 96oC for 1 minute followed by 30 cycles of denaturation at 96oC for 10 seconds, annealing at 55˚C for c.524 C>T and 58˚C for c.66 A>G for 7 seconds and polymerization at 60oC for 4 minutes. After PCR amplification, the amplicons were purified by precipitation method. For precipitation, 25 μl of sodium acetate: ethanol (1:25) mixture was added to each well of the plate and incubated at room temperature for 10 minutes. Afterwards, the plate was centrifuged at 4,000 rpm for 20 minutes at 4oC. The supernatant was removed with slow motion of hand shaking. The precipitated PCR product was purified by washing with alcohol. The products were washed with 70% ethanol twice. The plate was left at room temperature to facilitate evaporation of the remaining alcohol. After 20 minutes, 10ul of 50% Hi-Di formamide was added to each well and the plate was loaded on ABI 3730 DNA analyzer for reading the sequences of the amplicons. The sequencing results were edited as required using the sequence analysis software (Applied Biosystems, USA). After editing, the sequences were aligned using Auto-assembler software (Applied Biosystems, USA) for identification of the mutations/ polymorphisms. Statistical Analysis Chi-square (χ2) test for categorial data and Student’s ‘t’ test for continuous data were applied. Continuous data were summarized as Mean ± SD (standard deviation) while discrete (categorical data) in number and percentage (%). The statistical significance for deviation from the Hardy-Weinberg Equilibrium (HWE) was tested using Pearson’s Chi-square test in the control population. Genotype distribution analysis between cases and controls was done using 2 x 3 chi-square contingency table calculator available online at VassarStats: Statistical Computation Web Site (http://www.vassarstat.net). With the significant difference of genotype analysis further corresponding Odds ratio (OR) and 95% confidence interval (CI) were calculated. P-value (two-tailed tests) and 95% CI were used to assess the strength of association. Multivariable logistic regression analysis was applied for adjusting significant confounder variables (Gautam et al., 2016). Haploview software (version 4.2) was used to perform pairwise Linkage Disequilibrium (LD) analysis of the eligible polymorphisms. D’ and r2 were used to observe the magnitude of LD between sequenced variants. Haplotype frequencies were calculated to find if allelic combination of these substitution influenced the risk of urinary bladder cancer Results Subjects Characteristic The mean age of urinary bladder cancer (58.28 ± 10.18) patients was significantly differed (p = 0.01) with healthy subjects (60.74 ± 11.50). The present study found a statistically significant difference of smoking tobacco and dietary habit of cases and controls that was p = 0.05, p = 0.001 and p = 0.02, respectively (Table 1). There was no difference of other parameters including sex, BMI, occupation and alcohol consumption habit between cases and controls. Table 1 shows the selected characteristics of cases and controls. MTRR c.66A>G and c.524C>T substitution and urinary bladder cancer risk Polymorphisms at the two positions were analyzed and c.66A>G was in the HWE (p = 0.99) , while c.524C>T genotype distribution did not consistent with the HWE law at the level of significance that is 0.05. The present study found that MTRR (c.66 A>G) substitution was statistically significantly differed between cases and controls (p = 0.0001; OR = 2.03; 95% CI 1.41-2.92), it showed 2.03-fold higher risk of UBC in cases as compared to controls (Table 2). The distribution of MTRR (c.524 C>T) genotypes was also significantly associated with increased risk of UBC (p = 0.001; OR = 1.32; 95% CI 1.06-1.89) (Table 2). On applying multivariable logistic regression analysis for the significant confounder factors, we found that both genetic substitutions showed a protective association against the risk of urinary bladder cancer (Table 3). In c.66 A>G polymorphism for AA versus GG - unadjusted OR = 0.20, p = <0.001; adjusted OR = 0.21, p = <0.001 and for AG verses GG- unadjusted OR = 0.30, p = <0.001; adjusted OR = 0.30, p = <0.001 showed significant difference between cases and control (Table 3). Similarly, in c.524 C>T polymorphism for CC versus TT - unadjusted OR = 0.15, p = <0.001; adjusted OR = 0.15, p = <0.001 and for CT verses TT- unadjusted OR = 0.15, p = <0.001; adjusted OR = 0.16, p = <0.001 showed significant difference between the cases and controls although the association was protective against the risk of urinary bladder cancer (Table 3). MTRR haplotypes and risk of urinary bladder cancer risk Haplotyping of the two substitutions of MTRR generated four different haplotypes as detailed in Table 4. The distribution of the haplotype ‘GC’ was significantly different between cases and controls (p = 0.001), and associated with high risk of UBC as its frequency was higher in cases than controls. The frequencies of other haplotypes were almost similar between cases and controls. Further, the c.66A>G and c.524C>T polymorphisms of MTRR were not in significant LD (D’ = 0.02, LOD = 0.05, r2 = 0.0 Figure 1. Table 1 Demographic and Subject’s Personal Characteristics of Urinary Bladder Cancer Cases and Healthy Controls Characteristics Controls Cases p value n = 250 (%) N = 232 (%) Age (years) Mean ± SD 60.74 ± 11.50 58.28 ± 10.18 0.01* Sex: Female 36 (14.4) 28 (12.1) 0.45 Male 214 (85.6) 204 (87.9) BMI (kg/m2) Mean ± SD 23.98 ± 3.36 24.44 ± 3.71 0.14 Occupation Sedentary 114 (45.6) 103 (44.4) 0.79 Hard 130 (54.4) 120 (55.6) Smoking: No 126 (50.4) 96 (41.4) 0.05* Yes 124 (49.6) 136 (58.6) Tobacco: No 165 (66.0) 98 (42.2) <0.001* Yes 85 (34.0) 134 (57.8) Alcohol: No 219 (87.6) 203 (87.5) 0.97 Yes 31 (12.4) 29 (12.5) Diet: Vegetarian 147 (58.8) 111 (47.8) 0.02* Non-vegetarian 103 (41.2) 121 (52.2) *Significantly differed between cases and controls Figure 1 Linkage Disequilibrium Plot. The number of each cell represents D' and white colour cells shows no LD between polymorphisms The rs numbers are SNP IDs taken from National Center for Biotechnology Information (NCBI) Table 2 Comparison of Frequency of Genotype Distribution of MTRR Gene Polymorphisms between Cases the Cases of Urinary Bladder Cancer and Healthy Controls SNPs Controls Cases p value (n=250) (%) (n=232) (%) MTRR c.66A>G AA 135 (54.0) 85 (36.6) <0.001* AG 97 (38.8) 91 (39.2) GG 18 (7.2) 56 (24.1) MTRR c.524C>T CC 135 (54.0) 109 (47.0) <0.001* CT 109 (43.6) 90 (38.8) TT 6 (2.4) 33 (14.2) *Significantly differed between cases and controls Table 3 Multivariable Logistic Regression Analysis of Urinary Bladder Cancer Risk and MTRR Genotypes SNP Genotype Unadjusted Adjusted* OR (95%CI) OR (95%CI) MTRR c.66 A>G AA 0.20 (0.11-0.37) 0.21 (0.11-0.40) AG 0.30 (0.17-0.55) 0.30 (0.16-0.56) GG Ref Ref MTRR c.524 C>T CC 0.15 (0.06-0.36) 0.15 (0.06-0.37) CT 0.15 (0.06-0.37) 0.16 (0.06-0.40) TT Ref Ref Table 4 Common MTRR Haplotypes Distribution in Relation with Urinary Bladder Cancer Haplotype Frequency (overall) Frequency (cases, controls) P value AC 0.47 0.39, 0.41 0.21 CG 0.24 0.29, 0.20 0.001* AT 0.18 0.15, 0.17 0.36 GT 0.1 0.14, 0.06 0.91 *Significantly differed between cases and controls Discussion Folate and methionine metabolism are the key component for DNA methylation (Sharp and Little, 2004) . Several studies have found evidences between hypomethylation and hypermethylation and link with different form of cancers including colorectal, gastric cancer, esophagus cancer (Frigola et al., 2005; Miranti et al., 2017; Waki et al., 2002). DNA synthesis, repair and methylation is an important mechanism of gene regulation and expression and may play crucial roles in urinary bladder cancer initiation and its development if abnormality in these mechanisms occur due to altered activity of associated enzymes (Sharp and Little, 2004). The present case-control study has been carried out to investigate the influence of genetic polymorphisms in MTRR gene on the risk of urinary bladder cancer risk. MTRR c.66 A>G polymorphism results an enzyme with lower affinity to MTR therefore; as a result, homocysteine levels increases and methionine levels decreases, ultimately affects the process of DNA methylation (Miller et al., 2013; Wang et al., 2007). Till date only two genetic polymorphisms studies on MTRR c.66A>G (Moore et al., 2007; Rouissi et al., 2009) and only one study on MTRR c.524C>T (Rouissi et al., 2009) has investigated the association with urinary bladder cancer risk. Moore et al., in 2007 conducted a study on Caucasian population with a sample size of 1150 cases and 1149 controls, to investigate the role of c.66 A>G polymorphism and risk of urinary bladder cancer and found that variant genotype GG or AG did not have any significant association with urinary bladder cancer risk (Moore et al., 2007). A similar association was observed by Rouissi et al., in 2009 on Tunisian population however; the sample size (185 cases and 191 controls) was relatively very small from the previous study (Rouissi et al., 2009). For the pooled data, the present study showed a contradictive result from previously published reports. The study observed a statistically significant association of c.66 A>G polymorphism with the risk of urinary bladder cancer in which variant genotype (GA+GG) showed a 2.03-fold higher risk of urinary bladder cancer in cases as compared to controls. Although, this finding was totally contrary when we applied adjusted analysis for the confounder factors, now this substitution showed a protective association with the risk of urinary bladder cancer. A study reporting c.524 C>T polymorphism and risk of urinary bladder cancer did not find any relationship between c.524 C>T polymorphism and risk of urinary bladder cancer (Rouissi et al., 2009). In contrast, the present study found a statistically significant association of c.524 C>T polymorphism with the risk of urinary bladder cancer. Although, the effect did not remain same after applying the multivariable logistic regression analysis. The finding of the present study showed a strong affect of confounders that might be associated with the risk of urinary bladder cancer and may support the cumulative effect of genetic polymorphisms with other associated risk factors. The present study is first to identify the effect of haplotyping analysis and linkage disequilibrium between the selected substitution. These two polymorphisms of MTRR gene did not show any LD between them. Nevertheless, four haplotypes were generated in haplotype analysis and one (GC) of them was significantly differed between cases and controls. Present study considered some limitations like the subjects were recruited from the single hospital, therefore, the genotypes in the cases and controls may not be the true representatives of the population at large. In addition, pathological characteristics of the tumor were not studied with reference to the genotype distribution among the cases of urinary bladder cancer. In conclusion, the pooled data results of this study showed statistically significant evidence that supports the interaction between genetic polymorphism in MTRR gene and risk of urinary bladder cancer, although this association deviate after applying multivariable logistic regression analysis for the significant confounder factors. This may support the joint effect of genetic polymorphisms and other risk factors. Further studies with a greater number of subjects of different ethnicity and polymorphisms are recommended for the better understanding urinary bladder cancer etiology and to screen the population who are at higher risk of developing urinary bladder cancer. Author Contribution Statement Singh R and Sankhwar SN: supervision, conceptualization and methodology. Gautam KA and Raghav A: experiment, result interpretation, analysis. Gautam KA and Tripathi P: manuscript writing. All authors have read and approved the final draft of manuscript Acknowledgements The study was funded by University Grants Commission for graduate fellowship (F.16-1936 (SC)/2010(SA-III). We thank all the subjects for participation in the study. Conflict of interest Authors have no conflict of interest to declare. ==== Refs References Bray F Ferlay J Soerjomataram I Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J Clin 2018 68 394 424 30207593 Dong H Chao S Xiangning M Methionine synthase reductase A66G polymorphism contributes to tumor susceptibility: evidence from 35 case-control studies Mol Bio Reps 2012 39 801 Freedman ND Silverman DT Hollenbeck AR Association Between Smoking and Risk of Bladder Cancer Among Men and Women JAMA 2011 306 737 45 21846855 Frigola J Sol´e X Paz MF Differential DNA hypermethylation and hypomethylation signatures in colorectal cancer Hum Mol Genet 2005 14 319 26 15574462 Gaughan DJ Kluijtmans LA Barbaux S The Methionine Synthase Reductase (MTRR) A66G polymorphism is a novel genetic determinant of plasma homocysteine concentrations Atherosclerosis 2001 157 451 6 11472746 Gautam KA Tripathi M Sankhwar SN Functional polymorphisms in the IL-6 gene promoter and the risk of urinary bladder cancer in India Cytokine 2016 77 152 6 26605964 Hannibal L Blom HJ Homocysteine and disease: Causal associations or epiphenomenons? Mol Aspects Med 2017 53 36 42 27876556 Longe J Gale Encyclopedia of cancer: A guide to cancer and its treatments 2005 Detroit Thomson Gale 137 ISBN-13: 978-1414403625 Miller JW Beresford SA Neuhouser ML Homocysteine, cysteine, and risk of incident colorectal cancer in the Women’s Health Initiative observational cohort Am J Clin Nutr 2013 97 827 34 23426034 Miranti EH Stolzenberg-Solomon R Weinstein SJ Low vitamin B12 increases risk of gastric cancer: A prospective study of one-carbon metabolism nutrients and risk of upper gastrointestinal tract cancer Int J Cancer 2017 141 1120 9 28568053 Moore LE Malats N Rothman N Polymorphisms in one-carbon metabolism and trans-sulfuration pathway genes and susceptibility to bladder cancer Int J Cancer 2007 120 2452 8 17311259 Ravel C Chantot-Bastaraud S Chalmey C Lack of association between genetic polymorphisms in enzymes associated with folate metabolism and unexplained reduced sperm counts PLoS One 2009 4 e6540 19657388 Rouissi K Ouerhani S Oliveira E Polymorphisms in one-carbon metabolism pathway genes and risk for bladder cancer in a Tunisian population Cancer Genet Cytogenet 2009 195 43 53 19837268 Sharp L Little J Polymorphisms in genes involved in folate metabolism and colorectal neoplasia: A HuGE Review Am J Epidemiol 2004 159 423 43 14977639 Waki T Tamura G Tsuchiya T Promoter methylation status of E-cadherin, hMLH1, and p16 genes in nonneoplastic gastric epithelia Am J Pathol 2002 161 399 403 12163364 Wang L Ke Q Chen W Y et al Polymorphisms of MTHFD, plasma homocysteine levels, and risk of gastric cancer in a high-risk Chinese population Clin Cancer Res 2007 13 2526 32 17438114 Wang M Zhu H Fu G Polymorphisms of methylenetetrahydrofolate reductase and methionine synthase genes and bladder cancer risk: a case-control study with meta-analysis Clin Exp Med 2009 9 2009 Wilson A Platt R Wu Q A common variant in methionine synthase reductase combined with low cobalamin (vitamin B12) increases risk for spina bifida Mol Genet Metab 1999 67 317 23 10444342

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 10.31557/APJCP.2023.24.4.1101 Letter to Editor TikTok Content Analysis on Smokeless Tobacco and Betel Quid: Perception Versus Reality Anand Rahul * Sarode Gargi S Sengupta Namrata Sarode Sachin C Department of Oral Pathology and Microbiology, Dr DY Patil Dental College and Hospital, Dr DY Patil Vidyapeeth, Pune, India. * For Correspondence: rahul.anand303@gmail.com 2023 24 4 11011102 4 1 2023 8 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) ==== Body pmc Dear Editor We read with great interest the paper by Senevirathna and colleagues titled ‘TikTok- potential impact on the use of smokeless tobacco and betel quid by young people.’ (Senevirathna et al., 2022). We take this opportunity to discuss some of the intricacies associated with the keyword terminologies used and its probable impact on the data interpretation. The author on multiple occasions refer to the use of betel quid (BQ) and smokeless tobacco (SLT) synonymously with ‘meethapaan’, ‘sweet-paan’ and ‘paan’ which in our opinion cannot be considered as completely accurate as the food items are different in terms of ingredients used in them. The latter (meetha-paan/sweet-paan) does not include areca nut or tobacco as a constituent and uses merely herbal spices and components including and not limited to fennel seeds, cardamom, coconut, dates etc. wrapped in betel leaf and is popularly consumed as a mouth freshener cum digestive aid. On the other hand, the former usually contains tobacco and areca nut additionally in different concentrations with the preparations being usually customizable. The latter preparation of BQ without added areca nut or SLT poses negligible hazard to oral or general health and the literature provides no data on any ill effects of consuming betel leaf. On the contrary, plenty of literature is available on its medical benefits (Rai et al., 2011, Ali et al., 2022, Aara et al., 2020). On the other hand, the preparation constituting areca nut and/or SLT has been categorised as a class I carcinogen by WHO. (IARC Monographs, 2004) Furthermore, literature has categorized BQ into categories; ones containing areca nut or tobacco or both (Zain et al., 1999). Placing these various substances under the same umbrella group despite having a difference in the constituents with variable degree of carcinogenic potential is preposterous and will only lead to more ambiguity in this regard. As is evident from the data provided by the authors, majority of the posts (88.7%) analysed do not describe the ingredients used in the products. In the same context, ‘fire-paan’, ‘fire-paan-challenge’, ‘cool-lip’, ‘cool-lip-challenge’ are variations of the ‘sweet-paan’ with added flavours and tinctures but seldom includes areca nut or SLT. Considering that majority of the keywords used by the authors correspond with the hashtag terminologies of ‘#paan, #meetapaan, #bengalipaan, #firepaan, #paanlover, #sweetpaan, #firepan, #firepaanchallenge, #coollipchallenge, #coollip’, which should not be considered in the same category as other products containing areca nut and or SLT. Despite the ambiguities, the data provided by the authors is highly commendable as it provides an insight into popular trends on such media platforms and the potential impact it may have on the younger generation. Undeniably, the influence of social media platforms like ‘TikTok’ and other content sharing applications on the attitude build-up of younger generation cannot be overlooked especially when there exist no age restrictions for access on such platforms. While majority of the content being posted on the application is monitored and goes through stringent filters before being shared and any violation of the terms and guidelines can lead to suspension of the individuals account temporarily or permanently, there still exists gaps in the system which are manipulated or exploited for personal gains such as increasing views or likes on one’s post. Photograph or videograph posts imitating a restricted product (tobacco or cigarette smoking) are still available on such social media platforms in bulk and can be considered to fall into the grey area of being restricted. The real outcome of the influence of social media in this aspect can only be assessed at an individual level through KAP (knowledge, attitude, and practice) analysis. Nonetheless, the potential impact of Tik-Tok and other social media platforms in shaping a young mind cannot be disregarded and warrants prompt amendments. Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran APJCP-24-1101 Letter to Editor Reply to the letter to the editor: TikTok Content Analysis on Smokeless Tobacco and Betel Quid: Perception Versus Reality 2023 24 4 11011102 4 1 2023 8 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Dear Editor We intend to publish an article as letter to the editor in response to a recent article published in your journal titled ‘TikTok – Potential Impact on the Use of Smokeless Tobacco and Betel Quid by Young People’. On behalf of all the contributors, I will act as guarantor and correspond with the journal from this point onward. I hereby declare that all the authors have participated substantially in the intellectual content, conception, designing and writing this article. This work has not been published before, is not being considered for publication elsewhere and has been read and approved by all the authors. There is no conflict of interest and financial interest. ==== Refs References Aara A Chappidi V Ramadas MN Antioxidant activity of eugenol in Piper betel leaf extract J Family Med Prim Care 2020 28 327 31 Ali MZ Elbaz WFA Adouri S Effect of a Novel Betel Leaf Dentifrice on Commonly Seen Oral Hygiene Parameters-A Randomized Clinical Crossover Study Dent J (Basel) 2022 10 166 36135162 IARC Working Group on the Evaluation of Carcinogenic Risks to Humans “Betel-quid and areca-nut chewing and some areca-nut derived nitrosamines,” IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 2004 1 334 Rai MP Thilakchand KR Palatty PL Piper betel Linn (betel vine), the maligned Southeast Asian medicinal plant possesses cancer preventive effects: time to reconsider the wronged opinion Asian Pac J Cancer Prev 2011 12 2149 56 22296348 Senevirathna K Hettiarachchi K Warnakulasuriya S Jayasinghe R TikTok - Potential Impact on the Use of Smokeless Tobacco and Betel Quid by Young People Asian Pac J Cancer Prev 2022 23 3665 71 36444578 Zain RB Ikeda N Gupta PC Oral mucosal lesions associated with betel quid, areca nut and tobacco chewing habits: consensus from a workshop held in Kuala Lumpur, Malaysia, November 25-27, 1996 J Oral Pathol Med 1999 28 1 4 9890449

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116129 10.31557/APJCP.2023.24.4.1105 Review Article Risk Factors Associated with Nasopharyngeal Cancer Incidences in Indonesia: A Systematic Review and Meta-Analysis Romdhoni Achmad Chusnu 1* Rejeki Purwo Sri 2 Guo How Ran 3 Milla Clonia 4 Melbiarta Rezy Ramawan 4 Visuddho Visuddho 4 Nugraha David 4 1 Department of Otorhinolaryngology-Head and Neck Surgery, Faculty of Medicine, Universitas Airlangga, Indonesia. 2 Department of Physiology and Medical Biochemistry, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia. 3 Department of Environmental and Occipational Health, College of Medicine, National Cheng Kung University, Taiwan. 4 Medical Program, Faculty of Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia. * For Correspondence: romdhoni-a-c@fk.unair.ac.id 2023 24 4 11051111 24 11 2022 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: To determine the risk factors associated the incidence of NPC, particularly in Indonesia. Methods: This systematic review and meta-analysis was conducted according to PRISMA statement. Database including PubMed, Scopus, Science Direct, Web of Science, and GARUDA were retrieved. Newcastle-Ottawa scale was used to assess the quality of published study and analyse the risk of bias of included study. Random-effect model and reported pooled Odds Ratio (OR) with 95%CI was carried out in our meta-analysis. Results: A pooled of 7 studies were included in our study which included 764 participants. We found that female gender was not associated with the incidences of NPC (OR 1.45, 95% CI: 0.61-3.45, p=0.40), and smoking was highly increased the incidence of NPC (OR 4.39 95% CI (0.79-24.40), but not statistically significant (p=0.09). Furthermore, salted fish consumption and some HLA alleles were associated with increased risk. Conclusion: The incidence of NPC is not associated with female gender nor smoking habits. However, the risk of NPC is higher for those who consume salted fish and have some susceptible HLA alleles. Further investigations in larger studies are needed to confirm these findings. Key Words Nasopharyngeal cancer Indonesia gender smoking salted fish ==== Body pmcIntroduction Nasopharyngeal cancer (NPC) has always been one of the global burdens. In 2019, the incidence of NPC reached 176,500 number of cases globally. Indonesia became a country with the third highest mortality rate due to nasopharyngeal cancer, reaching 3,220 deaths in 2019 alone (Yu et al., 2022). Many socioeconomic and demographic factors contributed towards the higher incidence of NPC, starting from diet, lifestyle, to race (Okekpa et al., 2019). These risk factors, however, differ between populations and countries. Being a heterogenous country itself, Indonesia has varieties of ethnicities, culture, and dishes. As a result, risk factors of NPC among Indonesians become diverse and cannot be generalized with other countries. For such reason, it is necessary to assess these risk factors independently. As no country is not affected by globalization, people in Indonesia have also been experiencing change of dietary habits, gradually changing from consuming the natural local goods to the chemically-preserved foods, such as instant meals and salted fishes in their dishes (Vasan, 2020). The current economic growth and the variety of geographical landscapes may also contribute to the changing flow of lifestyle. For instance, Indonesia has a high rate of tobacco consumption. The trend seems to never stop increasing as cigarette is one of the most substantial contributors towards Indonesia’s national economy (Audrine, 2020). Not only that Indonesia provides many foods and culture, it also holds a heterogeneity of ethnicities, creating a special diversity of genetic predispositions toward certain diseases including NPC (Pradana et al., 2020). As the risk factors of NPC in Indonesia has not yet been established and was found to be inconsistent throughout many studies, therefore, we aimed to conduct a systematic review and meta-analysis of the mentioned scope to assess the possible distinguished risk factor of NPC in Indonesia from pooled case-control studies. Materials and Methods Searching Strategy This is PROSPERO-registered study (CRD42022316497) was written according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Figure 1) (Page et al., 2021). A literature search was conducted on 5 online databases (PubMed, ScienceDirect, Scopus, Web of Science, and Garuda) on March 12th, 2022 using the keywords presented in Supp. 1. As recommended in the Meta-Analysis of Observational Studies in Epidemiology (MOOSE) guideline (Stroup et al., 2000), collected all searches and removed the duplicates using Rayyan. Afterward, the title and abstract were screened independently to exclude articles with irrelevant topics. Finally, all aforementioned authors evaluated the available full texts based on the pre-established eligibility criteria and resolved any disagreement by discussion. Eligibility Criteria All case-control studies regarding lifestyle, dietary, and sociodemographic factors in adult patients with nasopharyngeal carcinoma within the past fifteen years were included into the analysis. On the other hand, there were some criteria to exclude the studies: (1) preclinical studies, case reports, case series, review papers, or editorial comments; (2) not published in English nor Indonesia; or (3) no wanted data. Data Extraction Data regarding authors, publication year, location, study design, sample size, and primary outcomes (NPC) were extracted by the four aforementioned authors and checked by other authors. The data were compiled into an online spreadsheet. The primary outcomes were the number of populations with risk factors of NPC (i.e., lifestyle, dietary, or sociodemographic factors) in the NPC patients versus the control subjects. Quality Assessment The risk of bias in all case-control studies were appraised using the Newscale-Ottawa Scale (NOS) tool. The reported outcome would be in numeric scale and classified according to the number of stars attributed for each item into high (seven to eight), medium (five to six), and low (less than five). Four prementioned authors contributed and resolved any disagreements over the risk of bias. Data Analysis Dichotomous data (with versus without risk factors) were presented as odds ratio (OR) and 95% confidence interval (95%CI). Heterogeneity analysis were conducted using the I2 test. Subsequently, if the I2 test were ≤ 50%, the analysis is considered to be homogenous and a Mantel–Haenszel (M-H) fixed-effect is applied. However, if the data were considered to be heterogenous, a M-H random-effect is applied. These statistical analyses are performed in Review Manager 5.4. Results Study selection The overall study selection process is illustrated in Figure 1. The initial electronic database searches yielded 572 records. After deduplication process, the total studies were 436. Then 339 records were excluded based on the titles and abstracts. Total of 12 reports were excluded because of full-text not retrieved, 13 reports were excluded because of irrelevant study design, 19 reports were excluded because of unsuitable outcome, 14 reports were excluded because of wrong population and 7 reports were excluded because of inapt publication type. Finally, there were 7 studies included for qualitative analysis and 4 studies for quantitative analysis. All these variables were reviewed for qualitative analysis of NPC risk factor in Indonesia. Study characteristic The characteristics of the included studies are summarized in Table 1. A total of 764 participants were included with a total of 368 were NPC subjects and 396 were healthy controls. The males dominated with percentages ranging from 47.8% to 70.1%. There were only three studies which reported smoking status as a result. One study disclosed about alcohol consumption, one study reported about salted fish consumption, and two studies described the distribution of HLA allele (Amtha et al., 2009; Arania R, Puji S, 2014; Judajana, 2018; Kurniawan et al., 2019; Nuaba et al., 2020; Purwanto, 2015; Tao Li et al., 1995). Quality assessment of the studies According to the Newcastle Ottawa Scale score for case-control studies, 4 studies have 7 scores, 1 study has 6 scores, 2 studies have 4 scores, given a satisfactory quality of bias among all studies. The item-specific quality assessment of the studies was showed in the Table 2. Risk factor for NPC in Indonesia Out of four studies, there were three reported higher incidences of NPC in male gender compared to woman (Amtha et al., 2009; Arania R, Puji S, 2014; Purwanto, 2015). The pooled analysis revealed that gender was not a significant risk factor of NPC in Indonesian population (OR 1.45, 95% CI: 0.61-3.45, p=0.40). However, a high level of heterogeneity was observed (I2 = 72%). Smoking status analysis included three studies for meta-analysis (Amtha et al., 2009; Kurniawan et al., 2019; Nuaba et al., 2020). The overall pooled analysis revelaed that smoking increased the odds of NPC, although no statistical emerge as risk factor (OR 4.39, 95% CI: 0.79-24.40) the risk of NPC; however, it was not statistically significant (p=0.09). The random effect model was used since the result showed a high level of heterogeneity (I2 = 93%). Nuaba et al. (2020) reported a significant result (p=0.036) regarding salted fish consumption, with OR 2.438 (1.051-5.654), meaning an increased risk to develop into NPC8. Meanwhile, Amtha et al., 2009 was the only study which compared the consumption of alcohol in NPC patients (1.2%) to healthy subjects (0.6%)(Amtha et al., 2009). Two studies reported the association between HLA-antigen types with NPC. Judajana et al. (2009) disclosed a significant association between HLA–A24 (RR 2.25; p<0.001), HLA A2 (RR 1.635; p<0.001) and HLA A11 (RR 1.065; p<0.05), while study by Delfitri et al. (2011) revealed no association between HLA-DQB1 genotypes with NPC (Judajana, 2018; Tao Li et al., 1995). Figure 1 PRISMA Flowchart Figure 2 Forest Plot for Smoking Status as a Risk Factor for Nasopharyngeal Cancer Figure 3 Forest Plot for Female Gender as a Risk Factor for Nasopharyngeal Cancer Table 1 Pooled Data of Included Studies Study; Country Population, n (case vs control) Study design Age (Mean±SD/ n (%)) Gender, male n (%) Outcome NPC Control Nuaba, 2020; Indonesia NPC (46) vs non-NPC (46) Retrospective study 47.35±11.89 38.04±10.58 44 (47.8%) Salted fish consumption, OR 2.438 95%CI (1.051-5.654), p=0.036 Smoking, OR 4.7 95%CI (1.959-11.389), p<0.001 Amtha et al., 2009; Indonesia NPC (81) vs non-NPC (162) Retrospective study > 49 years, 35 (43.2%) > 49 years, 69 (42.6%) 150 (61.7%) Alcohol consumption, OR 2.012 95%CI (0.123-32.977), p=0.616 Smoking, OR 17.875 95%CI (9.096-35.126), p=0.000 Purwanto, 2015; Indonesia NPC (81) vs non-NPC (6) Retrospective study ≥ 40 years, 65 (80.3%) ≥ 40 years, 5 (83.3%) 61 (70.1%) N/A Arania et al., 2014; Indonesia NPC (41) vs non-NPC (39) Retrospective study > 40 years, 34 (82.9%) > 40 years, 7 (17.95%) 54 (67.5%) N/A Kurniawan, 2019; Indonesia NPC (40) vs healthy controls (39) Retrospective study 49.58 ± 2.56 50.58 ± 2.38 N/A Smoking, OR 0.975 95%CI (0.420-2.262), p=0.91 Smoking duration, mean ± SD 28.89 ± 13.07 vs 33.21 ± 2.99, p=0.47 Cigarette/day, mean ± SD 8.95 ± 1.30 vs 12.89 ± 1.93, p=0.21 Judajana et al., 2009; Indonesia NPC (24) vs non-NPC (N/A) Retrospective study N/A N/A N/A HLA-A2, RR 1.635, p<0.001 HLA-A11, RR 1.065, p<0.05 HLA-A24, RR 2.25, p<0.001 HLA-B16, RR 1.632, p<0.05 Delfitri, 2011; Indonesia Bataknese NPC patients (55) vs bataknese non-NPC patients (104) Retrospective study N/A N/A N/A HLA-DQB1 *02, OR 1.65 95% CI (0.64-4.27), p=0.3 HLA-DQB1 *0301, OR 0.77 95% CI (0.37-1.7), p=0.49 HLA-DQB1 *0302, OR 0.62 95% CI (0.12-3.14), p=0.56 HLA-DQB1 *0303, OR 0.62 95% CI (0.06-6.14), p=0.68 HLA-DQB1 *0501, OR 0.67 95% CI (0.3-1.51), p=0.33 HLA-DQB1 *0502, OR 2.11 95% CI (0.85-5.25), p=0.1 HLA-DQB1 *0503, OR 1.02 95% CI (0.38-2.73), p=0.97 HLA-DQB1 *0601, OR 1.19 95% CI (0.52-2.75), p=0.68 HLA-DQB1 *0603, OR 1.93 95% CI (0.26-14.05), p=0.51 HLA-DQB1 *0609, OR 1.92 95% CI (0.12-31.1), p=0.65 NPC, nasopharyngeal cancer; N/A, not available; SD, standard deviation; CI, confidence interval; OR, odds ratio; RR, relative risk; HLA, human leukocyte antigen Table 2 Risk of Bias Analysis Using Newcastle-Ottawa Scale (NOS) for Case-Control Newcastle-Ottawa Scale Nuaba, 2020 Amtha,2009 Purwanto, 2015 Arania et al, 2014 Kurniawan, 2019 Delfitri, 2019 Judajana et al, 2019 Study Design Case-Control Case-Control Case-Control Case-Control Case-Control Case-Control Case-Control Selection Is the case definition adequate 1 1 0 0 1 1 1 Representativeness of the cases 1 1 1 1 0 0 1 Selection of Controls 1 1 0 0 0 1 0 Definition of Controls 0 0 0 0 1 1 1 Comparability Comparability of cases and controls on the basis of the design or analysis 1 1 1 1 1 1 1 Exposure Ascertainment of exposure 1 1 0 0 1 1 1 Same method of ascertainment for cases and controls 1 1 1 1 1 1 1 Non-Response Rate 1 1 1 1 1 1 1 Total Score 7 7 4 4 6 7 7 Discussion The results of our meta-analysis showed that male gender and smoking habits increased the risk of developing NPC in adult patients in Indonesia, though not statistically significant. Smoking habit was identified as a risk factor for NPC development in various regions(Long et al., 2017; Okekpa et al., 2019). Our study revealed that smoking habit increases NPC risk with the OR 1.45; however, the result was not statistically significant. In contrary to a meta-analysis conducted by Okekpa et al., (2019), the study showed similar but significant risk of smoking habit among people from Asia (OR 1.41, 95% CI: 1.27-1.57). Previous studies in several regions have shown a significant association of tobacco consumption, including in Indonesia (Amtha et al., 2009; Chang et al., 2017; Nuaba et al., 2020), although some were insignificant(Adoga et al., 2018; Kurniawan et al., 2019). Given a larger population in genetic diversity will present more representative results; however, limited studies and high heterogeneity might explain the difference in the current pooled analysis. We speculate that different susceptibility to smoking-related-DNA methylation among ethnicity could be one of the reasons. Even so, further studies are still needed to confirm such hypothesis (Elliott et al., 2014; Huang et al., 2015; Risanti et al., 2018). The contact between smoke and nasopharynx has a direct chemical effect that irritates the epithelium. Moreover, nicotine present in tobacco contains nitrosamine-derived carcinogenic agents which promote carcinogenesis (Mydin and Okekpa, 2019).The regulation between cell-stimulating and cell-inhibitory factors played by α4β2 nicotinic-acetylcholine receptors (nAChRs) and α7nAChRs, respectively, were impaired. The level of nAChR which contributes to the regulation of various cellular activities was upregulated in smokers. Altered cellular signalling pathways will then result in disrupted microenvironment balance. Hence, enhanced tumor cell activities leading to carcinogenesis were seen(Mydin and Okekpa, 2019). Lifestyle has been linked to correlate with NPC development, especially in maritime countries such as Indonesia where fish commodities were common(Mydin and Okekpa, 2019). Studies in Indonesia showed that salted fish consumption increased the risk of NPC significantly (OR 2.438; 95% CI: 1.051-5.654). This finding was correlated with nitrosamine, which could lead to carcinogenesis as discussed before(Mydin and Okekpa, 2019). Another lifestyle identified as a risk factor was alcohol consumption. Studies in various regions showed that heavy alcohol consumption significantly increases the risk of getting NPC (Chen et al., 2009; Du et al., 2019; Polesel et al., 2011), contrasting to a study in Indonesia that showed insignificant results (Amtha 2019). Increase in reactive oxygen species (ROS), lipid peroxidation, and acetaldehyde as alcohol by-products exert immune reactions, mutation, and DNA lesions (Mydin and Okekpa, 2019). However, Indonesia is a country with a majority of the citizens who are Muslims. It was found that alcohol had a dose-response relationship, hence, the risk was more extrusive in heavy compared to low alcohol consumption. This statement also explains the lack of data between alcohol consumption and NPC development in Indonesia, thus, pooling analysis was not possible for us to conduct (Chen et al., 2009; Polesel et al., 2011). As our strength, this is the first systematic review and meta-analysis that identified the risk factors of NPC patients by evaluating case controls studies in Indonesia. However, we acknowledge several limitations in our study. First, given the small number of studies, we cannot provide the pooled estimated effect of alcohol and salted fish consumption on the risk of NPC. Second, there was a high level of heterogeneity in both gender and smoking habits risk, so the random-effects model has been employed to minimize the variability effects. In conclusion, the incidence of NPC is not associated with female gender nor smoking habits. However, the risk of NPC is higher for those who consummate salted fish and have some HLA alleles susceptibility. Further investigated in larger study are needed to confirm these findings. Abbreviations NPC: Nasopharyngeal Cancer, PRISMA: Preferred Reporting Items for Systematic Review and Meta-Analysis, GARUDA: Garba Rujukan Digital, OR: Odds Ratio, CI: Confidence Interval, HLA: Human Leucocyte Antigen, NOS: Newscale-Ottawa Scale, M-H: Mantel–Haenszel, nAChRs : Nicotinic-Acetylcholine Receptors, DNA: Deoxyribo Nucleic Acid, ROS: Reactive Oxygen Species, MOOSE: Meta-Analysis of Observational Studies in Epidemiology. Author Contribution Statement ACR, PSR, and HRG conceptualizing and finding ideas; checking and revising the manuscript. CM and RRM screening, extracting, investigating, writing, editing, checking, and revising the manuscript. V and DN screening, extracting, analyzing, and writing the manuscript. All the authors have read and approved the final manuscript. Acknowledgements The authors would like to thank them for the invaluable assistance provided to them by Mentors, Seniors and Dr. Soetomo General Hospital and Parahita Dharmawangsa laboratory in East Java, Indonesia, and Medical Faculty of Universitas Airlangga Indonesia in the management of the patient and the preparation of this paper for presentation and publication. Funding Statement The research was supported by “Lembaga Penelitian dan Pengabdian Masyarakat, Universitas Airlangga, Surabaya” in the SATU Program (Southeast and South Asia and Taiwan Universities) Joint Research Scheme (JRS) Data Availability Statement This manuscript use data previously published by other authors and all data are presented into results section. Conflict of Interest All authors declared no conflict of interest. ==== Refs References Adoga AA Kokong DD Nimkur TL Environmental and Life-Style Related Risk Factors for Sinonasal and Nasopharyngeal Malignancies among a Prospective Cohort in Jos, Nigeria Int J Otolaryngol 2018 2018 1 6 Amtha R Zain R Razak IA Dietary patterns and risk of oral cancer: A factor analysis study of a population in Jakarta, Indonesia Oral Oncol 2009 45 49 53 Arania R Pujilestari SM Jayanti I Hubungan Faktor Usia, Jenis Kelamin dan Gejala Klinis dengan Kejadian Karsinoma Nasofaring di RSUD Dr H Abdul Moeloek J. Ilmu Kedokt. Dan Kesehat 2014 1 1 13 Audrine P A Policy Perspective on Tobacco Farming and Public Health in Indonesia Cent. Indones. Policy Stud Policy Pap 2020 29 Chang ET Liu Z Hildesheim A Active and Passive Smoking and Risk of Nasopharyngeal Carcinoma: A Population-Based Case-Control Study in Southern China Am J Epidemiol 2017 185 1272 80 28459936 Chen L Gallicchio L Boyd-Lindsley K Alcohol consumption and the risk of nasopharyngeal carcinoma: A systematic review Nut Cancer 2009 61 1 15 Du T Chen K Zheng S Association Between Alcohol Consumption and Risk of Nasopharyngeal Carcinoma: A Comprehensive Meta-Analysis of Epidemiological Studies Alcohol. Clin Exp Res 2019 43 2262 73 31436869 Elliott HR Tillin T McArdle WL Differences in smoking associated DNA methylation patterns in South Asians and Europeans Clin Epigenetics 2014 6 1 10 24382160 Huang T Chen X Hong Q Meta-analyses of gene methylation and smoking behavior in non-small cell lung cancer patients Sci Rep 2015 5 1 8 Judajana FM Asosiasi Human Leukocyte Antigen (Hla) Karsinoma Nasofaring (Knf) Indones. 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Biomed J 2018 10 123 127 Stroup DF Berlin JA Morton SC Meta-analysis of observational studies in epidemiology: a proposal for reporting Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group JAMA 2000 283 2008 12 10789670 Tao Li PK Poon ASY Tsao SY No association between HLA DQ and -DR genotypes with nasopharyngeal carcinoma in southern Chinese Cancer Genet. Cytogenet 1995 81 42 45 7773959 Vasan Consumer’s Preference and Consumption Towards Instan Food Products Think India J 2020 22 8333 37 Yu H Yin X Mao Y The global burden of nasopharyngeal carcinoma from 2009 to 2019: an observational study based on the Global Burden of Disease Study 2019 Eur. Arch. Oto-Rhino-Laryngology 2022 279 1519 33

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116164 10.31557/APJCP.2023.24.4.1401 Research Article Outcomes and Prognostic Factors of Patients with Platinum-Resistant or Refractory Epithelial Ovarian Cancer, Fallopian Tube Cancer and Peritoneal Cancer Hamontri Suttha Tantitamit Tanitra * Department of Obstetrics and Gynecology, Faculty of Medicine, Srinakharinwirot University, Nakhonnayok, Thailand. * For Correspondence: ttanitra@gmail.com 2023 24 4 14011405 6 1 2023 17 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: To determine the survival outcomes and prognostic factors of the patients with recurrent platinumresistant and refractory epithelial ovarian cancer (EOC), tubal, and peritoneal cancer. Methods: Women with recurrent platinum-resistant and refractory EOC, tubal and peritoneal cancer who received treatment at the HRH Princess Maha Chakri Sirindhorn Medical Center (MSMC) between January 2010 and December 2019 were included. Demographic data, serum marker, surgical factors, pathological factors and response of treatment were reviewed. Kaplan-Meier was used to calculate survival outcome. Result: Forty patients were recruited in this study (platinum-resistant 24 patients and refractory 16 patients). The median survival times were 19 and 21 months in and platinum-resistant and platinumrefractory patients, respectively. There were no significant differences in overall survival according to age, comorbidity, tumor grading, primary treatment, and secondary surgery. However, histology of clear cell carcinoma may associate with increased risk of death. The median overall survival of patients with clear cell carcinoma, serous carcinoma, and others were 14.4, 22.9, and 32.2, respectively (p = 0.003). Conclusion: Almost 10 years, the survival rate of the patients in these group has not increased despite new treatments option. Novel strategies should be considered in National policy of the treatment for ovarian cancer in our country. Key Words Ovarian cancer recurrence survival prognosis ==== Body pmcIntroduction Ovarian cancer is the most lethal gynecological cancer. In 2020, ovarian cancer was the sixth most common cancer in Thai females with age-standardized incidence rates of 7.9 per 100,000 and age-standardized mortality rates of 4.9 per 100,000. There were 4,475 new cases diagnosed in Thailand; however, in more than 50% (2,941 cases) of those cases, deaths were reported (Sung et al., 2021). Almost all ovarian cancer cases are diagnosed at an advanced stage due to unspecific symptoms and lack of screening, leading to early recurrence and poor prognosis. Late-stage presentation has a 5-year relative survival rate of 29%, in contrast with 92% for early-stage disease (Lheureux et al., 2019). Although most patients receive standardized surgery and chemotherapy, the status of recurrent disease is heterogeneous. Recurrence is incurable in about 75% of women with advanced disease (Lheureux et al., 2019). Intrinsic resistance is seen in 10% to 15% of patients defined as platinum-refractory and is associated with a poor median survival of less than 9 months. Approximately 20% to 30% of patients have cancer which will recur or progress within 6 months of completing chemotherapy (termed platinum-resistant) and have a median overall survival of approximately 12-18 months (Lheureux et al., 2019). For patients with platinum-resistant or platinum-refractory disease, single-agent nonplatinum-based chemotherapy is used until subsequent progression or unacceptable toxicity is observed. However, the expected response rate in this setting is low (about 10–15%) (Lheureux et al., 2019; Pujade-Lauraine et al., 2019). The choice of agent for an individual patient depends on the history of prior treatment, residual toxicities, availability, cost, the convenience of treatments, and patient preferences after being fully informed (Pujade-Lauraine et al., 2019). Many prognostic indicators must be considered when determining expected prognosis and rate of response to future lines of therapy in recurrent EOC. These factors include tumor volume and histology, prior treatment and platinum-free intervals, BRCA mutation, symptoms, performance status, and comorbidity (Dockery et al., 2019). In Thailand, there is limited data regarding outcomes and prognosis factors predicting the survival of patients with platinum-resistant or refractory epithelial ovarian cancer. The latest data published reported the median overall survival of a patient with refractory disease was 20 months, which was higher than in other countries (Foley et al., 2013; Jansaka and Suprasert, 2014). Due to the inconclusive results and outdated data in our country, we conducted a study to determine the outcomes of these patients and to identify the prognosis factors of a good response after therapy. Materials and Methods This study was a retrospective design, conducted between August, 2021, and July, 2022. All women with recurrent platinum-resistant or refractory epithelial ovarian cancer, fallopian tube cancer, and peritoneal cancer who received treatment at the gynecologic oncology unit, HRH Princess Maha Chakri Sirindhorn Medical Center (MSMC) between January 2010 and December 2019 were included. Those with incomplete documents were excluded. The sample size was calculated based on the results of Pitakkamkul (2013). The minimum number of patients required to determine the survival outcome was 42. However, we studied all patients who had received treatment within the last 10 years. Approval was obtained from the ethics committee of Srinakharinwirot University. Demographic data (age, parity, comorbidity), the stage at diagnosis according to FIGO staging, the start and end of chemotherapy (first and second line), and chemotherapy protocol were documented. Eastern Cooperative Oncology Group (ECOG) performance status and the serum levels of CA125 collected before the start of second-line chemotherapy were also documented. Computed Tomography (CT scan) results before and after complete treatment were used to assess the response. The response was classified as complete or partial response (CR, PR), stable disease (SD), or progressive disease (PD) according to the radiologist’s evaluation. Descriptive data are presented as mean and standard deviation (SD) for continuous variables, and as numbers and percentages for categorical variables. Survival time was calculated from the date of the first diagnosis until death or the last follow-up visit. Kaplan-Meier was used to calculate the survival outcome. All statistical analyses were performed with STATA version 14 (Stata, College Station, TX). Results There were 196 patients diagnosed with EOC, fallopian tube cancer, and peritoneal cancer during 2010-2019. The numbers of patients in the platinum-refractory group, platinum-resistant group, and platinum-sensitive group were 16, 24, and 44 respectively. The patients’ characteristics are presented in Table 1. Most patients were treated by using single chemotherapy agents (64%), which were gemcitabine, weekly or triweekly paclitaxel, and liposomal doxorubicin. Most of them received three lines of chemotherapy. The most common reasons for stopping treatment were poor performance status and death. Seven patients (17.5%) received secondary surgery, which aimed to eliminate macroscopic disease (cytoreductive surgery) and to alleviate the symptoms in five and two cases, respectively. However, only one patient had cancer which was optimally debulked (no residual disease). The overall survival curve is shown in Figure 1. The median survival times were 21 months and 19 months in platinum-refractory and platinum-resistant patients, respectively (p 0.89). Table 2 shows response rates and adverse effects of treatment. The overall response rate (complete and partial response) was 12.5% in both groups of patients. Two-thirds of patients had minor toxicities from chemotherapy. There were no significant differences in overall survival according to age, comorbidity, tumor grading, primary treatment, and secondary surgery. However, the median overall survival of patients with clear cell carcinoma, serous carcinoma, and others were 14.4, 22.9, and 32.2, respectively (p 0.003) (Figure 2). Table 1 Clinical Characteristics of Platinum-Refractory and Platinum-Resistant Ovarian Cancer Patients Characteristics Total (%) Mean age (range) 55 (21-76) Co-morbidity 19 (47%) Diagnosis Epithelial ovarian cancer 39 (97.5%) Primary Peritoneal cancer 1 (2.5%) Mean total number of chemotherapy regimens (range) 2.5 (1-5) Nulliparity 18 (45%) Advanced stage (stage III, IV) 33 (82.5%) Mean CA 125 level, before treatment (SD) 1,781.76 (2583) Mean CA 125 level, at recurrent (SD) 200 (698) Histology Serous 15 (37.5%) Mucinous 1 (2.5%) Endometrioid 5 (12.5%) Clear cell 12 (30%) Adeno NOS 3 (7.5%) Mixed (serous-clear, clear-mucinous) 2 (5%) Others (Brenner tumor) 2 (5%) High grade or grade3 18/22 (81%) Neoadjuvant chemotherapy 17/40 (42%) Complete surgical staging 15/23 (65%) Optimal surgery 22/40 (55%) Treatment Surgery + chemotherapy 7 (17.5%) Chemotherapy 33 (82.5%) Table 2 Treatment Outcomes of the First Episode of Tumor Progression/Recurrence Total (%) Refractory (%) Resistant (%) Response of treatment Complete response (CR) 4 (10) 2 (12.5) 2 (8.3) Partial response (PR) 1 (2.5) 0 (0) 1 (4.2) Stable disease (SD) 1 (2.5) 0 (0) 1 (4.2) Progressive disease (PD) 34 (85) 14 (87.5) 20 (83.3) Adverse effect from chemotherapy None/ only G1-2 28 (70) 11 (68.8) 17 (70.8) Hematologic effect 11 (27.5) 5 (31.2) 6 (25) Non-hematologic effect 1 (2.5) 0 (0) 1 (4.2) Further treatment (Third line chemotherapy) 22 (55) 14 (35) 8 (20) Number of survivors at the last contact 9 (22.5) 3 (7.5) 6 (15) Figure 1 Overall Survival Time of Patients with Platinum-Refractory and Platinum-Resistant Ovarian Cancer Figure 2 Overall Survival Curves for Patients with Platinum-Refractory and Platinum-Resistant Ovarian Cancer, Classified by A) age at diagnosis, B) comorbidity, C) histological type, D) grade, E) primary treatment, and F) secondary surgery. *NACT, Neoadjuvant Chemotherapy Discussion The patients with refractory and platinum-resistant disease had a median overall survival time of 19-21 months. This result was similar to another previous study in Thailand. Jansaka (2014) reported the median overall survival times were 20 and 14 months in platinum-refractory and platinum-resistant patients, respectively. However, they were slightly longer than those found in other studies (Foley et al., 2013; Pujade-Lauraine et al., 2019; Kerio et al., 2022). This might be due to the small number of patients. In our center, the patients were followed up every 3 months during the first 2 years after primary treatment. They were diagnosed with refractory or recurrent disease when the disease was detected by physical examination or rising CA125 values outside the normal range, regardless of their symptoms. Imaging including USG or CT scan was used to confirm the diagnosis in some cases. Using elevated CA125 levels alone could result in platinum-sensitive patients being falsely classified as platinum resistant. This might explain why the survival time found in this study was longer. A multi-institute European trial evaluated the benefits of early treatment based on elevated CA125 levels versus delaying treatment until clinically indicated. The results showed no evidence of a difference in overall survival between the two groups (Rustin, 2011). Regarding the response to treatment, the overall response rate was 12.5%, which was similar to a previous study in Thailand (Pitakkarnkul et al., 2013). Most of the patients received subsequent chemotherapy with gemcitabine and paclitaxel. Use of other types of chemotherapy, including targeted therapy, was limited by Thailand’s Universal Health Coverage. The mean total number of chemotherapy regimens was 2.5 (range 1-5). None of the patients in this study had received target therapy. Our practice in the treatment of recurrent ovarian cancer was similar to that found in the survey of Thai gynecologic oncologists. The results of the survey taken by 170 responders showed best supportive care was given more frequently after a failure of second-line chemotherapy. Targeted therapy was prescribed to only 5% of patients (Manchana et al., 2020). Regarding factors influencing the survival of patients, one study revealed that prolonged post progression survival of recurrent ovarian cancer patients was associated with post recurrence treatment, including secondary debulking surgery, favorable response to second-line therapy, and more than two chemotherapy regimens after the first recurrence (Soyama et al., 2017). Hilal (2016) reported that long-term survival of recurrent ovarian cancer was characterized by a young age, low tumor stage, long recurrence-free interval, and combined modality treatment with optimal cytoreductive surgery and systemic chemotherapy. Multiple retrospective studies suggested that a greater number of neoadjuvant chemotherapy cycles (more than 3 or 4 cycles) may be associated with worse outcomes (Colombo et al., 2014; Altman et al., 2017; Lui et al., 2020). In our study, the number of neoadjuvant cycles ranged from 2-6. Thirty five percent (6/17) of patients receiving more than 4 cycles. This might be another factor that influence survival after treatment. Due to the small number of patients, we couldn’t accurately estimate the effect of treatment to identify the prognostic factors. However, we found that patients with histology of ovarian clear cell carcinoma (OCCC) had the worst survival outcome. A large prospective randomized trial found that in the early stages, OCCC had a better outcome than serous carcinoma, however, in late-stage patients, OCCC was significantly associated with decreased overall survival (OS) compared to serous carcinoma (Oliver et al., 2017). The results of a recent study from Korea also showed that advanced-stage OCCC had a worse OS than advanced-stage serous carcinoma (Oliver et al., 2017; Kim et al., 2022). The proportion of patients with clear cell carcinoma was higher in East Asian women while there was no improvement in overall survival (Matz et al., 2017; Park et al., 2017; Kim et al., 2022). Therefore, the development of innovative strategies and precision medicine is important and further clinical trials are still needed. Only a few studies have presented the survival outcomes of the patients in this group. Studies on these patients have been published since 2014. The strength of our study is that it keeps researchers up to date with the real situation of patients with refractory and platinum-resistant EOC in Thailand. We found that for almost 10 years, the survival rate of the patients in these two groups has not increased despite a new treatment option. Current practice has been limited by Thailand’s Universal Health Coverage. Our data could be used to improve the policy of treatment for ovarian cancer in Thailand. There may be some possible limitations of this study. First, its retrospective design, missing data and the small number of patients may have influenced the results. A multi-center study covering every region in the country could provide more accurate results on patient outcomes. Second, we didn’t assess the quality of life (QoL), which is an important factor to consider before deciding on the treatment option. One study in Thailand reported that in the setting of this group of patients, salvage chemotherapy has a QoL score comparable with that of palliative care alone (Srisuttayasathien and Khemapech, 2013). Further studies regarding cost-effectiveness of chemotherapy, new strategies or targeted therapy, and palliative treatment will have an impact on the decision-making of treatment for recurrent ovarian cancer in a developing country. In conclusion, the survival outcome of patients with refractory and platinum-resistant EOC remains poor. Novel strategies based on molecular biologics have been studied in clinical trials with encouraging results. This may offer longer survival and better quality of life to the patients. Author Contribution Statement All authors contributed to the study’s conception and design. The first draft of the manuscript was written by Tanitra T. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements This study is based on the dataset from HRH Princess Maha Chakri Sirindhorn Medical Center provided and produced by the Obstetric and Gynecological Department, Srinakharinwirot University. Funding statement This work was supported by a research grant from HRH Princess Maha Chakri Sirindhorn Medical Center, Faculty of Medicine, Srinakharinwirot University. Ethical Declaration Approval was obtained from the ethics committee of Srinakharinwirot University. The procedure used in this study adheres to the tenets of the Declaration of Helsinki. Availability of data The datasets used and analysed during the present study are available from the corresponding author on reasonable request. Competing interests The authors have declared no conflicts of interest. ==== Refs References Altman AD McGee J May T Neoadjuvant chemotherapy and chemotherapy cycle number: A national multicentre study Gynecol Oncol 2017 147 257 61 28800940 Colombo PE Labaki M Fabbro M Impact of neoadjuvant chemotherapy cycles prior to interval surgery in patients with advanced epithelial ovarian cancer Gynecol Oncol 2014 135 223 30 25220627 Docker LE Rubenstein AR Ding K Extending the platinum-free interval: The impact of omitting 2nd line platinum chemotherapy in intermediate platinum-sensitive ovarian cancer Gynecol Oncol 2019 155 201 6 31522837 Foley OW Rauh-Hain JA del Carmen MG Recurrent epithelial ovarian cancer: an update on treatment Oncology (Williston Park) 2013 27 288 94 Hilal Z Schultheis B Hartmann F What Characterizes Long-term Survivors of Recurrent Ovarian Cancer? Case Report and Review of the Literature Anticancer Res 2016 36 5365 71 27798900 Jansaka N Suprasert P Survival outcomes of recurrent epithelial ovarian cancer: experience from a Thailand northern tertiary care center Asian Pac J Cancer Prev 2014 15 10837 40 25605186 Kerio P Abid K Hassan A Shah A Manzoor A Treatment Outcomes of Epithelial Ovarian Cancer-A Longitudinal Study Asian Pac J Cancer Care 2022 7 253 9 Kim SI Ha HI Eoh KJ Trends in the Incidence and Survival Rates of Primary Ovarian Clear Cell Carcinoma Compared to Ovarian Serous Carcinoma in Korea Front Oncol 2022 12 874037 35463304 Lheureux S Braunstein M Oza AM Epithelial ovarian cancer: Evolution of management in the era of precision medicine CA Cancer J Clin 2019 69 280 304 31099893 Lheureux S Gourley C Vergote I Oza AM Epithelial ovarian cancer Lancet 2019 393 1240 53 30910306 Lui YL Zhou QC Lasonos A Pre-operative neoadjuvant chemotherapy cycles and survival in newly diagnosed ovarian cancer: what is the optimal number? A Memorial Sloan Kettering Cancer Center Team Ovary study Int J Gynecol Cancer 2020 30 1951 21 33082239 Manchana T Charakorn C Lertkhachonsuk A-a Treatment of Recurrent Ovarian Cancer: Survey of Practice among Thai Gynecologic Oncologists J Med Assoc Thai 2020 103 90 7 Matz M Coleman MP Sant M The histology of ovarian cancer: worldwide distribution and implications for international survival comparisons (CONCORD-2) Gynecol Oncol 2017 144 405 13 27931752 Oliver KE Brady WE Birrer M An evaluation of progression free survival and overall survival of ovarian cancer patients with clear cell carcinoma versus serous carcinoma treated with platinum therapy: An NRG Oncology/Gynecologic Oncology Group experience Gynecol Oncol 2017 147 243 9 28807367 Park HK Ruterbusch JJ Cote ML. Recent Trends in Ovarian Cancer Incidence and Relative Survival in the United States by Race/Ethnicity and Histologic Subtypes Cancer Epidemiol Biomarkers Prev 2017 26 1511 8 28751475 Pitakkarnkul S Tangjitgamol S Srijaipracharoen S Treatment outcomes of paclitaxel for refractory or recurrent epithelial ovarian cancer patients in Thailand Asian Pac J Cancer Prev 2013 14 2421 7 23725151 Pujade-Lauraine E Banerjee S Pignata S Management of Platinum-Resistant, Relapsed Epithelial Ovarian Cancer and New Drug Perspectives J Clin Oncol 2019 37 2437 8 31403868 Rustin GJ Follow-up with CA125 after primary therapy of advanced ovarian cancer has major implications for treatment outcome and trial performances and should not be routinely performed Ann Oncol 2011 22 viii45 8 22180399 Soyama H Takano M Miyamoto M Factors favouring longterm survival following recurrence in ovarian cancer Mol Clin Oncol 2017 7 42 6 28685073 Srisuttayasathien M Khemapech N Quality of life in ovarian cancer patients choosing to receive salvage chemotherapy or palliative treatment Asian Pac J Cancer Prev 2013 14 7669 74 24460351 Sung H Ferlay J Siegel RL Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries CA Cancer J Clin 2021 71 209 49 33538338

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116143 10.31557/APJCP.2023.24.4.1217 Research Article Clinical and Molecular Characteristics of Patients with Mixed Phenotype Acute Leukemia Kandeel Eman Zaghloul Hassan Naglaa M. El Ashry Mona Shafik * Department of Clinical Pathology, National Cancer Institute, Cairo University, Cairo, Egypt. * For Correspondence: mon282@cu.edu.eg 2023 24 4 12171223 8 11 2022 24 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Introduction: Mixed phenotype acute leukemia (MPAL) is a rare heterogeneous disease with a poor prognosis. This study analyzed the clinical, immunophenotypic, molecular, and cytogenetic characteristics of a group of patients with MPAL. Methods: This prospective study included 75 patients diagnosed with MPAL according to the World Health Organization (WHO)-2016 diagnostic criteria, using cytochemistry, conventional cytogenetics, and molecular studies. Screening of BCR::ABL1 fusion gene was performed by Fluorescent in-situ hybridization (FISH) and polymerase chain reaction (PCR). Results: Children represented 49.3% of MPAL patients. The main phenotype was B-lymphoid/myeloid (80%). Molecular alterations were detected in 17 patients (22.7%). The BCR::ABL1 fusion gene was detected in 10 patients (13.3%).. Myeloid protocols were used to treat 58 patients (77.3%), and lymphatic protocols in 17. By the end of the follow-up, 57 patients (76%) achieved complete remission (CR). There was no association between BCR::ABL1 and response to treatment. The cumulative overall survival (OS) at 12 months was 47.8%. The bone marrow transplantation (BMT) was associated with better OS (p = 0.027). The disease-free survival (DFS) was not affected by all tested prognostic factors. Conclusion: MPAL is a complex entity with heterogeneous features. BCR::ABL1 is a common abnormality. BMT is associated with better OS. Key Words Mixed Phenotype Acute Leukemia MPAL Cytogenetic Analysis BCR::ABL1 KMT2A ==== Body pmcIntroduction Mixed phenotype acute leukemia (MPAL) is acute leukemia of ambiguous lineage (ALAL) that does not fit within lineage-specific leukemia categories (Gerr et al., 2010). It is a rare disease that comprises 2% to 5% of all acute leukemias (Weinberg et al., 2014). MPAL is a high-risk class with a poor prognosis. Survival proportions of 15-35% were reported before the transplant era (Shi and Munker, 2015). In a large study multicenter study including 519 adult patients diagnosed with de novo MPAL who underwent allo-SCT and were reported to the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT), the overall survival was 56.3% at three years (Munker et al., 2017). MPAL can be biphenotypic presenting cross-lineage myeloid, B-lymphoid, T-lymphoid antigen expression, or bilineal with a distinct single-lineage blast population (Weinberg and Arber, 2010). The term MPAL was introduced in the World Health Organization (WHO) classification in 2008. This classification was updated in 2016 by adding subcategories, i.e., MPAL with t(9;22)(q34;q11.2) BCR::ABL1 fusion gene and MPAL with t(v;11q23.3) KMT2A-rearranged (KMT2A-r) (Swerdlow et al., 2017). BCR::ABL1 fusion gene (Philadelphia chromosome) resulting from the t(9;22)(q34;q11.2) translocation can be detected in chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), or acute myeloid leukemia (AML) (Soverini et al., 2019). It encodes a tyrosine kinase, p190, p210, or p230, promoting cell proliferation and suppressing apoptosis (Ayatollahi et al., 2018). Given that there is no consensus on a therapeutic strategy to assign patients with MPAL to lymphoid- or myeloid-directed treatment, a better understanding of the characteristics of MPAL is crucially needed. This study aimed to analyze the clinical, immunophenotypic, molecular, and cytogenetic characteristics of a group of MPAL patients as defined according to WHO-2016 criteria. Materials and Methods This prospective study included newly diagnosed patients with MPAL from January 2016 to December 2018. All patients were recruited from the outpatient clinics of the pediatric and medical oncology departments of the National Cancer Institute (NCI), Cairo University, Egypt. Patients with incomplete data were excluded from the analysis. All patients or patients´ guardians had provided informed consent to share in scientific research on admission. The study was performed following the 2013 declaration of Helsinki and was approved by the institutional review board of the NCI. The diagnosis of MPAL was based on WHO diagnostic criteria (Béné and Porwit, 2012), morphological examination of peripheral blood (PB) and bone marrow (BM) smears, cytochemistry, conventional cytogenetics, and molecular studies. BCR::ABL1 screening performed by FISH and PCR. Eighty patients were diagnosed as MPAL based on immunophenotyping, but five having blasts with a mixed phenotype and t(8;21) were excluded later because they would be reclassified as having AML, according to 2016 WHO classification (Swerdlow et al., 2017). Analytical Methods Flow Cytometry Peripheral blood and BM samples were tested for immunophenotyping. A complete panel of monoclonal antibodies was done, including myeloid markers MPO, CD13, CD33, CD117, CD64, CD15, CDIIB, CD36, CD14; B-cell markers CD19, CD10, CD22, CD20, cytoplasmic m, cytoplasmic CD79a, kappa, lambda; T-cell markers cytoplasmic CD3, CCD7, CD1, CD2, CD4, CD8, CD56; other markers CD45, CD11C, CD34, NG2, MHC CLASS II and CD135. Analysis was done on six colors Navios cytometer (Beckman Coulter Diagnostics, USA). Minimal residual disease (MRD) was assessed on days 14, 28, and 42 post-induction. Cytogenetic analysis Following the standard techniques, pre-treatment diagnostic BM samples were subjected to conventional karyotyping using G-banded metaphase cells from unstimulated 24-hour cultures. In most cases, at least 20 metaphases were analyzed using an IKAROS imaging system (Metasystems, Altlußheim, Germany). The karyotypes were interpreted using the International System for Human Cytogenetic Nomenclature (ISCN 2016). Fluorescence in situ hybridization (FISH) was performed following the manufacturer’s instructions. For detecting BCR::ABL1 fusion gene, LSI BCR-ABL1 dual color dual fusion probe (Vysis, Abbott, Maidenhead, UK) was used. A minimum of 10 metaphases and 200 interphase nuclei were analyzed using a fluorescence microscope (AxioImager.Z1 mot, Carl Zeiss Ltd., Hertfordshire, UK) equipped with appropriate filter sets. Image capture and processing were performed using an ISIS imaging system (Metasystems, Altlußheim, Germany). Molecular detection of fusion gene transcripts According to the manufacturer’s instructions, total RNA was extracted from bone marrow or peripheral blood samples using Qiagen RNA Blood Mini Kit (cat#5723, Germany). RNA was reverse transcribed using a high-capacity complementary DNA reverse transcription kit (cat#4368814, USA) Applied biosystems. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for all samples for the detection of fusion transcripts t(9;22)(q34;q11) p190 and p210, t(4;11)(q21;q23), t(12;21)(p13;q22), t(1;19) (q23;p13) and t(8;21)(q22;q22), according to the BIOMED-1 guidelines. Treatment Lymphatic protocol All pediatric patients received total XV protocol (modified from St. Jude total XV protocol), while adults received German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) protocol. Risk classification to low, standard, and high risk was based on initial presenting features. Response to treatment was based on the minimal residual disease (MRD) at specific time points (day 15 and day 42 from the start of induction chemotherapy). The treatment protocol consists of three phases, induction of remission, consolidation, and maintenance. The induction phase (42 days) involved four-drug regimens (prednisone, vincristine, doxorubicin, and L-asparaginase), the consolidation therapy (8 weeks) consisted of 4 cycles of high-dose methotrexate, and the maintenance phase (120 weeks for females and 146 weeks for males). Patients with t(9;22) BCR-ABL1 started tyrosine kinase inhibitor (imatinib) at a dose of 260 mg/m2 per day once molecular results were available and continued till the end of treatment. If the patient had an MRD < 0.1% by flow cytometry at the end of induction and more than 3 log reduction (major molecular response), or MRD PCR at week 7, the patient was not considered eligible for hematopoietic stem cell transplantation. Allogeneic hematopoietic stem cell transplantation is indicated for patients with high-risk leukemia (poor response to induction treatment; MRD > 1%). Myeloid Protocol All patients received standard induction chemotherapy with 3+7 protocol (idarubicin as a short infusion for three days with cytarabine 100 mg/m2 continuous infusion for seven days). Patients, who achieved CR, according to their risk stratification, were offered consolidation with high dose cytarabine and HLA matching followed by allogeneic bone marrow transplantation. Refractory cases received re-induction with a high-dose cytarabine-based regimen. Response to treatment was based on MRD at specific time points (day 14 and day 28 from induction chemotherapy). The outcome of the induction treatment was assessed at day 28, where patients were categorized into complete remission (CR) or refractory group. Statistical analysis Statistical analysis was done using IBM© SPSS© Statistics version 25 (IBM© Corp., Armonk, NY, USA). Numerical data were expressed as mean and standard deviation or median and range as appropriate. Qualitative data were expressed as frequency and percentage. Chi-square test (Fisher’s exact test) was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was made using independent sample t-test or Mann-Whitney test. Survival analysis was done using the Kaplan-Meier method, and comparison between two survival curves was made using the log-rank test. A p-value < 0.05 was considered significant. Results Out of 75 patients, 37 (49.3%) were 18 years old or younger, i.e., children. Manifestations of BM failure were noticed in 36 patients (48%). Fifty-six patients (74.7%) had hypercellular BM at presentation. Sixty patients (80%) had B-lymphoid/myeloid type. Baseline demographic, clinical, and laboratory characteristics are shown in Table 1. All patients with T/myeloid phenotype were positive for cytoplasmic CD3. Molecular alterations were detected in 17 patients (22.67%). Using the FISH technique, BCR::ABL1 was seen in 10 patients (13.3%). However, molecular analysis missed one of these cases. KMT2A-r were detected in 3 patients (4%); 2 patients had t (4;11)(q21.3;q23.3) and one had an unidentified partner. Cytogenetic data were available only for 37 patients, of which 33 had abnormal karyotypes (89%). Chromosome 21 abnormalities (ch.21.abn), including translocations, gains, or loss, were the most frequently encountered (n=15). The BCR::ABL1 fusion gene was detected more frequently in adults (p=0.046). Otherwise, there was no significant difference between BCR::ABL1 positive and negative cases in sex or different clinical characteristics (Table 2). Chromosome 21 abnormalities had no significant relation with age, sex, BM cellularity, or immunophenotypes (Table S1) but were associated with hyperdiploidy (p=0.005). Myeloid protocols were used to treat 58 patients (77.3%), while the remaining 17 received lymphatic protocols. Ten patients received imatinib, and five were treated with BM transplantation (BMT). Table 3 shows the response to treatment on days 14, 28, and 42. By the end of the follow-up, 57 patients (76.0%) achieved CR. Six patients died earlier than 28 days. Although BCR::ABL1 positivity was not associated with CR, it was noted that all positive BCR::ABL1 cases had MRD of 0.1% or more on day 42. Still, the relation was not statistically significant (p = 0.094) (Table 2). In addition, there was no association between ch.21.abn and response to treatment (Table S1). The median follow-up period was 8.2 months (range: 2-60 months). Forty-six patients died during follow-up. The median overall survival (OS) was 10 months (95%CI: 4-16 months). The cumulative OS proportion at 12 months was 47.8%. The OS was not affected by BCR::ABL1 positivity or ch.21 abn (Table 4), while it was better in patients treated with BMT (p = 0.027). Although the OS was worse in KMT2A-r positive cases (p = 0.001), the conclusion may not be reliable with the low number of the cases (Figure 1). The median disease-free survival (DFS) of patients who achieved CR (n=57) was 9.4 months (95%CI: 4.4-14.3). The cumulative DFS proportion at 12 months was 42.7%. DFS was not affected by all tested prognostic factors (Table S2). Table 1 Baseline Demographic, Clinical, and Laboratory Characteristics of the Studied Group Parameter Value Age (years) 19 (1-63) Children 37 (49.3%) Adults 38 (50.7%) Sex Male 56 (74.7%) Female 19 (25.3%) Clinical Manifestations Hepatomegaly 31 (41.3%) Splenomegaly 30 (40.0%) Lymphadenopathy 33 (44.0%) Central nervous system infiltration 9 (12.0%) Fever 39 (52.0%) Laboratory Characteristics Total leukocytic count (x103/mL) 28.0 (1.0-883.2) Hemoglobin concentration (gm/dL) 8.0±2.4 Platelet count (x103/mL) 48.0 (5.0-393.0) Peripheral blood blast cells (%) 25.0 (0.0-98.0) Bone marrow blast cells (%) 80.0 (22.0-99.0) Bone marrow cellularity Normocellular 19 (25.3%) Hypercellular 56 (74.7%) Immunophenotyping B-lymphoid/myeloid 60 (80.0%) T-lymphoid/myeloid 15 (20.0%) Myeloid Markers MPO 74 (98.7%) CD33 70 (93.3%) CD13 69 (92.0%) B-cell Markers CD19 57 (76.0%) CD10 58 (77.3%) CD22 49 (65.3%) T-cell Markers CD7 21 (28.0%) CD2 16 (21.3%) CD3 15 (20.0%) Molecular alterations BCR::ABL1 fusion 10 (13.3%) KMT2A rearrangements 3 (4.0%) t (12;21) 2 (2.7%) JAK2 V617F mutation 1 (1.3%) FLT3-ITD mutation 1 (1.3%) Modal chromosome number Diploid 22/37 (59.5%) Hypodiploid 3 /37 (8.1%) Hyperdiploid 12/37 (32.4%) Structural Chromosomal abnormalities Yes 22/37 (59.5%) No 15/37 (40.5%) Chromosome 21 abnormalities Yes 15/37 (40.5%) No 22/37 (59.5%) Data are expressed as median (range), mean±SD, or number (%) Table 2 Response to Treatment on Days 14, 28, and 42 Parameter Value Day 14 Response Complete Remission 43 (57.3%) Partial Remission 5 (6.7%) Refractory 27 (36.0%) Bone marrow cellularity Hypocellular 52 (69.3%) Normocellular 18 (24.0%) Hypercellular 5 (6.7%) Bone marrow blasts < 5% 41 (54.7%) ≥ 5% 34 (45.3%) Minimal Residual Disease < 0.1% 5 (6.7%) ≥ 0.1% 71 (93.3%) Day 28 (n=69) Bone marrow blasts < 5% 18 (26.1%) ≥ 5% 51 (73.9%) Minimal Residual Disease < 0.1% 11 (15.9%) ≥ 0.1% 58 (84.1%) Day 42 (n=60) Bone marrow blasts < 5% 7 (11.7%) ≥ 5% 53 (88.3%) Minimal Residual Disease < 0.1% 14 (23.3%) ≥ 0.1% 46 (76.7%) Complete Remission at the end of follow up 57 (76.0%) Data are expressed as number (%) Figure 1 Overall Survival of Patients with MPAL in Relation to (A) treatment with bone marrow transplantation and (B) the presence of KMT2A rearrangement Table 3 Demographic, Clinical Characteristics, and Response to Treatment of Patients with and without BCR::ABL1 Fusion Gene Parameter BCR-ABL Positive BCR-ABL Negative p-value Age Children 2 (5.4%) 35 (94.6%) 0.046 Adults 8 (21.1%) 30 (78.9%) Sex Male 8 (14.3%) 48 (85.7%) 0.677 Female 2 (10.5%) 17 (89.5%) Immunophenotyping B-lymphoid/myeloid 9 (15.0%) 51 (85.0%) 0.396 T-lymphoid/myeloid 1 (6.7%) 14 (93.3%) BM cellularity Normocellular 2 (10.5%) 17 (89.5%) 0.677 Hypercellular 8 (14.3%) 48 (85.7%) Modal chromosome number  Hypodiploid 0 (0.0%) 3 (100.0%) 0.255 Diploid 8 (36.4%) 14 (63.6%) Hyperdiploid 2 (16.7%) 10 (83.3%) Complete Remission Yes 8 (80.0%) 49 (75.4%) 0.75 No 2 (20.0%) 16 (24.6%) MRD on day 42 < 0.1% 0 (0.0%) 14 (26.9%) 0.094 ≥ 0.1% 8 (100.0%) 38 (73.1%) Data are presented as number (%) Table 4 Overall Survival of the Studied Group in Relation to Prognostic Factors n n events Cumulative survival proportion (%) Median survival (months) p-value All cases 75 46 47.8 10 (4-16) Age group Children 37 23 50.2 12.6 (1.2-24.0) 0.951 Adults 38 23 45.6 10.1 (3.5-16.7) Sex Male 56 33 54.2 13.8 (6.5-21.0) 0.249 Female 19 13 28.5 6.4 (3.0-9.9) Immunophenotype B/myeloid 60 35 51.5 12.6 (6.0-19.2) 0.292 T/myeloid 15 11 33.3 5.5 (1.6-9.3) BCR::ABL1 Positive 10 4 58.3 * 0.412 Negative 65 42 46.4 8.0 (2.5-15.7) KMT2A-r Yes 3 3 0 2.1 (0.2-4.0) No 72 43 49.8 10.8 (5.3-16.3) 0.001 Cytogenetics (n=37) Normal 4 3 50 6.4 (0.0-30.8) 0.684 Abnormal 33 17 50.1 15.4 (0.0-31.6) Ch.21 abn. (n=37) Yes 15 6 64.6 * No 22 14 40 4.1 (0.0-10.5) 0.131 CTH Myeloid 58 38 46.3 10.1 (3.7-16.5) 0.131 Lymphatic 17 8 52.9 * Imatinib Yes 11 5 51.1 * No 64 41 47.1 10.1 (2.5-17.7) 0.36 BMT Yes 5 0 100 * No 70 46 44.2 * 0.027 *, Median survival cannot be calculated; BMT, Bone marrow transplantation Discussion This study shows the characteristics of 75 patients with MPAL. They were 37 children and 38 adults. The main phenotype was B/myeloid type (80%). However, according to the current WHO 2016 classification (Swerdlow et al., 2017), the present series included 13 cases of the two genomic categories. Three cases were KMT2A-r positive, and 10 were BCR::ABL1 fusion-positive. Therefore, they can be classified into four classes: BCR::ABL1 fusion-positive (n=10), KMT2A-r positive (n=3), B/myeloid NOS (n=48), and T/myeloid NOS (n=14). B/myeloid immunophenotype was also more common in previous studies in adults and children. Matutes et al., (2011) found 59%, and Yan et al., (2012) found 70% B/myeloid type in adults. Half of the children in other series had B/myeloid type (Lee et al., 2019; Chang et al., 2021). It is assumed that MPAL evolves from multipotent stem cells, capable of differentiating into myeloid and lymphoid lineages during the development of acute leukemia (Lee et al., 2019). It has been shown that CD34 is primarily considered a marker of hematopoietic stem cells and hematopoietic progenitor cells (Sidney et al., 2014). We found CD34 in 82.7% of patients, and this finding supports the view that MPAL cells originate at the early stages of hematopoietic differentiation. Among 37 patients with available karyotype, only four had a normal karyotype, and four had a complex one defined as ≥3 structural abnormalities. Therefore, about 11% of our patients had complex karyotypes. These figures are lower than those reported by Matutes et al., (2011) and Yan et al., (2012), who found 32% and 24% complex karyotypes in their series, respectively. This discrepancy could be explained by the different used techniques to detect the fusion genes and other cytogenetic aberrations and due to ethnic variations; Matutes et al., (2011) performed FISH analysis for the screening of BCR::ABL1, ETV6::RUNX1, and KMT2A rearrangements while in our study only BCR::ABL1 fusion gene was screened by FISH. The higher incidence of complex karyotypes reported by Yan et al., (2012) is due to the fact of using multiplex reverse transcription-polymerase chain reaction (RT-PCR) to detect 29 acute leukemia-related fusion genes in adult Chinese patients which may explain the higher incidence of reporting abnormal cytogenetic results and subsequently complex karyotype. In addition, the cohort assessed in the mentioned study included adult patients only while our cohort included pediatric and adult patients with MPAL. On the other hand, Weinberg et al., (2014) found 44% of their series with normal karyotypes. MPAL has no specific chromosomal abnormalities. Owaidah et al. (2006) reported clonal abnormalities in 68% of MPAL patients; KMT2A-r was the most common, followed by BCR::ABL1 (Alexander et al., 2018). In agreement with Chang et al., (2021), clonal cytogenetic abnormalities were found in 89% of MPAL patients in the present series. The BCR::ABL1 fusion gene was detected in 8 adults compared to two children (p=0.046). Besides, all KMT2A-r positive cases were adults. Previous studies have shown that BCR::ABL1 translocation is uncommon in children, while 15% of MPAL cases in children harbor KMT2A-r (Alexander et al., 2018). Lee et al., (2019) detected one BCR::ABL1 positive patient, and two had KMT2A-r. In another study, BCR::ABL1 and KMT2A-r were mainly found in B/myeloid MPAL (Cao et al., 2019). However, the most frequent genetic abnormality in this series was ch.21.abn found in 15 patients (20%), mainly in B/myeloid cases (n=11). Previous studies reported different frequencies of ch.21 abn ranging from 8% to 26%. In contrast, Chang et al., (2021) reported that 50% of pediatric MPAL patients were associated with ch. 21 abn. However, most of these studies had a small sample size (Yan et al., 2012; Takahashi et al., 2018; Quesada et al., 2018; Eckstein et al., 2016). Chromosome 21 abnormalities were detected in patients with B/myeloid phenotype (Cao et al., 2019). In a sample of 23 adult and pediatric MPAL patients, ch.21 abn were found in 2 patients: one child and one adult (Eckstein et al., 2016). The largest series of Alexander et al., (2018) found 11% ch.21 abn. Thus, the present study detected the highest frequency of ch.21.abn with a relatively large sample size. Despite its rarity, MPAL has a poor prognosis compared to other subtypes of acute leukemia. Therefore, proper diagnosis of MPAL is essential for a successful outcome in these patients (Porwit and Béné, 2019). Precise diagnosis necessitates a careful assessment of the clinical, immunophenotypic, and genetic data (Khan et al., 2018). We defined the disease according to the WHO criteria in the current series. The WHO criteria used fewer parameters than the European Group for the Immunological Classification of Leukemia (EGIL) scoring system (Weinberg et al., 2014); however, it used measures from cytometry and cytochemistry (Lee et al., 2019). The WHO 2016 classification defined three categories: associated with BCR::ABL1 fusion gene, associated with KMT2A rearrangements, and non-otherwise specified (Béné and Porwit, 2012). At present, there is no consensus about the treatment of MPAL. It is controversial to use single chemotherapy or combined therapy for lymphoid and myeloid leukemia (Zheng et al., 2021). A systematic review investigated the efficacy of different regimens on disease response and survival. The study included data from 97 reports, including 1351 patients. The meta-analysis showed that AML induction was less likely to attain a CR than ALL regimens. Also, OS was better for patients beginning with ALL versus AML therapy (Maruffi et al., 2018). Cellular and molecular genetic abnormalities can help for guiding the proper treatment strategy of MPAL patients (Matutes et al., 2011; Yan et al., 2012). Nevertheless, lacking robust prospective trials is the main obstacle to reaching a standard approach for treating MPAL as a heterogeneous disease. In summary, this study demonstrates that MPAL is a complex entity with heterogeneous clinical, immunophenotypic and genetic characteristics. The main immunophenotype was B/myeloid type, and BCR::ABL1 and ch.21 abn are the most frequent associated abnormalities. The treatment outcome is generally poor, and patients subjected to BMT had better overall survival. Accurate diagnostic criteria are essential for tailoring specific treatment strategies. Other trials are needed to standardize the optimal therapeutic modality of MPAL cases. Author Contribution Statement All authors have contributed to the manuscript in significant ways have reviewed and agreed upon the manuscript content. Eman Z. Kandeel: Conceptualization, Methodology and Validation; Naglaa M. Hassan: Investigation, data curation and reviewing; Mona S. El Ashry: Investigation, Data curation, Writing- Original draft preparation. Acknowledgements The authors would like to thank all the patients who were included in this study. Declarations Ethics approval and consent to participate: Every patient gave a written informed consent. The study was conducted in accordance with the Helsinki declaration of 2011 and was approved by the National Cancer Institute’s internal review board. Consent for publication Not applicable. Availability of data and material The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ==== Refs References Alexander TB Gu Z Iacobucci I The genetic basis and cell of origin of mixed phenotype acute leukaemia Nature 2018 562 373 9 30209392 Ayatollahi H Keramati MR shirdel A BCR-ABL fusion genes and laboratory findings in patients with chronic myeloid leukemia in northeast Iran Casp J Intern Med 2018 9 65 70 Béné MC Porwit A Acute leukemias of ambiguous lineage Semin Diagn Pathol 2012 29 12 8 22372202 Cao P Wang M Wang F Deciphering Common Gene Fusions and Mutations in Acute Leukemia of Ambiguous Lineage Blood 2019 134 3793 Chang TY Chen SH Jaing TH Cytogenetic aberration in mixed-phenotype acute leukemia in children: A single-center retrospective review Pediatr Neonatol 2021 62 21 5 32859541 Eckstein OS Wang L Punia JN Mixed Phenotype Acute Leukemia (MPAL) Exhibits Frequent Mutations in DNMT3A and Activated Signaling Genes Exp Hematol 2016 44 740 4 27208809 Gerr H Zimmermann M Schrappe M Acute leukaemias of ambiguous lineage in children: characterization, prognosis and therapy recommendations Br J Haematol 2010 149 84 92 20085575 Khan M Siddiqi R Naqvi K An update on classification, genetics, and clinical approach to mixed phenotype acute leukemia (MPAL) Ann Hematol 2018 97 945 53 29546454 Lee HG Baek HJ Kim HS Biphenotypic acute leukemia or acute leukemia of ambiguous lineage in childhood: clinical characteristics and outcome Blood Res 2019 54 63 73 30956966 Maruffi M Sposto R Oberley MJ Kysh L Orgel E Therapy for children and adults with mixed phenotype acute leukemia: a systematic review and meta-analysis Leukemia 2018 32 1515 28 29550836 Matutes E Pickl WF Van’t Veer M Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification Blood 2011 117 3163 71 21228332 Munker R Labopin M Esteve J Mixed phenotype acute leukemia: outcomes with allogeneic stem cell transplantation A retrospective study from the Acute Leukemia Working Party of the EBMT Haematologica 2017 102 2134 40 28971902 Owaidah TM Al Beihany A Iqbal MA Elkum N Roberts GT Cytogenetics, molecular and ultrastructural characteristics of biphenotypic acute leukemia identified by the EGIL scoring system Leukemia 2006 20 620 6 16437134 Porwit A Béné MC Multiparameter flow cytometry applications in the diagnosis of mixed phenotype acute leukemia Cytometry B Clin Cytom 2019 96 183 94 31033213 Quesada AE Hu Z Routbort MJ Mixed phenotype acute leukemia contains heterogeneous genetic mutations by next-generation sequencing Oncotarget 2018 9 8441 9 29492206 Shi R Munker R Survival of patients with mixed phenotype acute leukemias: A large population-based study Leuk Res 2015 39 606 16 25858895 Sidney LE Branch MJ Dunphy SE Dua HS Hopkinson A Concise Review: Evidence for CD34 as a Common Marker for Diverse Progenitors STEM CELLS 2014 32 1380 9 24497003 Soverini S Bassan R Lion T Treatment and monitoring of Philadelphia chromosome-positive leukemia patients: recent advances and remaining challenges J Hematol Oncol J Hematol Oncol 2019 12 39 31014376 Takahashi K Wang F Morita K Integrative genomic analysis of adult mixed phenotype acute leukemia delineates lineage associated molecular subtypes Nat Commun 2018 9 2670 29991687 Weinberg OK Arber DA Mixed-phenotype acute leukemia: historical overview and a new definition Leukemia 2010 24 1844 51 20844566 Weinberg OK Seetharam M Ren L Alizadeh A Arber DA Mixed Phenotype Acute Leukemia: A Study of 61 Cases Using World Health Organization and European Group for the Immunological Classification of Leukaemias Criteria Am J Clin Pathol 2014 142 803 8 25389334 Yan L Ping N Zhu M Clinical, immunophenotypic, cytogenetic, and molecular genetic features in 117 adult patients with mixed-phenotype acute leukemia defined by WHO-2008 classification Haematologica 2012 97 1708 12 22581002 Zheng X Shen H Zhu M Mixed phenotype acute leukemia with PML-RARα positive: a case report and literature review Mol Cytogenet 2021 14 10 33573663

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116141 10.31557/APJCP.2023.24.4.1199 Research Article Health-Related Quality of Life (HRQoL) Using EQ-5D-5L: Value Set Derived for Indian Breast Cancer Cohort Wadasadawala Tabassum 1* Mohanty Sanjay K 2 Sen Soumendu 2 Khan Pijush Kanti 3 Pimple Sharmila 4 Mane Jaykumar V 4 Sarin Rajiv 4 Gupta Sudeep 4 Parmar Vani 4 1 Advanced Center for Treatment Research and Education in Cancer, Tata Memorial Center, Mumbai, Homi Bhabha National Institute, India. 2 International Institute for Population Sciences, Mumbai, India. 3 International Institute of Health Management Research, New Delhi, India. 4 Tata Memorial Center, Homi Bhabha National Institute, Mumbai, India. * For Correspondence: twadasadawala@actrec.gov.in 2023 24 4 11991207 23 10 2022 6 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: The purpose of this study was to report quality of life of newly diagnosed breast cancer patients from India in a large cohort using the EQ-5D-5L instrument. Methods: The study used longitudinal data of 500 breast cancer and 200 non-cancer subjects registered at our centre, during June 2019 and March 2022. The EQ-5D-5L and EQ-VAS instruments were used to measure and compare utility scores among cancer and non-cancer subjects. Descriptive statistics were analyzed and Tobit regression model were used to confirm the predictors of the utility score. Results: The cancer subjects had a mean EQ-ED-5L utility score of 0.8703 (SD=0.121), 0.8745 (SD=0.094) and 0.8902 (SD=0.107) at the time of baseline, completion and follow up surveys respectively. EQ-5D-5L values had significantly worsened after diagnosis of cancer as compared to the non-cancer cohort (0.87 vs. 0.93, p value 0.000). EQ-5D-5L utility scores as per stage for the cancer cohort were 0.88, 0.86 and 0.83 respectively for stage I-II, III and IV. Similarly, the EQ-VAS scores for stage I-II, III and IV were 74.9, 72.6 and 73.2 respectively. Multivariate analysis confirmed strong association of age, religion and income with the utility-values. Conclusion: This is the first longitudinal study reporting the utility scores derived from a large cohort of breast cancer patients demonstrating lower utility scores compared to non-cancer cohort. The utility scores also improve post treatment completion for cancer patients and decrease with higher stage at diagnosis. This information will be useful for future health economic research in India pertaining to breast cancer. Key Words EQ-5D-5L quality of life health state utility breast cancer India ==== Body pmcIntroduction Breast cancer incidence in India is rising at an alarming rate. The age-standardised incidence rate of breast cancer has increased from 0.5 million to 1 million over the past two and half decades and thereby making breast cancer the most common female malignancy both in urban as well as rural India (Dhillon et al., 2018). Breast cancer is not only the commonest cause of cancer death in women but also most common contributor to disability-adjusted life years (DALYs) as per the 2016 Lancet report (Dhillon et al., 2018). Late diagnosis and morbidity from cancer treatment affect the length and quality of life of cancer survivors. Though stage by stage cancer survival is increasing with the improvement in cancer care, the adverse impact of cancer and its treatment cannot be estimated without patient-reported outcomes (PRO) and health-related quality of life (HRQoL) data. The inequitable access to cancer care has promoted comparative economic evaluations of health interventions that aid the government in decision-making for the inclusion of various cost-effective interventions in the public health insurance schemes. Moreover, health economic research has also gained popularity in any interventional clinical trials as it allows investigators to evaluate the direct cost-effectiveness of different treatment modalities. Among different methods of economic evaluation, cost-utility analysis is preferred for comparative analysis. In this analysis, outcome is measured in the form of quality-adjusted life years (QALY) which is a utility-based index. QALY has two essential components, viz, longevity or life span and utility scores for the health condition under consideration. QALY is the preferred measure for economic evaluation and the EQ-5D-5L is the most recommended instrument to derive utility scores (Huang et al., 2018; Jyani et al., 2020b). The EQ-5D was developed by the EuroQol Group in the 1980s to provide a concise, generic instrument that could be used to measure, compare and value health status across disease areas (EuroQol, 2022). However, disease-specific utility scores are more valued over the generic value set (derived from general population) as these are based on the target population (in this case breast cancer patients). Hence it is advisable to obtain utility-based value set from the target population in order to calculate QALYs. Though utility value sets for breast cancer have been published earlier from other countries, it is unlikely that these values will be useful for Indian patients because of the differences in the sociodemographic profile, per capita income, insurance coverage and country requirements (Cheung et al., 2014; Yousefi, 2016). The aim of this study was to report Health State Utility (HSU) in a large cohort of newly diagnosed breast cancer patients from India using the EQ-5D-5L. Previous studies report QoL of Indian women with breast cancer using FACT B instrument as well as of cancer caregivers using Caregiver Quality of Life Cancer (CQoLC) index (Amarsheda & Bhise 2021, Vashistha et al., 2019). Recently, the Indian value sets of EQ-5D-5L instrument for the general population have been published and made available in the public domain (Jyani et al., 2020a). However, the use of EQ-5D-5L instrument has been reported in very few oncological studies from India (Jyani et al., 2020b). The current study reports the EQ-5D-5L data collected for newly diagnosed breast cancer women who were registered at Tata Memorial Center (TMC) and seeking cancer directed treatment. Materials and Methods Study design and accrual This study was carried out as a collaborative research project by Tata Memorial Center (TMC) and International Institute for Population Sciences (IIPS), Mumbai. The project was approved by the institutional ethics committee at TMC as well as the Academic Research Council at IIPS. The study was also registered on the Clinical Trial Registry of India (CTRI/2019/07/020142). This study is a comprehensive study evaluating the health expenditure on breast cancer treatment as well as estimating the HSU values in a large prospective cohort of breast cancer patients evaluated at TMC and seeking cancer directed treatment. The project accrued study subjects from two different cohorts: cancer cohort and non-cancer cohort. The target accrual of 500 breast cancer patients based on convenient sampling was completed over a period from September 2019 to December 2021. Similarly, target accrual of 200 non-cancer subjects from the individuals visiting the prevention oncology department at TMC was also accomplished during the same period. All adult histologically confirmed breast cancer patients of stage I to IV who were willing to share relevant socio-demographic information, details of expenditure on cancer treatment and were able to fill or respond to the quality of life (QOL) instruments were screened for the study. Written informed consent was obtained from all the study participants. The data collection was locked on 31st March 2022. At the time of start of the project, the Indian value set for the general population was not available. Hence, cohort of non-cancer subjects was also enrolled in order to get comparative estimates of health status of cancer vs. non-cancer female adult in India. Measures The EQ-5D-5L instrument: Aiming at valuing health states gave the potential for the instrument to estimate quality-adjusted life-years (QALYs) for use in cost effectiveness analysis. With the adoption of health technology assessment (HTA) by various countries for decision making on health interventions, QALY has been increasingly used as an important tool in the health economic evaluations both for generating large-scale data covering the entire population as well as in clinical trials. The EQ-5D-5L first part consists of five descriptors related to mobility, self-care, usual activities, pain/discomfort, and anxiety/ depression. Patients are asked to rate the ability to perform the function from one of the five-responses: no problems, slight problems, moderate problems, severe problems, and extreme problems. The responses are taken in Likert scale, such as, 1 for no problem to 5 for extreme problem. This gives 1-digit number for each of the five dimensions and the combined 5-digit number describes the current health status of the respondent. For instance, if a respondent provides no problem for each of the five dimensions, the health state will be recorded as 11,111 and if respondent has extreme problem in all five dimensions, the health state would be 55,555. A total of 3,125 health states can be derived by the potential combinations of responses. Each of these combinations has a unique utility score value that is already validated and published for India (Jyani et al., 2020). For the Indian value set, health state 11,111 gets utility score value of 1 whereas for 55,555 it is -0.923. The second part of the EQ-5D-5L comprises of an EQ Visual Analogue scale (EQ VAS) on a 20-cm vertical scale with endpoints labelled ‘the best health you can imagine’ and ‘the worst health you can imagine’ at the two ends of the scale. For this study, already available validated instruments of EQ-5D-5L in English, Hindi and Marathi languages were used. The QOL instrument was served to cancer patients at three time points: baseline within 4 weeks of cancer diagnosis, within 4 weeks from treatment completion) and during the controlled state i.e during the first follow up visit (6 months from treatment completion). QOL data from the non-cancer cohort was collected only once at the time of registration. This paper describes the baseline utility values for the cancer cohort, impact of transition from one state to another on utility values and compares with the non-cancer cohort using the Indian general utility value set. In our future work, disease specific quality of life assessed using EORTC-QLQ-C30 and BR 23 and its mapping with the EQ-5D-5L will be reported. Statistical analysis The EQ-5D-5L utility values depending upon the rating of the five health dimensions done by the patients were derived from the recently published Indian value set (Jyani et al., 2020a). The EQ-VAS scores were analysed directly from the scale served to the patients. We assess the correlation of EQ-5D-5L and EQ-VAS scores using Pearson’s correlation coefficient. We used bivariate analyses and Tobit regression model to understand the predictors of quality of life. Two-sided p-values were used and value of < 0.05 was considered statistically significant. STATA version 17 was used for statistical analysis. Results Demographic characteristics of the study sample In the current paper, 500 cancer patients were compared with the non-cancer cohort of 200 subjects. The non-cancer females who visited the preventive oncology department for their preventive health check-up formed the non-cancer cohort. The baseline demographic details were recorded at the time of accrual for both the cancer and heathy (non-cancer) cohort. The comparison of the two cohorts implies that the non-cancer cohort and the cancer cohort are naturally expected to have different epidemiological and socio-economic background. The non-cancer cohort predominantly belonged to the Mumbai and sub-urban region thereby resulting in the difference between the demographic variables as shown in Table 1. The median time of treatment completion of the cancer cohort was 9 months. Comparison of EQ-5D-5L between cancer and non-cancer cohort In the analysis of EQ-5D-5L data, the five domains mobility, self-care, usual activity, pain/discomfort and anxiety/depression were studied. Proportion of subjects in the two groups having problems on a 5-point scale (no, slight, moderate, severe, extreme) were considered. This is shown in Table 2 below. All the three positive health states (mobility, self-care and usual activity) were reported to a higher extent in the non-cancer cohort while the negative health states (pain/discomfort and anxiety/depression) were more frequent in the cancer cohort. Figure 1 shows the comparative data between the two cohorts. Figure 2 shows the distribution of the baseline EQ-5D-5L and EQ-VAS scores for cancer cohort and non-cancer cohort respectively. EQ-5D-5L utility scores as per stage for the cancer cohort were 0.88, 0.86 and 0.83 respectively for stage I-II, III and IV. Similarly, the EQ-VAS scores for stage I-II, III and IV were 74.9, 72.6 and 73.2 respectively. The temporal trend with respect to cancer stage is shown in Figure 3. While the quality of life improves for stage I-II and III, it reduces for stage IV cancer. compared to post treatment assessment. The stage IV cohort primarily comprised of patients who had limited (oligo) metastatic disease and were treated with curative intent. The mean utility value for the baseline, completion and follow up states of cancer cohort and non-cancer cohort have been reported in Table 3. Thus, the difference in the two cohorts was statistically significant by both the methods of assessment of utility scores. The utility values showed a significant positive trend in the controlled state compared to baseline while the completion values were not statistically significant from baseline as measured by EQ-5D-5L but significantly worse as assessed by VAS. However, there was positive correlations between mean utility score and VAS for each of the time period (for baseline it was 0.5, conclusion it was 0.4 and for follow up it was 0.6). In order to study the effect of various socio-economic variables, we carried out bivariate analysis independently for both the cohorts (baseline data was used in the cancer cohort). Age, religion, social group, educational and marital status, wealth quintile, family type, household size, health insurance, place of residence, annual income and cancer stage were considered for the analyse. The results are shown in Table 4 below. It was observed that younger age, Hindu religion and higher income had positive impact on the utility values in both the cohorts but the difference was statistically significant only for the religion and income while the association with age showed a decreasing trend for statistical significance in both the cohorts. In addition, cancer stage also impacted utility scores showing a trend towards statistical significance. Multivariate analysis also confirmed the strong association of age, religion and income with the utility values (Table 5). Table 1 Demographic Profile of the Cancer and Non-Cancer Cohort Socio-demographic characteristics Cancer (in %) Non-cancer (in %) p-value Age (in years) Mean age in years 46.9 41.5 0.000 Below 40 30.4 46.8 0.000 41 to 59 57.4 46.8 60+ 12.2 6.5 Education Mean years of schooling 7 10 0.000 Illiterate 26.6 13.9 0.000 Primary 21.2 13.9 Secondary 25.4 24.4 Higher secondary and above 26.8 47.8 Religion Hindu 78.8 89.6 0.001 Others 21.2 10.5 Social group Unreserved 51.8 59.2 0.114 Scheduled Caste/Scheduled Tribe 33.8 25.9 Other Backward Class 14.4 14.9 Wealth quintile Poorest 20 20.4 1.000 Poorer 20 19.9 Middle 20 19.9 Richer 20 20.4 Richest 20 19.4 Household size 1 to 4 49.6 63.7 0.002 5 to 6 35.8 28.4 7 and more 14.6 8 Health insurance Yes 9 28.9 0.000 No 91 71.1 Place of residence Urban 46.4 76.6 0.000 Rural 53.6 23.4 Annual household income Less than 50,000 22.4 8 0.000 50,000- 1lac 23.4 16.1 1 lac- 2 lac 22.2 25.6 More than 2 lacs 32 50.3 Clinical stages I & II 33.6 NA NA III 60.8 NA IV 5.6 NA Note: Chi-square test for categorical variables and t-test for continuous variables were performed. Figure 1 Comparative EQ-5D-5L Data from Cancer and Non-Cancer Cohort on a) mobility b) self-care c) usual activity d) pain or discomfort e) anxiety or depression at different treatment stages Table 2 Comparative EQ-5D-5L Data from Cancer and Non-Cancer Cohort Mobility (%) Self - Care (%) Usual Activity (%) Pain/Discomfort (%) Anxiety/Depression (%) Problems Cancer Non-Cancer Cancer Non-Cancer Cancer Non-Cancer Cancer Non-Cancer Cancer Non-Cancer No 66.8 85.6 83.4 95.5 54.8 89.6 25.6 52.2 16.6 42.8 Slight 27.0 13.4 16.2 4.5 37.8 10.0 60.6 36.8 66.2 39.3 Moderate 6.0 1.0 0.4 0.0 6.6 0.5 12.4 11.0 15.2 15.4 Severe 0.2 0.0 0.0 0.0 0.4 0.0 1.4 0.0 2.0 2.5 Extreme 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 Table 3 Comparative EQ-5D-5L and VAS Scores from Non-Cancer and Cancer Cohort at Different Time Points Mean utility score VAS score Cancer Mean± SD p-value* Mean ±SD p-value* Baseline 0.8703±0.121 NA 73.43 ± 12.66 NA Completion 0.8745 ± 0.094 0.557 69.81 ± 10.14 0.000 Follow up 0.8902 ± 0.107 0.043 71.56 ± 10.50 0.063 Non-cancer 0.9323 ± 0.082 0.000 78.88 ± 13.65 0.000 *t-test was done using baseline value; NA, Not applicable Table 4 Socio-Economic Differential of Mean EQ-5D-5L Score among Baseline Cancer Patients and Non-Cancer Cohort (bivariate analysis) Socio-economic characteristics Cancer Non-cancer Mean EQ-5D-5L score p-value Mean EQ-5D-5L score p-value Age (in years) Below 40 0.882±0.117 0.0642 0.947±0.071 0.0722 41 to 59 0.870±0.121 0.920±0.090 60+ 0.839±0.127 0.918±0.094 Education Illiterate 0.857±0.122 0.3512 0.908±0.103 0.1875 Primary 0.870±0.120 0.922±0.091 Secondary 0.872±0.113 0.929±0.076 Higher secondary and above 0.883±0.129 0.944±0.075 Marital status Currently married 0.871±0.123 0.6781 NA NA Others 0.865±0.108 NA Religion Hindu 0.877±0.117 0.0148 0.937±0.083 0.0288 Others 0.845±0.132 0.895±0.072 Household size 1 to 4 0.878±0.111 0.3995 0.935±0.080 0.803 5 to 6 0.862±0.133 0.928±0.090 7 and more 0.866±0.125 0.925±0.077 Health insurance Yes 0.892±0.130 0.2065 0.942±0.076 0.2982 No 0.868±0.120 0.928±0.085 Place of residence Urban 0.872±0.126 0.7276 0.939±0.078 0.0406 Rural 0.869±0.117 0.911±0.093 Annual household income Less than 50,000 0.843±0.138 0.0364 0.881±0.091 0.0242 50,000- 1lac 0.869±0.101 0.923±0.082 1 lac- 2 lac 0.877±0.124 0.927±0.090 More than 2lac 0.886±0.117 0.945±0.075 Cancer stage I & II 0.884±0.093 0.0894 NA NA III 0.866±0.126 NA IV 0.836±0.187 NA p-value: *<0.1, ** <0.05, *** <0.01; NA, Not applicable Table 5 Socio-Economic Determinants of EQ-5D-5L Score among Baseline Cancerous and Non-Cancerous Group (multivariate analysis) Cancer Non-cancer Co-efficient Confidence interval (CI) Co-efficient Confidence interval (CI) Age (in years) Below 40® 41 to 59 -0.018 [-0.045, 0.008] -0.049*** [-0.082, -0.015] 60+ -0.053** [-0.094, -0.013] -0.032 [-0.1, 0.035] Education Illiterate® Primary 0.007 [-0.027, 0.04] 0.011 [-0.049, 0.071] Secondary 0.005 [-0.028, 0.038] 0.019 [-0.037, 0.075] HS and above 0.002 [-0.033, 0.037] 0.023 [-0.032, 0.077] Religion Hindu® Others -0.033** [-0.061, -0.005] -0.059** [-0.11, -0.008] Household size 1 to 4® 5 to 6 -0.013 [-0.038, 0.013] 0.011 [-0.025, 0.048] 7 and more -0.01 [-0.045, 0.026] -0.001 [-0.065, 0.063] Health insurance Yes® No 0.009 [-0.034, 0.052] -0.005 [-0.045, 0.034] Place of residence Urban® Rural 0.001 [-0.023, 0.024] -0.03 [-0.07, 0.01] Annual household income Less than 50000® 50000- 1lac 0.022 [-0.011, 0.055] 0.046 [-0.021, 0.113] 1 lac- 2 lac 0.038** [0.004, 0.073] 0.049 [-0.017, 0.116] More than 2lac 0.045** [0.011, 0.079] 0.07** [0.006, 0.135] Cancer stage I & II® III -0.017 [-0.042, 0.008] N.A. IV -0.048* [-0.1, 0.004] N.A. Constant 0.885*** [0.835, 0.934] 0.926*** [0.856, 0.995] p-value: *<0.1, ** <0.05, *** <0.01; NA, Not applicable Figure 2 Temporal Trend of Mean Utility Score by Stage of Cancer Diagnosis Table 6 Review of Literature of Available Health State Utility Studies for Breast Cancer Author Country (N) Mean Utility value Median Utility value SD of utility value Setting Instrument Eun-ju Kim et al. (2012) Korea (509) 0.82 - 0.16 Metastatic EQ-5D-5L-3L and EORTC C-30 and BR23 Mahmood Yousef et al. (2016) Iran (163) 0.685 0.761 0.216 Primary, recurrent, stable and metastatic states EQ-5D-3L and SF-6D Cheung et al (2014) China (238) 0.777 (Japan) 0.785 (UK) 0.740 (Japan) 0.777 (UK) 0.163 (Japan) 0.200 (UK) Curative. Palliative, follow up EQ-5D-5L and FACT-B Takeru Shiroiwa et al (2011) Japan (300) 0.720 to 0.843 in different types of chemo# - - Curative on chemotherapy and follow up EQ-5D-3L and FACT-B Seon-Ha Kim et al (2017) Korea (199) 0.352 to 0.804 in different states 0.300 to 0.900 0.255 to 0.275 8 hypothetical states, metastatic and non-metastatic VAS and SG Tzu-Chun Chou et al (2020) Taiwan, (104) 0.04 to 0.62 in different states of MBC* -0.02 to 0.68 0.33 to 0.47 Metastatic breast cancer VAS and TTO method Current study India (500) 0.87 0.88 0.121 Stage I to IV breast cancer, , measured at baseline, endline and follow up EQ-5D-5L and VAS *Progression-free 0.43, responding 0.62, progressing 0.22 and palliative 0.04; #ACP 0.764, ACD 0.843, PTX 0.742 and DTX 0.720; UVAS i = (Raw score of health state i - Raw score of dead)/ Raw score of perfect health - Raw score of dead) Discussion Public health systems of developing countries like India have to tackle infectious diseases as well as non-communicable diseases (NCD) which are increasing at an exponential rate. The factors contributing to the increased prevalence of NCD include early age at diagnosis, lack of health awareness resulting in late diagnosis and lack of access to health care facilities. These issues are particularly more relevant in oncology as cancer treatment which involves multidisciplinary care is available mainly in the urban areas. The increased vulnerability of the rural population is the direct manifestation of poor access and low insurance coverage. Under such circumstances, India has launched the ambitious public health insurance scheme to bridge the gap between access to care and financial risk. However, it is important to link health packages with clinically approved treatment guidelines to deliver optimal cancer care. This requires sound scientific knowledge about the clinical benefit of various health interventions and their impact on patient reported outcomes (PRO). Though data is available from the clinical trials, very often the instruments used are not directly useful in calculation of utility of various health states. They require mapping to the commonly recommended tools like the EQ-5D-5L. Moreover, the clinical trial data may not be fully representative of the real-world population based on the eligibility criteria employed in various trials. Hence, the current study for estimating utilities from real-world population was undertaken using previously validated and recommended tools. The EQ-5D-5L instrument has been applied in a variety of health sector settings like population health studies and health technology assessments wherein it captures the patient-reported outcome from patients suffering from varied clinical conditions. In 2009, NHS England introduced its Patient Reported Outcome Measures (PROMs) programme which comprised of the EQ-5D alongside condition-specific PROs for a variety of medical conditions (NIHCR, 2022). These data are used to monitor the performance of healthcare providers, incentivize quality by linking reimbursement to performance, and inform patients in making appropriate choice of the service provider. Similar uses of EQ-5D are underway, or planned, in the healthcare system like the private insurance companies in Sweden, Canada, Australia and New Zealand. In this article we report the HSU derived from a large cohort of breast cancer women taking treatment at TMC. To the best of our knowledge, such a report of utility value sets from India for breast cancer has not been published earlier. It was observed that the EQ-5D-5L values had numerically worsened after diagnosis of cancer as compared to the non-cancer cohort. However, the difference was not significant for EQ-5D-5L scores. A discrepancy was noted in the values derived by the two methods i.e. EQ-5D-5L and EQ-VAS. This observation was noted for both cancer as well as non-cancer cohort. The mean difference in percentage distribution of the 5 dimensions in moderate-extreme problem category between the cancer and non-cancer cohort was 5.2% (19), 0.4% (12), 6.9% (35), 2.8% (27) and -0.7% (26) for mobility, self-care, usual activity, pain/discomfort and anxiety/depression respectively. Similarly, cancer affected all domains of the EQ-5D-5L as observed from the lower percentage of cancer patients in the “no problem” category and the difference between cancer and non-cancer ranged from 12 to 35% for this category. A similar trend was also noted for the VAS-derived values. The utility values among cancer patients improved over 6 months which was significant compared to the baseline estimate. It however did not reach the values comparable to the non-cancer cohort. The recently published Indian value set also reported a mean value of 0.84 (SD 0.209) for the overall female participants (Jyani et al., 2022). Though utility values for other demographic variables have been reported, no separate gender-wise data is available. The socio-economic determinants of the EQ-5D-5L scores found in this study corroborate with the results from the DIVINE study published recently by Jyani et al., (2020). We found statistically significant association of utility scores with age, religion, marital status, annual income and cancer stage but not for educational status and place of residence as reported in the DEVINE study. The health state utility data sets on breast cancer have been reported from several countries but there is heterogeneity in the methods of deriving the utility scores as well as in the population studied. The available literature has been tabulated below (Table 6). It can be noted that EQ-5D-5L is the most commonly used tool, but the earlier studies have used the EQ-5D-3L in which only 243 health states can be analysed as against the EQ-5D-5L in which 3125 health states can be studied. The only study comparable to the current study is the Chinese study that evaluated breast cancer patients in combined curative treatment, palliative treatment and follow up phases (Cheung et al., 2014). The Indian value seen in this study is higher (0.87) compared to the Chinese (0.777 or 0.785) value and could be related to the limited proportion of stage IV patients in our study (6% vs 33%). To assess whether the non-cancer cohort was representative of general population, we compared the key socio-demographic determinants like age, education, marital status, social group and religion with the 2011 census report of the country. Similarly, we compared the cancer cohort with the breast cancer 2020 report published by the National Cancer Registry Program (NCDIR) and the large breast cancer multi-institutional cohort study of non-metastatic breast cancer (NCRP, 2020). While health economic evaluations require generic measures like EQ-5D-5L, SF-6, HUI etc. for broader generalizability across different health conditions, they fail to capture all the domains of health-related quality of life. Hence, these are not preferred or commonly used measures in clinical practice. This issue is more pertinent to oncology which is a complex disease due to its natural history, primary organ involved, staging, multimodal treatment, morbidity and expected survival. As QALYs cannot be calculated from the disease specific measures like the EORTC QLQ C30, FACT-G etc., there is a need of mapping of disease specific measures onto the generic measures of utility. We intend to study this by mapping of EORTC QLQ C-30 and BR23 modules with the EQ-5D-5L and will be the subject for are upcoming publications. Moreover, as the current study is first of its kind reporting the utility scores from a large cohort of breast cancer patients seeking treatment at the country’s largest cancer facility, it will guide clinicians as well as policy makers for economic evaluations in future. An important observation was the discordance between the utility values derived based on the EQ-5D-5L and VAS. The values obtained by VAS method were lower in our data as opposed to the EQ-5D-5L value which contrasts with the finding reported by Peasgood et al., (2010), and also highlights the lower reliability of the VAS method. Hence stand-alone use of the VAS method is generally not recommended (Torrance et al., 2001). However, we have found moderate to high correlations between utility score and VAS. The EQ-5D-5L values on the other hand are prone to lesser bias due to the inclusion of a wide range of health states that increases the sensitivity and reduces the ceiling effect that is reported for the EQ-5D-3L values (Kim et al., 2012). Strengths and limitations The large sample size of breast cancer patients taking treatment at a tertiary cancer centre gives the opportunity to include patients from diverse socio-economic background and different parts of the country. As all the breast cancer patients were accrued uniformly and consecutively over the study period, the convenience sampling is likely to represent the overall breast cancer population in the country. Due to lack of availability of the Indian value at the time of initiation of the study, the cancer cohort has been compared with the non-cancer cohort in order to get non-skewed data. In this study, we haven’t captured the utility values separately for the different types of treatment like surgery, chemotherapy and radiotherapy. Moreover, the side-effects, recurrence or progression states have also not been captured. The metastatic stage is also under-represented in our data. Author Contribution Statement TW & SKM conceptualized the research and decided the study design. SP, JM, RS, SG, VP were involved in screening of subjects and data collection, SS and PKK performed the data analysis. TW & SS were involved in writing the first draft of the manuscript. All the authors read and edited the final draft. TW & SKM provided overall supervision of the study. Acknowledgements Funding statement The authors extend their deepest gratitude to the Women’s Cancer Initiative, the Nag Foundation, the International Institute for Population Sciences, and the Tata Memorial Centre for providing financial support. Availability of data Data is not publicly available, and it may be available upon reasonable request to corresponding author. Corresponding author has all rights in sharing the data. Ethics approval The study has obtained approval from the institutional ethics committee of TMC and was registered on the Clinical Trial Registry of India with CTRI No CTRI/2019/07/020142 on 10/07/2019. This was an investigator initiated study. Consent to participants Informed written consent was obtained from all the participants included in the study. Competing interests The authors have no relevant financial or non-financial interests to disclose. ==== Refs References Amarsheda S Bhise A Association of Fatigue, Quality of Life and Functional Capacity in Breast Cancer Patients Receiving Adjuvant Chemotherapy Asian Pac J Cancer Care 2021 6 59 4 Cheung YB Luo N Ng R Lee CF Mapping the functional assessment of cancer therapy-breast (FACT-B) to the 5-level EuroQoL Group’s 5-dimension questionnaire (EQ-5D-5L) utility index in a multi-ethnic Asian population Health Qual Life Outcomes 2014 12 1 10 Chou TC Chiang SC Ko Y Health state utilities for metastatic breast cancer in Taiwan Breast J 2020 51 57 4 Department of Health Research Health technology assessment in India: a manual., Health Technology Assessment in India A Manual, MOHFW, Govt of India 2018 https://htain.icmr.org.in/documents/publications/htain-manual Dhillon PK Mathur P Nandakumar A The burden of cancers and their variations across the states of India: the Global Burden of Disease Study 1990–2016 Lancet Oncol 2018 19 1289 6 30219626 Huang W Yang J Liu Y Assessing health-related quality of life of patients with colorectal cancer using EQ-5D-5L: a cross-sectional study in Heilongjiang of China BMJ Open 2018 8 e022711 Jyani G Chauhan AS Rai B Health-related quality of life among cervical cancer patients in India Int J Gynecolo Cancer 2020b 30 Jyani G Prinja S Kar SS Valuing health-related quality of life among the Indian population: a protocol for the Development of an EQ-5D Value set for India using an Extended design (DEVINE) Study BMJ Open 2020a 10 e039517 Jyani G Sharma A Prinja S Development of an EQ-5D value set for India using an extended design (DEVINE) study: The Indian 5-level version EQ-5D value set Value Health 2022 25 1218 6 35779943 Kim EJ Ko SK Kang HY Mapping the cancer-specific EORTC QLQ-C30 and EORTC QLQ-BR23 to the generic EQ-5D in metastatic breast cancer patients Qual Life Res 2012 21 1193 3 22012023 Kim SH Jo MW Ock M Lee HJ Lee JW Estimation of health state utilities in breast cancer Patient Prefer Adherence 2017 2017 531 6 Kim SH Kim HJ Lee SI Jo MW Comparing the psychometric properties of the EQ-5D-3L and EQ-5D-5L in cancer patients in Korea Qual Life Res 2012 21 1065 3 21947656 National Cancer for Disease Informatics and Research Report of National Cancer registry Programme (2012-2016) 2020 Retrieved July 18, 2022 https://ncdirindia.org/All_Reports/Report_2020/resources/Chapter7CancerBreast.pdf National Institute for Health and Cancer Research Health Technology Assessment 2022 Retrieved May 29, 2022 https://www.nihr.ac.uk/explore-nihr/funding-programmes/health-technology-assessment.htm Peasgood T Ward SE Brazier J Health-state utility values in breast cancer Expert Rev Pharmacoecon Outcomes Res 2010 10 553 6 20950071 Shiroiwa T Fukuda T Shimozuma K Long-term health status as measured by EQ-5D among patients with metastatic breast cancer: comparison of first-line oral S-1 and taxane therapies in the randomized phase III SELECT BC trial Qual Life Res 2017 26 445 3 27517267 Torrance GW Feeny D Furlong W Visual analog scales: do they have a role in the measurement of preferences for health states? Med Deci Making 2001 21 329 4 Vashistha V Poulose R Choudhari C Kaur S Mohan A Quality of Life among Caregivers of Lower-Income Cancer Patients: A Single-Institutional Experience in India and Comprehensive Literature Review Asian Pac J Cancer Care 2019 4 87 3 Yousefi M Najafi S Ghaffari S Mahboub-Ahari A Ghaderi H Comparison of SF-6D and EQ-5D scores in patients with breast cancer Iran Red Crescent Med J 2016 18

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116148 10.31557/APJCP.2023.24.4.1257 Research Article Implication of Dynamin-2 (DNM2) Mutations in Adult T-cell Acute Lymphoblastic Leukemia Sobh Marwa 1 Aref Salah 2* Al Agdar Mohamed 2 El Zafrany Maha 3 El-Sokkary Ahmed M.A 1 1 Biochemistry Subdivision, Chemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt. 2 Hematology Unit, Clinical Pathology Department, Mansoura University Oncology Center, Mansoura University, Mansoura, Egypt. 3 Medical Oncology Department, Mansoura University Oncology Center (MUOC), Mansoura, Egypt. * For Correspondence: salaharef@yahoo.com 2023 24 4 12571264 27 11 2022 23 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: The objective of the present study was to improve the risk stratification of T-cell Acute Lymphoblastic Leukemia (T-ALL) patients. It aimed to identify the frequency and clinical impact of DNM2 gene mutations among adult T-ALL cases. Methods: The current study included 25 T-ALL patients before starting their treatment. Mutational analysis of DNM2 gene (exons 18 and 22) was performed for all patients using Macrogen 3730 apparatus. Results: We identified DNM2 gene mutations in 19 out of 25 (76%) patients. The detected mutations were either missense or deletion. Only active mutations (deletion) were associated with poor induction remission response and high frequency of relapse. Two novel mutations were addressed among the studied cohort of patients. They included c.1866G>C (p.V596L) and c.1872delA in exon 18. A high frequency of silent mutations was also found in T-ALL patients, but with no impact on clinical features. Conclusion: The DNM2 mutations were prevalent among adult T-ALL patients and might have a role in the pathogenesis of the disease. Active DNM2 mutations were associated with poor clinical outcome. Moreover, high frequency of DNM2 mutations indicated that these mutations could be utilized in detection of minimal residual disease in T-ALL patients. Key Words DNM 2 mutations adult T-cell acute lymphoblastic leukemia prognosis ==== Body pmcIntroduction T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive, malignant disorder that affects both pediatrics and adults (Bridges et al., 2023; Pui et al., 2008). It represents about 15% of pediatrics and 25% of adults (Chennamadhavuni et al., 2022; Iacobucci and Mullighan, 2017). In 2023, it was reported 6540 T-ALL new cases and 1390 deaths in a project done in the United States (Siegel et al., 2023). The diagnosis of T-ALL requires the presence of 20% or more blasts in bone marrow (Paul et al., 2016). Clinical features of T-ALL patients usually include elevation of the total leukocyte count (TLC) and may show organomegaly and CNS involvement (Chiaretti and Foà, 2009; Del Principe et al., 2014; Ikonomidou, 2021). T-ALL is divided into 5 subtypes according to molecular biology, gene expression and FISH results. These subtypes include pro-T, pre-T, cortical, mature T-ALL and ETP (Ferrando et al., 2002; Ge et al., 2016). T-ALL is associated with high risk of treatment failure (Trochet and Bitoun, 2021). Previous studies indicated that genetic mutations might have an adverse impact on patient with T-ALL (Chiaretti and Foà, 2009; Neumann et al., 2015; Roberts and Mullighan, 2015). More than one hundred genes were identified to be mutated in ALL but with variable frequency. The most popular mutated genes were NOTCH1 and CDKN2A/2B. It was reported that mutations of these genes appeared in more than 50% of patients with T-ALL (Aref et al., 2020). According to the available genomic data, it was noticed that each T-ALL case contained more than 10 genes defects (Girardi et al., 2017, Aref et al., 2016, Aref et al., 2021). Dynamin-2 (DNM2) is a large GTPase (100kDa) located on short arm of chromosome 19 (19p13.2). Its product was specified as a micro-tubule binding protein which composed of four isoforms 1, 2, 3 and 4. The DNM2 gene is composed of 22 exons and consists of 5 domains: GTPase domain, intermediate domain (MD), pleckstrin homology domain (PH), GTPase effector domain (GED) and proline-arginine-rich domain (PRD) (Cao et al., 1998; Durieux et al., 2010; Ramachandran and Schmid, 2018; Sambuughin et al., 2015; Xu et al., 2014). The GTPase DNM2 is essential for a lot of physiological functions; it is responsible for membrane trafficking, cytokinesis and receptor endocytosis. It is also acting as a regulator of cytoskeletons. Overexpression of DNM2 gene is associated with some cancer types and their progression. DNM2 mutations are observed in early precursor T-ALL (EPT-ALL) with a high risk of induction failure or not achieving complete remission (Ge et al., 2016; Sambuughin et al., 2015). This study aimed to determine the frequency of DNM2 gene mutations in adult T-ALL patients and relate them to clinical and pathological characters. Materials and Methods The study was approved by Mansoura University IRB and was done according to Helsinki regulation. Patients and Samples Five mls of EDTA blood sample were collected from 25 T-ALL patients (17 male, 8 females), with mean age 34.76 (age range 17-62), who were newly diagnosed with recruitment at Mansoura University Oncology Center (OCMU), Mansoura University, Mansoura, Dakahlia Governorate, Egypt. (Table 1). The examined T-ALL patients were followed up for 24 months. Treatment protocol Induction remission protocol for adult T-ALL cases, was by either augmented Berlin-Frankfurt-Münster (aBFM) protocol (Chang et al., 2008) or modified hyper CVAD (cyclophosphamide, vincristine, dexamethasone, doxorubicin) (anthracycline intensification) (Thomas et al., 2010) in relapsed or high risk ALL cases and gemcitabine was used in pediatric T-ALL cases (Angiolillo et al., 2006). Follow up and prognostic criteria Hematological remission was identified when the blast cells < 5% in bone marrow. Complete remission was addressed by minimal residual disease cut off levels of blast cells count < 0.01%. Relapse is identified as reappearance of blasts in blood smear or the presence of > 5% blast cells in BM smear. Disease free survival (DFS) is the time from complete remission to time of relapse, death or date of last contact with patient. Overall survival (OS) is the time from study entry until death from any cause or last contact with patient (Aref et al., 2020). Methods T-ALL diagnosis was done by morphological examination of bone marrow smear (Blast cells equal to or more than 20%) and immunophenotyping using flowcytometry panel including cytoplasmic CD3, CD5, CD7, CD2, CD4, CD8, CD34, TDT, CD19, CD79a, MPO, and CD117. DNA Extraction and Amplification Mini QIA amp DNA isolation kit (QIAGEN) (Cat. no.51104) was used to extract genomic DNA from T-ALL patient’s bone marrow following the standard procedures according to the manufacturer’s guidelines. Isolated DNA fragments were amplified using conventional PCR technique. Conventional Polymerase Chain Reactions (PCRs) were done in a total volume of 50 µL containing 25 μL of Dream Taq master mix, 0.2 μl from the both primers (100 pmol), and 2 μL of extracted DNA and completed to 50 μL with distilled water. PCR was done using Thermo Scientific Arktik Thermal Cycler. Cycling conditions were 35 cycles with annealing temperature 54ºC for exon 18 and 56ºC for exon 20. The primers used for PCR amplification of DNM2 exons (18 and 20) were as follow: Exon 18 forward, CTAGAGCCCATTCCTCTCGG and reverse, CATGATTTCAGAGACTCCTGGC and exon 20 forward, CCCGCCCTGTGAGAGATG and reverse, AGGACCCTGCAGGACACAC The process included the following steps: initial denaturation at 95oC for 2 minutes, 35 cycles at 95oC for 30 seconds, 54oC (for exon 18) and 56oC (for exon 20) for 30 seconds, and 72oC for 30 seconds, and a final extension at 72oC for 10 minutes. DNA Purification Purification of PCR products was done using the DNA Clean and Concentrator®-25 PCR Purification Kit (Cat.no.D4005-USA) according to the manufacturer’s guidelines. Cycle sequencing of the purified PCR products was performed using the Big Dye Terminator, version 3.1, Cycle Sequencing Kit. DNA Sequencing Macrogen3730 Applied Biosystem apparatus was used for performing mutational analysis of DNM2 exons (18 and 22). Statistical Analysis The sample size of the T-ALL patients group was calculated statistically. The analysis of the data was performed using Excel and Statistical Package for Social Sciences (SPSS version 22). Qualitative data were presented in the form of numbers and percentages. Quantitative data were presented in the form of mean (M) ± standard deviation (SD). For evaluation of survival analysis (OS and DFS), we used Kaplan-Meier test. A p value was considered significant at level p<0.05. Results Demographic Data and Patients’ Characteristics This study was conducted upon 25 patients who were newly diagnosed with T-cell Acute Lymphoblastic Leukemia (T-ALL). The study group was formed of 8 females and 17 males, with mean age 34.76 (age range 17-62). Demographic data and laboratory findings of the studied T-ALL patients’ group are shown in (Table 1). Clinical Criteria and Laboratory Data of Adult T-ALL Patients with Active DNM2 Mutations Clinical and laboratory findings of adult T-ALL patients (with active DNM2 mutations) showed organomegaly (hepatomegaly and splenomegaly) (50%), lymphadenopathy (60%), fever (60%), CNS infiltration (25%) and pallor (70%) (Table2). DNM2 Mutations Characterization Eighteen patients exhibited silent mutation (c.2219C>T) (p.A713A) in exon 20, one patient showed active mutation (c.1866G>C) (p.V596L) in exon 18 and 4 patients had frame-shift “deletion” mutations in exon 18 (c.1872delA). There were 4 patients who gained 2 kinds of mutations, one patient with (c.1866G>C and c.2219C>T) and 3 patients with (c.1872delA and c.2219C>T) (Figure 1, Table 3). Frequency of Detected DNM2 Mutations In the current study, 25 newly diagnosed T-ALL adult patients were tested for mutations in the DNM2 gene for both exons 18 and 20. We found that 19 out of 25 (76%) patients had one or more kinds of mutations (point or frame shift) (Table 3, Figure 2). Impact of DNM2 Mutations on T-ALL Patients’ Outcomes In the current study, we observed that 17 patients were induction remission responders and 8 patients were non-responders. During the follow up process, 12 patients were relapsed and 13 patients continued in complete remission. After 24 months, 15 patients (60%) died and 10 patients (40%) were still alive (Table 4). Clinical Criteria of Patients with DNM2 novel Mutations Two novel detected mutations include: (c.1866G>C) (p.V596L) and (c.1872delA) in exon 18 (Figure 1) were detected in 6 patients. Clinical criteria of patients with DNM2 novel mutations are shown in (Table 2). These patients exhibited high white blood cells count and increased initial LDH, CNS infiltration and organomegaly (Table 2). Impact of DNM2 Silent Mutations on T-ALL Patients’ Overall Survival We analyzed the impact of DNM2 silent mutations on T-ALL patients overall survival (OS) by Kaplan Miere Curve. This analysis revealed that there were no significant differences between mutated and non-mutated T-ALL cases regarding OS (Figure 3). Impact of DNM2 Active Mutations on T-ALL Patients’ Overall Survival The impact of DNM2 active mutation was evaluated by Kaplan Miere Curve. The results showed that patients with active mutations exhibited shorter OS compared to those with silent mutations or non mutated ones. T-ALL patients with active mutations had significantly shorter OS compared to those with silent or non-mutated (Figure 4). Impact of DNM2 Mutations on T-ALL Patients’ Disease Free Survival The statistical analysis revealed that the mutated T-ALL patients displayed high frequency of relapse compared to non-mutated ones (Figure 5). Table 1 T-ALL Patients’ Characteristics Parameters No (Mean)Range Age (year) Mean (range) 25 34.76 (17- 62) TLCs (109/L) 25 84.56 (11.1-328.00) Hemoglobin (g/dl) 25 9.98(5.09- 13.77) Platelets count (109/L) 25 78.1 (9.00 – 328.00) LDH (U/L) 25 1848.2 (263.32-11134.60) Bone Marrow blast cells (%) 25 83.0 (40-95) Table 2 Clinical criteria of adult T-ALL patients with active DNM2 Mutations Case no TLC X109/L Hb g/dl PLT X109/L LDH IU/ml BM Blasts% CNS infiltration Abdominal ultrasound Lymphadenopathy Mutations 1 37.8 8.4 34 638 90% Positive Normal live and spleen with enlarged para aortic lymph nodes Negative c.1866G>C c.2219C>T 2 149 13 40.4 598 90% Free Mild enlarged liver Enlarged spleen Few enlarged lymph nodes at portahepatis; largest one about 2 cm c.1872delA c.2219C>T 3 164 8.8 32.52 1300 95% Free Normal Liver and spleen size Negative c.1872delA c.2219C>T 4 58 8.5 58.7 5623 90% Positive Enlarged liver and spleen Multiple bilateral small axillary LNs c.1872delA 5 328 8 24 4967 90% Free Enlarged liver and moderate splenomegaly. Bilateral enlarged cervical L.N.s the largest in right posterior triangle. Few enlarged supraclavicular L.N.s left side. Enlarged bilateral axillary L.N.s right side c.1872delA c.2219C>T TLC, Total leucocyte count; Plat, Platelets count; Hb,Hemoglobin; Abd.US, Abdominal ultrasound; LN, lymph nodes Figure 1 Types of DNM2 Mutations in Adult T-ALL (active point mutation; Silent point mutation, and deletional mutation) Figure 2 Types and Frequency of Detected Mutations in T-ALL patients Table 3 The Detected MutationsTypes and Frequency in T-ALL Patients Types of mutations Mutation: nucleotide Mutation: amino acid No. of mutated samples (19/25) Point mutation (19/25 )(1 active and 18 silent) Exon 18: 1 Samples(active) c.1866G>C p.V596L Exon 20: 18 Samples (silent) c.2219C>T p.A713A Deletion mutation in Exon 18 ( 4 Samples) c.1872delA Frame shift mutation with unknown protein. Among the detected mutations 2 novel ones were detected in exon 18, all of them were active, one was point mutation (c.1866G>C), and one was frame-shift mutation (c.1872delA) was detected in 4 samples. Table 4 Impact of DNM2 Mutation on Adult T-ALL Patients’ Outcome Induction of remission response Relapse During Follow up Outcome after 24 months Responder Non-responder Relapsed Non-relapsed Died Alive (n=17) (n=8) (n=12) (n=13) 15 10 68% 32% 48% 52% 60% 40% Figure 3 Kaplan Meier Curve for Overall Survival (OS) of T-ALL patients with DNM2 Mutations Compared to Non-Mutated Ones. The analysis revealed that there were no significant differences between mutated and non- mutated T-ALL cases regarding OS Figure 4 Kaplan Meier Curve for Overall Survival (OS) of T-ALL Patients with DNM2 Active Mutation vs. Non Active (Silent) vs. Non Mutated Ones. Patients with active mutations exhibited shorter OS compared to those with silent mutations or non mutated ones. T-ALL patients with active mutations had significantly shorter OS compared to those with silent or no-mutation Figure 5 Impact of DNM2 Mutation on DFS of T-ALL Patients. The mutated patients displayed high frequency of relapse compared to non-mutated ones Discussion The percentage of DNM2 mutations detected in exons 18 and 20 was 76%. This high frequency of DNM2 mutations indicated that these mutations might have an important role in the pathogenesis of T-ALL. Among the detected mutations, there are 2 active mutations detected in 5 out of 25 patients and 18 silent mutations were detected in 25 (72%) T-ALL patients. Previous report stated that DNM2 mutations induced elevation of the content of Interleukin 7 (IL-7) receptor in the plasma membrane in pre-leukemic thymocytes leading to enhancing IL-7 signaling and development of more immature T-ALL (Trochet and Bitoun, 2021). Also, it was reported that T-ALL was promoted by DNM2 loss-of-function mutations resulted from enhancing IL-signaling. These mutations included: L354P, K562del, S698L and P791T. These mutations were found along the gene functional domains (Tremblay et al., 2016). In the current study, 2 novel mutations were detected in 5 cases (20%). They included c.1866 A>C in one case and c.1872delA in 4 cases. Previous reported mutations included c.1081C>T, c.1453T>C, c.1609G>A and c.1801C>T, which were located in exons 8, 13, 16 and 18, respectively. Moreover, two silent amino acid mutations included Ala713Ala and Asp720Asp were reported in exon 20 (Ge et al., 2016). In the current study, the silent mutations were not associated with characteristic clinic-pathological features. Similar finding was previously reported by (Ge et al., 2016) who suggested that the sites of silent mutations might represent the hot spots and the nucleotides at these sites could be quickly corrected and were easily changed. Similar to DNM2 mutations that located in functional domain, inherited DNM2 mutations were associated with degenerative neurologic diseases like peripheral neuropathy, Charcot– Marie–Tooth, autosomal dominant Centro nuclear myopathy, lethal congenital contracture syndrome and Siberian family with hereditary spastic paraplegia (Sambuughin et al., 2015; Trochet and Bitoun, 2021). Recurrent DNM2 mutations were identified in patients with T-ALL through exons (18 and 20). Previous study found that there were two silent amino acid mutations including Ala713Ala and Asp720Asp in exon 20. The first mutation was found in 31 out of 42 patients and the other one was found in 3 out of 42 patients with 80.95% for both in the T-ALL patients. The two kinds of mutations of exon 20 were found in only one case (Ge et al., 2016). Consequently, we found additive novel mutations [(c.1866 G>C) (p.V596L), (c.1872delA)]. In another study, 13 ETP-ALL and 4 non-ETP ALL with DNM2 mutations had been reported, 4 out of 17 mutations were reported in PH domain of the gene (Zhang et al., 2012). The impact of DNM2 mutations on induction of remission response revealed that 30% of patients were non-responders and 70% responded during 24 months of therapy. Responders included 5 patients with no mutations and 12 patients with mutations. Non-responders included 7 mutated and 1 non-mutated. Similar findings were reported in another study which stated that certain mutations were appeared in patients that exhibited induction failure and relapse (Ge et al., 2016). During 24 months follow up after induction therapy, 12 out of 19 patients with DNM2 mutations were relapsed. Three patients relapsed after 6 months, four after 8 months and two after 17 months. In addition, three patients relapsed three times, the first relapse was after 6 months, the second was after 11 months and the third was after 19 months. Previous studies reported that only one patient (1/4) with point mutations get relapsed, one patient had an increased TLC count, and 2 patients had lymph node metastasis and complex karyotypes (Ge et al., 2016). In the current study, we assessed the impact of DNM2 mutations on overall survival. The results revealed that 13 out of 19 mutated patients were died with ratio of 68.42% and with ratio of 52% of whole 25 patients diagnosed with T-ALL. Similarly, patients with DNM2 mutations exhibited high-risk leukemia and possessed a poor prognosis (Ge et al., 2016). We had evaluated the impact of DNM2 active mutations on overall survival. From 19 mutated samples, there were only 5 samples with active mutations. We reported 4 deaths from 5 patients. It was reported that it needed more than 4 weeks for a complete remission in patients with DNM2 mutations (Ge et al., 2016). Previous reports stated that treatment of T-ALL with chemotherapy came with high overall survival especially for younger patients. However, this treatment showed more side effects and relapse with poor prognosis. Girardi et al., (2017) suggested that the whole-exome sequencing of relapsed patients could make a detailed view on the genomes of relapsed T-ALL cases. This result can help in the introduction of new targeted drugs and to reduce the toxicity of current chemotherapies. DNM2 mutations are frequent in our cohort of T-ALL cases. Similar to our results, previous study reported that 34 out of 42 cases exhibited DNM2 gene mutations with frequency of 80.95% (Ge et al., 2016). High frequency of DNM2 mutations in T-ALL patients suggested that these mutations might have a role in the pathogenesis of the disease. Furthermore, these mutations could be a candidate for minimal residual disease detection in T–ALL patients. In conclusion, The DNM2 mutations were prevalent among adult T-ALL patients and might have a role in the pathogenesis of the disease. Active DNM2 mutations were associated with poor clinical outcome. Moreover, high frequency of DNM2 mutations indicated that these mutations could be used in detection of minimal residual disease in T-ALL patients. Author Contribution Statement Marwa Sobh: Laboratory Work and Analysis of Data; Salah Aref: Study Idea and Design; Mohamed Al Agdar: Laboratory Work; Maha El Zafrany: Patients’ Follow up and Clinical Management; Ahmed M.A El Sokkary: Supervision of the Study. Acknowledgements The present study is the result of MSc thesis of the postgraduate student, Marwa Sobh and approved by the ethical committee of Faculty of Science, Mansoura University. The authors would like to thank the technicians in the Hematology laboratory at Mansoura University Oncology Center (MUOC). Funding Statement This study was funded in whole by the authors themselves. The authors did not receive any financial support from any organization for the submitted paper. Ethical Statement This study was approved by Mansoura Faculty of Medicine ethical committee and done according to declaration of Helsinki. Availability of Data The data that support the finding of the present study are available upon request from the corresponding author. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of the present paper. ==== Refs References Angiolillo A Whitlock J Chen Z Krailo M Reaman G Phase II study of gemcitabine in children with relapsed acute lymphoblastic leukemia or acute myelogenous leukemia (ADVL0022) a Children’s Oncology Group Report Pediatric Blood Cancer 2006 46 193 7 15929131 Aref S El Menshawy N El-Ghonemy M Abou Zeid T El-Baiomy M Clinicopathologic Effect of DNMT3A Mutation in Adult T-Cell Acute Lymphoblastic Leukemia Clin Lymphoma Myeloma Leuk 2016 16 43 8 26711182 Aref S El Agdar M Salama O Abouzeid Zeid T Sabry M Significance of NOTCH1 mutations détections in T-acute lymphoblastic leukemia patients Cancer Biomark 2020 27 157 62 31796666 Aref S Fawzy E Darwish A Aref M Al Agdar M Cortactin Expression is a Novel Biomarker for Risk Stratification of T-Cell Acute Lymphoblastic Leukemia J Pediatr Hematol Oncol 2021 43 e798 e803 33235155 Bridges CS Chen TJ Puppi M Rabin KR Lacorazza HD Antileukemic properties of the kinase inhibitor OTSSP167 in T-cell acute lymphoblastic leukemia Blood Adv 2023 7 422 35 36399528 Cao H Garcia F McNiven MA Differential distribution of dynamin isoforms in mammalian cells Mol Biol Cell 1998 9 2595 609 9725914 Chennamadhavuni A Lyengar V Mukkamalla SKR Shimanovsky A Leukemia 2022 StatPearls. Treasure Island (FL) StatPearls Publishing Chang J Medlin S Kahl B Augmented and standard Berlin-FrankfurtMunster chemotherapy for treatment of adult acute lymphoblastic leukemia Leuk Lymphoma 2008 12 2298 307 Chiaretti S Foà R T-cell acute lymphoblastic leukemia Haematologica 2009 94 160 2 19181788 Del Principe MI Maurillo L Buccisano F Central nervous system involvement in adult acute lymphoblastic leukemia: diagnostic tools, prophylaxis, and therapy Mediterr J Hematol Infect Dis 2014 6 e2014075 25408861 Durieux AC Prudhon B Guicheney P Bitoun M Dynamin 2 and human diseases J Mol Med (Berl) 2010 88 339 50 20127478 Ferrando AA Neuberg DS Staunton J Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia Cancer Cell 2002 1 75 87 12086890 Ge Z Li M Zhao G Xiao L Novel dynamin 2 mutations in adult T-cell acute lymphoblastic leukemia Oncol Lett 2016 12 2746 51 27698851 Girardi T Vicente C Cools J De Keersmaecker K The genetics and molecular biology of T-ALL Blood 2017 129 1113 23 28115373 Iacobucci I Mullighan CG Genetic Basis of Acute Lymphoblastic Leukemia J Clin Oncol 2017 35 975 83 28297628 Ikonomidou C Cerebrospinal Fluid Biomarkers in Childhood Leukemias Cancers 2021 13 438 33498882 Neumann M Vosberg S Schlee C Mutational spectrum of adult T-ALL Oncotarget 2015 6 2754 66 25595890 Paul S Kantarjian H Jabbour EJ Adult Acute Lymphoblastic Leukemia Mayo Clin Proc 2016 91 1645 66 27814839 Pui CH Robison LL Look AT Acute lymphoblastic leukaemia Lancet 2008 371 1030 43 18358930 Ramachandran R Schmid SL The dynamin superfamily Curr Biol 2018 28 R411 6 29689225 Roberts KG Mullighan CG Genomics in acute lymphoblastic leukaemia: insights and treatment implications Nat Rev Clin Oncol 2015 12 344 57 25781572 Sambuughin N Goldfarb LG Sivtseva TM Adult-onset autosomal dominant spastic paraplegia linked to a GTPase-effector domain mutation of dynamin 2 BMC Neurol 2015 15 223 26517984 Siegel RL Miller KD Wagle NS Jemal A Cancer statistics, 2023 CA Cancer J Clin 2023 73 17 48 36633525 Thomas D O’Brien S Faderl S Chemoimmunotherapy with a modified hyper-CVAD and rituximab regimen improves outcome in de novo Philadelphia chromosome-negative precursor B-lineage acute lymphoblastic leukemia J Clin Oncol 2010 28 3880 9 20660823 Tremblay CS Brown FC Collett M Loss-of-function mutations of Dynamin 2 promote T-ALL by enhancing IL-7 signalling Leukemia 2016 30 1993 2001 27118408 Trochet D Bitoun M A review of Dynamin 2 involvement in cancers highlights a promising therapeutic target J Exp Clin Cancer Res 2021 40 238 34294140 Xu B Teng LH Silva SD The significance of dynamin 2 expression for prostate cancer progression, prognostication, and therapeutic targeting Cancer Med 2014 3 14 24 24402972 Zhang J Ding L Holmfeldt L The genetic basis of early T-cell precursor acute lymphoblastic leukaemia Nature 2012 481 157 63 22237106

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116160 10.31557/APJCP.2023.24.4.1367 Research Article Evaluation of Health-Related Quality of Life in Patients Receiving Treatment for Penile Cancer: A Single-Center Cross-Sectional Study Firmansyah Firmansyah 1 Prapiska Faurizki Febrian 2 Siregar Ginanda Putra 2 Kadar Dhirajaya Dharma 2 Warli Syah Mirsya 23* 1 RSUP H. Adam Malik Medan, Indonesia. 2 Urology Division, Department of Surgery, Faculty of Medicine, Universitas Sumatera Utara/ H. Adam Malik Hospital Medan, Indonesia. 3 Department of Urology, Faculty of Medicine, Universitas Sumatera Utara Hospital, Universitas Sumatera Utara Medan, Indonesia. * For Correspondence: warli@usu.ac.id 2023 24 4 13671371 28 12 2022 17 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Introduction: Penile cancer is one of the uncommon types of cancer in men. The treatment could significantly impact a patient’s quality of life (QOL), leading to difficulties in fulfilling life functions. Methods: This descriptive observational study aimed to describe a situation using a cross-sectional design objectively. The population of this study was all patients with a diagnosis of penile cancer who underwent therapy at the Haji Adam Malik Hospital from September 2020 to September 2021. Quality of life was assessed using EORTC QLQ-C30. Results: The respondents’ mean age and standard deviation were 54.44 and 8.647 years, respectively. The youngest was 38 years, while the oldest age was 64 years. Most respondents had no history of circumcision (55.6%). All respondents had a poor QOL based on the 28 components in the questionnaire. This study showed that erectile function, changes in sexual function, and overall sexual function were correlated with health-related quality of life (HRQoL) post-treatment. In general, lack of sexual activity is the primary factor responsible for decreasing HRQoL in penile cancer patients. It has been reported that 70% of patients experienced a negative impact on sexuality post-treatment. Conclusion: The quality of life in patients receiving treatment for penile cancer at RSUP H. Adam Malik, Medan, was poor. It is associated with a lack of sexual activity. Key Words Quality of life penile cancer penile cancer therapy EORTC QLQ-C30 ==== Body pmcIntroduction Penile cancer in men is rare but quite detrimental to sufferers and is often a diagnostic and therapeutic challenge for urologists (Pettaway et al., 2021). Penile cancer is still rare in Western countries, with an incidence of <1 per 100,000 men in Europe and the United States. However, it is more common in African, Asian, and South American countries and accounts for about 10% of all cancers in men in the United States (Van Poppel et al., 2013). For example, Prayoga and Tranggono (2016) found 35 cases of penile cancer in 2006-2013 at Sardjito Hospital, with the dominant age being 40-60 years and 91.4% being the type of squamous cell carcinoma. However, these numbers may vary especially in Medan, as epidemiologic data regarding penile cancer is still unavailable. There are several risk factors associated with penile cancer, which includes phimosis, balanitis, chronic inflammation, penile trauma, smoking, poor hygiene (Clark et al., 2013). Despite that, circumcision has been found to be a protective factor towards penile cancer, especially if circumcised since birth (Van Poppel et al., 2013). On the other hand, history of sexually transmitted diseases, particularly human immunodeficiency virus (HIV) and human papillomavirus (HPV), is also a significant risk factor to penile cancer (Clark et al., 2013). This corresponds to previous literatures as HPV infection is involved in the pathogenesis of penile cancer, as E6 and E7 proteins are associated with penile cancer carcinogenesis processes that influence cell apoptosis and cell cycle regulation (Rodney et al., 2016). Cancer and its treatment have a significant impact on the patient’s life which can result in difficulties in fulfilling family roles, ability to work, or participation in usual social activities. Even when successfully treated, cancer can cause long-term physical and psychological morbidity (Velikova et al., 2012). Therefore, a robust family support system must be established before proceeding with therapy. In addition, the patient’s psychological well-being and partner support are essential in the patient’s follow-up process (Cassell et al., 2020). As cancer patients are required to undergo massive burden throughout its management, there is a need to consider their impact on health-related Quality of Life (HRQoL) when making patient management or treatment decisions (Fayers and Bottomley, 2002). In order to reliably assess quality of life among penile cancer patients, usage of an internationally-accepted assessment tool is required. Additionally, the assessment tool needs to have been validated in the local language, Bahasa Indonesia. However, there are currently no such questionnaires. Meanwhile, one of the most commonly used measuring tools in the HRQoL assessment of cancer patients is the EORTC-QLQ C-30 questionnaire developed by the European Organization for Research and Treatment of Cancer (EORTC) (Sosnowski et al., 2016). The EORTC-QLQ C-30 consists of five functional scales (Physical, Social, Role, Cognitive, and Emotional Functions), a scale for the Global QoL, and nine symptom scales (Fatigue, Pain, Nausea/Vomiting, Shortness of Breath, Loss of Appetite, Sleep Disorders, Constipation, Diarrhea, and Financial Difficulties) (Giesinger et al., 2014). Additionally, the EORTC-QLQ C-30 questionnaire has also been validated and translated into Indonesian by Perwitasari et al., (2011) so that it can be used to help assess the quality of life of cancer patients, in this case, especially penile cancer patients. As such, this study aims to further investigate the quality of life, specifically those health-related, for patients receiving penile cancer treatment in Haji Adam Malik General Hospital, Medan. Results from this study are hoped to be incorporated into the health services for penile cancer in order to develop a more accurate, and holistic patient-centered care for penile cancer patients. Materials and Methods The method used in this research is a descriptive observational research method with the primary objective of accurately describing a situation with the type of research design, namely a cross-sectional study. The study was conducted at Haji Adam Malik (HAM) General Hospital, Medan. The study was carried out after the ethics committee approved the proposal in September 2021. The population of this study was all patients with a diagnosis of penile cancer who underwent therapy at the HAM Hospital from September 2020 to September 2021, while the sample of this study was taken based on the total sampling technique considering the very high incidence of cases. Therefore, in total sampling, the number of samples is the same as the total population. The inclusion criteria consisted of penile cancer patients who underwent therapy from September 2020 to September 2021, who are willing to be included in the study and are competent to make such decision, marked by signing the informed consent. The exclusion criteria consisted of penile cancer patients who had undergone therapy before September 2020, penile cancer patients receiving second-line chemotherapy, patients experiencing penile cancer, not as the primary tumor, and uncooperative patients experiencing decreased consciousness. The research was conducted after obtaining a research permit from the Universitas Sumatera Utara (USU) Medical Faculty and then sending the permit to the Haji Adam Malik Hospital as the place for the research. After getting permission, the researcher can collect data. Furthermore, before asking about the patient’s willingness to be a respondent, comprehensive information regarding the study was given. If willing, participants will then be asked to sign the consent form, and to fill out the questionnaire. Participants would be accompanied by a researcher in case of any need of assistance. The instrument used in this study is a questionnaire consisting of several questions filled in directly by the respondent. The questionnaire has two parts; the first part contains patient demographic data, namely age, ethnicity, religion, marital status, education, occupation, and history of circumcision. The second part contains a list of questions in the standardized EORTC-QLQ C-30 from the European Organization for Research and Treatment of Cancer (EORTC), which has been validated by previous studies (Sosnowski et al., 2016). Data analysis is done using IBM SPSS Statistics v.26, which will be carried out when all data has been collected and the data has been checked and re-assures that the demographic information and the answers to the questionnaire have been filled in according to the instructions. Demographic data and each questionnaire assessment variable will be presented using descriptive analysis presenting the frequency and percentage distribution table. Results This research is a descriptive observational, cross-sectional study. The study was conducted at Haji Adam Malik General Hospital Medan, carried out in September 2021. In this study, the number of respondents studied was nine respondents. The following presents the distribution of frequencies and percentages based on the variables studied. Based on Table 1 related to age, it is found that the average age of the respondents is 54.44±8,647 (38-64) years old. Table 2 shows that most of the respondents are Bataknese, with as many as 5 (55.6%) respondents. Respondents with Javanese ethnicity of as many as 2 (22.2%), whereas respondents with Malay and Nias ethnic groups each as many as one people (11.1%).Based on Table 3, it is known that the religion consists of 4 Muslims (44.4%), Catholics (33.3%), and Protestants (22.2%). Based on Table 4, it is known that all respondents are married. Based on Table 5, respondents with junior high school education were 2 (22.2%), high school were 6 (66.7%), and vocational diploma were 1 (11.1%). Based on Table 6, it is known that the number of respondents with government employees is 1 (11.1%), private employees are 2 (22.2%), laborers are 1 (11.1%), and self-employed are 3 (33.3%), and not working as much as 2 (22.2%). Based on Table 7, respondents with a history of circumcision were 4 (44.4%), with no history of circumcision, and as many as 5 (55.6%). Table 8 shows that six respondents had a history of partial penectomy (66.7%), and 3 had a history of total penectomy (33.3%). Based on Table 9, regarding how participants rate their overall health, as many as 1 (11.1%) respondents assessed 4, as many as 6 (66.7%) respondents assessed 5, and 2 (22.2%) respondents gave an assessment of 6. Based on Table 10, regarding how participants rate their quality of life, 6 (66.7%) respondents assessed 5, and 3 (33.3%) respondents assessed 6. Based on Table 11, regarding the conclusion of the EORTC-QLQ C-30, it is known that all respondents have a poor quality of life. Table 1 Frequency Distribution of Research Respondents by Age Variable Mean+SD (Min-Max) Age 54.44+8.647 years (38-64 years) Table 2 Frequency Distribution of Research Respondents by Ethnicity Ethnic group Frequency Percentage (%) Java 2 22.2 Batak 5 55.6 Malay 1 11.1 Nias 1 11.1 Total 9 100.0 Table 3 Distribution of Frequency and Percentage by Religion Religion Frequency Percentage (%) Islam 4 44.4 Catholic 3 33.3 Protestant 2 22.2 Total 9 100.0 Table 4. Distribution of Frequency and Percentage Based on Marital Status Marital status Frequency Percentage (%) Marry 9 100.0 Total 9 100.0 Table 5 Distribution of Frequency and Percentage by Education Education Frequency Percentage (%) Junior High School 2 22.2 Senior High School 6 66.7 Diploma 1 11.1 Total 9 100.0 Table 6 Distribution of Frequency and Percentage by Occupation Work Frequency Percentage (%) Government officials 1 11.1 Private employees 2 22.2 Laborer 1 11.1 Self-employed 3 33.3 Does not work 2 22.2 Total 9 100.0 Table 7 Distribution of Frequency and Percentage Based on History of Circumcision Circumcision History Frequency Percentage (%) Found 4 44.4 Not Found 5 55.6 Total 9 100.0 Table 8 Distribution of Respondents' Answers: How would you rate your overall health during the past week? How would you rate your overall health condition during the past week? Frequency Percentage (%) Rating 4 from intervals 1-7 1 11.1 Rating 5 from intervals 1-7 6 66.7 Rating 6 from intervals 1-7 2 22.2 Total 9 100.0 Table 9 Distribution of Respondents' Answers: How would you rate your quality of life during the past week? How would you rate your quality of life during the past week? Frequency Percentage (%) Rating 5 from intervals 1-7 6 66.7 Rating 6 from intervals 1-7 3 33.3 Total 9 100.0 Table 10 Frequency Distribution by Quality of Life Quality of Life Frequency Percentage (%) Bad 9 100.0 Total 9 100.0 Table 11 Frequency Distribution by History of Therapy Therapy History Frequency Percentage (%) Partial penectomy 6 66.7 Total penectomy 3 33.3 Total 9 100% Discussion According to the National Comprehensive Cancer Network (NCCN), penile cancer accounts for less than 1% of all cancers affecting men, representing 0.91 per 100,000 population. In general, treatment includes surgery, which significantly impacts the patient, considering that part of the penis is removed, affecting the quality of life and daily functions. The quality of life of patients treated for penile cancer is not always mentioned in the medical literature, considering that the success of treatment is measured by healing the disease. However, quality of life an important part of its management (Montes Cardona and García-Perdomo, 2017). The areas with the highest incidence worldwide are characterized by a high prevalence of human papillomavirus infection and phimosis, which, together with low socioeconomic status, are major predisposing factors for developing penile cancer. The incidence of penile cancer peaks in the sixth decade of life, but penile cancer also affects younger men. For obvious reasons, organ-sparing penile cancer treatment is the preferred method of non-invasive and localized treatment of the disease, which can be managed in most cases with topical chemotherapy, laser ablation, or resurfacing of the gland. (Harju et al., 2021). Radiotherapy modalities may be considered for particular cases of invasive penile cancer; however, surgery remains the most reliable option for providing long-term healing (Kieffer et al., 2014). Most studies report a compromised quality of life among patients with penile cancer and a correlation between surgery rates and symptoms. In addition, as many as 50% of penile cancer patients may suffer from symptoms resembling traumatic stress disorder, which underscores the disease’s impact on the patient’s psyche (Suarez-Ibarrola, Cortes-Telles, and Miernik, 2018). However, more consensus has yet to be reached regarding the optimal metric to measure the quality of life among these patients. A recent systematic review highlighted this shortcoming and reported the many unmet needs experienced by patients with penile cancer. Therefore, further investigation is needed into the specific deficits that have the most profound effects on the quality of life of patients with penile cancer (Sosnowski et al., 2016a). The average age of the respondents was 54.44 years with a standard deviation of 8,647 years, where the youngest age was 38 years, and the oldest age was 64 years. The results of previous studies showed that the average age of penile cancer patients was 67 years with an interquartile range of 53.1–72.0, and patients were followed up for an average of 26.4 months (12.0–62.8). Another study in India on penile cancer patients aged from 22 years to 88 years with a mean age of 58.75 ± 12.30 years. Another study also found that the mean age of the sample was 54.3 years (range 37-77) at the time of diagnosis (Troiano and Nante, 2018). Our results are comparable with other analyses, notably the Maddineni et al. study in which treatment was reported to harm 40% of patients. However, the only consideration used in the investigator’s study, the only one validated for the Mexican population, was EORTC-QLQ-30. Although valuable evaluation tools, other instruments may be used for various cancers or other pathologies but have not been developed specifically for patients with penile cancer in Indonesia, which would require a validated translated version. (Troiano and Nante, 2018; Kusumajaya and Safriadi, 2022). Concerning sexual function, the mean age of the patients was 63.4 years. Therefore, it is essential to ask whether the absence of sexual activity is due to the disease itself, as indicated in previous analyses, such as the study by Opjordsmoean et al., or external factors, such as age-associated metabolic changes or marital problems (Sosnowski et al., 2016b). However, as this was not included in the questionnaire, personal investigation regarding sexual function towards each patient is important to note. Another study on penile cancer patients in Germany showed that the mean self-reported global QoL score was 54.0 (SD 5.9), which corresponds to the mean QoL (0-100 score) and is significantly below the mean age standard for German patients. For general function scores, the following mean scores were determined: physical (n=73), social (n=61), emotional (n=60), cognitive function (n=69), and role function (n=63). Compared with the German reference group, there were significant differences in role function (P < 0.001) and emotional (P < 0.001), social (P < 0.001), and cognitive (P < 0.001) functions, that is, to be expected from a significant reduction in this patient area by disease (Sosnowski et al., 2016b; Pardo et al., 2020). Concerning general symptom scores and individual items, the following scores were found: fatigue (n=35), nausea (n=6), pain (n=27), dyspnea (n=23), insomnia (n= 41), loss of appetite eating (n=25), constipation (n=19), diarrhea (n=10), and financial difficulties (n=25). These scores further represent healthcare-related aspects. Again, there were differences in the reference groups, especially in the areas of dyspnea (P < 0.005), insomnia (P < 0.001), loss of appetite (P < 0.001), constipation (P < 0.001), diarrhea (P < 0.001). 0.001), and financial difficulties (P < 0.001)(Vieira et al., 2020; Monteiro et al., 2021). It is mostly considered a classic side effect of systemic treatment. In particular, diarrhea was described as part of the 5-fluorouracil treatment. Furthermore, about 50% of the included patients were under 65 years of age, so some of these patients were employed. However, having to get back to work before diagnosis is no longer possible due to cancer and treatment, resulting in a loss of livelihood (Draeger et al., 2018; Sosnowski et al., 2019). In conclusion, the average age of the respondents was 54.44±8,647 (38-64). Most respondents are Bataknese and Muslims. Education level of most respondents were high school. Patients have various employments, however all patients were married. Patients who were circumcised are approximately equal to those who are not. Additionally, most underwent parnial penectomy as therapy. From our study, it can be concluded that the quality of life is poor among all penile cancer patients according to the EORTCQLQ-C30. Author Contribution Statement All authors contributed equally in this study. Acknowledgements None. ==== Refs References Draeger DL Sievert KD Hakenberg OW Cross-Sectional Patient-Reported Outcome Measuring of Health-Related Quality of Life With Establishment of Cancer- and treatment-specific Functional and Symptom Scales in Patients With Penile Cancer Clin Genitourin Cancer 2018 16 e1215 20 30201215 Harju E Pakarainen T Vasarainen H Health-Related Quality of Life, Self-esteem and Sexual Functioning Among Patients Operated for Penile Cancer – A Cross-sectional Study J Sex Med 2021 18 1524 31 Kieffer JM Djajadiningrat RS van Muilekom EAM Quality of life for patients treated for penile cancer J Urol 2014 192 1105 10 24747092 Kusumajaya C Safriadi F Characteristics of Penile Cancer at Tertiary Center Hospital: A Nine Years Study from 2010-2019 Indones J Cancer 2022 16 28 Monteiro LL Skowronski R Brimo F Erectile function after partial penectomy for penile cancer Int Braz J Urol 2021 47 515 22 33620995 Montes Cardona CE García-Perdomo HA Incidence of penile cancer worldwide: systematic review and meta-analysis Rev Panam Salud Pública 2017 2017 1 10 Pardo Munevar CA Mina Riascos S García- Perdomo HA Evaluación de la calidad de vida en pacientes con cáncer de pene: Una revisión sistemática Ciencia e Innovación en Salud 2020 2020 Sosnowski R Kulpa M Kosowicz M Quality of life in penile carcinoma patients – Post-total penectomy Cent Eur J Urol 2016a 69 204 11 Sosnowski R Kulpa M Kosowicz M Quality of life in penile carcinoma patients – Post-total penectomy Cent Eur J Urol 2016b 69 204 11 Sosnowski R Wolski JK Talewicz UZ Assessment of selected quality of life domains in patients who have undergone conservative or radical surgical treatment for penile cancer: An observational study Sex Health 2019 16 32 8 30532994 Suarez-Ibarrola R Cortes-Telles A Miernik A Health-Related Quality of Life and Sexual Function in Patients Treated for Penile Cancer Urol Int 2018 101 351 7 30149391 Troiano G Nante N Quality of Life After Surgical Treatment for Penile Carcinoma Int J Sex Health 2018 30 141 8 Vieira CB Feitoza L Pinho J Profile of patients with penile cancer in the region with the highest worldwide incidence Sci Rep 2020 10 32001736

PMC010xxxxxx/PMC10352740.txt

==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116161 10.31557/APJCP.2023.24.4.1373 Research Article Different Clinicopathological Characteristics in Indonesian Colorectal Patients with NRAS Mutations and HER2 Over-Expression Lukman Kiki 1 Reza Ade Tan 1 Hasibuan Lisa Y. 1 Sribudiani Yunia 2 Dewayani Birgitta Maria 3 Rudiman Reno 1 Primastari Etis 3 Nugraha Prapanca 1* 1 Department of Surgery, Faculty of Medicine, Universitas Padjadjaran / Dr. Hasan Sadikin General Hospital, Bandung, Indonesia. 2 Department of Basic Medical Sciences, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia. 3 Department of Pathologic Anatomy, Faculty of Medicine, Universitas Padjadjaran / Dr. Hasan Sadikin General Hospital, Bandung, Indonesia. * For Correspondence: rudiman@unpad.ac.id 2023 24 4 13731377 3 1 2023 17 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: This study aims to assess the association of subject characteristics and NRAS mutations with HER2 expression in CRC. Methods: This research is a cross-sectional study. The research subjects in this study were colorectal cancer patients in the Digestive Surgery division at Dr. Hasan Sadikin Hospital. There were 58 study subjects. Examination of NRAS mutations was carried out by Polymerase Chain Reaction (PCR) from fresh tumour tissue obtained from surgery or colonoscopy. Meanwhile, HER2 examination used the Immunohistochemistry (IHC) method of paraffin blocks for anatomical pathology examination of the same patients. Result: HER2 overexpression was found in 6/58 (10.3%) patients with CRC, and from 8 subjects with NRAS mutations, only 1 subject (1.7%) showed overexpression of HER2. Univariate analysis of HER2 expression showed no significant associations to age, sex, histologic feature, tumor location, and NRAS mutations. A significant association was found between HER2 expression and stage of the CRC with p=0.001. Conclusion: There is no association between NRAS mutations and HER2 overexpression in colorectal cancer patients. Key Words Colorectal cancer- HER2- IHC- NRAS- PCR ==== Body pmcIntroduction Colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women. According to GLOBOCAN 2020 data, there were 1.15 million new cases of colon cancer, 0.7 million new cases of rectal cancer, and 50,000 new cases of anal cancer in 2020 globally (Bray et al., 2018). With continued progress, these figures are predicted to increase to 1.92 million, 1.16 million, and 78,000 by 2040. Globally, CRC is one of the cancers with a steadily increasing incidence, accounting for 11% of all cancer diagnoses (Xi and Xu, 2021). Data in Indonesia based on GLOBOCAN in 2020 shows CRC in the fourth position with around 35,000 new cases each year, and an incidence number of 19.1 in men and 15.6 in women per 100,000 population (Bray et al., 2018; Erida et al., 2015). Colorectal cancer is a complex and genetically heterogeneous disease that drives multiple oncogenic signaling pathways. Pathogenic mechanisms, including microsatellite instability (MSI), CpG island methylation phenotype (CIMP), and chromosomal instability (CIN), represent 80–85% of the causes of all CRC cases (Porru et al., 2018). RAS/RAF/MEK/ERK/MAPK or MAPK/ERK (mitogen-activated protein kinases / extracellular signal-regulated kinases) is the most well-known pathway in the pathogenesis of KKR (Gong et al., 2018). RAS and BRAF are members of the MAPK/ERK pathway that mediates cellular responses to growth signals and are members of the multigene family, of which RAS is composed. of KRAS, NRAS and HRAS, while RAF consists of BRAF, RAF1 (c-Raf), and ARAF (Jafari et al., 2022). Members of the RAS family are frequently found in mutated, oncogenic forms in human tumors. Because it causes reduced intrinsic GTPase activity and insensitivity to GTPase-activating proteins, the mutated RAS protein is constitutively active, resulting in a constitutively active GTP-bound state and activation of pro-proliferative and tumorigenesis signaling pathways (Irahara et al., 2010; Schirripa et al., 2015). In total, activating mutations in the RAS gene occur in about 20% of all human cancers, mainly at codons 12, 13, or 61. Mutations in KRAS account for about 85% of all RAS mutations in human tumors, NRAS for about 15%, and HRAS for less than 1% (Irahara et al., 2010). The NRAS proto-oncogene (Neuroblastoma Rat Sarcoma Viral Oncogene Homolog) (locus 1p13.2) is a member of the RAS gene family that encodes proteins involved in signal transmission in cells and participates in the regulation of cell growth. NRAS gene mutations have also been associated with KKR tumors (Jafari et al., 2022; Prior et al., 2020). NRAS is commonly mutated in melanoma and hematopoietic cancers via mapping to chromosome 1. In the tumorigenesis pathway, NRAS mediates both MAPK and PI3K/AKT/MYC signaling.NRAS-induced classical MAPK signaling leads to cyclin D1 expression, cell cycle dysregulation, and promotion of prosurvival pathways. In addition, NRAS effectively prevents Glycogen Synthase Kinase-3 (GSK3)-mediated MYC phosphorylation via PI3K/AKT (phosphoinositide 3-kinase), resulting in increased activity of endogenous MYC protein. NRAS mutations cause RAS GTP to be continuously activated, resulting in malignant proliferation and metastasis (Wang et al., 2014). Research states that mutations in NRAS, BRAF, and KRAS are mutually exclusive (Meriggi et al., 2014; De Roock et al., 2010). One explanation for the phenotypic differences between KRAS and NRAS is that high expression of KRAS mutants can promote proliferation, while low expression of mutant NRAS can suppress apoptosis (Schirripa et al., 2015; Meriggi et al., 2014; De Roock et al., 2010). NRAS mutations may impair response to anti epidermal growth factor receptor (EGFR) or MoAbs (monoclonal antibodies), and significant differences in median OS (overall survival) were observed in NRAS-WT (wild-type) tumors compared with NRAS mutations (Schirripa et al., 2015). Sullivan et al. (2011) reported that NRAS mutations were associated with a lack of response to cetuximab. In addition, because of the high percentage of resistance to therapy in the same CRC patients, an additional predictive marker for cetuximab-targeted therapy was considered, namely human epidermal growth factor receptor 2 (HER2). Human epidermal growth factor receptor 2 is a monomeric receptor present on the cell surface. After the ligand binds to its extracellular domain, the HER protein undergoes dimerization and transphosphorylation from its intracellular domain. HER2 lacks a direct activating ligand and may be in a constitutively activated state or become activated after heterodimerization with other HERs, such as HER1 and HER3. Homo- or heterodimerization results in autophosphorylation of tyrosine residues in the cytoplasmic domain of their receptors and induces various signaling pathways, especially MAPK, PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase), and PKC (protein kinase C), triggering cell proliferation, differentiation, angiogenesis, and invasion (Iqbal et al., 2014). Overexpression or amplification of HER2 is an established therapeutic target in breast and gastric cancer. The role of HER2 in CRC is less clear (Muzny et al., 2012). Several studies have shown that HER2 gene amplification is significantly associated with resistance to cetuximab or panitumumab and is associated with significantly worse progression-free survival (PFS) and overall survival (OS) trends (Bertotti et al., 2011). In CRC, HER2 overexpression and amplification have also been used as potential therapeutic targets. Although several studies have reported the incidence of HER2 overexpression or amplification in CRC, it varies widely, ranging from 0% to 83% (Muzny et al., 2012; Lee et al., 2014). This study aims to assess the relationship between the NRAS mutation and HER2 expression in colorectal cancer patients. Materials and Methods Study design and setting This research is a cross-sectional study. The research subjects in this study were colorectal cancer patients in the Digestive Surgery division at Dr. Hassan Sadikin Hospital. There were 58 study subjects. Examination of NRAS mutations was carried out by polymerase chain reaction (PCR) from fresh tumor tissue obtained from surgery or colonoscopy. Meanwhile, the HER2 examination used the immunohistochemistry (IHC) method of paraffin blocks for anatomical pathology examination. The Dr. Hasan Sadikin Hospital ethics committee approved the study with a waiver of informed consent. Tumour Tissue Collection Blood sampling and PCR Analysis Tissue samples were collected from a colonoscopy biopsy or surgical resection. Part of the tissue was immediately sent to the Pathology Anatomy Laboratory for Hematoxylin-Eosin (HE) staining, and the remaining tissue was stored in DNA/RNA shield solution (Zymo Research, CA, USA) for further genomic DNA isolation. A maximum of 10 milligrams of fresh colon tissues were dissected into single cells by vortexing them for 40 seconds using the ZR BashingBead Lysis Tube (Zymo Research, CA, USA). The cell suspension was centrifuged at 14000 rpm for 30 seconds. Cell pellets were used for genomic DNA isolation using Quick-DNATM Miniprep (Zymo Research, CA, USA) according to the manufacturer’s protocol. The quality of DNA was measured using a NanoDropTM 2000 spectrophotometer (Thermofisher). A polymerase chain reaction of all exons of NRAS was performed. All exons were amplified using the touch-down PCR method with annealing temperatures ranging from 65 oC to 55 oC. Sequencing was performed forward, and identified mutations were validated in reverse. The DNA amplification step in pre-sequencing was performed using the Big Dye Terminator V3.3 kit (Applied Biosystem, Foster City, CA, USA) on an ABI 3500 automated sequencer. In Silico, an analysis of identified mutations was performed to predict the functional impact of the mutations. The analysis was performed using three online software programs: Mutation Taster 2021, PolyPhen-2, and SIFT. HER2 Protein Expression using IHC Analysis Tissue samples were examined histologically by HE staining for the diagnosis of CRC. The subject’s block paraffin was immunohistochemically stained using the monoclonal antibody for HER2 (Brand Cell Marque, catalogue 237R-24). Two experienced pathologists (B.S.H. and H.Y.) scored independently, following the consensus recommendations for HER2 scoring for CRC, on a 4-point scale (0, 1+, 2+, 3+). As far as HER2 localization is concerned, 2+ and 3+ showed predominantly membrane localization, while 1+ and 0+ showed more staining in the cytoplasm of the tumor cells. Our study focused on the assessment of membranous HER2 expression. +3 HER2 expression was identified as highly positive or over-expression. Statistical analysis. The statistical analysis of the variable was performed using the SPSS 26 software (SPSS Inc., Chicago, IL, USA). The comparison of the variables was based on the chi2 test. Results Subject characteristics A total of 58 patients were included in the study. There were 37 female and 21 male patients. The mean age was 56.5 years ± 1.41, and 74.1% of patients were older than 50. Most of the patients showed adenocarcinoma histologically (87.9%), and 63.8% showed well differentiation of the tumor. There were 37 (63.8%) rectal cancer, and 21 (36.2%) colon cancer patients. NRAS mutation was shown in 8 (13.8%) patients, and over-expression of HER2 was shown in 6 (10.3%) patients. Univariate analysis of variables and HER2 expression One variable affecting HER2 expression was statistically significant (p < 0.05) in univariate analysis, as shown in Table 3. In stage II, there were 5 (8.6%) subjects with HER2 overexpression, with p = 0.001. Table 1 Characteristics of Research Subjects Variable Proportion (%) Age Mean 56.83 ± 1.41 year Median 56.5 year Age < 50 15 (25.9%) > 50 43 (74.1%) Sex Male 21 (36.2%) Female 37 (63.8%) Histologic Adenocarcinoma 51 (87.9%) Mucinous Adenocarcinoma 5 (8.6%) Signet Ring Cell 1 (1.7%) Neuroendocrine 1 (1.7%) Grade Well Diff 37 (63.8%) Moderately Diff 7 (12.1%) Poorly Diff 7 (12.1%) Specific 7 (12.1%) Tumor location Colon 21 (36.2%) Rectum 37 (63.8%) Stage I 1 (1.7%) II 12 (20.7%) III 17 (29.3%) IV 28 (48.3%) Metastases Liver 16 (27.6%) Lung 3 (5.2%) Bone 1 (1.7%) Omentum 1 (1.7%) Perforation 1 (1.7%) Uterine 1 (1.7%) Liver and bone 2 (3.4%) Liver and lung 1 (1.7%) NRAS Mutation 8 (13.8%) Wild type 50 (86.2%) HER2 0 23 (39.7%) + 12 (20.7%) ++ 19 (29.3%) +++ 6 (10.3%) HER2 Normal expression 52 (89.7%) Over Expression (+++) 6 (10.3%) Table 2 Univariate Analysis for Subject Characteristics with NRAS Mutations Mutation Wild Type Chi-square (p.value) Age 0.952 Early onset (<50) 2 (3.4%) 13 (22.4%) Late onset (>50) 6 (10.3%) 37 (63.8%) Sex 0.935 Male 3 (5.2%) 18 (31.0%) Female 5 (8.6%) 32 (55.2%) Histologic 0.923 Adenocarcinoma 7 (12.1%) 44 (75.9%) Mucinous Adenocarcinoma 1 (1.7%) 4 (6.9%) Signet Ring Cell 0 1 (1.7%) Neuroendocrine 0 1 (1.7%) Grade 0.113 Well Diff 3 (5.2%) 34 (58.6%) Moderately Diff 1 (1.7%) 6 (10.3%) Poorly Diff 3 (5.2%) 4 (6.9%) Specific 1 (1.7%) 6 (10.3%) Tumor location 0.096 Colon 5 (8.6%) 16 (27.6%) Rectum 3 (5.2%) 34 (58.6%) Stage 0.435 I 0 1 (1.7%) II 1 (1.7%) 11 (19.0%) III 1 (1.7%) 16 (27.6%) IV 6 (10.3%) 22 (37.9%) HER2 0.829 Normal expression 7 (12.1%) 45 (77.6%) Over Expression (+++) 1 (1.7%) 5 (8.6%) Table 3 Univariate Analysis for Subject Characteristics with HER2 Expression Over Expression Normal Chi-square (p.value) Age 0.659 Early onset (<50) 2 (3.4%) 13 (22.4%) Late onset (>50) 4 (6.9%) 39 (67.2%) Sex 0.293 Male 1 (1.7%) 20 (34.5%) Female 5 (8.6%) 32 (55.2%) Histologic 0.862 Adenocarcinoma 5 (8.6%) 46 (79.3%) Mucinous Adenocarcinoma 1 (1.7%) 4 (6.9%) Signet Ring Cell 0 1 (1.7%) Neuroendocrine 0 1 (1.7%) Grade 0.789 Well Diff 4 (6.9%) 33 (56.9%) Moderately Diff 0 7 (12.1%) Poorly Diff 1 (1.7%) 6 (10.3% Specific 1 (1.7%) 6 (10.3% Tumor location 0.291 Colon 1 (1.7%) 20 (34.5%) Rectum 5 (8.6%) 32 (55.2%) Stage 0.001 I 0 1 (1.7%) II 5 (8.6%) 7 (12.1%) III 0 17 (29.3%) IV 1 (1.7%) 27 (46.6%) NRAS 0.829 Wild type 5 (8.6%) 45 (77.6%) Mutation 1 (1.7%) 7 (12.1%) Discussion NRAS mutations in this study showed eight subjects (13.8%) with positive mutation results. In this study, the proportion of NRAS mutations was higher than in several other studies. A study by Irahara et al., (2010) found NRAS mutations in 2.2% of the 225 CRC patients. A study by Schiripa et al., (2015) found NRAS mutations in 6% of the 785 included CRC patients. A study by Levi et al., (2016) showed NRAS mutations in 6 (5%) of 121 patients with CRC. Ibarra et al., (2020) reported that of 500 CRC patients, only 20 (4%) showed NRAS mutations. In this study, more NRAS mutations appeared in the colon than the rectum (5 vs. 3), and more NRAS mutations were found in metastatic CRC (mCRC) than early stage CRC (6 vs. 2), but statistically, this difference was not significant. Similar to the studies of Irahara et al., (2011) and Levi et al,.(2016), univariate analysis of NRAS mutations did not show a significant association between NRAS mutations and age, sex, type of anatomic pathology, grade, stage, or location of the tumor. A study by Ibarra et al., (2020) showed that more NRAS mutations appeared in the rectum than the colon (3 vs. 2) and showed better tumor differentiation, but statistically the difference was not significant. HER2 expression in this study showed that there were six subjects (10.3%) with overexpression results. In this study, the proportion of overexpression of HER2 appeared to be higher than in several other studies. In the study of Seo et al., (2014), there were two HER2 assessment cohorts, the first cohort involved 365 patients with CRC, and HER2 overexpression was found in 8 subjects (2.2%), while the second cohort involved 174 patients with stage IV CRC, and HER2 overexpression was present in 5 subjects. (2.9%). Valtorta et al., (2015) conducted a study on 304 patients with CRC. 14 subjects (4.6%) showed HER2 overexpression. In the study by Ross et al., (2018), 148 subjects (1.6%) of 8,887 patients with CRC showed excess HER2 expression. Razzaq et al., (2021) assessed HER2 expression in patients with CRC, out of 17 patients with CRC, there were four subjects (23.52%) with excess HER2 expression. Similar to them, HER2 expression did not show a significant relationship with age, sex, or type of anatomic pathology. In this study, a significant association was only seen at the tumor stage, most of the HER2 overexpression was found at stage II (p = 0.001). Meanwhile, in Seo et al., (2014), HER2 overexpression was associated with tumor location and was more frequently found in the rectum than in the colon (p =  0.013 in cohort 1, p =  0.009 in cohort 2). In study by Razzaq et al., (2021), HER2 overexpression was associated with tumor grade (p = 0.03). A univariate analysis of NRAS mutations with HER2 overexpression showed p = 0.829. This indicated that there was no significant relationship between NRAS mutations and HER2 overexpression. A cross-tabulation of HER2 expression and NRAS mutations showed that out of 8 subjects showing NRAS mutations, only one subject had excess HER2 expression. In a similar study conducted by Ross et al., (2018), out of 8,887 patients with CRC, there were 4.3% of subjects with NRAS mutations and 1.6% of subjects with HER2 overexpression. The cross-tabulation of HER2 expression and NRAS mutations showed that 3.3% of NRAS mutation patients showed over-expression of HER2. In this study, the number of patients with mCRC was 28 (48.7%), and six of them (21.4%) had NRAS mutations, and 22 (78.6%) had wild-type NRAS. In contrast, only one (3.6%) of the 28 mCRC patients had HER2 overexpression. In the study of Valentini et al., (2018), out of 29 mCRC patients, two (7%) were found with NRAS mutations, and only one (3.4%) had HER2 overexpression. Our study, similar to those of Ross et al., (2018) and Valentini et al., (2018), showed no significant relationship between NRAS mutations and HER2 overexpression in colorectal cancer patients. NRAS mutations and HER2 are not the only deterministic carcinogenic factors for CRC, other carcinogenic and clinicopathological factors also contribute, such as patient sex, age, molecular subtype, and tumor stage. This study showed that NRAS mutations and HER2 overexpression were not related to clinicopathological factors, this may be related to the small number of research samples in this study. However, it is important to examine NRAS and HER2 mutations as a consideration for continuing therapy with EGFR inhibitors and anti-HER2 in patients with CRC. In conclusion , there is no association between NRAS mutations and HER2 overexpression in colorectal cancer patients. Author Contribution Statement Ade Tan Reza and Prapanca Nugraha participated in collecting the patient’s information. Yunia Sribudiani participated in analyzing the NRAS mutation. Birgitta M. Dewayani and Etis Primaswati participated in the histologic examination of the colorectal diagnosis and the IHC examination of HER2. Kiki Lukman, Lisa Y Hasibuan, and Reno Rudiman analyzed the data, drafted the manuscript, and finalized the manuscript. Acknowledgements We thank all the patients who agreed to participate in this study and the trainees and surgical residents who helped carry out this study. Funding Statement The preparation of this study was supported by internal funding from Riset Kompetensi Dosen Unpad (RKDU) Faculty of Medicine, Universitas Padjadjaran with register number 2203/UN.6.3.1/PT.00/2022. Approval This study was approved as Ade Tan Reza’s thesis Ethical Declaration The Dr. Hasan Sadikin Hospital ethics committee approved the study with No. Ethical Approval LB.02.01/X.6.5/327/2022. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. Conflict of Interest The authors have no conflicts of interest to declare. ==== Refs References Bray F Ferlay J Soerjomataram I GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J Clin 2018 68 394 424 30207593 Bertotti A Migliardi G Galimi F A molecularly annotated platform of patient- derived xenografts (“xenopatients”) identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer Cancer Discov 2011 1 508 23 22586653 De Roock W Claes B Bernasconi D Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: A retrospective consortium analysis Lancet Oncol 2010 11 753 62 20619739 Erida Y Aminah H Yulianti H Hernowo BS Vitamin D Receptor (VDR) and Phosphatidylinositol 3-Kinase (PI3K) Independently Affected Colorectal Adenocarcinoma Differentiation Indones J Clin Pharm 2015 4 264 74 Gong S Xu D Zhu J Efficacy of the MEK Inhibitor Cobimetinib and its Potential Application to Colorectal Cancer Cells Cell Physiol Biochem 2018 47 680 93 29794421 Jafari M Laraqui A Baba W Prevalence and patterns of mutations in RAS/RAF/MEK/ERK/MAPK signaling pathway in colorectal cancer in North Africa BMC Cancer 2022 22 1 4 34979993 Ibarra HE Jiang X Gallegos-Gonzalez EY KRAS, NRAS, and BRAF mutation prevalence, clinicopathological association, and their application in a predictive model in Mexican patients with metastatic colorectal cancer: A retrospective cohort study PLoS One 2020 15 Irahara N Baba Y Nosho K NRAS mutations are rare in colorectal cancer Diagnostic Mol Pathol 2010 19 157 63 Iqbal N Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications Mol Biol Int 2014 2014 1 9 Lee WS Park YH. Lee JN Comparison of HER2 expression between primary colorectal cancer and their corresponding metastases Cancer Med 2014 3 674 80 24668895 Levi M Prayogi G Sastranagara F Clinicopathological Associations of K-RAS and N-RAS Mutations in Indonesian Colorectal Cancer Cohort J Gastrointest Cancer 2018 49 124 31 28044264 Meriggi F Vermi W Bertocchi P The Emerging Role of NRAS Mutations in Colorectal Cancer Patients Selected for Anti-EGFR Therapies Rev Recent Clin Trials 2014 9 8 12 24758538 Muzny DM Bainbridge MN Chang K Comprehensive molecular characterization of human colon and rectal cancer Nature 2012 487 330 7 22810696 Porru M Pompili L Caruso C Targeting kras in metastatic colorectal cancer: Current strategies and emerging opportunities J Exp Clin Cancer Res 2018 37 1 10 29301578 Prior IA Hood FE Hartley JL The frequency of ras mutations in cancer Cancer Res 2020 80 2669 974 Razzaq EA Venkatachalam T Bajbouj K HER2 overexpression is a putative diagnostic and prognostic biomarker for late-stage colorectal cancer in North African patients Libyan J Med 2021 16 Ross JS Fakih M Ali SM Targeting HER2 in colorectal cancer: The landscape of amplification and short variant mutations in ERBB2 and ERBB3 Cancer 2018 124 1358 73 29338072 Schirripa M Cremolini C Loupakis F Role of NRAS mutations as prognostic and predictive markers in metastatic colorectal cancer Int J Cancer 2015 136 83 90 24806288 Seo AN Kwak Y Kim DW HER2 status in colorectal cancer: Its clinical significance and the relationship between HER2 gene amplification and expression PLoS One 2014 9 Sullivan KM Kozuch PS Impact of KRAS Mutations on Management of Colorectal Carcinoma Patholog Res Int 2011 2011 1 11 Valentini AM Cavalcanti E Di Maggio M Caruso ML RAS-expanded Mutations and HER2 Expression in Metastatic Colorectal Cancer: A New Step of Precision Medicine Appl Immunohistochem Mol Morphol 2018 26 539 44 30199395 Valtorta E Martino C Sartore-Bianchi A Assessment of a HER2 scoring system for colorectal cancer: Results from a validation study Mod Pathol 2015 28 1481 91 26449765 Wang Y Velho S Vakiani E Mutant N-RAS Protects Colorectal Cancer Cells from Stress-Induced Apoptosis and Contributes to Cancer Development and ProgressionN-Ras in Colorectal Cancer Cancer Discovery 2013 3 294 307 23274911 Xi Y Xu P Global colorectal cancer burden in 2020 and projections to 2040 Transl Oncol 2021 14 101174 34243011

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116167 10.31557/APJCP.2023.24.4.1419 Research Article Prediction and Detection of Cervical Malignancy Using Machine Learning Models Devi Seeta 1* Gaikwad Sachin Ramnath 2 R Harikrishnan 2 1 Symbiosis College of Nursing (SCON), Symbiosis International Deemed University (SIDU), Pune- 412115, India. 2 Symbiosis Institute of Technology (SIT), Symbiosis International Deemed University (SIDU), Pune- 412115, India. * For Correspondence: drseetadevi1981@gmail.com 2023 24 4 14191433 18 1 2023 23 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: Human papillomavirus and other predicting factors are responsible causing cervical cancer, and early prediction and diagnosis is the solution for preventing this condition. The objective is to find out and analyze the predictors of cervical cancer and to study the issues of unbalanced datasets using various Machine Learning (ML) algorithm-based models. Methods: A multi-stage sampling strategy was used to recruit 501 samples for the study. The educational intervention was the video-assisted counseling which is consisted of two educational methods: a documentary film and face-to- face interaction with women followed by reminders. Following the collection of baseline data from these subjects, they were encouraged to undergo Pap smear screening. Women having abnormal Pap tests were sent for biopsy. Machine learning classification methods such as Decision Tree (DT), Random Forest (RF), Logistic Regression (LR), Multi-layer Perceptron (MLP) and Naive Bayes(NB) were used to evaluate the unbalanced input and target datasets. Result: Merely 398 women out of 501 showed an interest to participate in the study, but only 298 stated a willingness for cervical screening. Atypical malignant cells were discovered on the cervix of 26 women who had abnormal pap tests. These women had guided for further tests, such as a cervical biopsy, and seven women had been diagnosed with cervical cancer. LR in models 1, 2, and 4 showed 88% to 94% sensitivity with 84% to 89% accuracy, respectively for cervical cancer prediction, whereas DT in models 3, 5, and 6 algorithms exhibited 83% to 84% sensitivity with 84% to 88% accuracy, respectively. The NB and LR algorithms produced the highest area under the ROC curve for testing dataset, but all models performed similarly for training data. Conclusion: In current study , Logistic Regression and Decision Tree algorithms were identified as the best-performed ML algorithm classifiers to detect the significant predictors. Key Words Prediction and detection Cervical malignancy Machine learning algorithms Prediction Cervical screening ==== Body pmcIntroduction Deep Learning (DL) and Machine Learning (ML) are effective algorithm classifiers in predicting brain tumours, breast cancer, thermal sensation, dementia evaluation, COVID-19, renal disorders, heart problems, and cervical cancer (Abbas et al., 2021; Ayoub et al., 2021; Khamparia et al., 2021). Because of technological improvements in the health care system, several medical disorders can now be predicted at an earlier stage based on identifying critical factors than traditional diagnostic approaches (Chen H et al., 2021; Javed et al., 2021; Javed et al., 2020; Sarwar et al., 2019). According to Global Statistics (2020), around 604,127 cervical malignant cases were detected (Parkin et al., 2005). Whereas, Cancer.Net Editorial Board survey stated that across the globe, about 341,831 women died from cervical cancer in 2020. Cervical cancer is primarily found in developing countries, accounting for 83% of deaths (Salmi et al., 2019). Cervical malignant growths are the 4th utmost leading cause of death in women worldwide. It is one of the most serious malignancies that endangers women’s health, and initial symptoms are difficult to detect until the disease has progressed to stage II (Aminisani et al., 2012). The diseased cells damage the cervical cells, and these cells migrate to other organs, including the lungs, heart, liver, kidneys etc. (Do et al., 2001). The most common cause of the rising prevalence of cervical cancer among women, particularly in developing countries, is a lack of awareness about screening measures that aid in early diagnosis (MacCosham et al., 2020). The burden of cervical cancer is being reduced in many countries, particularly in developed countries, by implementing predictive, detective, preventive, and systematic treatment techniques. Cervical cancer deaths decreased from 4% per year in 1996-2003 to 1% per year from 2010 to 2019. (Cancer.Net Editorial Board). Screening tests enable clinicians to treat precancerous lesions at an early stage, preventing them from progressing to malignant tumours. Although, the death rate in developing nations continues to rise due to a lack of resources, insufficient preventive methods, a lack of freely available Human papillomavirus (HPV) vaccine programmes, and a lack of awareness initiatives. HPV contamination is one of the responsible factors for the development of cervical malignancy. HPV is primarily transmitted through sexual contact. It’s acquisition has become increasingly associated with unusual sexual behaviours such as early sexual exposure, multiple sexual partners, and so on. Precancerous lesions take approximately 5 to 10 years to develop into malignant cells (MacCosham et al., 2020; Schiffman et al., 2007). Thereby providing an accessible time for women to go for cervical screening at least once in every three years with a Pap smear or visual inspection with acetic acid (VIA) or HPV DNA test, which assists them in diagnosing cervical cancer at an early stage. Cervical cancer is a preventable disease condition because it can be detected early by employing predictive and screening models. However, in developing nations, only 5% of women participate in cervical screening (Aminisani et al., 2012). Furthermore, cytological factors in Pap smear test are considered as diagnostic- predictive aspects because they identify the structure of the gland cell, squamous epithelial tissue, metaplastic cells, aberrant polymorphic cells, and dysplasia cells, as well as the existence of blood, bacteria, and fungus in the client’s samples (Do et al., 2001). Numerous susceptible predictors of cervical carcinoma have been identified, include smoking by the individual or their partner, inadequate nutrition, immunosuppression, use of immunomodulatory drugs, prolonged contraception utilization, racial groups, deficiency of vitamin A, C and folate, having multiple sex partners, subsequent pregnancies, childbearing at a young age, sexually-transmitted genital tract infections, low socioeconomic status, and illiteracy (Latha et al., 2014; Mandelblatt et al., 1991; Randall et al., 2016; Workowski et al., 2015). Daily, health sector creates huge volumes of data that can be utilized to forecast future sickness based on a patient’s treatment history and health information. Furthermore, by incorporating essential healthcare data, these areas can be enhanced. Non-invasive classification technologies, such as supervised machine learning (ML), are critical for cervical carcinoma prediction (Ramondetta, 2013; Workowski et al., 2015). ML in health care allows researchers to process massive amounts of complex medical data and evaluate it for curative ideas. Physicians use this material to extend medical treatment to patients. Patient satisfaction may enhance due to ML in healthcare coverage. Among the methods utilized are the Decision Tree (DT), Random Forest (RF), Logistic Regression (LR), Neural Network (NN), Naive Bayes, Artificial Neural Network Decision tree, and Support Vector Machines (SVM) (Mukhopadhyay et al., 2016; Nematollahi et al., 2017; Rezaianzadeh et al., 2019). The aim of this study was to deploy the various ML algorithm based models to understand the issues in uneven datasets. Following were the objectives of this study 1. To find out and analyze the predictors of cervical cancer using machine learning classifiers. 2. To study the issues of unbalanced datasets using various ML algorithm-based models. 3. To conduct the survey in view to find out the concerns of women regarding cervical cancer screening and that provides an accurate message to the readers. This paper includes the various sections namely, Related work, Research methodology, Results, Discussion and Conclusion. Related work As per the literature section criteria (LSC), the authors examined the relevant research papers from various databases. The current study investigated several electronic resources, including SCOPUS, PubMed, Institute of Electrical and Electronics Engineers (IEEE) Xplore, and Springer. The article listed below provides existing studies on the current study. Literature Selection Criteria It is one of the most critical aspects of the literature selection criterion since it enables researchers to arrange their work systematically, especially while downloading articles. Regarding search criteria, the authors ensured that the chosen publication was at least published in SCOPUS indexed journals. Inclusion criteria followed in the review of the current study • The purpose of the study was included in the researcher article. • The duration of this survey was established between 2015 and Nov 2022, and it attempted to comprehend the insights of earlier studies. Recent studies focused on several methodologies based on standard machine learning approaches, such as k-nearest neighbors (KNN), K-means clustering and RF, for early detection of cervical malignancy. A study was conducted to examine the effectiveness of several means in artificial neural networks to detect malignancy, and non-malignant cells (Singh et al., 2020). The researchers aimed to demonstrate a way of screening for cervical cancer utilizing cervigram pictures and the directed local histogram methodology (OLHT) (Asadi et al., 2020). Nithya et al., 2019 attempted to determine the level of cervical infection using the UCI data repository and six categorization models. In this study, physicians verified pre-processing data to confirm some risk factors and perform validation. The models’ performance was further evaluated using 10-fold cross-validation . A study had examined various measurements, including accuracy, sensitivity, specificity, and area under the curve (AUC). In that sequence, the QUEST values were 95.55, 90.48, 100, and 9.20, respectively. The authors employed an integrated learning technique to diagnose machinery faults. Each model training was applied at an indigenous level to improve learning performance (Lu et al., 2020). For the prediction of cervical cancer (Jahan et al., 2021), authors applied various types of ML classification algorithms. This study aimed to identify the topmost features that can cause cervical cancer utilizing eight well-known classification algorithm methods, including Multilayer Perceptron, Random Forest and k-Nearest Neighbor, Decision Tree, Logistic Regression, SVC, Gradient Boosting, and AdaBoost. The measures used to assess the performance of those classifications were accuracy, recall, precision, and f1-score. The MLP classification method worked admirably in detecting a wide range of relevant features in the datasets (Jahan et al., 2021). Authors (Jaswinder Singh et al., 2019) attempted to provide a model for cervical cancer prediction utilizing the UCI data repository and ML classification models. The data was pre-processed, and then the repository data was updated by extraction and validation. This study model included ten data elements connected with four stages. The pre-processed data were made available to the physician for verification before training the ML classifiers. Six classifiers were utilized in this research, with the decision tree classifier confirming the suitable stage prediction in terms of false-positive rate, f1-measure, and precision (Jaswinder Singh et al., 2019). A study had described the numerous LM classifiers for the early prediction of cervical cancer, including multi-layer perceptron, decision trees, random forest, K-Nearest Neighbor, and Naive-Bayes. The authors of this study examined the performance of various ensemble methods (AdaBoost, Stochastic Gradient Boosting, Random Forests, and Extra Trees) and ML classifiers (SVM and K-Nearest Neighbor) for predicting cervical cancer based on risk factors. This study’s measurement metrics are F1 score, Area Under Curve, and Recall. The extra trees classifier performed the best, with 96% accuracy (Ahishakiye et al., 2021). Al Mudawi et al.,2022 presented a report in which they used ML classifiers to predict cervical cancer. The study is divided into four phases: dataset, data pre-processing, predictive model selection, and pseudo-code assignment. This work utilized algorithm classifiers such as decision tree, logistic regression, K-nearest neighbor’s algorithm, adaptive boosting, gradient boosting, random forest, and XGBoost. Adaptive boosting, Random forest (RF), decision tree, and gradient boosting algorithms performed best with the highest accuracy score of 100%, while SVM also performed with 99% accuracy. Studies attempted to employ five machine learning algorithms in those investigations: random forest, KNN, C5.0, SVM, and RPart, they achieved the highest accuracy rates of 97, 96.9, 96, 88, and 88, respectively. ML approaches such as decision tree, random forest, and logistic regression have been utilized in combination with the voting method (Alyafeai et al., 2020; Mukama et al., 2017) and assimilated carcinomas of the cervix prediction models and a cervical screening pipeline based on cervical imaging. A deep neural network-learning model was used to automate the detection and diagnosis of cervical malignant tumours. This finds the union intersection (IoU) 1,000 times faster than state-of-the-art data-driven simulation, with a detecting accuracy of 0.68. By automating the diagnosis of cervical cancer from Pap-drug pictures, (William et al., 2019) used a model to reduce the likelihood of errors. For image improvement, a local adaptive histogram was applied. Authors (Unlersen et al., 2017) collected data from 858 patients with 33 variables employed in predictive analysis to detect cervical cancer. A unique machine learning techniques such as Multilayer Perceptron, BayesNet, and k-Nearest Neighbor were used for accurate prediction. The performance of this algorithm is measured using a confusion matrix and the proportion of correctly recognized occurrences. The performance measurements of confusion matrix are precision call, recall score, F1 score and accuracy. The confusion matrix and cost-effectiveness in terms of CPU time are used to analyze the performance of numerous approaches. This proposed approach obtains the best feature while decreasing process time to aid clinicians in the early detection of cervix carcinoma. Logistic Regression (LR) yields 100% accuracy but requires additional CPU time. Nonetheless, 99% accuracy is obtained in exchange for reduced CPU time (Singh et al., 2020). This paper deals with unbalanced datasets using a combination of attribute selection methods and evaluates the performance of ML algorithms-based models. In contrast, previous studies focused on splitting the balanced dataset, and some studies focused on finding the best ML algorithms for predicting cervical cancer. The authors of this paper also employed the Gini index analysis to determine the percentage of predictors that appeared in the target categories. Materials and Methods A prospective study was conducted, which consists of four phases, as listed below 1. Researchers reviewed various databases, recent research studies, and other library resources to identify risk factors and critical variables likely to lead to cervical cancer. Machine learning techniques were also found as important for predicting cervical cancer. 2. Researchers developed a questionnaire based on a literature review and expert opinion, focusing on risk factors for cervical cancer. Cronbach’s alpha is used to calculate and confirm the reliability, and its value was 0.92, indicating that the tool’s reliability was determined to be effective. 3. Researchers sought permission from various Pimpri Chinchwad Municipal Corporation (PCMC) hospitals to collect data and obtain ethics approval from Symbiosis Independent Ethics Committee, Pune. The hospital and volunteers were recruited using a multistage sampling strategy from various PCMC facilities. A total of 501 women participated in the study; after collecting baseline data from the women, they were motivated to undergo the Pap smear screening. Of 501 women, 298 expressed interest in the study and participated in cervical screening. Atypical malignant cells were found on the cervix among 26 women with abnormal pap tests. These women had guided further investigations, such as a cervical biopsy, of those 7 women were diagnosed with cervical cancer. 4. Data Preprocessing: The data was normalized using the Continuize discrete variables technique with a single feature value. Due to class imbalance in the dataset, we adopted the methodology of dividing the data into six models. Initially, we had 501 samples; out of this sample, 298 samples had undergone screening. Seven samples were diagnosed with cervical cancer, while others had negative screening results. Due to the class imbalance, this data cannot be used for accurate prediction as per medical considerations. This imbalanced class may provide wrong prediction values. To overcome this issue, we have reduced the class imbalance by dividing the dataset into six models and keeping the less-numbered samples. Samples in these models were selected randomly from 298 participants, the five models consisted of 50 samples, while 6th model entailed 45 samples, and each of these models mechanized with seven positive samples. Figure 1 indicates a similar workflow for all six models. The prediction model was evaluated using 5 folds of cross validation with stratification method and the target class was chosen as the average over classes. 5 algorithms’ hyperparameters are fine-tuned. Challenges of data collection The collection of data from women for cervical screening (Pap test) became challenging due to several reasons. Some of the common challenges are included below Lack of awareness Many women were not aware of the importance of regular cervical screening and were not ready for testing. This resulted in a low participation percentage; despite the fact that the authors recruited 501 subjects for the study, only 298 had undertaken screening. Fear and embarrassment Many women felt uncomfortable or embarrassed about undergoing a Pap test. Accessibility and cost In all health centres, cervical screening services were not readily accessible, or the cost of screening was too high. Thus, the authors had decided to collect the data only from YCM hospital where the testing charges were less and for some women charges are waved off. Lack of trust Some women had the problems of trusting healthcare providers, the healthcare system, and results of test. To overcome these challenges, we had adopted some strategies such as education and awareness campaigns, outreach programs, and the provision of language support were implemented to encourage women to participate in cervical screening programs. Machine learning algorithms for prediction of medical dieases This section explains the methods of machine learning techniques used to predict cervical cancer in the current study. Decision Tree (DT) algorithm DT trails the rules of divide and conquer. In DT algorithm, the features will take up the different values called as classification trees. To resolve classification and regression issues, the classification and regression tree to be used. DT purports to have a lot of tree branches, which is why it has the Tree in its name. The DT starts with the root nodes, just as a tree originates through its roots. The leaves represent special categories, while the branches represent the mix of characteristics that result in the categorical variables. DT can also accept the continuous variables known as the regression trees. The commonly used DT algorithms in medical field are C4.5 and EC4.5 (Lilhore et al., 2022; Tiwari et al., 2018). Random Forest (RF) Utilizing various learners, ensemble methods improve performance of the models. RF is a form of ensemble intelligence as well. The RF tagging approach decreases the possibility of aberrations influencing findings. This is effective for both categorical and continuous data. In this, scaling of the datasets is not required. For the more learners, the higher computational resources are needed for complicated methods. The decision in this method is determined by polling. This type of method is known as ensemble learning. Random forests are composed of an array of trees and plants. Random forests have numerous decision trees, similar to the number of trees in the forest. The decision made by the majority of trees is regarded as the right conclusion (Al Mudawi et al., 2022; Kaur et al., 2018). Logistic Regression Logistic regression (LR) is a machine learning (ML) method used to tackle class imbalance problem. The LR model is built on a conditional framework, with data values that ranges from zero to one. Email spam detection, fraudulent financial transactions identification, and malignant tumour diagnosis are all instances of LR-based ML. LR employs the cost function, sometimes known as the sigmoid function. Any actual number from zero and one is transformed by the sigmoid function (Al Mudawi et al., 2022; Wright, 1995). Naïve Bayes (NB) The NB model is a classification algorithm based on Bayesian concepts. It anticipates membership likelihood for every category according to a specific record or data point. The most likely category is the one with the highest likelihood. Rather than just making predictions, the NB classification projects probabilities (Ahsan et al., 2022). The existence of a feature in a category is assumed to be unconnected with any other characteristic in that category, and if they’re connected, then remain independent of each other’s occurrence. Because each characteristic contributes to the likelihood individually. An advantage of this approach is that it can be used on both binary and multi-dimensional data. Also, unlike other machine learning methods, we need fewer training datasets (Khalil et al., 2019). Multi-layer Perceptron (MLP) A Multi-layer Perceptron is a type of neural network that consists of multiple layers of interconnected nodes or neurons. The MLP receives input data, processes it through multiple hidden layers, and produces an output. MLP is a supervised learning algorithm that is used for classification and regression tasks (Mohd Fakharuddin Zorkafli et al., 2019) . The prediction models are created using the input dataset and fine-tuning each algorithm’s hyper-parameter. After clicking on each algorithm, we could fine-tune the hyper-parameters to maintain better prediction accuracy. To develop prediction models, a 5-fold cross-validation model was adopted. Process of descriptive statistics One of the data mining methods involving mathematics and related data collection and elucidation is descriptive statistics for predicting a high cervical cancer data set (Ramnath et al., 2021). The JASP is a simple tool to use for researchers with limited computer skills. Figure 2 depicts the four sections of descriptive statistics in this tool. The below image depicts descriptive statistics such as central tendency (median), and dispersion. Table 1 presents the major risk factors used for cervical cancer prediction based on data from recent research studies and library resources. Table 1 shows that the majority of the data is categorical data, which is organised by a set of categories rather than evaluated on a continuous numerical scale. Just smoking duration falls within the category of numerical data. All components’ roles were treated as input variables, while screening results were considered as target variables for data processing. Results Thus seventeen significant variables were used in the study to predict cervical cancer. These variables were measured in each participant. The numerical and categorical value of each target variable susceptible to cervical carcinoma was either negative/positive results. Results of descriptive statistics Table 2 illustrates the median, dispersion and missing values of the predictors used in the current study. The data provided appears to be a set of predictors and their corresponding median and dispersion values. Each predictor represents a factor or characteristic that may be related to the risk of developing cervical cancer. The study’s median age was between 30 and 35 years, and the majority of participants had married and living with their partners, with a median age at marriage larger than 15 years and a dispersion value of 0.53. The median age of first sexual activity is between 16 and 20 years’ age, and the majority of participants had 0-1 lifetime sexual partners, with a dispersion value of 0.318. Surprisingly, the majority of participants had never been screened for cervical cancer, with a dispersion value of 0.24. There was no missing data among the selected predictors. Figure 3 Showed the data of distribution of the positive cases for cervical cancer. A total of 298 women participated in the current study for cervical screening (Pap smear and Biopsy). The scatter diagram depicts, 7 positive and 291 negative results for cervical cancer. Finding Predictors of cervical cancer using machine learning classifiers Table 3 shows that, in the initial stages of modeling, four variables are categorized under exclusion criteria based on at least one variable being depicted in each ML algorithm; none of those listed in this table fit this criterion. As a result, researchers have exempted these four variables from further data analysis. Table 4 presents the classification performance of algorithms in the second stage of modelling with tuned hyper-parameters. The comparison of all six models is shown in Table 4. Models 1, 2, and 4 of the logistic regression algorithm and models 3, 5, and 6 of the Decision Tree algorithm produced better classification prediction outcomes for cervical cancer prediction. Table 4. Classification performance of algorithms in the second stage of modelling with tuned hyper-parameters. Figure 4. Presents the ROC curve algorithm classification. The relevant variables considered for cervical cancer prediction were carried out at the second stage of modeling (Table 4), and the parameters were evaluated individually for each model. Based on the evaluation criteria of specificity, sensitivity, and area under the ROC curve, Random Forest, Naive Bayes, MLP, Logistic Regression, and Decision Tree machine algorithms correspondingly performed the finest. Figure 4 exhibits the results of evaluating ROC curve and the area under the ROC curve for the algorithms run in the second stage of modelling revealed that the Naive Bayes (in 1, 3, and 5 models), Logistic Regression, (in 2,4, and 6 models) had the utmost area under the ROC curve for the test data while other others have performed similarly for the training data. Figure 5 Shows the significant predictors appeared by tree viewer Gini Index. Table 5 depicts the significant final predictors used in the current study. The 14 variables that are confirmed in the second stage of modeling as the predictors for prediction of the cervical cancer. The women’s participation in cervical screening was also evaluated in the current study. Merely 29 (4.79%) women out of 501 actively participated in cervical cancer screening prior to the start of our study. The majority of women have never heard of cervical screening or its advantages. Following the evaluation of women’s participation in cervical screening, the researcher expressed a keen interest in analyzing the barriers to non-participation in cervical screening among the participants. The vast majority (51.70%) of women were unaware of cervical.cancer screening and had never had it done (94.21%). Study subjects had reported a variety of reasons for nonparticipation, including embarrassment over the test (40.32%), the belief that the test is expensive (33.93%), non-acceptance by family members (16.13%), not knowing where the test would be administered (30.14%), and the assumption that she is healthy and does not need the test (50.10%). Figure 1 Workflow Diagram for Classification Prediction Models Table 1 Important Variable Collected from the Various Library Resources Raw Factors Type of the data Role 1 Age Categorical Input 2 Marital status Categorical Input 3 Ag a marriage Categorical Input 4 Age at onset of the sex Categorical Input 5 Age at the first child Categorical Input 6 Life time sexual partners Categorical Input 7 Life time pregnancies Categorical Input 8 Screened for cervical cancer Categorical Input 9 Number of times screened Categorical Input 10 Reasons for not screening Categorical Input 11 Smoking consumption Categorical Input 12 Duration of smoking Numerical Input 13 Temporary family planning methods Categorical Input 14 Type of the family planning methods Categorical Input 15 Family history cervical cancer Categorical Input 16 Infection of the cervix Categorical Input 17 Screening results Categorical Target Table 2 Distribution of the Samples based on Their Risk Factors Predictors Median Dispersion Missing Age 30-35 year 1.65 0% Marital status Married and living with partner 0.53 0% Age marriage > 15 years 0.53 0% Age at onset of the sex 16-20 year 1.04 0% Age at first child 15-20 years 0.919 0% Life time sexual partners 0-1 0.318 0% Life time pregnancies 0-2 0.772 0% Screened for cervical cancer No 0.246 0% If no why Not informed / no awareness 1.01 0% No. of times screening 0 0.229 0% Smoking consumption No 0.447 0% Usage of temporary family planning methods No 0.582 0% Family history of cervical cancer No 0.41 0% Infection of the cervix Candidiasis , and bacterial vaginosis 1.32 0% Figure 2 Descriptive Statistics in JASP Tool Figure 3 Distribution of the Positive Cases for Cervical Cancer Table 3 Categorization of Predictors in Exclusion Criteria during 1st Stage of the Modeling Predictor DT RF LR MLP Naïve Bayes Occurrences Age Nil Nil Nil Yes Nil 1 Age at marriage Nil Yes Nil Nil Nil 1 Number of times screened Nil Nil Nil Nil Nil 0 Life time pregnancies Nil Nil Nil Nil Nil 0 Table 4 Classification Performance of Algorithms in the Second Stage of Modelling with Tuned Hyper-Parameters Model Algorithm AUC CA F1 Precision Recall Sensitivity Specificity Accuracy Model 1 Decision Tree 0.59 0.79 0.79 0.79 0.79 0.88 0.14 0.79 Logistic Regression 0.55 0.84 0.82 0.81 0.84 0.94 0.14 0.87 Multi-layer Perceptron 0.54 0.82 0.81 0.8 0.82 0.92 0.14 0.82 Naive Bayes 0.68 0.72 0.76 0.84 0.72 0.74 0.57 0.72 Random Forest 0.47 0.68 0.72 0.77 0.68 0.76 0.14 0.68 Model 2 Decision Tree 0.49 0.74 0.76 0.78 0.74 0.74 0.14 0.74 Logistic Regression 0.7 0.88 0.85 0.84 0.88 0.88 0.14 0.84 Multi-layer Perceptron 0.64 0.84 0.82 0.81 0.84 0.84 0.14 0.84 Naive Bayes 0.69 0.68 0.74 0.86 0.68 0.68 0.71 0.68 Random Forest 0.48 0.82 0.81 0.8 0.82 0.82 0.14 0.82 Model 3 Decision Tree 0.67 0.84 0.82 0.81 0.84 0.84 0.14 0.84 Logistic Regression 0.51 0.84 0.82 0.81 0.84 0.84 0.14 0.84 Multi-layer Perceptron 0.44 0.79 0.79 0.79 0.79 0.79 0.14 0.79 Naive Bayes 0.75 0.75 0.79 0.85 0.75 0.75 0.57 0.75 Random Forest 0.51 0.79 0.8 0.81 0.79 0.79 0.29 0.79 Model 4 Decision Tree 0.81 0.86 0.83 0.82 0.86 0.86 0.57 0.86 Logistic Regression 0.83 0.89 0.89 0.89 0.89 0.89 0.14 0.89 Multi-layer Perceptron 0.77 0.81 0.8 0.79 0.81 0.81 0.14 0.81 Naive Bayes 0.71 0.7 0.75 0.84 0.7 0.7 0.57 0.7 Random Forest 0.54 0.82 0.81 0.8 0.82 0.82 0.14 0.82 Model 5 Decision Tree 0.59 0.84 0.82 0.81 0.84 0.84 0.14 0.84 Logistic Regression 0.56 0.74 0.76 0.78 0.74 0.74 0.14 0.74 Multi-layer Perceptron 0.47 0.75 0.77 0.78 0.75 0.75 0.14 0.75 Naive Bayes 0.66 0.72 0.76 0.82 0.72 0.72 0.43 0.72 Random Forest 0.66 0.82 0.81 0.8 0.82 0.82 0.14 0.82 Model 6 Decision Tree 0.48 0.83 0.82 0.81 0.83 0.83 0.29 0.88 Logistic Regression 0.69 0.75 0.78 0.83 0.75 0.75 0.57 0.75 Multi-layer Perceptron 0.49 0.79 0.79 0.79 0.79 0.79 0.29 0.79 Naive Bayes 0.72 0.75 0.78 0.83 0.75 0.75 0.57 0.75 Random Forest 0.57 0.73 0.75 0.77 0.73 0.73 0.29 0.73 Figure 4 (a) Model 1- ROC Curve-based Classification Prediction Figure 4 (b) Model 2- ROC Curve-based Classification Prediction Figure 4 (c) Model 3-ROC Curve-based Classification Prediction Figure 4 (d) Model 4- ROC Curve-based Classification Prediction Figure 4 (e) Model 5- ROC Curve-based Classification Prediction Figure 4 (f) Model 6- ROC Curve-based Classification Prediction Table 5 Significant Predictors Appeared in the Second Stage of the Modeling Sr. No. Predictor Random Forest Naive Bayes MLP Logistic Regression Decision Tree Occurrence 1.         Age at onset of the sex Yes Yes Yes Yes Yes 5 2.         Life time sexual partners Yes Yes Yes Yes Yes 5 3.         Screened for cervical cancer Yes Yes Yes 3 4.         Reasons for not screening Yes Yes Yes Yes Yes 5 5.         Smoking consumption Yes Yes Yes Yes Yes 5 6.         Family history cervical cancer Yes Yes Yes 3 7.         Infection of the cervix Yes Yes Yes Yes 4 8.         Duration of contraceptives used Yes Yes Yes Yes Yes 5 9.         Type of the family planning methods Yes Yes Yes Yes 4 10.     Family history cervical cancer Yes Yes Yes Yes 4 11.     Marital status Yes Yes Yes 3 12.     Age at the first child Yes Yes Yes Yes 4 13.     Duration of smoking Yes Yes Yes Yes 4 14.     Smoking Consumption Yes Yes Yes Yes 4 Figure 5 (a) Model 1- Significant predictors appeared by tree viewer Gini Index Figure 5 (b) Model 2- Significant Predictors Appeared by Tree Viewer Gini Index Figure 5 (c) Model 3- Significant Predictors Appeared by Tree Viewer Gini Index Figure 5 (d) Model 4- Significant Predictors Appeared by Tree Viewer Gini Index Figure 5 (e) Model 5- Significant Predictors Appeared by Tree Viewer Gini Index Figure 5 (f) Model 6- Significant Predictors Appeared by Tree Viewer Gini Index Discussion The current study focused on developing a model for predicting cervical cancer, considering the most probable susceptible variables. Cervical cancer does not display signs or symptoms until it has progressed to the later stages, where the prognosis is poor; therefore, predicting and detecting cervical cancer at an early stage is critical to reducing morbidity and mortality rates among middle-aged women. As a result, the current study’s researchers analyzed essential relevant predictors and the efficacy of the widely used algorithms in predicting cervical cancer. In our study, 17 risk factors were used as predictors of cervical cancer. Age, age at marriage, number of times screened, and lifetime pregnancies were excluded in the first step of modeling because they did not occur in more than two algorithms. However, in contrast, age, age at marriage, and lifetime pregnancies are influential factors in cervical cancer prediction (Kashyap et al., 2019). In the present study, due to the imbalance in the dataset, the authors divided the data into six models, each having 57 samples (except the sixth model, which contained 45 samples), and these were compared with samples of positive screening findings. Following the data preparation, five algorithms were statistically evaluated: Decision Tree, Logistic Regression, Multi-layer Perceptron, Naive Bayes, and Random Forest. In the first, second, and fourth modeling of the data, the Logistic Regression displayed 88 % to 94% sensitivity with 84% to 89% of accuracy for classification prediction results for cervical cancer, while in the third, fifth, and sixth models of the data, the Decision Tree method demonstrated 83% to 84% sensitivity and 84 % to 88% accuracy in predicting cervical cancer. When analyzing the Receiver Operating Characteristic (ROC) curve and the area under the ROC curve for the algorithms run in the second phase of the modeling techniques, the greatest area under the ROC curve was discovered to be linked with the Naive Bayes and Logistic Regression algorithms machine for the testing dataset. In contrast, all the techniques performed similarly for the training data in the following study. Authors (Asadi et al., 2020) conducted a cross-sectional study in Iran with participants of 145 and 23 testing features. The data were analyzed by machine learning algorithms which contain SVM, QUEST, C&R tree, MLP, and RBF. Accuracy, sensitivity, specificity, and area under the curve (AUC) were the measurement criteria to evaluate the algorithms. The accuracy, sensitivity, specificity, and AUC of MLP were 90.9%, 90%, 91.67%, and 91.5% respectively. Personal health, relationship status, social status, contraception dosage, education level, and frequency of cesarean births were the significant predictors in the Algorithms. Another study, conducted by (Vidya et al., 2006). segmented the data with the attributes of 500 datasets and 100 testing datasets; it showed the greatest results related to MLP with 98% of accuracy, 98% of sensitivity, and 99 % of the area under ROC curve when matching with other algorithms. In the present study, all five algorithms showed better performance. However, Logistic Regression and Decision Tree have shown great performance classification results out of all six models. Compared to the current study, MLP-ANN and SVM obtained the greatest results in all indicators and the area under the ROC curve. This difference in findings can be addressed by choosing a greater sample size, such as 500 training data and 100 testing datasets (Vidya et al., 2006). As per the results of Hemalatha et al., (2016). MLP algorithm showed the best results with 85.5% accuracy, a 78.94% sensitivity and a 60.72% precision, while in another study, Kusy et al., (2013) with a sample size of 107 displayed the results of 72% of accuracy, 69% of sensitivity, 74% of specificity, and 67% area under the ROC curve, where similar results are observed in the present study also. However, in all six models, the specificity percentage levels are less, possibly due to the significant difference in the data and target features. In Kusy et al., study, RBF neural network algorithm showed poorer performance with 55% of accuracy, 42% of sensitivity, 67% of specificity, and a 48% area under the ROC curve matched in the present study . Last but not restricted, this paper gives a glimpse of how Artificial Intelligence can help in predicting Cervical cancer. Despite recent breakthroughs in artificial intelligence (AI) and its applications in cancer, there are various constraints and obstacles that need to be overcome. Some of these are data access control, generalisation, building practical applications, explanations challenges, and obstacles related to education and competence in the subject. Even though numerous studies have shown Machine learning and artificial intelligence to be effective in the prediction of different types of cancers, the validation of specific ML algorithm is required to generalize the findings (Patil et al., 2020). The short comings of the current study include, the small sample size with the imbalanced input and target datasets. Another types of ML algorithms might be administered as in our study; we have used only five algorithms to predict the cervical cancer. Nevertheless, as per the results of review studies and current study, it is concluded that ML algorithm approaches are quite beneficial in predicting cervical cancer. Large sample size is recommended to resolve the issues of imbalanced datasets. In conclusion, the results of current study proved that machine learning algorithms could improve the cervical cancer predictions. Logistic Regression and Decision Tree have shown great performance classification results out of all six models. This study’s results indicated that all five algorithms showed better performance. However, Logistic Regression had shown great performance classification results of 88 % to 94% sensitivity with 84% to 89% of accuracy and the Decision Tree method demonstrated 83% to 84% sensitivity and 84 % to 88% accuracy in predicting cervical cancer. For testing dataset, the highest area under the ROC curve was observed to be associated with the Naive Bayes and Logistic Regression methods machine. This study proved that we could obtain accurate sensitivity and accuracy prediction values even when there is an imbalanced dataset of input and output criteria. Author Contribution Statement Conceptualization, S.D., G.S.R., and H.R; methodology, S.D. and G.S.R.; software, G.S.R.; validation, S.D. and G.S.R.; formal analysis, S.D. and G.S.R.; investigation, S.D. and G.S.R.; resources, S.D., G.S.R., and H.R; data curation, S.D., G.S.R., and H.R; writing, S.D.; writing, review and editing, S.D., G.S.R., and H.R.; visualization, S.D., G.S.R.; supervision, S.D., G.S.R., and H.R; project administration, S.D. Acknowledgements General The author expresses heartfelt gratitude to Symbiosis Centre for Research Innovation (SCRI) and Symbiosis International Deemed University for supporting authors throughout the study. The authors wish to express their sincere gratitude to the authorities of YCM hospital for data collection approval and also we would thank the women who participated in this study. Funding Statement Present study was funded by Symbiosis Centre for Research Innovation (SCRI) and Symbiosis International Deemed University (SIDU). Approval Obtained from Symbiosis College of Nursing, Symbiosis International Deemed University. Informed Consent Statement was obtained from the participants. Ethical Declaration Authors had obtained an ethical approval from Independent Ethics Committee (IEC) of Symbiosis International (Deemed University), Pune Maharashtra, India. Data Availability Data collected from the study subjects. The data are not publicly available due to privacy and ethical reasons. 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116165 10.31557/APJCP.2023.24.4.1407 Research Article Down-Regulation and Clinic-Pathological Correlation of SIK-1 and SIL-1-LNC in Non-Small Cell Lung Cancer Patients Anvarnia Alireza 12 Panahizadeh Reza 3 Zarredar Habib 1 Asadi Milad 4 Hashemzadeh Shahriyar 1 Vatankhah Mohammad Amin 3 Valizadeh Hamed 1* Raeisi Mortaza 5* 1 Tuberculosis and Lung Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2 Shohada Clinical Research Development Unit, Shohada Hospital, Tabriz University of Medical Science, Tabriz, Iran. 3 Students Research Committee, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran. 4 Department of Basic Oncology, Ege University, Institute of Health Sciences, Izmir, Turkey. 5 Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. * For Correspondence: Raiisy@yahoo.com, H_valley78@yahoo.com 2023 24 4 14071411 7 1 2023 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Non-small-cell lung cancer (NSCLC) is currently the leading cause of mortality cancer. Introducing noninvasive approaches to diagnose NSCLC, especially at an early phase, might improve the disease’s prognosis. Long noncoding RNAs (lncRNAs), which are important regulators of the expression genes inside the cells, have been linked to a range of biological processes, such as cancer progression and metastasis, including NSCLC. The present work aims to determine the potential involvement of SIK-1-LNC and SIK-1 in NSCLC pathogenesis and the possible use of these molecules as novel biomarkers or therapeutic targets. Methods: In this work, the expression levels of SIK-1-LNC and SIK-1 in 50 pairs of NSCLC tumor and tumor marginal tissues were evaluated. So, after total RNA extraction and complementary DNA synthesis, the SIK-1-LNC and SIK-1 expression levels were evaluated by real-time PCR. In the study groups, clinical and pathological characteristics of the NSCLC patients were also examined. Results: Our findings showed that tumor samples had much lower levels of SIK-1 and SIK-1-LNC expression than tumor margin samples. SIK-1-LNC expression was correlated with SIK-1 levels in NSCLC samples. Interestingly, both stage and lymph node metastasis features of the tumor were associated significantly with SIK-1 and SIK-1-LNC expression levels. A ROC curve analysis indicated a biomarker index of 0.69 and 0.74 for SIK-1 and SIK-1-LNC, respectively. Conclusion: Collectively, our study emphasized the role of SIK-1-LNC and SIK-1 downregulation in NSCLC oncogenesis. Additionally, SIK-1 and SIK-1-LNC, particularly the latter, have shown remarkable potential to be utilized as new NSCLC biomarkers and therapeutic targets. Key Words SIK-1- SIK-1-LNC non-small cell lung cancer metastasis ==== Body pmcIntroduction Lung cancer (LC) is the most highly prevalent carcinoma and the leading cause of cancer-related mortality globally, with over 2,090,000 new cases, 1,760,000 deaths, and a 5-year survival rate of 19.4%. Notably, the incidence rates of LC have grown up in the Northern and Southern regions of Africa, China, Eastern Europe, and Indonesia (Bade et al., 2020; de Groot et al., 2018). Small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC), the latter which also encompasses adenocarcinoma and squamous cell carcinoma, are the two subtypes of LC. About 85% of all LCs are diagnosed as NSCLC (Zarredar et al., 2018; Zarredar et al., 2019). The use of tobacco and smoking, familial history, drinking alcohol, chronic inflammation, ionizing radiation, occupational exposures, asbestosis, silica, mixed occupation exposures, and air pollution are the main risk factors for NSCLC (Gholi-Nataj et al., 2022; Malhotra et al., 2016). Surgical removal, chemotherapy, and radiation therapy are the conventional approaches in the treatment of NSCLC. Comparatively, NSCLC is more chemoresistant than SCLC and most patients with NSCLC have attained an advanced stage due to inadequacy of early diagnostic techniques (Arbour et al., 2019; Sharma et al., 2019). So, developing novel biomarkers and therapeutic targets could be of significance in NSCLC. Long noncoding RNAs (lncRNA) are a subtype of RNA molecules which are larger than 200 nucleotides. LncRNAs are implicated in several biological procedures, particularly in the progression and metastasis of different cancers. Also, the interaction between lncRNAs and protein, RNA, and DNA leads to carcinogenesis of colorectal (Wen et al., 2016), gastric (Chen et al., 2018), lung (Yao et al., 2016), osteosarcoma (Aftabi et al., 2021; Li et al., 2018), and esophageal (Congxiu Huang et al., 2016) cancers via targeting specific genes. Therefore, investigation of lncRNAs that are implicated in cancer signaling pathways might provide us with perspicuity into the evolution of neoplasms (Liang et al., 2020; Shuai et al., 2020). SIK1-LNC is identified at chromosome 21q22.3 and it indicated a positive tendency with salt-inducible kinases 1 (SIK1) in various cancerous cells. SIK1 is a serine/threonine kinase that has crucial functions in regulating the proliferation of cells (Yang et al., 2018). Previous studies recommend that SIK1 performs vital roles in several cancers including gastric cancer (Selvik et al., 2014) and epithelial ovarian cancer (Wu et al., 2017). It has been revealed that down-regulation of LKB1-SIK1 signaling increases EMT (epithelial to mesenchymal transition) in NSCLC, which causes metastasis enhancement (Yao et al., 2016). Thus, the low levels of SIK-1 expression in some cancer cells, could be linked to poor prognosis. However, the role and expression of SIK1-LNC and SIK-1 remain unclear in lung NSCLC cells. In the present work, we evaluated the expression level of SIK1-LNC and SIK-1 mRNA in biopsy samples of NSCLC patients and its critical function in the prognosis of this cancer. Materials and Methods Research population and sampling method We obtained tumor and margin healthy tissues from 50 NSCLC patients after surgery between 2019 and 2021. The samples were identified by surgical oncologists and validated by a board-certified pathologist at Imam Reza Hospital, Tabriz, Iran. The clinical and pathological features of the patients are demonstrated in Table 1. Following surgery, tissue samples were promptly placed in RNase inhibitor solution (Qiagen, Hilden, Germany) and transported to the laboratory. All patients signed a written informed consent form prior to sampling. Also, this research has been approved by the Ethical Committee of Tabriz University of Medical Sciences (Grant number: IR.TBZMED.REC.1400.756). RNA extraction RNX-PLUS Solution (SinaClon, Tehran, Iran) was used to extract total RNA in accordance with the manufacturer’s instructions. A NanoDrop (Thermo Fisher Scientific, USA) spectrophotometer was used to confirm the quality and quantity of total RNA. Gel electrophoresis on 1% agarose was also used to evaluate RNA samples. Then, until cDNA synthesis, the RNA samples were maintained in an -80°C refrigerator. cDNA synthesis and Real-time PCR Using cDNA synthesis kits, cDNA synthesis was carried out in a total volume of 20 μl. (Biofact, Seoul, South Korea). The house-keeping gene was GAPDH. The Roche real-time PCR Light Cycler 96 instrument (Roche Diagnostics, Mannheim, Germany) was used to perform RT-PCR using the master mix SYBER Green, and the findings were reported using the 2−ΔΔCT method. Primer sequences used for qRT-PCR and respective annealing temperatures are summarized in Table 2. Statistical analysis Data evaluation was carried out using GraphPad Prism 6 (San Diego, CA, USA). The normality distribution of variables was determined using the Kolmogorov-Smirnov test. Statistical comparison was made using Paired Student T-test (P-value < 0.05, mean ± SD). Results Expression of SIK-1-LNC in NSCLC tissues We have assessed the expression of SIK-1 and SIK-1-LNC in 50 NSCLC samples compared to the tumor margin samples. The expression of SIK-1 and SIK-1-LNC in tumor and tumor margin tissues was then evaluated. In parallel to RT-PCR analysis, clinical and pathological features of NSCLC patients were assessed in the research groups, including gender and age, and tumor-related attributes such as stage and lymph node metastasis. The results revealed a substantial reduction in SIK-1 and SIK-1-LNC expression in tumor samples as compared to tumor margin samples, with p values of 0.0025 and 0.0019, respectively (Figure 1). The expression level of SIK-1 and SIK-1-LNC were not significantly associated with the age and sex of the patients. However, both stage and lymph node metastasis features of the tumor were associated significantly with SIK-1 and SIK-1-LNC expression levels. The exact p values of the association are demonstrated in Table 3. The diagnostic competence SIK-1 and SIK-1-LNC in NSCLC The sensitivity and specificity of SIK-1 and SIK-1-LNC as potential novel biomarkers for NSCLC were determined using the Receiver operating characteristic (ROC) curve. In NSCLC patients, the result indicated a ROC area of 0.69 and 0.74 biomarker index for SIK-1 and SIK-1-LNC, respectively. ROC curve data are demonstrated in Figure 2. SIL-1-LNC expression was correlated with SIK-1 expression levels in NSCLC samples We also evaluated the relationship between SIK-1-LNC with SIK-1 expression levels. According to Fig. 3, Pearson’s correlation analysis indicated a significant (p-value = 0.0071) positive correlation between SIK-1-LNC and SIK-1 expression in NSCLC samples (r = 0.4046), indicating that SIK-1-LNC may play a role in NSCLC pathogenesis via modifying SIK-1 expression. Figure 1 In NSCLC Tissues Relative to the Marginal Zone, SIK1-LNC (A) and SIK (B) expression levels were significantly downregulated Figure 2 The Correlation between SIK1-LNC and SIK1 Expression Levels Figure 3 The Correlation between SIK1-LNC and SIK1 Expression Levels in NSCLC Samples. Correlation analysis indicated a significant (p-value = 0.0071) positive correlation between SIK-1-LNC and SIK-1 expression in NSCLC samples (r = 0.4046) Table 1 Clinicopathological Features of NSCLC Patients Number of Samples Total:50 Age < 55 23 > 55 27 Sex Male 24 Female 26 Stage Stage 2 23 Stage 3 25 Stage 4 2 Lymphnod metastasis Negative 21 Positive 29 Table 2 List of Primer Sequences Used for qRT-PCR Analysis Genes Forward (5′-3′) Reverse (5′-3′) Annealing temp. (°C) Product Size SIK-1 5′-CTCCGGGTGGGTTTTTACGAC-3′ 5′-CTGCGTTTTGGTGACTCGATG-3′ 59 93bp SIK-1-LNC 5′-GAATGGGAGGGGAAGGAAGAAGC-3′ 5′-TCAGCAGAGCAAGAAGAGGTCAG-3′ 60 283bp GAPDH 5′-CAAGATCATCAGCAATGCCTCC-3′ 5′-GCCATCACGCCACAGTTTCC-3′ 59 166bp Table 3 a Relation of the Clinical and Demographic Features of the Studied NSCLC Patients with the Expression of SIK-1 and SIK-1-LNC Features Age Sex Stage Lymph node metastasis Genes Change in expression level p-value SIK-1 Downregulated 0 None-significant None-significant Significant (p-value = 0.0031) Significant (p-value = 0.001) SIK-1-LNC Downregulated 0.05 None-significant None-significant Significant (p-value = 0.0015) Significant (p-value = 0.029) Table 3 b Clinicopathological Features of Patients Coloration with Genes SIK-1 SIK-1-LNC Age P value:0.42 P value:0.23 Sex P value:0.18 P value:0.29 Stage P value:0.0031 P value:0.0015 Lymphnod metastasis P value:0.001 P value:0.029 Discussion Considering the satisfactory outcome of therapeutic strategies used in the treatment of NSCLC patients in the clinic, understanding the molecular mechanisms and recognition of new diagnostic biomarkers and therapeutic targets implicated in NSCLC is of significant importance. Previous studies revealed that lncRNAs have crucial functions in the proliferation, metastasis, apoptosis, and cell differentiation of cancerous cells. Changing cytoplasmic localization of proteins, and modulating the action of corresponding proteins are some biological functions of lncRNAs (Wang et al., 2011; Wilusz et al., 2009). SIK-1 as a member of the AMP-activated protein kinase-related kinases has main roles in the cell metabolism and distal metastasis of cancers (Chen et al., 2016). In this work, we demonstrated that the expression levels of SIK1-LNC and SIK-1 were significantly reduced in NSCLC tissues in comparison with adjacent normal tissues. SIK-1-LNC expression was correlated with SIK-1 levels in NSCLC samples and SIK-1-LNC through modulating SIK-1 is involved in NSCLC pathogenesis. Furthermore, SIK-1-LNC was shown to be a more efficient potential biomarker of NSCLC than SIK-1 with a biomarker index of 0.74 versus 0.69. Similar to our study, but in a different population, Yang et al., (2018) showed that SIK-1 and SIK-1-LNC are downregulated in NSCLC and also induction of SIK-1-LNC expression could suppress proliferative and invasive abilities of cancerous cells. Consequently, SIK-1-LNC may be used in NSCLC as both a prognostic marker and a therapeutic target. Also, recent studies reported that down-regulation of SIK1-LNC and SIK-1 enhances proliferative characteristics of various other malignancies. Wang et al. showed that the expression level of SIK1-LNC was attenuated in AML patients and the down-regulation of SIK1-LNC leads to leukemogenesis. They indicated that SIK1-LNC improves the anti-cancer effects of retinoic acid in AML. Moreover, SIK1-LNC induces autophagy by targeting E2F1 in NB4 AML cells (Wang et al., 2022). Chen et al., (2016) demonstrated that miR-141 acts as an oncomir and enhances the cell growth of ovarian cancer cells. They also showed that down-regulation of miR-141 causes the elevation of SIK-1 expression in ovarian cancer. In another study, Ren et al., (2016) revealed that the expression of SIK-1 was reduced in pancreatic cancerous cells and this down-regulation of SIK-1 is correlated with gemcitabine chemoresistance in pancreatic cancerous cells. Qu et al., (2016) showed that the expression level of SIK-1 was down-regulated in hepatocellular carcinoma (HCC) and the over-expression of SIK-1 causes EMT suppression and cell proliferation inhibition in HCC. They also showed that Twist-1 negatively regulates the promotor activity of SIK-1 and the expression levels of SIK-1 are inversely associated with Twist-1 in HCC. Huang et al., (2019) established that SIK-1 expression was attenuated in colorectal cancer cells. They ascertained that the expression level of miR-17 was enhanced in colorectal cancer and the up-regulation of miR-17 induced the cell growth by targeting SIK-1. Ponnusami et al., (2021) demonstrated that the expression level of SIK-1 was reduced in human breast cancer. They also demonstrated that SIK-1 interacted with p53 and facilitates oxidative phosphorylation in breast carcinoma and leads to proliferation and invasion in breast cancer. Collectively, our study in parallel with previous studies emphasized the role of SIK-1-LNC and SIK-1 downregulation in NSCLC oncogenesis. It has been shown that SIK-1 and SIK-1-LNC, particularly the latter, have the potential to be utilized as both new biomarkers and therapeutic targets in NSCLC. Author Contribution Statement MR, HV, and HZ took responsibility for design of the study, data acquisition, statistical analysis and wrote the paper; AA, SH, MAV, and RP participated in the data acquisition and drafted the manuscript; MA made substantial contributions to the conception or design of the work and English edit. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank the Shohada Clinical Research Development Unit, Shohada Hospital, Tabriz University of Medical Science, Tabriz, Iran. Study Approval This work was supported by a thesis grant (IR.TBZNED.REC.1400.756) from Tabriz University of Medical Sciences. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Availability of Data On reasonable request, the associated author will release the datasets used in this work. Conflict of Interest None. ==== Refs References Aftabi Y Ansarin K Shanehbandi D Long non-coding RNAs as potential biomarkers in the prognosis and diagnosis of lung cancer: A review and target analysis IUBMB Life 2021 73 307 27 33369006 Arbour KC Riely GJ Systemic therapy for locally advanced and metastatic non–small cell lung cancer: a review JAMA 2019 322 764 74 31454018 Bade BC Cruz CSD Lung cancer 2020: epidemiology, etiology, and prevention Clin Chest Med 2020 41 1 24 32008623 Chen JF Wu P Xia R STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells via mTOR signal-mediated inhibition of autophagy Mol Cancer 2018 17 1 16 29304823 Chen JL Chen F Zhang TT Suppression of SIK1 by miR-141 in human ovarian cancer cell lines and tissues Int J Mol Med 2016 37 1601 10 27081781 De Groot PM Wu CC Carter BW The epidemiology of lung cancer Transl Lung Cancer Res 2018 7 220 30050761 Gholi-Nataj M Rafieian S Barahman M A Meta-analysis for Prevalence of Lung Cancer Patients with SARS-CoV-2 Infection during the COVID-19 Pandemic Lung Cancer 2022 2022 Huang C Liu J Xu L MicroRNA-17 promotes cell proliferation and migration in human colorectal cancer by downregulating SIK1 Cancer Manag Res 2019 11 3521 31118777 Huang C Yu Z Yang H Increased MALAT1 expression predicts poor prognosis in esophageal cancer patients Biomed Pharmacother 2016 83 8 13 27470544 Li T Xiao Y Huang T HIF1αinduced upregulation of lncRNA UCA1 promotes cell growth in osteosarcoma by inactivating the PTEN/AKT signaling pathway Oncol Rep 2018 39 1072 80 29328452 Liang Y Song X Li Y LncRNA BCRT1 promotes breast cancer progression by targeting miR-1303/PTBP3 axis Mol Cancer 2020 19 1 20 31901224 Malhotra J Malvezzi M Negri E Risk factors for lung cancer worldwide Eur Respir J 2016 48 889 902 27174888 Ponnusamy L Manoharan R Distinctive role of SIK1 and SIK3 isoforms in aerobic glycolysis and cell growth of breast cancer through the regulation of p53 and mTOR signaling pathways Biochim Biophys Acta Mol Cell Res Bba-Mol Cell Res 2021 1868 118975 Qu C He D Lu X Salt-inducible Kinase (SIK1) regulates HCC progression and WNT/β-catenin activation J Hepatol 2016 64 1076 89 26778753 Ren ZG Dong SX Han P miR-203 promotes proliferation, migration and invasion by degrading SIK1 in pancreatic cancer Oncol Rep 2016 35 1365 74 26719072 Selvik L-KM Rao S Steigedal TS Salt-inducible kinase 1 (SIK1) is induced by gastrin and inhibits migration of gastric adenocarcinoma cells PLoS One 2014 9 e112485 25384047 Sharma P Mehta M Dhanjal DS Emerging trends in the novel drug delivery approaches for the treatment of lung cancer Chem Biol Interact 2019 309 108720 31226287 Shuai Y Ma Z Liu W TEAD4 modulated LncRNA MNX1-AS1 contributes to gastric cancer progression partly through suppressing BTG2 and activating BCL2 Mol Cancer 2020 19 1 20 31901224 Wang K Liu Jd Deng G LncSIK1 enhanced the sensitivity of AML cells to retinoic acid by the E2F1/autophagy pathway Cell Prolif 2022 55 e13185 35092119 Wang KC Chang HY Molecular mechanisms of long noncoding RNAs Mol Cell 2011 43 904 14 21925379 Wen J Xu J Sun Q Upregulation of long non coding RNA PCAT-1 contributes to cell proliferation, migration and apoptosis in hepatocellular carcinoma Mol Med Rep 2016 13 4481 6 27035680 Wilusz JE Sunwoo H Spector DL Long noncoding RNAs: functional surprises from the RNA world Genes Deve 2009 23 1494 504 Wu DD Chen X Sun KX Role of the lncRNA ABHD11-AS1 in the tumorigenesis and progression of epithelial ovarian cancer through targeted regulation of RhoC Mol Cancer 2017 16 1 10 28093071 Yang L Xie N Huang J SIK1-LNC represses the proliferative, migrative, and invasive abilities of lung cancer cells Onco Targets Ther 2018 11 4197 30050311 Yao YH Cui Y Qiu XN Attenuated LKB1-SIK1 signaling promotes epithelial-mesenchymal transition and radioresistance of non–small cell lung cancer cells Chin J Cancer 2016 35 1 9 26728009 Zarredar H Ansarin K Baradaran B Potential molecular targets in the treatment of lung cancer using siRNA technology Cancer Invest 2018 36 37 58 29336624 Zarredar H Farajnia S Ansarin K Synergistic effect of novel EGFR inhibitor AZD8931 and p38α siRNA in lung adenocarcinoma cancer cells AntiCancer Agents Med Chem 2019 19 638 44 30827261

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116132 10.31557/APJCP.2023.24.4.1125 Research Article Mapping EORTC-QLQ-C30 onto EQ-5D-5L Index in Indonesian Cancer Patients Perwitasari Dyah Aryani 1* Purba Fredrick Dermawan 2 Candradewi Susan Fitria 1 Marwin Marwin 1 Permata Agung 3 Faza Muhammad Barik Ulfa 4 Septiantoro Bayu Priyo 5 Kaptein Adrian A. 6 1 Faculty of Pharmacy, Universitas Ahmad Dahlan, Yogyakarta, Indonesia. 2 Faculty of Psychology, Universitas Padjadjaran, Sumedang, Jawa Barat, Indonesia. 3 Department of Clinical Pharmacy and Community, Institute of Technology, Science and Health, Dr Soepraoen Hospital, Malang, East of Java, Indonesia. 4 Farmaza Pharmacy, Demak, Centre of Java, Indonesia. 5 Karyadi Hospital, Semarang, Indonesia. 6 Leiden University Medical Centre, Netherlands. * For Correspondence: dyah.perwitasari@pharm.uad.ac.id 2023 24 4 11251130 19 6 2022 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: This study aims to develop a mapping algorithm for EORTC QLQ-C30 to EQ-5D-5L which can produce utility values in patients with cancer. Methods: We used a cross sectional study design with 300 cancer patients. The research instruments used were EORTC QLQ-C30 and EQ-5D-5L. Data were collected by interviewing cancer patients who were hospitalized in the Kasuari Installation of Dr Kariadi Hospital Semarang, Indonesia. The Ordinary Least Squares (OLS) regression method was used to predict the utility value of EQ-5D-5L. This study uses two models to predict utility values, namely model 1 with all domains, and model 2 with domains that affect the EQ-5D-5L. The predictive power of regression on the model is evaluated by calculating the mean absolute error (MAE) and root mean square error (RMSE) values. Result: The highest score in the functional domain is the ‘emotional function’ domain (mean: 85.89; SD: 16.04) and the highest symptom domain is ‘weakness’ (mean: 36.21; SD:21.69). The predicted utility values of models 1 and 2 are 0.683. The mean absolute error (MAE) and root mean square error (RMSE) values of model 1 are 0.128 and 0.173, while in model 2 the MAE and RMSE values obtained are 0.125 and 0.168. Conclusion: The development of the mapping algorithm from the EORTC QLQ-C30 to EQ-5D-5L instrument shows a predictive value of utility in a sample of patients with cancer at Dr. Kariadi Hospital, Semarang, Indonesia. The utility prediction in both model is similar, however model 2 involves fewer domains and symptoms. Key Words Mapping utility EORTC QLQ-C30- EQ-5D-5L cancer ==== Body pmcIntroduction Appraisal of the effects of available alternatives regarding health, health care costs, and other effects, namely health technology assessment (HTA), are important in addition to assessing clinical efficacy. Cost-utility analysis (CUA) is one of the most prevalent methods in HTA, by evaluating health-related quality of life (HRQOL) outcomes and comparing costs and outcomes between different health care programs in terms of cost per Quality Adjusted Life Years (QALY) (Drummond et al., 2015). To obtain a QALY, utilities of a health state are differentiated over a lifetime. This utility is measured by generic multi-attribute utility instruments (MAUIs). The three most frequently mentioned MAUIs in HTA guidelines across the world are the EQ-5D, Health Utility Index (HUI), and the Short-Form 6-Dimension (SF-6D) (Kennedy-Martin et al., 2020). Measures of HRQOL are often applied as a secondary outcome of studies in cancer patients. Researchers often prefer to use disease specific HRQOL questionnaires over generic ones in their studies. It has been argued that generic measures are not as sensitive to changes in quality of life in certain diseases as condition-specific measures (Lorgelly et al., 2017). However, these disease-specific questionnaires are not preference-based measures and unable to produce utilities of health states. Therefore, these questionnaires cannot be used directly for estimating the QALYs. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) is one of the most widely used instrument to measure the quality of life of cancer patients (Aaronson et al., 1993). The Indonesian version of the EORTC QLQ-C30 has been validated for use in Indonesian cancer patients (Perwitasari et al., 2011). For obtaining utility from a disease-specific questionnaire such as the EORTC QLQ-C30, one solution offered is mapping technique. This technique usually consists of two stages: (i) calculate the relationship between the preference-based questionnaires (as the outcomes) and the non-preference-based as the independent variables, and (ii) use this relationship in the datasets containing the non-preference-based measure for prediction of EQ-5D-5L index (Ameri et al., 2020; Chen et al., 2019). In this study, we used the EQ-5D-5L as the preference-based questionnaire, because it has been proven as valid in Indonesian cancer patients (Setiawan et al., 2018) and has a national value set to obtain utilities (Purba et al., 2017). Therefore, the aim of the present study was to map the EORTC QLQ-C30 responses to the EQ-5D-5L in cancer patients in Indonesia. Materials and Methods Respondents We collected data from three patient groups: breast cancer, nasopharyngeal cancer, and colorectal cancer. The inclusion criteria were: (i) diagnosed with breast cancer, nasopharyngeal cancer, or colorectal cancer, (ii) aged 18 years and above, (iii) having an adequate command of the Indonesian language (Bahasa Indonesia), and (iv) willing to participate in this study. Exclusion criteria were the following: (i) refusing to participate, (ii) having complications such as type 2 diabetes mellitus, cardiovascular disorders, impaired kidney function disorders, liver function disorders, or communication disorders, as assessed by physician which were reported in the medical records. The researcher assisted the patients in completing the questionnaires, thus we also involved the patients who illiterate. The clinical data were collected from patients’ medical records. Procedures The present study was approved by the Health Research Ethics Committee, Dr. Kariadi Hospital, Semarang (Number: .401/EC/KEPK-RSDK/2019). This study was conducted in RSUP Dr. Kariadi in Semarang, Indonesia from May to July 2020. The researcher approached patients in the waiting room of the hospital, introduced this study and asked for their participation. After informed consent was signed, the patients completed the socio-demographic data, EQ-5D-5L, and EORTC QLQ-C30. The researcher helped patients to complete the questionnaires when needed. Instruments We collected the demographic of the patients, including name, age, gender, education level, marital status, and monthly income. Their medical data were collected from the medical record: cancer type, stage, and treatment cycle. The cancer-specific health-related quality of life (HRQOL) questionnaire being used in this study was the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30). It consists of five function domains (physical, emotional, social, role, cognitive), three symptom scales (fatigue, nausea and vomiting, pain), and six single items for various symptoms (shortness of breath, insomnia, loss of appetite, constipation, diarrhea, and financial difficulties) and one general health status scale (Aaronson et al., 1993). Patients respond on a four-point scale from “not at all” to “very much” for most items. Most items use a “past week” recall period. The scores of each scale were calculated in two stages: first, the raw score is calculated. Then, a linear transformation is used to convert the score into a range of 0 to 100. Higher functional scale and global health status/QoL scale scores indicate better functioning and HRQOL, respectively, whereas higher symptom and single items scores depict worse status (Fayers et al., 2001). The EORTC QLQ-C30 has been translated into Bahasa Indonesia and validated to assess the quality of life in cancer patients in Indonesia (Perwitasari et al., 2011). The generic HRQOL instrument being used was the Bahasa Indonesia version of EQ-5D-5L provided by the EuroQol Group. [The EQ-5D-5L is a generic HRQOL instrument which consists of two parts: the descriptive system and the EQ-visual analogue scale (EQ-VAS). The descriptive system consists of five dimensions (mobility, self-care, usual activities, pain/discomfort, anxiety/depression), each of which can take one of five severity level responses (no problems, slight problems, moderate problems, severe problems, and unable/extreme problems). Combination of these five dimensions and five severity level resulted in 3125 (5) unique health states. Each health state is then translated into utility values using the Indonesian EQ-5D-5L value set (Purba et al., 2017). The EQ Visual Analogue Scale (EQ-VAS) records the respondent’s self-rated health on a 20 cm vertical visual analogue scale with endpoints labelled “the best health you can imagine” and “the worst health you can imagine” (Herdman et al., 2011). The EQ-5D-5L has been proven to be valid (Setiawan et al., 2018) and reliable (Purba et al., 2018) to be used on the Indonesian population. Data analysis The demographic and medical characteristics are described as percentages within the subgroups: i.e., age, gender, education level, marital status, monthly income, cancer types, cancer stage and cycle number. We divided our final dataset into derivation (n=266) and validation (n=34) data sets. The proportion of training data set was bigger than the proportion of validation data set The validation was applied to get the description that the conceptual model approached the real situation. To obtain the mapping algorithm, ordinary least squares (OLS) regression was used. EQ-5D-5L utility value served as the dependent variable and the EORTC QLQ-C30 scale and item scores were the explanatory or independent variables. We treated all variables as continuous. The model 1 (full model) was determined using all scales and items of the EORTC QLQ-C30. The second model (model 2) was developed using backward elimination with a significance level of 0.1 from the model 1. The model 2 was run to get the optimum performance after the backward elimination of model 1. The validation of the obtained algorithm was done by assessing the accuracy of the prediction: (i) mean absolute error (MAE) is the mean absolute difference between the observed and predicted values, (ii) and root-mean-squared error (RMSE) is the root of the mean squared difference, also reported as a percentage of the scale size (i.e., 1.865, the range of the EQ-5D-based utility according to the Indonesian value set), referred to as the normalized RMSE (Versteegh et al., 2012). Smaller MAE and RMSE values indicate better model performance. Statistical analyses were performed using SAS software (ver. 9.1; SAS Institute Inc., Cary, NC). P<0.05 was considered statistically significant. Results Our study has the aim to explore the mapping of utility value of cancer patients using EORTC-QLQ-C30 and EQ-5D-5L. We also describe the cancer patients’ quality of life measured by the two instruments. Table 1 shows the patients characteristics. Most of the patients are female (N=216; 72.0%), diagnosed with breast cancer (139; 46.33%), less than 60 yo (266; 88.67%), advanced stage of cancer (234; 78.00%), graduated from senior high school (97; 32.43%), having occupation (148; 49.33%) and married (246; 82.00%). Table 2 shows that the mean of EQ-5D-5L utility values is 0.68 (SD: 0.32) while the mean of self-rated health is 74.34 (SD: 12.22). For EORTC QLQ-C30, the general QoL mean is 72.19 (SD: 17.42). All domains had the score between 72.19- 87.78, which is in the category of good quality of life. The highest domain score is cognitive domain (mean: 87.78; SD: 16.24) and the lowest score domain is physical domain (mean: 72.51, SD: 24.92). Among the symptoms, the worst symptom reported by the patients is weakness (mean: 36.22; SD: 21.69). The EORTC QLQ overall mean score is lower than the score on its lowest domain (i.e., ‘physical domain’). Table 3 shows the results of OLS regression analysis for the mapping model from two models: i.e., Model 1 with all scales included and Model 2 with only scales with a significance level of 0.1 from the model 1. The statistically significant values are shown in the physical domain, role domain, emotional domain, pain symptom and financial difficulties score (‘financial difficulties’ are not a symptom). The most influential domain in both models is physical domain. The predicted utility scores are defined from individual patients in the validation sample (Table 4). Table 4 also shows the performance of the two models in predicting EQ-5D-5L utility values. The explanatory power for model 1 and model 2 is 73.7% and 74.4%, respectively. Model 2 shows better MAE and RMSE compared to Model 1. Table 5 shows the validation result of model analysis. Table 1 Cancer Patients’ Characteristics (n=300) in Dr. Kariadi Hospital from May to July 2020 Characteristics N % Age (yo) ≤60 266 88.67 >60 34 11.33 Sex Male 84 28.00 Female 216 72.00 Cancer type Breast 139 46.33 Colorectal 41 13.67 Nasopharing 120 40.00 Cancer stage 1-2 66 22.00 3-4 234 78.00 Last education No schooling 12 4.00 Elementary 76 25.33 Junior High School 43 14.33 Senior High School 97 32.33 Undergraduate 72 24.00 Work Yes 148 49.33 No 152 50.67 Marietal Status Married 246 82.00 Not married 54 18.00 Salary per month (IDR) < 2.500.000 108 35.00 ≥ 2.500.000 192 65.00 IDR, Indonesian Rupiah Table 2 Utility and VAS of EQ-5D-5L and Domains’ Scores of EORTC QLQ-C30 in Cancer Patients of Dr. Kariadi Hospital from May to July 2020 (N=300) EQ5D5L Mean SD Utility 0.68 0.32 Visual Analog Scale 74.34 12.22 EORTC QLQ-C30 Domains Physical 72.51 24.92 Role 73.17 29.64 Emotional 85.89 16.04 Cognitive 87.78 16.24 Social 79.67 25.41 Symptoms Weakness 36.22 21.69 Nausea-Vomiting 24.67 24.47 Pain 34.5 30.69 Dyspnea 4.78 14.53 Insomnia 33.00 35.28 Loss of appetite 32.67 30.9 Constipation 14.89 25.42 Diarrhea 7.67 18.4 Financial difficulties 30.67 33.26 General QoL 72.19 17.42 QoL, Quality of life Table 3 Performance of Model for Predicting the Utility Value from Derivation Dataset from Cancer Patients of Dr. Kariadi Hospital from May to July 2020 (n=266) Model 1 Model 2 β S.E p-value β S.E p-value Intercept -0.105 0.114 0.359 -0.033 0.760 Physical domain 0.008 0.001 0.000 0.008 0.001 0.000 Role domain 0.001 0.001 0.032 0.001 0.001 0.029 Emotional domain 0.001 0.001 0.098 0.001 0.001 0.020 Cognitive domain 0.001 0.001 0.175 Social domain 0.001 0.005 0.210 Weakness 0.000 0.001 0.645 Nausea-Vomiting 0.000 0.005 0.320 Pain -0.002 0.005 0.001 -0.002 0.000 0.000 Dyspnea 0.000 0.001 0.971 Insomnia 0.000 0.000 0.449 Loss of appetite 0.000 0.000 0.919 Constipation 0.000 0.000 0.931 Diarrhea 0.000 0.001 0.814 Financial difficulties -0.001 0.000 0.023 -0.001 0.000 0.008 General QoL -0.001 0.001 0.365 QoL, Quality of life Table 4 The Models for Predicting the Utility Value in Cancer Patients of Dr. Kariadi Hospital from May to July 2020 Model Actual EQ-5D-5L mean utility (SD) Predicted EQ-5D-5L mean utility (SD) Adjusted R2 MAE RMSE Model 1: full 0.682 (0.323) 0.683 (0.279) 0.737 0.128 0.173 Model 2: Backward Method 0.682 (0.323) 0.683 (0.278) 0.744 0.125 0.168 Table 5 Results of Model Analysis in Validation in Cancer Patients of Dr. Kariadi Hospital from May to July 2020 (n=34) Mean Actual (SD) Mean Predicted (SD) Mean Absolute Error R-Square RMSE Validation data set Model 1 : EORTC 0.7109 (0.2703) 0.7076 (0.2207) 0.1095 0.6706 0.1528 Model 2 : EORTC (Backward Method) 0.7109 (0.2703) 0.7114 (0.2255) 0.1119 0.6793 0.1508 Derivation data set Model 1 : EORTC 0.7197 (0.2749) 0.7197 (0.2315) 0.1058 0.7089 0.1477 Model 2 : EORTC (Backward Method) 0.7197 (0.2749) 0.7197 (0.2307) 0.1075 0.7042 0.1488 Discussion Our study defines the algorithm of mapping the EORTC QLQ-C30 onto the EQ-5D-5L. Based on the regression analysis, model 2, consisted of the physical domain, role domain, emotional domain, pain symptom and financial difficulties symptoms as the predictors for EQ-5D-5L utility value. The parameters of predictability and consistency in both models are similar, presented by the lower MAE and RMSE and high R square. Our study finds that the two models show a good predictive power of regression performance. The results of the predictability and consistency values are similar to the previous studies in colorectal cancer and non-small cell lung cancer (Khan et al., 2016; Marriott et al., 2017; Versteegh et al., 2012). Another previous study in breast cancer, shows lower MAE and higher RMSE (E. Kim et al., 2012). The physical domain and role domain became the predictors of EQ-5D-5L, due to the physical condition after cancer treatment and the patients have to limit their role in some activities to keep their body fit (Zandbergen et al., 2019). Dyspnea was experienced by around 40% advanced cancer of patients. Its prevalence may be increased due to the worsening of other symptoms and can cause deterioration of quality of life (Damani et al., 2018). Furthermore, nausea-vomiting is the most frightening side effect for the cancer patients, due to the emetogenic effect of cytostatic drugs (Perwitasari et al., 2012). Our study shows that weakness is the most experienced symptom, which could be caused by the advanced stage of cancers as well. The previous study shows that the predictors of EQ-5D-5L were global health, physical, role, emotional functions and pain. This model has MAE of 0,069 and RMSE of 0.095 (Kim et al., 2012). The two models in our study, using the OLS with full model and backward elimination model, show similar performance. Other previous studies showed that the backward elimination model had a good performance (Crott and Briggs, 2010). Another study with stepwise regression model demonstrated a good performance with global, physical and emotional domains (Kontodimopoulos et al., 2009). With these results, we suggest the use of model 2 for predicting the utility value of EQ-5D-5L. One limitation from the present study is that we only collected data for the validation and derivation data set from one hospital. This might limit the generalizability of our findings. Further study involving patients from various hospitals is warranted. Another limitation pertains to the inclusion of patients with one of three types of cancer due to the available diagnosis during the study period. According to the Globocan 2018, the most frequent cancer in Indonesia are breast cancer both in male and female, lung cancer and colorectal cancer in males (GLOBOCAN, 2018) . Author Contribution Statement DAP and FDP are contributed in the conceptualization of the study, study design, data validation, data analysis, writing the manuscript and reviewing the manuscript; SFC contributed in data validation, data analysis, investigation, writing the manuscript and reviewing the manuscript; MM, AP and MBUF contributed to the data collection, investigation, data analysis, writing the manuscript and reviewing the manuscript; BPS and AAK contributed in study design, data collection, data validation, writing the manuscript and review the manuscript, AAK contributed to the conceptualizations and reviewing the manuscript. Acknowledgements General We would like to thank the Director of Kariadi Hospital and staffs for granting and assisting us in collecting data for this study, and Ministry of Research and Technology/ Badan Riset dan Inovasi Nasional No: 9/E1/KPT/2021 for the research grant. Funding Statement This study is funded by the Ministry of Research and Technology/ Badan Riset dan Inovasi Nasional No: 9/E1/KPT/2021 Approval All authors have approved the manuscript content Ethical Declaration This research is registered and approved by Ethics Committee of Dr. Kariadi Hospital No.401/EC/KEPK-RSDK/2019 Data Availability The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request Conflict of Interest All authors have no conflict of interest ==== Refs References Aaronson NK Ahmedzai S Bergman B The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology J Natl Cancer Inst 1993 85 365 76 8433390 Ameri H Yousefi M Yaseri M Mapping EORTC-QLQ-C30 and QLQ-CR29 onto EQ-5D-5L in Colorectal Cancer Patients J Gastrointest Cancer 2020 51 196 203 30977049 Chen J Sun M Adeyemo A Genome-wide association study of type 2 diabetes in Africa Diabetologia 2019 62 1204 11 31049640 Crott R Briggs A Mapping the QLQ-C30 quality of life cancer questionnaire to EQ-5D patient preferences Eur J Health Econ 2010 11 427 34 20473703 Damani A Ghoshal A Salins N Deodhar J Muckaden M Prevalence and Intensity of Dyspnea in Advanced Cancer and its Impact on Quality of Life Indian J Palliat Care 2018 24 44 50 29440806 Drummond M Sculpher M Claxton K Stoddart G Torrance G Methods for the Economic Evaluation of Health Care Programmes (4 th) 2015 Oxford University Press Herdman M Gudex C Lloyd A Development and preliminary testing of the new five-level version of EQ-5D (EQ-5D-5L) Qual Life Res 2011 20 1727 36 21479777 Kennedy-Martin M Slaap B Herdman M Which multi-attribute utility instruments are recommended for use in cost-utility analysis? A review of national health technology assessment (HTA) guidelines Eur J Health Econ 2020 21 1245 57 32514643 Khan I Morris S Pashayan N Comparing the mapping between EQ-5D-5L, EQ-5D-3L and the EORTC-QLQ-C30 in non-small cell lung cancer patients Health Qual Life Outcomes 2016 14 60 27072351 Kim E Ko SK Kang HY Mapping the cancer-specific EORTC QLQ-C30 and EORTC QLQ-BR23 to the generic EQ-5D in metastatic breast cancer patients Qual Life Res 2012 21 1193 03 22012023 Kim SH Jo MW Kim HJ Ahn JH Mapping EORTC QLQ-C30 onto EQ-5D for the assessment of cancer patients Health Qual Life Outcomes 2012 10 151 23244763 Kontodimopoulos N Aletras VH Paliouras D Niakas D Mapping the cancer-specific EORTC QLQ-C30 to the preference-based EQ-5D, SF-6D, and 15D instruments Value Health 2009 12 1151 7 19558372 Lorgelly PK Doble B Rowen D Brazier J Condition-specific or generic preference-based measures in oncology? A comparison of the EORTC-8D and the EQ-5D-3L Qual Life Res 2017 26 1163 76 27830513 Marriott ER van Hazel G Gibbs P Hatswell AJ Mapping EORTC-QLQ-C30 to EQ-5D-3L in patients with colorectal cancer J Med Econ 2017 20 193 9 27676291 Perwitasari DA Atthobari J Dwiprahasto I Translation and validation of EORTC QLQ-C30 into Indonesian version for cancer patients in Indonesia Jpn J Clin Oncol 2011 41 519 29 21266448 Perwitasari DA Atthobari J Mustofa M Impact of chemotherapy-induced nausea and vomiting on quality of life in indonesian patients with gynecologic cancer Int J Gynecol Cancer 2012 22 139 45 22080888 Purba FD Hunfeld JAM Iskandarsyah A Quality of life of the Indonesian general population: Test-retest reliability and population norms of the EQ-5D-5L and WHOQOL-BREF PLoS One 2018 13 e0197098 29750806 Purba FD Hunfeld JAM Iskandarsyah A The Indonesian EQ-5D-5L Value Set Pharmacoeconomics 2017 35 1153 65 28695543 Setiawan D Dusafitri A Galistiani GF van Asselt ADI Postma MJ Health-Related Quality of Life of Patients with HPV-Related Cancers in Indonesia Value Health Reg Issues 2018 15 63 9 29474181 Versteegh MM Leunis A Luime JJ Mapping QLQ-C30, HAQ, and MSIS-29 on EQ-5D Med Decis Making 2012 32 554 68 22114301 Zandbergen N de Rooij BH Vos MC Changes in health-related quality of life among gynecologic cancer survivors during the two years after initial treatment: a longitudinal analysis Acta Oncol 2019 58 790 800 30698069

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116159 10.31557/APJCP.2023.24.4.1359 Research Article Follow-Up Strategies and Detection of Recurrent Breast Cancer in the Modern Era Ithimakin Suthinee 1* Luengwatthanakit Natthakit 2 Wongkraisri Concord 1 1 Division of Medical Oncology, Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand. 2 Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand. * For Correspondence: aesi105@yahoo.co.th 2023 24 4 13591366 23 12 2022 17 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Regular history assessments and physical examination with annual breast imaging have been recommended as the standard surveillance protocol for breast cancer patients who underwent curative-intent therapy. Based on randomized studies conducted in the 2000s, surveillance with regular chest or abdominal imaging, chemistry panels, or tumor marker measurements does not improve survival in such patients. Given the remarkable recent improvements of systemic therapy, we hypothesized that more intensive surveillance may lead to early detection and improve treatment outcomes in the modern era. Methods: We retrospectively evaluated the follow-up strategies and benefits of investigations used in usual practice. Breast cancer patients who had initial adjuvant therapy were recruited and classified according to the receipt of standard follow-up (history, physical examination, and annual breast imaging) or alternative follow-up (surveillance with at least annual chest or abdominal imaging or biannual liver function testing). The primary outcome was overall survival. Secondary outcomes included disease-free survival and the indicator of recurrence detection. Results: Of 412 recruited patients, 213 (51.7%) and 199 patients (49.3%) were included in the standard follow-up group and alternative follow-up group, respectively. Among 90 patients (21%) with disease recurrence, the most frequent indicators of recurrence were newly reported symptoms or physical examination abnormalities (64%), followed by abnormal breast imaging (23%) and abnormal chest X-ray (10%). After a median follow-up of 85 months, approximately 90% of patients remained alive after 5 years in both groups. The mean overall survival was similar between the standard and alternative follow-up groups (154.5 months vs. 151.9 months, p = 0.54). There was no difference in terms of the proportion of interval visits, specific cancer treatment received, and disease-free survival. Conclusion: Standard follow-up with history assessments, physical examination, and annual breast imaging remains the recommended surveillance strategy in the modern era. Alternative follow-up strategy did not improve oncologic outcomes. Key Words Breast cancer follow-up surveillance recurrence detection ==== Body pmcIntroduction Breast cancer is the most common cancer in Thai woman. Approximately 80 percent of Thai patients with breast cancer were diagnosed with non-metastatic disease amenable to curative treatment (Sangkittipaiboon et al., 2015). After surgery and adjuvant therapy, surveillance is critical for detecting disease recurrence and long-term treatment complications in breast cancer survivors to optimize outcomes. Breast cancer is a heterogeneous disease with a varied clinical course. Breast cancer is classified into three main molecular subtypes, namely luminal tumors (estrogen receptor-positive [ER+] or progesterone receptor-positive), triple-negative, and human epidermal growth factor receptor 2-positive (HER2+). Luminal tumors are typified by slow progression, but late recurrence occasionally occurs. Meanwhile, patients with HER2+ and triple-negative tumors more frequently experience rapid progression. The optimal follow-up measures for detecting disease recurrence might differ among the breast cancer subtypes. The American Society of Clinical Oncology (ASCO) recommends clinical surveillance including regular history assessments, physical examination, and annual breast imaging as a proper surveillance protocol among patients with breast cancer who completed primary therapy with curative intent (Khatcheressian et al., 2013). Scheduled visits consisting of symptom assessment and physical examination should be performed every 3-6 months in the first 3 years after treatment, every 6-12 months up to 5 years, and annually thereafter, together with annual breast imaging. Complete blood counts, chemistry panels, bone scan, chest or abdominal imaging, and/or tumor marker measurements are not recommended for routine follow-up in asymptomatic patients without specific findings on clinical examination (Cardoso et al., 2019). Consistent with previous studies from Western countries published in the past decade, the aforementioned investigations do not improve survival in patients with early breast cancer (Investigators, 1994; Rosselli Del Turco et al., 1994; Palli et al., 1999; Kokko et al., 2005). Although clinical studies and recommendations from Western organizations support the use of only clinical examination with annually breast imaging as a proper surveillance breast cancer schedule, the application of this policy in Thai patients remains controversial. Because of a lack of awareness or limited opportunity to urgently reach cancer experts in Thailand, some patients developed rapid and extensive symptomatic metastasis requiring unscheduled emergency visits. Some of these patients cannot undergo chemotherapy because of organ dysfunction or poor performance status due to the widespread disease. Consequently, in Thai clinical practice, some experts prefer chest or abdominal imaging as well as blood chemistry panels as surveillance tools to better detect recurrence and increase the chance for patients to receive specific breast cancer treatment. Contrarily, intensive monitoring may lead to unnecessary investigations, causing anxiety, higher patient costs, and a greater burden on medical personnel. However, with recent remarkable improvements in systemic therapy for advanced breast cancer, the earlier detection of asymptomatic breast cancer recurrence and earlier treatment may lead to better outcomes in the modern era. The recent recommendations for breast cancer surveillance are mainly based on the results of clinical studies performed in the 1990s and 2000s. Therefore, this retrospective study compared outcomes among patients with breast cancer between standard clinical surveillance and more intensive surveillance measures. Materials and Methods Patients The data of patients with stage 1–3 breast cancer who completed surgical treatment and received adjuvant systemic treatment between 2010 and 2013 and who underwent follow-up at Siriraj Hospital, Bangkok, Thailand were retrospectively reviewed. Patients with questionable metastasis at diagnosis, other primary cancer, or incomplete data were excluded from the study. Baseline characteristics, breast cancer subtypes, stage, treatment, surveillance schedules, site of recurrence, date of recurrence or death, and follow-up data were obtained from electronic medical records. Surveillance measures of interest included breast imaging, chemistry panels, chest or abdominal imaging, and serum tumor markers. The patients were categorized into the standard or alternative follow-up group. Standard follow-up measures included history assessments and physical examination by a physician every 3–4 months in the first 2 years, every 6 months in the following 3 years, and annually thereafter, as well as annual breast imaging. Patients who underwent other investigations because of suspicion of recurrence based on standard surveillance were categorized into standard follow-up group. Patients with alternative follow-up were defined as asymptomatic patients who completed the same scheduled visits as those in the standard follow-up group in addition to annual chest or abdominal imaging or biannual liver function testing. The two follow-up and surveillance patterns were performed according to physician’s preference. The study was approved by the Siriraj Institutional Review Board (Protocol number 134/2018). The patients were not required to obtain consent form due to its retrospective method. Statistical analysis Comparisons of clinico-pathological parameters, breast cancer subtypes, and surveillance patterns between the groups were performed using the chi-squared test, Mann-Whitney U test and Fisher’s exact test. Categorical data were reported as percentages, whereas continuous variables were reported as the median and range. Overall survival (OS) was defined as time from diagnosis to death from any cause. Death date was retrieved from hospital database and civil registration. Disease-free survival (DFS) was defined as time from diagnosis to disease progression or death. Overall survival and DFS were analyzed using the Kaplan-Meier survival method. Comparisons of overall survival, disease-free survival, and time to events between the groups were performed using the log-rank test. p values were two-sided, and 95% confidence intervals were presented. Multivariate analysis was assessed included only those variables that were positive in univariate analysis and were assessed using multiple binary logistic regression analysis. Statistical analysis was performed using SPSS version 20 (licensed by Mahidol University, Bangkok, Thailand). From the literature review, the 5-year mortality rate of early-stage breast cancer is 13.2% (Chairat et al., 2014). We hypothesized that alternative or more intensive follow-up would improve the survival rate by approximately 10%. At least 197 patients in each follow-up pattern were required to achieve statistical power of 80% and a two-sided alpha error of 0.05. The primary endpoint was overall survival, defined as the time from diagnosis to death. Secondary outcomes included disease-free survival and patterns of recurrence detection. Results In total, 412 patients with non-metastatic breast cancer were included in the study. The cohort included 269 (65%), 37 (9%), 33 (8%) and 42 patients (10.2%) with ER+/HER2−, ER+/HER2+, triple-negative, and ER−/HER2+ breast cancer, respectively (Table 1). Meanwhile, 213 (51.7%) and 199 patients (49.3%) were included in the standard and alternative follow-up groups, respectively. Most patients had stage 2–3 breast cancer. There was no significant difference in breast cancer treatment modalities between the two groups. There was no difference in terms of chemotherapy used among patients in standard and alternative follow-up group. Of 96 patients with HER2+ breast cancer, 23 patients (46%) in alternative follow-up group and 12 patients (26%) in standard follow-up group received adjuvant trastuzumab (p=0.04). All patients with luminal breast cancer received hormonal therapy with mainly aromatase inhibitor in both follow-up groups (74% in standard follow-up group vs 87% in alternative follow-up group, p=0.4). All patients were reviewed for new or abnormal symptoms or submitted to physical examination during each scheduled visit. All patients received appropriate annual breast imaging, i.e., breast ultrasound, mammography, or MRI. Approximately 40% of patients underwent surveillance chest X-ray annually, whereas 30% of patients underwent semiannual liver function testing. Among the patients in the alternative follow-up group, some underwent both chest imaging and blood chemistry analysis per their oncologists’ discretion. Fifty-three patients (24.9%) with standard follow-up experienced disease recurrence, compared to 37 patients (18.6%) who received alternative follow-up (p = 0.12). Among the 90 patients (21.8%) with disease recurrence, the most common site of recurrence was the ipsilateral breast or chest wall (40%), followed by the lungs or pleura (33.3%) and bone (26.7%), Table 2. Considering the site of first relapse according to the mode of surveillance, no association was detected. The lungs were comparably identified as the first site of recurrence despite annual chest X-ray in most patients in the alternative follow-up group. The most frequent sign of recurrence was new symptoms detected via history assessment and physical examination (64%), followed by breast imaging (23.3%) and abnormal surveillance chest X-ray results (10%, Table 2). Meanwhile, 39 of 90 patients with disease recurrence (39%) experienced with symptomatic recurrence requiring an urgent visit. There was no significant difference in the rate symptomatic or urgent visits between the standard and alternative follow-up groups (35.8% and 42.1%, respectively, Table 2). After a median follow-up of 85 months, the median overall survival was not reached. Approximately 90% of patients in both groups survived for 5 years. The mean overall survival was similar between the standard and alternative follow-up groups (154.5 and 151.9 months, respectively, p = 0.54, Tables 3–4). However, disease-free survival tended to be shorter in the standard follow-up group (111.4 months vs. 139.3 months, p = 0.07). Multivariate analysis adjusted for known prognostic factors revealed that disease-free survival was significantly longer among patients who underwent liver function testing (Table 5). Considering follow-up patterns in breast cancer subtypes, multivariate analysis adjusted for known prognostic factors and treatment received showed that alternative follow-up did not associate with longer overall survival and disease-free survival in subgroup of ER-positive. Interestingly, alternative follow-up pattern especially annual chest-x-ray associated with longer disease-free survival in patients with HER2-positive breast cancer (Table 6). Patients with recurrent breast cancer received specific cancer treatment, excluding three patients who did not receive treatment because of their poor performance status. Of these three patients, two patients who underwent standard surveillance required additional visits because of the appearance of new symptoms. The median time from the suspicion of recurrence to the start of treatment tended to be longer in the standard follow-up group (50 days vs. 42 days, p = 0.81, Table 7). In addition, there was no meaningful difference in the time from suspicion of recurrence to disease progression and the time from definite recurrence to disease progression between the groups. Table 1. Baseline Characteristics Baseline characteristics Standard follow-up Total = 213 n (%) Alternative follow-up Total = 199 n (%) p Menstrual status Pre-/perimenopausal 98 (46) 88 (44.2) 0.73 Postmenopausal 111 (52.1) 105 (52.8) Undetermined 4 (1.9) 6 (3) Stage 1 23 (10.8) 16 (8) 0.37 2 130 (61) 116 (58.3) 3 60 (28.2) 67 (33.7) Pathology type Invasive ductal carcinoma 198 (93) 186 (93.4) 0.57 Invasive lobular carcinoma 10 (4.7) 10 (5.1) Others 5(2.3) 2 (1) Breast cancer subset Luminal 138 (64.8) 131 (65.8) 0.52 Luminal HER-2 29 (13.6) 8 (11.6) Triple-negative 11 (16.4) 22 (11.1) ER−, HER2+ 18 (8.5) 24 (12.1) Grade 1 21 (9.9) 17 (8.5) 0.93 2 118 (55.4) 112 (56.3) 3 59 (27.7) 58 (29.1) Missing 15 (7) 12 (6) Resection Mastectomy 154 (72.3) 148 (74.4) 0.64 Breast conservative surgery 59 (27.7) 51 (25.6) Axillary node surgery Sentinel node biopsy 87 (40.8) 66 (33.2) 0.11 Axillary node dissection 126 (59.2) 133 (66.8) Neoadjuvant chemotherapy No 187 (87.8) 170 (85.4) 0.48 Yes 26 (12.2) 29 (14.6) Radiotherapy No 84 (39.4) 67 (33.3) 0.23 Yes 129 (49.4) 132 (50.6) n, number of patients; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2 Table 2 Breast Cancer Recurrence and Patterns of Recurrence Detection Breast cancer recurrence Standard follow-up Total = 53 n (%) Alternative follow-up Total = 37 n (%) p Breast cancer recurrence Yes 53 (24.9) 37 (18.6) 0.12 No 160 (75.1) 162 (81.4) First site (s) of recurrence Ipsilateral breast or chest wall 14 (26.4) 9 (24.3) 0.82 Contralateral breast 5 (9.4) 4 (10.8) 0.83 Ipsilateral axillary node 13 (24.5) 4 (10.8) 0.1 Non-regional nodes 3 (5.7) 1 (2.7) 0.5 Bone 12 (22.6) 12 (32.4) 0.3 Liver 6 (11.3) 6 (16.2) 0.5 CNS 3 (5.7) 5 (13.5) 0.2 Lung or pleura 15 (28.3) 15 (40.5) 0.23 Period of recurrence detection 0.45 Follow-up visit 34 (64.2) 21 (57.9) Emergency room/ interval visit 19 (35.8) 16 (42.1) Indicator of recurrence History assessment or abnormal physical examination 38 (71.7) 20 (54.1) 0.029 Surveillance breast imaging 13 (24.5) 8 (21.6) Surveillance chest X-ray 2 (3.8) 7 (18.9) Others 0 2 (5.4) n, number of patients; CNS, central nervous system Table 3 Overall Survival Depends on the Follow-up Strategy Follow-up strategy Five-year OS (%) OS (months) Mean (95% CI) p Follow-up type Standard (n = 219) 90.9 154.5 (146.8, 162) 0.538 Alternative (n = 199) 90.8 151.9 (146.4, 157.4) Surveillance chest X-ray Yes (n = 167) 90.9 151.86 (145.9, 157.8) 0.569 No (n = 245) 90.8 154.8 (147.6, 162) Surveillance LFT Yes (n = 125) 92.6 155.97 (150.25,161.68) 0.116 No (n = 287) 90.4 153.96 (147.7, 160.2) OS, overall survival; CI, confidence interval; LFT, liver function test Table 4 Univariate and Multivariate Factors Associated with Overall Survival Factors Event/total Univariate Multivariate Hazard ratio (95% CI) p Hazard ratio (95% CI) p Stage 1 2/39 1 1 2 20/246 1.30 (0.30–5.59) 0.722 1.24 (0.28–5.42) 0.776 3 22/127 3.18 (0.75–13.55) 0.117 2.51 (0.57–11.04) 0.222 Breast cancer subset Luminal 28/269 1 1 Luminal HER2 5/51 0.96 (0.37–2.48) 0.929 0.82 (0.31–2.16) 0.689 Triple-negative 6/50 1.22 (0.51–2.95) 0.656 0.95 (0.36–2.36) 0.855 ER−/HER2+ 5/42 1.19 (0.46–3.07) 0.727 0.95 (0.36–2.51) 0.91 Grade 1 1/38 1 1 2 21/230 3.65 (0.49–27.15) 0.206 3.19 (0.42–24.27) 0.262 3 19/117 6.89 (0.92–51.45) 0.06 5.81 (0.74–45.38) 0.093 Unavailable 3/27 4.12 (0.43–39.67) 0.22 3.46 (0.34–34.74) 0.292 LVI Negative 13/182 1 1 Positive 13/95 1.86 (0.86–4.02) 0.113 1.32 (0.58–2.96) 0.503 NA 18/135 1.84 (0.90–3.76) 0.094 1.62 (0.78–3.36) 0.198 Follow-up type Standard 24/213 1.21 (0.67–2.18) 0.539 0.67 (0.18–2.45) 0.541 Alternative 20/199 1 1 Surveillance CXR Yes 17/167 0.84 (0.47–1.54) 0.569 0.68 (0.21–2.22) 0.525 No 27/245 1 1 Surveillance LFT Yes 9/125 0.56 (0.27–1.17) 0.122 0.49 (0.20–1.21) 0.122 No 35/287 1 1 CI, confidence interval; HER2, human epidermal growth factor receptor 2; ER, estrogen receptor; NA, not available; LVI, lympho-vascular invasion; CXR, Chest X-ray: LFT, Liver function test Table 5 Univariate and Multivariate Factors Associated with Disease-Free Survival Factors Event/total Univariate Multivariate Hazard ratio (95% CI) p Hazard ratio (95% CI) p Stage 1 6/38 1 1 2 44/246 0.95 (0.40–2.23) 0.901 0.88 (0.37–2.10) 0.773 3 40/127 1.93 (0.82–4.57) 0.133 1.69 (0.69–4.13) 0.248 Breast cancer subset Luminal 57/269 1 1 Luminal HER2 13/51 1.26 (0.69–2.30) 0.453 1.02 (0.55–1.90) 0.948 Triple-negative 10/50 0.99 (0.50–1.93) 0.966 0.95 (0.46–1.94) 0.881 ER−/HER2+ 10/41 1.26 (0.65–2.48) 0.494 1.14 (0.57–2.27) 0.715 Grade 1 4/38 1 1 2 51/229 2.78 (0.82–6.30) 0.113 2.01 (0.71–5.70) 0.191 3 25/117 2.28 (0.79–6.60) 0.125 1.97 (0.65–5.95) 0.228 Unavailable 10/27 4.34 (1.36–13.84) 0.013 3.75 (1.13–12.45) 0.031 LVI Negative 33/181 1 1 Positive 27/95 1.58 (0.95–2.63) 0.078 1.19 (0.69–2.05) 0.525 NA 30/135 1.21 (0.74–1.99) 0.448 1.13 (0.68–1.86) 0.647 Follow-up type Standard 53/213 1.47 (0.97–2.24) 0.072 0.80 (0.30–2.13) 0.655 Alternative 37/198 1 1 Surveillance CXR Yes 31/166 0.69 (0.45–1.07) 0.099 0.66 (0.28–1.59) 0.357 No 59/245 1 1 Surveillance LFT Yes 17/124 0.50 (0.29–0.84) 0.01 0.48 (0.24–0.96) 0.037 No 73/287 1 1 CI, confidence interval; HER2, human epidermal growth factor receptor 2; ER, estrogen receptor; NA, not available; LVI, lympho-vascular invasion; CXR, chest X-ray; LFT, liver function test Table 6 Univariate and Multivariate Factors Associated with Disease-Free Survival in HER2-Positive Breast Cancer Patients Factors Event/total Univariate Multivariate Hazard ratio (95% CI) p-value Hazard ratio (95% CI) p-value Follow up patterns Standard 17/47 3.28 (1.29-8.32) 0.013 4.24 (1.54-11.68) 0.005 Alternative 6/45 1 1 Surveillance chest X-ray Yes 5/38 0.34 (0.12-0.90) 0.031 0.31 (0.11-0.88) 0.027 No 18/54 1 1 Surveillance liver function test Yes 3/26 0.33 (0.10-1.12) 0.076 0.30 (0.09-1.03) 0.055 No 20/66 1 1 Neo-Adjuvant regimen Yes 5/15 1.56 (0.58-4.20) 0.38 2.20 (0.66-7.33) 0.2 No 18/77 1 1 Anti HER2 Yes 9/35 1.01 (0.44-2.33) 0.986 0.67 (0.26-1.73) 0.407 No 14/57 1 1 CI, confidence interval; HER2, human epidermal growth factor receptor 2; Multivariate adjusted hazard ratio for stage, breast cancer subsets, grade, and lympho-vascular invasion. Table 7 Time to Recurrence and Disease Outcomes in Patients with Recurrence Standard follow-up Total = 53 Alternative follow-up Total = 37 p Median time to recurrence 39.4 months 45.16 months 0.236 median (IQR) (19.13–62.30) (29.17–65.60) Time from suspicion of recurrence to treatment 50 days 42 days 0.525 Median (IQR) (25.00–91.00) (16.00–77.00) Time from suspicion of recurrence to progressive disease 13.07 months 10.97 months 0.715 Median (IQR) (4.23–29.90) (5.77–23.43) Time from definite recurrence to progressive disease 12.33 months 7.87 months 0.709 Median (IQR) (3.00–27.70) (4.40–23.10) Comparisons of medians between the two groups were performed using the Mann–Whitney U test. IQR, interquartile range Discussion Although convincing scientific evidence is lacking to date, most physicians assume that the early detection of recurrent breast cancer will have a favorable impact on survival. Most available data regarding surveillance recommendations were generated in an era with less advanced radiographic or tools for breast cancer recurrence as well as less efficacious therapy. However, the hypothesis regarding possibility of cure with aggressive multimodality therapy has arisen for patients with oligo-metastases (Hortobagyi, 2002). To support this approach, effective surveillance protocol for early detection of limited metastasis is crucial. According to our study, chest X-ray is the most common additional diagnostic test, followed by liver function testing. However, indicators of breast cancer recurrence primarily included symptoms recorded during clinical examinations and breast imaging, in line with previous reports from Western countries (Loomer et al., 1991). In our study, the standard follow-up group underwent follow-up as recommended by ASCO in 2013, which recommended only clinical examination and annual breast imaging. Both overall survival and disease-free survival were similar between the groups. Our standard surveillance approache corresponds with a guideline developed from European Society Medical Oncology (ESMO) which also supported clinical evaluation and annual breast imaging only (Cardoso et al., 2019). The recurrence rate in our study was similar that in a previous study conducted in Thailand (Chairat et al., 2014). Therefore, our study indicated that patients with breast cancer in Thailand do not benefit from alternative diagnostic tests performed in addition to routine medical surveillance. Our study is the first to demonstrate the follow-up patterns of patients with breast cancer in Thailand in the modern era with markedly advanced and effective systemic therapy. Although limited patients with recurrent breast cancer were included in our study, we described a retrospective cohort with an adequate follow-up duration. Additional diagnostic tests such as chest X-ray or blood chemistry might increase the workload of radiologists and laboratory technicians. The routine use of more intensive follow-up both results in the consumption of additional resources and leads to additional tests because of equivocal results or insignificant lesions. Moreover, intensive follow-up possibly causes unnecessary anxiety in patients with false-positive results. According to a study in France, the cost of intensive follow-up was 2.2–3.6-fold higher than that of standard follow-up (Mille et al., 2000). Further analysis of the financial impact of the follow-up strategy in Thailand might result in cost savings. Conversely, intensive follow-up might enhance early disease detection rates, thereby permitting proper and timely management. In our retrospective study, three patients with disease recurrence could not undergo systemic treatment because of their poor performance status. However, this phenomenon was not significantly different between the two groups. Based on previous randomized trials together with our retrospective cohort reflecting the modern era, the earlier detection of metastatic disease using any investigation modality has not improved oncologic outcomes. Larger populations and randomized controlled studies are needed to reassess whether proper intensive follow-up strategy increases the likelihood of breast cancer-specific treatment or leads to improved survival among patients with breast cancer in the modern era. Since biology of breast cancer impacts patterns of recurrence including sites of metastasis and timing of recurrence (Kennecke et al., 2010; Jatoi et al., 2011; Metzger-Filho et al., 2013), it is interesting to determine optimal follow up or surveillance schedule designed more specifically for individual breast cancer subtypes. Subset analysis looking at ER+ breast cancer patients showed no additional benefit of alternative surveillance measure compared to standard surveillance protocol. Patients with HER2+ breast cancer in our cohort recruited in standard follow-up group experienced higher recurrence compared to alternative follow-up group. This phenomenon could be a result of higher proportion of patients in alternative follow up group received adjuvant trastuzumab (46% vs 26%, p = 0.04). Multivariate analysis demonstrated association of alternative follow-up protocol and better disease-free survival among patients with HER2+ breast cancer. However, the advantage of alternative follow-up protocol in HER2+ subgroup did not translate to overall survival benefit. Based on our report, alternative follow-up surveillance protocol might provide meaningful benefit to early disease recurrence detection for patients with HER2-positive breast cancer. The limitations of this study included its retrospective design, which resulted in variation of the follow-up strategies and limited the sample size. Patients categorized into the alternative follow-up group were heterogeneous. Moreover, the result from our study reflected oncologic practice during the past decade when the targeted therapy was widely available, but it may not totally reflect the current practice when numerous newly approved targeted drugs have emerged. Another unknown issue is the intensity of surveillance needed to detect disease recurrence. The alternative surveillance measures used in our hospital may not be sufficient to detect early disease recurrence. Although PET/CT scan is more sensitive to detect metastatic disease (Vogsen et al., 2021), the value of PET/CT evaluation to detect recurrence in asymptomatic patients is still low (Taghipour et al., 2016). In the future, sensitive imaging or emerging molecular diagnostics, such as circulating tumor cells or circulating DNA detection, might play role to the early detection of relapse. Another limitation of this study was its single-institutional nature, which could limit the applicability to patients in other institutions. In conclusion, standard follow-up schedules with history assessments, physical examination, and annual breast imaging should remain the standard follow-up strategy in this era. Additional follow-up with chest X-ray, chemistry panels, or liver ultrasound did not improve overall survival and disease-free survival among patients with breast cancer who had completed curative-intent therapy. However, there is a trend of benefit of using alternative follow-up protocol for patients with HER2-positive breast cancer. Further research is needed to identify potential efficacious tools and determine specific subgroup that will benefit the most from more aggressive follow-up protocol in order to better detect disease recurrence or facilitate early treatment, which may improve breast cancer treatment outcomes. Author Contribution Statement Study concepts: SI. Study design: SI, NL. Data acquisition: SI, NL, CW. Quality control of data and algorithms: SI, CW. Data analysis and interpretation: All authors. Statistical analysis: SI, CW, Manuscript preparation: SI. Manuscript editing: All authors. Manuscript review: All authors. All authors have read and approved the manuscript. Acknowledgements We thank Miss Khemjira Karnkejklang, Department of Medicine, Faculty of Medicine Siriraj Hospital, for her statistical review. Study approval and funding sources The study was approved by Siriraj Institutional Review Board. It is not the part of an approved student thesis. There was no funding for the study. Data availability The data analyzed in this study are available from the corresponding author, (Suthinee Ithimakin, E-mail: aesi105@yahoo.co.th) on reasonable requests. Conflict of interest The authors have no conflict of interest to declare. ==== Refs References Ghezzi P Magnanini S Rinaldini M Kyriakides S Ohno S Early breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up† Ann Oncol 2019 30 1194 220 31161190 Chairat R Puttisri A Pamarapa A Recurrence and death from breast cancer after complete treatments: an experience from hospitals in Northern Thailand J Med Assoc Thai 2014 97 932 8 25536710 Hortobagyi GN Can we cure limited metastatic breast cancer? J Clin Oncol 2002 20 620 3 11821439 Investigators TG Impact of follow-up testing on survival and health-related quality of life in breast cancer patients A multicenter randomized controlled trial The GIVIO Investigators. JAMA 1994 271 1587 92 8182811 Jatoi I Anderson WF Jeong JH Breast cancer adjuvant therapy: time to consider its time-dependent effects J Clin Oncol 2011 29 2301 4 21555693 Kennecke H Yerushalmi R Woods R Metastatic behavior of breast cancer subtypes J Clin Oncol 2010 28 3271 7 20498394 Khatcheressian JL Hurley P Bantug E Breast cancer follow-up and management after primary treatment: American Society of Clinical Oncology clinical practice guideline update J Clin Oncol 2013 31 961 5 23129741 Kokko R Hakama M Holli K Follow-up cost of breast cancer patients with localized disease after primary treatment: a randomized trial Breast Cancer Res Treat 2005 93 255 60 16132530 Loomer L Brockschmidt JK Muss HB Postoperative follow-up of patients with early breast cancer Patterns of care among clinical oncologists and a review of the literature Cancer 1991 67 55 60 1985723 Metzger-Filho O Sun Z Viale G Patterns of Recurrence and outcome according to breast cancer subtypes in lymph node-negative disease: results from international breast cancer study group trials VIII and IX J Clin Oncol 2013 31 3083 90 23897954 Mille D Roy T Carrère MO Economic impact of harmonizing medical practices: compliance with clinical practice guidelines in the follow-up of breast cancer in a French Comprehensive Cancer Center J Clin Oncol 2000 18 1718 24 10764432 Palli D Russo A Saieva C Intensive vs clinical follow-up after treatment of primary breast cancer: 10-year update of a randomized trial National Research Council Project on Breast Cancer Follow-up JAMA 1999 281 1586 10235147 Rosselli Del Turco M Palli D Cariddi A Intensive diagnostic follow-up after treatment of primary breast cancer A randomized trial National Research Council Project on Breast Cancer follow-up. JAMA 1994 271 1593 7 7848404 Sangkittipaiboon S Leklob A Sriplung H Breast Cancer in Lopburi, a Province in Central Thailand: Analysis of 2001-2010 Incidence and Future Trends Asian Pac J Cancer Prev 2015 16 8359 64 26745085 Taghipour M Sheikhbahaei S Trahan TJ Value of fourth and subsequent post-therapy follow-up 18F-FDG PET/CT scans in patients with breast cancer Nucl Med Commun 2016 37 602 8 27110955 Vogsen M Jensen JD Christensen IY FDG-PET/CT in high-risk primary breast cancer-a prospective study of stage migration and clinical impact Breast Cancer Res Treat 2021 185 145 53 32920740

PMC010xxxxxx/PMC10352745.txt

==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116145 10.31557/APJCP.2023.24.4.1231 Research Article Association of Interactions between Metabolic ‘Caretaker’ Genes, p53, MDM2, and Tobacco Use with the Risk of Oral Cancer: A Multifactor Dimensionality Reduction Approach Singh Ragini D 1* Patel Kinjal A 2 Patel Jayendra B 2 Patel Prabhudas S 2 1 Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), Rajkot, Gujarat, India. 2 Molecular Oncology Laboratory, Cancer Biology Department, The Gujarat Cancer and Research Institute, Asarwa, Ahmedabad, Gujarat, India. * For Correspondence: singh.ragini28@yahoo.com 2023 24 4 12311237 12 11 2022 7 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: The present study investigated the association of interactions between gene polymorphisms in metabolic ‘caretaker’ genes (Phase I: CYP1A1, CYP2E1; Phase II: GSTM1, GSTT1), the cell cycle regulatory gene, p53, along with its negative controller, MDM-2, and the environment variable (tobacco). A nonparametric model, multifactor dimensionality reduction (MDR), was applied to analyse these interactions. Materials and Methods: This case-control study was carried out on 242 subjects. Genomic DNA was extracted from peripheral blood lymphocytes.11 gene variants with an exposure variable (tobacco use) were analysed using MDR to identify the best locus model for gene-gene and gene-environment interactions. Statistical significance was evaluated using a 1000-fold permutation test using MDR permutation testing software (version 1.0 beta 2). The value of p<0.05 was considered statistically significant. Results: The best three-locus model for gene-gene interaction included two of the p53 gene polymorphisms; rs17878362 (intron 3) and rs1042522 (exon 4) and rs6413432 in the Phase I gene, CYP2E1(DraI). The three-locus model to evaluate the gene-environment interaction included two intronic polymorphisms of the p53 gene, that is, rs17878362 (intron 3) and rs1625895 (intron 6), and rs4646903 in the Phase I gene CYP1A1*2C. The interaction graphs revealed independent main effects of the tobacco and p53 polymorphism, rs1042522 (exon 4), and a significant additive interaction effect between rs17878362 (intron 3) and rs1042522 (exon 4). Conclusions: The nonparametric approach highlighted the potential role of tobacco use and variations in the p53 gene as significant contributors to oral cancer risk. The findings of the present study will help implement preventive strategies in both tobacco use and screening using a molecular pathology approach. Key Words Oral cancer CYP1A1 CYP2E1 - GST p53 MDM-2- multifactor dimensionality reduction ==== Body pmcIntroduction In India, oral cancer is the most common malignancy among men and the fifth most common among women (Saini, 2021). Approximately 77,000 new cases are added annually, contributing to 26% of the global cancer burden (Borse et al., 2020). It has become a major health concern due to an alarming trend of an increase in oral cancer cases in <40-45 years of age in recent years (Hussein et al., 2017). The malignancy is also associated with high mortality and disfigurement. This can be attributed to the increased use of various tobacco-chewing products such as gutkha and pan masala. 60% of tobacco users in India use only smokeless tobacco (Sankhla et al., 2018). The Gujarat region (western part of India) is showing a serious growing trend in the use of areca nut-based tobacco products, according to the Global Adult Tobacco Survey (GATS) conducted in India (Sharma et al., 2018). Well-established risk factors for oral cancer include tobacco use in various forms, alcohol, and HPV16/18 infection. However, all tobacco users do not acquire cancer, implying the role of genetic predisposition. In the past two decades, the role of the genomic constitution/genetic makeup of individuals has emerged in oral cancer susceptibility (Damani et al., 2020), suggesting the existence of differences in risk between individuals and between populations (Xie et al., 2016). Single-nucleotide polymorphisms (SNPs), the most abundant variations in the human genome, represent individual inherited differences. These SNPs affect DNA stability, transcriptional factor binding stability, DNA processivity, and nucleosome assembly functions. Consequently, these SNPs in a number of genes affect a number of processes, including cell proliferation, immunological function, inflammation, transcription, DNA repair, and xenobiotic metabolism. Therefore, genetic variations represent significant risk factors for the growth and transformation of a normal cell to a malignant phenotype. These SNPs may be synonymous or non-synonymous, directly or indirectly affecting gene expression and phenotype (Zeng et al., 2019). Oral cancer is preceded by well-established premalignant disorders that undergo several molecular changes on the path to invasive oral cancer (Speight et al., 2018). The interaction between an individual’s genetic predisposition and environmental variables (i.e., tobacco and alcohol use) substantially impacts these molecular changes (Batta et al., 2019). Accumulating published evidence indicates the association of several SNPs that involve numerous pathways with the risk and progression of oral cancer. However, there are inconsistencies in the results, even in studies that analyze similar SNPs and their association with this complex multifactorial disease. Most of these studies involve a single SNP analysis, which does not provide a clear picture of the association. Comprehensive analysis of gene-gene interaction is necessary to characterize multigenic and multifactorial diseases, as interactions between gene polymorphisms can show a synergistic or nonadditive effect on the pathogenesis of oral cancer (Ritchie et al., 2018). Furthermore, recent meta-data have suggested that variants of Phase I: CYP1A1, CYP2E1; Phase II: GSTT1, GSTM1, p53, and MDM-2 show regional, ethnic, and geographical differences in distribution and can modify the risk of oral cancer. These studies have particularly emphasized the inclusion of tobacco in analyses (Tang et al., 2010; Guo et al., 2015; Yang et al., 2015; Zhuo et al., 2016; Li et al., 2018; Zeng et al., 2019). The tobacco components undergo biotransformation by Phase I and Phase II xenobiotic metabolising enzymes. The generated metabolites lead to the formation of DNA adducts and cause DNA damage. These damages are detected by the guardian of the genome, p53, which in turn is regulated by its negative regulator, MDM-2. Each individual has inherited differences in their ability to metabolise these carcinogens and effectively repair the damage caused by them. However, it is challenging to create and identify the susceptibility and predictive biomarker panel that may be used to identify high-risk individuals for this complicated disease. Robust estimation of gene-gene and gene-environment interaction effects requires a large number of samples, thereby incurring a prohibitive expense. Additionally, large sample collection and analysis yield high-dimensional data. Analysing these high-dimensional data using parametric statistical methods, such as logistic regression, has limitations (Ritchie et al., 2018). Taking into account this background, we investigated the role of SNPs in genes associated with tobacco metabolism (CYP1A1, CYP2E1, GSTM1 and GSTT1) and cell cycle regulation (p53 and MDM2) using a nonparametric statistical tool; Multifactor Dimensionality Reduction method (MDR). This tool has been effectively used to characterize gene-gene and gene-environment interactions in various other diseases in a relatively small sample size (Fu et al., 2017). Materials and Methods This study was approved by the Institutional Ethical Committee, (no. EC/35/2012). Written informed consent was obtained from all subjects (cases and controls) after a proper explanation of the blood collection procedure and the purpose of the study in the local language. Subjects We included 121 cases of histopathologically confirmed oral squamous cell carcinoma that attended the outpatient department of the institute. Genetically unrelated healthy subjects from the same region with similar socioeconomic status (n=121) were also included as controls. Controls were blood donors who attended the institute’s blood bank. The inclusion criterion for controls was the absence of a history of cancer, precancer, or other significant health hazards. A standard questionnaire was designed to collect epidemiological data, including details on age, sex, occupation, ethnicity, tobacco consumption habits, and family history of the study population. 92.3% and 81.2% were males in the control and case/patient groups, respectively. 47.9% were tobacco users in the control group, while 85.1% were in the patient group. The mean age of the subjects was 40.2±0.62 years in controls and 42.6±0.42 years in cancer cases. DNA Isolation and Genotyping Genomic DNA was isolated from peripheral blood lymphocytes using commercially available DNA isolation kits (Qiagen, CA) according to the manufacturer’s instructions and stored at -20°C until analysis. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) (Eppendorf master cycler gradient, Germany) to determine the polymorphic genotypes of the genes mentioned in Table 1 according to the procedures previously reported by our laboratory (Singh et al., 2014; Patel et al., 2013). Multifactor-dimensionality reduction (MDR) MDR is a statistical tool that uses a non-parametric approach (Fu et al., 2017) to generate the best locus model for gene-gene and gene-environment interactions (Moore et al., 2002). The entire case-control data set for the SNPs studied is divided into ten equal portions, or subsets, by default, with one subset for testing and the rest for training. The algorithm uses the training part for all combinations of factors/locus to create contingency tables based on cases and controls. The multilocus genotypes are then pooled into high- and low-risk groups. Subjects in cells with a case/control ratio greater than 1 are labeled high risk, while the other cells are labeled a low risk. Based on this new categorization of the training part (9/10), the training error is calculated. With the testing part (1/10), the prediction error is calculated. Therefore, with the algorithm, genotype predictors are effectively reduced from dimensions ‘n’ (high-dimensional genotype data) to one dimension, that is, a single character with two levels of risk: high and low. These steps are repeated for each cross-validation fold (by default, 10-fold cross-validation). Cross-validation consistency (CVC) and permutation tests are used to assess the potential of this unique one-dimensional multilocus genotype to classify and predict the disease status. CVC is defined as the number of times a particular SNP combination is identified across the k-fold cross-validation (CV) or the number of times MDR found the same model when it divided the data into different segments. Significant models will have CVC≥9 based on the fact that the data have been cross-validated 10 times by MDR. Testing balanced accuracy (TBA) is the measure of the average of sensitivity and specificity. The CVC and TBA can then be used to choose the best SNP combination (best-model), which should have the highest CVC and/or TBA. If two or more models have similar TBA, then CVC can be used to decide on the best model (Figure 1). A 1,000-fold permutation test is used to compare observed testing accuracies with those expected under the null hypothesis of no connection to determine the statistical significance of the best model’s testing accuracy. By repeating the entire analysis on 1,000 data sets that are compatible with the null hypothesis, the permutation testing corrects for multiple testing. In permutation testing, the case-control labels of a data set are randomly generated thousands of times, and the MDR method is repeated on each permuted data set. The MDR algorithm also creates interaction graphs/models/dendrograms, which can be used to visualize the nature of the dependencies or interactions effect between polymorphic genes (additive and non-additive) (Pattin et al., 2009). The interaction model describes the percentage of entropy (information gain or IG) by each factor or two-way interaction. In the interaction graphs, a node (point) represents a gene/SNP, and a line represents the interaction between two nodes. The percentage in the nodes expresses the amount of uncertainty in the label eliminated by the node attribute. The IG in the case-control status is eliminated by each variable (independent main effect), and each pairwise combination of characteristics (interaction effect) is indicated by nodes and connections, respectively (Moore et al., 2002). Positive entropy values indicate additive/synergistic interaction, while negative entropy values indicate redundancy between polymorphic genes. This study used MDR software v.3.0.2 and MDR permutation testing software (version 1.0 beta 2) (www.epistasis.org). High- and low-risk genotypic combinations were determined based on the threshold value of 1 (121/121) for the present data. A statistically significant difference is indicated by a p<0.05. Results Gene-Gene Interaction The best two-way gene-gene interaction model generated by the MDR algorithm involved two SNPs in the p53 gene, that is, rs17878362 (intron 3) and rs1042522 (exon 4). This model exhibited a CVC of 6/10 (p=0.68) (Table 2). However, for the best three-way gene-gene interaction model, the SNP in the CYP2E1 gene (DraI), rs6413432, the SNPs in exon 4 (rs1042522), and intron 3 (rs17878362) of the p53 gene had the highest balanced test precision (0.61) and training (0.66) and a high CVC (9/10) (Table 2) (Figure 2a). For the gene-gene interaction, the network plot (dendrogram) revealed a significant additive interaction between intron 3, rs17878362, and exon 4, rs1042522 of the p53 gene (IG=1.46%) (indicated by the entropy values marked on the line, red, connecting the intron 3 and exon 4 nodes). Additionally, an interactive/additive effect was observed between the nodes, CYP2E1 (DraI), and SNP in exon 4. The entropy values in cells (Figure 3a) show a significant independent effect (positive IG) of the p53 exon 4 polymorphism (IG=2.04%) followed by the intron 3 polymorphism (IG=0.22%) (Figure 3a). Gene-Environment Interaction The probable gene-environment interactions in patients were also analyzed using the MDR approach, taking tobacco as a factor/variable (Figure 2b and Figure 3b). Tobacco exposure showed the most significant univariate effect, with the highest TBA (68.2%) and CVC (10/10). The two-way interactions between CYP1A1*2C (Ile/Val) and tobacco exposure exhibited the highest TBA (66.9%) and CVC (7/10). The best-three-way model had interactions between the SNPs present in the intronic regions of the p53 gene, i.e. rs 17878362 (intron 3) and rs1625895 (intron 6), Phase I, CYP1A1*2C (Ile/Val) rs4646903, and tobacco (Table 3). The interaction graph showed a significant independent effect of tobacco (IG=11.19%), less synergy between CYP1A1 * 2C and the intronic polymorphisms of p53. CYP1A1*2C exhibited an additive effect with tobacco. Table 1 Gene Variants Included in the Present Study (MDR Model) Gene Function Polymorphism Metabolic pathway CYP1A1 (Xenobiotic metabolising enzyme) Phase I oxidative and reductive CYP1A1*2A (MspI), rs1048943 CYP1A1*2C(Ile/Val), rs4646903 CYP2E1 (Xenobiotic metabolising enzyme) Phase I oxidative and reductive CYP2E1*5B (c2) (PstI restriction, position: −1019), rs3813867 CYP2E1*5A (c1) (RsaI restriction, position: −1259), rs2031920 CYP2E1*6 (DraI restriction, intron 6), rs6413432 GSTT1(Xenobiotic metabolising enzyme) Phase II Null polymorphism GSTM1(Xenobiotic metabolising enzyme) Phase II Null polymorphism p53 Maintenance of genomic integrity, Regulation of cell cycle progression, apoptosis, autophagy, differentiation, senescence, DNA repair and oxidative metabolism Intron 3 duplication, rs17878362 Exon 4 Pro72Arg, rs1042522 Intron 6, rs1625895 MDM2 p53 negative regulator rs2279744 Figure 1 MDR Algorithm: Stepwise process to identify the best model to predict oral cancer risk for multiple genes Figure 2 Interaction of Attributes: the best 3-locus genotype combinations associated with oral cancer. (a) gene-gene interaction; (b) gene-environment interaction. In each box, the distribution of cases (left bars) and controls (right bars) for each of the genotype combinations are shown. High-risk combinations are depicted as dark-shaded cells and low-risk combinations as light-shaded cells; empty cells are left blank Table 2 MDR Interaction Analysis between SNPs (Gene-Gene Interaction) MDR model Balanced accuracy CV training Balanced accuracy CV testing CV consistency Permutation test 'p' value p53 exon 4, rs1042522 0.5786 0.5404 10/10 0.51 p53 intron 3, rs17878362-p53 exon 4, rs1042522 0.6147 0.5214 6/10 0.68 CYP2E1 DraI, rs6413432- p53 intron 3, rs17878362 - p53 exon 4, rs1042522 0.6623 0.612 9/10 0.06 Table 3 MDR Interaction Analysis between SNPs and Tobacco (Gene-Environment Interaction) MDR model Balanced accuracy CV training Balanced accuracy CV testing CV consistency Permutation test ‘p’ value Tobacco 0.682 0.682 10/10 <0.001 Tobacco - CYP1A1*2C, rs4646903 0.6835 0.6694 7/10 <0.001 Tobacco,- CYP1A1*2C, rs4646903, -p53 Intron 3 rs17878362 - Intron 6 rs1625895 0.7047 0.6333 7/10 0.01 CVC, Cross Validation Consistency; p-values as calculated after 1000 permutations Figure 3 Interaction Entropy Graphs (Network Plots) Highlighting the Amount of Information Gain (IG) in Cells and on Lines as a Percentage: a) gene-gene interaction; (b) gene-environment interaction. Entropy values marked on the lines connecting two SNPs represent the entropy of interaction. Positive percent entropy represents synergy or information gain; negative percent entropy represents redundancy or lack of information gain. . Entropy values in the cells of individual SNPs indicate the main independent effects. Schematic coloration used in graphs represents a continuum from synergy to redundancy. Red represents a high degree of synergy (positive IG); Orange a lesser degree of positive IG; Brown a midway point between synergy and redundancy; Green and blue represent a redundancy Discussion The balance in the expression and activities of Phase I enzymes such as CYP1A1 and CYP2E1 and Phase II enzymes such as GSTs play a critical role in the biotransformation and metabolism of tobacco procarcinogens. This balance, if lost, can potentially cause DNA damage. To deal with the damage caused by tobacco procarcinogens, we have p53, a well-reported tumor suppressor gene with pleiotropic activities (Ghosh et al., 2022) with the negative regulator of its activity, MDM-2. With this as a rationale, research studies on polymorphic variability in xenobiotic metabolizing genes, CYP1A1, CYP2E1, GSTT1, and GSTM1, and the cell cycle regulator, p53 with its negative controller, MDM-2, were reported from our laboratory (Patel et al., 2013; Singh et al., 2014). The variability in the candidate genes studied with oral cancer risk was analyzed using a binary logistic regression model. However, no statistically significant risk of oral cancer was observed. For the tumor suppressor gene, p53, the proline allele of the SNP rs1042522 in exon 4 conferred protection against oral cancer development (Patel et al., 2013). Furthermore, no association was observed with oral cancer risk for SNPrs2279744 in the MDM-2 gene (Patel et al., 2013; Singh et al., 2014,). Analysis of gene-gene interaction revealed that of the ‘caretaker’ genes studied, only GSTM1 and GSTT1, in combination, exhibited a statistically significant higher odds ratio (Singh et al., 2016). In addition to this, a gene-tobacco interaction analysis was also performed on the same population of subjects. The analysis revealed that tobacco use, particularly tobacco chewing, is a significant risk modifier in subjects harboring variant genotypes of CYP1A1, CYP2E1, GSTM1, GSTT1, p53, and MDM-2 (Singh et al., 2012; Patel et al., 2015). Unfortunately, due to the limitation of the small sample size in these studies, a combined gene-gene and gene-environment interaction analysis could not be performed (Patel et al., 2013; Singh et al., 2014). However, these results were successful in (i) providing a tentative SNP fingerprint for oral cancer susceptibility and emphasizing the role of SNPs in these candidate genes in the development of oral cancer, and (ii) highlighting the importance of the association between genes and the environment in modulating susceptibility to oral cancer in the population of Gujarat. Interestingly, the results indicated a limitation, ie the inability to analyse gene-gene and gene-environment interactions involving multiple genes in the same set of patients. Considering this limitation, MDR, an alternative statistical tool with a nonparametric approach, was used for gene-gene and gene-environment analysis in our case-control data set. The results of the MDR algorithm successfully generated the best two-way and three-way interactive models for both gene-gene and gene-environment predicting the risk of oral cancer. The best interactive model between genes included SNPs in exon 4 (rs1042522) and intron 3 (rs17878362) of the p53 gene. These SNPs also exhibited an additive effect. Notably, strong linkage disequilibrium between intron 3 and exon 4 of p53 has also been reported (Wu et al., 2002). Interestingly, exon 4 SNP, rs1042522, showed a significant independent effect on oral cancer risk. On the other hand, the results of the best gene-environment interactive model reinforced that tobacco use is the most important environmental factor that affects oral cancer susceptibility. The model also included both intronic polymorphisms of p53, ie rs17878362 and rs1625995. The results for gene-gene and gene-environment interactions share an interesting finding, ie, the putative role of the p53 gene polymorphisms in oral cancer susceptibility. In the analysis of gene-gene and gene-environment interactions, genomic variations in p53 were a common factor. These genomic variations allow for the generation of p53 isoforms through alternate splicing. These isoforms are involved in creating an immunosuppressive environment, which helps to trigger tumour initiation and progression (Eiholzer et al., 2020). Furthermore, at the mechanistic level, there is some evidence that these polymorphisms may have an impact on the structure of the p53 protein. Therefore, the variants differ in their biochemical and biological activities, such as DNA repair capacity and apoptosis (Wu et al., 2002; Gemignani et al., 2004; Sullivan et al., 2004; Pietsch et al., 2006; Marcel et al., 2011). This makes these loci a potent hotspot region for mutations and increases cancer risk (Sagne et al., 2013). The results of the current analysis also support the findings of our previous study on p53 mutations and oral cancer risk. We reported that p53 exon 4 exhibited a maximum cluster of mutations (Singh et al., 2016), and p53 alterations play an important role in the risk of oral cancer. In conclusion, the MDR application successfully identified a significant interaction between three polymorphisms of two genes, CYP1A1*2C (Ile/Val; rs4646903), intron 3 (rs17878362), intron 6 of p53 (rs1625895), and gene-environment interactions between tobacco and p53 polymorphisms in the oral cancer case-control data set. Such analysis becomes meaningful in the absence of any statistically significant independent main effects of the candidate genes, mainly when there is a limitation to performing gene-environment interactions owing to a low sample size. Importantly, this analysis assessed SNPs involving critical genes for specific pathways in the same case-control data set. Therefore, these results can be used to design and implement preventive tobacco consumption strategies and screen the ‘at risk’ population. Abbreviations CVC, cross-validation consistency; GATS, Global adult tobacco survey; IG, information gain; PCR-RFLP, Polymerase chain reaction-restriction fragment length polymorphism; PCR-SSCP, Polymerase chain reaction-single strand conformation polymorphism; SNP, Single nucleotide polymorphism; TBA, Testing balance accuracy. Author Contribution Statement Ragini Singh: conceptualized and planned the experiments, analysis, and interpretation of the results, and writing of the manuscript; Kinjal Patel: Planned and performed the experiments, analysed and interpreted the results, and prepared the manuscript; Jayendra Patel: supervision of experiments; Prabhudas Patel: provided critical feedback and helped shape the research, analysis, and manuscript. Acknowledgements The authors are thankful to The Gujarat Cancer and Research Institute and Gujarat Cancer Society for financial support. The study was approved by the Institutional Ethics committee (no. EC/35/2012). Conflict of Interest Statement The authors declare that they have no conflict of interest. ==== Refs References Batta N Pandey M Mutational spectrum of tobacco associated oral squamous carcinoma and its therapeutic significance World J Surg Oncol 2019 17 198 31775759 Borse V Konwar AN Buragohain P Oral cancer diagnosis and perspectives in India Sens Int 2020 1 100046 34766046 Damani SH Saranath D Pradhan S Single nucleotide polymorphisms in transcription factor genes associated with susceptibility to oral cancer J Cell Biochem 2020 121 1050 60 31452252 Eiholzer RA Mehta S Kazantseva M Intronic TP53 Polymorphisms Are Associated with Increased Δ133TP53 Transcript, Immune Infiltration and Cancer Risk Cancers (Basel) 2020 12 2472 32882831 Fu G Dai X Symanzik J Quantitative gene-gene and gene-environment mapping for leaf shape variation using tree-based models New Phytol 2017 213 455 69 27650962 Gemignani F Moreno V Landi S A TP53 polymorphism is associated with increased risk of colorectal cancer and with reduced levels of TP53 mRNA Oncogene 2004 23 1954 6 14647431 Ghosh S Bhattacharjee M Jana N Gene Regulation by p53 in Human Cancer System Asian Pac J Cancer Biol 2022 7 97 109 Guo Y Zhou S Liu F CYP2E1 RsaI/PstI polymorphisms contributed to oral cancer susceptibility: a meta-analysis Int J Clin Exp Pathol 2015 8 14685 92 26823792 Hussein AA Helder MN de Visscher JG Global incidence of oral and oropharynx cancer in patients younger than 45 years versus older patients: A systematic review Eur J Cancer 2017 82 115 27 28654785 Li JY Huang LN Xue HL Glutathione S-transferase mu-1, glutathione S-transferase theta-1 null genotypes, and oral cancer risk: A meta-analysis in the Chinese population J Cancer Res Ther 2018 14 1052 56 Marcel V Dichtel-Danjoy ML Sagne C Biological functions of p53 isoforms through evolution: lessons from animal and cellular models Cell Death Differ 2011 18 1815 24 21941372 Moore JH Hahn LW Ritchie MD Application of Genetic Algorithms to the Discovery of Complex Models for Simulation Studies in Human Genetics Proc Genet Evol Comput Conf 2002 2002 1150 55 23413413 Patel KR Vajaria BN Begum R Association between p53 gene variants and oral cancer susceptibility in population from Gujarat, West India Asian Pac J Cancer Prev 2013 14 1093 1100 23621193 Pattin KA White BC Barney N A computationally efficient hypothesis testing method for epistasis analysis using multifactor dimensionality reduction Genet Epidemiol 2009 33 87 94 18671250 Pietsch EC Humbey O Murphy ME Polymorphisms in the p53 pathway Oncogene 2006 25 1602 11 16550160 Ritchie MD Van Steen K The search for gene-gene interactions in genome-wide association studies: challenges in abundance of methods, practical considerations, and biological interpretation Ann Transl Med 2018 6 157 29862246 Sagne C Marcel V Amadou A A meta-analysis of cancer risk associated with the TP53 intron 3 duplication polymorphism (rs17878362): geographic and tumor-specific effects Cell Death Dis 2013 4 e492 23412385 Saini A Challenges of oral cancer in India J Dent Res Prac 2021 3 Sankhla B Kachhwaha K Hussain SY Genotoxic and Carcinogenic Effect of Gutkha: A Fast-growing Smokeless Tobacco Addict Health 2018 10 52 63 30627385 Sharma S Satyanarayana L Asthana S Oral cancer statistics in India on the basis of first report of 29 population-based cancer registries J Oral Maxillofac Pathol 2018 22 18 26 29731552 Singh RD Haridas N Shah FD Gene polymorphisms, tobacco exposure and oral cancer susceptibility: a study from Gujarat, West India Oral Dis 2014 20 84 93 23444898 Singh RD Patel KR Patel PS p53 mutation spectrum and its role in prognosis of oral cancer patients: A study from Gujarat, West India Mutat Res 2016 783 15 26 26687995 Speight PM Khurram SA Kujan O Oral potentially malignant disorders: risk of progression to malignancy Oral Surg Oral Med Oral Pathol Oral Radiol 2018 125 612 27 29396319 Sullivan A Syed N Gasco M Polymorphism in wild-type p53 modulates response to chemotherapy in vitro and in vivo Oncogene 2004 23 3328 37 15077186 Tang K Li Y Zhang Z The PstI/RsaI and DraI polymorphisms of CYP2E1 and head and neck cancer risk: a meta-analysis based on 21 case-control studies BMC Cancer 2010 10 575 20969746 Wu X Zhao H Amos CI p53 Genotypes and Haplotypes Associated With Lung Cancer Susceptibility and Ethnicity J Natl Cancer Inst 2002 94 681 90 11983757 Xie S Luo C Shan X CYP1A1 MspI polymorphism and the risk of oral squamous cell carcinoma: Evidence from a meta-analysis Mol Clin Oncol 2016 4 660 6 27073686 Yang XL Xie S Jiang YY Association between CYP1A1 Ile462Val Polymorphism and Oral Squamous Cell Carcinoma Susceptibility: Evidence from 13 Investigations J Cancer 2015 6 302 9 25767599 Zeng Z Bromberg Y Predicting Functional Effects of Synonymous Variants: A Systematic Review and Perspectives Front Genet 2019 10 914 31649718 Zhuo X Song J Liao J Does CYP2E1 RsaI/PstI polymorphism confer head and neck carcinoma susceptibility? A meta-analysis based on 43 studies Medicine (Baltimore) 2016 95 e5156 27787372

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116152 10.31557/APJCP.2023.24.4.1297 Research Article Anti-tumor Effects of IL-1β Induced TRAIL-Expressing hUCMSCs on Embelin Treated Breast Cancer Cell Lines Teng Jui-Wen 1 Hung Eric 1 Wu Jiann-Ming 2 Liang Ya-Han 1 Chiu Yun-Hsuan 1 Tyan Yeu-Sheng 3 Wang Hwai-Shi 1* 1 Institute of Anatomy and Cell Biology, School of Medicine, National Yang Ming University, Taipei, Taiwan, ROC. 2 General Surgery Division, Far Eastern Memorial Hospital, New Taipei City, Taiwan, ROC. 3 Department of Medical Imaging, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC. * For Correspondence: hswang@nycu.edu.tw 2023 24 4 12971305 6 12 2022 17 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Human umbilical cord mesenchymal stem cells (hUCMSCs) have high therapeutic value in cancer treatment. We have found that pre-activating hUCMSCs with IL-1β promotes tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression and facilitates anti-tumor effect. Furthermore, embelin has been found to induce apoptosis of different cancer cell lines by upregulating the expression of TRAIL receptor 1 (DR4) and TRAIL receptor 2 (DR5). This study investigated whether IL-1β induced TRAIL-expressing hUCMSCs, in combination with low-dose embelin, could further induce apoptosis in breast cancer cell lines. Materials and Methods: MTT assay was used to examine the cytotoxicity of embelin in MDA-MB-231 and MCF-7. To detect the interested protein expression in cells, Western blot and cell immunofluorescence were used to double-confirm the observed results. Annexin V/PI apoptosis assay was detected by flow cytometry to analyze the apoptosis rate of embelin treated breast cancer cell lines and the effect of co-culturing with breast cancer cells and hUCMSCs. Results: Using Western blot and immunofluorescence, we found that breast cancer cell lines treated with low-dose embelin (2.5-5 μM) increased the expression of apoptosis-related receptor DR4, DR5 and the cleaved caspase 8, 9 and 3. Moreover, TRAIL expression was enhanced in IL-1β induced hUCMSCs. Combining these observations, we expected that coculturing IL-1β induced hUCMSCs with low dose embelin treated MDA-MB-231 and MCF-7 cells might enhance the apoptosis of breast cancer cells. We confirmed via flow cytometry that coculture of IL-1β induced TRAIL-expressing hUCMSCs and embelin treated MDA-MB-231 and MCF-7 cells enhances the apoptosis rate of these breast cancer cells. Conclusion: We found that embelin upregulated the expression of DR4 and DR5 to increase the TRAIL-mediated apoptosis in breast cancer cell lines. Low dose embelin treated breast cancer cell lines in combination with IL-1β induced TRAIL-expressing hUCMSCs may become a potential anti-tumor therapy. Key Words Human umbilical cord mesenchymal stem cells (hUCMSCs) Interleukin-1β (IL-1β) ==== Body pmcIntroduction Therapeutic application of Human umbilical cord mesenchymal stem cells (hUCMSCs) in cancer treatment is due to its demonstrated characteristics of low tumorigenicity, homing capability, and tumoricidal activity. Human bone marrow-derived mesenchymal stem cells (hBMMSCs) have been found to suppress tumor growth (Secchiero et al., 2010). However, hBMMSCs have also been found to transform into tumor-associated fibroblasts (TAFs) which then promote tumor development (Paunescu et al., 2011). In contrast, human umbilical cord-derived mesenchymal stem cells (hUCMSCs) do not transform to TAFs after cocultured with breast and ovarian cancer cells (Subramanian et al., 2012) (Subra. As for homing ability, evidences have indicated that injured tissues are known to be associated with releasing cytokines and growth factors such as tumor necrosis factor-alpha (TNF-α), and interleukins (IL) (Egea et al., 2011). In our previous study we have found that IL-1β could enhance hUCMSCs adhesion to endothelial cells (Wu et al., 2019), as well as increasing the transendothelial migration ability (Guo et al., 2018). These studies suggest that through the process of adhesion to endothelial cells and transendothelial migration, hUCMSCs may migrate to the inflammation sites. The migration ability of hUCMSCs to sites of tumorigenesis and their capability as efficient cellular vehicles for tumoricidal activity support the therapeutic potential for MSCs in cancer therapy. Past studies have demonstrated the tumor-suppressive effect of engineered MSCs, specifically their capability of migrating to various tumors and locally secreting therapeutic proteins including interferon β (IFN-β), IFN-γ and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (Li et al., 2015). Embelin (2, 5-dihydroxy-3-undecyl-1, 4-benzoquinone), a naturally occurring benzoquinone found in the fruit of the Embelia ribes, possesses anti-inflammatory and anti-cancer properties (Xu et al., 2005). Embelin was known as a cell-permeable, small molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP) that releases caspase 3 and caspase 9 and initiates caspase pathways leading to apoptosis (Hussain et al., 2017). Numerous studies have shown that embelin modulates NF-kB signaling pathways and demonstrates cytotoxic effects on a variety of cancer cell lines (Ahn et al., 2007; Danquah et al., 2012). Recent investigations found that embelin enhances TRAIL-induced apoptosis through TRAIL-Receptor 1 (DR4) and TRAIL-Receptor 2 (DR5) upregulation in human leukemia cells (Hu et al., 2015). TRAIL or Apo 2 ligand (TRAIL/Apo2L) is a type II transmembrane protein and belongs to the tumor necrosis factor (TNF) superfamily of ligands capable of initiating apoptosis by binding to its receptors. TRAIL is able to trigger apoptosis in some tumor cell lines, such as breast cancer and colon cancer (Seki et al., 2003). By using TRAIL gene-targeted mice, it has been found that TRAIL plays an important role in suppressing tumor initiation and metastasis (Cretney et al., 2002). Recombinant soluble TRAIL exhibited no detectable cytotoxicity to normal tissues both in murine and humans, and has also demonstrated its safety as a delivery vector for cancer therapeutic agents in vitro on various cell lines (Ashkenazi et al., 1999). However, the short half-life of recombinant TRAIL in serum has limited its application potential in clinical use (de Miguel et al., 2016). Therefore, a novel therapeutic approach capable of overcoming the current treatment barrier is needed. Our previous studies show that pre-stimulating hUCMSCs with IL-1β increase protein level of TRAIL and enhance apoptosis ability on breast cancer cell lines (Liang et al., 2021). Five homologous human receptors for TRAIL have been found (Kimberley and Screaton, 2004). Both the death receptors DR4 and DR5 possess a death domain capable of transducing the apoptosis signal. Different cancers induce TRAIL-related apoptosis with different contributions of DR4 or DR5. Though colon and breast cancer cells have been found to use DR5 for apoptosis induction (Kelley et al., 2005), pancreatic cancer cell lines may trigger apoptosis primarily via DR4 (Lemke et al., 2010). TRAIL death receptors undergo constitutive apoptosis in some cancer cells, which makes them a promising target for cancer therapy. hBMMSCs have been used as delivery vectors to overcome the limitations posed by the short half-life of recombinant soluble TRAIL (Yuan et al., 2015). The prolonged half-life of TRAIL expression and the homing ability of hUCMSCs stimulated by IL-1β, make these cells excellent delivery vectors to overcome the limitations posed by the short half-life of soluble TRAIL in suppressing tumorigenicity. It has been demonstrated that the relative sensitivity of the breast cancer cell lines to TRAIL are different. MDA-MB-231 cells were found to be a TRAIL-sensitive breast cancer cell line, but MCF-7 cells displayed a certain degree of resistance to TRAIL (Zhang and Zhang, 2008). In this study, we found that embelin enhances the expression of TRAIL receptors DR4 and DR5 on both MDA-MB-231 and MCF-7 breast cancer cell lines. Additionally, we also examined embelin regulated caspase 8, caspase 9 or caspase 3 protein levels potentially leading to different apoptosis pathways in these two cell lines. Further, we confirmed the anti-tumor ability of IL-1β induced TRAIL-expressing hUCMSCs on embelin-treated MDA-MB-231 and MCF-7 breast cancer cell lines via flow cytometry. The aim of this study was to assess whether the combination of IL-1β induced TRAIL-expressing hUCMSCs with embelin-treated breast cancer cells could enhance the apoptosis of cancer cells. Results from this study may yield potential anti-breast cancer therapeutics. Materials and Methods Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) hUCMSCs were purchased from Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. hUCMSCs were maintained in low serum defined medium including 56% Low-glucose Dulbecco’s Modified Eagle Medium (Life technology, NY, USA), 37% MCDB 201 Medium (Sigma, MO, USA), 2% fetal bovine serum (Thermo, Logan, UT), 0.5 mg/ml AlbuMAX® I (Life technology, NY, USA), 1X Insulin-Transferrin-Selenium-A (Life technology, NY, USA), 10 nM Dexamethasone (Sigma, MO, USA), 10ng/ml Epidermal growth factor (PeproTech, NJ, USA), 50nM L-ascorbic acid 2-phosphate (Sigma, MO, USA), and 1 ng/ml of platelet-derived growth factor-BB (PeproTech, NJ, USA). Cells were incubated at 37oC and 5% CO2. When cells reached 70-80% confluence, the cells were detached with HyQtase (Thermo, Logan, UT) and replated in culture dishes at a ratio of 1:4. Breast Cancer Cells Human breast cancer cells MDA-MB-231, MCF-7 (purchased from BCRC, Hsinchu, Taiwan) were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (Life technology, NY, USA) consisting with 10% fetal bovine serum, and 1% HyClone® Penicillin-Streptomycin solution (Thermo, Logan, UT). Cells were incubated at 37°C and 5% CO2. When cells reached 70-80% confluence, the cells were detached with HyQtase (Thermo, Logan, UT) and replated in culture dishes at a ratio of 1:4. MTT assay To examine the cytotoxicity of embelin on hUCMSCs, 1×104 cells per well were seeded in 96-well plates with 2% culture medium (2% CM; DMEM (Low glucose) containing 2% FBS). Cells were treated with embelin at various concentrations (0-200μM) for 24 hrs. Following incubation, 1 mg/ml MTT reagent was added to each well for 4 hrs at 37°C. The cells were then suspended in dimethyl sulfoxide for 2 hrs at 37°C and detected using multimode microplate readers (Infinite 200, TECAN) at a wavelength of 545 nm. Annexin V/PI apoptosis assay Cells were collected and pelleted at 1000 rpm for 5 mins and washed twice with 1 mL PBS. The resulting pellets were resuspended in 100 μl of 1X Annexin V binding buffer and stained with 50 μg/mL annexin V-FITC and 100 μg/mL propidium iodide (Life, NY, USA). Samples were kept at room temperature (RT) for 20 mins and protected from light. After the incubation period, samples were analyzed immediately using flow cytometry (Beckman, IL, USA). Western Blotting Cells were washed with 1X PBS and lysed in M-PER Mammalian Protein Extraction Reagent (Thermo, IL, USA) with 1% Halt Protease Inhibitor Cocktail (Thermo, IL, USA). The extractions were gently shaken for 3 mins and centrifuged at 14,000 g for 10 mins at 4oC. Protein concentrations were determined by Coomassie Plus (Bradford) Protein Assay reagent (Thermo, IL, USA) and Multimode microplate readers (Infinite 200, TECAN). Protein samples were electrophoresed on 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride membranes. Membranes were blocked for 1 hour at RT using 5% Fish Gelatin Blocking Buffer (AMRESCO, OH, USA) in TBST buffer (136mM NaCl, 2.7mM KCl, 25mM Tris-base, 0.05% Tween-20). After blocking, membranes were incubated with primary antibody TRAIL R1, TRAIL R2 (Novus, St Charles, USA), caspase 8 (GeneTex, CA, USA), caspase 9 and caspase 3 (Cell Signaling, MA, USA) diluted at 1:1,000 and beta-actin antibody (GeneTex, CA, USA) diluted at 1:10,000 in TBST buffer at 4oC overnight. Then membranes were washed three times with TBST, and incubated with Rabbit IgG antibody (GeneTex, CA, USA) for 1 hour at RT. The results were detected by LAS-4000 Luminescence Imaging System (GE, CT, USA) and analyzed by AlphaEaseFC 4.0 software. Cell Immunofluorescence and Images Cancer cells were plated in 24-well plates and starved for 12-16 hours in DMEM F12 containing 0.5% FBS. After starvation, cells were treated with different concentrations of embelin for 24 hours. Cells were fixed with PBS containing 4% (v/v) paraformaldehyde for 15 mins and permeabilized with 0.1% Triton X-100 in PBS for 10 mins. Cells were then blocked with 2% BSA in PBS for 30 mins and incubated with primary antibody at 4oC overnight. After cells were washed three times with PBS, they were incubated with appropriate second antibodies for 1 hour at RT. Cells were stained with Hoechst 33258 (Sigma, MO, USA), washed three times with PBS and imaged using a fluorescent microscope (DM6000B, Leica, German). Pre-stimulation of hUCMSCs with Cytokines Before coculture, hUCMSCs were detached and plated at 1x105 cells in 2% culture medium (2%CM; DMEM (low glucose) containing 2% fetal bovine serum) in 6-well plates and incubated for 4-6 hrs. For pre-activation, hUCMSCs were incubated in 2% CM containing 100 ng/ml IL-1β (PeproTech, NJ, USA). Direct Co-culture of hUCMSCs and Breast Cancer Cells MDA-MB-231 or MCF-7 cells were pretreated with different concentrations of embelin for 24 hrs. After pretreatment, an equal number of breast cancer cells were added to hUCMSCs containing wells for direct coculture for 24 hours with embelin in 2% CM. Both types of cells were detached and collected into the same tubes. The mixtures were washed with PBS by centrifugation, suspended in 100μl PBS and labeled with CD90-PE (Beckman Coulter, France) for 45 mins on ice in order to distinguish hUCMSCs and breast cancer cells by flow cytometry. Statistical Analysis Quantitation data was analyzed by the student’s t-test and one-way ANOVA. P values <0.05 were considered statistically significant. Statistical analysis was performed with Prism 5 software. Results Effect of embelin on MDA-MB-231 and MCF-7 cell apoptosis To investigate the effect of embelin on apoptosis of MDA-MB-231 and MCF-7 breast cancer cell lines, cells were treated with 2.5, 5, 10, 50 μM of embelin for 24 hrs and the extent of apoptosis was quantified using flow cytometry. As shown in figure 1, there was no significant increase in apoptotic rate in both cell lines following treatment with 2.5, 5, 10 and 50 μM embelin for 24 hrs. In these experiments, the DR4 and DR5 ligand recombinant human TRAIL (rhTRAIL) induce the apoptosis of both cell lines. However, 2.5, 5, and 10 μM embelin treated for 24 hours did not induce the apoptosis rate of both cell lines (Figure 1). By contrast, treatment with 50 μM embelin resulted in an apoptosis rate of ~50% in MDA-MB-231 cells and ~25% in MCF-7 cells (Figure 1b, 1d). Treatment of MDA-MB-231 and MCF-7 with embelin enhances DR4 and DR5 expression In light of the fact that DR4 and DR5 expression play important roles in many TRAIL-related cancer cells apoptosis, we analyzed the protein levels of DR4 and DR5 through western blotting after 24 hours treatment with embelin. The results showed that both DR4 and DR5 protein expression were upregulated in MDA-MB-231 cells at 5μM and 10μM embelin treatment; there was no significant change at the concentration of 2.5μM (Figure 2a, b, d, e). We further examined surface expression of DR4 and DR5 on cell membrane by immunofluorescence. The results are consistent with the Western blot data that embelin treatment enhances DR4 and DR5 expression in MDA-MB-231 cells (Figure 2c, f). The expression of DR4 in MCF-7 cells increased significantly at 2.5 and 10μM of embelin treatment the same as in MDA-MB-231 cells (Figure 2g, h). Interestingly, in MCF-7 cells DR5 showed significant increase only at embelin concentrations of 2.5μM (Figure 2j, k). The immunofluorescence results are consistent with the Western blot data that low-dose embelin treatment enhances DR4 and DR5 expression in MCF-7 cell lines (Figure 2i, l). Embelin induces activation of caspase proteins in breast cancer cells To determine whether caspase activation was involved in embelin-treated breast cancer cells, we next examined the activation of caspase 8, caspase 9, the initiator of the intrinsic and extrinsic apoptosis pathways and caspase 3. MDA-MB-231 and MCF-7 were incubated with different concentrations of embelin (0, 2.5, 5, 10 μM) for 24 hrs. The activation of caspase 8, 9 and 3 were analyzed by western blotting. The results showed that cleaved caspase 8, cleaved caspase 9 and cleaved caspase 3 increased significantly in both MDA-MB-231 and MCF-7 treating with embelin at 5 μM and 10 μM (Figure 3). These results suggest that low-dose (5 μM and 10 μM) embelin-treated breast cancer cell lines lead to apoptosis through both intrinsic or extrinsic pathways. IL-1β-stimulated hUCMSCs induce apoptosis in embelin-treated MDA-MB-231 and MCF-7 In our previous study, we found that IL-1β (100 ng/ml) stimulation can induce TRAIL expression in hUCMSCs. To examine the effect of IL-1β induced TRAIL-expressing hUCMSCs on breast cancer cell apoptosis and whether treatment with embelin could further enhance apoptosis in breast cancer cell lines. MDA-MB-231 and MCF-7 cells were pre-treated with embelin of 5 and 10 μM for 24 hrs, then cells were directly co-cultured with naïve hUCMSCs or IL-1β induced TRAIL-expressing hUCMSCs with/without embelin for 24 hrs. Treatment with 5 or 10 μM embelin on hUCMSCs for 24 hours, the results showed no cytotoxic effect on hUCMSCs (Sl Figure). Cocultured 5 or 10 μM embelin pre-treated MDA-MB-231 cells with IL-1β induced TRAIL-expressing hUCMSCs, the apoptosis rate of the MDA-MB-231 cells was increased (Figure 4a, b). However, co-cultured the naïve hUCMSCs with 5 μM embelin treated MDA-MB-231, compared with control, the apoptosis rate did not change (Figure 4b). The apoptosis rate of 5 or 10 μM embelin treated MDA-MB-231 cells co-cultured with IL-1β induced TRAIL-expressing hUCMSCs is significantly higher than co-cultured with the naïve hUCMSCs (Figure 4a-d). As for 5 μM embelin pre-treated MCF-7 cells, co-culturing with naïve hUCMSCs or IL-1β induced TRAIL-expressing hUCMSCs and 5μM embelin did not alter the apoptosis rate in comparison to MCF-7 cells only. However, 10 μM embelin pre-treated MCF-7 cells co-cultured with IL-1β induced TRAIL-expressing hUCMSCs, the apoptosis rate was increased significantly (Figure 4g, h). These results showed that the apoptosis of 10 μM embelin pre-treated MDA-MB-231 and MCF-7 cells can be enhanced by co-culture with IL-1β induced TRAIL-expressing hUCMSCs for 24 hrs. Figure 1 Apoptosis Induced by Embelin in MDA-MB-231 and MCF-7 Cells. Annexin V/propidium iodide (PI) dye was used to determine the rate of apoptosis following treatment of (a) MDA-MB-231 and (b) MCF-7 cells with different concentrations of Embelin for 24hrs and statistically analysis (b and d). Breast cancer cells treated with 100 ng/ml rhTRAIL are regarded as the positive control. The data were represented as mean ± SD (n=3, *P<0.05, **P<0.01, *** P<0.001). Figure 2 Embelin Upregulated DR4 and DR5 Expressions. (a, d) MDA-MB-231 and (g, j) MCF-7 cells were incubated with embelin of 2.5, 5 and 10 μM for 24 hrs, and DR4 and DR5 protein expressions were determined by Western blot. Quantify of western blot of DR4, DR5 in (b, e) MDA-MB-231 and (h, k) in MCF-7. The data are presented as the mean ± SD of three independent experiments. (n=3, *P<0.05, **P<0.01, *** P<0.001). Immunofluorescence staining for DR4, DR5 (green) and DAPI (blue) in (c, f) MDA-MB-231 and (d, l) MCF-7 cells after treatment with embelin of 2.5 to 10 μM for 24 hrs (Scale bar = 50μM). Figure 3 Embelin Induces Activation of Caspase Proteins in Breast Cancer Cells. Following treatment of breast cancer cells with different doses of embelin (0, 2.5, 5 and 10 μM) for 24hrs, Western blotting was used to analyze the expression of cleaved caspase 8, cleaved caspase 9, cleaved caspase 3 and quantitative graphs of (a, b, c) MDA-MB-231 cells and (d, e, f) MCF-7 cells. (n=3, *P<0.05, **P<0.01, *** P<0.001). Figure 4 Stimulated hUCMSCs Combined with Embelin Induce Apoptosis in Breast Cancer Cell Lines. After co-culture of naïve hUCMSCs or IL-1β induced TRAIL-expressing hUCMSCs (IL-1β-hUCMSCs) with 5 and 10 μM embelin treated MDA-MB-231 or MCF-7 cells for 24hrs, cells were collected and labeled with anti-CD90 antibody to distinguish hUCMSCs from breast cancer cells. Flow cytometry was used to analyze the apoptosis rate in (a, b, c, d) MDA-MB-231 and (e, f, g, h) MCF-7 by staining with Annexin V-FITC and PI. (n=3, *P<0.05, **P<0.01, *** P<0.001). Discussion TRAIL is a member of the TNF family proteins, capable of inducing apoptosis in most cancer cells but not in normal cells (Daniels et al., 2005). TRAIL induces apoptosis by activating cleaved caspase 8, cleaved caspase-9 and BAX in TRAIL-sensitive cancer cells (Johnstone et al., 2008). Previous studies indicated that TRAIL-resistant cancer cell lines expressed NF-kB in nuclear extracts, which could inhibit apoptosis induced by its death receptor’s action (Keane et al., 2000). Embelin (25-50 μM) has been found to inhibit cell proliferation and induce apoptosis in several human cancer cells (Siegelin et al., 2009; Park et al., 2015; Hussain et al., 2017). Low dose (1.5-5μM) embelin can enhance the sensitivity of renal cancer cell lines to an anti-renal cancer agent-axitinib by inhibiting the HIF pathway (Fang et al., 2023). Recently, we found that the antitumor effect of embelin (in a concentration of 50 μM) in breast cancer cell lines occurred via downregulating cellular FADD-like IL-1ß-converting enzyme inhibitory protein (cFLIP), a regulator in TRAIL-mediated apoptosis, which is correlated with TRAIL resistance in cancer cells (Liang et al., 2021). Low-toxicity embelin has also been found to enhance TRAIL-induced apoptosis via DR4 and DR5 upregulation and caspase activation in human leukemia HL-60 cells (Hu et al., 2015). Many drugs have been found to sensitize tumor cells to TRAIL by DR4 and/or DR5 upregulation. Doxorubicin and etoposide have been found to increase DR4 expression on small cell lung carcinoma cells to sensitize TRAIL induced apoptotic effect of TRAIL through increasing DR5 on cell surfaces (Vaculova et al., 2010). Treatment with cisplatin led to the upregulation of DR5 in glioblastoma-derived stem cells and restored TRAIL apoptotic pathway in the neurospheres (Ding et al., 2011). While research on embelin is ongoing, we focused on two common breast cancer cell lines including MDA-MB-231 (TRAIL-sensitive) and MCF-7 (TRAIL-resistant) in this study to elucidate the weather embelin-induced apoptosis in human breast cancer cells involves DR4 and DR5 upregulation. In our analysis, we demonstrated that low dose embelin (5 μM) upregulated both DR4 and DR5 expression in TRAIL sensitive and TRAIL resistant breast cancer cell lines using western blotting and Immunofluorescence staining. There are two main signaling pathways that trigger apoptosis, the intrinsic pathway and extrinsic death receptor pathway (Igney and Krammer, 2002). In the present study, we found that after MDA-MB-231 and MCF-7 cells were treated with different doses of embelin for 24hrs, both cleaved caspase 8, cleaved caspase 9 and cleaved caspase 3 levels increased significantly. These results indicate embelin-induced apoptosis in these two cancer cell lines through both the extrinsic and intrinsic apoptosis pathways. Given its ability to induce the apoptosis of cancer cells selectively, rhTRAIL has been used as anti-cancer therapy. However, the efficacy was poor (Herbst et al., 2010; Quintavalle and Condorelli, 2012). Our previous studies found that stimulated hUCMSCs with IL-1β increased TRAIL expression (Liang et al., 2021). Pre-stimulated hUCMSCs with IL-1β induced TRAIL can overcome the short half-life when compared with rhTRAIL. In addition to the limitation of short half-life, the resistance to TRAIL-induced apoptosis may also be involved in poor efficacy of TRAIL anti-cancer treatment. Low dose embelin induced DR4 and DR5 may be a good strategy to improve the TRAIL induced apoptosis of cancer cells. In this study, we found that the apoptotic rate of MDA-MB-231 cells increased during co-culture with IL-1β induced TRAIL-expressing hUCMSCs. Most importantly, a combination of low-toxicity embelin with IL-1β induced TRAIL-expressing hUCMSCs could enhance the apoptosis rate of MDA-MB-231 and MCF-7 cells. Moreover, cytokines secreted by cancer cells enhance the homing ability of hUCMSCs, which could improve the clinical efficacy of stem cell therapy (Kang et al., 2012). We assumed that IL-1β induced homing ability and TRAIL expression, suggesting that IL-1β may be a key molecule to modulate hUCMSCs in inhibiting the growth of cancer cells. In summary, our data indicated that low dose embelin promoted DR4 and DR5 expression in breast cancer cell lines. IL-1β induced TRAIL-expressing hUCMSCs combined with embelin can enhance apoptosis more significantly in TRAIL-sensitive (MDA-MB-231) than in TRAIL-resistant (MCF-7) cell lines. As a result, we suggest that low dose embelin combined with IL-1βinduced TRAIL-expressing hUCMSCs may possess the advantage of homing ability and increased clinical efficacy in TRAIL-related cancer therapies. These results suggest a different path for the clinical use of mesenchymal stem cells in cancer therapy. Author Contribution Statement H-SW contributed to conception and design, development of methodology. J-WT, EH, Y-HL, Y-HC, Y-SC contributed to acquisition of data. J-MW, Y-ST and H-SW contributed to material support and interpretation of data. J-WT, Y-HL and H-SW contributed to writing of the manuscript. Part of the data are from J-WT’s master thesis. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank Wen-Yi Yang for editing the manuscript and the National Science and Technology Council (Taiwan) for funding this research. Funding Statement This work is financially supported by a research grant from National Science and Technology Council, Taiwan, ROC (MOST 107-2320-B-010-029-MY3 to H.-S. Wang). Ethics Approval and Consent to Participate Not applicable. Data Availability All data needed to evaluate the conclusions in the paper are present in the paper. Conflicts of Interest The authors declare no conflicts of interest. ==== Refs References Ahn KS Sethi G Aggarwal BB Embelin, an inhibitor of X chromosome-linked inhibitor-of-apoptosis protein, blocks nuclear factor-kappaB (NF-kappaB) signaling pathway leading to suppression of NF-kappaB-regulated antiapoptotic and metastatic gene products Mol Pharmacol 2007 71 209 19 17028156 Ashkenazi A Pai RC Fong S Safety and antitumor activity of recombinant soluble Apo2 ligand J Clin Invest 1999 104 155 62 10411544 Cretney E Takeda K Yagita H Increased susceptibility to tumor initiation and metastasis in TNF-related apoptosis-inducing ligand-deficient mice J Immunol 2002 168 1356 61 11801676 Daniels RA Turley H Kimberley FC Expression of TRAIL and TRAIL receptors in normal and malignant tissues Cell Res 2005 15 430 8 15987601 Danquah M Duke CB Patil R Combination therapy of antiandrogen and XIAP inhibitor for treating advanced prostate cancer Pharm Res 2012 29 2079 91 22451249 De Miguel D Lemke J 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116154 10.31557/APJCP.2023.24.4.1313 Research Article Genotyping and Phylogenetic Analysis of Human Papillomaviruses in Formalin Fixed Paraffin Embedded Sections from Cervical Lesions in Duhok-Iraq Othman Adil Abdulsalam 1 Goreal Amer Abdalla 2* Pity Intisar Salim 3 1 Blood Bank Directorate, Ministry of Health, Duhok, Iraq. 2 Department of Medical Microbiology, College of Medicine, University of Duhok, Duhok, Iraq. 3 Department of Pathology, College of Medicine, University of Duhok, Duhok, Iraq. * For Correspondence: amer.goreal@uod.ac 2023 24 4 13131319 7 12 2022 23 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: Human papillomavirus (HPV) has been identified as an important causative factor in cervical cancer development. Cervical cancer is the fourth most prevalent malignant tumor among women globally. The purpose of this study was to investigate the prevalence and genotyping sequences of HPV in formalin-fixed paraffin-embedded (FFPE) cervical tissue using conventional polymerase chain reaction (PCR), and HPV-DNA sequencing. Material and Methods: Retrospective cross-sectional study. Forty (FFPE) blocks with different cervical lesions were taken; patients’ ages ranged from 24 to 65 years. Detection and sequencing of HPV DNA were done by conventional PCR (L1 gene), which was achieved by universal PCR primers (MY09/11 oligonucleotides). Then sequencing and phylogenetic tree was constructed. Results: Nine samples were found positive and detected by conventional PCR, they were identified in CIN1 and SCC at 7.5% (n=3) for each, 5.0% (n=2) KA, and 2.5% (n=1) in CIN3 cases, after sequencing were submitted to GenBank and accession numbers were obtained. The phylogenetic tree was constructed and the aligned sequences showed high homology with the nucleotide sequence of the references from the Genbank database. HPV 11, 16, 18, 22, 33, 52, and 58 were found to have little nucleotide heterogeneity and thus no amino acid heterogeneity. Conclusion: Sequencing and phylogenetic analysis of circulating HPV types in Duhok provides very essential data about nucleotides and amino acid heterogeneity, to reveal genetic diversity with strains included in the vaccines that have not been introduced to Iraq yet. Key Words Human papilloma virus cervical lesions molecular detection DNA-sequencing phylogenetic tree ==== Body pmcIntroduction HPV identified as an important causative factor in cervical cancer development (Jalilian et al., 2017; Kamal et al., 2021). Cervical cancer is women’s fourth most prevalent malignant tumor globally (Bray et al., 2018). More than 200 genotypes of HPV have been identified based on DNA sequences (Mühr et al., 2018). HPV genotypes are divided into several categories. High-risk types (HR–HPV) are mainly 16 and 18, as well as 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. HPV types 26, 53, and 66 were categorized as probably high-risk, and HPV types 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81 were categorized as low-risk types (LR-HPV) (Ehteram et al., 2019). Different studies from neighbor countries revealed that most frequent HR-HPV and LR-HPV associated with genital lesions are HPV16, HPV 6 respectively (Chalabiani et al., 2017). Infection with HPV is suspected by the presence of clinical lesions and by cytology, histology, and colposcopy, which are all subjective and often inaccurate (Liao and Li., 2019). Most cervical HPV infections are asymptomatic and the immune system naturally eliminated 70% of them within 12-24 months (Giuliano et al., 2011). The understanding that HPV is the major cause of cervical malignant tumors has led to the creation of advanced screening methods. Early diagnosis and treatment of precancerous conditions can restrain the progress of cervical cancer (Venkatas and Singh., 2020). The risk of precancerous lesions of the cervix in women is primarily explained by a cytological screening of cells. Molecular diagnostic tests that distinguish between high and low-risk HPV genotypes are highly sensitive and specific, detect HPV DNA, and are used as additional tests in cytological studies (Ghosh et al., 2014). DNA sequencing of purified DNA which is amplified by conventional PCR using consensus primers MY09/11 is another technique to detect the genotype and alignment with reference strains (Depuydt et al., 2007). Since the HPV vaccine is proved to be an efficient strategy to prevent cervical cancer in different countries therefore it is the next approach for control of these infections. The phylogenetic analysis wil help alignment of circulating strains with those included in HPV vaccine (Sopian etal., 2019). This study aimed to investigate the prevalence and genotyping sequences of HPV in samples obtained from FFPE cervical tissue by HPV-DNA sequencing. Materials and Methods Study design and population This retrospective cross-sectional study included FFPE block samples of 40 patients with different cervical lesions in the period from July 2020 to December 2021. The samples were taken from archives of histopathology departments in Duhok central lab public health and VIN private laboratories. Patients’ ages ranged from 24 to 65 years (Mean: 42 years STD ± 10.5). Sample collection The FFPE-fixed biopsy was sectioned. Five continuous sections with a thickness of 10 µm were taken from each biopsy, with a new blade for each one to avoid cross-contamination. Two outer sections were stained with hematoxylin and eosin and examined microscopically to confirm the diagnosis that was classified as; 27.5% (n=11) and showed Koilocytotic atypia (KA), 10.0% (n=4) unremarkable (non-neoplastic) pathology negative for Intraepithelial lesions or malignancy (NILM), 20.0% (n=8) Squamous cell carcinoma (SCC) Malignant, 22.5% (n=9) Cervical intraepithelial neoplastic (CIN1), 12.5% (n=5) CIN2, and 7.5% (n=3) CIN3. DNA extraction For Human papillomavirus (HPV) DNA extraction, three internal sections were placed in a 1.5 ml Eppendorf tube. Xylene (1ml) was added to the microtube containing the tissue section, deparaffinized, and vortexed for 10 seconds to mix vigorously. Then centrifuged at 20,000 rpm for 2 min and the supernatant was removed. This procedure was repeated 3 times to remove paraffin residue. The pellet was then washed 3 times with 1ml of ethanol (96-100%), centrifuged at 20000 rpm for 2 min, and the supernatant was then discarded. The pellet was air-dried at 37°C for 10-15 minutes until all residual ethanol had evaporated. After tissue deparaffinization, DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) according to the manufacturer’s instructions. HPV DNA detection and sequencing The PCR was performed using consensus MY09/11 oligonucleotide primers to amplify the 450pb sequence from the L1 region of HPV. Amplification generally consists of a denaturation step were 40 cycles at 94°C for 1 min, followed by, an annealing step for 1 min at 55°C, an extension step for I min at 72°C, a final extension of 1 cycle at 72°C for 5 min (Venceslau et al., 2014) Table1. To confirm the presence of HPV DNA fragments, 10μL of each PCR product was visualized on a 2% agarose gel electrophoresis using RedSafe Nucleic Acid (iNtRON Biotechnology) staining. The PCR product samples were then sent for DNA sequencing by universal PCR primers (MY09/11) to Macrogen (Macrogen Co., Seoul, Korea). Multiple alignments were performed using BioEdit (version 7.2.5.0) software and submitted in Fasta format to the GenBank database and analyzed based on the best sequence homology search was conducted using BLAST ( Basic Local Alignment Search Tool ), which is available at the National Center Biotechnology Information (NCBI) online web site, and accession numbers (ACCN) were obtained. The phylogenetic tree was constructed by the maximum likelihood using MEGA 11.0 software and identified the evolutionary relationships between the analyzed sequences. Results In this study, among (n=40) FFPE cervical specimens, the L1 gene HPV was amplified by specific primers MY09/11. Nine samples were found positive and detected by PCR (Figure 1). Patients’ ages ranged from 24 to 65 years (Mean: 42 years STD ± 10.5), and participants were classified into five age groups. HPV was most prevalent in the 31–40 age group and there were no significant differences (p= 0.909) (Table 2). The virus was identified in CIN1 and SCC at 7.5% (n=3) for each, 5.0% (n=2) KA, and 2.5% (n=1) in CIN3 cases (Table 3). Sequence analysis and phylogenetic tree The positive PCR products were sent for DNA sequencing, then sequencing was analyzed and submitted to GenBank in which the accession numbers (ACCN), OM963133, ON210811, ON221505, ON221506, ON221508, ON221509, ON221510, ON367531, and ON191582 were obtained. The results for all samples; showed the evolutionary relations between the studied strains with the nearest similar species of the HPV genus found in the Genbank data. Sequences were compared with the data available at the NCBI-Genbank based on the highest percentage or lowest value. The ratio of similarity to the identified strains in the study with other strains from NCBI-Genbank ranged from 99-100% as in Table 4. Two isolates of HPV16 (ON367531 and ON191582) were detected and the aligned sequence showed high homology with the nucleotide sequence of the references from the Genbank database (Figure 2). HPV18 was detected in one sample (ON210811) representing, the aligned sequence that showed high homology with the nucleotide sequence of HPV18 (MH057749) isolated in Saudi Arabia (Figure 3). The results of the phylogenetic analysis showed that the isolated DNA of HPV33 in Duhok/Iraq (one sample) ON221508 has a kinship with those in GQ479014 Canada and GU797222 in the USA. The HPV52 identity in one case (ON221506) was the match found with the HPV52 L1 region (KT799936) isolated from Chinese patients having cervical cancer. The two HPV58 (OM963133 and ON221509) identity matches were found with the HPV58 L1 region sequence (MT267729) isolated from Iran among patients with cervical cancer. The HPV11 (ON221510) identity match was found with L1 region sequence HPV11 (KU721777) isolated from China patients, while, HPV22 isolated in one case (ON221505) was found to match identity with HPV22 isolated from macules on the chest of an Italian epidermodysplasia verruciformis patient (Table 4). A phylogenetic tree was constructed using the genomic sequence deposited under the ACCN, OM963133, ON210811, ON221505, ON221506, ON221508, ON221509, ON221510, ON191582, and ON367531 and analyzed against HPV genotype references sequences. Bootstrap test analysis was performed (1000 replicates) to estimate the reliability of the obtained branching patterns in the phylogenetic trees (Figure 4). Table 1 Genetic Oligonucleotide Primers Used for DNA of HPV Detection (Venceslau et al., 2014) Primers Sequence Fragment Size Protocol (PCR) Conditions (PCR) MY09 MY11 5’CGTCCMARRGGAWACTGATC3’ 5’GCMCAGGGWCATAAYAATGG3’ 450pb 15 mM MgCl2 (buffer 1×), 800 µM of dNTPs, 50 pmol/µl of each oligonucleotide primer, 1.25 U from Hot Star Taq DNA polymerase 40 cycles 94°C/1min 55°C/1min 72°C/1min 1 cycle 72°C/1min Figure 1 Agarose Gel Electrophoresis of Products of HPV PCR. Lane L, 1000bp DNA ladder; lanes 2,3,4,6,8,9,11, and 12, HPV positive samples contained 450bp amplicon from the HPV genome Table 2 HPV and Age Groups Result Age Groups N (%) Total 21-30 31-40 41-50 51-60 61-70 Positive 1 (2.5%) 4 (10.0%) 3 (7.5%) 0 (0.0%) 1 (2.5%) 9 (22.5%) Negative 4 (10.0%) 12 (30.0%) 9 (22.5%) 3 (7.5%) 3 (7.5%) 31 (77.5%) Total 5 (12.5%) 16 (40.0%) 12 (30.0%) 3 (7.5%) 4 (10.0%) 40 (100.0%) χ2 test used, P=0.909 Figure 2 Two isolates of HPV16 (ON367531 and ON191582) were performed by sequence analysis after alignment (ClustalW) of the sequences with reference strains using BioEdit (version 7.2.5.0). The point mutation (base substitution) occurred at positions 6693, 6719, 6852, 6860, 6863, 6968, and 6992, to the reference sequence (ACCN: MH057735) isolated in Saudi Arabia Figure 3 One Isolate of HPV18 (ON210811) was Performed by Sequence Analysis after Alignment (ClustalW) of the Sequences with Reference Strains Using BioEdit (version 7.2.5.0). The point mutation (base substitution) occurred at positions 6719, 6749, and 6917 to the reference sequence (ACCN: KT365828) isolated in Iraq Table 3 Human Papilloma Virus Results and Morphologic Diagnosis Results morphologic diagnosis N (%) Total NILM KA CIN1 CIN2 CIN3 SCC Positive 0 (0.0%) 2 (5.0%) 3 (7.5%) 0 (0.0%) 1 (2.5%) 3 (7.5%) 9 (22.5%) Negative 4 (10.0%) 9 (22.5%) 6 (15.0%) 5 (12.5%) 2 (5.0%) 5 (12.5%) 31 (77.5%) Total 4 (10.0%) 11 (27.5%) 9 (22.5%) 5 (12.5%) 3 (7.5%) 8 (20.0%) 40 (100.0%) χ2 test used, p= 0.471 Figure 4 Phylogenetic Analysis of L1 Gene Using Selected HPV: The phylogenetic tree was constructed by the maximum likelihood method of MEGA 11.0 software, the percentage of the replication tree in which the relevant taxa are clustered together in the bootstrap test (1000 replications) is shown next to the branches Table 4 Representative Genotyping Analysis of HPV Sample Isolates based on Capsid Protein (L1) Gene According to a Phylogenetic Tree and NCBI –BLAST Genotypes Identity (%) Analysis No. Accession Number Country %Identities Author, year 1 ON221510:HPV11 Duhok/Iraq Current study, 2022 2 M14119 Germany 100.00% (Dartmann et al., 1986) 3 JN644142 Iran 100.00% (Eftekhaar et al., 2017) 4 MW404328 Hungary 100.00% (Nagy et al., 2021) 5 JQ773408 Thailand 100.00% (Chansaenroj et al., 2012) 6 EU056630 Ireland 99.74% (Menton et al., 2009) 7 ON191582: HPV16 Duhok/Iraq Current study, 2022 8 ON367531 Duhok/Iraq 100.00% Current study, 2022 9 LC456183 Japan 100.00% (Galati et al., 2019) 10 MH937413 Italy 100.00% (Hirose et al., 2019) 11 MZ484662 Pakistan 100.00% (Minhas et al., 2022) 12 MN966564 Iran 100.00% (Mahmoudvand et al., 2022) 13 MH057735 Saudi 97.97% (Sait et al., 2019) 14 ON210811: HPV18 Duhok/Iraq Current study, 2022 15 MH057749 Saudi 100.00% (Sait et al., 2019) 16 MH028437 Italy 100.00% (Frati et al., 2020) 17 KY595177 Serbia 100.00% (Kovacevic et al., 2019) 18 MF288727 Netherlands 100.00% (van der Weele et al., 2018) 19 LC508994 Japan 100.00% (Yamaguchi-Naka et al., 20 KT365828 Iraq 99.05% (Al-Malkey M. K, 2018) 21 ON221505: HPV22 Duhok/Iraq Current study, 2022 22 U31780 Germany 99.74% (Kremsdorf et al., 1984) 23 L38912 Netherlands 99.62% (Berkhout, 1995) 24 U21866 Singapore 99.60% (Chan et al., 1995) 25 ON221508:HPV33 Duhok/Iraq Current study, 2022 26 GU797244 USA 100.00% (Boyd et al., 2011) 27 GQ479014 Canada 100.00% (Cornut et al., 2010) 28 KU550674 China 99.71% (Chen et al., 2016) 29 FJ202006 Brazil 99.71% (Raiol et al., 2009) 30 HM596529 Greece 99.12% (Ntova et al., 2012) 31 EU056641 Ireland 99.12% (Menton et al., 2009) 32 ON221506:HPV52 Duhok/Iraq Current study, 2022 33 KJ676085 Croatia 100.00% (Zhang et al., 2014) 34 KT799936 China 100.00% (Zhang et al., 2016) 35 EU077220 Canada 100.00% (Gagnon et al., 2007) 36 KU050117 Ecuador 100.00% (Bedoya-Pilozo et al., 2018) 37 LC270063 Japan 100.00% (Tenjimbayashi et al., 2017) 38 OM963133: HPV58 Duhok/Iraq Current study, 2022 39 ON221509 Duhok/Iraq 100.00% Current study, 2022 No. Accession Number Country %Identities Author, year 40 KU721777 China 99.76% (Wang et al., 2016) 41 LR861919 Luxembourg 99.76% (Latsuzbaia et al., 2020) 42 DQ057325 USA 99.76% (Calleja-Macias et al., 2005) 43 AJ621382 Cyprus 99.28% (Neophytou P.I., 2004) 44 HM639621 South Korea 99.04% (Chan et al., 2011) Discussion There are 12.2 million females over 15 years of age in Iraq who are at risk of developing cervical cancer. Current estimates that 286 women are diagnosed with cervical cancer each year and 193 die from the disease. Cervical cancer is the 15th most common cancer in Iraqi women and the 12th most common cancer in women between 15-44 years of age (Bruni el al., 2021). Human papillomavirus (HPV) distribution varies in different populations, countries, and even in different parts of a country. Data on the burden of HPV on the Iraqi population are not yet available. However, in West Asia, including Iraq, an estimated 2.5% of women in the general population will always be infected with HPV 16/18 in the cervix at a given time, and 72.4% of (invasive cervical cancers will result in HPV 16 or HPV18 (Bruni el al., 2021). The prevalence and detection of HPV in cervical cancer specimens using various technical tools have been published in many reports from Iraq, but an analysis of genetic diversity based on the L1 region in this study was done for the first time. We found HPV DNA in just 22.5% of the specimens in our research, which is substantially lower than in previous PCR investigations. This result might be explained by inadequate DNA extraction from fixation. Fixing pathology specimens and embedding them in paraffin wax are critical stages in preparing tissues for microscopic examination and long-term storage The MY09/MY11 consensus primer, which targets the conserved 450bp conserved sequence of the HPV L1 region, is one of the most commonly used in conventional PCR assays, and previous studies have shown that it has some limitations, especially a low sensitivity (Gravitt et al., 2000; Depuydt et al., 2007; Venceslau et al., 2014), therefore only nine cases were detected that means this method is less sensitive to detect multiple infections compared to other methods. Remmerbach et al, reported that HPV detection was higher for GP5+/GP6+ oligonucleotides than for MY09/MY11 oligonucleotides which can be explained by the small size of the DNA fragments amplified by the GP-PCR system (Remmerbach et al., 2004). The sensitivity and specificity of the PCR method depend primarily on the choice of oligonucleotide primers to be used, the number of base pairs in the product amplified by PCR, the performance of the DNA polymerase used in the reaction, the amplified DNA-HPV type spectrum, and the ability to recognize different types of infection (Iftner and Villa, 2003). Considering the age, although statistically not significant, higher HPV rates were observed among young women (31–40 years of age). It represents the highest incidence at marriage age and after starting sexual activity due to some cultural customs and religious beliefs such as not having illegitimate sex, increased marriage age, and lack of immunity to HPV in the absence of a national vaccination program (Oztürk et al., 2004; Muderris et al., 2019). The gradual decrease in HPV infection rates among middle-aged and older individuals can be explained that young women’s infections with HPV are transient and the immune system would eliminate the virus in most cases (Garbuglia et al., 2020; Ferrall et al., 2021). Considering different morphologies, HPV DNA was identified in those with abnormal morphology. In Erbil-Iraq, 71% of cases with abnormal morphology were HPV DNA positive with the absence of this DNA among negative cases (Ismail et al., 2014), while in a previous local study (Duhok-Iraq), 46.2% of NILM cases and 53.8% of abnormal Pap cases were positive for HPV (Pity et al., 2019). Nevertheless, heterogeneous findings have been obtained among unremarkable and abnormal cases in different neighboring countries such as Saudi Arabia (28.6% and 53.1%) respectively and Kuwait (36.8% and 63.2%) respectively (Al-Awadhi et al., 2013; Al-Ahdal et al., 2014). The highest identity (100.0%) of HPV16 detected in this study was observed with HPV16 (LC456183, MH937413) reported from Japan and Italy respectively. In addition, comparative nucleotide sequence analysis of the HPV18 L1 gene with retrieved HPV18 from Genbank showed variable sequence similarity ranging from 100.00% in Saudi Arabia to 99.05% in Iraq. This demonstrates that there was very little nucleotide heterogeneity, and therefore no amino acid heterogeneity, concerning HPV 16, 18, which is a very important result obtained from this study to align the circulating viruses with that included in the vaccine which is not introduced to Iraq yet. In conclusions, the current work extends previous observations by providing baseline data on the circulation of HPV types among Duhok’s women, and their association with normal and abnormal cytology with age. Primer configurations used in HPV DNA amplification are important to determine the sensitivity and specificity of PCR-based assays. This study provides very important information and a practical approach to genetic diversity and phylogenetic analysis, which can be very helpful in linking epidemiological studies as well as the natural history and evolution of HPV in Iraq and deciding the type of vaccine that would be introduced in the future. Author Contribution Statement OAA: Table development, editing the manuscript and writing; GAA: conceptualization, editing the manuscript and writing review; PIS: supervision, visualization, and review, all authors have read and approved the final version. Acknowledgements Ethical Approval Ethical approval to carry out the study was obtained from the Medical Ethics Committee of the Duhok General Director of Health on 20/July/2020- Reference number (20072020-3). This study was approved by the scientific committee of the college of Medicine/ University of Duhok as Ph.D. project. Data Availability The data appeared in this research can be obtained from the corresponding author of the article Conflict of Interest We declare no conflict of interest associated with this research and there has been no significant financial support for this project that could influence its outcome. ==== Refs References Al-Ahdal MN Al-Arnous WK Bohol MF Human papillomaviruses in cervical specimens of women residing in Riyadh, Saudi Arabia: a hospital-based study J Infect Dev Ctries 2014 8 320 5 24619263 Al-Awadhi R Chehadeh W Al-Jassar W Viral load of human papillomavirus in women with normal and abnormal cervical cytology in Kuwait J Infect Dev Ctries 2013 7 130 6 23416659 Bray F Ferlay J Soerjomataram I Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J Clin 2018 68 394 424 30207593 Bruni L Albero G Serrano B ICO/IARC Information Centre on HPV and Cancer (HPV Information Centre). 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Summary Report 2021 Chalabiani S Khodadad Nazari M Shabani M Retrospective Analysis of Prevalence of High-risk and Low-risk Human Papillomavirus (HPV) Genotypes in Iranian Women During 2013-2016 Asian Pac J Cancer Biol 2017 2 85 90 Depuydt CE Boulet GA Horvath CA Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types J Cell Mol Med 2007 11 881 91 17760847 Ehteram H Mousavian MS Mazoochi T Khamehchian T Karimian M Association of Some High-Risk Mucosal Types of Human Papillomavirus with Cutaneous Squamous Cell Carcinoma in an Iranian Population Iran J Pathol 2019 14 313 31754361 Ferrall L Lin KY Roden R Hung CF Wu TC Cervical Cancer Immunotherapy: Facts and Hopes Clin Cancer Res 2021 27 4953 73 33888488 Garbuglia AR Lapa D Sias C Capobianchi MR Del Porto P The use of both therapeutic and prophylactic vaccines in the therapy of papillomavirus disease Front Immunol 2020 18 188 Ghosh S Seth S Paul J Evaluation of Pap smear, high-risk HPV DNA testing in detection of cervical neoplasia with colposcopy guided or conventional biopsy as a gold standard Int J Healthcare Biomed Res 2014 2 192 7 Giuliano AR Lee JH Fulp W Incidence and clearance of genital human papillomavirus infection in men (HIM): a cohort study Lancet 2011 377 932 40 21367446 Gravitt PE Peyton CL Alessi TQ Improved amplification of genital human papillomaviruses J Clin Microbiol 2000 38 357 61 10618116 Iftner T Vill LL Chapter 12: Human papillomavirus technologies J Natl Cancer Inst Monogr 2003 31 80 8 Ismail AT Ahmed NY AbdulHameed RA Prevalence of HPV Immunostaining in Benign, Preneoplastic & Neoplastic Cervical Lesions of Kurdish Women in Erbil City/Kurdistan of Iraq Am J Res Commun 2014 2 67 74 Jalilian S Izadi B Madani S Mohajeri P The Prevalence and Genotype Distribution of Human Papillomavirus Types in the General Female Population in West of Iran Jundishapur J Microbiol 2017 10 e40855 Kamal M Lameiras S Deloger M Human papillomavirus (HPV) integration signature in Cervical Cancer: identification of MACROD2 gene as HPV hot spot integration site Br J Cancer 2021 124 777 85 33191407 Liao Y Li Q Profile of MeltPro® HPV test for human papillomavirus genotyping and cervical precancer screening Exp Rev Mol Diagn 2019 19 857 62 Muderris T Afsar I Yıldız A Akpınar Varer C HPV genotype distribution among women with normal and abnormal cervical cytology in Turkey Rev Esp Quimioter 2019 32 516 24 31642640 Mühr LSA Eklund C Dillner J Towards quality and order in human papillomavirus research Virology 2018 5 74 6 Oztürk S Kaleli I Kaleli B Bir F Servikal orneklerde insan papillomavirus DNA varliğinin hibrid yakalama yöntemiyle araştirilmasi [Investigation of human papillomavirus DNA in cervical specimens by hybrid capture assay] Mikrobiyol Bul 2004 38 223 32 15490841 Pity IS Abdo HM Goreal AA Human Papillomavirus Genotyping among Different Cervical Smears in Duhok/Iraq Asian Pac J Cancer Prev 2019 20 2059 64 31350966 Remmerbach TW Brinckmann UG Hemprich A PCR detection of human papillomavirus of the mucosa: comparison between MY09/11 and GP5+/6+ primer sets J Clin Virol 2004 30 302 8 15163418 Sopian M Tuan Din S Hussin H Obstacles to Implementing the HPV Vaccine: Is it Worth Pursuing or Not? Asian Pac J Cancer Care 2019 4 165 9 Venkatas J Singh M Cervical cancer: a meta-analysis, therapy, and future of nanomedicine Ecancermedicalscience 2020 14 1111 33144879 Venceslau E Bezerra M Lopes A HPV detection using primers MY09/MY11 and GP5+/GP6+ in patients with cytologic and/or colposcopic changes J Bras Patol Med Lab 2014 50

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116147 10.31557/APJCP.2023.24.4.1249 Research Article Factors Associated with the Quality of Life among Family Caregivers of Cholangiocarcinoma Survivors in Northeastern Thailand Summart Ueamporn 1 Sangruangake Monthida 2* Songthamwat Metha 3 Wittayapun Yuwadee 4 Teinprasert Saranya T 5 Chaplik Napachun 5 Mongkolsawad Jariya 5 1 Faculty of Nursing, Roi Et Rajabhat University, Roi Et, Thailand. 2 Faculty of Nursing, Khon Kaen University, Khon Kaen, Thailand. 3 Department of Obstetrics and Gynecology, Ang Thong Hospital, Ang Thong, Thailand. 4 Movement Science and Exercise Research Center-Walailak University (MoveSE-WU), Walailak University, Nakhon Si Thammarat, Thailand. 5 Faculty of Nursing, Udon Thani Rajabhat University, Udon Thani, Thailand. * For Correspondence: montsa@kku.ac.th 2023 24 4 12491255 24 11 2022 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Cholangiocarcinoma (CCA) is a common and usually lethal liver cancer especially in Southeast Asia. Family caregivers (FCs) and their quality of life (QOL) is important for the care process to operate effectively. However, there are only few research articles about the QOL of CCA FCs. The goal of this study was to assess the QOL and its associated factors among CCA FCs. Material and Methods: This cross-sectional study was undertaken with 231 CCA FCs who were the primary FCs for CCA patients in a tertiary hospital in Northeastern Thailand. The QOL was measured using the Thai version of the World Health Organization’s Quality of Life Questionnaire. Multivariate regression models were developed to investigate the predictors of the QOL, including the demographic characteristics, symptoms, anxiety and depression, and support care need. Results: The CCA caregivers had moderate to high QOL for all of four domains: the mean score was 27.03 (SD=2.81) for physical, 23.13 (SD=2.81) for psychological, 11.32 (SD=1.08) for social relationships, and 28.08 (SD=2.81) for environment. Multivariable analysis shows that, symptoms, support care need, age and education level were significant predictors of FCs’s QOL. Moreover. The QOL was lower in younger FCs (p<0.001). Conclusion: Symptoms, support care needs, age, and education level were associated with QOL among FCs for CCA patients. A holistic strategy that includes caregiver training, psychosocial therapies, and proper support may help these FCs for a better QOL. Key Words Cholangiocarcinoma caregivers quality of life symptoms support care need ==== Body pmcIntroduction Cholangiocarcinoma (CCA) is a global public health issue since it is the leading cause of mortality. According to the World Health Organization’s (WHO) International Agency for Research on Cancer [IARC] report from 2020, there were 905,677 new CCA cases globally with 830,180 of them dying (Sung et al., 2021). However, the number of cancer patients surviving has increased as a result of advancements in; cancer screening and treatment modalities with a wider range of contributing CCA survival factors (Miller et al., 2019). In general, CCA survivors had at least one or more common symptom such as a feeling of tiredness, pain, and psychological distress, and these symptoms may reduce their quality of life (QOL) as well as the QOL of their family caregivers (FCs) (Wen et al., 2018). Therefore, these FCs experienced high levels of psychological and physical stress along with other conflicts while caring for cancer patients, resulting in their poor QOL (Lim et al., 2021). FCs have a significant impact on how well a patient manages his or her illness, and they are frequently the patient’s primary source of social and emotional support (Fumaneeshoat and Ingviya, 2020). Similarly FCs desired to know more about the condition and its progression in order to better manage their lives and make decisions (Hashemi et al., 2018), and they also needed additional instruction about what to do after the patient death (Cui et al., 2014). In addition. FCs’ unpleasant experiences can affect not only their personal QOL, but also their ability to provide care (Abdullah et al., 2019). Therefore, to improve the QOL for both patients and their carers. The patient’s treatment modalities, including; surgery, chemotherapy, and radiotherapy, should be communicated to them depending on the progression of the cancer and of the consequences of that communication. As the cancer metastasized, most FCs are asked to take on more responsibilities, such as longer periods of home care and more frequent hospital visits, putting them under more stress and compromising their QOL (Kilic and Oz, 2019). Even though prior studies have reported many factors influence FCs’ QOL including those that affect both patients and FCs, such as; their patient’s underlying diseases, the cancer progression, the number of hours they spend caring for them each day, their age, income, educational level, and their personal relationships that may suffer because of their responsibilities. (TIAN et al., 2012; Aun and Mohd, 2016; Ndikuno et al., 2016; Yu et al., 2017; Fumaneeshoat and Ingviya, 2020). Nevertheless, these studies included patients with various type of cancers, this approach has the advantage of a larger sample size, but it may obscure potential differences in individuals who care for cancer patients of various types (Turkoglu and Kilic, 2012; Kong and Guan, 2019; Abbasi et al., 2020). There is a scarcity of information in the literature about FC’s cancer specific QOL, and the few current studies have targeted on liver cancers (Hansen et al., 2021). In Thailand, only a few studies have been conducted concerning FCs of cancer patients (Meecharoen et al., 2013; Fumaneeshoat and Ingviya, 2020). However, no research has specifically examined CCA patients and their FCs’ QOL and workloads, as well as associated factors. Therefore, the aim of this study was to assess the QOL and its associated factors among FCs. of CCA patients. Materials and Methods Study design and sample In this cross-sectional study, 231 primary CCA FCs from a public tertiary care university with a teaching facility were enrolled. Only one family member was recruited for each CCA patient. Questionnaires were administered in February 2021 to April 2021.The inclusion criteria were a family member of a CCA survivor who assumes primary responsibility for the patient’s care and devotes the most amount of time to it, 18 years of age or older, ability to speak and understand Thai language, full literacy (reading and writing in Thai and attending the hospital, Permission to collect the data was obtained from the head of each hospital unit, and all participants provided their informed consent. The ethics committees of Khon Kaen University (HE631628) approved the study protocol. Instrument The first part of the questionnaire consisted of FCs’; demographic data including gender, age, marital status, income, educational level, number of family members, and relationship status with the CCA survivor. The Thai version of the World Health Organization’s Quality of Life Questionnaire (WHOQOL-BREF-THAI), a questionnaire which contains 26 items including 2 items, concern QOL and general health and 24 items that deal with degrees of satisfaction, which were; physical health (7 items), psychological health (6 items), social relationships (3 items), and environmental health (3 items) are covered in this section (8 items). Each item is graded on a scale of 1 to 5 on a 5-point Likert scale. The domain score was calculated using the mean score of items within each domain and translated into 4-10 and 0-100 scores according to the questionnaire rules. Higher scores indicated a higher standard of living (Gholami et al., 2013). The WHO has officially recognized the WHOQOL-BREF-dependability, and this tool has a Cronbach’s alpha coefficient of 0.841 and a content validity score of 0.652. The Memorial Symptom Assessment Scale Short Form (MSAS-SF), a 32-items questionnaire with a 5-point Likert scale, is aimed to measure the frequency, severity, and discomfort associated with 32 different multidimensional symptoms that patients experience. The MSAS-SF has been used to diagnose a wide spectrum of conditions and can be utilized in both clinical and research contexts (Menezes et al., 2017). The Hospital Anxiety and Depression Scale (HADS) (Zigmond and Snaith, 1983) is commonly used to examine anxiety and depression symptoms. In Thailand. It has 14 questions which seven questions are for assessing anxiety symptoms (all odd numbers) and seven questions for measuring depression symptoms (all even numbers). Each response has a score on a Likert scale ranging from 0 to 3. The anxiety and depressive episode scores are divided into parts ranging from 0 to 21, with the following categories: Three scales are used to determine the score range: non-anxiety and depression are defined as 0-7 points. 8-10. The Supportive Care Needs Survey for Partners and Caregivers (SCNS-P&C) was created to measure the multi-dimensional supportive care needs of FCs at various of the illness of those receiving the care (Girgis et al., 2011). The SCNS-P&C items were translated from English to Thai in our study. The 45 items in this tool are divided into four categories: communication and relationships (10 items), health care and information (16 items), social and work (11 items), and psychological (8 items). A five-point rating scale is used for all the products (1-no need: not applicable; 2-no need: satisfied; 3- low need; 4-moderate need; 5-high need). The overall score is calculated by adding all the need items together, with higher scores indicating more unmet requirements. Statistical analysis Study outcome The mean scores of each domain of the WHOQOL-BREF, including physical, psychological, social relationship, and environmental, were treated as dependent variables. Descriptive statistics were used, with continuous variables being described using means and standard deviation (SD) and categorical variables being represented using numbers and percentages. Pearson’s correlation coefficient was used for investigating the relationships among demographic factors, MSAS-SF, HADS, the SCNS-P&C, and WHOQOL-BREF domains. The differences between demographic characteristics and WHOQOL-BREF domains were explored using an independent t-test and one way ANOVA (followed by a Bonferini test) as appropriate to determine which specific factors that affect FCs’ QOL. The four QOL domains were included as dependent variables in multivariate linear regression models, with the factors that demonstrated a significant correlation (p<0.05) with QOL. All independent variables were coded or translated into categorical measurements before being put into the regression models. We used an enter method, with statistical significance defined as a p value less than 0.05. The tolerance and variance inflation factor (VIF) were used to check for multicollinearity in all models. A multicollinearity problem is indicated by a tolerance of less than 0.1 and/or a VIF of more than 5, however none of the variables demonstrated severe multicollinearity. Results Family caregiver Characteristics Most FCs were female (69.7%) with a mean age of 46.11 years (SD=11.47). The majorities of FCs were son/daughter (56.7%) or spoused (41.1%), married (70.5%), under academic study (73.2%), and the household income was low compared with the local average, more than two third (74.4%) earning less than 15,000 thb. The FCs had a prominent level of emotional distress, with 50.2 % having severe anxiety. In contrast, for depression, only 4 FCs (1.7%) reported having severe depression (Table 1). Quality of life of family caregivers Most FCs of CCA survivors had a moderate to high level QOL scores in both a total QOL and all four domains. For the total QOL scores, two third of FCs (70.1%) reported having a high QOL including Physical Health (71.9%), Psychological (70.1%), and Social Relationships (57.5%). Conversely, for the Environment domain, the majority of FCs (71.9%) expressed that they had a moderate level of QOL (Table 2). Symptom of family caregivers The top 5 symptoms are presented in Table 3, and the mean total score of symptoms was 7.76 (SD= 5.38). Sleep problem was the highest symptom occurrences among FCs with a mean score of 1.23 (SD= 0.48), followed by tiredness with a mean score of 0.95 (SD= 0.30) and depression with a mean score of 0.38 (SD= 0.77) (Table 3). Descriptive Statistics and Correlations among Study Variables The means, SDs, and intercorrelations across multiple variables are shown in Table 3. The mean anxiety score was 11 ± 3.23, with 116 FCs (50.2%) exceeding the scale cut-off for severe anxiety. WHOQOL-BREF domains showed significantly negative correlations (p<0.05) among the MSAS-SF, Social Relationships QOL, Environment QOL, and SCNS-P&C. The QOL and its associated factors among FCs for CCA survivors Physical Health domain According to the standardized regression coefficient in Table 5, the factors that are significantly associated with Physical Health domain of FCS were age 40-49 years (B = 1.57; p < 0.05), academic level (B = 2.95; p <0.001), income 5,000-9,999 baht per month (B =1.32; p < 0.05) and marital status (B = 0.98; p < 0.05), respectively. On the other hand, both the SCNS-P&C subscales and MSAS-SF were marginally negatively correlated with the Physical Health domain (B=-0.01 and -0.07; p < 0.001). Furthermore, when the SCNS-P&C and MSAS-SF subscales were added to the model, there was a considerable improvement in the model (R2 adj=32.3%; p < 0.001). Psychological Domain For the Psychological domain, age 40-49 years and age 50-59 years (B =1.79, B=1.79; p < 0.001), secondary and Bachelor/ higher education level were significantly related to this domain (B=3.50, and B=3.99; p < 0.001). In contrast, both the MSAS-SF and SCNS-P&C subscales were marginally negatively correlated with Psychological (B =−0.02, and B=-0.02; p < 0.001). When the SCNS-P&C and MSAS-SF subscales were added to the model, there was a reasonable enhancement in the model (R2 adj = 44.5%; p < 0.001). Social Relationships Domain Age less than 40 years and age more than 60 years (B = 0.61 and B= 0.91; p<0.05), secondary and Bachelor/ higher education level (B = 0.51, and B=0.87; p <0.05), and income 5,000-9,999 and more than 20,000 baht (B=0.59 and 0.74; p<0.05) were all significantly positively associated with the Social Relationships Domain of the FCS. Marital status (B = -0.43, p =0.019), the SCNS-P&C subscales (B=-0.01, p<0.001), and MSAS-SF (B=-0.07, p<0.001), on the other hand, were significantly negatively linked with this domain. In the Social Relationships model, there was a considerable improvement (R2 adj = 29.7%, p< 0.001). Environment Domain Secondary and bachelor/ higher education levels (B = 2.62 and 1.33; p <0.001 and p = 0.028), income 5,000-9,999, 15,000-19,999 baht, and more than 20,000 baht (B = 1.48, 2.83, and 4.52, p= 0.001), and income 5,000-9,999, 15,000-19,999 baht (B =1.48, 2.83, and 4.52, p=0.001). Furthermore, the Environment domain was slightly negative linked with the SCNS-P&C subscales and MSAS-SF (B =-0.02 and -0.01; p =0.008 and p=0.001). When the SCNS-P&C subscales and MSAS-SF were included to the model, the Social Relationships model improved significantly (R2 adj = 46.1%; p <0.001). (Table 5) Table 1 Demographic Characteristics of Family Caregivers (n=231) Baseline characteristics Number Percent Gender Male 70 30.3 Female 161 69.7 Age Mean (±SD) 46.11(±11.47) Median (min: max) 45(19:74) Education level Primary 73 31.6 Secondary 96 41.6 Bachelor or higher 62 26.8 Household income Less than 5000 53 22.9 5,000-9,999 53 22.9 10,000-14,999 66 28.6 15,000-19,999 33 14.3 ≥ 20,000 26 11.3 Marital status Single 45 19.5 Married 186 70.5 Relationship status Partner 95 41.1 Son/Daughter 136 58.9 Family member Less than 5 96 41.6 ≥ 5 135 58.4 Anxiety Normal 12 5.2 Doubtful case 103 44.6 Case 116 50.2 Mean anxiety score (±SD) 11.02 (± 3.23) Depression Normal 193 83.5 Doubtful case 34 14.8 Case 4 1.7 Mean depression score (±SD) 8.03 (± 2.96) MSAS-SF score (±SD) 7.76 (± 5.38) SCNS-P&C score (±SD) 154.63 (± 20.36) Table 2 Distribution of Family Caregiver’s Quality of Life Scores WHOQOL-BREF Domains Mean (±SD) Average score (1-5) Number (%) Low Medium High Physical Health 27.03(±2.81) 3.87 0 (0.0) 65 (28.1) 166 (71.9) Psychological 23.13(±2.81) 3.86 6 (2.6) 63 (27.3) 162 (70.1) Social Relationships 11.32(±1.08) 3.77 8 (3.5) 90 (39.0) 133 (57.5) Environment 28.08(±2.81) 3.51 0 (0.0) 166 (71.9) 65 (28.1) Total 97.13(±9.28) 3.74 0 (0.0) 69 (29.9) 162 (70.1) Table 3 Distribution of Family Caregiver’s Symptoms Symptom Mean SD Sleep problem 1.23 0.48 Fatigue 0.95 0.3 Depression 0.38 0.77 Weight lost 0.3 0.67 Pain 0.16 0.42 Total 7.76 5.38 Table 4 Means Standard Deviations, and Correlation among Study Variables (n=231) Variables 1 2 3 4 5 6 7 8 1. Anxiety 1 2. Depression 0.33* 1 3. MSAS-SF 0.01 -0.06 1 4. SCNS-P&C 0.11 0.01 0.18* 1 5. Physical Health QOL -0.07 0.04 -0.32* -0.12 1 6. Psychological QOL -0.04 -0.08 -0.36* -0.03 0.83* 1 7.SocialRelationships QOL -0.02 0.09 -0.33* -0.20* 0.58* 0.51* 1 8. Environment QOL 0.05 -0.03 -0.21* -0.21* 0.64* 0.53* 0.62* 1 Mean 11 8.03 7.76 154.63 27.03 23.13 11.32 28.08 SD 3.23 2.96 5.38 20.36 2.81 2.81 1.08 2.81 *p-value<0.05 Table 5 Results of Multiple Linear Regressions, Including Factors Related WHOQOL-BREF Domain of CCA Caregivers Variables Physical Health Psychological Social Relationships Environment B (SE) p B (SE) p B (SE) p B (SE) p Age Less than 40 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 40-49 1.57 (0.50) 0.002 1.79 (0.46) <0.001 0.61 (0.20) 0.002 0.72 (0.45) 0.11 50-59 0.48 (0.51) 0.341 1.70 (0.46) <0.001 0.03 (0.20) 0.883 0.36 (0.45) 0.432 ≥ 60 -0.38 (0.71) 0.594 1.23 (0.65) 0.064 0.91 (0.28) 0.001 0.76 (0.64) 0.234 Education level Primary 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Secondary 2.02 (0.56) <0.001 3.50 (0.52) <0.001 0.51 (0.22) 0.021 2.62 (0.50) <0.001 Bachelor/ higher 2.95 (0.68) <0.001 3.99 (0.62) <0.001 0.87 (0.27) 0.001 1.33 (0.60) 0.028 Income < 5,000 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 5,000-9,999 1.32 (0.51) 0.01 0.07 (0.47) 0.875 0.59 (0.20) 0.004 1.48 (0.45) 0.001 10,000-14,999 -0.53 (0.57) 0.348 -0.84 (0.52) 0.105 0.21 (0.22) 0.339 -0.49 (0.51) 0.33 15,000-19,999 0.11 (0.65) 0.866 -0.12 (0.60) 0.841 0.38 (0.26) 0.146 2.83 (0.58) 0.001 ≥ 20,000 0.67 (0.73) 0.363 0.30 (0.67) 0.651 0.74 (0.29) 0.011 4.52 (0.65) 0.001 Marital status Single 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Married 0.98 (0.47) 0.037 0.53 (0.43) 0.219 -0.43 (1.18) 0.019 0.12 (0.42) 0.771 SCNS-P&C -0.03 (0.31) <0.001 -0.02 (0.01) 0.008 -0.01 (0.01) <0.001 -0.02 (0.01) 0.008 MSAS-SF -0.11 (0.03) <0.001 -0.02 (0.29) <0.001 -0.07 (0.01) <0.001 -0.10 (0.02) 0.001 Model characteristic R2=32.3% R2=44.5% R2=29.7% R2=46.1% p<0.001 p<0.001 p<0.001 p<0.001 Discussion The relationship between FCs and their patients is widely acknowledged to have a significant impact on FCs’ quality of life. FCs’ poor QOL demands special attention, especially in a culture that emphasizes family contribution, but little dependence on social/professional care (Fadhilla et al., 2019). As a result, the goal of this study was to determine the CCA FCs’ QOL. In Thailand, family members play an important part on patient care in the hospitals. They do a variety of tasks that would normally be performed by nurses in Asian countries. This result shows that the level of QOL of FCs was Middle to High because of the superior quality of the health care team for CCA patients and their FCs. This result supports the results recorded by Abdullah et al. (Abdullah et al., 2019) In the present study, our findings illustrated that the level of QOL of FCs was middle to high as a result of a good quality of health care team for CCA patients and their FCs. As a consequence of this hospital care which delivered unique treatments directly to CCA patients may have contributed indirectly to the higher QOL in that study. This result is supported by Abdullah et al (Abdullah et al., 2019). However, CFs’ QOL in a research of leukemia patients was lower than ours because they were all at an advanced stage, whereas our cancer patients were in various stages. In comparison to prior studies, FCs in our study reported higher burden domain QOL scores (Yu et al., 2017). In our study, all WHOQOL domains including physical Health, psychological, social relationships and environment were negatively correlated with SCNS-P&C and MSAS-SF scores due to the experience of long-term stressors because of long-term care of cancer patients that impact on FCs’ physical symptom occurrences and their unmet needs. Moreover, this study shows that the CCA FCs have high levels of unmet needs which impact on all domain of their QOL. When considering with other developing countries, where FCs are typically the primary source of assistance for cancer patients, this conclusion is unsurprising (Almutairi et al., 2017). As the need for information related to cancer and their process of cares were the most prioritized unmet need of FCs. In other studies, the most essential demand of informal caregivers of cancer patients has been identified as information. The key needs of FCs in a study conducted by Sajadian et al. (Hydary and Mokhtari Hesari, 2015) were acquiring knowledge about breast cancer, self-care, and patient care. Likewise, Cui et al. (Cui et al., 2014) found that the most common unmet needs, among 649 family caregivers in Shanghai hospitals, were a knowledge about the disease and its treatment, as well as assistance from healthcare professionals. Most FCs were affected by stress related to unmet need deficit as evidenced by each of the variables examined. Over 75% of FCs experienced sleep difficulties, fatigue, pain, and depression, which is consistent with prior research findings (Lee et al., 2015; Valero-Cantero et al., 2021). These findings underscore our study’s unique contribution, as little previous research has been done on the impact of advanced cancer symptoms on carers’ QOL. We show that a higher overall severity of symptoms in advanced cancer patients is linked to a lower QOL for the caregiver. Only one previous study has looked into this relationship; in this case, the patient’s distress was directly linked to the caregiver’s symptom impairment (Otto et al., 2019). Financial assistance may be helpful to alleviate the financial strain of FCs. In connection to their physical, psychological, and environmental functioning, this study discovered that patient insurance coverage was a predictor of higher QOL of FCs. Tian and colleagues (TIAN et al., 2012) conducted a study on support and found a positive correlation between household income and the QOL of FCs. Because medical treatments for CCA patients are expensive, it places financial stress on patient families. Even though many care responsibilities are delegated to FCs or hired care workers, due to nursing shortages, these costs are not covered by social health insurance programs. Families in financial distress are not only limited in their treatment options, but they are also less likely to recruit paid caregivers to help relieve the load of caregiving (Son et al., 2012). The stress, that married FCs face, has a significant influence on their QOL, particularly for married women who leave their jobs to care for aging parents (Aun and Mohd, 2016). According to our research. Our findings were in line with those in Taiwan and Turkey, which found substantial links between caregiver burden and QOL (Turkoglu and Kilic, 2012). The multivariable regression models demonstrated a positive relationship between age and FCs’ QOL, implying that older FCs have a higher QOL. This is in direct contrast to the findings of a recent study on cancer patients, which found that younger FCs had a higher QOL than older FCs, resulting in a higher QOL for their patients (Shahi et al., 2014) . Kim et al. (Kim et al., 2009) discovered that when patients and FCs grew older, they were better able to adapt to new settings and develop the knowledge and abilities needed to manage their critical illnesses. Several studies reported that older carers had a lower QOL compared to younger FCs (Rivera-Navarro et al., 2009; Alptekin et al., 2010). This can be explained by the fact that older carers may have had more experience of adapting to change and overcoming challenges in their life. Also, older caregivers reported better psychosocial adjustment than younger caregivers. Older caregivers may have understood the changes in their lives more than those who were younger (Kim et al., 2012). As stated in the literature, various degrees of schooling had different influences on QOL in the tested sample, Participants with lower educational levels were found to have poorer physical and social effects, with statistically significant disparities. Many studies found that low levels of education are associated with poorer QOL outcomes, acting as a risk factor for psychosocial diseases such as depression and a growing dissatisfaction with health care services (Calvo et al., 2011; Ndikuno et al., 2016). However, some of the conditions that cause patients and FCs to become associated may be beyond the competence or area of responsibility of physicians and nurses. As a result, multidisciplinary care teams and other systems that provide support and palliative services become more important, and they can play a vital role in reducing the burden of care for FCs and improving their QOL by implementing the right strategies and interventions. This study has some limitations. As a cross-sectional survey, it is not possible to make a causal inference about the associations between the investigated factors and the QOL of FCs. The participants were recruited from one hospital in Thailand, and therefore the findings, of this study, should be cautiously accepted. However, the findings on the factors associated with the QOL of FCs are unlikely to be seriously influenced by the sample selection bias. The research highlights on the QOL of FCs for CCA survivors. The majority FCs’ QOL is moderate to high, which is determined by both patient and FCs features. Symptoms, support care needs, age, and education level were associated with QOL among FCs for CCA patients. A holistic strategy that includes caregiver training, psychosocial therapies, and proper support may help these FCs for a better QOL Author Contribution Statement MS., MS., UP, and YW. envisioned the research idea. US. and MT. analyzed, interpreted the data, and helped to draft the manuscript. That was also discussed with MS, YW and US. who supervised the research project. ST, NC. who collected data and literature reviewed. All authors revised the manuscript critically and approved the final version to be published. Acknowledgements The authors would like to thank the caregivers who participated in this study, and Mr. Peter Barney for assistance for the English -language presentation of this manuscripts. Funding This research is funded by the Young Research Development Project of Khon Kaen University Year 2021. Ethical Approval This research was approved by the research ethics committee of Khon Kaen University, Thailand (HE631628). The informed written consent was obtained from each of the study participants of this study. Besides, every participant was assured about the confidentiality of his personal information. Availability of the data Due to ethical restrictions, the datasets are not publicly available but are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare that there is no conflict of interests regarding the publication of this paper. ==== Refs References Abbasi A Mirhosseini S Basirinezhad MH Relationship between caring burden and quality of life in caregivers of cancer patients in Iran Support Care Cancer 2020 28 4123 9 31872293 Abdullah NN Idris IB Shamsuddin K Health-related quality of life (HRQOL) of gastrointestinal cancer caregivers: the impact of caregiving Asian Pac J Cancer 2019 20 1191 Almutairi KM Alodhayani AA Alonazi WB Assessment of health-related quality of life among caregivers of patients with cancer diagnosis: a cross-sectional study in Saudi Arabia J Relig Health 2017 56 226 37 27236467 Alptekin S Gönüllü G Yücel İ Characteristics and quality of life analysis of caregivers of cancer patients Med Oncol 2010 27 607 17 19590990 Aun NSM Mohd RH Informal caregiving: empowering social support programs by employers Akademika 2016 86 3 9 Calvo A Moglia C Ilardi A Religiousness is positively associated with quality of life of ALS caregivers Amyotroph Lateral Scler 2011 12 168 71 21348787 Cui J Song L Zhou L Needs of family caregivers of advanced cancer patients: a survey in S hanghai of C hina Eur J Cancer Care 2014 23 562 9 Fadhilla A Nurhidayah I Adistie F The correlation between family functioning and quality of life of caregiver of children with leukemia Belitung Nurs J 2019 5 32 40 Fumaneeshoat O Ingviya T Quality of Life and Burden of Lung Cancer Patients’ Caregivers: A Cross-Sectional Study from Southern Thailand J Health Sci Med Res 2020 38 177 92 Gholami A Jahromi LM Zarei E Application of WHOQOL-BREF in measuring quality of life in health-care staff Int J Prev Med 2013 4 809 24049600 Girgis A Lambert S Lecathelinais C The supportive care needs survey for partners and caregivers of cancer survivors: development and psychometric evaluation Psychooncology 2011 20 387 93 20878835 Hansen L Chang MF Lee CS Physical and mental quality of life in patients with end-stage liver disease and their informal caregivers Clin Gastroenterol Hepatol 2021 19 155 61 32289544 Hashemi M Irajpour A Taleghani F Caregivers needing care: the unmet needs of the family caregivers of end-of-life cancer patients Support Care Cancer 2018 26 759 66 28952034 Hydary L Mokhtari Hesari P Common breast cancer family care giving problems Iran J Breast Dis 2015 8 7 14 Kilic ST Oz F Family caregivers’ involvement in caring with cancer and their quality of life Asian Pac J Cancer 2019 20 1735 Kim M-D Hong S-C Lee C-I Caregiver burden among caregivers of Koreans with dementia Gerontology 2009 55 106 13 19023194 Kim Y Spillers RL Hall DL Quality of life of family caregivers 5 years after a relative’s cancer diagnosis: follow-up of the national quality of life survey for caregivers Psychooncology 2012 21 273 81 22383269 Kong SV Guan NC Burden in family caregivers of cancer patients: the association with depression, religiosity and religious coping Asian Pac J Cance Care 2019 4 171 82 Lee KC Yiin JJ Lin PC Sleep disturbances and related factors among family caregivers of patients with advanced cancer Psychooncology 2015 24 1632 8 25871014 Lim J Cho H Bunds KS Cancer family caregivers’ quality of life and the meaning of leisure Health Care Women Int 2021 42 1144 64 32490741 Meecharoen W Northouse LL Sirapo-ngam Y Family caregivers for cancer patients in Thailand: An integrative review Sage Open 2013 3 2158244013500280 Menezes JRd Luvisaro BMO Rodrigues CF Test-retest reliability of Brazilian version of Memorial Symptom Assessment Scale for assessing symptoms in cancer patients Einstein (São Paulo) 2017 15 148 54 28767911 Miller KD Nogueira L Mariotto AB Cancer treatment and survivorship statistics CA Cancer J Clin 2019 69 363 85 31184787 Ndikuno C Namutebi M Kuteesa J Quality of life of caregivers of patients diagnosed with severe mental illness at the national referral hospitals in Uganda BMC Psychiatry 2016 16 1 9 26739960 Otto AK Gonzalez BD Heyman RE Dyadic effects of distress on sleep duration in advanced cancer patients and spouse caregivers Psychooncol 2019 28 2358 64 Rivera-Navarro J Benito-León J Oreja-Guevara C Burden and health-related quality of life of Spanish caregivers of persons with multiple sclerosis Mult Scler J 2009 15 1347 55 Shahi V Lapid MI Kung S Do age and quality of life of patients with cancer influence quality of life of the caregiver? 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116144 10.31557/APJCP.2023.24.4.1225 Research Article Early-Onset Colorectal Cancer Survival among Unscreened Population -Multicenter Cohort Retrospective Analysis Alsiary Rawiah 1 Aboalola Doaa 1 Alsolami Mona 1 Alsaiari Turki 2 Ramadan Majed 1* 1 King Abdullah International Medical Research Center (KAIMRC), King Saud Bin Abdulaziz University for Health Sciences, Ministry of National Guard - Health Affairs, Kingdom of Saudi Arabia. 2 College of Medicine, King Abdulaziz University, Kingdom of Saudi Arabia. * For Correspondence: ramadhanma@NGHA.MED.SA 2023 24 4 12251230 9 11 2022 8 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: In Saudi Arabia and across the world, the incidence of early-onset colorectal cancer (< 50 years) has increased. The diagnosis of EOCRC, on the other hand, is frequently delayed. It is critical to implement a national screening program to identify those group of patients who might benefit from early diagnosis. Method: A retrospective search was conducted using data from the Ministry of National Guard Health Affairs’ (MNG-HA) Cancer Registry. The population of 1440 CRC patients were eligible for the analyses. Patients’ demographics including age at diagnosis, gender, and marital status, were all reported. The demographic and clinical characteristics were assessed across early-onset and late-onset groups using Chi-square and Fisher exact test where appropriate. Results: CRC patients, early-onset CRC (18-50 years) was reported in 23.26%, mainly with advance disease. Late-onset (>50 years) CRC individuals have worse survival rate and higher probability of dying compared to early-onset CRC individuals. After age at diagnosis classification into three categories (18-40 years), (41-50 years), and (>50 years) the Kaplan-Meier Survival curve show that early-onset (18-40 years) CRC individuals had significantly better survival than (41-50 years), and (>50 years) CRC patients. Conclusions: Comparing our data to another screened population using US SEER datasets, we discovered a substantial difference in survival rates, with the SEER population having a considerably greater chance of survival. There is very little research on the significance of screening for Saudi CRC patients, and this is an issue that needs to be looked into more. Limitations: A study’s drawback is the lack of data for a variety of risk variables linked to colorectal cancer incidence, such as the KRAS mutation and environmental risk factors including BMI and smoking. More research with a nationally representative sample and comprehensive demographic and clinical data accessible is needed. Key Words Colorectal cancer screening early-onset late-onset survival ==== Body pmcIntroduction Colorectal cancer (CRC) is considered as second leading cause of cancer mortality worldwide and the third most prevalent cancer. Poor nutrition, alcohol consumption, smoking, and visceral fat are some of the risk factors for CRC. Males are more likely than females to experience greater rates of CRC mortality and morbidity. CRC is the most frequent malignancy among men and the third most common malignancy in women in Saudi Arabia, accounting for 2,047 new cases and 1,090 deaths in 2014. On the other hand, Obesity and low rates of physical exercise are related with the worst CRC outcomes in Saudi women (Alyabsi et al., 2021). Colorectal cancer survivors’ quality of life was also harmed. Following cancer therapy, the most prevalent symptoms are fatigue, sleep loss, and urine frequency (Magaji et al., 2019). The patient’s age, the presence of comorbidities, sex, mode of presentation, tumor grading, and the timing of symptom onset are all factors that influence survival (Hansen et al., 2012). Furthermore, the laterality of colon cancer may affect survival outcomes (Beart et al., 1983; Benson et al., 2017; Jaruhathai et al., 2022; Lim et al., 2017). Colorectal cancer (CRC) identified before the age of 50 is known as early-onset colorectal cancer (EOCRC). It is becoming more common all around the world (Siegel et al., 2019). Consequently, CRC research has switched to look at the molecular signature, incidence, clinical performance, risk factors, and pathological features of early-onset colorectal cancer comparing to late-onset colorectal cancer (CRC diagnosed beyond the age of 50). Because of a lack of patient and physician knowledge, EOCRC is often misdiagnosed as more benign illnesses. General incidence rate of CRC has reduced recently as a result of breakthrough in screening and treatment. However, the prevalence of EOCRC has increased significantly worldwide including Saudi Arabia (Agazzi et al., 2021; Alyabsi et al., 2021; Siegel et al., 2019). This is possibly related to misconceptions about colon function, colon cancer prevalence, and when patients should be tested for colon cancer (Alsulaim et al., 2021). According to the 2018 update, American Cancer Society’s Colorectal Cancer Screening for Adults at Average Risk, individuals aged 45 and older should begin screening with visual assessment and a high-sensitivity stool-based test, all positive non-colonoscopy results are followed by a colonoscopy (Rosato et al., 2013). The bowel cancer screening program (BCSP) of the National Health Service (NHS) in the United Kingdom and The Australian National Bowel Cancer Screening Program (NBCSP), have not modified their target range remains constant, proposing screening every two years for people aged between 50 and 74 (Archambault et al., 2021). The objective of this research is to emphasize the alteration in survival rates CRC early-onset and CRC late-onset, as well as to compare survival rates by stage between screened CRC populations utilizing (SEER) and unscreened CRC populations (MNG-HA). Materials and Methods Study design, Setting, and Population This current study adapted a retrospective cohort design utilizing the data for the Ministry of National Guard Health Affairs’ (MNG-HA) Cancer Registry in the major two cities in Saudi Arabia, Riyadh and Jeddah city from 1/1/2009 to 31/12/2017 with follow-up time started at the date of CRC diagnosis in the registry and ended on the date of death, date of last contact or when study ended on December 31, 2017. The registries, stored information regarding demographic, clinical characteristics such as type of cancers, stage, and grade at the first diagnosis. The registries stored the data for treated patients at King Abdulaziz Medical City (KAMC) in Riyadh and Jeddah city. In each visit, patients’ data were structured to store follow-up information, the date of last contact, and other information required to estimate patient survival. Inclusion criteria included patients aged ≥18 years diagnosed with colorectal cancer using International Classification of Diseases (ICD) (ICD-10 C18– C20) who treated at MNG-HA hospitals from January first, 2009, to December 31, 2017. Total patients who included in the study was 1,560. About 120 (7.6) patients were excluded from the study because of missing of required information such as unknown admission or contact dates. The total eligible patients for the analysis were 1440. Patient and Tumor Characteristics Demographic information such as age at diagnosis, gender, and marital status were retrieved from the cancer registries. Patient demographics including age at diagnosis, gender, and marital status, were all extracted from electronic health records. When classifying age at diagnosis groups, we accounted for the time of the study and the existing colorectal cancer screening guidelines (Burt et al., 2010). The recommended age for screening is 50, so we defined early-onset CRC as CRC diagnosed at age 50 or younger. The compared group (late-onset) included patients diagnosed with CRC at ages 51 years or older (Cheng et al., 2021). We further classify age at diagnosis into three groups (18-40, 41-50, and >50) (Chen et al., 2014; Lee et al., 2013; Young, 2006). Statistical Analysis We used Chi-square when we have sufficient observation in each cell and Fisher exact test when we have fewer than 5 observation per cell to examine the demographic and clinical characteristics across early-onset and late-onset groups. To build the multivariate model, we used purposeful variable backward selection technique to include only variables with P< 0.05. We used the Kaplan-Meier product limit method and the log-rank test to estimate the survival rates. Cox proportional hazards regression adjusted models were used to estimate the hazard ratio associated with early onset of CRC. To validate our finding, and to minimize potential confounder bias, we performed a sensitivity analysis with multiple and different age groups for each testing model. We used SAS statistical software version 9.4 (SAS Institute Inc. Cary, NC) to perform the analysis. Results The cohort included 1,440 individuals diagnosed with CRC, 826 (57.36%) males and 614 (42.64%) females, Table 1 represent patients’ demographical and clinical characteristics. Among EOCRC, the proportions of diagnosed CRC males and female were approximately similar (50.75% vs 49.25%), while among late-onset patients, the proportions of males were 20% higher than females. When comparing between individuals with EOCRC to those with late onset CRC, incidence in male were more likely to increase at late onset (50.75% vs 59.37%; P < .005). EOCRC individuals were more likely to go through chemotherapy and/or surgical treatment (60%, and 55.85%; P < 0.01) compared to late-onset (44.52%, and 48.55%; P <0.0001). The overall median survival for early-onset CRC individuals was significantly higher than late onset CRC individuals (P = 0.03) (Table 1). The median survival on the Kaplan-Meier Survival curve was substantially lower among people with late-onset CRC (69 months) (95% CI, 67-73 months) in comparison to early-onset CRC individuals 113 months (95% CI, 77-115 months) (Figure 1). However, when the early onset cohort was further sup-grouped into (18-40 years) and (41-50 years), the Kaplan-Meier Survival curve show that early-onset (18-40 years) CRC individuals had significantly higher survival than early-onset (41-50 years), and late onset (>50 years) (P =0 .03) (Figure 2). Late-onset CRC individuals had higher probability of dying compared to early-onset CRC individuals (23.28% vs 28.33%; P < 0.01) during the follow-up period (Table 1). In the multivariate Cox regression both unadjusted and adjusted models, showed a statistically significant difference in survival between EOCRC vs late-onset CRC (HR, 0.82; 95% CI, 0.68-0.98; P 0.03), (HR, 0.78; 95% CI, 0.64-0.96; P 0.01) (Table 2). EOCRC patients was associated with lower mortality as compared to late-onset CRC individuals. Similarly, after age at diagnosis classification into three categories, both unadjusted and adjusted models showed that patients who aged between 18-40 years, relative to late-onset onset (>50years), showed statistically significant improved survival (HR, 0.81; 95% CI, 0.64-0.99; P 0.03), (HR, 0.77; 95% CI, 0.61-0.97; P 0.01). In terms of overall survival rate, the MNG-HA and SEER cohorts were compared. EOCRC patients had a better overall survival rate than late-onset CRC patients across all stages in both populations (Table 3). Among localized and regional stages, the survival rate was significantly varied between MNG-HA and SEER. Survival rate among SEER population was 30% to 40 % significantly higher than MNG-HA for localized stages (93.7 vs 58.97) and (92.4 vs 47.75), and 10% to15% higher among regional stages (78.5 vs 65.27), and (76.55 vs62.09) (Table 3). Figure 1 Kaplan-Meier Mortality Estimates for Early-Onset (18-50) and Late-Onset (>50) Colorectal Cancer (CRC) Figure 2 Kaplan-Meier Mortality Estimates for Early-Onset (18-40), (4-50), and Late-Onset (>50) Colorectal Cancer (CRC) Table 1 Demographic, Clinical and Pathological Characteristics of Collateral Cancer Patients at MNG-HA Hospitals a (N=1440) Patients Characteristics Early-onset (18-50 years) N (%) a Late-onset (51 or older) N (%) P b Total 335 (23.26) 1105 (76.74) 5-year overall survival 67.53% 59.65% 0.03 Died 78 (23.28) 313 (28.33) 0.01 Gender 0.005 Male 170 (50.75) 656 (59.37) Female 165 (49.25) 449 (40.63) Marital status Married 239 (71.34) 865 (78.28) 0.008 Unmarried 96 (28.66) 240 (21.72) Stage at diagnosis 0.17 Distance metastasis 133 (39.7) 398 (36.02) Localized 39 (11.64) 182 (16.47) Regional 146 (43.58) 468 (42.35) Unknown 17 (5.07) 57 (5.16) Pathological Grading 0.87 Well differentiated 15 (4.47) 49 (4.43) Moderate differentiated 249 (74.32) 820 (74.2) Poor differentiated 27 (8.05) 100 (11.05) Others 44 (13.13) 136 (12.3) Tumor Morphology (Histotype) 0.34 Adenocarcinoma (AC), NOS 273 (81.49) 954 (86.33) Mucinous AC 30 (8.96) 73 (6.61) Mucin-producing AC 3 (0.9) 8 (0.72) Signet ring cell carcinoma 5 (1.49) 10 (0.90) AC in villous/tubuvillous adenoma 7 (2.09) 13 (1.18) Others g 17 (5.07) 47 (4.25) Tumor location 0.21 Left side 238 (71.04) 770 (69.68) Right side 57 (17.01) 228 (20.63) Unknown 40 (11.94) 107 (9.68) Chemotherapy <0.0001 Yes 201 (60.00) 492 (44.52) No 134 (40.00) 613 (55.48) Surgical treatment 0.01 Yes 188 (55.85) 536 (48.55) No 148 (44.15) 568 (51.45) Radiotherapy 0.26 Yes 26 (7.76) 67 (6.06) No 309 (92.24) 1038 (93.94) a,Sample size and percentage N(%); b, P value of the chi-square, and fisher exact test where appropriate Table 2 Hazard Ration ComparingA Early-Onset versus Late-Onset Age at diagnosis Model 1 a Model 2 b HR (95% CI) d P e HR (95% CI) P Early-onset vs late-onset <50 years 0.82 (0.68, 0.98) 0.03 0.78 (0.64, 0.96) 0.01 >50 Reference Reference Early-onset vs late-onset <40 0.81 (0.64, 0.99) 0.04 0.77 (0.61, 0.97) 0.03 41-50 0.95 (0.77, 1.17) 0.66 0.99 (0.79, 1.24) 0.96 >50 Reference Reference a, Unadjusted; b, Adjusted for demographics, clinical factors and treatment factors; d, Hazard ratio (HR), confidence interval (CI); e, P-value (P) Cox regression model Table 3 Five-Year Colorectal Cancer Survival by Stages in MNG-HA compared to US SEER All stages Localized Regional Survival (%) 95% CI Survival (%) 95% CI Survival (%) 95% CI Early-onset US SEER 63.6 (62.8, 63.5) 93.7 (93.2, 94.1) 78.5 (77.9, 79.05) MNG-HA 67.53 (77, 115) 58.97 (57, 78) 65.27 (67, 114.83) Late-onset US SEER 61.71 (64.5, 65.3) 92.4 (92.0, 92.8) 76.55 (86.7, 77.2) MNG-HA 59.65 (67, 73) 47.75 (53, 63) 62.09 (67, 82) Discussion In this study, we observed that EOCRC patients’ overall median survival rate was markedly higher than late-onset CRC individuals. Prior research from the United States that supports our findings was recently published (Cheng et al., 2021). According to statistics, early-onset colon cancer accounts for 11% and 10% of all CRC cases in European and Western nations, respectively (Agazzi et al., 2021; Alyabsi et al., 2021). The findings of the current study demonstrated that early-onset cancer accounts for around 23% of all CRC cases. The alarming discovery in the current study revealed that nearly 23% of CRC patients in our community were young individuals identified at an advanced stage. Although, there are minimal Saudi studies investigating early-onset incidence, in alignment with our finding, a study conducted by Alyabsi et al., (2021) demonstrated that the early-onset CRC incidence in Saudi Arabia, increased (Alyabsi et al., 2021). Hence, an urgent monitoring system need to be implementing to lower the general incidence rates of CRC (Siegel et al., 2019). Definitions of EOCRC is still heterogenous with some variation across countries in terms of screening protocols (Archambault et al., 2021; Rosato et al., 2013). A National Bowel Cancer Screening Program must be created in Saudi Arabia, with a focus on those aged 40 and over. This cutoff number was suggested based on our findings that CRC patients aged (41-50) and patients above 50 exhibit a similar pattern in the Kaplan-Meier Survival curve, with a considerably worse survival rate than patients aged less than 40. Cheng et al., (2021) prior findings are precisely in accord with the conclusions of this study. However, one of the most significant non-modifiable contributors of EOCRC development is age between 40 and 50 years, as contrast to 40 years (Rosato et al., 2013). We have definitely revealed that patients under the age of 40 have a greater chance of surviving. The necessity of screening is emphasized here because over 75% of our population is diagnosed late in life, with a worse survival rate. EOCRC patients’ may have distinct biological characteristics comparing to old CRC patients’ which possibly result in a different prognosis. It is essential to comprehend the age-related variability of CRC in order to inform treatment strategies and comprehend the distinctive biological differentiation within early-onset CRC. Advances in screening, monitoring, and therapy have reduced overall CRC death rates in developed countries as represented by US SEER. Despite the fact that certain high-income nations, like as Saudi Arabia, have a robust and free healthcare system with all treatment regimens available to all patients, the survival rate in local and regional CRC is lower than in the United States. This is owing to the absence of a CRC screening program. The paper’s recommendation is to underline the need of screening for Saudi patients. In conclusion, the current study revealed that nearly two times more young individuals CRC patients with advanced stage compared to the Western and the US countries. The absence of screening in Saudi Arabia added more burden to patients diagnosed with CRC. The finding of the current study is a call for health policy makers in the kingdom explaining the vivid need for CRC screening for individuals aged 40 years or older in Saudi Arabia. However, more studies in a national level population are need it to confirm these findings. Author Contribution Statement M.R: Conceptualization, Methodology, Software M.R, R.A Data curation, Writing- Original draft preparation. M.R, R.A, M.A, Visualization, Investigation. M.R, R.A Supervision.: M.R, D.A, T.A: Software, Validation.: M.R: Writing- Reviewing and Editing M.R, R.A, D.A, M.A, and T.A. Acknowledgements Funding statement We gratefully acknowledge the financial support of King Abdullah International Medical Center (KAIMRC), King Saud bin Abdulaziz University for Health Sciences (KSAU-HS) Ethics approval Ethical approval was obtained from the Local Institutional Review Board of the Saudi National Guard Health Affairs, Jeddah (IRBC/1346/17). All patients’ identifying variables were omitted before the analysis. Data availability The data that support the findings of this study are available from King Abdullah International Medical Research Centre (KAIMRC) but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of King Abdullah International Medical Research Centre (KAIMRC) Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ==== Refs References Agazzi S Lenti MV Klersy C Incidence and risk factors for preneoplastic and neoplastic lesions of the colon and rectum in patients under 50 referred for colonoscopy Eur J Intern Med 2021 87 36 43 33610414 Alsulaim L AlOdhaybi G AlSalamah M Awareness and Knowledge of Colorectal Cancer in Qassim Region, Saudi Arabia Asian Pac J Cancer Care 2021 6 397 405 Alyabsi M Algarni M Alshammari K Trends in Colorectal Cancer Incidence Rates in Saudi Arabia (2001-2016) Using Saudi National Registry: Early- Versus Late-Onset Disease Front Oncol 2021 11 730689 34568065 Archambault AN Lin Y Jeon J Nongenetic Determinants of Risk for Early-Onset Colorectal Cancer JNCI Cancer Spectr 2021 5 Beart RW Melton LJ Maruta M Trends in right and left-sided colon cancer Dis Colon Rectum 1983 26 393 8 6851801 Benson AB Venook AP Cederquist L Colon Cancer, Version 1 201 NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw 2017 15 370 98 28275037 Burt RW Barthel JS Dunn KB NCCN clinical practice guidelines in oncology Colorectal cancer screening J Natl Compr Canc Netw 2010 8 8 61 20064289 Chen VW Hsieh MC Charlton ME Analysis of stage and clinical/prognostic factors for colon and rectal cancer from SEER registries: AJCC and collaborative stage data collection system Cancer 2014 120 3793 806 25412391 Cheng E Blackburn HN Ng K Analysis of Survival Among Adults With Early-Onset Colorectal Cancer in the National Cancer Database JAMA Netw Open 2021 4 e2112539 34132794 Hansen IO Jess P Possible better long-term survival in left versus right-sided colon cancer - a systematic review Dan Med J 2012 59 A4444 22677242 Jaruhathai S Santidamrongkul A Wiwitkeyoonwong J The Impact of Colon Cancer Sites on Survival Rates after Curative Resection and Adjuvant Chemotherapy: A SingleCenter Study Asian Pac J Cancer Care 2022 7 615 20 Lee YC Lee YL Chuang JP Differences in survival between colon and rectal cancer from SEER data PLoS One 2013 8 e78709 24265711 Lim DR Kuk JK Kim T Comparison of oncological outcomes of right-sided colon cancer versus left-sided colon cancer after curative resection: Which side is better outcome? Medicine (Baltimore) 2017 96 e8241 29049212 Magaji B Moy F-M Law C Pattern of Health-Related Quality of Life and its Association among Patients with Colorectal Cancer Asian Pac J Cancer Care 2019 4 45 52 Rosato V Bosetti C Levi F Risk factors for young-onset colorectal cancer Cancer Causes Control 2013 24 335 41 23224326 Siegel RL Torre LA Soerjomataram I Global patterns and trends in colorectal cancer incidence in young adults Gut 2019 68 2179 85 31488504 Young JL SEER summary staging manual, 2000 : codes and coding instructions 2006 Bethesda, MD. Dept. of Health and Human Services, National Institutes of Health, National Cancer Institute

PMC010xxxxxx/PMC10352750.txt

==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116146 10.31557/APJCP.2023.24.4.1239 Research Article Optimal Combination of Pitch, Modulation Factor and Dosimetric Considerations in Treatment Planning for Total Body Irradiation Using Helical Tomotherapy Kaliyaperumal Venkatesan * Kataria Tejinder Tamilselvan Singaravelu Gupta Deepak Abraham Susan K Narang Kushal Banerjee Susovan Dayanithi Kamaraj Rajesh Selvaraj Bisht Shyam S Shishak Sorun Division of Radiation Oncology, Medanta Cancer Institute, Medanta The Medicity, Gurgaon, Haryana, India. * For Correspondence: venkphysics@gmail.com 2023 24 4 12391248 13 11 2022 7 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: The aim of this study is to makethe standard total body irradiation (TBI) protocol for Helical tomotherapy© (HT) and to analyze the optimal pitch and modulation factor (MF) with respect to dose homogeneity index (HI), target dose coverage, target overdose, beam on time (BOT) and mean lung dose. Materials and Methods: Ten patients who underwent high-dose TBI were taken for this study. For each patient, 35 dose plans were created by different combination of pitch and MF. The optimal pitch and MF were deduced using scatter plot and regression methodology based on target coverage, HI, target volume receiving 103%(V103%), 105%(V105%) and 107% (V107%) of the prescription dose and BOT. Using these optimal pitch and MF, the final dose plan was made and the planning aim and achieved dose was compared using two tailed student’s t-test. Radiochromic films and ionization chambers were used to measure the delivered dose using anthropomorphic phantom on various points for the head and pelvis regions to verify the skin flash margin and its effect on skin dose. Results: The optimal pitch and MF value were 0.287 and 2.4 respectively. Based on optimal pitch and MF, the mean BOT was 1692 seconds with optimal inhomogeneity (7.4%). For target, D95 and D98 were 97.09% (range:94.7-99.6%, p=0.002) and 93.9% (range:91.5-94.4%,p=0.007) respectively, and mean D2 was within 107% with SD of ±1.22% (p=0.04). The mean of PTV receiving V103, V105 and V107 was 24.48% (range=7.7-36.6%, p=0.03), 5.76% (range=1.4-12.1%, SD=±3.3%), 1.93% (range=0.1-4.6%, p=0.008) respectively. Our measurements show that the flash margin did not increase the skin dose. Conclusion: In our study, the optimal combination of pitch value of 0.287 and MF value of 2.4 provided acceptable plans for all patients planned for TBI in HT. The flash margin can provide adequate coverage during patient position uncertainty without increasing the skin dose. Key Words Helical tomotherapy total body irradiation pitch modulation factor skin dose ==== Body pmcIntroduction Total body irradiation (TBI) is an important part of the conditioning regimen for stem cell transplant for multiple myeloma, leukemias, lymphomas and some solid tumors (Gruen et al., 2013; Poonsombudlert et al., 2019). The purpose of TBI is to bring about bone marrow depletion, to destroy any cancer cells left behind after cytotoxic chemotherapy and to prevent rejection of donor cells using immunosuppression, all of which together result in a successful stem cell engraftment (Halperin et al., 2013). In TBI, a uniform radiation dose with megavoltage photon beams is delivered to the entire body, including the central nervous system (CNS) and testis, where traditional chemotherapy is not effective (Seung et al., 2013). Generally, the TBI schedules include twice daily 2-Gy fractions given over 3 days (total dose 12 Gy), 1.5-Gy fractions (twice-daily) over 4-4.5 days (total dose 12-13.5 Gy), or 1.2-Gy fractions (thrice-daily) over 4 days (total dose 12-13.2 Gy (Wong et al., 2018). Uniform dose distribution to the whole body during TBI is mandatory as recommended by American Association of Physicist in Medicine Task group-29 (AAPM TG-29) and the dose variation throughout the body should be within ±10% of the prescription dose (Van Dyk et al., 1986). Cleuziou et al., (2021) discussed the methodology to set up the protocol, dosimetric evaluation and pretreatment QA and in-vivo dosimetry of TBI using Helical TomoTherapy® (HT). TBI in HT provides good dose homogeneity and conformity with an acceptable organ at risk (OAR) tolerance which leads to reduced severity and less late complications (Sarradin et al., 2018, Zarghani et al., 2021, Peñagarícano et al., 2011). As a result, excellent conditioning is attained with minimal side-effects for lung and other body organs (Gruen et al., 2013). Each gantry rotation in the HT field is divided into 51 projections with 7.06° of gantry rotation per projection and this single projection is divided into 64 beamlets. These 64 MLC binary leaves open time with inverse planning provides a target dose from its projection (Boyd et al., 2019). Pitch, modulation factor (MF), and the treatment slice width can play important roles in beam-on time (BOT) and acceptable plan quality, and the selection of a lesser pitch value does not increase BOT because the gantry rotational speed is variable (15–60 seconds per rotation) (Langen et al., 2010). Inverse-planning optimization in tomotherapy needs to select MF, which is defined as MF= (Max leaf open time)/(average non-zero leaves open time). The pitch is the ratio between the couch travel per gantry rotation and the treatment slice width. Kissick et al., (2005), proposed a magic number for determining the pitch value ie., = 0.86/n, n; integer; for reducing the thread effect, and it is indicated by ripples in the longitudinal dose profile. During the treatment plan in Tomotherapy, the final MF value was less than the optimization value for reducing the treatment time using the increased average leaf opening time which provides a less OAR dose, and the optimal range of MF for head and neck was between 3.0 and 1.8. But the MF could be increased (up to 2.4) for sparing the critical organ where the target was very close to it (Ryczkowski et al., 2013). The MF directly contributes to the treatment time and the BOT is defined using the following formula, Beam on time (BOT)= gantry period x active gantry rotations. Complex targets and OARs close to the target require a high MF to modulate the dose distribution to the target (Fenwick et al., 2004, Binny et al., 2015). The suggested optimal pitch and MF were 0.215 and 0.25 respectively for prostate cancer patients which provides a balance between BOT and dose distribution (Skórska et al., 2013). Meyer (2015) studied the optimal treatment planning parameters (pitch and MF) for extremity soft tissue sarcomas and stated that the 0.43 pitch value is not optimal, and that for a pitch value of 0.215, optimum MF is more than 2. This same study (Meyer et al., 2015) suggests using pitch 0.287 which could provide an acceptable plan quality and reduced treatment time. Salz (2015) discussed intensity modulated TBI with TomoDirect® method using various MF and pitch and stated that IMRT with TomoDirect®allows a superior homogeneity compared to conventional methods with lung sparing. The optimal pitch and MF have been evaluated for various sites by several authors (Skórska et al., 2013; Meyer et al., 2015), but to our knowledge, the optimal pitch and MF for TBI using HT mode, has not been analyzed. The aim of this study to make the standard TBI protocol for HT and to analyze the optimal pitch and MF with respect to dose homogeneity index (HI), target dose coverage, target overdose, BOT and lung sparing for TBI in HT. Further, this study aims to validate the flash margin and its effect with skin dose using radiochromic External Beam Therapy 3 (EBT3) and ionization chamber in anthropomorphic phantom. Materials and Methods Patient selection, Immobilization and CT simulation Ten patients who underwent high-dose TBI in our center from 2019 to 2021 were taken for this study (Table 1). Patients were positioned in the supine position in a vacuum cushion with a headrest. A thermoplastic cast was used to immobilize the head and both the arms were positioned by the side. Leg separation between the two legs was fixed within 40 cm so that TomoDirect® (AP/PA) could be used for below thigh treatment. For patient setup, the laser alignment markings were fixed at head, thorax, pelvis and knee region. Fiducials were placed in the thorax and knee region for the reference position. 5mm slice thickness was acquired in computed tomography (Siemens Biograph 64, Siemens Healthinners, Erlangen, Germany) scanner. If the height of the patient was more than 120 cm, then two scans, one each in the head-first supine (HFS) and feet-first supine (FFS) positions, were acquired. Radio-opaque catheters were placed at the thigh (5cm from knee) level in both HFS and FFS scans for summation of dose plans. The acquired image was exported to the treatment planning system (Precision Version 3.2.0, Accuray incorporated Sunnyvale USA). Target and Normal structure delineation The planning target volume (PTV) of total body, created by using the body structure at 3 mm inside the body was contoured as a PTV and this PTV was split into PTV_HFS (up to radio-opaque (RO) marker at thigh level) for HFS scan and PTV_FFS for FFS scan. The step wedge technique was used to create the uniform distribution at junction. Total 7 structures (step wedge) with 1cm thickness were drawn at the level of junction for both HFS and FFS scan (Figure 1). The normal structures including lungs, eyes, kidney and others were contoured as per standard institutional protocol. The PTV was taken inside the lung for 1cm to avoid the under dosage for rib regions. The avoidance volume was drawn by subtracting PTV from lung as an OAR. The body structure was created using auto segmentation of skin creation tool which provides the outer edge of the patient body. The manual correction of body structure was done for removing the immobilization devices which were overlapping with body contour. For taking care of setup uncertainties, 2cm margin from PTV was created and named as PTV_Flash. The right and left hand were separately contoured for removing high / low dose during optimization. The planning process started with choosing the density model for the calculation which provided mass density value using the Hounsfield units. Subsequently machine parameters like treatment machine, delivery mode, plan mode and jaw mode were selected. In our center, for single fraction TBI, the direct delivery mode with 3DCRT is preferred if the width of the patient is less than 40cm. For high dose TBI/multiple fractions, Helical mode with IMRT planning technique is used for sparing the lung. For keeping the account of patient setup uncertainties, fixed jaw mode is kept for direct as well as helical mode. The couch replacement was performed as per vendor recommendation for modeling the couch in Treatment Planning System (TPS). The patient position in TPS, as guided by green lasers, was kept at the center of the patient volume, which comes at chest level in HFS scans in anterior-posterior (AP), superior-inferior (SI), and left-right (LR) directions. The red laser was aligned with the fiducial markers kept at the chest-level of the patient. Before moving to the optimization process, the machine geometry was carefully verified from head to thigh, for any missing or out-of-field geometry parameters. Treatment Planning In this study, the helical mode with IMRT was used for HFS position in all plans. The optimization process was performed by choosing four target structures, PTV_HFS, PTV_Flash (2cm margin to PTV_HFS), Rt hand, and Lt hand. The target and OAR objectives were provided in such a way that the 95% PTV_HFS volume should get at least 95% of the prescription dose. The dummy structures at the junction area were assigned a dose constraint in such a way that it could reduce a dose by 1Gy on each strip. The initial optimization started with these parameters and if the high or low dose region were in the hand region, then separate target objectives were assigned for those regions. For sparing the lung, lung - PTV (Right & Left) was assigned a critical structure constraint with a high penalty. Once an optimal solution was achieved, the process was repeated for various pitch and modulation factors. Starting the pitch value from 0.2 to 0.5 with an increment of 0.05 with a combination of modulation factors of 2 to 4 (increment with 0.5). By using the above combination each patient had 35 plans and all the other planning parameters were kept constant (unchanged). The initial iterations of the plan were approximately 80,000 with medium optimization resolution. The dose distribution for each beamlet was calculated and the number of beamlets depended on slice width, pitch, target volume, and shape. The least-square optimization method was used to optimize the objective function (Mackie et al., 2003) and the final dose calculation was performed at a high resolution with a grid size of 3.91x5x3.91 mm using a convolution superposition algorithm. For FFS plan, if the target had more than 40 cm separation in LR direction then HT was used otherwise Tomo-Direct (TD) was preferred. For FFS, a similar planning process was used as explained in HFS plan. Plan evaluation Initially, the HFS plan and FFS plan was summed up using the Medical Image Merge software (MIM use with Precise RTX version 6.8) and the under- or over-dosage at the junction was assessed (Figure 2). The HFS plan selection criteria were based on the target dose homogeneity, target volume getting 103% (V103%),105% (V105%) and 107% (V107%) of the PD, beam-on time, and fractional monitor units. The target dose homogeneity was calculated as per the international commission on Radiation Unit and measurements (ICRU) report 83,using the following equation: HI=D2%-D98%D50%×100 (1) Where D2%, D98% and D50% were dose received by 2%, 98% and 50% of PTV_HFS volume respectively. If the HI value is lesser, then the plan provides a more homogeneous distribution to PTV_HFS. V103% and V105% of the target were evaluated using a Dose-Volume histogram (DVH). Beam-on time was noted for each of the HFS plan with gantry period and arc rotation. The minimum beam-on time provides a lesser monitor unit with lesser patient setup uncertainty. The goal of this plan selection process is the minimum beam-on time with acceptable target inhomogeneity, V103%, and V105% Using the x-y scatter plot. The pitch and modulation factor was kept in the x-axis and the t homogeneity was kept in Y-axis. The quadratic equation is created by choosing the feasible pitch and modulation factor using the plan number and minimum HI. The same process was used for beam-on time, V103% and V105%. In this evaluation process, the spare lung volume mean dose was kept at less than 6.5Gy for all the plans). From above method, the suitable pitch and MF were calculated for each one of the scatter plots. Finally, the suitable pitch and MF were selected as per minimum BOT at appropriate clinical needs. By using this pitch and MF, the final plan was optimized for treatment delivery verification with the same other planning parameters. Once the deliverable plan was verified, the quality assurance of the above plan was processed to verify the delivery uncertainty. Delivery verification and Quality Assurance The Quality Assurance (QA) of the final deliverable plan was split into two parts. 1. Point dose verification with cylindrical Tomotherapy Phantom (Cheese Phantom, Med-Cal Inc, Verona, WI, USA) for all the patients. 2. Surface dose measurements on anthropomorphic phantom using radiochromic film. Each of the deliverable final plans for all the patients were transferred to cheese phantom® with the same planning parameters. Three different points were chosen (head, thorax, and pelvis) to verify the TPS calculated dose on cheese phantom using 0.053 cm3 ionization chamber (A1SL, Exradin®, Standard Imaging, Middleton, WI, USA). By using the final pitch and MF, the dose plan was created in anthropomorphic phantom. In TBI, as the total body is the target, patient positioning in one area may not provide the cumulative patient setup and if the target (patient body) deviates from its position, even by a millimeter, there may be a dosimetric deviation from the planned dose. For avoiding the above uncertainty, the skin flash should be used in TBI treatment plans. In our study, we a created 2 cm flash margin from the PTV and assigned as a target volume with dose constraints. This was verified with films and chamber. Radiochromic films and ionization chambers were used to measure the delivered dose on anthropomorphic phantom with various points for the head-neck (Vertex, Forehead, Right Buccal mucosa, Left Buccal Mucosa, Sternum) and Pelvis regions (Superior, inferior, left, right and Pubis). Radiochromic films are placed in the surface of the anthropomorphic phantom to verify the skin doses. Apart from these measurements, the flash margin validation and its importance were verified with anthropomorphic phantom using radiochromic films and cylindrical ionization chamber (Figure 3a-c). The anthropomorphic phantom (both Head&Neck and Pelvis) was scanned in CT scanner with the slice thickness of 3mm and the PTV was generated to mimic the TBI. Two type of dose plans were created with and without flash margin (2cm from PTV). The customized bolus which has the dimension of 8 cm (length) x 9cm (width) x 2cm (height) has been attached in the head&neck and pelvis phantom during the dosimetric measurements (for both the plans) to verify the effect of flash margin. The pre-measurement image was acquired using Mega-Voltage on-board CT (TrueCT®) images and it was co-registered with reference planned CT to verify the setup uncertainty. Both the plans were executed to anthropomorphic phantom with attached customized bolus. Statistical Analysis Plan selection process was made using the scatter plot and regression methodology (using the quadratic equation) as mentioned earlier. The comparison between dosimetric objectives and achieved dose was done using two tailed Student’s T-test using IBM SPSS Statistics for Windows, version 24 (IBM Corp., Armonk, N. Y., USA). The significance level was set at 0.05. Results For analyzing the HI with respect to MF and pitch, the homogeneity decreased (HI increased) on increasing pitch and MF (Figure 4a). A minimal increase in HI was observed when the pitch value was increased from 0.2 to 0.3 but increasing the pitch value beyond 0.3 resulted in more inhomogeneous plans with respect to MF. For a constant pitch value, the BOT increased with increasing MF. A sharp decrease of BOT was observed when increasing the pitch and its corresponding MF (Figure 4b). The V103% of PTV increased with pitch and constant MF (Figure 4c) but for the constant pitch value, the increasing MF provided a lesser V103%. The above pattern was observed for V105% and V107% also (Figure 4d, 4e). From the above analysis, the optimal pitch and MF was evaluated, and the quadratic equation was plotted to find the optimal pitch and MF for V103%, V105%, and V107% without increasing the BOT and HI. The optimal pitch and MF value were 0.287 and 2.4 respectively. Based on this pitch and MF, the treatment plans were generated for all patients. The dosimetric outcomes were compared with other pitch and MF values. The minimum BOT was found when the pitch was 0.45 and MF was 2.0 (Table 3). But the dose inhomogeneity was increasing at the level of 20% and this inhomogeneity was not acceptable for treatment. Based on optimal pitch and MF, the mean BOT was 1692 seconds with optimal inhomogeneity (7.4% %). The planning aim and achieved doses were compared in HT. For PTV_HFS, D95% and D98% were 97.09% (range: 94.7-99.6%, SD=±1.77% and p=0.002) and 93.9% (range:91.5-94.4%, SD=±1.7%, p=0.007) respectively, and mean D2% was within 107% with SD of ±1.22% (p=0.04). The mean of PTV receiving V103%, V105%, and V107% was 24.48% (range=7.7-36.6%, SD=±10.44, p=0.03), 5.76% (range=1.4-12.1%, SD=±3.3%, p=0.005), 1.93% (range=0.1-4.6%, SD=±1.19%, p=0.008) respectively and all the values were within the planning constraints. The mean dose to the combined lung was 8.62 Gy (range=8.4-8.7 Gy, p=0.01) for 12 Gy prescription and 5.74Gy (range=5.2-6.2Gy, p=0.04) for an 8 Gy prescription (Table 2). Dose to eyes and lenses were within the planning aim. The plan specific QA for all the dosimetric plans were within 2% from TPS calculated dose. The measured dose using the ionization chambers with flash margin plans shown closer to 2 Gy but no-flash plans were provided the less than 2 Gy in all points. The mean deviation between these flash and no-flash margin was 26.18% and 26.66% for head-neck and pelvis respectively. In skin dose measurements using the EBT3 films, the average difference between flash and no-flash plans were 0.9 % and 0.43% for head-neck and pelvis plans respectively in anthropomorphic phantom (Table 4). Table 1 Patient Demographics, PTV Volumes, Prescription Dose and Lung Volume Patient No # Age (Years) Sex Diagnosis Height (cm) Max Width (cm) PTV (HFS) Volume (cm3) PTV Total Volume (cm3) PTV+2cm (HFS) Volume (cm3) Prescribed DosePD for Total Body (Gy) Total fractions HFS FFS 1 13 M ALL 100 62 36 25,506 30,835 41,736 12 6 2 18 M ALL 111 66 44 42,415 52,895 66,545 8 8 3 40 F ALL 105 62 44.5 43,187 49,836 63,153 13.2 6 4 22 M ALL 116 72 59 70,524 110,688 92,445 8 8 5 21 M ALL 109 73 45 40,515 46,663 62,199 8 8 6 39 M ALL 109 69 53 68,843 80,191 95,517 8 8 7 24 F ALL 107 64 42 32,929 39,679 53,778 12 6 8 24 M CML 109 67 50 50,945 68,605 81,264 8 8 9 19 M ALL 121 40 44.1 39,273 47,071 62,576 8 8 10 32 F MS 102  52 39.4 33,021 41,904 50,534 8 8 ALL, Acute Lymphoblastic Leukemia; MS, Myeloid sarcomaTable 1 Patient demographics; PTV volumes, prescription dose and lung volume; ALL, Acute Lymphoblastic Leukemia; MS, Myeloid sarcoma; CML, Chronic Myeloid Leukemia Figure 1 Target and OAR Delineation for TBI_HFS scan. Figure 1 a. axial section shows the sparing Lung volume and margin taken for covering the rib without underdose. Figure 2 b and 2 c shows the 2cm flash margin throughout body to take care the setup uncertainty and 7 dummy strips with 1cm thickness in the inferior direction used for creating step wedge for an uniform dose at the junction Figure 2 Dose Planning of HFS and FFS and Its Summation was Shown. 2a. PTV_HFS plan was shown with step wedge dose distribution in inferior part 2b. PTV_FFS plan shows step wedge dose distribution in superior part 2c. Summation of HFS and FFS plan with uniform dose distribution at junction Table 2 Planning Aim and Achieved Dose for the Target and Organ at Risks Structures Planning Aim Achieved (mean, range, SD) p PTV D 95% >95% 97.09, 94.7-99.6, 1.77 0.002 D98% >90% 93.9, 91.5-94.4, 1.70 0.007 D2 <107% 106.82, 104.8-109.2, 1.22 0.04 V 103 <30% 24.48, 7.7-36.6, 10.44 0.03 V 105 <10% 5.76, 1.4-12.1, 3.30 0.005 V 107 < 2% 1.93, 0.1-4.6, 1.19 0.008 Combined Lung D mean <8.5 Gy (for 12 Gy PD) 8.62, 8.4-8.7, 0.2 0.01 <6.5Gy (for 8Gy PD) 5.74, 5.2-6.2, 0.34 0.04 Eyes D mean ≤100% 100.46, 89-103, 4.17 0.04 Eye Lens D mean ≤100% 98.08, 70-102.5, 10.06 0.047 Figure 3 a, Anthropomorphic head-neck phantom with and gaffchromic film at 5 position (Vertex, Rt Buccal mucosa, Lt Buccal mucosa, sternum); b, Anthropomorphic head-neck phantom with chamber insertion in wax bolus; c, Anthropomorphic pelvis phantom with chamber insertion gaffchromic film at 8 positions (Superior, Superior-Left, Superior-Right, left, right, left thigh and right thigh) Table 3 Pitch, MF and Beam on Time (Mean) for the PTV_HFS Plan. The Beam on time for optimal pitch and MF was also shown Pitch MF Beam on time (Sec) p Pitch MF Beam on time (Sec) p Pitch MF Beam on time (Sec) p 0.2 2 2,354 0.25 2 1,883 0 0.3 2 1,569 2.5 2,354 0 2.5 1,883 2.5 1,583 0 3 2,354 3 1,935 3 1,715 3.5 2,382 3.5 2,045 3.5 1,845 4 2,484 4 2,176 4 1,982 0.35 2 1,376 0.148 0.4 2 1,214 0.45 2 1,138 2.5 1,453 2.5 1,335 0.149 2.5 1,268 3 1,589 3 1,465 3 1,414 0 3.5 1,722 3.5 1,610 3.5 1,606 4 1,879 4 1,801 4 1,722 0.5 2 1,087 2.5 1,222 0.287 2.4 1,692 3 1,395 0 3.5 1,648 4 1,834 Figure 4a Pitch and MF Versus Homogeneity Index; Increasing the Pitch (0.2 to 0.5) with MF (2 to 4) could Increase the Inhomogeneity Figure 4b Pitch and MF Versus Beam on Time; Increasing the Pitch (0.2 to 0.5) with MF (2 to 4) could Decrease the Beam on Time Figure 4c Pitch and MF Versus V103% Received by PTV_HFS; increasing the pitch (0.2 to 0.5) with MF (2 to 4) could increase the V103% of PTV_HFS Figure 4d Pitch and MF Versus V105% Received by PTV_HFS; increasing the pitch (0.2 to 0.5) with MF (2 to 4) could increase the V105% of PTV_HFS Figure 4e Pitch and MF Versus V107% Received by PTV_HFS; increasing the pitch (0.2 to 0.5) with MF (2 to 4) could increase the V107% of PTV_HFS Figure 5 PTV Received by 90%, 95% and 120% Isodoses were Shown for the PTV HFS Plan. 5a. Dose distribution for the Pitch value of 0.2 and with MF 2.0 ; 5b. Dose distribution for the Pitch value of 0.287 and with MF 2.4 ; 5c. Dose distribution for the Pitch value of 0.5 and with MF 4.0 Table 4 Chamber and Film Verification of Flash Margin and Skin Dose. The use of flash margin was verified with cyinderical ionization chamber with customized wax slab and the skin dose was verified with Gaffchromic film in anthropomorphic phantom Chamber/Film Position HN-Flash HN-Flash Skin dose Film Position Pelvis-Flash Pelvis-Flash Skin dose Flash and Without Flash Chamber measurement Flash and Without Flash Film measurement Flash and Without Flash Chamber measurement Flash and Without Flash Film measurement Flash (Gy) No Flash (Gy) % Diff Flash (Gy) No Flash (Gy) % Diff Flash (Gy) No Flash (Gy) % Diff Flash (Gy) No Flash (Gy) % Diff Scalp 1.99 1.45 27.14 2.05 1.98 3.41 Superior-Up 1.74 1.4 19.95 1.94 1.92 0.85 Vertex 2.11 1.55 26.54 2.11 2.05 2.84 Superior left 2.14 1.54 28.22 2.08 2.06 0.87 Chin 2.09 1.39 33.49 1.98 1.99 -0.51 Superior-right 1.73 1.47 14.88 1.95 1.91 2.14 Rt BM 1.94 1.48 23.71 1.88 2.01 -6.91 Inferior- Left 2.18 1.39 36.46 2.03 2.07 -1.9 Lt BM 2.08 1.55 25.48 2.08 2.05 1.44 Inferior- Right 2.27 1.5 33.82 2.09 2.07 1.04 Rt Shoulder 1.96 1.52 22.45 1.98 2.11 -6.57 Pubis 2.11 1.4 33.65 1.96 1.94 1.09 Lt Shoulder 1.94 1.39 21.65 2.05 2.04 -2.93 Rt Thigh 2.08 1.52 26.92 1.71 1.82 -6.17 Sternum 2.07 1.47 28.99 2.01 1.97 1.99 Lt Thigh 2.01 1.62 19.4 1.88 1.91 -1.33 Discussion Hui et al., (2005) reported the dose homogeneity was worsened by 2% with every 10% change in the pitch from 0.46 to 0.556, and the higher pitch values impacted the average dose to the critical structures. The above effect was obtained in our study also and increasing the pitch value increased the HI approximately by 10% (more inhomogeneity) on increasing the pitch from 0.3 to 0.5, independently of MF. The V103 gradually increased from 35% to 65% when the pitch is increased from 0.35 to 0.5, again independent of MF. The same study (Hui et al., 2005), also showed that with increase in MF from 2 to 2.5, the homogeneity was slightly greater (DHI decreased 2%). In our results, the HI was increased (more inhomogeneous) mainly with pitch value. The MF value has lesser impact when comparing the pitch values in view of dose homogeneity but the particular pitch value with increasing MF could reduce the inhomogeneous at the edges of the body. Standard TBI protocols recommend the median lung dose to be less than 10Gy, and lung toxicity is minimal if the lung doses are kept less than 8Gy (Buchstab et al., 2020). In our study, patients planned for 12 Gy and 8Gy prescription dose received mean lung doses less than 8.6 Gy and 5.7Gy, respectively. For analyzing the lung dose with change in pitch and MF, the deviation in lung dose was minimal but the dose coverage at the rib region was less (cold spot) when the pitch values was higher. To reduce the lung dose, the central lung region was contoured separately and assigned as complete block (full block), which helped provide a uniform gradient from rib to central lung. We observed that this type of planning could provide a lesser mean lung dose when comparing with normal planning methodology. Takahashi et al., (2013) described about thread effect and peripheral dose heterogeneity for total marrow irradiation in HT. In this study, they found that pitch value of 0.2 improves the target homogeneity arms for extremely large body width. But in our study, the pitch value was kept as 0.86/n (n=3, pitch=0.287) for all patient plans, but the MF has been deduced from the HI and from analysis of other dosimetric parameters. We found that the homogeneity varies at the left and right arm, not only with pitch value but also with MF. For correlation between homogeneity and plan analysis, we chose 2.4 as the optimal pitch value, and the same could provide a suitable HI and treatment time. Figure 4 shows the dose distribution of three different pitch and MF values (Plan a= pitch 0.2 and MF 2.0 (Figure 5a), Plan b=Pitch 0.287 and MF 2.4 (Figure 5b) and Plan c= pitch 0.5 and MF 4.0 (Figure 5c). The homogeneity and dose coverage for both the arms are superior in plan a, but the treatment time is marginally high (2354 seconds) and in plan c, the treatment time is lesser, but the homogeneity and coverage are sub-optimal. In plan b, the homogeneity and coverage are optimal with suitable treatment time (1692 seconds) (Table 3). In a previous study (Takahashi et al., 2013), the authors found that the thread effect was minimal in the central region if the MF was used higher. In our findings, the higher MF could provide the high homogeneity, but it could increase the treatment time. The radiochromic EBT films have been used by several authors to measure the superficial skin dose in tomotherapy (Hardcastle et al., 2008, Ramsey et al., 2007). Avanzo et al., (2013) studied the skin dose in HT treatments and found that radiochromic EBT films are suitable for surface dose measurements, but the TPS overestimates the skin dose when comparing with the measured dose. Uncertainty in the dose delivered to the patient ranged from 1% to 25% or overdosage of up to 10% when there was a positioning or volume uncertainty in the target volume (Avanzo et al., 2013). The skin flashes can be used to avoid this uncertainty which increases in the target volume closer to the surface. Our measurements show that the flash margin did not increase the skin dose (Table 4), and at the same time, it could provide adequate coverage to the target if the target deviated from its position. In conclusion, the overall planning workflow for TBI in HT with its dosimetric verification was analyzed and the optimal combination of pitch and MF was deduced, using homogeneity index, beam-on time and dosimetric parameters. In our study, the optimal combination of pitch value of 0.287 and MF value of 2.4 provided acceptable plans for all patients planned for TBI plan in HT. The flash margin can provide adequate coverage during patient position uncertainty without increasing the skin dose. Author Contribution Statement All authors reviewed the data, results and approved the manuscript. Study design, data collection, statistical analysis, results verification and manuscript draft. Acknowledgements General The first author thanks to Mr Christy Alekchander, Chief Medical Physicist, Patel Hospital, Jalandar, Punjab, India for his technical support. Data Availability The data of this study are available on request. Ethical Declaration Ethical approval is not required for this study as this is a dosimetric study not used for patients’ treatments. Conflicts of Interest The authors declare no conflict of interest. ==== Refs References Avanzo M Drigo A Ren Kaiser S Dose to the skin in helical tomotherapy: results of in vivo measurements with radiochromic films Phys Med 2013 29 304 11 22575703 Binny D Lancaster CM Harris S Effects of changing modulation and pitch parameters on tomotherapy delivery quality assurance plans J Appl Clin Med Phys 2015 16 87 105 26699293 Boyd R Jeong K Tomé WA Determining efficient helical IMRT modulation factor from the MLC leaf-open time distribution on precision treatment planning system J Appl Clin Med Phys 2019 20 64 74 30957967 Buchstab WT Leitzen C Schmeel LC Total body irradiation: Significant dose sparing of lung tissue achievable by helical tomotherapy Med Phys 2020 30 17 23 Cleuziou JP Desgranges C Henry I Total body irradiation using helical tomotherapy: Set-up experience and in-vivo dosimetric evaluation Cancer Radiother 2021 25 213 21 33402290 Fenwick J Tome W Jaradat HA Quality assurance of a helical tomotherapy machine Phys Med Biol 2004 49 2933 53 15285257 Gruen A Ebell W Wlodarczyk W Total Body Irradiation (TBI) using Helical Tomotherapy in children and young adults undergoing stem cell transplantation Radiat Oncol 2013 8 92 23587349 Halperin E Wazer D Perez C Brader L Principles and Practice of Radiation Oncology 6th Edition 2013 Philadelphia Lippincott Williams & Wilkins Hardcastle N Soisson E Metcalfe P Rosenfeld AB Tome WA Dosimetric verification of helical tomotherapy for total scalp irradiation Med Phys 2008 35 5061 8 19070240 Hui SK Kapatoes J Fowler J Feasibility study of helical tomotherapy for total body or total marrow irradiation Med Phys 2005 32 3214 24 16279075 Hodapp N Der ICRU-Report 83: The ICRU Report 83: prescribing, recording and reporting photon-beam intensity-modulated radiation therapy (IMRT) Strahlenther Onkol 2012 188 97 9 22234506 Kissick MW Fenwick J James JA The helical tomotherapy thread effect Med Phys 2005 32 1414 23 15984692 Langen KM Papanikolaou N Balog J QA for helical tomotherapy: Report of the AAPM Task Group 148a Med Phys 2010 37 4817 53 20964201 Mackie TR Olivera GH Kapatoes JM Helical Tomotherapy in Intensity-Modulates Radiation Therapy The State of the Art, AAPM Summer School Proceedings Med Phys Madison 2003 2003 247 84 Meyer P Bouhours H Dehaynin N The optimal tomotherapy treatment planning parameters for extremity soft tissue sarcomas Phys Med 2015 31 542 52 26032005 Poonsombudlert K Limpruttidham N Total Body Irradiation and Risk of Diabetes Mellitus; A Meta-Analysis Asian Pac J Cancer Prev 2019 20 885 91 30912408 Peñagarícano JA Chao M Van Rhee F Clinical feasibility of TBI with helical tomotherapy Bone Marrow Transplant 2011 46 929 35 20935684 Ramsey CR Seibert RM Robison B Mitchell M Helical tomotherapy superficial dose measurements Med Phys 2007 34 3286 93 17879792 Ryczkowski A Piotrowski T Influence of the modulation factor on the treatment plan quality and execution time in Tomotherapy in head and neck cancer: In-phantom study J Cancer Res Ther 2013 9 618 23 24518706 Salz H Bohrisch B Howitz S TG Intensity-modulated Total Body Irradiation (TBI) with TomoDirect™. Radiat Oncol 2015 6 58 Sarradin V Simon L Huynh A Total body irradiation using Helical Tomotherapy®: Treatment technique, dosimetric results and initial clinical experience Cancer Radiother 2018 22 17 24 29395854 Seung S Larson D Galvin J American College of Radiology (ACR) and American Society for Radiation Oncology (ASTRO) Practice Guideline for the Performance of Stereotactic Radiosurgery (SRS) Am J Clin Oncol 2013 36 310 5 23681017 Skórska M Piotrowski T Optimization of treatment planning parameters used in tomotherapy for prostate cancer patients Phys Med 2013 29 273 85 22521735 Takahashi Y Verneris MR Dusenbery KE Peripheral dose heterogeneity due to the thread effect in total marrow irradiation with helical tomotherapy Int J Radiat Oncol Biol Phys 2013 87 832 9 24011657 Van Dyk J Galvin JM Glasgow GP Podgorsak EB AAPM Report No 17: the Physical Aspects of Total and Half Body Photon Irradiation Radiation therapy committee task group 29 American Association of Physicists in Medicine by the American institute of Physics 1986 New York Wong JYC Filippi AR Dabaja BS Total Body Irradiation: Guidelines from the International Lymphoma Radiation Oncology Group (ILROG) Int J Radiat Oncol Biol Phys 2018 101 521 9 29893272 Zarghani G Geraily T Hadisinia Comparison of different TBI techniques in terms of dose homogeneity – review study Cancer/Radiothérapie 2021 25 380 9 33431295

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116151 10.31557/APJCP.2023.24.4.1289 Research Article Saudi Nursing Students’ Knowledge and Perception of Testicular Cancer Assessment and Management: A Cross-Sectional Study Saleh Zyad T 1 Elshatarat Rami A 2 Almarwani Abdulaziz Mofdy 3 Alahmadi Hanadi A 4 Elneblawi Nora H 2 Al-Za’areer Majed S 45 Alhujaili Abdullah D 2 Saleh Ahmad M 6* Abdel-Aziz Hassanat R 6 Al-Sayaghi Khaled M 27 1 Department of Clinical Nursing, School of Nursing, The University of Jordan, Amman, Jordan. 2 Department of Medical and Surgical Nursing, College of Nursing, Taibah University, Madinah, Saudi Arabia. 3 Department of Psychiatric Nursing, College of Nursing, Taibah University, Madinah, Saudi Arabia. 4 College of Health Science and Nursing, Al- Rayan Colleges, Madinah, Saudi Arabia. 5 Faculty of Medicine, University Sultan Zainal Abidin (UniSZA), Kuala Terengganu, Malaysia. 6 College of Applied Medical Sciences, Prince Sattam bin Abdulaziz University, AlKharj, Saudi Arabia. 7 Nursing Division, Faculty of Medicine and Health Sciences, Sana’a University, Sana’a, Yemen. * For Correspondence: am.saleh@psau.edu.sa 2023 24 4 12891295 6 12 2022 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Introduction: Testicular cancer (TC) incidence is increasing worldwide. This study aimed to investigate Saudi nursing students’ knowledge and perception about TC. Method: This cross-sectional study was done using convenience sampling method. In this study, 280 nursing students from different nursing schools in six cities of Saudi Arabia were recruited. A structured self-report questionnaire was used to collect data. Result: About 49.2% of the participants received education about TC in their nursing schools. The findings showed lack of enough knowledge about TC among Saudi nursing students. Mostly, the participants reported that heredity factor and having family history of TC (48.9%) and age between 56 and 70 years (41.8%) were the most common risk factors of TC. According to the participants, physical examination was the most common diagnostic test usually used for early detection of TC (40.4%) and biopsy test was the most accurate test to confirm TC diagnosis (45.4%). Only one third of the participants (34.6%) knew that between 75% and 100% of TC cases can be cured in case of early detection. About half of the participants (51.8%) reported that surgical procedure was the most common treatment for TC. The nursing students who had high GPA (r=0.86, p<0.001), were unwilling to get more information on TC (r=0.24, p=0.04), had family history of TC (r= 0.53, p=0.02), medical problems with testicles (r= 0.69, p=0.01), received education about TC in their school of nursing (r=0.65, p=0.02), and were more self-confident in assessing and managing TC (r=0.38, p=0.03) had higher level knowledge about TC. Conclusion: Despite the importance of nurses’ roles in assessing and managing TC, nursing students in Saudi Arabia still did not have enough knowledge about TC. Improving nursing programs’ curricula and conducting health education programs are recommended. Key Words Testicular cancer nursing students nursing students’ knowledge Saudi Arabia ==== Body pmcIntroduction Testicle as a male sexual organ is responsible for the production of testosterone as well as production and storage of semen. Testicular cancer (TC) is defined as an abnormal growth of cells in one or both testicles (Albers et al., 2015; Baird et al., 2018). Fortunately, TC is a rare malignant cancer accounting for about 1% of all males’ cancers and has an excellent cure rate. In addition, the mortality rate of patients with TC is very low. In other words, The 5-year survival rate for TC patients is 95% (Albers et al., 2015; Farmanfarma et al., 2018; Gilligan et al., 2019). However, the recent statistics have shown that TC incidence is increasing worldwide (Farmanfarma et al., 2018; Gilligan et al., 2019). TC mostly affects young adults aged between 15 and 34 years old (Albers et al., 2015; Farmanfarma et al., 2018). Nevertheless, TC diagnosis can be distressing for the patient and his family. Literature documented that most cases of TC are discovered by testicular self-examination (TSE). According to previous studies, the chance of TC treatment is very high even in cases where it is detected at an advanced stages (Albers et al., 2015; Baird et al., 2018). Nevertheless, there is not enough knowledge about TC and TSE among men in Arab communities, including Saudi Arabia and Bahrin (Abd Algany et al., 2015; Alamri et al., 2021; Alaradi et al., 2020; Saab et al., 2016; Salati et al.) and even in developed countries (Albers et al., 2015; Farmanfarma et al., 2018; Gilligan et al., 2019; Ugboma et al., 2011). The importance of improving young males’ knowledge and awareness about TC prevalence, TC symptoms, and how to perform TSE is documented in previous studies (Aksoy et al., 2022; Albers et al., 2015; Marks, 2017; Saab et al., 2016; Ugboma et al., 2011; Wanzer et al., 2014). Health professionals, particularly nurse educators, play major roles in providing health education and counseling people in public about TC and the importance of doing TSE as a method for detecting and preventing TC. Moreover, bedside nurses, who are directly assigned for taking care of patients with TC, have an important role in assessing, managing, and providing care to these patients (Akers, 2018; Albers et al., 2015; Saab et al., 2016). To fulfill these roles efficiently, nurses have to be knowledgeable and competent about assessment and management of TC. However, there is few studies investigating nursing staff and students’ knowledge about TC and TSE in Middle East countries, specifically in Saudi Arabia (Ahmed et al., 2019; Akca et al., 2021; Alaradi et al., 2020). Nursing schools can play an efficient role in enhancing nurses’ knowledge and awareness of TC and TSE. Moreover, nursing schools have major roles in preparing nursing students to carry out their nursing responsibility in providing health education about TC and TSE and to do health assessment and management as well as take care of patients with TC. To the best of our knowledge, no study has yet investigated nursing students’ knowledge and awareness about TC in Saudi Arabia. Therefore, this study was conducted to assess knowledge and perceptions of nursing students from different Saudi regions about TC. The findings of this study may contribute health professionals and educators in developing guidelines for preventing TC and provide baseline information for further clinical research to discover the effect of health education programs on improving nurses’ knowledge about TC in Saudi Arabia and other Arab countries. Study objectives This study aimed to identify: 1) the source of nursing students’ knowledge and their willingness to participate in educational programs about TC, 2) the nursing students’ knowledge about TC, 3) the nursing students’ perceptions about TC, and 4) factors affecting knowledge and perception about TC Materials and Methods Design This correlational cross-sectional study was conducted to describe Saudi nursing students’ knowledge and perceptions about TC. Sample and Setting The convenience sampling method was used to select 280 male nursing students from 6 different cities in Saudi Arabia. Nursing students were recruited from Al-Rayan Colleges, Medina city, (N= 73 students) and six campuses of nursing colleges at Al-Ghad International Colleges for Applied Medical Sciences (N= 207 students), which are located in Riyadh (N= 40 students), Medina (N= 37 students), Dammam (N= 31 students), Jeddah (N= 30 students), Qassim (N= 38 students), and Tabuk (N= 31 students) cities in Saudi Arabia. The inclusion criteria were being a full time male nursing student, successfully completed physical examination course and three courses of medical-surgical nursing, and being able to understand English language. Any nursing student who previously was diagnosed with TC was excluded from the study. G * Power was used to calculate the effective sample size. The researchers considered power of 0.80, moderate effective size of 0.30, and alpha value of 0.05 to conduct correlation analysis. The effective sample size was estimated as 268 students. The researchers distributed 300 questionnaires among the recruited participants. A total of 280 questionnaires were fully completed by the participants. Data Collection Procedure and Ethical Considerations Before conducting this study, formal approval was obtained from the Institutional Review Board (IRB) at Al-Ghad International Colleges. To identify the potential eligible participants, the researchers obtained the names of eligible students and their class timetables from the heads of nursing departments. Then, the researchers met the eligible nursing students in the nursing colleges’ classrooms at the selected settings. The researchers explained the objectives of the study to the eligible participants and invited them to participate in the study. All nursing students who accepted to participate in the study signed written consent form. The participants were informed that their participation is voluntary and their personal information would be confidential and anonymous. The researchers collected the data directly through structured self-report questionnaire. No physical measurement or educational interventions were provided. Completing the questionnaire took about 15 minutes. Instruments The researchers used structured self-report questionnaire, which extracted data about respondents’1) demographic and academic achievement information, 2) source of information about TC and willingness to improve knowledge about TC, 3) health status and possible risk factor to develop TC, and 4) knowledge and perception about TC. Demographic information included respondents’ marital status, accumulated Grade Point Average (GPA), and level of education in nursing program. To evaluate the participants’ health, they were asked to report their history of chronic disease and rate their health status using 5-point Likert scale (1=poor, 2=fair, 3=good, 4=very good, and 5=excellent). In addition, they were asked about history of using tobacco and having medical problems with testicles and family history of TC. The participants were also asked about their source of information about TC, willingness to get more information on TC, and self-confidence in their knowledge of assessing and managing TC. The participants’ response about self-confidence in their knowledge ranged between 0 (no self-confidence) and 10 (highest self-confidence). To determine participants’ knowledge level about TC, the researchers used ten multiple choice questions adopted from previous studies (Ahmed et al., 2019; Alamri et al., 2021; Avci et al., 2018; Baird et al., 2018). Specifically, these questions are developed to examine the knowledge about the prevalence, risk factors, common diagnostic test of TC, and TC treatment Furthermore, four-point Likert scale, with these responses (1=strongly disagree, 2=disagree to some extent, 3=agree to some extent, and 4=strongly agree), was used to identify the participants’ perception about TC. Higher scores indicate higher perception of TC. Data Analysis SPSS (version 25) was used for statistical analysis. Descriptive statistics and frequencies were used to analysis participants’ demographic information, self-rated health status, as well as knowledge and perception about TC. Correlation analyses were used to identify the bivariate associations between participants’ knowledge about TC and participants’ perception of TC variables and students’ GPA, level of education in nursing program, health status, source of information about TC, and self-confidence in their knowledge of assessing and managing TC. Results Participants’ demographic information and health status Table 1 shows that majority of the participants (86.4%) were single and in third year of nursing program (44%). The mean of cumulative GPA was 3.42±0.39 out of 5. Most of the participants rated their health status as excellent (76.1%) and reported that they had no chronic disease (90.7%). Approximately, one-third of the participants were current tobacco users. Only 6.4% of the participants had family history of TC and 2.9% experienced medical problem with testicles. Source of participants’ knowledge of TC and their willingness to improve their knowledge about TC Most of the participants were aware of TC (89.6%). Approximately, 64% of the participants learned about TC. About half of the participants (49.2%) did not receive information about assessment and management of TC during their study as nursing students. Two-thirds of the participants were willing to participate in educational programs about TC. Most of the participants (84.6%) preferred to take courses (as source of knowledge) to improve their knowledge about TC in the future. Based on our findings, mean participants’ self-confidence in knowledge of assessing and managing TC was 5.3±1.7 out of 10 (Table 2). Participants knowledge about TC One third of the participants reported that the prevalence of TC was between 10% and 19.9% among all male cancers. Mostly, the participants reported that heredity factor and having family history of TC (48.9%) and age between 56 and 70 years (41.8%) were the most common risk factors of TC. According to the participants, physical examination was the most common diagnostic test usually used for early detection of TC (40.4%) and biopsy test was the most accurate test to confirm TC diagnosis (45.4%). Most of the participants believed that pain or discomfort in the testicle or scrotum (32.9%) was the earliest symptom of TC. Only one third of the participants (34.6%) knew that between 75% and 100% of TC cases can be cured in case of early detection. About half of the participants (51.8%) reported that surgical procedure was the most common treatment for TC (Table 3). Participants’ perception about TC Mostly, the participants agreed or strongly agreed that family history of TC (80%) and being black and from Asian countries (61.8%) were risk factors to get TC. The participants believed that a man might have TC without symptoms (62.5%), TC can develop in one or both testicles (86.1%), TC causes infertility (70.7%), and TC can be prevented (75.7%). On the other hand, most of the participants disagreed or strongly disagree that self-examination for TC is needed only when the person has symptoms (69.3%), testicular tumors are metastatic (70.7%), TC can lead to serious illness or death (75.4%), there is no cure for TC (78.2%), and doing TSE is enough to detect TC (62.9%) (Table 4). Correlated factors of participants’ knowledge and perception about TC The findings revealed significant associations between participants’ knowledge about TC and GPA (r=0.86, p<0.001), level of education in nursing program (r=0.43, p=0.03), studying in Al-Rayan Colleges (r=0.43, p=0.03), not willing to get more information on TC (r=0.24, p=0.04), family history of TC (r= 0.53, p=0.02), history of having medical problems with testicles (r= 0.69, p=0.01), received information about TC from nursing school (r=0.65, p=0.02), self-confidence to assess and manage TC (r=0.38, p=0.03), and having correct perception about TC (r=0.91, p<0.001). Moreover, significant associations were found between participants’ correct perception about TC and GPA (r=0.72, p<0.001), education in nursing program (r=0.51, p=0.02), studying at Al-Rayan Colleges (r=0.37, p=0.04), not willing to get more information on TC (r=0.31, p=0.04), family history of TC (r= 0.56, p=0.02), history of having medical problems with testicles (r= 0.64, p=0.01), received information about TC (r=0.62, p=0.02), and self-confidence to assess and manage TC (r=0.54, p=0.02) (Table 5). Table 1 Participants’ Characteristics and Health Information Variable n (%) Marital status Single 242 (86.4) Has been married 38 (13.6) Setting of nursing school Al-Rayan Colleges 73 (26.1) Al-Ghad International Colleges 207 (73.9) Level of education in nursing program Third year 123 (43.9) Fourth year 98 (35) Internship 59 (21.1) In general, how would you rate your health status Excellent 213 (76.1) Very good 31 (11.1) Good 27 (9.6) Fair 8 (2.9) Poor 1 (0.4) Have you been diagnosed with any chronic disease Yes 26 (9.3) No 254 (90.7) Risk factors Tobacco user (yes) 91 (32.5) Family history of TC (yes) 18 (6.4) History of medical problem with testicles Yes 8 (2.9) Mean (± SD) GPA 3.42 (±0.39) Table 2 Source of Participants’ Knowledge and Participants’ Willingness to Participate in Educational Programs about TC Variables n (%) Having heard of TC (yes) 251 (89.6) Have been learned about TC (yes) 179 (63.9) Does your knowledge about assessment and management of TC depend upon the information you have received from the school of nursing? (N=179) No, not at all 88 (49.2) Yes, to some extent 33 (18.4) Yes, to high extent 24 (13.4) Yes, at all 34 (19.0) Willing to participate in educational programs about TC Yes 189 (67.5) No 91 (32.5) Preferred source of knowledge to improve knowledge of TC assessment and management. * Professional education (collage of nursing) 237 (84.6) Seminar or/and workshop 79 (28.2) Conferences 21 (7.5) Internet or media (e.g. TV, radio) 64 (22.9) Friends and relative 35 (12.5) Participants’ self-confidence in their nowledge to assess and manage TC (Ranging between 0 and 10) Mean (± SD) 5.3 (±1.7) *, The participants may select one or more choices Table 3 Participants’ Knowledge about TC Variables n (%) 1. How common is TC in men? 1% - 4.9% of all male cancers 52 (18.6) 5% -9.9% of all male cancers 89 (31.8) 10%-19.9% of all male cancers 94 (33.6) 20%-25% of all male cancers 33 (11.8) > 25% of all male cancers 12 (4.3) 2. What is the most common risk factor of TC? Old age 53 (18.9) Never having children 15 (5.4) Non-descending testicle (cryptochidism) 43 (15.4) Heredity and family history of TC  137 (48.9) Testicular atrophy 21 (7.5) Twinship or abnormal semen parameters 11 (3.9) 3. At which age are men at the greatest risk of TC? 71 years or older 74 (26.4) Between 56 and 70 years 117 (41.8) Between 36 and 55 years 54 (19.3) Between 15 and 35 years 21 (7.5) < 15 years 14 (5) 4. Which of these diagnostic tests is the most common one for early detection of TC? Physical examination (including TSE) 113 (40.4) Scrotal ultrasound 52 (18.6) Computerized tomography (CT) scan 39 (13.9) Magnetic resonance image (MRI) 25 (8.9) Biopsy 10 (3.6) Blood tests or tumor markers 11 (3.9) 5. Which of these diagnostic tests is the most accurate one to confirm a diagnosis of TC? Physical examination 9 (3.2 ) Scrotal ultrasound 16 (5.7) Computerized tomography (CT) scan 34 (12.1) Magnetic resonance image (MRI) 63 (22.5) Biopsy procedure 127 (45.4) Blood tests or tumor markers 31 (11.1) 6. Which of these clinical manifestations is the most earliest sign or symptom of TC? Enlargement or hardening of the testicle 47 (16.8) Pain or discomfort in a testicle or in the scrotum 92 (32.9) A feeling of heaviness in the scrotum 78 (27.9) A dull ache in the lower abdomen, back or in the groin 24 (8.6) A sudden collection of fluid in the scrotum 25 (8.9) Dysuria, burning sense on urination, or hematuria 14 (5.0) 7. Which of these clinical manifestations is the most common sign or symptom of TC? Enlargement or hardening of the testicle 83 (29.6) Pain or discomfort in a testicle or in the scrotum 75 (26.3) A feeling of heaviness in the scrotum 38 (13.6) A dull ache in the lower abdomen, back or in the groin 36 (12.9) A sudden collection of fluid in the scrotum 30 (10.7) Dysuria, burning sense on urination, or hematuria 18 (6.4) Variables n (%) 8. What percentage of TC cases can be cured in case of early detection ? 0 – 9% 17 (6.1) 10-24% 53 (18.9) 25 – 49% 36 (12.9) 50 – 74% 77 (27.5) 75 – 100% 97 (34.6) 9. After treating TC, should the affected testis be removed ? Yes, always 65 (23.2) Yes, sometimes 38 (13.6) No, not at all 147 (52.5) Do not know 30 (10.7) 10. What is the most common TC treatment method? Surgery 145 (51.8) Chemotherapy 74 (26.4) Radiotherapy 44 (15.7) Others (e.g. biotherapy, herbal treatment, and dietary treatment) 17 (6.1) Table 4 Participants’ Perception about TC Variables n (%) 1. Men who have blood relatives with TC are more likely to get TC Strongly agree 82 (29.3) Agree to some extent 142 (50.7) Disagree to some extent 38 (13.6) Strongly disagree 18 (6.4) 2. Black and Asian men are at higher risk of TC than white men * Strongly agree 82 (29.3) Agree to some extent 91 (32.5) Disagree to some extent 57 (20.4) Strongly disagree 50 (17.9) 3. A man may have TC without symptoms Strongly agree 38 (13.6) Agree to some extent 137(48.9) Disagree to some extent 49 (17.5) Strongly disagree 56 (20.0) 4. Self-examination for TC is needed only when the person has symptoms * Strongly agree 24 (8.6) Agree to some extent 62 (22.1) Disagree to some extent 114 (40.7) Strongly disagree 80 (28.6) 5. TC can develop in one or both testicles Strongly agree 85 (30.4) Agree to some extent 156 (55.7) Disagree to some extent 27 (9.6) Strongly disagree 12 (4.3) 6. Testicular tumors are commonly metastatic (have the ability to spread to other organs such as the lungs and brain) * Strongly agree 39 (13.9) Agree to some extent 43 (15.7) Disagree to some extent 94 (33.6) Strongly disagree 104 (37.1) Variables n (%) 7. TC is commonly responsible for serious diseases or death * Strongly agree 23 (8.2) Agree to some extent 46 (16.4) Disagree to some extent 75 (26.8) Strongly disagree 136 (48.6) 8. If someone is diagnosed with TC, he may become infertile Strongly agree 83 (29.6) Agree to some extent 115 (41.1) Disagree to some extent 48 (17.1) Strongly disagree 34 (12.1) 9. TC can be prevented Strongly agree 129 (46.1) Agree to some extent 83 (29.6) Disagree to some extent 37 (13.2) Strongly disagree 31 (11.1) 10. There is no treatment for TC * Strongly agree 15 (5.4) Agree to some extent 46 (16.4) Disagree to some extent 125 (44.6) Strongly disagree 94 (33.6) 11. Doing testicular self examination is enough to diagnose TC * Strongly agree 37 (13.2) Agree to some extent 67 (23.9) Disagree to some extent 85 (30.4) Strongly disagree 91 (32.5) * The most correct answer on these items is “Strongly disagree.” So, these items were reversed coding as (4=strongly disagree), (3=disagree to some extent), (2=agree to some extent), and (1=strongly agree). Table 5 Correlated Factors of Participants’ Knowledge and Perception about TC Variables Students’ knowledge about TC Students’ perception about TC Correlation (r) p value Correlation (r) p value participants’ GPA 0.86 < 0.001 0.72 < 0.001 Setting of nursing school (Taibah University) 0.43 0.03 0.37 0.04 Level of education in nursing program 0.43 0.03 0.51 0.02 Willingness to get more information on TC (No) 0.24 0.04 0.31 0.04 Rate of participants ‘health status 0.14 0.28 0.25 0.16 Tobacco use (Yes) 0.18 0.09 0.23 0.08 Family history of TC (yes) 0.53 0.02 0.56 0.02 Medical problems with testicles (Yes) 0.69 0.01 0.64 0.01 Participants’ knowledge about TC depends upon the information that have received from the school of nursing 0.65 0.02 0.62 0.02 Participants’ self-confidence in their knowledge to assess and manage TC 0.38 0.03 0.54 0.02 Participants’ perception about TC 0.91 < 0.001 Discussion This study was conducted to identify source of nursing students’ knowledge about TC, and nursing students’ willingness to participate in educational programs about TC, nursing students’ knowledge and perceptions about TC, and factors affecting nursing students’ knowledge and perception about TC. The majority of participants reported that they had heard and learned about TC were willing to improve their knowledge of TC, specifically before graduation. Only about half of the participants were not educated about TC in their nursing school. In addition, it was found that the participates had moderate self-confidence in their knowledge to assess and manage TC (mean=5.3 out of 10), which was in line with previous studies (Ahmed et al., 2019; Akca et al., 2021; Saab et al., 2016). Generally, the findings suggested the importance of improving knowledge of nursing students about assessment and management of TC. Moreover, considering the findings of this study and previous studies, it is recommended to use a wide range of educational resources (e.g. social media, courses, conferences, and workshops) and develop nursing programs’ curricula on the assessment and management of TC and TSE in Saudi Arabia (Ahmed et al., 2019; Akca et al., 2021; Altunkurek, 2020; Marks, 2017; Saab et al., 2016). Literature review showed that TC commonly affects young men age between 15 and 35 years old. The prevalence of TC is 1% worldwide. However, recent statistics showed that TC incidence has increased globally. Heredity and family history of TC as well as cryptorchidism are the major risk factors of TC. The treatment rate of TC is very high (> 90%), particularly if it is detected or diagnosed in early stage. TSE is an important method for early detection of TC, and it is recommended to do TSE once a month during or after hot or warm shower (Baird et al., 2018; Cheng et al., 2018; Farmanfarma et al., 2018; Shanmugalingam et al., 2013). The findings of this study also showed that the participants had not enough knowledge about the characteristics, symptoms, risk factors, and management of TC. For example, about 81% of the participants overestimated the global prevalence of TC. Although non-descending testicle (cryptochidism) is considered as one of the common risk factor of TC (Cheng et al., 2018), only 15.4% of the participants noted it as a common risk factor of TC. Despite the act that all the nursing students participated in this study are considered as high risk age group to get TC, only about 7.5% of them were not aware that they fell within this age group. Literature showed the importance of physical examination for early detection of TC and biopsy test for the confirmation of TC (Baird et al., 2018); however, only about half of the participants were aware of these diagnostic tests. Moreover, it was found that the participants did not have enough knowledge about the treatment rate and the most common treatment methods of TC, which was in line with findings reported in previous studies (Ahmed et al., 2019; Akca et al., 2021; El Mezayen et al., 2019). Furthermore, the findings of this study showed incorrect perception and awareness about risk factors, detection, and management of TC. For example, only about two-thirds of the participants believed that if someone was diagnosed with TC, he may become infertile. Despite the fact that white men are 4 to 5 times more likely to have TC (Akers, 2018; Shanmugalingam et al., 2013), only about one third of the participants were strongly disagree or disagree that black and Asian men are exposed to higher risk of TC than white men. Moreover, more than three-quarters of the participants incorrectly perceived that TC cannot be preventable and it is not curable. These findings were consistent with the findings reported in several previous studies conducted on nurses and nursing students in different countries (Ahmed et al., 2019; Akca et al., 2021; El Mezayen et al., 2019; Saab et al., 2016). Although our participants were passed most of basic courses in nursing program, including physical examination and medical–surgical courses, the findings showed that only half of them learnt about TC, suggesting that not all nursing schools in Al-Ghad International Colleges include medical disorders related to male reproductive system malignancies (e.g. TC) in their nursing program’s curricula. According to the findings of this study, to reduce lack of knowledge about TC, it is recommended to cover medical disorders related to male reproductive system malignancies in all nursing programs’ curricula or at least teach this topic to male nursing students. Study strengths and limitations To the best of our knowledge, there is no study conducted on Saudi nursing students to assess their knowledge and perception about TC. The study findings can provide baseline information about nursing students’ knowledge about TC, and consequently can help nursing schools to develop more comprehensive nursing programs’ curricula. Despite these strengths, there were some limitations. Using cross-sectional design limited the identification of causality relationships among the studied variables. Using convenience sampling methods also limited generalization of the study findings to other nursing students in Saudi regions. In conclusion, despite the importance of nurses’ roles in educating the public about TC, which is important in early detection and prevention of TC, and in assessing, managing, and taking care of patients with TC, the finding of this study and previous literature showed lack of enough knowledge about TC among nursing students. Therefore, all nursing schools in Saudi Arabia are recommended to develop comprehensive nursing programs focusing on characteristics, symptoms, detection, assessment, and management of TC. Furthermore, to improve nursing students’ confidence and skills to efficiently fulfill nursing roles regarding health education, it is recommended to motivate nursing students to participate in small group discussion (e.g. in nursing oncology course), design and develop health educational materials about TC and TSE such as audio-video media and written materials (e.g. brochures), and conduct health education classes about TC and TSE for public. Author Contribution Statement All authors conceived and designed the study, conducted research, provided research materials. RAE and ZTS analyzed and interpreted data. All authors wrote initial and final draft of article, and provided logistic support. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript. Acknowledgements The authors would like to thank all nursing students who participated in this study. The authors thank the vice president for scientific research affairs of Al-Ghad International Colleges for Applied Medical Sciences to provide us the formal approval to conduct this study at the selected settings. This study is supported via funding from Prince Sattam Bin Abdulziz University project number (PSAU/2023/R/1444). Availability of data Data are available upon official request from the corresponding author Ethical approval Permission was obtained from the ethical research committee of Al-Ghad International Colleges (approval number: AGICAMS-19/328) Conflict of interest There is no conflict of interest to be declared. ==== Refs References Abd Algany MM Soliman SM Abdel-Raoof SEH Self-awareness of testicular cancer among Mansoura male university students Alexandria Sci Nurs J 2015 17 89 106 Ahmed S Ali LA Ahmed MA Testicular cancer preventive behavior among nursing males’ students: intervention guidelines Evid-Based. Nurs Res 2019 1 1 7 Akca SO Cengiz T Aydin D Evaluation of Training Program for Nursing Students on Testicular Cancer and Testicular Self-Examination Iran Red Crescent Med J 2021 23 Akers C Aetiology, clinical presentation and treatment of testicular cancer Nurs Stand 2018 32 50 Aksoy C Ihrig A Reimold P Better Knowledge about Testicular Cancer Might Improve the Rate of Testicular (Self-) Examination: A Survey among 1,025 Medical Students in Germany Urol Int 2022 106 1056 60 35901778 Alamri A Alqahtani YM Al-Mudhi MM Knowledge, Attitudes and Practice toward Testicular Cancer and Testicular Self-Examination among adolescents and young adults in Aseer region, Saudi Arabia Middle East J Fam Med 2021 19 Alaradi M Almuqamam A Awareness of testicular cancer and testicular self-examination among men in Bahrain Asian Pac J Cancer Care 2020 5 259 63 Albers P Albrecht W Algaba F Guidelines on testicular cancer: 2015 update Eur Urol 2015 68 1054 68 26297604 Altunkurek SZ Testicular cancer and the importance of early diagnosis Male Reproductive Health 2020 IntechOpen Avci IA Altinel B The validity and reliability of health belief scale for testicular cancer self-examination Am J Mens Health 2018 12 531 8 26483292 Baird D Meyers GJ Hu JS Testicular cancer: Diagnosis and treatment Am Fam Physician 2018 97 261 8 29671528 Cheng L Albers P Berney DM Testicular cancer Nat Rev Dis Primers 2018 4 1 24 29930242 El Mezayen SE Abd El-Hay SA Effect of educational guidelines based on health belief model regarding testicular cancer knowledge, practice and beliefs among male nursing students Clin Nurs Stud 2019 7 27 41 Farmanfarma KK Mahdavifar N Mohammadian-Hafshejani A Testicular cancer in the world: an epidemiological review J Canc Res 2018 5 1 5 Gilligan T Lin DW Aggarwal R Testicular cancer, version 2 2020, NCCN clinical practice guidelines in oncology J Natl Compr Canc Ne 2019 17 1529 54 Saab MM Landers M Hegarty J Testicular Cancer Awareness and Screening Practices: A Systematic Review Oncol Nurs Forum 2016 43 8 23 Shanmugalingam T Soultati A Chowdhury S Global incidence and outcome of testicular cancer Clin Epidemiol 2013 5 417 24204171 Ugboma HA Aburoma H Public awareness of testicular cancer and testicular self-examination in academic environments: a lost opportunity Clinics 2011 66 1125 8 21876962 Wanzer MB Foster SC Servoss T Educating young men about testicular cancer: support for a comprehensive testicular cancer campaign J Health Commun 2014 19 303 20 24117344

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116162 10.31557/APJCP.2023.24.4.1379 Research Article Artificial Intelligence Role in Subclassifying Cytology of Thyroid Follicular Neoplasm Aly Alabrak Mona Mohamed 1* Megahed Mohammad 2 Alkhouly Asmma Abdulaziz 2 Mohammed Ammar 2 Elfandy Habiba 1 Tahoun Neveen 1 Ismail Hoda Abdel-Raouf 1 1 Department of Pathology, National Cancer Institute, Cairo University, Egypt. 2 Department of computer science, Faculty of Graduate Studies for Statistical Research, Cairo University, Egypt. * For Correspondence: Mona.al-abrak@nci.cu.edu.eg 2023 24 4 13791387 31 12 2022 24 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: Fine needle aspiration cytology has higher sensitivity and predictive value for diagnosis of thyroid nodules than any other single diagnostic methods. In the Bethesda system for reporting thyroid, the category IV, encompasses both adenoma and carcinoma, but it is not possible to differentiate both lesions in the cytology practice and can be only differentiated after resection. In this work, we aim at exploring the ability of a convolutional neural network (CNN) model to sub-classifying cytological images of Bethesda category IV diagnosis into follicular adenoma and follicular carcinoma. Methods: We used a cohort of cytology cases n= 43 with extracted images n= 886 to train CNN model aiming to sub-classify follicular neoplasm (Bethesda category IV) into either follicular adenoma or follicular carcinoma. Result: In our study, the model subclassification of follicular neoplasm into follicular adenoma (n = 28/43, images n = 527/886) from follicular carcinoma (n = 15/43, images n= 359/886), has achieved an accuracy of 78%, with a sensitivity of 88.4%, and a specificity of 64% and an area under the curve (AUC) score of 0.87 for each of follicular adenoma and follicular carcinoma. Conclusion: Our CNN model has achieved high sensitivity in recognizing follicular adenoma amongest cytology smears of follciualr neoplasms, thus it can be used as an ancillary technique in the subcalssification of Bethesda Iv category cytology smears. Key Words Thyroid gland Cytolopathology AI- convolutional neural network Diagnosis ==== Body pmcIntroduction Thyroid nodules is a common clinical problem, where the incidentally discovered nodules widely range between 20% and 76% (Ogmen et al., 2020). Thyroid fine needle aspiration cytology is a reliable diagnostic technique which identifies benign thyroid nodules, sparing many patients unnecessary surgery (Ali and Cibas, 2018). The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) is a uniform tiered reporting system for thyroid cytology, which stratifies the thyroid lesions into categories. Each diagnostic category is related to a certain risk of malignancy and accordingly guides towards the proper clinical management. The six categories are as follow: non-diagnostic (category I), benign (category II), atypia of undetermined significance/follicular lesions of undetermined significance (category III), follicular neoplasm/suspicion of follicular neoplasm (category IV), suspicion of malignancy (category V), and malignant (category VI) (Ogmen et al., 2020). The thyroid nodules diagnosed as follicular neoplasm / suspicious for follicular neoplasm (Bethesda category IV) has a risk of malignancy of 25-40% and is considered one of the cytologically indeterminate nodules, as it encompasses two lesions follicular adenoma and follicular carcinoma (Khan and Zeiger, 2020). The main goal of such category is the identification of potential malignancies (follicular carcinomas), rather than identifying all follicular neoplasm, as follicular adenomas are clinically innocuous, and there is little if any evidence that suggests the progression from follicular adenoma to follicular carcinoma. However, the diagnosis of follicular adenoma or carcinoma can only be guaranteed after surgical excision by histopathological examination and determination of tumor capsular and lympho-vascular state (Ali and Cibas, 2018). The standard of care for Bethesda IV category diagnosis is not proceeded before considering the clinical and sonographic features. The molecular testing may also be used as an ancillary test as an additional assessment of malignancy risk before surgical excision (Ali and Cibas, 2018). However, molecular testing has shown variable successes, and most were related to papillary thyroid carcinoma rather than other follicular lesions (Savala et al., 2018). Artificial intelligence (AI) has deeply impacted our lifestyle by imitating human intelligence using the computers, it is to execute tasks more efficiently, rapidly, and at a lower cost in solving complex problems than humans (Kamal and Kumari, 2020)A subset of artificial intelligence (AI) called machine learning includes mathematical algorithms that learn from input data to develop the capacity to generate predictions without being explicitly trained to do so (Landau and Pantanowitz, 2019). Machine learning is categorized into supervised and unsupervised learning based on labelling of the dataset. In supervised learning, the input is the value of the object analyzed and the output is the label representing ground-truth. After training phase, the model is able to classify the inputs into their correct label, for example in pathology image classification by assigning a tumor image to it’s correct class or prediction the outcome/prognosis of a disease (Zhou, 2018). On the other hand, unsupervised learning does not use predefined labels or values. These algorithms attempt to group similar inputs together by understanding the associations or identifying data patterns. Unsupervised learning solves clustering problem either, for example a new functional or structural subgroupings of breast cancer based on subtle histologic variations (Harrison et al., 2021). Until recently, machine learning used hand-crafted input data, where raw data is manipulated into a set of features / values before presenting to the model, for example a squamous cell images as an input data, is changed into set of features as cell size, shape, nucleus, cytoplasm, background and etc. Expert labeling of training data can be challenging, time-consuming, and susceptible to errors, especially on detailed labeling as with pathological images (Harrison et al., 2021). Deep learning technique is a special type of machine learning, which led to significant advances in computer vision. It has allowed skipping the expert labeling of features, and instead the model requires only a data set and it’s label for training (learning), for example the image of squamous cell with their corresponding diagnosis (label), then the model “learn” the features by itself. Although deep learning is more accurate, however one of the trade-offs is being a “black box”, where the model extract features by a complex interaction and those learned features are not understandable by humans, and it is essentially unknown how it arrives to the output (answer), and with each model run there may be a different result, and therefore any slight change in data quality or quantity may alter the learning process during model training and hence the answer the model on validation (Landau and Pantanowitz, 2019). One of the most popular learning models for machine learning is an artificial neural network (ANN), it is mathematical model that is inspired by functioning of the brain (Emmert-Streib et al., 2020). The sequential layers of the artificial neural network (ANN) are organized as an input layer, then a hidden layer, and finally an output layer. When the architecture has more than two hidden layers is commonly considered as as a deep learning model (Emmert-Streib et al., 2020). Convolutional neural network (CNN) is a special type of artificial neural network, that uses deep learning algorithms, and it is widely used in image and video recognition, due to it’s high performance in recognizing patterns (Ismael et al., 2020). CNN is a mathematical architecture composed of three types of layers (or building blocks): convolution, pooling, and fully connected layers. The convolution and pooling layers are responsible for feature extraction, whereas fully connected layer maps the extracted features into final output, such as classification (Yamashita et al., 2018) Early detection and accurate diagnosis are very vital aspect for the improvement of cancer prognosis. Applying artificial intelligence as a tool in these domain has shown promising potential for early detection and diagnosing with high precision, and it is just one of many applications of AI in cancer research (Patil et al., 2020). Several studies were published in thyroid imaging used CNN architecture for classifying being and malignant nodules, however, few studies in thyroid histopathology and cytopathology fields were published. (Wang et al., 2019) trained two CNN models using histological images of thyroid nodules to classify them according to their histologic types, achieving an accuracy of 97.34%. Two studied used CNN to distinguish papillary thyroid carcinomas from non-papillary thyroid carcinoma, one study by (Guan et al., 2019) achieved a 100% sensitivity and 94.91 % specificity, where (Sanyal et al., 2018) achieved a 90.48 % sensitivity and 83.33% specificity. (Elliott Rang et al, 202) have used CNN to classify thyroid nodules into benign vs malignant, achieving 92.0% sensitivity and 90.5% specificity. Other studies have applied CNN in the field of cytopathology as (Garud et al., 2017) who classified malignant vs benign breast lesions achieving accuracy of 80.76%, while (Martin et al., 2018) classified cervical cytology according to Bethesda system categories achieving an overall average accuracy of 60%. (Rahaman et al., 2021) trained several CNNs to classify cervical cytology images into benign and malignant achieving accuracies of 98%. In this work, we aim at exploring the ability of a CNN model to subclassifying cytological images of Bethesda category IV diagnosis into follicular adenoma and follicular carcinoma. A CNN model is trained using microscopic images of thyroid cytology smears, that was later resected with established histopathological diagnosis as the ground truth. Materials and Methods This is a retrospective study on glass smear slides of thyroid nodules cytology, diagnosed at the cytopathology unit, NCI, Cairo university from the year 2015 to 2021, after which a surgical resection was done and diagnosed histopathologically at surgical pathology unit, NCI, Cairo university. These cases were sampled/aspirated with by an experienced radiologist under sonographic guidance mostly, or free-handed by an experienced cytopathologist, for clinically prominent and palpable thyroid nodules. The study was carried out on the images for the cases, our cohort was 886 images captured from 43 follicular neoplasm cases. Cases collection Inclusion criteria Only the cases diagnosed cytologically as follicular neoplasm or suspicious for follicular neoplasm (Bethesda category IV) and after surgical resection confirmed the diagnosis of follicular adenoma or follicular carcinoma. Exclusion criteria Cases with an obscuring element as blood or dense inflammation Cases with dryness artifact or Diff-Quik stained slides (one slide per case), because the first step of stain is induction of dryness artifact to augment the cytoplasmic size and concurrently nuclear size, thus could be hindering the training process of the model. Cases cytologically misdiagnosed as Bethesda category II (nodular colloid hyperplasia and thyroiditis), or Bethesda category V (suspicious for papillary carcinoma), or diagnosed as Bethesda III (Follicular lesion of undetermined significance). To avoid training of the model on cases, that represent a challenge to the expert cytopathologists. Cases diagnosed after resection with double pathology as (follicular adenoma with nodular colloid hyperplasia), to avoid the error of sampling from the adjacent lesion, either free handed by the cytopathologist or by the radiologist under sonar -guided as some both lesions might mimic the other radiologically. Cases histologically diagnosed as Hürthle cell variant of follicular adenoma, as the abundant eosinophilic cytoplasm can be considered as variation in cellular morphology, thus might hinder the training process of the model. Smear slides revision Every PAP-stained smear glass slide for each case was microscopically reviewed to fix any artifact caused by the storage as staining artifact or broken slides cover, example of pre and post fixation artifact in as in Figure 1. Fixing processes Slides with air bubble artifacts were fixed either by replacing the slide cover as in Figure 1 or re-staining when needed with caution to avoid losing the material. The smear slides that were unrepairable were excluded from the study. Selecting the slides Smear slides with the most appropriate diagnostic fields as region of interest (ROI) in the images was selected and placed in a different box for image acquisition. Image acquisition Two different microscopes equipped with a microscopic camera were used for image acquisition: (1) An Olympus BX43 microscope equipped with inserted camera EP50, with image dimensions (2592x1944 pixels), (2) a Nikon (eclipseE600) microscope equipped with an inserted camera with image dimensions (1920X1080 Pixels). Using two different cameras and microscopes provides the required variations in color of the acquired images and the illumination as well, ultimately leading to a different sets of pixel values captured. Thus, the CNN can be trained to classify the lesions in varying conditions of illumination. Imaging setting were checked to ascertain that the microphotograph is mirroring the conventional microscopic image and by selecting the best setting that achieve the best quality of image e.g., focus, illumination, sharpness, saturation etc. However, some of the images underwent adjustment of saturation or lightening after capture, due to non-perfect staining or bad illumination at time of image acquisition. The images of the region of interest (ROI) on the slide was captured at microscopic magnifications 10x and 40x, however only 40x was used to simplify the learning process / training of the model. The images were acquired blindly with no prior knowledge of the case diagnosis to avoid any bias during image acquisition. With the retrieval of the captured images, they were then annotated/classified (labelled) according to histopathological diagnosis as follicular adenoma, as in Figure 2 and follicular carcinoma, as in Figure 3. Images / data cleaning Some of the images (n= 80 image) underwent adjustment of saturation or lightening after capture, due to non-perfect staining or bad illumination, that could not be adjusted at time of image acquisition, as illustrated in Figure 4. While the images with an artifact as haziness or obscuring agent were excluded. Data preprocessing Image color and size Images used were colored using three-channel. A color image is a three-dimensional array of size width × height × 3, where the values of the red, green, and blue channels are the depth of the image. All of the images were normalized into 256x256 pixels. Data augmentation techniques Deep learning models have a better performance with large datasets. Since medical images are always limited, data insufficiency represents a problem that hinder the model training. Augmentation is one of the ways to overcome such problem, which increases the number of images through manipulation of these images (as flipping and rotation), creating new images with the same label. Furthermore, augmentation solves class imbalance by enriching the diversity of the training samples and oversampling the minority class in the set, thus bringing balance to the data. Therefore, augmentation is known to allow the CNN to perform better, through enhancing the training of the model, which is the root problem, thus allowing the generalization of the results and avoid overfitting problem of the model. The techniques used for augmentation in this study are rotation_range = 2, zoom_range = 0.1, horizontal_flip = True. Data Split The images were split randomly into “training” samples from which the model learns, and “test / validation” samples that are used for evaluation of its performance. The split was with the ratio of (4:1), where 80% and 20% are training and validation samples, respectively. Proposed convolutional neural network model The architecture of the network is of feed-forward, convolutional neural network, illustrated in Figure 5 and detailed as follows: The proposed convolutional neural network is composed of several layers. The first three layers are convolutional layers, which are responsible for feature extraction from the data/images. Each convolutional layer is followed by max-pooling, and all have ReLU as an activation function, where each has a different filter as follows 32 size 2x2, 64 size 2x2 and 128 size 2x2, first, second and third layers respectively. All max-pooling layer size was 2x2. The following layer is a flattened layer. Then the deep network are three layers of network, which is responsible for classification. the first layer with 128 nodes, the second layer with 64 nodes, and the third layer with 32 nodes. And finally, the fourth and final layer with 2 nodes and activation function SoftMax. As earlier mentioned, CNN takes a 256x256 color image as input, then convolution and pooling layers extract features of that image, then by the final layer, CNN confers an output for binary classification of follicular adenoma versus follicular carcinoma. All previous steps are summarized in Figure 6. Evaluation metrics Instead of using statistical methods, the evaluation of convolutional neural network model performance is measured by different evaluation metrics. The evaluation metrics used in this study are the accuracy, sensitivity, specificity, precision, F1 score and recall as calculated in equations 1, 2, 3, 4, 5 and 6 respectively. These metrics are derived from the confusion matrix in Figure 7, which is plots the actual class versus the predicted class as follow; true positive (tp) and true negative (tn) denoting the number of positive and negative instances that are correctly classified. Meanwhile, false positive (fp) and false negative (fn) denoting the number of misclassified negative and positive instances, respectively. Accuracy=tp+tntp+fp+tf+fn Equation 1 Sensitivity=tptp+fn Equation 2 Specificity=tntn+fp Equation 3 Percision=tptp+fp Equation 4 F1-score=2+p+rp+r Equation 5 Recall=tptp+tn Equation 6 The other evaluation metrics is area under the curve (AUC) also known as ROC curve., simply it is the percentage of the plot box that lies under the ROC curve. The closer it is to 1 (100%), the better. It is calculated in Area under the curve=Sp-np(nn+1)/2npnn Equation 7 Sp is the sum of all positive examples ranked, while np and nn denote the number of positive and negative examples respectively. Results The number of follicular neoplasm cases n = 43 cases including follicular adenoma (n = 28; 65.11%) and carcinomas (n = 15; 34.88%). Number of images / regions of interest (ROI) n = 886 images; including follicular adenoma images (n=527; 59.48%) and follicular carcinoma images (n = 359; 40.51%). A convolutional neural network was trained using training sample (n=708; 80%) and validation samples (n= 178; 20 %) to measures model performance. Several evaluations metrics were calculated through confusion matrix, illustrated in Figure 7, where class 1 represents follicular adenoma and class 2 represents follicular carcinoma. Where model classification of follicular adenoma (represented as class 1), showed an accuracy 78.0% illustrated in Figure 8, a sensitivity of 88.4%, and a specificity of 64%, while F1-score of 82.5%, precision of 77.3% and recall of 88.46%. While model classification of follicular carcinoma (represented by class 2), showed an accuracy 78%, a sensitivity of 64%, a specificity of 88.4%, while F1-score of 70.6%, and precision of 79.6% and recall of 63.5%. The area under the curve (AUC) score for follicular adenoma vs follicular carcinoma is 0.87, each, where class 0 represents follicular adenoma and class 1 represents follicular carcinoma illustrated in Figure 9. Table 1 Summary of the Number and Percentages of Cases and Region of Interest Bethesda category Number of cases Percentages of cases Number of images Percentages of images Category IV Follicular adenoma 28 65.11% 527 59.48% Category IV Follicular carcinoma 15 34.88% 359 40.51% Total 43 100% 886 100% Table 2 Summary of Different Metrics for Evaluation of Model Performance Evaluation metrics Follicular adenoma Follicular carcinoma Accuracy 78.00% 78.00% Sensitivity 88.4%, 64.00% Specificity 64.00% 88.40% Recall 88.4%. 63.50% Precision 77.30% 79.60% F1-score 82.50% 70.60% Figure 1 An Example of Fixation Process. (a), Smear slide with an air bubble artifact mascaraing the follicular group of cells underneath; (b), The smear slide after changing the cover slip (Papanicolaou stained, 10X magnification) Figure 2 Case of Follicular Adenoma. Cytological diagnosed as follicular neoplasm and diagnosed after resection as follicular adenoma. (a)and(b), Cytological smears shows follicular cell arrangement in mainly in microfollicles pattern within a hemorrhagic background (Papanicolaou stain, 40X magnification); (C), Histological section shows Follicular adenoma with the surrounding intact capsule (HandE stained, 10X magnification) Figure 3 A Case of Follicular Carcinoma. A case cytologically diagnosed as follicular neoplasm and diagnosed after resection as follicular carcinoma. (a)and(b), Cytological smears are relatively cellular with follicular cell arranged mainly in microfollicular pattern. (Papanicolaou stain, 40X magnification); (C), Histological section shows widely invasive follicular carcinoma with capsular invasion and invasive groups at RED Arrowhead (HandE stained, 10X magnification) Figure 4 An Example of Image Editing. A case of follicular neoplasm, smears since 2015 (Papanicolaou stain, 40X magnification). (a), Shows hazy follicular groups with fade discoloration; (b), shows the image after edit (color enhancement and increasing the sharpness), which highlighted the follicular group as well Figure 5 An Illustration of the Proposed Convolution Neural Network. The architecture, showing number of layers and type and specifications of each layer with its activation function Figure 6 Summary of the Steps Starting with Primary Data Collection Till Training the CNN Model Figure 7 An Illustration of Confusion Matrix where Class 1 Represents Follicular Adenoma and Class 2 Represents Follicular Carcinoma (upper right are the cases of predicted correctly predicted as follicular adenoma, lower right are the cases falsely predicted as follicular carcinoma , upper left are the cases wrongly predicted as follicular carcinoma and lower left are the cases predicted correctly as follicular carcinoma) Figure 8 Graph Plotting the Achieved Accuracy during Model Training (represented by blue line) and model validation (represented by green line) Figure 9 A Graph Plotting Area under the Curve / ROC Curve. The vertical line represents true positive and horizontal line represents false positive rate, class 0 represents follicular adenoma and class 1 represents follicular carcinoma Discussion Out of all six categories of Bethesda system for reporting thyroid cytopathology, follicular neoplasm / suspicious for follicular neoplasm (Bethesda category IV) is considered one of the indeterminate nodules, as it constitutes both follicular adenoma and carcinoma, where each has different biologic behavior and sometimes requires different management. This study is exploring the utility of applying artificial intelligence represented by a convolutional neural network (CNN) model, for subclassifying thyroid cytopathology images of Bethesda category IV into follicular adenoma and follicular carcinoma. In our study, differentiating follicular adenoma from follicular carcinoma achieved an accuracy of 78.0%, with sensitivity 88.4%, and specificity 64% and AUC = 0.87. A previous work that classified thyroid cytology into follicular adenoma vs follicular carcinoma by applying an artificial neural network, conducted by Savala and his colleague, they achieved an accuracy of 100% and ROC = 1 in the training set (Savala, et al., 2018). Compared to their study, our work stems on convolutional neural network while theirs stems on a backpropagation neural network. The backpropagation neural network, used morphologic cytologic nuclear features (nuclear area, diameter, perimeter, pleomorphism and cellularity .. etc) extracted by using another software, with the integration of clinico-morphometric parameters as age. But in our study, the convolutional and max pooling layers were responsible for feature extraction directly from cytological images only labeled by their own diagnosis. Few researchers have applied CNN for the purpose of classifying thyroid nodules. Elliott Range and his colleagues used CNN to classify thyroid lesions into benign vs malignant and their work achieved AUC score of 0.93, 92.0% sensitivity, and 90.5% specificity (Range et al., 2020). Compared to our results, they achieved relatively better performances than ours that could be attributed to larger sample size (n=659 comparted to our study n=43). Other studies by Guan and his colleagues in 2019 and Sanyal and his colleagues in 2018, both studies were conducted to differentiate papillary thyroid carcinoma from non-papillary thyroid carcinoma using CNN. (Guan et al., 2019) used two different CNN architectures (VGG-16 and Inception-v3) achieving a better performance by 100% sensitivity and 94.91% specificity by applying VGG-16 model and 98.55% sensitivity and 86.44% specificity with Inception-3 model. (Sanyal et al., 2018) achieved relatively better performance achieving 90.48 % sensitivity of and 83.33% specificity and an accuracy of 85.06% with the use of one objective magnification images for training (either 10X or 40 X). However, the model achieved lower sensitivity of 33.33% with still a better specificity of 98.48% and accuracy 82.76% on using two objective magnifications (x10 and x40). Discrepant results between our study and previous studies of (Elliott Range et al., 2020), (Guan et al., 2019) and (Sanyal et al., 2018) can be attributed to diverse source of images in our work compared to a single source of images in all the previous works. However, the greatest attribution to the discrepant results is the nature of thyroid lesions. Elliott Range et al., (2020) included malignant category such as medullary carcinoma, anaplastic carcinoma and papillary carcinoma to be compared to benign thyroid nodule that usually does not represent a large burden to the cytopathologist to diagnose. As well papillary thyroid carcinoma has distinctive nuclear morphology with main differential diagnosis is non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). The studies were designed to distinguish papillary carcinoma from lesions other than NIFTP such as colloid nodular goiter, lymphocytic thyroiditis and follicular neoplasm representing Bethesda II and IV (Sanyal et al., 2018) and benign nodules (Bethesda II) categories as differential lesions (Guan et al., 2019). Consequently, the CNN is expected to achieve high performance, whereas our approach was challenged by a category (Bethesda IV) that is already challenging to the highly experienced cytopathologists on cytology smears. Our study concludes that convolutional neural network model can be used as an ancillary technique in the diagnosis of thyroid cytopathology. Although the differentiation between follicular adenoma and carcinoma is challenging even to artificial intelligence model and our cohort was not large, however, the model has achieved a high sensitivity in recognizing follicular adenoma. The CNN model can be used as an additional factor in risk classification, as part of pre-surgical intervention assessment for follicular neoplasms. Artificial Intelligence (CNN models) are a long-term investment in the field of pathology, although it requires data (which is not always available), however, with good training of the model it offers an affordable and sustainable ancillary technique, rather than the other costly alternatives. Our recommendations for future studies for applying artificial intelligence in thyroid FNAC are as follows: (1) A larger dataset is to be used for training the CNN model with better performance. (2) A dataset with higher quality would be better to be used to enhance the performance of the model, even without a larger dataset. (3) Different architectures of a convolutional neural network can be trained on the available dataset, to explore the ability of each architecture to exploit the current dataset of cytology images, regardless of their quality and quantity, to achieve higher performance of classification. Author Contribution Statement All the authors have contributed to this study. Data collection, review of the slides and image acquisition were performed by Dr Mona Alabrak, and under supervision of Dr Habiba Elfandy, Prof. Dr Neveen Tahoun and Dr Hoda Ismail. A Pilot CNN model run was done by Asmma A. Alkhouly, to allow better image acquisition of region of interest and the proper magnification to be applied in the study, under supervision of professor Dr Ammar Mohammed. The construction of CNN architecture with the proper hyperparameters as well as CNN model training was executed by Mohammad Megahed. The validation of CNN model results was done by Professor Dr Ammar Mohammed. The preliminary draft of the manuscript was written by Dr Mona Alabrak and Dr Habiba Elfandy. All authors revised and commented on primary version of the manuscript and approved the final one. Acknowledgements We would like to acknowledge our great National Cancer Institute and its workers, technicians and staff members, for all the support and help in this study. Approved student thesis This paper is a part of an approved PHD thesis at National Cancer Institute (NCI), Cairo University. Ethical approval The study was approved by the Institutional Review Board (IRB) no. IRB00004025 of National Cancer Institute (NCI), Cairo University. Oral and written informed consents were obtained from all patients or from their eligible relatives. Availability of data (if apply to your research) The data are available from the corresponding author, on a reasonable request. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. ==== Refs References Ali SZ Cibas ES The Bethesda System for Reporting Thyroid Cytopathology. Definitions, criteria and explanatory notes, In follicular neoplasm/suspicious for follicular neoplasm 2018 Switzerland Springer international publishing AG 72 Emmert-Streib F Yang Z Feng H Tripathi S Dehmer M An Introductory Review of Deep Learning for Prediction Models With Big Data Front Artif Intell 2020 3 4 33733124 Garud H Karri SPK Sheet D High-Magnification Multi-views Based Classification of Breast Fine Needle Aspiration Cytology Cell Samples Using Fusion of Decisions from Deep Convolutional Networks 2017 IEEE Computer Society Conference on Computer Vision and Pattern Recognition Workshops 33 Guan Q Wang Y Ping B Deep convolutional neural network VGG-16 model for differential diagnosing of papillary thyroid carcinomas in cytological images: a pilot study J Cancer 2019 10 4876 31598159 Harrison JH Gilbertson JR Hanna MG Introduction to Artificial Intelligence and Machine Learning for Pathology Arch Pathol Lab Med 2021 145 1228 54 33493264 Ismael SA Mohammed A Hefny H An enhanced deep learning approach for brain cancer MRI images classification using residual networks Artif Intell Med 2020 102 101779 31980109 Kamal VK Kumari D Use of Artificial Intelligence/Machine Learning in Cancer Research During the COVID-19 Pandemic Asian Pac J Cancer Care 2020 5 251 3 Khan TM Zeiger MA Thyroid Nodule Molecular Testing: Is It Ready for Prime Time? Front Endocrinol (Lausanne) 2020 11 809 Landau MS Pantanowitz L Artificial intelligence in cytopathology: a review of the literature and overview of commercial landscape J Am Soc Cytopathol 2019 8 230 41 31272605 Martin V Kim TH Kwon M A More Comprehensive Cervical Cell Classification Using Convolutional Neural Network J Am Soc Cytopathol 2018 7 S66 Ogmen B Aydin C Kilinc I Can Repeat Biopsies Change the Prognoses of AUS/FLUS Nodule Eur Thyroid J 2020 9 92 8 32257958 Patil S Moafa IH Alfaifi MM Reviewing the Role of Artificial Intelligence in Cancer Asian Pac J Cancer Biol 2020 5 189 99 Rahaman MM Li C Yao Y DeepCervix: A deep learning-based framework for the classification of cervical cells using hybrid deep feature fusion techniques Comput Biol Med 2021 136 104649 34332347 Range DD Dov D Kovalsky SZ Application of a machine learning algorithm to predict malignancy in thyroid cytopathology Cancer Cytopathol 2020 128 287 95 32012493 Sanyal P Mukherjee T Barui S Das A Gangopadhyay P Artificial Intelligence in Cytopathology: A Neural Network to Identify Papillary Carcinoma on Thyroid Fine-Needle Aspiration Cytology Smears’ J Pathol Inform 2018 9 43 Savala R Dey P Gupta N Artificial neural network model to distinguish follicular adenoma from follicular carcinoma on fine needle aspiration of thyroid Diagn Cytopatho 2018 46 244 9 Wang Y Guan Q Lao I Using deep convolutional neural networks for multi-classification of thyroid tumor by histopathology: a large-scale pilot study Ann Transl Med 2019 7 45 30906749 Yamashita R Nishio M Do RKG Togashi K Convolutional neural networks: an overview and application in radiology Insights Imaging 2018 9 611 29 29934920 Zhou ZH A brief introduction to weakly supervised learning Natl Sci Rev 2018 5 44 53

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 10.31557/APJCP.2023.24.4.1103 Letter to Editor Estimating the Cancer Treatment Cost for 5 Common Types of Cancer with Separating Out-of-Pocket and Governmental Costs in Afghanistan, 2020 Abdullah Maihan 1* Hashimi Sayed Ikram 2 Masoom Mohammad Khalid 3 1 T. H. Chan School of Public Health. Harvard University, MA, U.S.A. 2 National Cancer Control Program, Ministry of Public Health, Kabul, Afghanistan. 3 Pfizer, North Carolina, U.S.A . * For Correspondence: maihanabdullah22@gmail.com. 2023 24 4 11031104 16 3 2023 17 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) ==== Body pmc Dear Editor The recently published article (Rohani et al., 2022) estimated the government and out-of-pocket treatment costs for five cancers (breast, esophagus, colorectal, stomach, and lung) in Afghanistan. We argue that some of the statements and findings expressed in this article are not consistent with the literature of the Ministry of Public Health (MoPH), nor is the methodology adequate to correctly measure the various components of treatment cost. This article reported the out-of-pocket cost of cancer treatment as 13.97% (82,537.66) while the National Health Accounts (NHA) has consistently reported the out-of-pocket cost in health sector as more than 70% (National Health Accounts 2018, 2019). One of the reasons for this significant difference could be the inadequate use of the research methodology and inaccurate analysis of cost and expenses of various cancer care services in this study. For example, the article mentioned that except for CT scan, MRI, and mammography, other services (Figure 3) were provided for free by the Cancer Center in Jamhuriat Hospital. Even though chemotherapy was administered for free, the National Cancer Control Program (NCCP) had not procured chemotherapeutic drugs but patients bought from pharmacies. Similarly, limited sonography services were provided to female patients only. Moreover, though the article mentioned the cost of biopsy procedure, it did not include the cost of cyto-histopathology examination. Therefore, if the costs of CT scan, MRI, mammography, and chemotherapy are added together, without even including the cost of cyto-histology and sonography, the out-of-pocket cost will increase from 13.9% to 43%. Furthermore, the article mentioned that 2,180 patients were registered with the Cancer Center in 2020. Based on the Center’s report, however, 16,058 visits/consultations were recorded in 2020. Those consultations resulted in the admission of 3,200 patients to the medical and surgical oncology wards (IPD) and admission of 2,180 patients in the day-care unit (OPD). It is very likely that the article has analyzed only these 2,180 patients that were treated in the day-care unit while overlooking the other 3,200 patients from the IPD. Also, the article stated that the possible causes of increased cancer prevalence were lack of awareness, late diagnosis, lack of access to medical services, and lack of financial resources. However, most of these factors are not associated with the increase in prevalence or incidence of cancer. These factors contribute to late diagnosis and subsequently premature deaths of cancer patients. In fact, the referenced article (Bhandari et al., 2021) implied that most of those factors were associated with low breast cancer screening intentions. Besides, the article provided detailed information about the educational status of patients, and listed the educational status of patients into six groups such as two patients had master’s, 18 patients had bachelor’s, 12 patients graduated from 12th grade, one person each had primary and secondary level education, and 735 (96%) patients had no education (illiterate). How did the study acquire information on the educational status of these patients when the patients’ files at the Cancer Center do not contain such data? Moreover, the article misidentified the Afghanistan Cancer Foundation (ACF) as Action Against Cancer Foundation. In the same paragraph, some other information is either incorrect or outdated. For example, the article stated that the NCCP was established in 2016 in Jamhuriat Hospital as Cancer Diagnosis and Treatment Project (CDTP). In fact, the CDTP and the NCCP were established in the MoPH in 2016 and 2017 respectively. On the other hand, it was the Cancer Center which was set up in Jamhuriat Hospital in 2016 by the CDTP. Last but not least, the article mentioned that the study’s ethical approval was taken from the Afghanistan National Charity Organization for Special Diseases (ANCOSD), which is a small non-government organization (NGO). Based on the Ministry of Public Health’s regulations, the Institutional Review Board (IRB) of Afghanistan National Public Health Institute (ANPHI) has the authority to approve biomedical research proposals. Even though the Ministry of Higher Education and Kabul University have their respective IRBs, they are limited to the research work in their own institutions (Health Research Policy, 2012). Therefore, even they are not in a position to give approval to such research. Given the above stated reasons, the article needs to be corrected. A well-designed and well-executed research is crucial to find out the economic cost of cancer, especially the catastrophic out-of-pocket cost of cancer treatment in Afghanistan. Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran APJCP-24-1103 Letter to Editor Reply to the letter to the editor: Estimating the Cancer Treatment Cost for 5 Common Types of Cancer with Separating Out-of-Pocket and Governmental Costs in Afghanistan, 2020 Hashemy Tayeba * Economic Research Center, Kateb University, Kabul, Afghanistan. * For Correspondence: tayeba.hashemy@gmail.com 2023 24 4 11031104 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Dear Editor The author team is glad to bring in to consideration which completing field research in Afghanistan is a great challenge and need to meet many regulations and policies. Altogether, the following responses were provided Although the out-of-pocket cost of health is 70% for all health services, this article is focused on 5 common types of cancer and since it focuses on one disease, the existing difference is completely reasonable and any argument related to this issue by presenting valid and similar researches can be acceptable. As mentioned in the article, the estimation of the costs of this disease only includes the service costs and not the costs of the drugs used to receive medical services. There were two logical reasons for this issue: 1) It was not possible to access the data related to patients in the system of this hospital and all the information was obtained from the patients’ hard files, which was extremely messy and inaccurate. Although the collection of these data was extremely time-consuming, it is a reliable reason for the severe lack of professionality of in charge individuals. 2) The medicines used by the patients were not available in their files, and according to repeated requests, no documents were available regarding the amount of medicine used by each patient. In addition, in the reviewed files, only 3 cases of mammography, CT scan and MRA were included in the patient files, and items such as sonography and cyto-histology were not included. The point that can be discussed here is based on which data did the author state that out-of-pocket costs for patients will increase by 43%? Moreover, it should be mentioned again that this article examines the cost of 5 common types of cancer and all the cancer cases registered in Jamhuriyat Hospital are more than 30 types. It is also necessary to mention that out of the 2180 registered cases, only 769 cases could be included with complete information, which shows the huge weakness of this department and the in-charge individuals in documenting the patients’ information. In addition, the educational status of the patients is not the key point in the examination of the costs and there is no need to add fake information. While, the questionnaire was designed after a pilot period. Meanwhile, If it is mentioned in the article that some concepts are mentioned incorrectly or old, based on any argument, being old does not mean that the words and meaning of the words are wrong. It is important to bring in to consideration that the smallness of NCOSD is not based on any logical argument that it is invalid. While it is officially registered in the government and is officially active and has had many activities in the field of research and publication of valid articles. In addition, the access to the information was done with the written order of the Minister of Public Health, under no circumstances the collected data has harmed third person. ==== Refs References Afghanistan National Health Accounts 2018 Islamic Republic of Afghanistan Ministry of Public Health Afghanistan National Health Accounts 2019 Islamic Emirates of Afghanistan Ministry of Public Health Bhandari D Shibanuma A Kiriya J Factors associated with breast cancer screening intention in Kathmandu Valley, Nepal PLoS One 2021 16 Health Research Policy and Strategy for Afghanistan 2012-2020 2013 Afghanistan National Public Health Institute National Cancer Control Program (NCCP) 2019 Ministry of Public Health Rohani H Mousavi SH Hashemy SM Estimating the Cancer Treatment Cost for 5 Common Types of Cancer with Separating Out-of-Pocket and Governmental Costs in Afghanistan, 2020 Asian Pac J Cancer Prev 2022 23 3273 9 36308349

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116140 10.31557/APJCP.2023.24.4.1187 Research Article Genetic Characterization of Ovarian Tumor Tissues from Patients with Epithelial Ovarian Cancer in a Philippine Tertiary Hospital: A Descriptive Study Lintao Ryan C V 12 Padua Ana Joy P 12 Nakura Yukiko 2 Llamas-Clark Erlidia F 34 Yanagihara Itaru 2* 1 College of Medicine, University of the Philippines Manila, Ermita, Manila 1000 Philippines. 2 Department of Developmental Medicine, Research Institute, Osaka Women’s and Children’s Hospital, Izumi, Osaka, Japan. 3 Department of Obstetrics and Gynecology, College of Medicine and Philippine General Hospital, University of the Philippines Manila, Manila, Philippines. 4 Institute of Child Health and Human Development, National Institutes of Health, University of the Philippines Manila, Manila, Philippines. * For Correspondence: itaruy@wch.opho.jp. Ryan C. V. Lintao and Ana Joy P. Padua have equal contribution in this study. 2023 24 4 11871197 11 10 2022 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: This study identified genetic variations in ovarian tumor specimens from Filipino epithelial ovarian cancer (EOC) patients using next-generation sequencing. Methods: Genomic DNA was isolated from formalin-fixed paraffin-embedded ovarian specimens from 8 chemosensitive and 8 chemoresistant EOC patients. Targeted next-generation sequencing was done to identify mutations in hotspot regions of common oncogenes and tumor-suppressor genes. The mutations were cross-referenced with dbSNP and ClinVar databases to identify previously reported alterations, and potentially damaging variants were predicted using PolyPhen-2. Results: Our study has identified 85 unique variants, 35 in chemosensitive EOC, 22 in chemoresistant EOC, and 28 in both. Chemosensitive EOC specimens had more exonic single nucleotide variants than chemoresistant EOC specimens. Of the 50 oncogenes and tumor suppressor genes, KDR gene had the most frequent variations in EOC patients. Two of the unique KDR variants identified were novel mutations. Thirty-nine unique protein-modifying genetic variants were identified in all specimens, the majority of which have been previously reported in dbSNP and ClinVar. Conclusion: This study was the first non-BRCA genetic analysis done on ovarian cancer in Filipino patients. Next-generation sequencing was able to identify previously reported alterations with known therapeutic implications which may benefit from targeted therapy instead of standard chemotherapy regimen. Key Words Epithelial ovarian cancer Philippines targeted next-generation sequencing chemotherapy resistance ==== Body pmcIntroduction Cancer of the ovaries is the fifth most diagnosed cancer in women from the Philippines, following breast, cervix, colorectal, and lung cancers, and the second most common gynecologic malignancy (Laudico et al., 2015). Most ovarian cancer diagnosed worldwide are of epithelial type, comprising 90% of the cases, occurring primarily in postmenopausal women (Jelovac & Armstrong, 2011). Globally, quarter of ovarian cases are associated with germline mutations, with the rest arising from sporadic mutations. Of the hereditary ovarian cancers, 20% of the cases in the Philippines and worldwide are attributed to mutations in BRCA 1/2 (De Leon Matsuda et al., 2002; Konstantinopoulos et al., 2020). Early-stage ovarian cancer usually does not present with any symptoms; if any, it presents as vague symptoms usually attributed to gastrointestinal pathology (Lheureux et al., 2019). Pelvic examination may detect ovarian masses, with serum tumor markers such as CA-125 and transvaginal ultrasound aiding in diagnosis. These modalities, however, are not ideal as screening tools because usage in average-risk women did not decrease the risk of mortality, and was associated with increased harm ranging from minor procedure-related adverse events (e.g., nausea, fainting) to more severe complications from cancer diagnosis or false-positive results (e.g., infection, bowel injury) (Buys et al., 2011). Non-specific symptomatology combined with a lack of effective screening tools contributes to late diagnosis of ovarian cancer, making it the most lethal gynecologic malignancy in terms of case-fatality rate (Doubeni et al., 2016). Epithelial ovarian cancer (EOC) is treated with debulking surgery to remove the primary tumor and other masses detected in the fallopian tube and the peritoneum during exploratory laparotomy, with a standard regimen of platinum-based chemotherapeutic drug and taxane (e.g. carboplatin-paclitaxel) as adjuvant therapy (Berek et al., 2018). The response to chemotherapy is classified as either resistant or sensitive, with chemoresistant ovarian cancer recurring within 6 months from the end of chemotherapy. In contrast, chemosensitive ovarian cancer recurs beyond 1 year (Stuart et al., 2011). Due to heterogeneity in chemotherapeutic response, there is a need to custom-fit treatment with the genetic profile of patients as part of precision medicine. In the Philippines, however, only a few genetic studies have been done on ovarian cancer, focusing on BRCA 1/2 mutations associated with hereditary breast-ovarian cancer (De Leon Matsuda et al., 2002; Nato, 2003; Que et al., 2018). This pioneering study described genetic variations in chemosensitive and chemoresistant ovarian specimens from Filipino patients that may be associated with pathogenesis or chemotherapeutic response via targeted next-generation sequencing of hotspot regions of common tumor suppressor genes and oncogenes. Materials and Methods Patient selection and sample preparation Deidentified formalin-fixed paraffin-embedded (FFPE) ovarian tissues composed of 8 chemosensitive (CS) and 8 chemoresistant (CR) from randomly selected patients were retrieved from University of the Philippines - Philippine General Hospital. The inclusion criteria were (1) pathologic diagnosis of epithelial ovarian based on routine hematoxylin and eosin-stained histopathology, and (2) no family history of ovarian cancer. The response to chemotherapy was defined as (a) Resistant, progression-free interval since the last line of platinum-based chemotherapy of less than 6 months; and (b) Sensitive, progression-free interval since the last line of platinum-based chemotherapy of more than 12 months (Stuart et al., 2011). Clinical follow-up until at least 1 year should be completed to be included. This study was conducted upon approval of University of the Philippines Manila Research Ethics Board. Targeted next-generation sequencing platform DNA was isolated from FFPE blocks with high tumor percentage using Maxwell® RSC DNA FFPE Kit (Promega) as described in the product manual. DNA samples were quantified using Qubit® 2.0 fluorometer with dsDNA BR assay kit (ThermoFisher Scientific) and stored at -20°C. About 10 ng of DNA for each sample was used for library preparation using Ion AmpliseqTM Kit for Chef DL8 and Ion ChefTM Instrument (ThermoFisher Scientific). Ion AmpliSeqTM Cancer Hotspot Panel v2 (ThermoFisher Scientific) was used for amplification of hotspot regions, including approximately 2,800 COSMIC mutations of 50 oncogenes and tumor suppressor genes such as APC, KDR, KIT, KRAS, PIK3CA, PTEN, and TP53 composed of 207 indexed, adaptor ligated, hybridization-captured primer pairs with average amplicon length 154 bp. Sequencing was performed using the Ion PGMTM Sequencer (ThermoFisher Scientific) and Ion PGMTM Hi-QTM View Chef Kit with the Ion 316TM Chip Kit described by the manufacturer. Bioinformatics analysis Data analysis of BAM files was carried Ion Torrent Sequencing platform (Life Technologies). Generated reads were aligned to the GRCh37 (hg19) human reference genome. Torrent Suite Software V.5.12 (Life Technologies) was used to call variants such as somatic single-nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs), insertions, deletions, and block substitutions. The same software package was used to filter and annotate variants. Annotation for each variant included the type of variant, gene location, type of transcript, and amino acid change due to the variant. Annotation was cross-referenced with UCSC Genome Browser (Kent et al., 2002) (https://genome.ucsc.edu/). PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) was used to predict functional effect of an amino acid substitution in a certain protein (Adzhubei et al., 2010). dbSNP (https://www.ncbi.nlm.nih.gov/snp/) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) were used to identify previously reported genetic variants (Sherry et al., 2001) and their relationship to human diseases (Landrum et al., 2018), respectively. Statistical analysis Descriptive analysis using tables, frequency (%), and median values were used to summarize and analyze the data collected. Mann-Whitney test was done to compare the median age of the two groups. For other baseline characteristics, chi-square test for independence was done to determine the association between the two groups. P-values of 0.05 or less were considered statistically significant. Proportions and frequencies were reported for genetic variants due to limitations in the analysis due to small sample size. Data were analyzed using GraphPad Prism 9.3.1 (https://www.graphpad.com) (San Diego, CA, USA). Results Patient demographics and tumor characteristics The study participants had a median age of 50.0 at the time of cytoreductive surgery, with age ranging from 23 to 57 years in chemosensitive group, and from 24 to 62 in chemoresistant group (Table 1). This was consistent with median age at diagnosis of 50-79 years reported by Momenimovahed et al (2019). The difference in median age between the two groups was not statistically significant (p=0.4564). Both groups had comparable gravidity (p=0.3149), number of abortions (p=0.5218) and number of preterm deliveries (p=0.5218). Majority of the patients (11 of 16) had advanced-stage disease at the time of cytoreductive surgery. The ovarian tumors surgically removed from patients were mostly serous, the most common histologic subtype in epithelial ovarian cancer (Torre et al., 2018). There was no statistically significant difference between chemosensitive and chemoresistant EOC groups in the cancer stage (p=0.3666) and histologic subtype (p=0.2276). Chemosensitive EOC specimens have more exonic SNVs A total of 305 genetic variants were detected in the EOC specimens, as shown in Table 2, of which 85 were unique. Supplementary Table A shows a list of all genetic variants identified via targeted next-generation sequencing. Of the 168 genetic variants detected in chemosensitive EOC specimens, 63 were unique. Similarly, 50 of the 137 genetic variants detected in chemoresistant EOC specimens were unique. Of the unique variants, 35/63 in chemosensitive EOC specimens and 22/50 in chemoresistant EOC specimens were exclusive to their corresponding groups. Twenty-eight unique variants were shared between chemosensitive and chemoresistant EOC specimens. These 28 common variants were mostly single nucleotide variants or SNVs (26, 92.86%), with 1 insertion-deletion and 1 multi-nucleotide variant. Eighteen SNVs were exonic, of which 11 were silent mutations, and 7 were missense mutations. In the chemosensitive EOC group, 33 of the 35 exclusive variants were SNVs, while 2 were insertion-deletion. Of the SNVs, 28 (84.85%) were exonic, of which 18 were missense mutations, 3 were nonsense mutations, and 7 were silent mutations. On the other hand, 20 of the 22 variants exclusive to the chemoresistant EOC group were SNVs, of which 12 (60.00%) were exonic. Five exonic SNVs were silent mutations, while seven were missense mutations. Genetic variants common to all EOC specimens Of the 305 non-unique genetic variants across 16 EOC specimens, KDR genetic variants had the highest frequency with 41 (13.44%), followed by APC (23, 7.54%), PDGFRA (20, 6.56%), and STK11 (19, 6.23%). FGFR3, FLT3, PIK3CA, RET, and TP53 each had 16 variants, accounting for 5.25% each. The frequency of the variants per gene in each specimen is shown in Figure 1. Table 3 shows the genetic variants common to chemosensitive and chemoresistant EOC specimens. SNVs in PDGFRA, APC, and FLT3 were present in all 16 specimens, followed by STK11 and FGFR3 (n=15 each). The most common SNVs which resulted in alteration in protein sequence were missense mutations in TP53 (n=13), KDR (n=11), and SMARCB1 (n=9). The gene with the most variants was KDR, with 1 insertion-deletion and 3 SNVs, of which 1 was a missense mutation. KDR was followed by PDGFRA, KIT, MET, RET, STK11 and SMARCB1 with 2 genetic variants each. Of the five genes, only MET and SMARCB1 had a genetic variant that resulted in a missense mutation. There were 7 variants that resulted in protein modification due to missense mutation in KIT, KDR, MET, KRAS, TP53, STK11 or SMARCB1. Exclusive genetic variants in chemosensitive and chemoresistant EOC There were 35 genetic variants found only in chemosensitive EOC specimens, as shown in Table 4. Twenty-three exclusive variants resulted in protein modification, 18 of which were due to missense mutations, 3 to nonsense mutations, and 2 to frameshift deletion. A frameshift deletion in APC was the most common genetic variant in this group (n=7), followed by a frameshift deletion in PTEN and an SNV in SMO (both n=4 each). The gene with the most variants was PTEN with 1 frameshift deletion as mentioned and 5 SNVs, of which 3 were missense mutations and 1 was a nonsense mutation. PTEN was followed by PIK3CA and ATM with 3 genetic variants each. In terms of variants that resulted in protein modifications, PTEN was followed by KDR at 1 missense and 2 nonsense mutations, followed by PIK3CA, EGFR, SMAD4 and TP53 with 2 missense mutations each. MLH1, CTNNB1, RB1, and AKT1 variants were found only in chemosensitive EOC group. On the other hand, of the 22 chemoresistant EOC-exclusive genetic variants, 9 resulted in protein modification. All genetic variants were present in 3 specimens or less. The gene with the most variants was PIK3CA with 6 SNVs. Interestingly, all 6 SNVs were found in one specimen. PIK3CA (2 missense) and PTEN (1 non-frameshift deletion, 1 missense) were the genes with most variants resulting in protein modification. MPL, NRAS, FBXW7, NOTCH1, ABL1 and JAK3 variants were exclusive to chemoresistant EOC group. In silico functional prediction reveals potentially damaging gene variants Table 5 shows the 39 unique protein-modifying genetic variants identified in all EOC specimens. Seven protein-modifying genetic variants were common to both EOC groups, all of which have been previously reported in dbSNP. Using ClinVar as reference database, 4 of these variants were deemed benign or likely benign, 1 was pathogenic, and 1 was not previously reported in ClinVar. The remaining variant had no interpretation provided. Of the 23 protein-modifying variants in chemosensitive EOC group, 13 were previously deposited in dbSNP. Eight of these 13 variants were reported as pathogenic or likely pathogenic, 1 was reported to be benign, 3 were of uncertain significance, and 1 was not previously reported in ClinVar. In particular, PIK3CA, PTEN, and TP53 variants described in this study were found to be associated with pathogenic conditions. Using PolyPhen-2 to predict functional effect of amino acid substitution in 8 previously unreported SNVs, 6 variants were predicted to be deleterious while 2 were predicted to be benign. On the other hand, of the 9 protein-modifying variants in chemoresistant EOC group, 7 were reported in dbSNP. Four variants were reported in ClinVar as pathogenic or likely pathogenic, while 3 were not previously reported in ClinVar. Of the 2 unreported SNVs, the FBXW7 variant was predicted to be damaging while the JAK3 variant was predicted to be benign. Table 1 Summary of Patient Demographics and Tumor Characteristics in EOC Total EOC group (n=16) Chemosensitive EOC group (n=8) Chemoresistant EOC group (n=8) P-value Median age (range) 50.0 (23-62) 49.5 (23-57) 55.5 (24-62) 0.4564 Gravidity 0.3149 Nulligravid 5 2 3 Primigravid 2 2 0 Multigravid 9 4 5 Abortion 3 1 2 0.5218 Preterm delivery 3 1 2 0.5218 FIGO Stage 0.3666 I 3 2 1 II 2 2 0 III 8 3 5 IV 3 1 2 Histologic type 0.2276 Serous 12 5 7 Endometrioid 2 2 0 Clear cell 1 0 1 Mucinous 1 1 0 Figure 1 Frequency Heatmap of Genetic Variations Across Chemosensitive and Chemoresistant EOC Specimens Table 2 Summary of Genetic Variants in both Chemosensitive and Chemoresistant EOC Groups Chemosensitive EOC group (n=8) Chemoresistant EOC group (n=8) Total genetic variants 168 137 Unique variants 63 50 Variants exclusive to the group 35 22 Type of alteration Insertion-deletion (Indel) 2 2 Single nucleotide variant (SNV) 33 20 Multi-nucleotide variant (MNV) 0 0 SNVs by location Exonic 28 12 Non-exonic 5 8 Exonic SNVs by effect Synonymous 7 5 Missense 18 7 Nonsense 3 0 Table 3 Frequency of Genetic Variants Common to Both Chemosensitive and Chemoresistant EOC Specimens Position Type Geno-type Gene Location Functional Consequence Frequency CS CR chr2:209113192 SNV G/A IDH1 exonic synonymous 3 3 chr2:212812097 SNV T/C ERBB4 intronic - 5 3 chr2:29432625 SNV C/A ALK intronic - 1 2 chr4:1807894 SNV G/A FGFR3 exonic synonymous 8 7 chr4:55141055 SNV A/G PDGFRA exonic synonymous 8 8 chr4:55152040 SNV C/T PDGFRA exonic synonymous 1 2 chr4:55593464 SNV A/C KIT exonic missense 1 2 chr4:55593481 SNV A/G KIT exonic synonymous 5 2 chr4:55946354 SNV G/T KDR intronic - 3 4 chr4:55962545 INDEL -/G KDR intronic - 3 4 chr4:55972974 SNV T/A KDR exonic missense 4 7 chr4:55980239 SNV C/T KDR intronic - 6 6 chr5:112175770 SNV G/A APC exonic synonymous 8 8 chr5:149433596 MNV TG/GA CSF1R 3’ UTR - 7 7 chr7:55249063 SNV G/A EGFR exonic synonymous 3 1 chr7:116339672 SNV C/T MET exonic synonymous 1 2 chr7:116340262 SNV A/G MET exonic missense 1 2 chr10:43613843 SNV G/T RET exonic synonymous 5 5 chr10:43615633 SNV C/G RET exonic synonymous 3 2 chr11:534242 SNV A/G HRAS exonic synonymous 2 2 chr12:25398284 SNV C/T KRAS exonic missense 1 2 chr13:28610183 SNV A/G FLT3 intronic - 8 8 chr17:7579472 SNV G/C TP53 exonic missense 8 5 chr18:48586344 SNV C/T SMAD4 intronic - 2 2 chr19:1220321 SNV T/C STK11 intronic - 8 7 chr19:1223125 SNV C/G STK11 exonic missense 2 2 chr22:24134064 SNV C/A SMARCB1 exonic missense 6 3 chr22:24176287 SNV G/A SMARCB1 intronic - 4 3 Table 4 Frequency of Exclusive Genetic variants in Chemosensitive and Chemoresistant EOC Specimens Position Type Genotype Gene Location Functional Consequence Freq. Chemosensitive EOC specimens only chr2:212576848 SNV T/C ERBB4 exonic missense 1 chr2:212578395 SNV G/A ERBB4 intronic 1 chr2:29443617 SNV C/G ALK exonic synonymous 1 chr3:178916876 SNV G/A PIK3CA exonic missense 2 chr3:178921547 SNV C/T PIK3CA exonic synonymous 2 chr3:178927980 SNV T/C PIK3CA exonic missense 1 chr3:37067240 SNV T/A MLH1 exonic missense 1 chr3:41266113 SNV C/A CTNNB1 exonic missense 1 chr4:55144628 SNV C/T PDGFRA exonic missense 1 chr4:55597497 SNV C/T KIT 5’ splice site 1 chr4:55972955 SNV G/A KDR exonic missense 1 chr4:55979624 SNV G/A KDR exonic nonsense 1 chr5:112175952 INDEL A/- APC exonic frameshift deletion 7 chr7:116339662 SNV G/A MET exonic missense 1 chr7:116340176 SNV C/T MET exonic synonymous 1 chr7:128845088 SNV A/G SMO exonic synonymous 4 chr7:55211110 SNV C/T EGFR exonic missense 1 chr7:55241701 SNV G/A EGFR exonic missense 1 chr10:43617372 SNV C/T RET intronic 1 chr10:89692905 SNV G/A PTEN exonic missense 2 chr10:89711899 SNV C/T PTEN exonic missense 2 chr10:89711910 SNV T/G PTEN exonic nonsense 1 chr10:89720725 SNV T/C PTEN exonic synonymous 1 chr10:89720812 INDEL A/- PTEN exonic frameshift deletion 4 chr10:89720870 SNV T/G PTEN exonic missense 1 chr11:108137941 SNV C/A ATM exonic nonsense 1 chr11:108180917 SNV T/C ATM exonic synonymous 1 chr11:108236264 SNV C/G ATM 3’ UTR 1 chr12:25378656 SNV T/C KRAS exonic synonymous 1 chr13:48942677 SNV G/T RB1 exonic missense 1 chr14:105246565 SNV C/T AKT1 intronic 1 chr17:7577108 SNV C/A TP53 exonic missense 1 chr17:7577570 SNV C/A TP53 exonic missense 1 chr18:48591907 SNV C/T SMAD4 exonic missense 1 chr18:48603030 SNV A/T SMAD4 exonic missense 1 Chemoresistant EOC specimens only chr1:115256542 SNV C/T NRAS exonic missense 1 chr1:43815034 SNV C/T MPL intronic 1 chr2:212578389 SNV A/G ERBB4 intronic 1 chr3:178917005 SNV A/G PIK3CA intronic 3 chr3:178936091 SNV G/A PIK3CA exonic missense 2 chr3:178952020 SNV C/T PIK3CA exonic synonymous 2 chr3:178952085 SNV A/G PIK3CA exonic missense 1 chr3:178952151 SNV G/A PIK3CA exonic synonymous 1 chr3:178952202 SNV T/C PIK3CA 3’ UTR 1 chr3:178952228 SNV G/A PIK3CA 3’ UTR 1 chr4:153250867 SNV G/A FBXW7 exonic missense 1 chr4:1807864 SNV C/T FGFR3 exonic synonymous 1 Chemoresistant EOC specimens only chr4:55953852 SNV G/A KDR exonic missense 1 chr4:55973048 SNV G/A KDR intronic 1 chr7:128845966 SNV A/G SMO intronic 1 chr9:133738374 SNV G/A ABL exonic synonymous 1 chr9:139399411 INDEL CCA/- NOTCH1 exonic non-frameshift deletion 1 chr10:89692902 SNV G/A PTEN exonic missense 1 chr11:108225611 SNV T/C ATM intronic 1 chr11:108236232 SNV G/A ATM exonic synonymous 1 chr17:7579437 INDEL C/- TP53 exonic frameshift deletion 1 chr19:17954206 SNV C/T JAK3 exonic missense 1 Table 5. In silico Functional Prediction of Exonic Genetic Variants in Chemosensitive and Chemoresistant EOC Specimens Position Genotype Gene Change PolyPhen-2 HumDiv score dbSNP ClinVar Interpretation Var ID Common to chemosensitive and chemoresistant EOC specimens chr4:55593464 A/C KIT M541L Benign (0.009) rs3822214 Benign/Likely benign​ 41599 chr4:55972974 T/A KDR Q472H Benign (0.003) rs1870377 Not provided 134603 chr7:116340262 A/G MET N375S Benign (0.038) rs33917957 Benign 41611 chr12:25398284 C/T KRAS G12D Benign (0.380) rs121913529 Pathogenic 12582 chr17:7579472 G/C TP53 P72R Benign (0.083) rs1042522 Benign 12351 chr19:1223125 C/G STK11 F354L Benign (0.025) rs59912467 Benign/Likely benign​ 7461 chr22:24134064 C/A SMARCB1 T72K Benign (0.008) rs1568936152 Not Reported in ClinVar n/a Chemosensitive EOC specimens only chr2:212576848 T/C ERBB4 S351G Probably Damaging (0.985) n/a n/a n/a chr3:178916876 G/A PIK3CA R88Q Probably Damaging (1.000) rs121913287 Pathogenic 376049 chr3:178927980 T/C PIK3CA C420R Probably Damaging (0.997) rs121913272 Pathogenic 31945 chr3:37067240 T/A MLH1 V384D Probably Damaging (1.000) rs63750447 Benign 41632 chr3:41266113 C/A CTNNB1 S37Y Probably Damaging (1.000) rs121913403 Pathogenic/Likely pathogenic​ 375894 chr4:55144628 C/T PDGFRA P701L Benign (0.027) n/a n/a n/a chr4:55972955 G/A KDR P479S Probably Damaging (0.999) n/a n/a n/a chr4:55979624 G/A KDR R275* n/a rs1720648779 Not Reported in ClinVar n/a chr5:112175952 A/- APC E1554fs n/a n/a n/a n/a chr7:116339662 G/A MET C175Y Probably Damaging (1.000) rs1584877055 Uncertain significance 948372 chr7:55211110 C/T EGFR A118V Probably Damaging (1.000) n/a n/a n/a chr7:55241701 G/A EGFR V717M Probably Damaging (0.998) n/a n/a n/a chr10:89692905 G/A PTEN R130Q Probably Damaging (1.000) rs121909229 Pathogenic 7829 chr10:89711899 C/T PTEN R173C Probably Damaging (1.000) rs121913293 Pathogenic 189500 chr10:89711910 T/G PTEN Y176* n/a rs1057522285 Likely pathogenic 492732 chr10:89720812 A/- PTEN N323fs n/a rs121913291 Likely pathogenic​ 928675 chr10:89720870 T/G PTEN F341V Probably Damaging (1.000) rs1554825652 Uncertain significance 495806 chr11:108137941 C/A ATM S837* n/a n/a n/a n/a chr13:48942677 G/T RB1 R355I Probably Damaging (0.959) n/a n/a n/a chr17:7577108 C/A TP53 C277F Probably Damaging (1.000) rs763098116 Uncertain significance​ 458567 chr17:7577570 C/A TP53 M237I Probably Damaging (1.000) rs587782664 Likely pathogenic 634770 Position Genotype Gene Change PolyPhen-2 HumDiv score dbSNP ClinVar Interpretation Var ID Chemosensitive EOC specimens only chr18:48591907 C/T SMAD4 S357F Probably Damaging (1.000) n/a n/a n/a chr18:48603030 A/T SMAD4 H444L Benign (0.109) n/a n/a n/a Chemoresistant EOC specimens only chr1:115256542 C/T NRAS D57N Probably Damaging (0.996) rs1465850103 Not Reported in ClinVar n/a chr3:178936091 G/A PIK3CA E545K Probably Damaging (0.970) rs104886003 Pathogenic/Likely pathogenic 13655 chr3:178952085 A/G PIK3CA H1047R Benign (0.263) rs121913279 Pathogenic 13652 chr4:153250867 G/A FBXW7 S398F Probably Damaging (1.000) n/a n/a n/a chr4:55953852 G/A KDR P1195L Possibly Damaging (0.780) rs748810441 Not Reported in ClinVar n/a chr9:139399411 CCA/- NOTCH1 V1578del n/a rs761020817 Not Reported in ClinVar  n/a chr10:89692902 G/A PTEN G129E Probably Damaging (1.000) rs121909218 Pathogenic 7812 chr17:7579437 C/- TP53 A84fs n/a rs1597374343 Pathogenic 646068 chr19:17954206 C/T JAK3 E135K Benign (0.004) n/a n/a n/a Discussion Based on a small follow-up prevalence study of protein-modifying FBXW7, KDR, NOTCH1 and PTEN variants seen exclusively in chemoresistant EOC (Supplementary Table B), no samples harbored the selected genetic variants, implying that these variants exist in low frequency i.e. sporadic. However, there were some interesting observations obtained from this study. KDR, which encodes for vascular epithelial growth factor receptor 2 or VEGFR-2, was found to be the gene with the most common alterations in all EOC specimens in this study. As KDR has the most significant prevalence of alterations in lung adenocarcinoma, colon adenocarcinoma, cutaneous melanoma (American Association for Cancer Research (AACR) Project GENIE Consortium, 2017), the role of KDR variants in epithelial ovarian cancer needs further scrutiny. Six of the KDR variants were already described in dbSNP, of which 4 did not have clinical significance yet as reported in ClinVar database. A missense mutation (genotype A>T) at chr4:55972974, which resulted in Q472H alteration of VEGFR-2, has been implicated in cancer susceptibility, but was not previously reported in ovarian cancer (Bodian et al., 2014). Two SNVs, one at chr4:55972955 and one at chr4:55973048 have not been deposited in dbSNP, thus this study is the first to report these mutations. In particular, the missense mutation at chr4:55972955 resulted in P479S alteration, which might increase cancer susceptibility. Of the protein-modifying genetic variants in chemoresistant EOC, three have been described in Personalized Cancer Therapy Knowledge Base for Precision Oncology. This knowledge base provides information on the functional effects of these genetic variants and their therapeutic implications (Dumbrava and Meric-Bernstam, 2018). A missense mutation in PIK3CA resulted in E545K alteration, an activating alteration associated with increased cell proliferation, colony formation, and invasiveness (Bader et al., 2006; Dogruluk et al., 2015; Gymnopoulos et al., 2007; Ikenoue et al., 2005; Kang et al., 2005; Ng et al., 2018; Samuels et al., 2005; Zhang et al., 2008). Another missense mutation in PIK3CA resulted in H1047R alteration within the kinase domain. The most frequently encountered alteration in somatic cancer results in the activation of PI3K/AKT/mTOR pathway to induce cell proliferation and survival, colony formation, and anchorage-independent growth (Berenjeno et al., 2017; Chang et al., 2016; Hart et al., 2015; Zhang et al., 2008). On the other hand, a missense mutation in PTEN resulted in G129E alteration deficient in lipid phosphatase activity (Furnari et al., 1998; Han et al., 2000; Leslie et al., 2007; Myers et al., 1998). This alteration, which has been reported in gliomas, endometrial cancers, melanomas as a somatic mutation, and Cowden syndrome as a germline mutation, is associated with colony formation and increased cell growth (Byron et al., 2008; Furnari et al., 1998; Hansen-Kiss et al., 2017; Steelman et al., 2008; Van Allen et al., 2014; Wang et al., 2000). The two PIK3CA alterations were responsive to PI3K/AKT/mTOR inhibitors, while the PTEN alteration was responsive to mTOR inhibitor rapamycin, implying an opportunity for targeted therapy given the resistance of cancerous lesions to platinum-based chemotherapy (Beaver et al., 2013; Elkabets et al., 2013; Garnett et al., 2012; Gonzalez-Angulo and Blumenschein, 2013; Janku et al., 2014; Liu et al., 2011; Mayer et al., 2014; Ng et al., 2018; Rashmi et al., 2014; Sangai et al., 2012; Steelman et al., 2008; Zhao et al., 2016). Two deletions were found in chemoresistant EOC specimens, both at exonic locations. A non-frameshift deletion at NOTCH1 gene resulted to deletion of one valine residue in the LVVVL sequence between residue 1570 and 1580. On the other hand, a deletion at TP53 gene resulted in a frameshift starting at residue 84 of p53. While TP53 mutations were long implicated in platinum chemoresistance in ovarian cancer (Reles et al., 2001), the relationship of Notch signaling with platinum chemoresistance has only been described previously in non-small cell lung cancer and colon cancer (Kukcinaviciute et al., 2018; Zhang et al., 2017). Of the genes with variants exclusively ascribed to chemoresistant EOC in this study, JAK3 is notable as mutations in JAK could affect downstream response of tumor cells to various interleukins such as IL-6 and IL-7 leading to platinum chemoresistance (Meng et al., 2020; Sun et al., 2019). Although our study includes a small sample of patients, to our knowledge, this is the first study conducted to study the genetic profiling of Filipino ovarian cancer patients outside of BRCA1 and BRCA2. As this is a descriptive study, we recommend functional studies looking into the effect of these unreported mutations not previously ascribed to cancer. Future direction includes conducting a prospective cohort study with larger sample size to identify potential prognostic and predictive biomarkers in conjunction with sonographic findings to help improve clinical outcomes in women with EOC. Author Contribution Statement A.J.P.P., E.F.L.C., I.Y. conceptualized and designed the study, with additional input from R.C.V.L.; A.J.P.P., Y.N., I.Y. performed the experiments and collected the data; R.C.V.L., A.J.P.P., Y.N., I.Y. analyzed the data; R.C.V.L., A.J.P.P., Y.N., E.F.L.C., I.Y. interpreted the results; R.C.V.L. prepared the initial manuscript; R.C.V.L., A.J.P.P., Y.N., E.F.L.C., I.Y. reviewed and approved the final version of manuscript. Acknowledgements General Two of the authors (R.C.V.L. and A.J.P.P.) are current and past trainees, respectively, of the MD-PhD in Molecular Medicine Program, supported by Department of Science and Technology - Philippine Council for Health Research and Development (DOST-PCHRD), and administered through University of the Philippines Manila. Funding Statement This study was supported by research grants from JSPS KAKENHI, Grant Number JP20H03564 (I.Y.). Approval This study was approved as an independent research work by the Technical Review Board of the Department of Obstetrics and Gynecology, College of Medicine and Philippine General Hospital, University of the Philippines Manila. Ethical Declaration This study underwent ethical review by the University of the Philippines Manila Research Ethics Board (UPMREB), and was performed in accordance with the principles stated in the Declaration of Helsinki. Data Availability Any information needed to reanalyze the data is available from the corresponding author upon reasonable request. Conflict of Interest All authors have no personal and/or financial conflicts of interest to declare. ==== Refs References Adzhubei IA Schmidt S Peshkin L A method and server for predicting damaging missense mutations Nat Methods 2010 7 248 9 20354512 American Association for Cancer Research (AACR) Project GENIE Consortium AACR Project GENIE: Powering Precision Medicine through an International Consortium Cancer Discov 2017 7 818 31 28572459 Bader AG Kang S Vogt PK Cancer-specific mutations in PIK3CA are oncogenic in vivo Proc Natl Acad Sci U S A 2006 103 1475 9 16432179 Beaver JA Gustin JP Yi KH PIK3CA and AKT1 mutations have distinct effects on sensitivity to targeted pathway inhibitors in an isogenic luminal breast cancer model system Clin Cancer Res 2013 19 5413 22 23888070 Berenjeno IM Piñeiro R Castillo SD Oncogenic PIK3CA induces centrosome amplification and tolerance to genome doubling Nat Commun 2017 8 1773 29170395 Bodian DL McCutcheon JN Kothiyal P Germline variation in cancer-susceptibility genes in a healthy, ancestrally diverse cohort: implications for individual genome sequencing PLoS One 2014 9 e94554 24728327 Buys SS Partridge E Black A Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial JAMA 2011 305 2295 303 21642681 Byron SA Gartside MG Wellens CL Inhibition of activated fibroblast growth factor receptor 2 in endometrial cancer cells induces cell death despite PTEN abrogation Cancer Res 2008 68 6902 7 18757403 Chang MT Asthana S Gao SP Identifying recurrent mutations in cancer reveals widespread lineage diversity and mutational specificity Nat Biotechnol 2016 34 155 63 26619011 De Leon Matsuda ML Liede A Kwan E BRCA1 and BRCA2 mutations among breast cancer patients from the Philippines Int J Cancer 2002 98 596 603 11920621 Dogruluk T Tsang YH Espitia M Identification of Variant-Specific Functions of PIK3CA by Rapid Phenotyping of Rare Mutations Cancer Res 2015 75 5341 54 26627007 Doubeni CA Doubeni AR Myers AE Diagnosis and Management of Ovarian Cancer Am Fam Physician 2016 93 937 44 27281838 Dumbrava EI Meric-Bernstam F Personalized cancer therapy-leveraging a knowledge base for clinical decision-making Cold Spring Harb Mol Case Stud 2018 4 a001578 29212833 Elkabets M Vora S Juric D mTORC1 inhibition is required for sensitivity to PI3K p110α inhibitors in PIK3CA-mutant breast cancer Sci Transl Med 2013 5 196ra199 Furnari FB Huang HJ Cavenee WK The phosphoinositol phosphatase activity of PTEN mediates a serum-sensitive G1 growth arrest in glioma cells Cancer Res 1998 58 5002 8 9823298 Garnett MJ Edelman EJ Heidorn SJ Systematic identification of genomic markers of drug sensitivity in cancer cells Nature 2012 483 570 5 22460902 Gonzalez-Angulo AM Blumenschein GR Defining biomarkers to predict sensitivity to PI3K/Akt/mTOR pathway inhibitors in breast cancer Cancer Treat Rev 2013 39 313 20 23218708 Gymnopoulos M Elsliger MA Vogt PK Rare cancer-specific mutations in PIK3CA show gain of function Proc Natl Acad Sci U S A 2007 104 5569 74 17376864 Han SY Kato H Kato S Functional evaluation of PTEN missense mutations using in vitro phosphoinositide phosphatase assay Cancer Res 2000 60 3147 51 10866302 Hansen-Kiss E Beinkampen S Adler B A retrospective chart review of the features of PTEN hamartoma tumour syndrome in children J Med Genet 2017 54 471 8 28526761 Hart JR Zhang Y Liao L The butterfly effect in cancer: a single base mutation can remodel the cell Proc Natl Acad Sci U S A 2015 112 1131 6 25583473 Ikenoue T Kanai F Hikiba Y Functional analysis of PIK3CA gene mutations in human colorectal cancer Cancer Res 2005 65 4562 7 15930273 Janku F Hong DS Fu S Assessing PIK3CA and PTEN in early-phase trials with PI3K/AKT/mTOR inhibitors Cell Rep 2014 6 377 87 24440717 Jelovac D Armstrong DK Recent progress in the diagnosis and treatment of ovarian cancer CA Cancer J Clin 2011 61 183 203 21521830 Kang S Bader AG Vogt PK Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic Proc Natl Acad Sci U S A 2005 102 802 7 15647370 Kent WJ Sugnet CW Furey TS The human genome browser at UCSC Genome Res 2002 12 996 1006 12045153 Konstantinopoulos PA Norquist B Lacchetti C Germline and Somatic Tumor Testing in Epithelial Ovarian Cancer: ASCO Guideline J Clin Oncol 2020 38 1222 45 31986064 Kukcinaviciute E Jonusiene V Sasnauskiene A Significance of Notch and Wnt signaling for chemoresistance of colorectal cancer cells HCT116 J Cell Biochem 2018 119 5913 20 29637602 Landrum MJ Lee JM Benson M ClinVar: improving access to variant interpretations and supporting evidence Nucleic Acids Res 2018 46 D1062 7 29165669 Leslie NR Yang X Downes CP Weijer CJ PtdIns(3,4,5)P(3)-dependent and -independent roles for PTEN in the control of cell migration Curr Biol 2007 17 115 25 17240336 Lheureux S Gourley C Vergote I Oza AM Epithelial ovarian cancer Lancet 2019 393 1240 53 30910306 Liu R Liu D Trink E The Akt-specific inhibitor MK2206 selectively inhibits thyroid cancer cells harboring mutations that can activate the PI3K/Akt pathway J Clin Endocrinol Metab 2011 96 577 85 Mayer IA Abramson VG Isakoff SJ Stand up to cancer phase Ib study of pan-phosphoinositide-3-kinase inhibitor buparlisib with letrozole in estrogen receptor-positive/human epidermal growth factor receptor 2-negative metastatic breast cancer J Clin Oncol 2014 32 1202 9 24663045 Meng J Liu K Shao Y ID1 confers cancer cell chemoresistance through STAT3/ATF6-mediated induction of autophagy Cell Death Dis 2020 11 137 32080166 Momenimovahed Z Tiznobaik A Taheri S Salehiniya H Ovarian cancer in the world: epidemiology and risk factors Int J Womens Health 2019 11 287 99 31118829 Myers MP Pass I Batty IH The lipid phosphatase activity of PTEN is critical for its tumor supressor function Proc Natl Acad Sci U S A 1998 95 13513 8 9811831 Ng PK Li J Jeong KJ Systematic Functional Annotation of Somatic Mutations in Cancer Cancer Cell 2018 33 450 62 29533785 Que FVF Ang D Andal JJ Incidence of germline BRCA1 and BRCA2 mutations among Filipinos J Clin Oncol 2018 36 e13628 Rashmi R DeSelm C Helms C AKT inhibitors promote cell death in cervical cancer through disruption of mTOR signaling and glucose uptake PLoS One 2014 9 e92948 24705275 Reles A Wen WH Schmider A Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in ovarian cancer Clin Cancer Res 2001 7 2984 97 11595686 Samuels Y Diaz LA Schmidt-Kittler O Mutant PIK3CA promotes cell growth and invasion of human cancer cells Cancer Cell 2005 7 561 73 15950905 Sangai T Akcakanat A Chen H Biomarkers of response to Akt inhibitor MK-2206 in breast cancer Clin Cancer Res 2012 18 5816 28 22932669 Sherry ST Ward MH Kholodov M dbSNP: the NCBI database of genetic variation Nucleic Acids Res 2001 29 308 11 11125122 Steelman LS Navolanic PM Sokolosky ML Suppression of PTEN function increases breast cancer chemotherapeutic drug resistance while conferring sensitivity to mTOR inhibitors Oncogene 2008 27 4086 95 18332865 Stuart GC Kitchener H Bacon M 2010 Gynecologic Cancer InterGroup (GCIG) consensus statement on clinical trials in ovarian cancer: report from the Fourth Ovarian Cancer Consensus Conference Int J Gynecol Cancer 2011 21 750 5 21543936 Sun CY Nie J Huang JP Zheng GJ Feng B Targeting STAT3 inhibition to reverse cisplatin resistance Biomed Pharmacother 2019 117 109135 31226634 Torre LA Trabert B DeSantis CE Ovarian cancer statistics, 2018 CA Cancer J Clin 2018 68 284 96 29809280 Van Allen EM Wagle N Sucker A The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma Cancer Discov 2014 4 94 109 24265153 Wang X Gjörloff-Wingren A Saxena M The tumor suppressor PTEN regulates T cell survival and antigen receptor signaling by acting as a phosphatidylinositol 3-phosphatase J Immunol 2000 164 1934 9 10657643 Zhang H Liu G Dziubinski M Comprehensive analysis of oncogenic effects of PIK3CA mutations in human mammary epithelial cells Breast Cancer Res Treat 2008 112 217 27 18074223 Zhang Y Xu W Guo H NOTCH1 Signaling Regulates Self-Renewal and Platinum Chemoresistance of Cancer Stem-like Cells in Human Non-Small Cell Lung Cancer Cancer Res 2017 77 3082 91 28416482 Zhao S Cao Y Liu SB The E545K mutation of PIK3CA promotes gallbladder carcinoma progression through enhanced binding to EGFR J Exp Clin Cancer Res 2016 35 97 27317099

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116130 10.31557/APJCP.2023.24.4.1113 Research Article Comparing the Effects of Sesame Oil vs. Nitroglycerin Ointment on the Incidence of Chemotherapy-Induced Phlebitis: A Single Blind Clinical Trial Safikhani Mohammadzadeh Neda 1 Elahi Nasrin 1* Molavynejad Shahram 1 Maraghi Elham 2 Ehsanpor Ali 3 1 Nursing Care Research Center in Chronic Diseases, School of Nursing & Midwifery, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 2 Department of Biostatistics and Epidemiology, Faculty of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 3 School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. * For Correspondence: elahi-n@ajums.ac.ir 2023 24 4 11131117 24 9 2021 24 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Phlebitis is a severe inflammatory response in patients undergoing chemotherapy that can lead to complications and increased length of hospitalization. Objective: This study was conducted to examine the effects of sesame oil and nitroglycerin ointment on the incidence of chemotherapy-induced phlebitis in patients with cancer. Methods: This clinical trial study involved 138 cancer patients who were randomly assigned into three groups. The three groups received nitroglycerin ointment, sesame oil, or betadine alcoholic solution that were applied on the distal catheter area at a length of 1.5 centimeters and width of 2 × 4 cm using graded paper. The site was then dressed and fixed with anti-allergenic adhesives. The research samples were examined for 72 hours for the incidence of phlebitis. Results: No statistically significant difference was observed between the incidence of phlebitis in the sesame oil, nitroglycerin ointment and alcohol-betadine groups in the first 24 hours (p=0.2), the second 24 hours (p=0.13) and the third 24 hours (p=0.13). Conclusion: External use of both sesame oil and nitroglycerin is effective in reducing chemotherapy-induced phlebitis. Due to its anti-inflammatory effect and low cost, however, using sesame oil is recommended. Key Words Phlebitis nitroglycerin sesame oil chemotherapy nursing ==== Body pmcIntroduction Cancer is a disease process that results from abnormal or acquired mutations causing abnormal cell behavior, and a set of different causes are involved with different manifestations, treatments, and prognoses (Wing and Schiffman, 2022). Chemotherapy as a cancer treatment has an important role in extending the lifespan of patients. However, chemotherapy drugs have multiple adverse effects. One of these adverse effects is phlebitis (Harris et al., 2020). Chemical phlebitis causes symptoms such as redness, erythema, pain, swelling, fever, as well as hardening and sclerosis of the vein, resulting in a palpable venous cord and thrombosis of the upper extremity veins (Bigdeli Shamloo et al., 2015, Harris et al., 2020). Currently, pharmacological and non-pharmacological treatments are being recommended to prevent or reduce the severity of phlebitis symptoms. Therapeutic approaches such as performing chemotherapy through a port (Mosavi et al., 2020), saline lock (Eghbali-Babadi et al., 2015), topical nitroglycerin (Avaze et al., 2004), or other methods such as rapid injection and dilution of chemotherapy agents, immediate catheter removal, heparin intermittent flushing, prophylactic antibiotics, transparent dressings, topical application of anti-inflammatory agents or corticosteroids, and application of a hot and/or wet compress (Annisa et al., 2017, Marsh et al., 2015). Herbal medicines such as sesame oil (Bigdeli Shamloo et al., 2015) and aloe vera (Sadoyu et al., 2021) are also used for this purpose. The published data shows that SO contains predominantly mono and polyunsaturated fatty acids (70–80 %) and 2–3% of unsaponifiable matter such as lignans (0.2 to 0.4 %), tocopherols, phytosterols and phenols. The health benefits of SO are associated with sesame lignans such as sesamol and sesamin, tocopherols and phytosterols due to their anti-oxidant properties. These lipophilic molecules (lignans, tocopherols and phytosterols) have been reported to be present in organic solvents (methanol, ethyl acetate, acetone, chloroform and hexane), extracts from SO, sesame seed (SS), or defatted SO (Deme et al., 2018). However, no definitive methods have been proposed to treat and prevent chemotherapy-induced phlebitis (Kohno et al., 2015). Therefore, there is a dire need for appropriate, low-cost, and novel methods to prevent or alleviate the symptoms associated with chemotherapy-induced phlebitis. The present study aimed to compare the effects of nitroglycerin ointment versus sesame oil on the incidence of chemotherapy-induced phlebitis in cancer patients. Materials and Methods Study design This was a single-blind randomized, controlled, parallel-group clinical trial. Participants and setting This study was conducted on 138 patients admitted to the oncology wards of Shaid Baghaei Hospital affiliated to Avahz Jundishapur University of Medical Sciences, Ahvaz, southwest of Iran, from May to October 2018. The inclusion criteria were as follows: being aged between 18-65 years old, having healthy upper extremities (absence of phlebitis), experiencing minimum hospital stay of 72 hours, having healthy and natural skin around the venipuncture site, having no history of dermatological and cardiovascular diseases, not having any combination therapies (i.e., chemotherapy in addition to radiotherapy or surgery), having no history of diabetes, vascular diseases, musculoskeletal diseases, verbal or mental disorders, autoimmunity, any signs of fever and neutropenia, allergic reactions to herbal oils, addiction to drugs or alcohol, taking no antibiotics, analgesics, and narcotics for pain relief, drugs or herbal remedies to treat phlebitis, and absence of port catheter to receive chemotherapy. The participants were excluded from the study if they: wished to withdraw from the study at any phase of the intervention for clinical or personal reasons, missed more than two follow-up sessions during the intervention, and experienced any signs of allergy to SO (sesame oil) or Nitroglycerin Ointment 2 % (NO) during the trial. The sample size was estimated based on a recent trial (Bigdeli Shamloo et al., 2015) in which the optimal sample size was estimated to be 41 patients per group at a confidence level of 95% and power of 0.80. Finally, considering a 10% attrition rate, the sample size was determined to be 46 patients for each group. Randomization Due to the nature of this study, blinding of the researchers and participants was not possible. A statistician who was not involved in the study prepared a randomized allocation table. Patients were randomly assigned (1:1) to receive sesame oil, nitroglycerin ointment, or betadine alcoholic solution using randomized permuted blocks (block size 6). After randomization, the codes allocated to each participant were kept by the oncology ward clerk for preserving allocation concealment. Therefore, neither the participant nor the researcher was aware of the allocation order until the commencement of the intervention (Fig 1). Ethical Considerations This study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (Ref. ID: IR.AJUMS.REC.1396.555). Ethical considerations were observed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. The study was registered in the Iranian Registry of Clinical Trials (No. IRCT20171203037737N1). Intervention After washing hands and wearing latex gloves, the researcher performed venipuncture on all participants. To make the intervention convenient, all catheters were placed in the anterior forearm and backside of the participants’ hands. At a physician’s discretion, the nitroglycerin ointment (made by Zawaira Co. in Poland) or Varuna sesame oil (made by Kimiagar Company in Kerman, Iran; with the license number of 4408 issued by Iran’s Ministry of Health and Medical Education) was then applied. After aseptically prepping the site, nitroglycerin or sesame oil ointment was applied to the distal catheter area: 1.5 cm in depth and in an area of 2 x 4 cm, using a graded paper. A sterile dressing was placed on it and was secured using hypoallergenic glue. The selection of the distal catheter area is due to the fact that ointments are more likely to enter the vessels thanks to the penetration of the angiocath needle into the vessels, which increases the possibility of severe side effects of ointments, especially nitroglycerin ointment. Even the simultaneous application of nitroglycerin ointment and alcohol can cause a drop in blood pressure once every 12 hours (in a 72-hour period). In case of drug leakage into the patient’s vein and presentation of symptoms of phlebitis such as pain, the patients were excluded from the study. Data Collection The data collection tool in this study included two sections. The first section elicited demographic information (gender, age, occupation, and education) and information about the absolute number of neutrophils, the duration of the disease, the duration of chemotherapy, and the chemotherapy regimen (type, dosage, frequency, and administration). The second section included a checklist to record the frequency of phlebitis in the first 24 hours, the second 24 hours, and the third 24 hours after the intervention. Data Analysis All tests were two-sided and p<0.05 was considered statistically significant. The Kolmogorov–Smirnov was used to examine the normal distribution of continuous variables. Categorical data were summarized as numbers (percentages). Continuous variables were reported as mean±SD. Kruskal-wallis test, Chi-square and Fisher’s exact tests were applied to compare study variables across the three groups. Friedman test was applied to examine the proportion of phlebitis over time in each groups. Results Demographic and clinical characteristics The differences between the experimental and control groups in terms of demographic and clinical information are shown in Table 1. Fifty-six participants (40.6%) were female, and 82 (59.4%) were male. The age range of the participants was 46.93 ± 14.70, and most of them were aged 44-55 years old. Venipuncture was done on the backside of 75 participants’ hands (54.3%) and in the forearms of 63 other participants (45.7%). The Kruskal-Wallis test results indicated no significant differences between the three groups in terms of demographic information such as age group, gender, duration of illness, and length of hospital stay. These findings showed that diabetes (n=9; 35.5%) and hypertension (n=25; 18.11%) were more prevalent among the participants. Results of the chi square test revealed no statistically significant difference between the incidence of phlebitis in the sesame oil, nitroglycerin ointment and alcohol-betadine groups in the first 24 hours (p=0.2), the second 24 hours (p=0.13) and the third 24 hours (p=0.13). Figure 1 Consort Flowchart of Randomization Table 1. Demographic and Medical Details of Patients in the Intervention and Control Groups Variable Alcohol swabs Nitroglycerin Sesame oil Total P Age(year) 45.80± 15.83 47.81± 13.81 46.90±14.73 46.93±14.70 0.899k gender Male 29.(63.0%) 28 (60.9%) 25 (54.3%) 82 (59.4%) 0.677c Female 17 (37.0%) 18 (39.1%) 21 (45.7%) 56 (40.6%) Injection location Hand 25 (54.3%) 28 (60.9%) 22 (47.8%) 75 (54.3%) 0.454c Forearms 21 (45.7%) 18 (39.1%) 24 (52.2%) 63 (45.7%) c, Chi-square test; K, Kruskal-Wallis test. Table 2 Comparison of Phlebitis in the Groups Treated with Alcohol Swabs, Nitroglycerin Ointment and Sesame Oil Time Alcohol swabs Nitroglycerin Sesame oil P¥ Number Percent Number Percent Number Percent First 24 hours 3 6.5 0 0 0 0 0.04 Second 24 hours 1 2.2 1 2.2 0 0 0.6 Third 24 hours 0 0 0 0 0 0 - Pair-wise comparisons. First 24 hours Second 24 hours Third 24 hours Alcohol-Nitroglycerin 0.242 > 0.99 - Alcohol- Sesame oil 0.242 > 0.99 - Nitroglycerin- Sesame oil NA > 0.99 - ¥Chi-square test. Discussion This study showed that the use of sesame oil and nitroglycerin 2% had no positive effect on the incidence of chemotherapy-induced phlebitis. Hence, sesame oil and nitroglycerin ointment had the same effects on the chemotherapy-induced phlebitis. According to the findings, there were no significant differences between the three groups in terms of age (P= 0. 899), and gender (P=0.677). Although some phlebitis symptoms were observed in the groups treated with routine alcohol swabs and nitroglycerin ointment in the first and second 24-hour periods, no signs of phlebitis were observed in the group treated with sesame oil during the three 24-hour periods after venipuncture. However, there were no significant differences between the three groups in terms of the incidence of phlebitis. In the first 24-hour period, three cases of phlebitis were observed in the group treated with alcohol swabs. In the second 24-hour period, one case of phlebitis was found in the group treated with alcohol swabs, and whereas one case was detected in the nitroglycerin ointment group. Nevertheless, there were no signs of phlebitis in the sesame group in all the three follow-up periods. Nor were there any significant differences between the frequency of phlebitis symptoms and the use of routine alcohol swabs, nitroglycerin ointment, or sesame oil. Although the research results indicated that there were no correlations between the severity and incidence of phlebitis in all the three studied groups during the three 24-hour periods (P-value> 0.05), the absence of phlebitis in the sesame oil group could exert beneficial effects on the incidence of phlebitis. Consistent with this finding, Bigdeli et al., 2015 indicated that the application of sesame oil was efficient in reducing the symptoms and severity of phlebitis in patients. Bagheri et al., 2015 also reported that the sesame oil significantly reduced the incidence of phlebitis in their studied patients. The sesame oil can protect the skin because it contains the essential fatty acids of phytosterols and antioxidants (Borchani et al., 2010). The sesame oil can also be absorbed through the skin; therefore, it plays a key role in preventing phlebitis (Shafipour et al., 2017). Regarding the effects of nitroglycerin ointment on phlebitis, a previous study was conducted to compare the effects of clobetasol and nitroglycerin ointments on the incidence of angiocath-induced superficial phlebitis. They reported that nitroglycerin ointment reduced the catheter-induced phlebitis and recommended it to be used to prevent the incidence of phlebitis in patients requiring the long-term use of angiocath (Akbari et al., 2014). Avazeh et al., 2004 analyzed the effects of topical application of nitroglycerine on the incidence and severity of catheter-induced phlebitis. They reported the beneficial effects of nitroglycerin on phlebitis and recommended it to be used at venipuncture sites where catheters needed to be kept for more than 48 hours (Avaze et al., 2004). The research findings showed that the effects of nitroglycerin ointment were not significantly different from those of the routine alcohol swabs and sesame oil. However, only one case of sensitivity was observed in the second 24 hours after the nitroglycerine ointment was used. This finding confirmed the results reported by Akbari et al., 2014 and Avazeh et al., 2004 with respect to the ability of nitroglycerin ointment to reduce phlebitis in patients requiring long-term catheters . According to the literature, there has been a significant reduction in the number of patients with phlebitis after catheter insertion in recent years. This may be due to the use of new hand rap solutions on the part of nurses as well as the training and emphasis on proper hand washing procedures, which have been implemented in hospitals as a patient safety principle in recent years. Given the complications of using alcohol swabs, Betadine alcoholic solution, and nitroglycerine ointment along with the high costs of using these substances compared with sesame oil, it is recommended to use sesame oil to treat patients with skin problems. Nurses should also be encouraged to use herbal remedies to prevent or reduce the severity of chemotherapy-induced phlebitis. The use of various ointments is also suggested for the management of phlebitis. In addition, clinicians can teach the patients and their families how to use herbal medicines, especially sesame oil, as confirmed in several studies. In conclusion, this study showed that using sesame oil, as opposed to alcohol, had a positive effect on the incidence and severity of chemotherapy phlebitis. Furthermore, sesame oil and nitroglycerin ointment left the same effects on chemotherapy phlebitis. Therefore, sesame oil can be used as a safe method to deal with chemotherapy-induced phlebitis. One limitation of this study was the small number of patients hospitalized for up to 72 hours. Author Contribution Statement Research conception and design: NS, NE, SM, EM and AE. Data collection: NS. Analysis and interpretation of data: NS and EM. Drafting of the manuscript: NS, NE, SM, EM and AE. Review and editing: NS, NE, SM, EM and AE. All authors contributed to the article and approved the submitted version. Acknowledgements This study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (Ref. ID: IR.AJUMS.REC.1396.555). The study was registered in the Iranian Registry of Clinical Trials (No. IRCT20171203037737N1). Funding This article was extracted from an MSc. thesis in nursing which was financially supported by the Research Deputy of Ahvaz Jundishapur University of Medical Sciences. The funder had no role in the design of the study, nor in the collection, analysis, and interpretation of the data or in writing the manuscript. Conflict of Interest All authors declared that there are no conflicts of interest. ==== Refs References Akbari H Raufi S Hekmat po D Anbari K Comparaing the effect of Nitroglycerin and Clobetazol Ointments on Prevention of Superficial Intravenous Catheter Induced Phlebitis Evide Based Care 2014 4 71 80 Annisa F Nurhaeni N Wanda D Warm Water Compress as an Alternative for Decreasing the Degree of Phlebitis Compr Child Adolesc Nurs 2017 40 107 13 29166201 Avaze A Elahi N Zaker M Rasekh A HajiHoseini F Effect of Topical Nitroglycerin on the Occurence and Severity of phlebitis due to Indwelling Intravenous Catheter in Hospitalized Patients J Adv Med Biomed Res 2004 12 15 23 Bagheri-Nesami M Shorofi S Hashemi-Karoie S Khalilian A The effects of sesame oil on the prevention of amiodarone-induced phlebitis Iran J Nurs Midwifery Res 2015 20 365 70 26120338 Bigdeli Shamloo MB Nasiri M Dabirian A The Effects of Topical Sesame (Sesamum indicum) Oil on Pain Severity and Amount of Received Non-Steroid Anti-Inflammatory Drugs in Patients With Upper or Lower Extremities Trauma Anesth Pain Med 2015 5 e25085 26161326 Borchani C Besbes S Blecker C Attia H Chemical Characteristics and Oxidative Stability of Sesame Seed, Sesame Paste, and Olive Oils J Agr Sci Tech 2010 12 585 96 Deme P Narasimhulu CA Parthasarathy S Identification and evaluation of anti-inflammatory properties of aqueous components extracted from sesame (Sesamum indicum) oil J Chromatogr B Analyt Technol Biomed Life Sci 2018 1087 61 9 Eghbali-Babadi M Ghadiriyan R Hosseini SM The effect of saline lock on phlebitis rates of patients in cardiac care units Iran J Nurs Midwifery Res 2015 20 496 501 26257807 Harris V Hughes M Roberts R Dolan G Williams EM The Development and Testing of a Chemotherapy-Induced Phlebitis Severity (CIPS) Scale for Patients Receiving Anthracycline Chemotherapy for Breast Cancer J Clin Med 2020 9 701 32150833 Kohno E Kanematsu S Okazaki S Methods of preventing phlebitis induced by infusion of fosaprepitant Gan To Kagaku Ryoho 2015 42 323 6 25812501 Marsh N Webster J Mihala G Rickard CM Devices and dressings to secure peripheral venous catheters to prevent complications Cochrane Database Syst Rev 2015 12 Cd011070 Mosavi SH Elahi N Asadizaker M Ahmadzadeh Deilami A Comparison of Care Outcomes Between Two Methods of Drug Injection Through Ports and Peripheral Veins in Patients Undergoing Chemotherapy Jundishapur J Chronic Dis Care 2020 9 e99315 Nekuzad N Ashke Torab T Mojab F Effect of External Use of Sesame Oil in the Prevention of Chemotherapy-Induced Phlebitis Iran J Pharm Res 2012 11 1065 71 24250538 Sadoyu S Rungruang C Wattanavijitkul T Aloe vera and health outcomes: An umbrella review of systematic reviews and meta-analyses Phytother Res 2021 35 555 76 32924222 Shafipour SZ Mosayebi N Asgari F Atrkarroushan Z Pasdaran A Effect of External Use of Sesame Oil in the Prevention of Chemotherapy-Induced Phlebitis in Children with Acute Lymphoblastic Leukemia(ALL) Iran J Ped Hematol Oncol 2017 7 198 206 Wing EJ Schiffman JF Cecil’s Essentials Medicine: oncologic diseases 2022 10th ed. edn Philadelphia Elsevier USA

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116157 10.31557/APJCP.2023.24.4.1343 Research Article Comparison of DPPIV Levels in Serum and Tumour of OSCC Patients and Its Correlation with Active Matrix Metalloproteinases 2 and 9 Talebi Taheri Abdolkarim 1 Lashkarbolouki Taghi 2 Karimi Abbas 3 Sirati-Sabet Majid 1 Karima Saeed 1 Goudarzi Afsaneh 1* 1 Department of Clinical Biochemistry, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2 School of Biology, Damghan University, Damghan, Iran. 3 Department of Oral and Maxillofacial Surgery, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran. * For Correspondence: Afsaneh.goudarzi@sbmu.ac.ir 2023 24 4 13431349 13 12 2022 24 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Introduction: The important role of Dipeptidyl Peptidase IV (DPPIV) has been reported in tumour progression of several human cancers. This study demonstrates the DPPIV mRNA expression level and activity in tumour and paired non-tumour tissues of oral squamous cell carcinoma (OSCC) patients and the potential modulation of DPPIV in the metastasis of tumour through regulating MMP2 and MMP9 activities. Materials and Methods: This study was conducted on 16 OSCC patients. The mRNA expression level of DPPIV was evaluated by RT-qPCR in tumour of OSCC patientsand compared with their paired non-tumour tissues. Additionally, DPPIV activity was measured in serum, tumour and paired non-tumour tissues of OSCC patients. Zymography was performed to measure and compare the activities of MMP2 and MMP9 between tumour and paired non-tumour tissues of OSCC patients. Results: The results showed significantly higher DPPIV mRNA level and activity in tumour of OSCC patients compared to their paired non-tumour tissues. Tumour DPPIV mRNA expression and activity were positively correlated with activities of MMP2 and MMP9, respectively. Serum DPPIV activity of OSCC patients was lower compared to healthy control and did not show correlation with tumour DPPIV mRNA level. Conclusion: These data indicate that secreted DPPIV may not originate from the tumour tissue of OSCC patients. Furthermore, increased DPPIV gene expression and activity in tumour of OSCC patients might be involved in the ECM degradation and invasion of OSCC through regulation of MMP2 and MMP9 activities. Key Words Oral Squamous Cell Carcinoma Dipeptidyl peptidase IV MMP ==== Body pmcIntroduction Head and neck squamous cell carcinoma (HNSCC) originates from mucosal epithelial cells and is the sixth most common cancer in the world, with 450,000 deaths and 890,000 new cases reported in 2018 (Johnson et al., 2020; Kiani et al., 2020). About half of HNSCC occurs in the oral cavity, of which OSCC is the most common form of oral cancer and has a high mortality rate and poor prognosis (Heroiu Cataloiu et al., 2013). OSCC arises in different sites of the oral cavity, including the floor of the mouth, the basal mucosa, the alveolar ridges, the retromolar trigone of the upper and lower gums, and the hard palate (Nazarian et al., 2019; Lin et al., 2021). Despite advances in cancer diagnosis and treatment, the overall 5-year survival rate for OSCC is the lowest among malignancies (Rashid et al., 2018; Sasahira and Kirita, 2018). Most patients with OSCC are diagnosed in late stages III or IV, which significantly reduces the chance of survival (Tsai et al., 2021). The invasion and metastasis of cancer cells rely on the activation of different proteolytic systems which modulate degradation and reorganization of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are key mediators of matrix degradation and tumour aggressiveness. MMP-2 and MMP-9 have gelatinase activity which cleave the basement membrane components, type IV collagen, elastin and laminin (Björklund and Koivunen, 2005). Upregulated expression and activity of fibroblast activation protein-α (FAP-α), DPPIV and MMps have been reported in esophageal cancer (Augoff et al., 2014). DPPIV is one of a six-member family of serine proteases that have amino peptidase activity after proline (Chen and Kelly, 2003). This enzyme is a transmembrane homodimeric glycoprotein complex with a molecular weight of 200 kDa whose gene is located on chromosome 2 (Chen and Kelly, 2003; Augoff et al., 2014). DPPIV as a multifunctional enzyme is involved in multiple cellular processes including cell growth, migration, invasion, angiogenesis, and immune activation (Boonacker and Van Noorden, 2003). These various functions of DPPIV depend on its intracellular or extracellular localization, ligand concentrations, cell type and cofactor (Boonacker and Van Noorden, 2003). Proteolytic cleavage of type I and IV collagens by DPPIV and MMPs, respectively, alter the structure and mechanics of extracellular scaffolds and facilitates metastasis and cell migration (Augoff et al., 2014). Additionally, HNSCC and colorectal studies have shown that DPPIV increases the expression of MMPs and its high level associates with increased levels of MMP2, MMP9, MMP13 and MT1-MMP, leading to invasion, metastasis and poor prognosis (Augoff et al., 2014; Bishnoi et al., 2019). Taking into account the various functions of DPPIV, the opposite effects of DPPIV have been shown in different cancers. For instance, overexpression of DPPIV has been shown to suppress the invasive potential of ovarian cancer cells whereas increased expression of DPPIV has been reported in prostate and thyroid cancers (Kotani et al., 1991; Wilson et al., 2000a; Kajiyama et al., 2003; Vitório et al., 2020). DPPIV inhibitors used by lung and colorectal cancer patients were associated with improvement of their overall survival, the mechanism underlying this improvement could be through its effect on cancer immunoregulation (Bishnoi et al., 2019). However, other studies have shown that the use of DPPIV increase increase increase the risk of metastasis in the cell lines of the colon, liver, lung, ovary and melanoma (Bishnoi et al., 2019). Considering the importance of DPPIV in cancer biology and its possible correlation with clinicopathological features of OSCC patients, we report DPPIV mRNA expression and activity in tumour and paired non-tumour tissues as well as DPPIV activity in the serum of OSCC patients. We also evaluate whether the serum DPPIV could originate from tumour tissue of OSCC patients. In addition, we investigate the correlation between DPPIV gene expression/activity and MMP2 and MMP9 activity. Materials and Methods Subjects and study design A total of 32 tissue (16 tumour and 16 paired non-tumour tissues) and serum from OSCC (n = 24) and healthy individuals (n = 16) were collected for this study, OSCC patients who went under surgery at Bahman and Shariati hospital of Tehran, Iran. Healthy subjects of control group for serum study were age and gendermatched without any history of major illness in the past. Blood samples were obtained from all the participants before surgery. Blood was collected into sterile BD Vacutainer tubes. Next, the tubes were centrifuged at 3000 rpm for 10 min and serum was separated immediately and stored at -70°C until use for DPPIV activity analysis. All of the tumours were diagnosed as grade I to III according to the WHO criteria. Subjects with diabetes, liver and autoimmune diseases were excluded. Informed consent from all patients for the analysis of their tissues was collected before surgery. This study was approved by the ethics committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1400.607). Table 1 represents the methods used for the evaluation of DPPIV in serum and tissue samples of OSCC patients. Zymography The extraction of total protein from frozen tissues was performed by homogenization 1:10 w/v in RIPA buffer supplemented with PMSF, sodium orthovanadate and protease inhibitors. Next, the homogenates were centrifuged for 15min at 12000 rpm at 4oC and the supernatant was collected. The protein concentration was determined with the Bradford method. For determining of gelatinolytic activity of the MMP2 and MMP9, substrate gel SDS-PAGE zymography was performed according to (Toth et al., 2012) with the following modifications: A total of 40 µg protein of each sample with none-reducing sample buffer 4x containing 0.25 M Tris-HCl (pH = 6.8) with 40% glycerol, 8% SDS and 0.01% bromophenol blue, was loaded in 10% SDS-polyacrylamide gel copolymerized with gelatin 0.1%. After electrophoresis, the enzymes were renatured by washing SDS out twice with a renaturing solution containing 2.5% Triton X-100 for 30 min at room temperature (RT). Next, the gel was incubated in developing buffer containing 50 mM Tris-HCl (pH = 7.5), 200 mM NaCl, 5 mM CaCl2, 1 µM ZnCl2 and 0.02% Brij-35 for 30 min at RT which was followed by another incubation in developing buffer at 37˚C overnight. To visualize the proteolytic bands, 5% v/v methanol, 10% v/v acetic acid, and 0.5% Coomassie blue R-250 were used to stain the gel, which was then destained with 10% acetic acid and 5% methanol. Densitometric measurements were conducted using an ImageJ gel analyzer to record the intensity of the pixels (inverted) of the bands. RNA isolation and real-time quantitative q-PCR (RT-qPCR) Total RNA was isolated from tumour and paired non-tumour tissues of OSCC patients using Trizol reagent (GENE All) and cDNA was synthetized with a First Strand cDNA Synthesis Kit (Takara, Japan) according to the manufacturer’s protocol. RT-qPCR was performed using Ampliqon master mix on StepOne Plus RT-PCR system. GAPDH was used as internal control. Each sample was performed duplicate and the data was normalized to GAPDH. Expression levels have been determined using 2-ΔΔCt method. The sequences of primers were as follows: DPPIV forward: 5’-AGTGGCGTGTTCAAGTGTGG-3’ and DPPIV reverse: 5’-CAAGGTTGTCTTCTGGAGTTGG-3’ and GAPDH forward: 5’-GCTCAGACACCATGGGGAAG-3’ and GAPDH reverse 5’-TGTAGTTGAGGTCAATGAAGGGG-3’. DPPIV activity in tumour, non-tumour and serum of OSCC patients DPPIV activity was measured colorimetrically using glycyl-prolyl-paranitroanilide (Gly-Pro-pNA) substrate based on method described in (Matheeussen et al., 2012). Briefly, 0.5 ml of 100 mM Tris-HCl (pH = 8) was added to 30-50 mg of tumour and non-tumour tissues and incubated on ice, then homogenates were centrifuged for 12min at 12,000 rpm at 4°C. After collection of the supernatant, 0.5 ml of 100 mM Tris-HCl (pH = 8) containing 2% Triton X-100 (Pierce) was added to the pellet and vortexed. Suspension was centrifuged for 12 min at 12,000 rpm, at 4°C. 10 μl of serum and supernatants recovered from tissue samples were incubated with substrate Gly-Pro-pNA 0.5 mM, in 50 mM Tris buffer (pH = 8.3) and the kinetic activity of DPPIV was assessed immediately by measuring the velocity of pNA release (405 nm) from the chromogenic substrate for 15min at 37°C. Based on the total protein content of the tissue, DPPIV activity was measured and expressed as nanomoles of substrate converted per milligram protein of tissue per minute. Statistical analysis The data was analyzed using GraphPad Prism version 6 and presented as an average standard deviation (SD). Data normality was evaluated by the Shapiro–Wilk test. Statistical analysis was done using the paired student’s t-test. The correlation coefficients were calculated based on Spearman’s correlation two tailed analysis. P-values <0.05 were considered statistically significant in all cases. Results Tissue DPPIV mRNA expression and activity To determine if the tumour and paired non-tumour tissues of OSCC patients could present difference in DPPIV gene expression, we performed RT-qPCR. As observed, tumour tissues presented higher DPPIV mRNA level (p = 0.0056) and activity (p = 0.0013) compared to their paired non-tumour tissues (Figures 1A and B). Next, we questioned if DPPIV mRNA level correlates with the enzyme activity, the results did not reveal correlation between expression level of DPPIV mRNA and its activity in tumour tissue of OSCC patients (Table 2). Serum DPPIV activity The serum DPPIV activity was significantly lower in OSCC patients compared to the normal group (p = 0.0354) (Fig. 1C). Next, we questioned if the higher tumour DPPIV mRNA and activity correlate with the serum DPPIV, in another words if the serum DPPIV could originate from OSCC tumour, the results obtained were against this correlation (r = 0.025, p = 0.943) (Table 2). Tissue enzyme activity of MMP2 and MMP9 The zymography data obtained from tumour and paired non-tumour of 16 OSCC patients showed higher active MMP2 (p = 0.0067) and MMP9 (p = 0.0002) in OSCC tumour tissues compared with their paired non-tumour tissues (Figures 2B and C). Correlation between tissue DPPIV mRNA level and activity of active MMP-2 and MMP-9 In this study, we have measured the tumour DPPIV mRNA level and activity and also analyzed whether DPPIV gene expression and activity is associated with the active form of MMP-2 and MMP-9. Table 2 shows that DPPIV activity in tumour tissues correlates positively with active-MMP-2 (r = 0.436, p = 0.013) and active MMP-9 activity (r = 0.448, p = 0.01). Additionally, tumour DPPIV mRNA expression was correlated only with active-MMP2 activities (r = 0.504, p = 0.049). Correlation between DPPIV mRNA level, DPPIV activity, MMP2 and MMP9 with clinical features of OSCC patients Bivariate correlation analysis of DPPIV gene expression, DPPIV activity and the levels of active-MMP2 and active-MMP9 with OSCC patients’ clinical features including age, gender, tumour size, grade and tumour location is shown in Table 3. There was no significant association between DPPIV mRNA level, DPPIV activity, active MMP2 and active MMP9 with grade, tumour size and tumour anatomical location. Serum DPPIV activity and active-MMP9 were significantly correlated with sex (r = 0.65, p = 0.001) and age (r = -0.515, p = 0.043), respectively. OSCC male patients had higher serum DPPIV activity than females (Table 3). Table 1 Methods Used for the Study of Proteolytic Enzymes Sample RT-qPCR DPPIV activity MMP2 and MMP9 (Gelatin zymography) Tumour and paired non-tumour tissues Yes Yes Yes Serum Yes DPPIV, dipeptidyl peptidase IV; MMP, matrix metalloproteinase Table 2 The Correlation between Tumour DPPIV Gene Expression and Activity with the Serum DPPIV Activity and the Levels of Active-MMP2 and Active-MMP9 Serum DPPIV activity Tissue MMP-9 activity Tissue MMP-2 activity Spearman r P-value Spearman r P-value Spearman r P-value Tissue DPPIV activity -0.053 0.848 0.448 0.01 0.436 0.013 Tissue DPPIV mRNA expression 0.025 0.943 0.118 0.663 0.504 0.049 Bold values represent significant correlations as evaluated by Spearman’s correlation analysis. Figure 1 Expression and Activity of DPPIV in Tumour and Paired non-Tumour Tissues (A and B) and Serum of OSCC Patients (C) (n = 16). Values represents mean ± standard deviation (SD). Table 3 The Correlation between DPPIV Gene Expression, Activity and the Levels of Active-MMP2 and Active-MMP9 with Clinical Features of OSCC Patients Grade Tumour site Size Sex Age Spearman r P-value Spearman r P-value Spearman r P-value Spearman r P-value Spearman r P-value Serum DPPIV activity 0.168 0.567 0.237 0.265 0.281 0.259 0.65 0.001 0.023 0.916 Tissue DPPIV activity 0.216 0.667 0.023 0.934 0 1 0.26 0.351 -0.304 0.251 Tissue DPPIV mRNA expression -0.378 0.25 0.093 0.731 0.426 0.147 -0.298 0.279 -0.046 0.867 Tissue MMP-9 activity -0.309 0.567 -0.306 0.245 -0.064 0.854 -0.315 0.242 -0.515 0.043 Tissue MMP-2 activity 0.031 0.967 -0.154 0.564 -0.114 0.735 0.301 0.262 0.204 0.446 Bold values represent significant correlations as evaluated by Spearman’s correlation analysis. Figure 2 Gelatinolytic Activity in Tumour and Paired Non-Tumour Tissues of OSCC Patients. The zymography gel showing pro- and active forms of MMP2 (lower bands) and MMP9 (upper bands) in 32 paired tumour and non-tumour tissues (n = 16 OSCC patients) (A). Semi-quantitative analysis of active MMP2 and active MMP9 levels in the tumour and paired non-tumour tissues (B and C) (n = 16), Zymograph of 8 OSCC patients is shown. PT and T represent peri-tumour and tumour tissues, respectively. Values represents mean ± standard deviation (SD) Discussion OSCC is an aggressive, locally dominant tumour which in recent decades has become a major concern for global public health. Although the diagnosis and treatment techniques of OSCC have improved in the past decades (Luo et al., 2020) but five years-survival rate is still lower than 50%. In order to have a better chance of survival, OSCC patients must be diagnosed and treated in the early stages (Luo et al., 2020). Therefore understanding the molecular mechanisms of OSCC to identify new therapeutic targets and developing reliable prognostic histological markers to predict biological behavior and improve stratification and management of OSCC patients is necessary (Ding et al., 2022; Li et al., 2022). It has been proved that different tumour markers, including lipids, enzymes, polyamines, immunoglobulins, viral markers, glycoproteins, circulating immune complexes (CIC), hormones, tumour-associated antigens, and oncofetal proteins have been useful in studying human cancers (Mishra et al., 2021). A cell surface glycoprotein known as DPPIV plays multiple roles, including the regulation of glucose metabolism, immunomodulation, and tumour progression (Pan et al., 2021). Deregulation of DPPIV has been shown in various cancers, DPPIV overexpression is reported in thyroid cancer, ovarian cancer, prostate cancer, colorectal cancer, osteosarcoma and malignant mesothelioma (Kotani et al., 1991b; Wilson et al., 2000b; Kajiyama et al., 2003b; Inamoto et al., 2007; Lu et al., 2013; Zhang et al., 2013). In contrast, DPPIV downregulation is observed in melanoma (Havre et al., 2008). DPPIV has been extensively investigated as a cancer biomarker and a therapeutic target (Ohnuma et al., 2018). Taking into account diverse biological functions of DPPIV, the mechanism through which DPPIV affects tumour cell behavior may be different depending on the type of cancer, possibly resulting in a divergent effect. Recent studies have demonstrated that DPPIV overexpression suppressed invasive potential in ovarian cancer cells through downregulation of MMPs, as well as upregulation of tissue inhibitors of metalloproteases (TIMPs) (Kajiyama et al., 2003a; Kikkawa et al., 2005). According to other studies, loss of DPPIV expression is associated with tumour progression and malignant transformation (Morrison et al., 1993; Pro and Dang, 2004). In contrast, prostate and thyroid malignancies were found to exhibit enhanced DPPIV expression and DPPIV knockdown decreased proliferation, migration and invasion in urothelial carcinoma cell lines (Kotani et al., 1991b; Wilson et al., 2000a). Considering the reported secretion of DPPIV from adipocytes and its contribution to the circulating DPPIV (Sell et al., 2013), we hypothesized on the possible secretion of DPPIV from tumour tissue of OSCC patients. Therefore, this research aimed to compare tissue DPPIV gene expression and activity with paired non-tumour tissues of OSCC patients as well as serum DPPIV enzyme activity between OSCC patients and healthy individuals and investigating the correlation between tissue DPPIV mRNA/ activity and serum DPPIV activity. Results showed that serum DPPIV is lower in OSCC samples compared with normal ones which is in agreement with findings reported by Fukasawa et al (Fukasawa et al., 1982; Urade et al., 1989), and Mishra et al (Mishra et al., 2021). The evaluation of tissue DPPIV mRNA expression and activity in tumour samples and their paired non-tumour samples showed higher tissue DPPIV mRNA expression and activity in tumour samples. In agreement to our findings Ding et al., (2019), and Augoff et al., (2014) demonstrated that the expression and activities of DPPIV in OSCC tissue were significantly higher than in their paired non-tumour samples. Furthermore, neither tissue DPPIV mRNA level nor tissue DPPIV activity showed correlation with their paired serum DPPIV activity which may indicate circulating DPPIV does not originate from tumour tissue of OSCC patients, considering low number of patients included in this study, additional researches with higher number of patients are needed to conclude whether OSCC tumour tissues secrete DPPIV or not. The results of the present study showed no significant correlation between serum DPPIV activity and age. This is in contrast with previous reported findings showing lower DPPIV activity in older adults (Neidert et al., 2016), which could be due to small sample size of our study. We also found higher serum DPPIV activity in men than women which is in contrast with the findings of Sanz et al., (2018). MMPs play a crucial role in cell migration, invasion and distant metastases by degrading the ECM (Kessenbrock et al., 2010). MMP-2 and MMP-9 are two members of the MMP family which are expressed in OSCC, and their expression has been shown to be significantly correlated with the invasion of OSCC (Lee et al., 2008; Yamada et al., 2016). Zymography of tumour samples and paired non-tumour tissues of OSCC patients showed that active MMP-2 and active MMP-9 were significantly higher in tumour samples. Published data have shown that DPPIV participates in the regulation of MMPs in ovarian carcinoma cells (Kajiyama et al., 2003a). Results of research conducted by Dings et al (Ding et al., 2019) on the assessment of DPPIV effects on the expression of E-cadherin, MMP2 and MMP9 in OSCC cell lines showed that high metastatic cell lines express significantly higher levels of DPPIV than non-metastasized ones, and siRNA suppression of endogenous DPPIV expression significantly reduced cell motility and invasion in highly metastatic cell lines through down regulation of MMP-9 expression. Correlating tissue DPPIV mRNA level and activity with the active MMP-2 and active MMP-9 levels, we observed that active MMP-2 and active MMP-9 had positive correlation with tissue DPPIV activity whereas only active MMP-2 showed positive correlation with tissue DPPIV mRNA level. The positive correlation between tumour DPPIV, MMP-2 and MMP-9 activities is in consistent with the findings of Ding et al on OSCC cell lines (Kajiyama et al., 2003; Ding et al., 2019). In conclusion, we found higher DPPIV gene expression, enzymatic activity, active MMP-2 and active MMP-9 in tumour tissue of OSCC patients compared to their paired non-tumour tissues. Additionally, we observed lower DPPIV enzymatic activity in serum of OSCC patients compared to healthy individuals. No correlation was found between DPPIV mRNA level and enzymatic activity with the serum DPPIV activity. We found a positive correlation of tumour DPPIV activity with the levels of active MMP-2 and active MMP-9. Here we report the possible correlation between the DPPIV enzymatic activity and active gelatinase including MMP-2 and MMP-9. These findings suggest that coordinate function of DPPIV, MMP-2 and MMP9 may contribute to regulation of OSCC’s behavior. Author Contribution Statement AG designed and supervised the study. ATT performed RT-qPCR, zymography and enzyme activity experiment under supervision of TL, MSS and AG. SK helped with enzyme activity experiment. AK provided patient’s samples. ATT and AG performed statistical analyses and wrote the manuscript. All authors critically reviewed and approved the final version of the manuscript Acknowledgements This article has been extracted from the thesis written by Abdolkarim Talebi Taheri in the School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. This study was approved (IR.SBMU.MSP.REC.1400.607) by the committee for ethics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Availability of data The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116158 10.31557/APJCP.2023.24.4.1351 Research Article Factors Associated with Demand for Medical Cannabis Use among Breast Cancer Patients in Northern Thailand: A Cross-Sectional Study Sukrueangkul Alongkorn 1* Sirisuwan Petcherut 2 Sanguankittiphan Pariyakorn 3 Prachaiboon Tiwakron 4 Anonjarn Kanlayawee 1 Boonkerd Sumonthip 1 Khongruangrat Yiam 1 1 Department of Adult and Gerontological Nursing, Faculty of Nursing, Nakhon Ratchasima Rajabhat University, Thailand. 2 Faculty of Liberal Arts and Science, Roi Et Rajabhat University, Roi Et, Thailand. 3 Department of Health Promotion, Faculty of Physical Therapy, Srinakharinwirot University, Nakhon Nayok, Thailand. 4 Faculty of Public Health, Valaya Alongkorn Rajabhat University under the Royal Patronage, Pathum Thani, Thailand. * For Correspondence: alongkorn.s@nrru.ac.th 2023 24 4 13511357 21 12 2022 17 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: Breast cancer is the leading cause of death among women worldwide. Although modern treatments are recognized as effective, they often cause side effects. In Thailand, medical cannabis (MC) was legalized in 2019 with limited research on demand for its use. Therefore, this study aimed to identify factors associated with demand for MC among breast cancer patients in the North of Thailand as a target group. Methods: This cross-sectional analytical study administered multistage random sampling to recruit 432 breast cancer patients in northern Thailand. Ethical approval and signed written informed consents were obtained from the patients, prior to the study. A standardized, self-administered structured questionnaire was used to obtain the sociodemographic characteristics, clinical characteristics, social support, attitudes toward MC, knowledge about MC, health literacy about MC, and questions on demand for MC use. The scores from all questionnaires were converted to percentages before analysis. Result: A total of 173 (40%) of patients with breast cancer reported demand to use MC. The factors that were significantly associated with demand to use MC included had high levels of health literacy about MC (adj.OR = 4.96; 95% CI: 2.77 to 8.87), higher levels of social support (adj.OR =4.56; 95% CI: 2.20 to 9.42), higher monthly household income (adj.OR =4.02; 95% CI: 2.33 to 6.94), and positive attitudes toward MC use (adj.OR = 3.52; 95% CI: 1.91 to 6.52) when controlling for effects of other covariates. Conclusion: We found substantial demand for MC use among breast cancer patients. Health literacy, social support, monthly household income, and attitudes about MC were significantly associated with demand for MC use. Therefore, improving health literacy, social support, and attitudes about MC, especially among breast cancer patients, could help increase demand for MC as a complementary and alternative medicine alongside cancer treatment. Key Words Alternative medicine ganja marijuana tumor ==== Body pmcIntroduction Breast cancer is a growing problem and one of the most important causes of death among women worldwide, accounting for an estimated 685,000 deaths and 2.3 million diagnoses (WHO, 2021). The burden of breast cancer in Thailand is similarly significant, with more than 22,158 new cancer cases and 8,266 deaths recorded in 2020, comprising over 6.6% of the premature deaths attributable to cancer (WHO, 2021) Currently, there are many methods to treat breast cancer, such as surgery, radiation therapy, chemotherapy, hormone therapy, and novel therapeutic methods, as well as combination therapies. The choice of the aggressiveness of the treatment depends on several factors, including the biologic aggressiveness of the tumor, progression of disease, potential long-term toxicities, the health status of patient, the preferences of the patients and family members, and the life expectancy of the patient (Das et al., 2019; Barzaman et al., 2020). However, despite many effective treatments, many of these treatments can cause side effects and reduced health-related quality of life (Fisusi and Akala, 2019). Therefore, the cannabis plant may be a potential alternative for reducing the side effects of modern treatments. Cannabinoids (CBs) from cannabis, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), have been shown to be an effective treatment for providing relief of symptoms associated with cancer and its treatment, including nausea, anorexia, cancer- and treatment-related cognitive impairment, anxiety, depression, fatigue, insomnia, and cancer-related pain in the palliative treatment of cancer patients. Additionally, in animal models, CBD has been shown to inhibit the tumor progression of several cancer types, including breast cancer (Pellati et al., 2018; Kleckner et al., 2019; Kisková et al., 2019). Many countries have recognized the effectiveness of cannabis and allow the legal use of cannabis for medicinal purposes. In addition, some countries allow the legal use of cannabis for recreational purposes (Hussain et al., 2021). In Thailand, The Thai government authorized the use of cannabis for medical purposes in 2019. The rapid introduction of medical cannabis (MC) has increased patients’ desire to use cannabis for treating illnesses, especially among cancer patients. MC use among breast cancer patients can be considered a form of complementary and alternative medicine (CAM). Sociodemographic factors that appear to be related to CAM use among breast cancer patients have shown some notable trends. For example, a systematic review about CAM use among breast cancer showed that younger age, higher education, higher income, being married, involvement in a support group, having health insurance, and having previously used CAM were all factors associated with use of CAM (Keene et al., 2019). Similarly, in Ethiopia, rural residency, higher educational status, higher average monthly income, and presence of co-morbidity were positively associated with the use of CAM (Ayele et al., 2017). These trends mimic trends in the broader population. For example, in Mongolia, female gender, younger age, higher education, shorter disease duration, and prior use of CAM were significantly associated with CAM use including herbal medicine (Oyunchimeg et al., 2017). Clinical characteristics are also important. Patients that have undergone or completed radiotherapy or have completed chemotherapy reported a high prevalence of CAM use (Chen et al., 2008). An advanced stage of cancer, longer time since diagnosis, and higher need of CAM information were also significantly associated with CAM use (Shin et al., 2012). Increased usage of CAM has also been associated with reduced trust in physicians (Azhar et al., 2016). Besides demographics and clinical characteristics, social support, attitudes toward MC, and health literacy can be an important factors in determining CAM usage. For example, in northern Thailand, health literacy, social support, and attitudes about MC were significantly associated with demand for MC use among cancer patients (Sukrueangkul et al., 2022). Similarly, another previous study in South Peninsular Malaysia found that social support can be an important factor in determining CAM usage. For example, in that study researcher found that patients’ families and friend encouraged them to use CAM, with the internet and social networks being the major sources of information on CAM (Razali et al., 2020). Additionally, health literacy regarding CAM is also important, with younger patients typically having higher levels of health literacy (Ahmad Sharoni et al.,2019). Therefore, given the importance of various factors that have been reviewed in previous studies, as well as the novelty of MC use in Thailand, it is imperative to study these factors to understand how these factors may affect the demand for MC use among cancer patients. Thus, to provide data that may direct MC policy, a target group was selected in Thailand. Given the higher incidence of cancer in northern Thailand relative to the rest of the country, we were interested in studying demand of MC use among breast cancer patients in the region. This study aimed to identify factors associated with demand for MC use among breast cancer patients in the North of Thailand. Materials and Methods Study design This cross-sectional study was conducted using an anonymous paper-based survey administered in out-patient cancer clinics located at six public hospitals that are cancer treatment centers within the north of Thailand (within Ministry of Public Health Regions 1 to 3). The six hospitals were multistage randomly selected. Participants Participants were eligible for inclusion based on the following criteria: Any cancer patient with a breast cancer diagnosis, receiving treatment at one of the studied hospitals, aged 18 or older, able to read and write in Thai, and mentally and physically able to answer the questionnaire were eligible for inclusion in the study. Recruitment took place between October 2020 and March 2021. Participants were recruited by registered nurse. Participants who received end stage cancer diagnosis or whose severe symptoms prevented them from providing information were excluded. Instruments Data were collected using a self-administered questionnaire that included 6 items with structured question format about MC. Social support was assessed using a social support questionnaire, which was coded into a score from 20 to 100. Attitudes about MC were assessed using a questionnaire, which was coded into a score from 15 to 45. Knowledge about MC was assessed using a questionnaire, which was coded into a score from 0 to 20. Finally, health literacy about MC was assessed using a health literacy instrument, which was coded into a score from 47 to 188. The scores from all questionnaires were converted to percentages before analysis. The questionnaire was constructed after reviewing the literature and was evaluated by a panel of five experts in the field of CAM, health behavior, pharmacology, research methodology and other medical sciences for validity. The questionnaire was trialed to test the reliability. The overall average Cronbach’s Alpha was 0.88 for the questionnaires. Data Analysis All data were analysed using the STATA software version 15.0 with 100% of data entry checked for accuracy. Descriptive statistics including frequency and percentage were used to describe categorical data, whereas mean and standard deviation were used for continuous data. Simple logistic regression was used to identify association between each individual independent variable and demand of MC use. The independent factors that had a p-value smaller than 0.25 (Bursac et al., 2008) were processed in the multivariable analysis using a generalized linear mixed model (GLMM) to identify factors associated with demand of MC use when controlling for the effect of other covariates. The magnitude of effects were presented as adjusted odds ratio (adj.OR) and 95% confidence interval (CI), using a statistical significance level α=0.05. Ethical considerations This research has been approved by the Lampang Cancer Hospital Ethics Committee in Human Research based on the Declaration of Helsinki and the ICH Good Clinical Practice Guidelines. Reference No. 8/2020. Results Patient Characteristics In total, 432 cancer patients were included in the final analysis (Table 1). Most of the participants were females (99.8%), and 46.0% were middle-aged with a mean age of 55.2 ± 10.9 years. Most participants reported being currently married or in a domestic partnership (75.0%), having completed only primary school (63.4%), and earning a monthly income less than or equal to 10,000 THB (around 350 USD). Almost one-third of participants lived in rural areas. Considering health coverage, 68.3% were covered under the Universal Coverage Scheme, which is the government welfare health insurance. Many participants (64.4%) had a comorbidity in addition to cancer. The average time from diagnosis of cancer was 12.14 months with a large amount of variability (±19.81 months). Almost half (57.6%) of participants were categorized into a group with early stage of breast cancer. Treatment included chemotherapy (66.7%), radiation therapy (13.9%), and surgery (12.5%). A large majority of respondents (86.8%) reported having received information about MC. The most common source of MC information was family (75.4%), television (53.7%), and social media (29.3%). Social Support, Attitudes, Knowledge, Health Literacy, and Demand for MC Almost one-third of participants had a low level of social support (Table 1), while 39.5% had a high level of positive attitudes about MC. Knowledge about MC was well distributed, with almost one-third of participants having an average level of knowledge about MC. Concerning health literacy, just over half were categorized as having problematic health literacy (56.2%). Overall, less than half of participants (40.0%) reported having a demand for MC. Bivariable analysis of factors associated with demand for MC use Simple logistic regression was used to identify association between each individual independent variable and demand of MC use (Table 2). The independent factors that had a p-value smaller than 0.25 included being employed/government officer/businesses (OR = 2.88; 95% CI: 11.90 – 4.35 : p-value <0.001), having a higher monthly household income (OR = 3.96; 95% CI: 2.59 – 6.03 : p-value <0.001), having health coverage under the Civil Servant Medical Benefit Scheme (OR = 1.76; 95% CI: 1.17 - 2.65: p-value = 0.007), having a moderate to high level of social support (OR = 6.86; 95% CI: 4.18-11.26; p-value <0.001), having a fair to good attitude toward MC use (OR = 5.22; 95% CI: 3.13 – 8.69; p-value <0.001), having an average to good knowledge about MC use (OR = 2.92; 95% CI: 1.84 – 4.63; p-value <0.001), and adequate to excellent levels of health literacy about MC (OR = 10.46; 95% CI: 6.29 – 17.39; p-value <0.001) (Table 2). Multivariable analysis of factors associated with demand for MC use The multivariable analysis using GLMM with backward elimination indicated that the factors significantly associated with demand to MC use were: adequate to excellent health literacy about MC (adj.OR = 4.96; 95% CI: 2.77 to 8.87), moderate to high levels of social support (adj.OR =4.56; 95% CI: 2.20 to 9.42), higher monthly household income (adj.OR =4.02; 95% CI: 2.33 to 6.94), and positive attitudes toward MC use (adj.OR = 3.52; 95% CI: 1.91 to 6.52) when controlling for effects of other covariates (Table 3). Table 1 Sociodemographic Factors among Breast Cancer Patients on Demand for Medical Cannabis use in the North of Thailand (n=432) Factors Number Percentage Gender Female 431 99.8 Male 1 0.2 Age group ≤45 (young adult) 79 18.3 45-59 (middle-aged adult) 199 46 ≥60 (elderly) 154 35.7 Mean ± S.D. = 55.2 ± 10.9 Marital Status Married/domestic partnership 324 75 Divorced/separated/widowed 62 14.3 Single 46 10.7 Highest education level Primary school 274 63.4 Junior high school and higher 158 36.6 Employment Status Unemployed/Retired 279 64.6 Employed/Government officer/Businesses 153 35.4 Monthly income (THB) ≤10,000 289 66.9 ≥10,000 143 33.1 Mean ± S.D. = 11,069.87 ± 11,902.01 Place of residence Rural area 272 63 Metropolitan area 160 37 Scheme Universal Coverage 295 68.3 Civil Servant Medical Benefit 66 15.3 Social Security 71 16.4 Health status Comorbidity 278 64.4 No Comorbidity 154 35.6 Time from diagnosis with cancer (month) < 12 354 81.9 ≥12 78 18.1 Mean ± S.D. = 12.14±19.81 Stage of Cancer Unknown 19 4.4 Early-stage 249 57.6 Advance stage 164 38 Current treatment received Chemotherapy 288 66.7 Radiation therapy 60 13.9 Surgery 54 12.5 Other 30 6.9 Received information about MC Yes 375 86.8 No 57 13.2 Factors Number Percentage Source of MC information (patients can choose more than one) Family 326 75.5 Television 232 53.7 Social media 127 29.4 Doctor, pharmacist, and medical staff 92 21.3 Newspaper/ brochures/ Academic article 77 17.8 Radio 59 13.7 Thai traditional medicine 43 10 Other 25 5.8 Social support Low (less than 60 percentage) 159 36.8 Moderate (60-79 percentage) 145 33.6 High (greater than or equal to 80 percentage) 128 29.6 Mean ± S.D. = 66.19 ± 12.97 Attitude toward MC Poor (less than 60 percentage) 133 30.8 Fair (60-79 percentage) 128 29.6 Good (greater than or equal to 80 percentage) 171 39.6 Mean ± S.D. = 69.29± 14.21 Knowledge about MC Low (less than 60 percentage) 131 30.3 Average (60-79 percentage) 157 36.3 Good (greater than or equal to 80 percentage) 144 33.4 Mean ± S.D. = 60.15 ± 11.20 Health Literacy for medicinal cannabis use dimensions Inadequate (0-50 percentage) 72 16.7 Problematic (51-65 percentage) 243 56.3 Sufficient (66-84 percentage) 58 13.4 Excellent (85 percentage and over) 59 13.6 Mean ± S.D. = 62.86 ±12.48 Demand to MC use No 259 60 Yes 173 40 Table 2 The Bivariable Analysis of Factors Associated with Demand to MC Use among Breast Cancer Patients in the North of Thailand (n=432) Factors Number % Demand to MC use Crude OR 95% CI P-value Occupation <0.001 Unemployed/Retired 279 31.83 1 - Employed/Government officer/Businesses 153 57.34 2.88 1.90 – 4.35 Monthly income (THB) <0.001 ≤10,000 289 29.41 1 - ≥10,001 143 62.24 3.96 2.59 – 6.03 Scheme 0.007 Universal coverage/Social security scheme 295 35.93 1 - Civil servant medical benefit 137 49.64 1.76 1.17 - 2.65 Social support <0.001 Low 159 15.09 1 - Moderate to high 273 54.95 6.86 4.18-11.26 Attitude toward MC <0.001 Poor 133 16.54 1 - Fair to Good 299 50.84 5.22 3.13 – 8.69 Knowledge about MC use <0.001 Low 131 23.66 1 - Average to good 301 47.51 2.92 1.84 – 4.63 Health literacy about MC <0.001 Inadequate- Problematic 315 26.03 1 - Adequate- Excellent 117 78.63 10.46 6.29 – 17.39 Table 3 The Multivariable Analysis of Factors Associated with Demand to MC Use among Breast Cancer Patients in the North of Thailand (n=432) Factors Number % Demand to MC use Crude OR Adjust OR 95% CI P-value Monthly income (THB) <0.001 ≤10,000 289 29.41 1 1 - ≥10,001 143 62.24 3.96 4.02 2.33 – 6.94 Social support < 0.001 Low 159 15.09 1 1 - Moderate to high 273 54.95 6.86 4.56 2.20 – 9.42 Attitude toward MC use < 0.001 Poor 133 16.54 1 1 - Fair to Good 299 50.84 5.22 3.52 1.91 – 6.52 Health literacy about MC < 0.001 Inadequate/ Problematic 315 26.03 1 1 - Adequate/ Excellent 117 78.63 10.46 4.96 2.77 – 8.87 Discussion We found that 40.0% of breast cancer patients in the North of Thailand reported having demand for MC use. This finding was comparable to actual CAM usage among breast cancer patients reported in Grömitz, Germany (Hammersen et al., 2020), and in New York, USA (Sura et al., 2022). Those countries have a longer history of legalized MC use compared to Thailand. In 2019, the Thai government legalized MC use as an alternative medicine to treat illnesses. Demand for MC use in Thai cancer patients, including breast cancer patients, may have been enhanced by recent attention. After controlling for covariates with backward elimination in the multivariate analysis, four variables were significantly associated with demand for MC use among breast cancer patients in the North of Thailand. Those variables were adequate to excellent health literacy about MC, higher levels of social support, higher monthly household income, and positive attitudes toward MC use. In our study, we also found that participants that had adequate to excellent levels of health literacy about MC were 4.96 times more likely to report having demand to use MC when compared with those with insufficient and problematic levels of health literacy about MC. This result is similar to that of a previous study in the North of Thailand by Sukrueangkul et al., (2022). Health literacy is a strong factor that has been shown to correlate with demand for MC usage among cancer patients. Another study reported that CAM usage was significantly associated with adequate levels of health literacy among patients with chronic diseases in Japan (Yukawa et al., 2015). People who reported moderate to high levels of social support were 4.56 times more likely to report demand for MC use when compared with those with low levels of social support. Previous research in Canada by Leos-Toro, Shiplo and Hammond (2018) has also shown that social support to be related to MC use in cancer patients. Another study among breast cancer patients in the northern region of peninsular Malaysia reported that CAM usage was positively correlated with receiving information about cancer and nutritional supplements from sources apart from doctors, including TV and radio (Knight, Hwa, & Hashim, 2015). Thus, a possible explanation of these results is that most of the participants who received social support had received information about MC products from sources that were mostly close friends and family members, television or radio, or social media. Breast cancer patients with a higher monthly household income were 4.02 times more likely to report demand for MC use when compared with those with lower monthly household income. This result was similar to that of a previous study in Hungary by Sárváry and Sárváry (2019), which reported higher monthly household income was positively correlated with CAM use in breast cancer patients. This finding may be explained by the added costs of MC. In Thailand, the price of MC is quite expensive and is not always covered by welfare health coverage. Thus, when participants have higher monthly household income, they can buy MC for alternative medicine in conjunction with conventional treatment. In some previous studies, such as one in Bandung, Indonesia by Azhar et al., (2016), CAM usage was more common in patients who had lower income, which was more a reflection of seeking alternative treatments in response to conventional treatment being unaffordable. Since Thailand has universal health coverage, this health seeking behavior is less common. Those participants who reported a fair to good attitude toward MC use were 3.52 times more likely to report demand for MC use when compared to those with poor attitudes toward MC use. This result is similar to that of a previous study in Malaysia by Islahudin et al., (2017), which reported that a positive attitude toward MC was positively correlated with CAM use in cancer patients. As reported in an earlier study in Pennsylvania, USA, by Bauml et al., (2015), attitude toward CAM was the factor that most influenced the decision to use CAM in cancer patients and explained much more variance in CAM use than clinical and demographic variables alone. When participants have a positive attitude towards MC, it reduces fears of the side effects. On the other hand, a lack of knowledge about MC use policy, reduces confidence in the decision to use MC. Similar to a previous study by Sukrueangkul et al., (2022), most patients (53.3%) have positive attitudes toward MC, and demand was intentioned to treat physical and mental symptoms that occur alongside cancer-related symptoms, relieve side effects from treatment, and a desire to live longer and improve their health. This cross-sectional study found that 40.0% of breast cancer patients in the North of Thailand reported demand to use MC. The factors significantly associated with reported demand to use MC were adequate to excellent health literacy about MC use, moderate to high levels of social support, higher monthly household income, and positive attitudes toward MC use when controlling for effects of other covariates. In conclusion, this study highlights the factors associated with demand for MC use among breast cancer patients in the of north Thailand. We found substantial demand for MC use among breast cancer patients. Health literacy, social support, monthly household income, and attitudes about MC were significantly associated with demand for MC use. Therefore, improving health literacy, social support, and attitudes about MC, especially among breast cancer patients, could help increase demand for MC as a complementary and alternative medicine to alleviate side effects and enhance cancer treatment. Author Contribution Statement All authors contributed equally in this study. Acknowledgements We extend our sincere thanks to the cancer patients who agreed to participate in the study. Limitations of the study This study used only data from breast cancer patients in the north of Thailand. Therefore, the results may not apply to breast cancer patients overall in Thailand. Study Implication The results showed that factors associated with demand for medical cannabis use among breast cancer patients in the of North Thailand included health literacy, social support, monthly household income, and attitudes about MC. Therefore, improving health literacy, social support, and attitudes about MC, especially among breast cancer patients, could help increase demand for MC as a complementary and alternative medicine use to alleviate side effects and enhance cancer treatment. Approval The current study deals with primary data, so approval of the scientific body is not needed. Ethical considerations This research has been approved by the Lampang Cancer Hospital Ethics Committee in Human Research base on the Declaration of Helsinki and the ICH Good Clinical Practice Guidelines. Reference No. 8/2020. Availability of data The datasets are not publicly available due to ethical restrictions but are available from the corresponding author on reasonable request. Conflict of interest All authors declared no conflict of interest. ==== Refs References Ahmad Sharoni SK Robani S Zaini SA Use of complementary and alternative medicine: Prevalence and health literacy among patients attending a Health Centre in Universiti Teknologi MARA Selangor. 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116133 10.31557/APJCP.2023.24.4.1131 Research Article The Potential of Expression of Cyclin-D1 on Neoadjuvant Chemotherapy in Invasive Breast Carcinoma Rustamadji Primariadewi 1* Wiyarta Elvan 2 Anggreani Ineke 1 1 Department of Anatomic Pathology, Faculty of Medicine, Universitas Indonesia, Dr. Cipto Mangunkusumo National Hospital, Jakarta, Indonesia. 2 Department of Medical Science, Faculty of Medicine, Universitas Indonesia, Dr. Cipto Mangunkusumo National Hospital, Jakarta, Indonesia. * For Correspondence: primariadewi.rustamadji@ui.ac.id 2023 24 4 11311136 26 6 2022 24 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: Patients undergoing neoadjuvant chemotherapy (NC) for invasive breast cancer (IBC) need indicators to track their progress during treatment. The goal of this research is to learn how cyclin D1 works in conjunction with taxane and non-taxane therapy for people with IBC. Methods: There were 31 examples divided into two groups, based on: those using a different type of NC (taxane- or non-taxane-based), and NC administration time (before or after). Tumor grade, age, PR, ER, Ki-67, HER2, and Cyclin D1 expression were among the factors considered. Using immunohistochemical labeling, we were able to categorize cyclin D1 levels according to a threshold value, and we supplemented this with data we found in our databases. To analyze the data, we used a modified linear model. Results: The expression of Cyclin D1 decreased after NC delivery (p=0.086). Cyclin D1 expression was reduced in the taxane group (p=0.792). The non-taxane group also saw no differences in outcomes (p = 0.065). There was a larger decrease in Cyclin D1 expression in the non-taxane group compared to the taxane group, but the difference was not statistically significant (p=0.200). Conclusion: Cyclin D1 expression, even if the differences are not statistically significant, may be a prognostic indicator of NC reaction in IBC. The involvement of Cyclin D1 in NC warrants more research with bigger IBC sample sizes. Key Words Cyclin-D1 taxane chemotherapy breast cancer invasive ==== Body pmcIntroduction Cancer deaths from breast cancer are the leading cause of mortality among women. According to Global Burden of Cancer (GLOBOCAN) statistics, women will be diagnosed with breast cancer at 11.7% of all cases and 6.9% of all deaths linked with the disease in 2020 (GLOBOCAN, 2020). Breast cancer can be classified as invasive (IBC) or non-invasive (nIBC) (GLOBOCAN, 2020). There are several subtypes of IBC, which is the most frequent form of breast cancer. Because of the high variation of IBC, the treatment must also be done early and aggressively (Rustamadji et al., 2021; Rustamadji et al., 2021; Sharma et al., 2010). Neoadjuvant chemotherapy (NC), which is administered before surgery, is a crucial part of modern IBC care (Lee et al., 2011). Currently, NC is the gold standard for patients with locally progressed breast cancer and is the therapy of choice for early-stage, possibly treatable diseases (Lee et al., 2011). There are two primary categories of NC: those founded on taxanes and those that do not (Zhang et al., 2019). In breast cancer therapy, the taxane is one of the most influential and extensively used systemic therapies. Resistance to NC, on the other hand, impacts breast cancer treatment (Zhang et al., 2019). A mechanism of self-protection has been devised by cancer cells in order to fight the effects of NC, for example, the NFkB activation pathway (Biliran et al., 2005). One of the NFkB protein complex’s most important functions is regulating gene expression(Biliran et al., 2005). By controlling many anti-apoptotic genes, NFkB may help cancer cells withstand chemotherapy(Biliran et al., 2005). Survival factors like Cyclin D1 are among them (Biliran et al., 2005; Garg et al., 2016; Pacifico et al., 2006). The involvement of cyclin D1 in the NC resistance pathway makes it a candidate for use as a prognostic indicator (Mohammadizadeh et al., 2013). Predictive indicators are ultimately used in therapy to improve total mortality after NC administration in IBC. Both taxane-based and non-taxane-based NC for IBC will be compared for their effects on cyclin-D1 expression before and after treatment. We hypothesized that cyclin-D1 expression is associated with NC delivery in IBC patients. Materials and Methods Data Collection and Design of Study This research was carried out between January and May of 2022 in the Pathological Anatomy Laboratory of the University of Indonesia’s Faculty of Medicine. The Universitas Indonesia Institutional Review Board approved the 21-11-1252 testing protocols in November 2021. Each participant gave a written agreement and understood the goal of the research. The research adheres to Declaration of Helsinki (Rickham, 1964). The data collection period ran from January 2014 through June 2016, and the five-year monitoring phase will run from January 2019 through May 2021. Data were collected, including tumor grade, age, tumor size, HER2 status, Ki-67, underarm lymph node spread, lymphovascular penetration, and NC type (taxane-based or non-taxane-based). Quantitative analysis of the IHC labeling findings on the paraffin sample was also performed to collect data on cyclin D1 expression. Samples Primary tumor paraffin samples were taken from female breast surgery patients who had initially been identified with IBC histopathologically. Specimens from individuals with non-IBC diseases, systemic illnesses, and damaged paraffin blocks are discarded. Both the NC treatment status (before or after) and the type of NC used (non-taxane or taxane) were used to categorize the data. The selected group represents the largest representative selection from the available records in the department. To avoid any potential for prejudice, only one researcher (E.W.) had access to the final groupings. Researchers didn’t have access to the studies’ classifications until after the analysis was done. Preparation of Samples Kusmardi et al., Wiyarta et al., and Primariadewi et al. all use this staining method (2022). In xylol (Brataco.inc, Bogor, Indonesia), we deparaffinized the paraffin block, then rehydrated it in 96%, 70%, and purified water for 5 minutes, as per protocol. Heat-induced antigen recovery in pH 9.0 Tris EDTA (Merk, Jakarta, Indonesia). was performed in a 96°C chamber for 20 minutes. There was a 15-minute period of peroxidase block (Merk, Jakarta, Indonesia) after antigen retrieval, then followed by 15 minutes of PBS pH 7.4 (Brataco Inc., Bogor, Indonesia) rinses. Post-primary and Novolink polymer incubations were conducted after anti-cyclin D1 antibodies (ab134175, Abcam, Cambridge, UK) were incubated for one hour. Hematoxylin and 5 percent lithium carbonate were used to counterstain the tissue slices before they were examined under a microscope for DAB staining (Abcam, Jakarta, Indonesia). Quantification of Cyclin D1 Expression Histopathology experts P.R. and I.A. assessed the immunohistochemistry stainings. A Leica DM750 microscope with a 400x total magnification was used to examine each specimen. Five sites were chosen at random, each containing 500 tumor cells, and their Cyclin D1 expression was analyzed. Each location had at least one hundred malignant cells. Cyclin D1 expression was detected by a dark smear in the tumor cytoplasm (Fusté et al., 2016). Cell counter were used to assess the brown hues and to classify the staining intensity as negative (0), weak (1+), moderate (2+), or strong (3+)(O’Brien et al., 2016). In order to measure the expression of cyclin D1, Peurala et al. developed a quantification formula (Ortiz et al., 2017). The percentage values of the very positive, positive, and low positive categories are combined to quantify the cyclin D1 expression (Ortiz et al., 2017). In addition, the cyclin D1 expression group was separated in to high and low expressions based on the prior quantification values. Samples with values higher than 0.4 were classified as having strong cyclin D1 expression (Ortiz et al., 2017). Two raters independently collected all of the results for the group. Until the entire sample has been examined, the results of the calculations that have already been performed are merged and sent to additional scholars (E.W.). The combined rating from the two raters will serve as the basis for further study. Statistical Analysis All data was been tabulated in Microsoft Excel (Microsoft Corp, Redmond, WA, USA) before research. Using SPSS (Statistical Package for the Social Sciences) 20, we examined and portrayed the collected data (IBM Corp, Armonk, NY, USA). To categorize cyclin D1 expression, the number 0.4 was used (Ortiz et al., 2017). The combined ratings from the two raters were then compared to the threshold to determine the overall rating (high or low). Each sample’s Cyclin D1 expression level is represented these overall rating. Results All thirty-one samples were stained for cyclin D1 by immunohistochemistry. Before and after administration of NC, each specimen exhibits the clinicopathologic features outlined in Tables 1 and 2. Figure 1 depicts the results of exemplary IHC staining. Negative, low positive, positive, and high positive samples of tumor cells are shown in each image, respectively. The images are composites of different parts of a single sample. These samples are analyzed further after the strength of the brown color has been measured and assigned a number. All 31 samples were assessed separately by two experts (I.A and P.R.). Following NC therapy, as shown in Figure 2 and Table 3, Cyclin D1 expression dropped, albeit not significantly (p=0.086). Both Table 4 and Figure 3 demonstrate the data’s separation into taxane and non-taxane group. Cyclin D1 was downregulated in the taxane-treated cohort. However, the numbers were too low to be considered significant (p=0.792). Similar results were observed in the control sample (p=0.065) that did not include taxanes. The level of cyclin D1 was also compared between the two groups. Cyclin D1 levels were significantly lower in the non-taxane group than in the taxane group. The difference between the two groups was not statistically significant (p=0.200). Figure 1 IHC Staining for cyclin-D1 Expression in IBC Tumor Cells at 400x Magnification before and after NC Administration. The scale bar represents 50 μm for all images Table 1 Clinicopathological Characteristics before Neoadjuvant Chemotherapy Administration Variables Category Cyclin-D1 Expression p-value High (%) Low (%) Age ≥50 y.o. 14 (83.9%) 2 (12.5%) 0.654 <50 y.o. 12 (80.0%) 3 (20.0%) Tumor grade 3 8 (88.9%) 1 (11.1%) 0.581 2 15 (78.9%) 4 (21.1%) 1 3 (100.0%) 0 (00.0%) ER status Positive 14 (93.3%) 1 (6.7%) 0.165 Negative 12 (75.0%) 4 (25.0%) PR status Positive 12 (85.7%) 2 (14.3%) 0.8 Negative 14 (82.4%) 3 (17.6%) HER2 status Positive 11 (78.6%) 3 (21.4%) 0.467 Negative 15 (88.2%) 2 (11.8%) Ki67 status Positive 4 (80.0%) 1 (20.0%) 0.797 Negative 22 (84.6%) 4 (15.4%) Taxane With 9 (90.0%) 1 (10.0%) 0.522 Without 17 (81.0%) 4 (19.0%) ER, estrogen receptor; PR, progesterone receptor, HER2, human epidermal growth factor receptor 2; Univariate analysis was performed using the chi-square test with continuity correlation;* p-value less than 0.05 is considered statistically significant Table 2 Clinicopathological Characteristics after Neoadjuvant Chemotherapy Administration Variables Category Cyclin-D1 Expression p-value High (%) Low (%) Age ≥50 y.o. 14 (87.5%) 2 (12.5%) 0.57 <50 y.o. 12 (80.0%) 3 (20.0%) Tumor grade 3 10 (90.9%) 1 (9.1%) 0.732 2 12 (80.0%) 3 (20.0%) 1 4 (80.0%) 1 (20.0%) ER status Positive 14 (93.3%) 1 (6.7%) 0.165 Negative 12 (75.0%) 4 (25.0%) PR status Positive 12 (85.7%) 2 (14.3%) 0.8 Negative 14 (82.4%) 3 (17.6%) HER2 status Positive 11 (78.6%) 3 (21.4%) 0.467 Negative 15 (88.2%) 2 (11.8%) Ki67 status Positive 4 (80.0%) 1 (20.0%) 0.797 Negative 22 (84.6%) 4 (15.4%) ALNM Yes 12 (80.0%) 3 (20.0%) 0.57 No 14 (87.5%) 2 (12.5%) LVI Yes 12 (75.0%) 4 (25.0%) 0.165 No 14 (93.3%) 1 (6.7%) Taxane With 9 (90.0%) 1 (10.0%) 0.522 Without 17 (81.0%) 4 (19.0%) ALNM, Axillary lymph node metastasis; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; LVI, Lymphovascular invasion PR, progesterone receptor; Univariate analysis was performed using the chi-square test with continuity correlation; * p-value less than 0.05 is considered statistically significant Figure 2 Individual before-after Line Showing Overall Changes in Cyclin-D1 Expression before and after Administration of Neoadjuvant Chemotherapy Table 3 Overall Changes in Cyclin-D1 Expression before and after Administration of Neoadjuvant Category n Cyclin-D1 Expression P value Before 31 0.700 ± 0.238 0.086 After 31 0.578 ± 0.308 Statistical analysis was performed using the generalized linear model Figure 3 Individual before-after Line Showing Changes in Cyclin-D1 Expression before and after Administration of Neoadjuvant Chemotherapy in Taxane-based vs. non-Taxane-based Group Table 4 Changes in Cyclin-D1 Expression before and after Administration of Neoadjuvant in Taxane vs non-Taxane Group Group Category n Cyclin-D1 Expression P value Cyclin-D1 Expression Mean Difference P value Taxane Before 10 0.735 ± 0.231 0.792 0.17 ± 0.29 0.2 After 10 0.706 ± 0.253 Non-Taxane Before 21 0.683 ± 0.245 0.065 0.03 ± 0.19 After 21 0.517 ± 0.318 Statistical analysis was performed using the generalized linear model Discussion Cyclin D1 expression was significantly changed in individuals with IBC who received NC. These alterations are detectable on an individual and collective level. Pre-NC administration, cyclin D1 expression was often higher than after administration of NC. This provides evidence for a causal relationship between NC and a reduction in cyclin D1 in IBC cells. Possible explanations for these observations involve the role of Cyclin D1 in drug resistance and the impact of it on the efficacy of therapy (Pysz et al., 2014). Consequently, several kinds of NC were created to target this biomarker (Villegas et al., 2018). Numerous investigations have shown that some anticancer drugs function by reducing cyclin D1 synthesis (Grillo et al., 2006). This is consistent with Grillo et al., (2006), which demonstrate that siRNA-mediated suppression of cyclin D1 in MCF-7 breast cancer cells has anticancer drug target potential. Overexpression of cyclin D1 was also linked to resistance to NC. Overexpression of cyclin D1 increases tumor cell proliferation and imparts resistance to cisplatin-mediated apoptosis, as reported by Biliran et al., (2005). All of these factors explain the involvement of NC in cyclin D1 expression modification. Pre- and post-NC groups and taxane and non-taxane groups showed differences in cyclin D1 expression. This research found that the non-taxane group experienced a greater shift in cyclin D1 than the taxane group. Although these findings are not statistically significant, they suggest a possibly more prominent decreasing tendency in the group using non-taxane-based NC. Some research may account for this discovery. Overexpression of cyclin D1 was also linked to IBC in patients who underwent surgery followed by anthracycline-based treatment, as was previously reported by Reis-Filho et al., (2006). In a separate investigation, cyclin-dependent kinase was linked with a more accurate prognostic model and lower pathological complete response rates (Wachter et al., 2013). This observation may be because anthracyclines (such as epirubicin and pirarubicin) interact with topoisomerase II and inhibit DNA transcription (Fisher et al., 1997; Li et al., 2011). In the context of neoadjuvant treatment, combining these two drugs is one of the most often reported chemotherapy regimens (Li et al., 2011; Tiezzi et al., 2007). Even though participants were recruited from an IBC referral center, the study’s sample size was insufficient. It’s possible that this is the case because NC has not been extensively adopted in the area where the research is taking place. Consequently, the expression of cyclin D1 was very minimally changed. Despite the need to consider clinical and laboratory importance, cyclin D1 expression is decreasing across all categories. As part of this initiative, more research on cyclin D1’s role in IBC and NC will need to be done. In conclusion, in IBC, cyclin D1 expression may be a prognostic indicator of NC response and clinical outcome, despite the fact that the differences are not statistically significant, as determined by an analysis of clinical and biochemical data. The involvement of cyclin D1 in NC warrants more research with bigger IBC sample sizes. Author Contribution Statement Conceptualisation, P.R., E.W., I.A.; methodology, P.R., E.W.; software, E.W.; validation, P.R., I.A.; formal analysis, P.R., E.W.; investigation, E.W., I.A.; resources, P.R.; data curation, P.R., E.W.; writing—original draft preparation, P.R., E.W.; writing—review and editing, all authors; visualization, E.W.; supervision, P.R.; project administration, E.W.; funding acquisition, P.R. The published version of the work has been reviewed and approved by all authors. Acknowledgements None. Ethics approval and consent to participate In November 2021, the Universitas Indonesia Faculty of Medicine Ethics Committee authorized the report procedures, with protocol number 21-11-1252. All participants engaged in the research provided informed permission for participation. Consent for publication All participants engaged in the research provided informed permission for publishing. Availability of data and materials This published paper includes all data produced or analyzed during the research. Competing interests The authors state that they have no conflicts of interest. ==== Refs References Biliran H Wang Y Banerjee S Overexpression of cyclin D1 promotes tumor cell growth and confers resistance to cisplatin-mediated apoptosis in an elastase-myc transgene-expressing pancreatic tumor cell line Clin Cancer Res 2005 11 6075 86 16115953 Fisher B Brown A Mamounas E Effect of preoperative chemotherapy on local-regional disease in women with operable breast cancer: findings from National Surgical Adjuvant Breast and Bowel Project B-18 J Clin Oncol 1997 15 2483 93 9215816 Fusté NP Castelblanco E Felip I Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer Oncotarget 2016 7 26979 91 27105504 Garg H Suri P Gupta JC Survivin: a unique target for tumor therapy Cancer Cell Int 2016 16 49 27340370 GLOBOCAN Estimated cancer incidence, mortality and prevalence in 2020 2020 Retrieved from https://gco.iarc.fr/today/data/factsheets/cancers/20-Breast-fact-sheet.pdf Grillo M Bott MJ Khandke N Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression Breast Cancer Res Treat 2006 95 185 94 16319987 Lee A Lim W Moon BI Chemotherapy response assay test and prognosis for breast cancer patients who have undergone anthracycline- and taxane-based chemotherapy J Breast Cancer 2011 14 283 8 22323914 Li Xr Liu M Zhang Yj Evaluation of ER, PgR, HER-2, Ki-67, cyclin D1, and nm23-H1 as predictors of pathological complete response to neoadjuvant chemotherapy for locally advanced breast cancer Med Oncol 2011 28 31 8 20467919 Mohammadizadeh F Hani M Ranaee M Role of cyclin D1 in breast carcinoma J Res Med Sci 2013 18 1021 5 24523791 O’Brien J Hayder H Peng C Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins JoVE 2016 2016 e54719 Ortiz AB Garcia D Vicente Y Prognostic significance of cyclin D1 protein expression and gene amplification in invasive breast carcinoma PLoS One 2017 12 e0188068 29140993 Pacifico F Leonardi A NF-kappaB in solid tumors Biochem Pharmacol 2006 72 1142 52 16956585 Pysz MA Hao F Hizli AA Differential regulation of cyclin D1 expression by protein kinase C α and ϵ signaling in intestinal epithelial cells J Biol Chem 2014 289 22268 83 24914206 Reis-Filho JS Savage K Lambros MBK Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: an immunohistochemical and chromogenic in situ hybridisation analysis Mod Pathol 2006 19 999 1009 16648863 Rickham PP Human Experimentation Code Of Ethics Of The World Medical Association Declaration Of Helsinki. Br Med J 1964 2 177 14150898 Rustamadji P Wiyarta E Bethania KA CD44 Variant Exon 6 Isoform Expression as a Potential Predictor of Lymph Node Metastasis in Invasive Breast Carcinoma of No Special Type Int J Breast Cancer 2021 2021 1586367 34925920 Rustamadji P Wiyarta E Bethania KA Potential of AKT2 expression as a predictor of lymph-node metastasis in invasive breast carcinoma of no special type J Pathol Transl Med 2021 55 271 8 34111909 Sharma GN Dave R Sanadya J Various types and management of breast cancer: an overview J Adv Pharm Technol Res 2010 1 109 26 22247839 Tiezzi DG Andrade JM Ribeiro-Silva A HER-2, p53, p21 and hormonal receptors proteins expression as predictive factors of response and prognosis in locally advanced breast cancer treated with neoadjuvant docetaxel plus epirubicin combination BMC Cancer 2007 7 36 17324279 Villegas SL Darb-Esfahani S von Minckwitz G Expression of Cyclin D1 protein in residual tumor after neoadjuvant chemotherapy for breast cancer Breast Cancer Res Treat 2018 168 179 87 29177689 Wachter DL Fasching PA Haeberle L Prognostic molecular markers and neoadjuvant therapy response in anthracycline-treated breast cancer patients Arch Gynecol Obstet 2013 287 337 44 22955249 Zhang Z Zhang X Chen S Taxane-based chemotherapy and risk of breast cancer-related lymphedema: Protocol for a systematic review and meta-analysis Medicine (Baltimore) 2019 98 e16563 31348280

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116136 10.31557/APJCP.2023.24.4.1151 Research Article Andersen’s Behavioral Model to Identify Correlates of Cervical Cancer Knowledge among American Indian Women Roh Soonhee 1* Lee Hee Yun 2 Jun Jung Sim 3 Lee Yeon-Shim 4 Won Cho Rong 2 1 Department of Social Work, University of South Dakota, 4801 North Career Ave, #145C, Sioux Falls, SD 57107, United States. 2 School of Social Work, The University of Alabama, 1022 Little Hall, Judy Bonner Drive, Tuscaloosa, AL 35401, United States. 3 Department of Sociology, Anthropology, and Social Work, 250 Waters Hall, 1603 Old Claflin Pl. Manhattan, KS 66506, United States. 4 School of Social Work, San Francisco State University, 1600 Holloway Avenue, San Francisco, CA, 94132, United States. * For Correspondence: Soonhee.Roh@usd.edu 2023 24 4 11511157 8 9 2022 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: Using the Andersen’s behavioral model of health services use as a framework, this study aims to examine factors (predisposing, needs, and enabling) related to American Indian (AI) women’s cervical cancer knowledge. Methods: Andersen’s behavioral model of health services was used to examine factors predisposing, needs, and enabling related to AI women’s cervical cancer knowledge. A sample of 259 AI women residing in the Northern Plains was recruited using a convenience sampling strategy. Cervical cancer knowledge was measured using guidelines from the American Cancer Society. Three predisposing factors, six enabling factors, and four need factors were observed. Result: The mean score of knowledge was 9.11 out of 13. Higher cancer knowledge was associated with 3 enabling factors (higher education, higher HPV knowledge, and use of TV/radio to gain health literacy) and one needs factor (experience in hospitalization). Conclusion: Our findings indicate that culturally sensitive educational interventions, especially those using media, to increase cervical cancer knowledge are needed among AI women. Key Words American Indian female Andersen’s behavioral model cervical cancer knowledge HPV Northern plains ==== Body pmcIntroduction Cervical cancer is highly treatable with early screening and detection. Cancer, in general, is the leading cause of death among American Indian (AI) women (National Center for Health Statistics, 2021). In recent trends reported by the National Cancer Institute (2021) AI women had higher incidence and mortality rates per 100,000 persons with 7.7 and 2.2, respectively than non-Hispanic White women with 6.7 and 2.0, respectively. Furthermore, AI women experience disparities in cancer incidence and mortality rates, which vary by tribe and region (Becker et al., 2008; White et al., 2014). When regional differences were observed, the incidence rates nearly doubled for Northern Plains (12.0 vs. 6.3), Alaska (10.9 vs. 6.8), Southern Plains (13.8 vs. 8.5), and Pacific Coast (13.8 vs. 6.9) (Melkonian et al., 2020). In South Dakota, the cervical cancer mortality rate among AI women is 79% higher than White women and 500% higher than the national average, resulting in a severe public health issue (Espey et al., 2014; Pandhi et al., 2011; Schmidt-Grimminger et al., 2013; Wienski, 2017). Because AI women tend to experience a higher prevalence of late-stage diagnosis, a lower survival rate, and a rapidly increasing incidence rate, it is critical to increase the cervical cancer knowledge of this population (Espey et al., 2005; Rogers and Petereit, 2011). Studies report a marginal level of cervical cancer knowledge among AI women. In a study conducted with AI women in Canada, O’Brien and associates (2009) reported that Cree First Nations women had inadequate information about cervical cancer knowledge and cancer screening. A systematic review on facilitators and barriers to cervical cancer knowledge and screening found a lack of awareness of disease progression and human papillomavirus (HPV) to be cited in many studies (Sethi et al., 2021). Previous studies report common predictors to knowledge related to cervical cancer, such as Pap smear or HPV. For example, one study assessing the effectiveness of cervical cancer education programs among AI women found that a higher level of Pap smear knowledge was associated with high educational level and income (Dignan et al., 1998). Another study on HPV knowledge, one of the leading infections causing cervical cancer, found that African Americans had lower knowledge than Whites, and those with less educational levels had lower knowledge than those with more education (Hughes et al., 2009). Other studies specifically on cervical cancer knowledge found similar predictors. For example, studies conducted in Sub-Saharan Africa found younger age, no birthing experience, low educational level, not being employed, low wealth levels, not having health insurance, far distance to health facility, rural residency, and not using radio or TV to be predictors of low cervical cancer knowledge (Kangmennaang et al., 2018; Moodley et al., 2020). Another study found rural residency and lower socioeconomic status among female medical university students in rural India to be associated with lower knowledge (Patel et al., 2021). Cervical cancer screening literacy, or knowledge in cervical cancer screening guidelines, is found to be related to cervical cancer screening behaviors among AI women (Kolahdooz et al., 2014; Lee et al., 2020; O’Brien et al., 2009). Thus, increasing knowledge about cervical cancer and screening guidelines is a significant factor in promoting screening behavior. Indeed, AI women have two barriers to receiving cervical cancer screening: they have less knowledge of cancer screening and are less likely to have a particular place to receive medical care than their White counterparts (Espey et al., 2014; Kolahdooz et al., 2014). The theoretical framework used for this study was Andersen’s behavioral model of health services. Some studies have applied this model on cancer related knowledge (Beltran et al., 2016; Lee et al., 2018; An et al., 2020). This model provides a relatively comprehensive guideline for conceptualizing the relationships between multiple explanatory factors (i.e., predisposing, enabling, and need factors) associated with health knowledge. Andersen’s behavioral model of health has been widely applied in studies on cancer screening behavior (Andersen, 1995). A systematic review of the model found that age, marital status, gender/sex, education, and ethnicity were used as predisposing factors (Babitsch et al., 2012). This same review found commonly used variables for enabling factors were income, health insurance, and having a primary care provider, while need factors included health status (Babitsch et al., 2012). Moreover, the model has been applied to various racial/ethnic minority populations, including the AI populations (Andersen et al., 2014; Babitsch et al., 2012; Roh et al., 2016). Some commonly found predisposing factors were age and gender, while income, education, and having a primary care physician were enabling factors (Jin et al., 2019b). Common needs factors included health status (Jin et al., 2019b), colorectal cancer screening adherence (Jin et al., 2019a), health literacy (Lee et al., 2015; Khuu et al., 2018), use of health service (Surood et al., 2010), and cancer literacy (Lee et al., 2014). Specifically, a study among AI women reported age, educational attainment, annual health checkups, awareness of mammograms, greater self-efficacy of colorectal cancer, and family and personal cancer history as predictors of mammogram uptake (Lee et al., 2020) and colorectal screening adherence (Roh et al., 2016). With 574 federally recognized AI tribes in the U.S. (U.S. Department of the Interior Indian Affairs, 2023) and the heterogeneity across tribes, it is critical to conduct regional investigations to consider unique tribal and cultural differences. Northern Plains AI women appear to differ from other region’s AI women in their cervical cancer knowledge. Moreover, little research has investigated AI women’s cancer knowledge with appropriate theoretical models that may guide more systematic intervention efforts. Therefore, this study aimed to use the Andersen’s behavioral model of health services as a framework to examine factors, predisposing, needs, and enabling related to AI women’s cervical cancer knowledge. Materials and Methods Sample and Data Collection After approval from the lead author’s institutional review board, a survey was conducted in rural areas with AI women between September 2013 and May 2014. The research team used a survey research design with convenience sampling to examine factors correlated with cervical cancer knowledge among AI women residing in the Northern Plains. Participants were recruited from multiple locations: local AI churches, other religious organizations, senior housing facilities, senior centers, an annual Indian art market, and three powwows in South Dakota. Although 269 AI women participated in the study, 10 participants were excluded due to missing data, yielding an analytic sample of 259. The study used a self-administered questionnaire; however, trained interviewers were available for anyone who asked for assistance in reading and understanding the questions (four participants required such assistance). The questionnaire took about 30 minutes to complete, and participants were offered $10 cash for their time. Measures and Variables Dependent variable Cervical cancer knowledge In order to measure cervical cancer knowledge, the research team modified 13 questions into a scale by following the guidelines from the American Cancer Society (American Cancer Society, 2021a; 2021b; Betancourt et al., 2010). Answers were coded in a binary format (1 = yes, 0 = no), ranging from 0 to 13. Higher scores indicated higher cervical cancer knowledge. Examples of the modified questions for the scale include: “I believe Pap test can help detect cervical cancer earlier”; “Human papilloma virus infection can cause cervical cancer”; and “Having several miscarriages can increase the risk of getting cervical cancer”. Cronbach’s alpha of this scale was .73 in this study. Independent variables Predisposing Factors Three demographic characteristics were collected to reflect predisposing factors, including age (in years, a continuous variable); marital status (1 = married, 0 = other); and religion (1 = yes, 0 = no). Enabling Factors This study included six variables as enabling factors: 1) monthly household income (ranged from 1 = less than $999 to 4 = $3,000 or over); 2) education (ranged from 1 = no high school diploma/GED to 4 = graduate or over); 3) appropriate methods for improving cancer health literacy: TV/radio (1 = yes, 0 = no); 4) awareness of Pap test (1 = yes, 0 = no); 5) health benefits regarding cancer screening; and 6) knowledge of HPV. This study employed one subscale (of the six subscales) of the Health Belief Model (HBM) constructs to measure health benefits regarding cancer screening (Glanz et al., 2008). The perceived health benefits subscale consists of 5 items with a 5-point Likert scale (from “1 = strongly disagree” to “5 = strongly agree”). The total scores ranged from 5 to 25. Higher scores indicated greater levels of perceived health benefits towards cancer screening. Examples of items from the subscale include: “When I participated in cancer screening, I feel good about myself” and “Participating in cancer screening will allow me to detect cancer early”. The coefficient of reliability was a = .82 in this study. This study used a nominal scale (1 = yes, 0 = no) with ten items regarding the knowledge of HPV to measure HPV knowledge (Buchwald et al., 2013). The total scores ranged from 0 to 10, and higher scores indicated more knowledge of HPV. Examples of items from the scale include: “HPV can cause cervical cancer” and “HPV is spread by sexual contact.” Cronbach’s alpha of this scale was .75 in this study. Need Factors This study used four variables to represent need factors: 1) personal cancer experience (1 = yes, 0 = no), “Has the doctor ever told you that you had cancer of any kind?”; 2) family cancer experience (1 = yes, 0 = no), “Have any of your family ever had cancer of any kind?”; 3) hospitalization (1 = yes, 0 = no), “Have you ever been hospitalized for last year?”; and 4) treatment without hospitalization (1 = yes, 0 = no), “Did you have treatment from hospitals or clinics, health centers, for the last three months without hospitalization?”. Data Analysis Three different data analysis methods were used in this study. First, the descriptive statistics method was used to understand participants’ socio-demographic characteristics. Second, a bivariate Spearman correlation matrix was employed to understand basic correlations among the main study variables. Third, the hierarchical multivariate regression method explored significant predictors influencing cervical cancer knowledge among the 259 AI women (Mertler and Vannatta, 2005). In addition, the regression analysis identified the specific amount of variance (R²) for three different hierarchical steps (George and Mallery, 2016) in the continuous dependent variable, cervical cancer knowledge. No multicollinearity problems were observed among all independent variables since the variance inflation factor scores were greater than 1.04 (Mertler and Vannatta, 2005). The IBM Statistical Package for the Social Sciences Program (SPSS version 28) was used for all the data analysis. Results Table 1 summarizes the socio-demographic characteristics of participants. The age of 259 AI women ranged from 18 to 65 years, with a mean age of about 42 years (SD = 13.38). Greater portions of participants were married (31%) or never married (38%). The majority of participants (89%) had a religious affiliation. Slightly more than 39% of participants’ monthly household income was less than $999, and about 21% reported not having a high school diploma/GED. About 39% of participants answered that TV/radio was an appropriate method for health literacy, while about 61% answered that it was not. Slightly over 95% indicated awareness of the Pap test. A large portion of participants, about 71%, had a family cancer history, while only 17% reported a personal cancer history. Almost 80% of participants experienced hospitalization in the previous year, while 39% reported receiving treatment without hospitalization. The mean score for health benefits was 17.78 (SD = 3.87; Range = 5 to 25), indicating that participants moderately agreed that taking a cancer screening will benefit their health. The mean score for HPV knowledge was 6.15 (SD = 1.90; Range = 0 to 10), indicating that participants had moderate HPV knowledge (Rashwan, Lubis, and Ni, 2009). Also, participants answered 64.8% of questions about HPV correctly. Finally, the mean score for cervical cancer knowledge was 9.11 (SD = 2.12; Range = 2 to 13), indicating that around 70% of answers for questions about cervical cancer knowledge were correct. The bivariate correlations among the main study variables are presented in Table 2. The main results indicated that cervical cancer knowledge was positively and significantly associated with monthly household income (r = 0.18, p ≤ 0.01), education (r = 0.27, p ≤ 0.01), and knowledge of HPV (r = 0.32, p ≤ 0.01). However, there was a negative association between cervical cancer knowledge and hospitalization (r = -0.15, p ≤ 0.05). In Table 3, the hierarchical multivariate regression results show the impact of predisposing, enabling, and need factors. In Step 1, findings indicated that predisposing variables explained 0.5% of the variance (R²) in cervical cancer knowledge. None of the predisposing factors were significant to cervical cancer knowledge. In Step 2, predisposing and enabling variables accounted for 21% of the variance (R²), increasing about 20.5% from Step 1 (adjusted R² = 18%). Education (B = 0.64, SE = 0.18, p ≤ 0.001) and knowledge of HPV (B = 0.29, SE = 0.07, p ≤ 0.001) were significant factors to cervical cancer knowledge in Step 2. In the final step, predisposing, enabling, and need factors accounted for 24% of the variance (R²), which increased about 3.5% from Step 2 (adjusted R² = 19%). Final results in Step 3 revealed that education was significantly associated with a higher level of cervical cancer knowledge (B = 0.58, SE = 0.18, p ≤ 0.01). People who answered that TV/radio were appropriate methods for health literacy were significantly related to a higher level of cervical cancer knowledge (B = 0.55, SE = 0.27, p ≤ 0.05). A higher level of HPV knowledge was significantly associated with a higher level of cervical cancer knowledge (B = 0.31, SE = 0.07, p ≤ 0.001). Participant hospitalization was negatively and significantly related to their cervical cancer knowledge (B = -0.73, SE = 0.36, p ≤ .05). However, age, married, religion, monthly household income, health benefits, awareness of Pap test, and need factors (personal cancer experience, family cancer experience, and treatment without hospitalization) were not significantly related to cervical cancer knowledge among this sample. Table 1 Socio-Demographics of Participants (in Percent or Mean, N = 259) n % Age Ranged from 18 to 65 M=42.30 (SD=13.38) 18-30 59 22.8 31-50 117 44.8 51-65 84 32.4 Marital status Married 80 31 Never married (single) 99 38.4 Divorced 41 15.9 Other 38 14.7 Religion None 29 11.4 Native American church 54 21.3 Traditional tribal spirituality 85 33.5 Protestant 33 13 Other 53 20.8 Monthly household income Less than $999 99 39.1 $1,000-$1,999 65 25.7 $2,000-$2,999 54 21.4 $3,000 and over 35 13.8 Education No high school diploma/GED 53 20.6 High school diploma/GED 149 58 Bachelor's degree 35 13.6 Graduate or over 20 7.8 Method of health literacy: TV/radio Yes 100 38.6 No 159 61.4 Awareness of Pap test Yes 243 95.3 No 12 4.7 Individual cancer experience Yes 44 17 No 215 83 Family cancer experience Yes 184 71.3 No 74 28.7 Hospitalization Yes 53 20.7 No 203 79.3 Treatment without hospitalization Yes 100 38.8 No 158 61.2 Health benefits Ranged from 5 to 25 M=17.78 (SD=3.87) Knowledge of human papillomavirus Ranged from 0 to 10 M=6.15 (SD=1.90) Cervical cancer knowledge Ranged from 2 to 13 M=9.11 (SD=2.12) Table 2 Correlations among Main Study Variables (N = 259) 1 2 3 4 5 6 7 8 9 10 11 12 13 Cervical cancer knowledge Age -0.04 Married 0.04 0.18** Religion 0.02 0.11 0.02 Monthly household income 0.18** 0.07 0.25** -0.03 Education 0.27** -0.08 0.06 -0.05 0.39** Methods for health literacy: TV/radio 0.12 0.05 -0.08 0.03 -0.02 0.05 Health benefits 0.07 0.15* -0.03 0.11 -0.01 -0.05 0.08 Awareness of Pap test 0.07 0.16* 0.11 0.04 0.12 0.03 0.14* 0.06 Knowledge of human papillomavirus 0.32** -0.09 -0.07 0.01 0.13 0.01 0.06 0.14* 0.04 Individual cancer experience -0.07 0.12 0.05 0 0.04 0.02 0.02 0 -0.05 0.04 Family cancer experience 0.08 0.13* 0.02 0.02 0.1 0.05 -0.01 0.11 0.19** 0.04 0.01* Hospitalization -0.15* 0.06 -0.06 0.05 -0.1 -0.20** -0.03 0.06 -0.11 0 0.28** -0.07 Treatment without hospitalization 0 0.18** -0.06 -0.04 -0.02 -0.03 -0.01 0.08 0.03 -0.07 0.18** 0.08 0.27** Note. *p ≤ .05, ** p ≤ .01, *** p ≤ .001 Table 3 Hierarchical Regression Model for Cervical Cancer Knowledge (N = 259) Cervical cancer knowledge β1 (SE2) Step 1 Step 2 Step 3 Predisposing Age -0.011 (0.011) -0.007 (0.010) ‒0.009 (0.011) Married 0.134 (0.323) 0.165 (0.304) 0.201 (0.306) Religion -0.029 (0.440) -0.008 (0.402) 0.040 (0.401) Enabling Monthly household income 0.077 (0.069) 0.072 (0.069) Education 0.638 (0.183)*** 0.577 (0.184)** Methods for health literacy: TV/radio 0.498 (0.268) 0.552 (0.268)* Health benefits 0.063 (0.036) 0.060 (0.036) Awareness of Pap test -0.360 (0.707) -0.637 (0.713) Knowledge of human papillomavirus 0.294 (0.070)*** 0.308 (0.070)*** Need Personal cancer experience -0.325 (0.375) Family cancer experience 0.245 (0.309) Hospitalization -0.727 (0.362)* Treatment without hospitalization 0.421 (0.286) F test (d.f. = 13) 0.332 6.062*** 4.822*** R² 0.005 0.21 0.238 Adjusted R² -0.009 0.176 0.188 Notes. *p ≤ .05, **p ≤ .01, ***p ≤ .001; 1, Unstandardized Beta coefficients; 2, Standard errors Discussion This study observed factors related to AI women’s cervical cancer knowledge by adopting Andersen’s behavioral model of health services use as a theoretical guide. Similar to previous literature on cancer screening adherence, education was significantly associated with higher knowledge (Kolahdooz et al., 2014; Lee et al., 2020). A possible explanation is that highly educated individuals may better understand cancer knowledge than their counterparts. Also, another enabling factor associated with cervical cancer knowledge was a higher level of HPV knowledge. Since HPV infection is one of the highest contributing factors to cervical cancer (Center for Disease Control and Prevention, 2020), those already aware of HPV may likely be aware of cervical cancer. Moreover, those who reported TV/radio as appropriate methods for health literacy had higher cervical cancer knowledge. A systematic review on cancer screening knowledge among an Indigenous population found that using media advertisements on cancer improved knowledge about cancer (Kolahdooz et al., 2014), indicating that media can be an excellent tool to distribute cancer information. Thus, an education program is highly needed among the AI community. Finally, this study found a negative association with participant hospitalization. A plausible explanation could be that historical oppression, discrimination, and culturally insensitive or inadequate services during hospitalizations may have caused mistrust in Western medical services (Gone and Trimble, 2012; Burnette, 2014), leading to a decrease in opportunities to obtain cancer-related knowledge. Hence, a culturally tailored education program is necessary. Limitations Some limitations to the present study should be noted. First, given its nonprobability sampling strategies, the present study focused on volunteer samples of three community-dwelling AI women. As such, findings are only suggestive, and their non-representative nature presents some limits on the generalizability of this study’s findings. Second, these data were based on a cross-sectional survey that hinders the researchers from identifying the causal directions of these associations. Additional exploration with a longitudinal study would help to draw causal inferences. Third, while this study successfully examined, an understudied area among rural AI women am hard-to-reach population, particular characteristics of the participants from this region may have influenced responses to our questions; and such characteristics may have changed since 2014. Therefore, similar research conducted in other parts of the U.S. and/or in more recent years may produce different results. Fourth, selection bias might have affected findings in several ways. Participants might have been more willing to discuss cervical cancer knowledge than AI women who did not choose to participate. Future studies should include a more representative sample and diverse tribal groups because cancer care may vary significantly by tribal affiliation, regional location, and rural/urban contexts. Fifth, all of the data were based on self-report, and participants could have provided answers they considered to be socially desirable. Finally, Andersen’s model does not include non-Western conceptualizations of health or preference for traditional healing modalities over biomedical treatments, which are relevant for many AIs (Beals et al., 2005; Hartmann and Gone, 2012). Implications for Health Research and Practice Despite these limitations, this study’s findings add to the limited body of literature providing critical information for promoting cervical cancer literacy among AI women. To our knowledge, this is the first study to examine factors associated with cervical cancer knowledge among AI women in the Northern Plains. Our findings suggest the importance of examining Andersen’s model as a meaningful conceptualization and framework for developing effective prevention and intervention strategies targeting AI women. AI women that were highly educated, had higher access to resources for health literacies such as TV/radio, greater knowledge of HPV, and less hospitalization experience had greater cervical cancer knowledge. To develop a more comprehensive intervention to improve cervical cancer knowledge in AI women, research topics should assess a broader spectrum of Andersen’s model constructs, including susceptibility, severity, cues to action, and self-efficacy. Moreover, health professionals working to reduce the disproportionate cervical cancer burden among AI women should work to promote cervical cancer literacy and health service use by targeting the population with associated factors found in our study. For example, as indicated by a systematic review (Kolahdooz et al., 2014), TV/radio can be an educational platform to approaching AI women on cervical cancer knowledge distribution. Other interventional studies using community-based participatory research and tribal community members to conduct the education workshops showed significant increase in cervical cancer knowledge before and after the education was given to AI (Christopher et al., 2008; Subrahmanian et al., 2011). Public health efforts should also be directed toward enhancing motivation in the importance and promotion of cervical cancer knowledge and eliminating perceived barriers to cervical cancer screening. For example, Turquoise Tuesday (third Tuesday on January) is a national cervical cancer awareness day for AI women and Congress has appointed January as the Cervical Health Awareness Month (National Indian Council on Aging, 2020). In cooperation with the American Indian Cancer Foundation which hosts events to promote screening and educate the AI community, more efforts should be directed towards community-wide campaign and education events via offline and online. Author Contribution Statement Dr. Soonhee Roh contributed to conception, design, and collection of data. Dr. Hee Yun Lee contributed to conception and design. Dr. Jung Sim Jun contributed to conception and data analysis. Dr. Yeon-Shim Lee contributed to conception, design, and collection of data. Rong Won Cho contributed to data analysis. Acknowledgements Funding Statement This work was supported by funding through the University of South Dakota Seed Grants at the School of Health Sciences. This work was also supported, in part, by Dakota Cancer Collaborative on Translational Activity (DaCCoTA) Scholars Award. The DaCCoTA is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number U54GM128729. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health (NIH). Ethical Declaration This study was approved by the University of South Dakota Institutional Review Board Data Availability (if apply to your research) Data is not available for the public. Study Registration Not applicable Conflict of Interest All authors declare that they have no potential competing interest. ==== Refs References American Cancer Society Cervical cancer quiz 2021a https://www.cancer.org/cancer/cervical-cancer/cervical-cancer-quiz.html Andersen R Revisiting the behavioral model and access to medical care: Does it matter? J Health Soc Behav 1995 36 1 10 7738325 Andersen RM Davidson PL Baumeister SE G. F. 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A case study of the determinants of cervical cancer knowledge and screening in Kenya Soc Sci Med 2018 197 203 12 29253722 Khuu BP Lee HY Zhou AQ Health Literacy and Associated Factors Among Hmong American Immigrants: Addressing the Health Disparities J Community Health 2018 43 11 8 28528527 Kolahdooz F Jang SL Corriveau A Knowledge, attitudes, and behaviours towards cancer screening in indigenous populations: a systematic review Lancet Oncol 2014 15 e504 16 25281469 Lee HY Choi J-K Lee MH Health literacy in an underserved immigrant population: New implications toward achieving health equity Asian Am J Psychol 2015 6 97 105 Lee HY Choi JK Park JH The Primary Care Physician and Cancer Literacy: Reducing Health Disparities in an Immigrant Population Health Educ J 2014 73 435 45 Lee HY Choi YJ Yoon YJ HPV Literacy: The role of English proficiency in Korean American immigrant women Clin J Oncol Nurs 2018 22 64 70 Lee YS Roh S Moon H Andersen’s Behavioral Model to Identify Correlates of Breast Cancer Screening Behaviors among Indigenous Women J Evid Based Soc Work 2020 17 117 35 Melkonian SC Weir HK Jim MA Incidence of and Trends in the Leading Cancers With Elevated Incidence Among American Indian and Alaska Native Populations, 2012–2016 Am J Epidemiol 2020 190 528 38 Moodley J Constant D Mwaka AD Mapping awareness of breast and cervical cancer risk factors, symptoms and lay beliefs in Uganda and South Africa PLoS One 2020 15 1 17 National Indian Council on Aging American Indians Twice As Likely To Develop Cervical Cancer [Online] 2020 Albuquerque: NICOA Available: https://www.nicoa.org/american-indians-twice-as-likely-to-develop-cervical-cancer / [Accessed October 16 2021] National Center for Health Statistics (U.S.) ortality Profile of the Non-Hispanic American Indian or Alaska Native Population, 2019. 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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116137 10.31557/APJCP.2023.24.4.1159 Research Article Integrative Analysis of the AMPK subunits in Colorectal Adeno Carcinoma T B Beena 1 A Jesil Mathew 1* K B Harikumar 2 E Safeeda 3 1 Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Science, Manipal Academy of Higher Education, Manipal, Udupi, Karnataka, India. 2 Cancer Research Program, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram 695014, India. 3 Centre for Professional and Advanced Studies, Department of Medical Biochemistry Gandhi Nagar, Kottayam, Kerala, India. * For Correspondence: jesil.m@manipal.edu 2023 24 4 11591171 20 9 2022 14 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Background: The 5-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an emerging cancer treatment and therapeutic target. Due to the enzyme’s complexity and dual nature, it is a confounding target in the treatment strategy. The study aimed to conduct an integrative analysis of the seven subunits and twelve isoforms of AMPK, which is not reported so far in colorectal adenocarcinoma patients. Methodology: The web-based tools UALCAN, Timer 2.0, KM Plotter, cBioPortal, COSMIC, and STRING were used to investigate the differential expression of AMPK subunits, protein-level Expression, promoter methylation status, survival analyses, Enrichment analysis, and protein-protein interaction. Results: The mRNA expression of AMPK subunits are upregulated in Colorectal Adeno Carcinoma (COAD), while the protein expression is comparatively reduced in colon tumors. The protein-level expression of α2 and β2 is decreased significantly in COAD patients. The γ3 subunit in colon tumor is hypermethylated. The study also reports that Liver Kinase B1 mutation in 7% of CRC patients, which might be the reason for downregulation of the gene and the protein expression of AMPK subunits in COAD. Conclusion: The Overall analysis of the subunits affirms that AMPK expression is beneficial in cancer. Key Words AMPK Colorectal cancer differential gene expression protein expression survival analysis ==== Body pmcIntroduction Colorectal Cancer (CRC) is the third most commonly diagnosed cancer which, accounts for 10% of global cancer incidence and 9.4% of cancer deaths in 2020, slightly behind lung cancer, which accounts for 18% of fatalities (Lu et al., 2021; Morris et al., 2018). Based on projections of aging, population expansion, and human progress, the global number of new CRC cases is expected to reach 3.2 million in 2040. The increased prevalence of CRC is primarily due to increased exposure to environmental risk factors because of a shift in lifestyle and food towards westernization (WHO, 2020). The energy homeostasis guardian, 5-adenosine monophosphate (AMP)-activated protein kinase (AMPK), is a well-known serine-threonine kinase family member. All eukaryotes have genes that code for AMPK subunits, making it an evolutionarily conserved set of enzymes. It is a heterotrimeric enzyme complex having catalytic α and regulatory β and γ subunits that are required for the AMPK to function (Guy and Hardie, 1981; Richter and Ruderman, 2010). α Subunit has two isoforms, α1 and α2, encoded by two distinct genes, PRKAA1(Protein Kinase AMP-Activated Catalytic Subunit Alpha 1) and PRKAA2 (Protein Kinase AMP-Activated Catalytic Subunit Alpha 2) in mammals. The N-terminus of the α subunit has a conventional Ser/Thr kinase domain (López et al., 2016). The kinase domain has a region called the activation loop or T-loop. The conserved Thr residue in this region should be phosphorylated to activate the complex. The C- terminal region of the α subunit forms a complex with the β and γ subunits (D Grahame Hardie, 2013). β Subunit has two isoforms, β1 and β2, encoded by different genes PRKAB1(Protein Kinase AMP-Activated Catalytic Subunit Beta 1) and PRKAB2 (Protein Kinase AMP-Activated Catalytic Subunit Beta 2), respectively. β Subunit is the protein scaffold of the AMPK complex since the C- terminal region of the β Subunit interacts with the α and γ subunits (Grahame Hardie et al., 2012). There are three isoforms for the γ subunit γ1, γ2, and γ3, encoded by PRKAG1, PRKAG2, and PRKAG3 (Protein Kinase AMP-Activated Catalytic Subunit γ1, γ2, γ3), respectively. γ subunits have four cystathionine β synthase sequences (CBS motifs). CBS1 and CBS3 are the binding sites for ATP, ADP, or AMP which binds competitively, depending on the cellular energy status. CBS2 and CBS4 strongly bind to AMP (Adenosine Mono Phosphate) and play the structural role (Steinberg and Carling). AMP, when attached to site 1, leads to activation, and site 3 regulates the phosphorylation state of Thr172 (Hardie and Alessi, 2013). The study aimed to analyze the expression of AMPK subunits in normal and primary tumors in each stage of Colorectal adenocarcinoma (COAD), expression during nodal metastasis, association with patient weight, and protein-level expression. The study also analyses the Overall Survival of AMPK subunits in Gastric Cancer patients. Materials and Methods The publicly available datasets are used for the analysis Colorectal Cancer Patient Data analysis The metastatic CRC patient data was collected from cBio Cancer Genomics Portal (http://www.cbioportal.org) based on the study of Memorial Sloan Kettering Cancer Center, Gastroenterology 2020. Four seventy-one patient data with unresectable CRC tumors were analyzed (accessed on January 23, 2022). The Pan-Cancer analysis of AMPK subunits The Pan-cancer differential expression (DE) of AMPK subunits in COAD and the normal tissues were studied using the TIMER 2.0 ( (http://timer.cistrome.org/ accessed on January 18, 2022) (Li et al., 2020). The Gene-DE module in the cancer exploration component was used for the analysis. Both the normal samples (n=41) and the tumor samples (n=451) were analyzed for the significant relation. The DE was generated as a box plot in which the significance was determined using edgeR (Tang et al., 2019; Warrier et al., 2021). The differential gene and total protein expression analysis of AMPK subunits UALCAN is a web tool that provides gene expression from The Cancer Genome Atlas (TGCA)which was used for the DE analysis. The protein expression analysis used data from Clinical Proteomic Tumour Analysis Consortium (CPTAC) Confirmatory/Discovery dataset (http://ualcan.path.uab.edu/index.html) (Accessed on November 13, 2021). Further analysis was done to investigate the expression in individual cancer stages, the association with the weight of the patient, and the nodal metastatic status. The screening conditions set in this study were “Gene: PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3”; “Analysis Type: Tumor vs. Normal Analysis”; “Cancer Type: Colorectal Adenocarcinoma”; “Data Type: TCGA and CPTAC dataset. The expression of the genes on average (n=41) was compared to the patient samples (n=286). The sample size for protein expression was normal=100 and primary tumor=97. The analysis was done using the student’s t-test. For each cancer type, Z-values denote standard deviations from the median across samples. CPTAC’s Log2 Spectral Count Ratio data were first standardized within each sample profile, then across samples (Chandrashekar et al., 2017). Promoter methylation of AMPK subunits The TCGA module of UALCAN was used to measure the promoter methylation level of the AMPK subunits. The Box Whisker plot showed the promoter DNA methylation. The average beta value of the TGCA samples is shown in the graph. The significance was calculated using the student’s t-test, which considers the unequal variance. The methylation status was compared to normal (n=37) vs. primary tumor (n=313). The ratio of methylation probe intensity to the sum of methylated and unmethylated probe intensity is known as beta values. The beta value is a number that ranges from 0 to 1. Unmethylation is shown by a value of zero, while complete methylation is indicated by a value of one. The beta values of CpG probes up to 1500 bp upstream of the gene’s start site are represented in the box plot (Chandrashekar et al., 2017). Identification and analysis of the association of the top twenty mutated genes in CRC, Gene enrichment, and Protein-protein interaction The top 20 genes mutated in CRC were taken from the Catalogue Of Somatic Mutations In Cancer (COSMIC) (https://cancer.sanger.ac.uk/cosmic) database, which provided the data by tissue type and histology. The query terms were Tissue selection: GIT; Sub tissue selection: includes all; histology also included all types like adenoma, carcinoid-endocrine tumor, carcinoma, and others and sub-histology types included adenocarcinoma, gastrinoma, hamartoma, etc. (Tate et al., 2019). ‘STRING’ (http://string-db.org) is a database that gathers, aggregates, and scores publicly available data to investigate possible functional protein interaction networks. The AMPK subunit genes, the prominent upstream and downstream members, and the top 20 mutated genes in CRC were selected to study the protein-protein interaction (Szklarczyk et al., 2019). ‘Metascape’ (http://metascape.org/gp/index.html#/main/) is a tool that combines information from more than 40 different knowledge bases with advanced capabilities such as interaction analysis, gene annotation, and member search. The Gene Ontology module was used to analyze the functional involvement of the top 20 mutated genes and the AMPK subunit genes in biological processes (BP), cellular components (CC), and molecular functions (MF), as well as KEGG pathways (Zhou et al., 2019). Terms with a p-value of less than 0.05, a minimum count of three, and an enrichment factor of more than 1.5 (the enrichment factor is the ratio between the observed counts and the counts expected by chance) were gathered and classified into clusters based on membership commonalities. To account for multiple testing’s, p-values were determined using the accumulative hypergeometric distribution, and q-values were calculated using the Benjamini-Hochberg procedure. When doing hierarchical clustering on the enriched phrases, Kappa score was used as the similarity measure, and sub-trees with a similarity of > 0.3 were considered a cluster. The cluster is represented by the statistically most significant term within it (Szklarczyk et al., 2019). Survival analysis Kaplan Meier plotter (KM) (https://kmplot.com/analysis/), a web-based tool capable of examining the effect of 54,675 genes on survival using 10,461 cancer samples, was used to investigate the prognostic significance of the AMPK subunits. It has 5,143 breast, 1,618 ovarian, 2,437 lung, and 1,065 GC patients with a mean follow-up of 69/40/49/33 months, respectively. The research was based on mRNA expression median values. The patients were categorized based on the high- and low-expression groups. KM Plotter doesn’t provide any particular group in CRC. So, the expression and analysis were done in Gastric cancer patients. The log-rank P-value and hazard ratio (HR) with a 95% confidence interval were used to calculate the overall survival (OS) of GC patients. Results Patient Data Analysis-cBioPortal The cancer type details of 471 CRC patients manifest 73.7% of patients have colon adenocarcinoma, 20.4% have rectal adenocarcinoma, 2.8% have colorectal adenocarcinoma, 2.1% have Mucinous Adenocarcinoma of colon and rectum, and 1.1% percent have Signet Ring Cell Adenocarcinoma of colon and rectum (Figure 1). The tumors were moderately differentiated in 73.2%, poorly differentiated in 25.7%, and well-differentiated in 1.1%. The overall survival rate of CRC patients was 66.0%, with 34% still alive. The primary site of metastasis of CRC is the Liver (63.5%), Lung metastasis ranks the next (38%), Bone metastasis is 7.2%, and metastasis to other sites represents 12.1% (Figure 2). Pan-Cancer Analysis The Pan-cancer analysis of the AMPK subunits didn’t show any consistent upregulation or downregulation of the genes (Figure 3). The expression was highly variable according to the cancer types. The COAD patients showed significant upregulation in PRKAA1, PRKAA2, PRKAB2, PRKAG1, and PRKAG2 expression. PRKAG3 gene encoding γ3 subunit expression was seen only in a few cancer types, including Head and Neck Squamous Cell Carcinoma (HNSC) (Figure 3E). The expression of the β2 subunit was non-significant in COAD patients. Differential Gene Expression of AMPK subunits PRKAA1 gene expression was not affected by the tumor condition. The individual stages of cancer also showed a consistent gene expression level. The nodal metastasis also didn’t affect the expression of the gene. The analysis of the DE based on the patient weight showed a significant result; the highly obese patients showed downregulation of the gene. The PRKAA2 gene expression was significantly reduced compared to normal. A consistent result was shown in individual stages of cancer. The expression increases as the cancer advances and as obesity increases in patients. N1 and N2 levels showed a comparatively increased expression of the gene. The PRKAB1 and PRKAB2 expression didn’t differ in normal and tumor tissues. PRKAG1 expression was increased in primary and individual cancer stages. A slight increase in the expression was seen as the patient’s weight increased, and the same pattern was seen in the nodal metastasis. PRKAG2 expression was decreased compared to the normal tissues, and down-regulation is observed in individual cancer stages and on various stages of nodal metastasis. The expression is slightly reduced in patients with average weight and obese patients (Figure 4). Protein Expression and Promoter methylation PRKAG3 gene, which codes for the AMPK γ 3 subunits, was not expressed in normal and COAD since the subunit is solely expressed in muscular tissues. All the other genes coding the subunits were statistically significant (p>0.05) compared to the expression of the gene in a normal colon. The protein-level expression decreased in all subunits compared to the normal (Figure 5). The γ2 and γ3 subunits were not reported to be expressed in colon cancer. Z values represent standard deviation from median across samples. The expression of α1 and β2 is significantly reduced (normal=1.3, tumor=0.003),( median normal=1.8, tumor=0.08) respectively (Table2). Analyzing the promoter methylation level of the AMPK subunits, only the AMPK γ2 in both normal and primary tumors was hypomethylated, and γ3 in tumor conditions was hypermethylated. Still, the hypomethylation did not affect the gene expression and protein expression (Figure 6). Prognostic role of AMPK subunits KM Plotter assessed the prognostic significance of AMPK subunits in GC. Survival curves were plotted for GC patients. KM plot for the AMPK subunits exhibited the median survival in months for both low expression and high expression cohorts of the subunits. High expression cohorts of PKAA1,PRKAA2,PRKAB1,PRKAB2 has shown improved OS than the low expression cohorts (Figure 7). The p- value for the above is statistically significant, whereas the PRKAG1 and PRKAG2 were not significant and are not related to the OS of the patients (Figure 7). Protein-Protein Interaction and network analysis The significant genes mutated in CRC were taken from the COSMIC database. The genes were KRAS (Kirsten rat sarcoma virus), TP53 (Tumor Protein), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) CDKN2A (cyclin-dependent kinase inhibitor 2A), MEN1(Multiple endocrine neoplasia, type 1), NOTCH1(Neurogenic locus notch homolog protein 1), ARID1A (AT-Rich Interaction Domain 1A), FAT1, APC (Adenomatous polyposis coli), NRG1 (Neuregulin 1), KMT2D (Histone-lysine N-methyltransferase 2D), STK11 (Serine/threonine kinase 11), SMAD4 (Suppressor of Mothers Against Decapentaplegic 2), ERBB4 (Erb-B2 Receptor Tyrosine Kinase 4), ERBB3 (Erb-B2 Receptor Tyrosine Kinase 3), ATM (Ataxia-telangiectasia), MSH6 (mismatch repair protein), RECQL4 (RecQ Like Helicase 4), BRAF (B-Raf murine sarcoma viral oncogene homolog B), and FBXW7 (F-box and WD repeat domain containing 7). Along with the 20 mutated genes PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, and mTOR (Mechanistic Target Of Rapamycin Kinase) were also included to analyze the PPI using STRING. The analysis showed that the genes that code for AMPK interact with STK11, TP53, PIK3CA, mTOR, and ULK1 (Unc-51-like autophagy activating kinase). All the other interactions were through mTOR. The upstream kinase of AMPK, STK11, strongly associates with mTOR, ERBB4, NOTCH, and ATM. Table 1 The Statistical Significance of DE, Promoter Methylation and Protein Expression of AMPK Subunits Encoding Gene DE of Subunits Promoter methylation Statistical significance protein expression PRKAA1 4.29E-01 3.13E-06 1.3308E-07888 PRKAA2 1.39E-06 <1E-12 8.25E-19 PRKAB1 3.65E-01 6.20E-03 6.00E-05 PRKAB2 3.73E-02 4.56E-02 4.42E-19 PRKAG1 8.23E-04 2.30E-03 4.30E-05 PRKAG2 2.06E-06 4.47E-10 Not available PRKAG3 Not expressed Not expressed Not available PRKAA1, protein kinase AMP-activated catalytic subunit alpha 1,PRKAA2, protein kinase AMP-activated catalytic subunit alpha 2, PRKAB1, protein kinase AMP-activated catalytic subunit beta 1; PRKAB1, protein kinase AMP-activated catalytic subunit beta 2; PRKAG1, protein kinase AMP-activated catalytic subunit gamma 1; PRKAG2, protein kinase AMP-activated catalytic subunit gamma 2; PRKAG3: protein kinase AMP-activated catalytic subunit gamma 3 Figure 1 (A), The Types of Colorectal cancer; (B), The pattern of differentiation; (C), Overall Survival (OS) status of patients; (D), KM plots of progression-free survival; and OS (E) of 471 patients from TCGA datasets of cBioPortal Figure 2 The Pie Diagram Showing the Metastasis Pattern of the 471 Colorectal Cancer Patients from TCGA Datasets Table 2 The Median Z-value for Protein Expression of AMPK Subunits Encoding Gene Median Z- value (Normal) Median Z-value (Tumor) PRKAA1 0.7 -0.043 PRKAA2 1.3 0.003 PRKAB1 0.4 0.029 PRKAB2 1.8 0.08 PRKAG1 0.96 0.015 PRKAA1, protein kinase AMP-activated catalytic subunit alpha 1,PRKAA2, protein kinase AMP-activated catalytic subunit alpha 2, PRKAB1, protein kinase AMP-activated catalytic subunit beta 1; PRKAB1, protein kinase AMP-activated catalytic subunit beta 2; PRKAG1, protein kinase AMP-activated catalytic subunit gamma 1; PRKAG2, protein kinase AMP-activated catalytic subunit gamma 2; PRKAG3: protein kinase AMP-activated catalytic subunit gamma 3 Figure 3 The PAN CANCER Analysis of AMPK Subunits (A-C) in Normal and Colon Tumors across the TCGA Datasets. The p- values are also represented as stars Figure 4 The Differential Expression of the Gene is Given across Tumor vs Normal Tissues, Individual Stages of Cancer, based on Patient Weight, and based on the Nodal Metastatic Status of Cancer. A: PRKAA1, B: PRKAA2, C: PRKAB1 Figure 5 The Figure Shows CPTAC Samples Used to Compare the Protein-Level Expression of AMPK Subunits in Normal and Tumor Tissues. A: PRKAA1, B: PRKAA2, C: PRKAB1:D, PRKAB2, E: PRKAG1 Figure 6 The Figure Represents the Promoter Methylation Level of the Subunits of AMPK. The data used to compare the samples are collected from TCGA. A: PRKAA1, B: PRKAA2, C: PRKAB1:D, PRKAB2, E: PRKAG1, F: PRKAG2, G: PRKAG3 Figure 7 The KM Plot of the AMPK Subunits. The figure depicts the OS of the patients in lower and higher expression of the subunits. HR: Hazard Ratio Figure 8 GO Analysis of AMPK (A) PPI Network analysis (B) by String of the most Mutated 20 Genes in COAD Patients Discussion The involvement of AMPK in cancer is debatable (Richter and Ruderman, 2010), even though new research has proven that AMPK is a tumor suppressor (Steinberg and Carling, 2019). AMPK is reported to have a dual nature in cancer mostly due to its complex structure-function and the context-dependent roles (Chuang et al., 2014). The tumor suppressor role of AMPK is believed to be mediated via LKB1. A recent study on B cells supports the tumor suppressive role, which has only one catalytic subunit, AMPK-α1. This has been knocked out, which accelerated the development of lymphomas in transgenic mice overexpressing c-Myc in the B cells. The tumor suppressor role of AMPK is mainly due to cell cycle arrest. It is associated with the stabilization of p53 and the cyclin-dependent kinase inhibitors p21WAF1 and p27CIP1. It also inhibits the anabolism of all the macromolecules and inhibits cell growth. The arrest in cell proliferation is by inhibiting mTORC1 phosphorylation. This leads to the ‘anti-Warburg’ effect by inhibiting hypoxia-inducible factor-1α (HIF-1α) (Baba et al., 2010). The present study analyzes the DE and protein expression of AMPK subunits using web tools. The subunit expression of AMPK is studied in different cancers, including CRC. The reported studies align with the current research that genes coding AMPK subunits are expressed in the tumor and normal tissues, with β2 and γ3 subunits an exemption. TGCA data from UALCAN reports that α2 expression is downregulated in COAD, but the data is not supported by the TIMER 2.0 data. According to TIMER 2.0 data, AMPK β1 is downregulated in COAD patients. Even though the mRNA is expressed in tumor samples, the protein level expression is decreased in COAD patients. As per the literature survey, the cancer cells downregulate the expression of AMPK through LKB1 mutation. 30% of non-small cell lung cancers and 20% of cervical cancers, and 10% of cutaneous melanomas report this mutation. There are shreds of evidence that AMPK activity is reduced in cancer. Reports show that enhanced tumor cell growth reduced the expression of the AMPK-α2 subunits in some cases of hepatocellular carcinoma. B-Raf mutation in melanoma cells also reported reduced AMPK activation. A more prominent mechanism of AMPK inhibition is phosphorylation at Ser 485 by Akt. This has been hypothesized to prevent the activating phosphorylation at Thr172, which is not yet studied in cancer. The possible hypothesis of downregulation of AMPK in cancer is its context-dependent role ( Hardie, 2011; Shackelford and Shaw, 2009). But the analysis of the study reports that AMPK subunits are expressed at the mRNA level. At the same time, the protein expression is reduced, which indicates that the AMPK complex formation and its functioning in the tumor microenvironment are to be studied in different types of cancer. AMPK is considered to be a target in obesity and metabolic syndrome. AMPKα1 expression is increased in patients with obesity and CRC. But the highly obese conditions inhibited the subunit expression, which needs further studies to unveil the mechanism. The gene expression of AMPKα2 was significantly reduced in COAD patients. Protein level expression of the subunits is also reduced in the tumor, which may be attributed to the peculiarities of AMPK like tissue specificity, Subcellular localization, tumor microenvironment, etc. The downregulation of gene and protein expression might be due to the mutation of the upstream kinase LKB1. The COSMIC data analysis for the major mutations in CRC shows that 7% of the CRCs manifest mutation of the STK11 gene encoding LKB1in CRC patients. The string analysis shows that the genes encoding the AMPK subunits are associated with P53 and PIK3CA genes which are also mutated in CRC. The gene enrichment analysis using metascape presents the significant functions of AMPK subunits and the top 20 genes mutated genes in CRC, which include positive regulation of biological processes, metabolic process, response to stimulus, growth, developmental process, signaling, rhythmic and cellular process, etc. This also provides the possible diseases associated with similar genes: Head and Neck Squamous cell carcinoma and their involvement in integrated breast cancer pathway, response to radiation, Cushing syndrome, and DNA recombination. Epigenetic modifications have a definite role in tumor formation and progression. DNA methylation is one of the significant epigenetic modifications that have a crucial role in cancer. DNA global hypomethylation is an early event in colorectal tumorigenesis, and the progression of the disease is associated with an increase in DNA methyltransferase activity (Habib et al., 2000). Global DNA hypomethylation and promoter-specific DNA methylation have been linked to genomic instability and tumor initiation in CRCs (Paz et al., 2005). The promoter methylation of the AMPK subunits is analyzed in normal and tumor tissues. The promoter DNA sequence of the γ3 subunits is hypermethylated, and this may not have any significance in CRC because the subunit is only expressed in muscle cells. The γ2 subunit is hypomethylated in the normal colon and tumor tissues. Many studies have shown that AMPK is an effective target in the therapy of CRC ( Honari et al., 2019; Hu et al., 2019; La et al., 2017; Li et al., 2015) Metformin’s usage in CRC has been studied extensively in vitro, in vivo, and in preclinical investigations (Bradley et al., 2018; Meng et al., 2017; Mogavero et al., 2017). The analysis conducted in the present study also affirms that AMPK activation benefits CRC patients’ overall survival. KM Plotter was used to analyze the predictive importance of AMPK subunits in GC. The survival study reveals that the PRKAB2 expression is not associated with the patient’s overall survival. However, overexpression of all other subunits has been linked to a better prognosis in CRC patients. Earlier studies also report that AMPKα1expression is associated with an improved OS in patients. In 37 renal cell carcinoma patients, total AMPKα1/α2 protein expression was linked with improved Progress Free Survival. Recent research suggests that overexpression of the genes encoding for AMPKα1, α2, β1, β2, and γ1 subunits was also related to improved OS in 417 clear-cell renal cell carcinoma patients, the study was based on the data from Cancer Genome Atlas (TCGA). Hoffman and colleagues found a link between higher expression of the genes encoding the regulatory AMPKα1 and α2 subunits and improved 5-year survival (P = 0.001 and 0.021, respectively) in diffuse large B-cell lymphoma patients using the Oncomine database (Zadra et al., 2015). As a concluding note, the genes encoding the AMPK subunits are upregulated in COAD, while the protein expression is comparatively reduced in colon tumors. The γ2 subunit in normal and colon tissue are hypomethylated, and the γ3 subunit in colon tumor is hypermethylated. The OS analysis of the subunits affirms that AMPK expression is beneficial in cancer. The downregulation of the gene and the protein of AMPK expression might be due to the mutation of the upstream kinase LKB1. Author Contribution Statement Study conception and design by KBH, JMA, and BTB. BTB and SE collected and processed the data. BTB has done the analysis. BTB prepared the first manuscript. All the authors commented on and revised the first draft of the manuscript. All the authors approved the final draft. Acknowledgements The authors want to acknowledge the support from the DST-INSPIRE Fellowship, Department of Science and Technology, Government of India, New Delhi, India, [DST/INSPIRE Fellowship/2018/IF 180900] Ethical approval No human or animal participation is involved in the study so the ethical approval doesn’t apply. Availability of Data The data that are extracted for the study is included in the current study. Conflict of Interest The authors declare no competing interest financially and non-financially. ==== Refs References Baba Y Nosho K Shima K Prognostic significance of AMP-activated protein kinase expression and modifying effect of MAPK3/1 in colorectal cancer Br J Cancer 2010 103 1025 33 20808308 Bradley MC Ferrara A Cohort Study of Metformin and Colorectal Cancer Risk among Patients with Diabetes Mellitus Cancer Epidemiol Biomarkers Prev 2018 27 525 531 29716927 Chandrashekar DS Bashel B UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses Neoplasia 2017 19 649 58 28732212 Chuang H Chou C Kulp SK Chen C AMPK as a Potential Anticancer Target - Friend or Foe? Curr Pharm Des 2014 20 2607 18 23859619 Guy PS Hardie DG Regulation of mammalian acetyl-CoA carboxylase FEBS Lett 1981 132 67 70 6117478 Habib M Dumollard J Barthelemy C Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis Gut 2000 47 689 93 11034586 Hardie DG Alessi DR LKB1 and AMPK and the cancer-metabolism link - ten years after BMC Biol 2013 11 1 11 23294804 Hardie D Grahame Ross FA Hawley SA AMPK: A nutrient and energy sensor that maintains energy homeostasis Nat Rev Mol Cell Biol 2012 13 251 62 22436748 Hardie DG AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function Genesdev 2011 25 1 14 Hardie DG Metabolism of inflammation limited by AMPK and pseudo-starvation Nature 2013 493 346 55 23325217 Honari M Shafabakhsh R Reiter RJ Resveratrol is a promising agent for colorectal cancer prevention and treatment : focus on molecular mechanisms Cancer Cell Int 2019 19 1 8 30622437 La X Zhang L Li Z Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells Oncotarget 2017 8 20909 24 28157699 Li T Fu J Zeng Z TIMER 0 for analysis of tumor-infiltrating immune cells Nucleic Acids Res 2020 48 W509 14 32442275 Li W Saud SM Young MR Targeting AMPK for cancer prevention and treatment Oncotarget 2015 6 7365 78 25812084 López M Nogueiras R Tena-sempere M Diéguez C Hypothalamic AMPK: a canonical regulator of whole-body energy balance Nat Rev Endocrinol 2016 12 421 32 27199291 Lu B Li N Luo CY Colorectal cancer incidence and mortality: the current status, temporal trends and their attributable risk factors in 60 countries in 2000-2019 Chin Med J 2021 134 1941 51 34238851 Meng F Song L Wang W Metformin Improves Overall Survival of Colorectal Cancer Patients with Diabetes: A Meta-Analysis J Diabetes Res 2017 2017 1 9 Mogavero A Maiorana MV Zanutto S Metformin transiently inhibits colorectal cancer cell proliferation as a result of either AMPK activation or increased ROS production Sci Rep 2017 7 1 12 28127051 Morris JS Bradbury KE Cross AJ Gunter MJ Murphy N Physical activity, sedentary behaviour and colorectal cancer risk in the UK Biobank Br J Cancer 2018 118 920 9 29520109 Paz MF Moreno V Esteller M Frigola J Sole X Differential DNA hypermethylation and hypomethylation signatures in colorectal cancer Hum Mol Genet 2005 14 319 26 15574462 Richter EA Ruderman NB AMPK and the biochemistry of exercise: Implications for human health and disease Biochem J 2010 418 261 75 Shackelford DB Shaw RJ The LKB1-AMPK pathway: Metabolism and growth control in tumour suppression Nat Rev Cancer 2009 9 563 75 19629071 Steinberg GR Carling D AMP-activated protein kinase: the current landscape for drug development Nat Rev Drug Discov 2019 18 527 51 30867601 Szklarczyk D Gable AL Lyon D STRING v11: Protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets Nucleic Acids Res 2019 47 607 13 30335158 Tang Z Kang B Li C Chen T Zhang Z GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis Nucleic Acids Res 2019 47 556 60 Tate JG Bamford S Jubb HC COSMIC: The Catalogue Of Somatic Mutations In Cancer Nucleic Acids Res 2019 47 941 7 30462257 Warrier NM Agarwal P Kumar P Integrative analysis to identify genes associated with stemness and immune infiltration in glioblastoma Cells 2021 10 1 20 Zadra G Batista JL Loda M Dissecting the dual role of AMPK in cancer: From experimental to human studies Mol Cancer Res 2015 13 1059 72 25956158 Zhou Y Zhou B Pache L Metascape provides a biologist-oriented resource for the analysis of systems-level datasets Nat Commun 2019 10 1 10 30602773

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116131 10.31557/APJCP.2023.24.4.1119 Research Article Low Expression of Mucin 2, High Expression of Mucin 13, and High Expression of Nuclear Factor Kappa-Light-Enhancer of Activated B Cells were Significant Pathways in Colorectal Cancer Development Mulyawan I Made * Division of Digestive Surgery, Department of Surgery, Udayana University, Prof. I.G.N.G. Ngoerah Hospital, Denpasar, Bali, Indonesia. * For Correspondence: mulyawan@unud.ac.id 2023 24 4 11191123 29 11 2021 6 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Objective: To prove the role of MUC2, MUC13, and NFκB protein expression as significant carcinogenesis pathways in colorectal cancer development. Methods: This is a nested case-control study conducted at the Udayana University, Sanglah General hospital, from September 2020 to 2021. All eligible subjects who visited the Digestive Surgery outpatient clinic with a colorectal surgery plan 2021 were included. The subjects were classified as case group (cancerous colonic mucosa) and control group (normal colonic mucosa), proved by histopathology examination. The parameters in this study were the expression of MUC2, MUC13, and NFκB by immunohistochemistry analysis. The data in this study will be collected and tabulated in SPSS 25.0 (Chicago, Illinois, USA). Results: A total of 36 subjects with colorectal cancer (case group) and 36 subjects with normal colonic mucosa (control group) were analyzed in this study. The cancerous colonic mucosa significantly had a lower MUC2, higher MUC13, and higher NFκB expression. After multivariate analysis for controlling the age variable, the result showed that only MUC2, MUC13, and NFκB expressions were still significant with p<0.05. The effect from MUC2, MUC13, and NFκB expression totally could assess up to 85.4% of the risk of developing colorectal cancer. Conclusion: There was a significantly lower MUC2, higher MUC13, and higher NFκB expression in the carcinogenesis of colorectal cancer, representing the influence of the inflammatory pathway and the abnormality of the protective barrier. Therefore, in the future, this result could remark a future early prediction or scoring system to assess colorectal cancer in clinical application. Key Words Mucin colorectal cancer carcinogenesis NFkB ==== Body pmcIntroduction Colorectal cancer is still reported to have high incidence and mortality until now. Most people with colorectal cancer in Indonesia were in an advanced stage, whereas the cases were usually unresectable and had bad prognoses (Ferlay et al., 2015). The carcinogenesis of normal colonic mucosa developing into colorectal cancer was related to inflammation and abnormality of the protective barrier system in the intestinal mucosa (Raisch et al., 2014). The main mucus layer that makes up the colonic mucosa is mucin types 2 and 13 (Kufe, 2009). In colorectal cancer, it was found that there was a decrease in MUC2 expression and an increase in MUC13 expression (Ahn et al., 2005). The relationship between inflammation and cancer has been studied intensively, and there is a role of the transcription factor NF-κB (Terzić et al., 2010). Thus, MUC2, MUC13, and NFκB were hypothesized to be involved in the pathways of colorectal cancer carcinogenesis. However, research results were still varied. There was no existing study in Indonesia that assessed those markers in colorectal cancer. Therefore, this study aimed to prove MUC2, MUC13, and NFκB protein expression’s role as significant carcinogenesis pathways in colorectal cancer development. Materials and Methods Study Design This is a nested case-control study conducted at the Division of Digestive Surgery, Department of Surgery, and the Department of Pathology of the Udayana University Medical School - Sanglah General Hospital. Data from medical records were collected from September 2020 to 2021. Study population The population of this study was all patients who visited the Digestive Surgery outpatient clinic of Sanglah Hospital with a colorectal surgery plan from 2020 to 2021. The included subjects were the population with the official histopathology examination after surgery. If the result was colorectal carcinoma, subjects were put in the case group, and subjects were put in the control group if the result was normal. The exclusion criteria were the unavailable demographic information from the medical records, the error of paraffin block due to a lot of necrotic and bleeding tissue, or the remaining amount being insufficient for further analysis. From the results of the calculation of sample 2 proportions for case-control research, the required sample size in this study was 36 subjects for each group. Study parameters The parameters in this study were the expression of MUC2, MUC13, and NFκB. The paraffin blocks from the included subjects were collected in the Pathology Laboratory of Sanglah General Hospital. They were then sliced and processed for immunohistochemistry examination. The specimen was fixed with 10% buffered formalin and then painted with each monoclonal antibody kit product. The painting process includes peroxidase block, primary antibody or negative control reagent, peroxidase-labeled polymer, substrate – chromogen, Hematoxylin Counterstain (Optional), and mounting. The specimen object glass was then read under Olympus CX21 microscope. The expression of MUC2, MUC13, and NfκB was interpreted as the sum of the intensity scores and the percentage scores. The intensity score was assessed based on the density of tumor cells stained positively with strong magnification (400x), scored negative (0), weak (+1), moderate (+2), and strong (+3). The percentage scores were interpreted as how much the tumor cells were stained in one field by the magnification of 40x, scored as no (0), <25% (+1), 26-50% (+2), 51-75% (+3), and >75% (+4). The cut-off point was determined from the median value of each variable. The determined median value for MUC was 6. If the score is less than 6, MUC2 or MUC13 expression is categorized as low and if the score is more than or equal to 6, the MUC2 or MUC13 expression is categorized as high. The determined median value for NFkB is 7. So if the score is less than 7, the expression of NFκB is categorized as low; if the score is more than or equal to 7, the expression of NFB is categorized as high. The demographic data were age, gender, the habit of smoking, drinking alcohol, red meat consumption, and presence of family history, taken from medical records. The respondent age was calculated from the date of birth to the year the research was conducted, categorized as less or more than 50 years. Gender was categorized as male or female. The habit of smoking was defined as using at least one cigarette per day, every day, categorized as yes or no. The habit of drinking alcohol was categorized as consuming alcohol at least one drink per day, every day, categorized as yes or no. The habit of consuming red meat was defined as eating more than three servings of red meat per week, with a single meal of about 350-500 grams of meat, categorized as yes or no. Family history is defined as the presence or absence of a family history of colorectal cancer. Statistical analysis The data in this study will be collected and tabulated in SPSS 25.0 (Chicago, Illinois, USA). The categorical variables will be distributed in the form of narration and tables. The numerical variables were expressed in terms of median, mean, and standard deviation, while categorical variables were expressed in terms of frequency and percentage. A statistical test was performed with Chi-Square to determine the difference in the expression of MUC2, MUC13, and NFκB on the occurrence of colorectal cancer. To determine the magnitude of the risk, an Odds ratio was estimated. The bias from demographic variables will be controlled by multivariate logistic regression analysis. The Hosmer-Lemeshow test assessed the feasibility of the logistic regression model and the model’s coefficient of determination was assessed by the Nagelkerke R square. The precision of the data was a 95% confidence interval, and the significance level was considered when p was less than 0.05. Results Characteristics of Subjects A total of 36 subjects with colorectal cancer (case group) and 36 subjects with normal colonic mucosa (control group) were analyzed in this study. The majority of subjects with colorectal cancer were >50 years old (90.9%), male (61.1%), did not have a smoking habit (58.3%), did not have an alcohol consumption habit (75%), had a habit of consuming meat (94.4%), and had no family history of colorectal cancer (90.9%). The only significant difference was found between the age groups >50 years o,d and <50 years old in the risk of developing colorectal cancer (p=0.004; Table 1). The MUC2, MUC13, and NFkB expression on the normal and cancerous colonic mucosa In this study, the cancerous colonic mucosa significantly had a lower MUC2 expression (p<0.001; OR 38.5 (95%CI 9.3-159.2)), a higher MUC13 expression (p<0.001; OR 55.0 (95%CI 12.6-239.6), and a higher NFκB expression (p<0.001; OR 40,0 (95%CI 10.3-155.8); Table 2; Figure 1). To control the age variable, multivariate analysis was done. The result showed that only MUC2, MUC13, and NFκB expressions were statistically significant, with p<0.05. The effect from MUC2, MUC13, and NFκB expression totally could assess up to 85.4% of the risk of developing colorectal cancer. Of the three variables, MUC13 expression had the most significant effect compared to MUC2 and NFκB expressions (Table 3). Table 1 Characteristics of Subjects Data Cases (Colorectal cancer) Control (Colitis) Total (n) p Age <50 years old 5 16 21 0.004* >50 years old 31 20 51 Gender Female 14 17 31 0.475 Male 22 19 41 Smoking Yes 21 28 49 0.077 No 15 8 23 Alcohol consumption Yes 27 22 49 0.126 No 9 4 13 Meat consumption Yes 2 4 6 0.394 No 34 32 66 Family history Yes 30 33 63 0.285 No 3 6 9 *Significant Figure 1 Immunohistochemical Analysis in Colon Mucosa. A, Low MUC2 expression; B, High MUC2 expression; C, High MUC13 expression; D, Low MUC13 expression; E, High NFκB expression; F, Low NFκB expression Table 2 The Difference in MUC2, MUC13, and NFκB Expression of the Colorectal Mucosa on the Risk of Developing Colorectal Cancer Data Cancerous colonic mucosa Normal colonic mucosa OR p MUC2 expression Low 33 8 38.5 <0.001* High 3 28 (95%CI 9.3-159.2) MUC13 expression High 33 6 55 <0.001* Low 3 30 (95%CI 12.6-239.6) NFκB expression High 30 4 40 <0.001* Low 6 30 (95%CI 10.3-155.8) *Significant Table 3 Regression Logistic Model for Risk of Developing Colorectal Cancer Variabel B SE p Exp (B) 95%CI MUC2 expression 3.693 1.33 0.005 40.17 2.96-544.29 MUC13 expression 3.817 1.268 0.003 45.47 3.79-545.84 NFκB expression 3.202 1.179 0.007 24.58 2.44-247.66 Constant -6.305 2.007 0.002 0.002 Discussion The development of colorectal cancer is an interaction between genomic and environmental factors that continues to evolve, associated with inadequate protection (MUC2 downregulation and MUC13 upregulation) and transcriptional pathways (NFκB) (Terzić et al., 2010). The epithelial lining of the intestine is covered by mucous layers, which consist mainly of secreted mucin. MUC2 is a significant component of the protective mucus layer of the colon and plays a role in the development of colorectal cancer (Byrd and Bresalier, 2004). Downregulation of MUC2 expression removes the barrier in epithelial cells so that bacteria can make contact with epithelial cells and trigger an inflammatory response (Hansson and Johansson, 2010). In cancer cells, the cytokines IL-4, IL-3, and TNF-alpha increased, resulting in the downregulation of MUC2. In addition, the carcinogenesis process directly triggers direct methylation of MUC2 in colorectal cancer cells in promoter area A (-289 and -274) and area C (-193 and -160), which in turn downregulate MUC2 and triggers transcription factors (Okudaira et al., 2010). In this study, it was shown that there was a significantly lower MUC2 expression in the cancerous colonic mucosa, which was consistent with other studies. Low MUC2 expression levels were significantly associated with poor, disease-free, or relapse-free survival (Al-Khayal et al., 2016). MUC13 is a mucin normally expressed at low levels at the apical boundary of epithelial cells containing phosphorylation sites. In the pathogenesis of metaplasia, IL-6 is known to increase the JAK/STAT5 pathway, complex with MAPK p44/42, increase P-ERK expression, then form complex with the MUC13 promoter to increase its expression (Maher et al., 2011). MUC13 increases the expression of several proteins, including SHH, TERT, BMI-1, GATA1, HER2/MAPK signaling, and Bcl-xl, which are involved in the development and metastasis of colon cancer. In addition, the MUC13 promotes NF-B activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase RIPK1, which triggers cell proliferation (Sheng et al., 2017). MUC13 can also interact directly with GSK-3β or via other complex members, preventing its ability to initiate catenin degradation, leading to catenin accumulation, increased Wnt/β-catenin signaling, increased transcriptional activity, and promoting the development of colorectal cancer. Overall, MUC13 is associated with activating several pathways that are essential for cancer progression. In this study, it was found that in the age group, the majority of the population had MUC13 upregulation, following other studies which reported that increased MUC13 production by colorectal cancer cells correlated with the ability of colon cancer cells to metastasize to the liver (Byrd and Bresalier, 2004). NF-κB factor is a transcription factor closely related to the process of carcinogenesis (Karin, 2006). In colorectal cancer cells, cancer stem cells trigger an increase in IL-6/STAT3 signaling, AMPK/mTOR activation, and NFκB directly. Epithelial cell NFκB activation directly promotes colitis progression to colorectal cancer. Cyclin D1 and cMyc induced by NFκB have important roles in cell growth and proliferation. In addition, various studies have shown that NFκB can block apoptosis by increasing anti-apoptotic proteins, such as inhibitors of apoptotic proteins (IAPs) and inhibiting activation (JNK). On the other hand, major angiogenic factors such as vascular endothelial growth factor (VEGF) and interleukin-8, directly or indirectly enhanced by NFκB activation, also play a role in cancer cell proliferation (Greten et al., 2004). This study found that there was an effect of high NFκB expression and the risk of colorectal cancer. From theory, the pathway that influenced a lot was the canonical pathway. Canonical pathways are associated with proinflammatory cytokines, IκBα phosphorylation, translocation to the nucleus, and promotion of transcription (Oeckinghaus et al., 2011). These results concluded that the expression of MUC2, MUC13, and NFκB plays an important role in the carcinogenesis pathway in colorectal cancer. In addition, this condition indirectly illustrates that the lower MUC2, higher MUC13, and high NFκB, the higher the cell proliferation and the more progressive cancer. MUC13 has the most decisive influence compared to others. This study showed a relationship between the three independent variables, MUC2, MUC13, and NFκB, even after controlling for the control variables, on colorectal cancer carcinogenesis. The novelty of this study is that there has never been a study that has assessed the expression of MUC2 and NFκB in colorectal cancer in the Indonesian population. Previous studies had only assessed MUC2, MUC13, and NFκB separately. However, there were several limitations of this study. First, this study only takes a subject of the Balinese population. Second, the analysis of smoking habits and meat consumption seems lacking because of the limitations of a more detailed analysis of the quantity consumed. In the future, an early colorectal cancer risk stratification system can be developed from these variables, both with markers and scoring systems. In conclusion, there was a significantly lower MUC2, higher MUC13, and higher NFκB expression in the carcinogenesis of colorectal cancer, representing the influence of the inflammatory pathway and the abnormality of the protective barrier. Therefore, in the future, this result could remark a future early prediction or scoring system to assess colorectal cancer in clinical application. Author Contribution Statement None. Acknowledgements We want to express our gratitude to all patients participating in this study. Funding statement This study did not receive funding from a third party or pharmaceutical company. Data availability Data sharing is applicable upon request by contacting the corresponding author. We did not register the data due to the privacy of the participant’s data. Ethical Issue Udayana University Local Ethical Committee has approved this study Conflict of interest The authors declare no potential conflict of interest. ==== Refs References Ahn DH Crawley SC Hokari R TNF-alpha activates MUC2 transcription via NF-kappaB but inhibits via JNK activation Cell Physiol Biochem 2005 15 29 40 15665513 Al-Khayal K Abdulla M Al-Obaid O Differential expression of mucins in Middle Eastern patients with colorectal cancer Oncol Lett 2016 12 393 400 27347157 Byrd JC Bresalier RS Mucins and mucin binding proteins in colorectal cancer Cancer Metastasis Rev 2004 23 77 99 15000151 Ferlay J Soerjomataram I Dikshit R Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012 Int J Cancer 2015 136 E359 86 25220842 Greten FR Eckmann L Greten TF IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer Cell 2004 118 285 96 15294155 Hansson GC Johansson ME The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria Gut Microbes 2010 1 51 4 21327117 Karin M Nuclear factor-kappaB in cancer development and progression Nature 2006 441 431 6 16724054 Kufe DW Mucins in cancer: function, prognosis and therapy Nat Rev Cancer 2009 9 874 85 19935676 Maher DM Gupta BK Nagata S Mucin 13: structure, function, and potential roles in cancer pathogenesis Mol Cancer Res 2011 9 531 7 21450906 Oeckinghaus A Hayden MS Ghosh S Crosstalk in NF-κB signaling pathways Nat Immunol 2011 12 695 708 21772278 Okudaira K Kakar S Cun L MUC2 gene promoter methylation in mucinous and non-mucinous colorectal cancer tissues Int J Oncol 2010 36 765 75 20198318 Raisch J Buc E Bonnet M Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation World J Gastroenterol 2014 20 6560 72 24914378 Sheng YH He Y Hasnain SZ MUC13 protects colorectal cancer cells from death by activating the NF-κB pathway and is a potential therapeutic target Oncogene 2017 36 700 13 27399336 Terzić J Grivennikov S Karin E Inflammation and colon cancer Gastroenterology 2010 138 2101 14 20420949

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==== Front Asian Pac J Cancer Prev Asian Pac J Cancer Prev APJCP Asian Pacific Journal of Cancer Prevention : APJCP 1513-7368 2476-762X West Asia Organization for Cancer Prevention Iran 37116153 10.31557/APJCP.2023.24.4.1307 Research Article Can Echinococcus Granulosus Infestation Prevent Pancreatic Cancer? An In vivo Experimental Study Doğan Serhat 1 Çakir Murat 2 Kartal Adil 2 Öztaş Haydar 3* Oltulu Pembe 4 1 Malatya Turgut Özal University, Faculty of Medicine, Department of General Surgery, Malatya, Turkey. 2 Necmettin Erbakan University, Meram Medical School, Department of Surgery, Konya, Turkey. 3 Necmettin Erbakan University, Ahmet Keleşoğlu Faculty of Education, Department of Biology, Konya, Turkey. 4 Necmettin Erbakan University, Meram Medical School, Department of Pathology, Konya, Turkey. * For Correspondence: haydar_oztas@yahoo.com.tr 2023 24 4 13071312 7 12 2022 18 4 2023 https://creativecommons.org/licenses/by-nc/4.0/ This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International License. (https://creativecommons.org/licenses/by-nc/4.0/) Hydatid cyst is a zoonotic infestation caused by Echinococcus granulosus, and it is known that some parasites found in humans cause cancer in humans or some may have a protective effect against cancer. Cancer is one of the most serious health problems of today and it has been shown in some studies that parasites such as Echinococcus granulosus can have an inhibitory effect. The aim of this study was determined as whether Echinococcus granulosus has an inhibitory effect on exocrine pancreatic cancer with the help of the azaserine-rat model used in different cancer studies. Material and Methods: During experimental process a total of 45 male Wistar rats used, 14-day-old male Wistar rats were divided into groups according to the experimental protocol, administered azaserine injection protocol or kept as a control group without azaserine injection. Animals are grouped as Group 1, Control Group (group not treated with Azaserine and not injected with protoscolex.) (E-A-) (n=7); Group 2, Group injected with (IP) Azaserine only (30mg/kg) (E-A+) (n=8);Group 3, Group injected (IP) with protoscolex suspension of 1 cc only (E+A-) (n=15);Group 4, Group injected both Azaserine (IP) and protoscolex suspension (IP) (E+A+) (n=15). Atypical Acinar Cell Foci (AACF) load in the exocrine pancreas of each rat was measured quantitatively with the help of a video image analyzer and the AACF load was calculated with the help of a mathematical model. Results: Findings showed that the Atypical Acinar Cell Foci (AACF) burden was statistically significantly lower in the Azaserine+ protoscolex (Azaserine-injected-protoscolex-implanted) rat group compared to the other groups, suggesting that Echinococcosis in the azaserine-rat model could inhibit the development of precursor foci of neoplastic changes in the exocrine pancreas. Conclusion: The most significant aspect of our study is that it contributes new insights into the controversy that Echinococcosis suppresses pancreatic cancer. Key Words Echinococcus granulosus azaserine rats pancreatic cancer ==== Body pmcIntroduction Hydatid cyst is a zoonotic infestation caused by Echinococcus granulosus (EG). It is widely seen in the Mediterranean countries, the Middle East, South America, New Zealand, Australia and South Eastern Asia (Karaman et al., 2011; Lynen et al., 2011). In addition to this, cancer is one of the most significant healthcare problems today. There are also articles reporting that parasites can lead to or inhibit cancer in humans (Akgül et al., 2003; Alvarez-Errico et al., 2001). However, it is not known exactly what kind of histopathological change parasites bring about in the pancreas in vivo animal experiments (Rhim and Stanger, 2010; Noya et al., 2013). It is assumed that pancreatic ductal adenocarcinoma (PDAC) completes its developmental process from a precursor lesion into cancer through multiple stages. Pancreatic intraepithelial neoplasia (PanIN) is a precursor lesion frequently detected in the pancreas of pancreatic cancer patients (Longnecker and Curphey, 1975; Rhim and Stanger, 2010; Vincent et al., 2011; Ji et al., 2009; Gourgou-Bourgade et al., 2011). It has been found that preneoplastic structures in the pancreas were seen in 80% of these cases (Hruban et al., 2008). KRAS oncogene is a well-known mutant gene that leads to pancreatic cancer. KRAS oncogene leads to the inactivation of tumor suppressor genes CDKN2A, TP53, DPC4 and BRCA2. Loss of chromosomes, gene amplification and telomere contraction are usually observed anomalies in pancreatic cancer cases in which activation of this gene is involved (Vincent et al., 2011). It has been reported that antigenic properties of tumor and EG infestation were similar (Alvarez et al., 2001). In another study, it has been demonstrated that mucin-like peptides occurring due to EG induce the increase of antitumor activity (Noya et al., 2000). EG is claimed to have antigenic properties similar to cancerous cells. This trait is thought to be protective against the development of cancer (Noya et al., 2013). The aim of this study was determined as whether Echinococcus granulosus has an inhibitory effect on exocrine pancreatic cancer with the help of the azaserine-rat model used in different cancer studies. It was main subject of research that whether experimentally induced Echinococcus granulosus in rats’ pancreas may inhibit pancreatic cancer precursor focus (AACF). Materials and Methods The study was carried out following the approval of the Experimental Animals Ethics Committee of the Experimental Medical Research and Implementation Center (Ethics Committee Approval No 2013-207). 45 male Wistar albino rats raised in Experimental Medical Research and Implementation Center were used. The study lasted 6 months. Male inbred Wistar strain rats were obtained from N. Erbakan University Animal House and kept five animals to a cage under standard conditions (room temperature 23 C; lighting 7 am-7pm), on sawdust bedding. Standard diet and tap water were supplied ad libitum. As experimental materials, azaserine (98% purity), hematoxylin, eosin were obtained from Sigma Chemical Co. Ltd (USA) acetone, formalin, HCl, methanol, and chloroform were obtained from Sigma-Aldrich Chemicals (USA). Experiment protocol In this study, using the azaserine-rat model previously developed by Longnecker and Curphey (1975), it was investigated whether Echinococcus granulosus, a common internal parasite in humans and animals, has an inhibitory effect on the development of AACF, which is known as a potential precursor of neoplastic changes in the exocrine pancreas. Pancreatic cancer rat model Azaserine, which is a chemical well known to have a mutagenic feature in the induction of cancer in the rat pancreas, was employed in order to induce hyperplastic differentiation in acinar cells of subjects (Noya et al., 2000). The azaserine-rat model, developed by Longnecker and Curphey (1975) to examine experimental exocrine pancreatic changes in rats, has recently been used by many researchers to investigate the origin of pancreatic acinar cell foci (AACF) (Khoo et al., 1991; Yıldız et al., 2013, 2019; Kalıpcı et al., 2012; 2013; Yener et al., 2013). In order to initiate neoplastic differentiation induced by means of EG infestation in the liver of rats, the rats were intra-peritoneally (IP) injected with 30 mg/kg azaserine for three weeks successively, dissolved in 0.9 % NaCl solution on the day of injection. After for three consecutive weeks azaserine injected and untreated rats randomly divided into experimental groups as below; Group 1: Control Group (group not treated with Azaserine and not injected with protoscolex.) (E-A-) (n=7); Group 2: Group injected with (IP) Azaserine only (30mg/kg) (E-A+) (n=8);Group 3: Group injected (IP) with protoscolex suspension of 1 cc only (E+A-) (n=15); Group 4: Group injected both Azaserine (IP) and protoscolex suspension (IP) (E+A+) (n=15). At the end of the 6-month period the rats in each experiment group were weighed on precision scales. Before sacrification, all animals were anesthetized with 50mg/kg (sc) Ketamine HCl and 15 mg/kg xylasin HCL (sc) and the rats were sacrificed by cervical dislocation. Each experimental animal was laparotomies. Pancreatic tissue was found and dissected. The entire rat pancreas was excised at autopsy and all adherent fat, mesentery and lymph nodes were carefully trimmed off. The wet weight of each pancreas was recorded before fixation in 10% buffered neural formalin. Before immersion in fixative solution each pancreas was spread out on a piece of porous paper to ensure maximal trans-sectional area for subsequent sectioning. Routine hematoxylin and eosin stains were applied to sections of 5 μm thicknesses from the prepared blocks with a rotary microtome (Thermo Scientific, Shandon Finesse 325). Histological sections were stained with haematoxylin & eosin and were examined by a light microscopy (ZEISS Axio Lab.A1). Foci in the sections were identified according to established criteria (Rao et al., 1982; Roebuck et al., 1984). According to the criteria’s of Longnecker (1987) carcinogen-induced pancreatic lesions were atypical acinar cell foci (AACF). Therefore, AACF has been evaluated as a possible precursor cancer cell growth in rat exocrine pancreas. Quantitative estimation of Atypical Acinar Cell Foci (AACF) load in pancreatic preparations stained with hematoxilen-eosin can provide useful information about precancerous lesions in the pancreas of rats used for experimental purposes. For this purpose the total area of exocrine pancreatic tissue was measured from each pancreas by means a video image analyzer (ZEISS Axio Lab.A1.) This system includes a microscope, video camera and data capture software. The same instrument was used to count acidophilic AACF via measure their transactional area. The observed data were proceed numerically by a computer software package (ZEISS Axio Lab.A1), which uses an algorithm based on the mathematical formula of Campel et al., (1982). As modified Pugh and his coworkers (1993). The total number of foci in whole pancreas was derived by multiplying the weight of pancreas. The number of foci, mean values and either standard errors of means or ranges were determined for all data. Non-parametric statistical analyses were performed using the Kruskal-Wallis one-way analysis of variance and Mann-Whitley U-test. Echinococcus granulosus infestation Live protoscoleces extracted from the cyst of animals with cystic hydatid to be sacrificed were brought to the laboratory in eau de roche in cold chain and secondary hydatidosis was formed by means of 1 ml IP injection. The vitality of protoscoleces was confirmed by means of hematoxylin–eosin stain (Burgu, 1975). After the protoscoleces were detected on the preparations, they were stained with eosin dye and it was decided whether they were alive or not according to the color change. The protoscoleces that were not stained were considered live (Figure 1a), those that were stained were considered dead (Figure 1b). The dead cell was stained since its cell membrane was not fully functional and thus the dye could permeate the membrane (Tezok et al., 1970; Kazez et al., 2000). Results The main body weights of experimental group are shown at Table 1. The differences were observed between only azaserine initiated group and others. There was a decrease in the mean body weight of Azaserine control with compared Untreated controls and Echinococus implanted rats. The mean weight of experimental animals at the end of the sixth month was 347.5 gr. A statistically significant loss of body weight was observed in the subjects in Group 2 (E-A+) compared to other groups (p<0.05) (Figure 2). The mean pancreas weight of the groups was 0.86 gram. There was no significant difference in the pancreas weights of the groups (p>0.05) (Figure 3). The number of AACF was higher only in the group injected with Azaserine (Group 2). The number of atypical acinar cell foci (AACF) was significantly different (p< 0.05) in the preparates developed in the pancreases of the group injected with Azaserine (Group 2) and the group injected with Azaserine + Protoscolex (Group 4) (Figure 4). Acidophilic AACF have been identified in the pancreas section in azaserine treatment groups. In the majority of these lesions the cells have a zymogen-rich cytoplasm and basal nucleus, round to oval in shape, which is slightly larger than normal and contains a prominent nucleolus (Figure 5a, b). The individual cells are usually smaller than normal parenchymal cells, resulting in an appearance of increased cellularity. Acidophilic foci are generally composed of larger acini then normal acinar cells. The number of AACF in Group 2 was established to be 25 on average for each preparate. The number of AACF in Group 4 (A+E+) was observed to be merely 6. However, no AACF foci were detected in Group 1 (Control Group) and Group 3 (E+ A-) (Figure 4). This led us to assume at least that EG infestation played no role in the development of pancreatic cancer. There was a clearly reduction AACF ration in Azaserine + Echinococcus group (Group 4).The rate of the mean area of AACFs with respect to the total area of pancreas in the group treated with Azaserine (Group 2) was found to be 10.23% while the same rate was 1.61% in the Azaserine + Echinococcus group (Group 4). There was a significant difference between the two (p<0.05). In the Azaserine group (Group 2) the total area of AACF was measured to be 27,765,107 µm2 (p<0.05), while in the Azaserine + Echinococcus group (Group 4) the total area of AACF was 4,360,084 µm2. There existed a statistically significant difference between the two (p< 0.05). Table 1 The Main Body and Pancreatic Weights of Experimental Groups Control (n=7) Azeserine (n=8) Echinococcus (n=15) Azaserine + Echinococcus (n=15) Mean Body weights (gr) 353.2857±2,13809 348.0±2,50713 * 353.0±2,61861 351.4667±3,13657 Mean pancreatic weights (g) 0.8614±0,02968 0.8613±0,02031 0.8621±0,02293 0.8607±0,02056 Figure 1 a, Live protoscoleces ; b, dead protoscoleces Figure 2 The Mean Body Weights of Experimental Groups (g). Figure 3 The Mean Pancreatic Weights of Experimental Groups (g). Figure 4. Distribution of the Mean Number of AACF among the Groups Figure 5 Atypical Acinar Cell Foci (AACF) in Azaserine (Group 2) Group, marked (in x50 magnification) (3a), AACF in Azaserine + Echinococcus group (Group 4) marked (in x50 magnification) (3b) Discussion Alterations that occur in acinar cells and ducts in the development of pancreatic ductal adenocarcinoma (PDAC) is critical in the development of pancreatic cancer (Rhim and Stanger, 2010). However, while altered acinar cells induce PanIN, Kras oncogene in vivo can also lead to pancreatic cancer (Ji et al., 2009; Gourgou-Bourgade et al., 2011). Longnecker et al., point out in a study that some of the cancers detected in human pancreas stem from acinar cells (Zurlo et al., 1982). In a study Flaks et al. argue that pancreatic cancer, for the most part, derives from acinar cells that mutate at the outset and transform into ducts or ductal structures and finally induce pancreatic cancer (D.E. et al., 1991). Azaserine (o-diazo acetyl-L-serin) is an antimetabolite that can be isolated from Streptomyces cultures. It has a chemical with mutagenic property according to Salmonella typhimurium test (Longnecker et al., 1979). It was first used for inducing pancreatic cancer in rats in 1975 (Longnecker et al., 1979). In many subsequent studies, the azaserine-induced rat model appears as a dependable model (Longnecker and Curphey, 1975; O’connor et al., 1989). We also used this model in our study and observed that the number of atypical cells increased in the group in which we used azaserine. Epidemiological studies demonstrate that there is a correlation between some parasites such as EG and development of cancers (Springer, 1997). Neoplastic cells induced in experimental animals are known to be inhibited by some bacteria (Listeria monocytogenes, Cornebacterium parvum) and parasites (Toxoplasma gondii, Besnoitia jellison). It is argued that these pathogens stimulate the immune system and thus inhibit neoplastic development. The fact that nonspecific macrophage activity resists the tumor with the immune response which is formed against parasitic infestation as well as the systemic inhibition of angiogenesis supports this observation (Hunter et al., 2001). In our study we demonstrated that the immune response against EG inhibits the development of AACF in the pancreas. There was a statically significant difference (Figure 5). The fact that the total area of AACF was 27,765,107 µm2 (p<0.05) in the Azaserine group (Group 2), while the total area of AACF in the group injected with Azaserine+Echinococcus (Group 4) was 4.360.084 µm2 also supports antitumor activity. Results obtained from surgical procedures in areas where EG infestations are common established that there was a negative correlation between the existence of hydatid cysts and development of cancers (Akgül et al., 2003). Interestingly, several studies have reported that antigenic properties of tumor and EG infestation were similar (Alvarez et al., 2001; Knapen, 1980). Mucin-like peptides derived from EG increases anti-tumor activity. There are also studies in literature which put forth that EG suppresses the immune system rather than cancer itself (Turhan et al., 2015). The results of our study demonstrate just the opposite of the thesis set forth in that study. Anti-tumor activity that appears against EG infestation inhibits the development of cancer. The number of AACF in the rats which were injected with only azaserine was 25, while the number of AACF in the subject injected with Echinococcus and azaserine was 6, which was statistically significant. Thus we believe that we have established anti-tumor activity in EG infestation. We assume that subjects do not have to have cysts in their peritonea or livers for the existence of anti-tumor activity. Even if the protoscoleces in the suspensions injected are dead, the subject’s immunity activates itself against the antigenic structure of protoscolex. We have observed this fact within the scope of our pilot study. However, we used protoscoleces suspension whose vitality was proved. In our study we have also observed that immunity that develops against scolex and hydatid fluid of Echinococcus granulosus suppresses cancer-precursor atypical cell foci. The limitations of this study include the fact that EG-specific antigenic structure has not been serologically established. In conclusion, findings from this experiment It has been shown in the well-known azaserine-rat model that azaserine-induced neoplastic changes in the exocrine pancreas can be inhibited by the parasitic Echinococcus granulosus in humans and animals. This supports the clinical and epistemological findings (Karaman et al., 2011; Turhan et al., 2015). The most significant aspect of present study is that it may contribute new insights into the controversy that Echinococcus granulosus suppresses pancreatic cancer. Author Contribution Statement All authors contributed equally in this study. Acknowledgements None. Conflicts of interest There are no conflicts of interest. ==== Refs References Akgül H Tez M Ünal A Echinococcus against cancer: why not? Cancer 2003 98 9 Alvarez-Errico Medeiros A Miguez M O-glycosylation in echinococcus granulosus: indentification and characterization of the carcinoma - associated to antigen Exp Parasitol 2001 98 100 9 11465993 Burgu A Secondary cyst of Echinococcus granulosus proloskolex in white mice The effect of radiation on their ability to form AÜ Vet Fac J 1975 22 137 48 Campbell HE Pitit HC Potter BR Laishes BA Application of quantitative stereology to evaluation of enzyme altered foci in rat liver Cancer Res 1982 42 465 6120037 Khoo DE Flaks B Oztas H Williamson RC Habib NA Effects of dietary fatty acids on the early stages of neoplastic in duction in therat pancreas Changes in fatty acid composition and development of atypical acinar cell nodules Int J Exp Pathol 1991 72 571 80 1742210 Gourgou-Bourgade S de la Fouchardière C Bennouna J FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer N Engl J Med 2011 364 1817 25 21561347 Hruban RH Maitra A Goggins M Update on pancreatic intra epithelial neoplasia Int J Clin Exp Pathol 2008 1 306 16 18787611 Hunter CA Yu D Gee M Cutting Edge: Systemic Inhibition of Angiogenesis Underlies Resistance to Tumors During Acute Toxoplasmosis J Immunol 2001 166 5878 81 11342601 Lynen I Sogut O Guldur ME Primary hydatid cyst: an unusual cause of a mass in the supraclavicular region of the neck J Clin Med Res 2011 3 52 4 22043272 Ji B Tsou L Wang H Gaiser S Ras activity levels control the development of pancreatic diseases Gastroenterol 2009 137 1072 82 Kalıpcı E Ozdemir C Oztas H Assessing eco-toxicological effects of industrial 2,4-D acidiso-octylester herbicide on rat pancreas and liver Biotech Histochem 2013 88 202 7 23398176 Kalıpcı E Yener Y Yıldız H Oztas H Investigationof possible ecotoxic effects of acrylamide on liverwiththeazaserine-rat model Pol J Environ Stud 2012 21 1243 7 Karaman E Yilmaz M Ada M Yilmaz RS Isildak H Unusual location of primary hydatid cyst: soft tissue mass in the parapharyngeal region Dysphagia 2011 26 75 7 20204411 Kazez K Sahin M Vatansev C Experimentally developed secondary Echinococcosis in pleural and peritoneal cavities and utility of serological tests during the follow-up Turk J Med Sci 2000 30 101 7 Khoo DE Flaks B Oztas H Williamson RCN Habib NA Effects of dietary fatty acids on the early stages of neoplastic induction in the rat pancreas, Changes in fatty acid composition and development of atypical acinar cell nodules Int J Exp Pathol 1991 72 571 80 1742210 Knapen FV Echinococcus granulosus infection and malignancy Br Med J 1980 281 195 6 7407519 Longnecker DS Curphey TJ Adenocarcinoma of the pancreas in azaserine-treated rats Cancer Res 1975 35 2249 58 1097106 Longnecker DS Lılja HS French J Transplantation of azaserine – induced carcinomas of pancreas in rats Cancer Lett 1979 7 197 202 509403 Noya V Bay S Festari MF Mucin-like peptides from Echinococcus granulosus induce antitumor activity Int J Oncol 2013 43 775 84 23817837 O’connor P Roebuck BD Peterson J Effect of dietary omega-3 and omega-6 fatty acids on development of azaserine-induced preneoplastic lesions of rat pancreas J Nat Cancer Inst 1989 8 858 63 Pugh TD King JH Koen H Reliable stereological method for estimating the number of microscopic hepatocellular foci from their transections Cancer Res 1983 43 1261 8 6825098 Rhim AD Stanger BZ Molecular biology of pancreatic ductal adenocarcinoma progression: aberrant activation of developmental pathways Prog Mol Biol Transl Sci 2010 97 41 78 21074729 Springer GF Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis and immunotherapy J Mol Med (Berl) 1997 75 594 602 9297627 Tezok UF Kılıçturgay K Toppare S Aktaş H Studies on experimental hydatid cyst formation and treatment 1 Secondary cyst implantation in experimental animals Microbiol Bult 1970 4 207 14 Turhan N Esendagli G Ozkayar O Co-existence of Echinococcus granulosus infection and cancer metastasis in the liver correlates with reduced Th1 immune responses Parasite Immunol 2015 37 16 22 25319434 Vincent A Herman J Schulick R Hruban RH Goggins M Pancreatic cancer Lancet 2011 378 607 20 21620466 Yener Y Kalıpcı E Oztas H Aydin AD Yıldız H Possible neoplastic effects of acrylamide on rat exocrine pancreas Biotech Histochem 2013 88 47 53 23101568 Yıldız H Oztas H Yıldız D Koc A Kalıpcı E Inhibitory effects of acetylsalicylic acid on exocrine pancreatic carcinogenesis Biotech Histochem 2013 88 132 7 23331184 Yıldız H Kalipci E Oztas H Yıldız D Effects of acetylsalicylic acid on rats: an ın vivo experimental study in azaserine-rat model J Cancer Res Ther 2019 15 231 6 30880783 Zurlo J Roebuck BD Rutkowski JV Curphey TJ Longnecker DS Effect of pyridoxal deficiency on pancreatic DNA damage and nodule induction by azaserine Proc Am Assoc Cancer Res 1982 23 68

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==== Front JAMA Netw Open JAMA Netw Open JAMA Network Open 2574-3805 American Medical Association 37459099 10.1001/jamanetworkopen.2023.23336 zoi230690 Research Original Investigation Online Only Oncology First-Line Genomic Profiling in Previously Untreated Advanced Solid Tumors for Identification of Targeted Therapy Opportunities First-Line Genomic Profiling in Advanced Solid Tumors for Identification of Targeted Therapy First-Line Genomic Profiling in Advanced Solid Tumors for Identification of Targeted Therapy Matsubara Junichi MD PhD 1 Mukai Kumi BS 1 Kondo Tomohiro MD 1 Yoshioka Masahiro MD PhD 1 Kage Hidenori MD PhD 2 Oda Katsutoshi MD PhD 2 Kudo Ryo MD 3 Ikeda Sadakatsu MD PhD 3 Ebi Hiromichi MD PhD 4 Muro Kei MD PhD 5 Hayashi Ryuji MD PhD 6 Tokudome Nahomi MD PhD 7 Yamamoto Nobuyuki MD PhD 7 Muto Manabu MD PhD 1 1 Department of Clinical Oncology, Kyoto University Hospital, Kyoto, Japan 2 Department of Clinical Genomics, The University of Tokyo Hospital, Tokyo, Japan 3 Department of Precision Cancer Medicine, Tokyo Medical and Dental University Hospital, Tokyo, Japan 4 Division of Molecular Therapeutics, Aichi Cancer Center Research Institute, Nagoya, Japan 5 Department of Clinical Oncology, Aichi Cancer Center, Nagoya, Japan 6 Department of Clinical Oncology, Toyama University Hospital, Toyama, Japan 7 Internal Medicine III, Wakayama Medical University, Wakayama, Japan Article Information Accepted for Publication: May 29, 2023. Published: July 17, 2023. doi:10.1001/jamanetworkopen.2023.23336 Open Access: This is an open access article distributed under the terms of the CC-BY-NC-ND License. © 2023 Matsubara J et al. JAMA Network Open. Corresponding Author: Manabu Muto, MD, PhD, Department of Therapeutic Oncology, Kyoto University Graduate School of Medicine, Department of Clinical Oncology, Kyoto University Hospital, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan (mmuto@kuhp.kyoto-u.ac.jp). Author Contributions: Dr Muto had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design: Matsubara, Kondo, Ikeda, Muro, Muto. Acquisition, analysis, or interpretation of data: All authors. Drafting of the manuscript: Matsubara, Mukai, Ikeda, Tokudome, Muto. Critical revision of the manuscript for important intellectual content: Matsubara, Kondo, Yoshioka, Kage, Oda, Kudo, Ikeda, Ebi, Muro, Hayashi, Yamamoto, Muto. Statistical analysis: Matsubara, Muto. Obtained funding: Matsubara, Muto. Administrative, technical, or material support: Matsubara, Mukai, Kondo, Yoshioka, Oda, Ebi, Hayashi, Tokudome, Yamamoto, Muto. Supervision: Matsubara, Ikeda, Muro, Yamamoto, Muto. Conflict of Interest Disclosures: Dr Yoshioka reported receiving personal fees from Chugai Pharmaceutical Co Ltd during the conduct of the study. Dr Kage reported receiving grants from Konica Minolta Inc outside the submitted work. Dr Oda reported receiving grants from Chugai Pharmaceutical Co Ltd during the conduct of the study and grants from Konica Minolta Co Ltd outside the submitted work. Dr Ikeda reported receiving personal fees from Chugai Pharmaceutical Co Ltd during the conduct of the study and outside the submitted work. Dr Ebi reported receiving grants from Chugai Pharmaceutical Ltd during the conduct of the study; personal fees from Guardant outside the submitted work; and honoraria from AMGEN, BMS, Taiho, Takeda, Ono Pharmaceuticals, Incyte, and Merck Serono. Dr Muro reported receiving grants from Chugai Pharmaceutical Co Ltd to the institution during the conduct of the study; and grants from Astellas, Amgen, Sanofi, Eisai, Daiichi Sankyo Taiho, MSD, Novartis, and Ono to the institution; and lecture fees from Eli Lilly, Taiho, Takeda, Daiichi Sankyo, Ono, and Bristol-Myers Squibb outside the submitted work. Dr Tokudome reported receiving personal fees from Chugai Pharmaceutical Co Ltd outside the submitted work. Dr Yamamoto reported receiving grants from Chugai Pharmaceutical Co Ltd outside the submitted work. Dr Muto reported receiving grants from Chugai Pharmaceutical Co Ltd the conduct of the study. No other disclosures were reported. Funding/Support: This research was funded by Chugai Pharmaceutical Co Ltd Japan. Role of the Funder/Sponsor: The sponsor reviewed and approved the manuscript but had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and decision to submit the manuscript for publication. Data Sharing Statement: See Supplement 3. Additional Contributions: We thank all the coinvestigators in the 6 participating institutions, all patients and their families, and the support team of this study. We also thank the following collaborators: Department of Biomedical Statistics and Bioinformatics, Kyoto University Graduate School of Medicine for statistical analyses, INTAGE Healthcare Inc for data management and monitoring, and KBBM Inc for administrative works. 17 7 2023 7 2023 17 7 2023 6 7 e232333621 2 2023 29 5 2023 Copyright 2023 Matsubara J et al. JAMA Network Open. https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the CC-BY-NC-ND License. jamanetwopen-e2323336.pdf Key Points Question Are comprehensive genomic profiling tests using next-generation sequencing clinically meaningful for patients with previously untreated metastatic or recurrent tumors of the gastrointestinal, pancreatic, biliary tract, lung, breast, gynecologic, and melanoma origin? Findings In this cohort study including 180 patients, 100% of patients with previously untreated advanced cancer had actionable genomic alterations and 61% had options of molecular-based recommended therapy (MBRT) determined by the molecular tumor board. Of these, 20% of the patients received MBRT. Meaning The findings of this study suggest that clinical benefits of comprehensive genomic profiling, which is currently limited to patients with cancer refractory to standard therapies in Japan, can be expanded to patients with previously untreated metastatic or recurrent solid tumors. IMPORTANCE Precision oncology using comprehensive genomic profiling (CGP) by next-generation sequencing is aimed at companion diagnosis and genomic profiling. The clinical utility of CGP before the standard of care (SOC) is still not resolved, and more evidence is needed. OBJECTIVE To investigate the clinical utility of next-generation CGP (FoundationOne CDx [F1CDx]) in patients with previously untreated metastatic or recurrent solid tumors. DESIGN, Setting, and Participants This multicenter, prospective, observational cohort study enrolled patients with previously untreated advanced solid tumors between May 18, 2021, and February 16, 2022, with follow-up through August 16, 2022. The study was conducted at 6 hospitals in Japan. Eligible patients were aged 20 years or older and had Eastern Cooperative Oncology Group performance status of 0 to 1 with previously untreated metastatic or recurrent cancers in the gastrointestinal or biliary tract; pancreas, lung, breast, uterus, or ovary; and malignant melanoma. EXPOSURE Comprehensive genomic profiling testing before SOC for advanced solid tumors. MAIN OUTCOMES AND MEASURES Proportion of patients with actionable or druggable genomic alterations and molecular-based recommended therapy (MBRT). RESULTS A total of 183 patients met the inclusion criteria and 180 patients (92 men [51.1%]) with a median age of 64 years (range, 23-88 years) subsequently underwent CGP (lung [n = 28], colon/small intestine [n = 27], pancreas [n = 27], breast [n = 25], biliary tract [n = 20], gastric [n = 19], uterus [n = 12], esophagus [n = 10], ovary [n = 6], and skin melanoma [n = 6]). Data from 172 patients were available for end point analyses. Actionable alterations were found in 172 patients (100.0%; 95% CI, 97.9%-100.0%) and druggable alternations were identified in 109 patients (63.4%; 95% CI, 55.7%-70.6%). The molecular tumor board identified MBRT for 105 patients (61.0%; 95% CI, 53.3%-68.4%). Genomic alterations included in the companion diagnostics list of the CGP test were found in 49 patients (28.5%; 95% CI, 21.9%-35.9%) in a tumor-agnostic setting. After a median follow-up of 7.9 months (range, 0.5-13.2 months), 34 patients (19.8%; 95% CI, 14.1%-26.5%) received MBRT. CONCLUSIONS AND RELEVANCE The findings of this study suggest that CGP testing before SOC for patients with advanced solid tumors may be clinically beneficial to guide the subsequent anticancer therapies, including molecularly matched treatments. This cohort study examines the use of comprehensive genomic profiling in patients with metastatic and/or recurrent carcinomas to guide anticancer therapies, including molecularly matched treatments. ==== Body pmcIntroduction Precision oncology using comprehensive genomic profiling (CGP) testing by next-generation sequencing has been introduced into clinical practice to effectively select the treatment based on druggable genomic alterations.1 Among several CGP tests, FoundationOne CDx software (Foundation Medicine) was approved by the Ministry of Health, Labour, and Welfare in Japan as a medical device for detection of substitutions, insertion and deletion alterations, and copy number alterations in 324 genes and selected gene rearrangements. This software has the function of both a CGP test and a companion diagnostics (CDx) test. Therefore, it could be ideally used in the early stage of treatments, thereby enabling the CDx-positive cases to be selected for molecularly targeted treatments and patients with druggable gene alteration–positive cancers to be enrolled in clinical trials. Even if the CDx or druggable gene alteration is negative, standard treatments can be provided in the early setting. The use of CGP tests was reimbursed by the National Health Insurance in 2019 in Japan2 as well as in the US and Europe.3,4,5 However, the indication in Japan is limited to patients with advanced solid tumors who had completed standard treatment or rare cancers that have no standard treatment, and CGP cannot be used before the initiation of systemic therapy, even though there are no timing restrictions on this indication in other countries. Thus, the full potential of CGP is not used. Moreover, recent advances in tumor-agnostic CDx and the corresponding effective treatments have supported the use of CGP before rather than after the standard of care (SOC) for advanced solid tumors.6,7,8 Given the indication in Japan, many patients may miss an opportunity to receive molecularly matched therapies early in their treatment. Furthermore, some patients who had completed the standard treatment could not enroll in the clinical trial of new drugs matched by CGP because of their deteriorated physical condition and restriction of the acceptable treatment line.9,10,11,12 In addition, CGP for patients who had completed the SOC did not achieve an improved prognosis.13,14 The National Comprehensive Cancer Network and the European Society for Medical Oncology recommend the use of CGP for selected cancers in daily practice,5,7 as well as for research purposes in academic centers. To our knowledge, the outcomes shown with CGP before first-line cancer treatments have not been reported and more evidence is needed.15,16 We previously reported that CGP for patients with chemotherapy-naive gastrointestinal cancer or cancer of unknown primary site had the potential to provide molecularly matched therapies in 23% of the patients,17 including targeted therapies for cancers in which the corresponding CDx test was not covered by the National Health Insurance. Thus, upfront comprehensive molecular profiling might be clinically important to provide an opportunity for such patients to receive potentially effective molecularly matched therapy. However, the usefulness of CGP before SOC has not been established.18 In the present study, we prospectively investigated the clinical utility of a CGP test in patients with previously untreated metastatic or recurrent tumors of the gastrointestinal, pancreatic, biliary tract, lung, breast, gynecologic, and melanoma origin (the FIRST-Dx study). Methods Study Design and Patient Selection This study was an investigator-initiated, multi-institutional, prospective cohort clinical study performed as part of Advanced Medical Care B, which is a program that is approved by the Ministry of Health, Labour, and Welfare in Japan, and conducted at the following 6 hospitals: Kyoto University Hospital, The University of Tokyo Hospital, Tokyo Medical and Dental University Hospital, Aichi Cancer Center, Toyama University Hospital, and Wakayama Medical University Hospital. Eligible patients were aged 20 years or older with previously untreated, histologically confirmed metastatic and/or recurrent carcinomas in the gastrointestinal or biliary tract; pancreas, lung, breast, uterus, or ovary; and malignant melanoma. Other inclusion criteria were an Eastern Cooperative Oncology Group performance status of 0 or 1 and the availability of an archival formalin-fixed, paraffin-embedded tissue sample for the CGP test. Patients who had received previous adjuvant or neoadjuvant chemotherapy, radiotherapy, or hormone therapy for the carcinoma concerned were eligible. Patients with other concurrent cancers were excluded. The study protocol and all amendments were approved by the institutional review boards of Kyoto University Hospital (Kyoto, Japan) and all participating hospitals. The study was conducted in accordance with the 2013 Declaration of Helsinki.19 All patients provided written informed consent before enrollment in the study; no financial compensation was provided. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline. The primary end point was the proportion of patients with actionable cancer genomic alterations detected using the next-generation CGP (FoundationOne CDx) test. Secondary end points were the proportion of patients with options of molecular-based recommended therapy (MBRT) determined by the molecular tumor board,16 the proportion of patients with druggable genomic alterations, the proportion of patients with genomic alterations covered by CDx, the success rate of the CGP test, the proportion of patients who received MBRT, matching score of MBRT calculated using a matching score system,20 and overall survival. While the proportion of patients who received MBRT was considered the most important secondary end point, we also considered the proportion of patients with options of MBRT as one of the most important secondary end points because of the lack of off-label use due to regulations and other factors, such as clinical trial and geographic availabilities. Definition of Outcomes Actionable cancer genomic alterations and druggable cancer genomic alterations were defined based on the CGP test report for each patient from Foundation Medicine. We defined alterations described in the sections of biomarker findings, genomic findings, and genomic findings with no reportable therapeutic or clinical trial options in the CGP test report as actionable cancer genomic alterations. If the alteration had a therapeutic drug option in the report, it was defined as a druggable cancer genomic alteration. Alterations reported as having only therapeutic resistance were not included in druggable alterations, for example, RAS mutations in colorectal cancer with resistance to antiepidermal growth factor receptor antibodies. Turnaround time was defined as the interval between the shipping date and the date on which we received the report from Foundation Medicine. The evidence level classification was decided according to the second edition of the clinical practice guidance for next-generation sequencing in cancer diagnosis and treatment.21 Overall survival was defined as the time from the date of registration to the date of death due to any cause. Molecular Tumor Board The molecular tumor board of this trial was mandatory and consisted of specialized physicians in the following 5 fields: medical oncology, clinical genetics, pathology, molecular genetics, and bioinformatics according to the requirements of Japanese medical insurance reimbursement. Attendance of at least 1 specialist from each field was required for the board conference. All patients started first-line treatment before the findings of the molecular tumor board were received to avoid the delay in the treatment timing (eFigure 1 in Supplement 1). Matching Score A matching score system was used for each patient as described previously.20 The matching score was calculated by dividing the total number of molecular alterations matched to the MBRT (numerator) by the total number of actionable cancer genomic alterations (denominator). We stratified patients based on matching scores (>50% designated as high or ≤50% designated as low). Statistical Analysis The sample size of the FIRST-Dx study was calculated to have the width of the 95% CI of the primary end point as 15% (±7.5%). The expected actionable alteration ratio of 70%, which was based on a previous report,17 required a total of 141 patients in this study. Because we assumed the success rate of CGP to be 80%, we planned to enroll 180 patients. The success rate of the CGP tests was calculated in the full analysis set of patients. All other end points were calculated in the per-protocol set of patients (Figure 1). Regarding the proportion of patients who received MBRT and overall survival, the interim analysis was prespecified at 6 months after the final patient enrollment. The Kaplan-Meier method was used to estimate the median for overall survival. All comparisons used a 2-sided, 5% type I error, and P value based on the Mann-Whitney test. Statistical analyses were performed using R, version 3.5.3 (R Foundation for Statistical Computing). Figure 1. Study Flowchart CGP indicates comprehensive genomic profiling. Results Patient Characteristics From May 18, 2021, to February 6, 2022, a total of 183 patients were registered in the study (Figure 1). Three patients were excluded because of consent withdrawal (n = 1), ineligibility due to low tumor cellularity (<20%) of the specimen (n = 1), and rapid disease progression (n = 1), resulting in 180 patients who underwent CGP (full analysis set) (92 men [51.1%]; 88 women [48.9%]; mean age, 64 [range, 23-88] years). Among them, 1 patient withdrew consent later and 2 patients were ineligible for the study due to their receipt of prior radiotherapy or chemotherapy. The CGP test was unsuccessful in 5 patients (stomach: 1, biliary tract: 2, pancreas: 2). Finally, 172 patients were eligible for end point analyses (per-protocol set; Figure 1). Patient characteristics are summarized in eTable 1 in Supplement 2. The 3 organs with the most primary tumors in the study were the lung (n = 28), colon/small intestine (n = 27), and pancreas (n = 27). The other sites affected were breast (n = 25), biliary tract (n = 20), gastric (n = 19), uterus (n = 12), esophagus (n = 10), ovary (n = 6), and skin melanoma (n = 6) (Figure 2A). Ninety-nine patients developed relapsed disease after surgery; follow-up was conducted through August 16, 2022. Figure 2. Genomic Alterations and Molecular-Based Recommended Therapies for Per-Protocol Set of Patients A, Number of patients by the organ of the primary tumor in the per-protocol set of patients. B, The proportion of patients with actionable cancer genomic alterations, druggable cancer genomic alterations, molecular-based recommended therapy (MBRT) determined by the molecular tumor board, MBRT available in clinical trials, and genomic alterations related to the companion diagnostics (CDx) regardless of the primary organ. C, Number of patients according to the evidence level classifications of MBRT. The highest evidence level per patient was counted. D, Evidence level classifications of MBRT according to the organs of primary tumors. Primary organs are listed in descending order of the number of patients. Comprehensive Genomic Profiling The success rate of CGP was 97.2% (175 of 180; 95% CI, 93.6%-99.1%) (eTable 2 in Supplement 2). The median turnaround time was 13 days (range, 8-26 days). Cancer Genomic Alterations and MBRT Actionable cancer genomic alterations were found in all patients of the per-protocol set (n = 172; 95% CI, 97.9%-100.0%) (Figure 2B). The gene list of actionable alterations and the frequency are shown in eFigure 2 in Supplement 1. Druggable cancer genomic alterations were found in 109 patients (63.4%; 95% CI, 55.7%-70.6%) (Figure 2B). The gene list of druggable alterations and the frequency according to primary tumors are shown in Figure 3. The top 3 most frequently altered genes were BRAF in skin melanoma, PIK3CA in breast cancer, and ERBB2 in gastric cancer, after excluding the KRAS/NRAS alterations in colon cancer that reportedly had only therapeutic resistance. Figure 3. Frequency of Druggable Cancer Genomic Alterations The vertical axis shows the list of druggable cancer genomic alterations categorized by pathway analysis. The heatmap value represents the frequency of the “number of druggable cancer genomic alterations” divided by the “number of patients” in each cancer type. The molecular tumor board discussed and determined the MBRT for each patient based on the CGP test report. A summary flowchart of patients after receipt of the CGP test report is shown in Figure 4A. Molecular-based recommended therapy was determined for 105 patients (61.0%; 95% CI, 53.3%-68.4%) (Figure 2B and Figure 4A). The highest proportion of patients with options of MBRT was those having breast cancer (21 of 25 [84.0%]), and the lowest proportion was those with esophageal cancer (3 of 10 [30.0%]) (eFigure 3A in Supplement 1). Among 109 patients with druggable genomic alterations, 86 patients had options of MBRT and 23 patients did not. However, among 63 patients without druggable genomic alteration, 19 patients had options of the following MBRT (Figure 4A; eFigure 3B in Supplement 1): palbociclib plus trametinib for patients with concomitant CDKN2A and/or CDKN2B alterations (upregulate CDK4/6) along with KRAS or BRAF alterations (activate the MEK pathway) (n = 12),22 anti-HER2 agents for HER2 amplification (n = 1),23,24 alpelisib for PIK3CA alteration (n = 1),25 selective FGFR 1-3 inhibitors for FGF amplifications (n = 1),26,27 trametinib for NRAS mutation (n = 1),28 immune checkpoint inhibitors for SMARCA4 alteration (n = 1),29 tazemetostat for SMARCB1 alteration (n = 1),30 and an MDM2 inhibitor for MDM2 amplification (n = 1)31 (eTable 3 in Supplement 2). Forty-nine patients (28.5%; 95% CI, 21.9%-35.9%) had 1 option of MBRT per patient and 56 patients (32.6%; 95% CI, 25.6%-40.1%]) had 2 or more options of MBRT per patient (eTable 4 in Supplement 2). Eighty-one patients with options of MBRT (47.1%; 95% CI, 39.5%-54.8%) could become candidates for clinical trials to access the recommended agents (Figure 2B). Figure 4. Summary of Patient Flow After Receipt of Comprehensive Genomic Profiling (CGP) Test Reports and Biological Targets of Molecular-Based Recommended Therapy (MBRT) in Tumor-Agnostic Setting A, Summary of the patient flow after the receipt of the CGP test reports, including those who received MBRT. Druggable alteration was defined as the alteration with a therapeutic drug option in the CGP test report. MBRT was decided by the molecular tumor board. MBRT in the CDx list was in a tumor-agnostic setting. B, Number of patient assignments to tumor-agnostic MBRT. Horizontal labels indicate each MBRT with its biological target in parentheses. CDx indicates companion diagnostics; HRR, homologous recombination repair; ICI, immune checkpoint inhibitor; MBRT, molecular-based recommended therapy; MSI, microsatellite instability; and TMB, tumor mutational burden. aCDx list is available in eTable 5 in Supplement 2. bPatients who received MBRT were identified at the time of interim analysis for prognosis. Evidence Levels of MBRT The evidence level of each MBRT was decided by the molecular tumor board based on the published criteria.21 Evidence level A MBRT was suggested to 49 patients (28.5%; 95% CI, 21.9%-35.9%) (Figure 2C). Evidence level B MBRT was suggested to 3 patients (1.7%; 95% CI, 0.4%-5.0%), evidence level C to 24 patients (14.0%; 95% CI, 9.2%-20.1%), and evidence level D to 29 patients (16.9%; 95% CI, 11.6%-23.3%). The proportion of MBRT evidence levels according to organs of the primary tumor is shown in Figure 2D. The proportion of patients with genomic alterations included in the CDx list of the FoundationOne CDx test was 28.5% (95% CI, 21.9%-35.9%; n = 49) when gene alterations in the tumor-agnostic setting were considered (Figure 2B). However, the proportion decreased to 15.1% (95% CI, 10.1%-21.4%; n = 26) when the primary cancer type was limited to the CDx indication of FoundationOne CDx in Japan (eTable 5 in Supplement 2). MBRT Corresponding Biological Target in Tumor-Agnostic Setting The number of patients assigned to each tumor-agnostic MBRT is shown in Figure 4B. The most recommended therapy was platinum agent therapy followed by poly(adenosine diphosphate-ribose) polymerase inhibitor (n = 25) therapy for patients with deleterious alterations in at least 1 of the 15 prespecified genes involved in homologous recombination repair, as reported previously.32 The second most tumor-agnostic MBRT was anti-HER2 agents for HER2-activating alterations (n = 20) and the third most tumor-agnostic MBRT was immune checkpoint inhibitor for tumor mutational burden (TMB)-high (n = 17). The results of discussions of the molecular tumor board are summarized in eTable 3 in Supplement 2. The number of patients with microsatellite instability (MSI)-high or TMB-high tumors according to primary cancer type is shown in eFigure 4A in Supplement 1. The most MSI-high and TMB-high tumors were gastric (n = 5) and lung (n = 6). The TMB scores in patients with MSI-high or microsatellite stable tumors are summarized in eFigure 4B in Supplement 1. All MSI-high tumors were categorized as TMB-high tumors. The molecular tumor board discussed the matching scores of MBRT for each patient (n = 105), using the published principle of scoring.20 The MBRT with matching scores greater than 50, which would provide higher efficacy than those with scores less than or equal to 50, were recommended to 27 patients (25.7%; 95% CI, 17.7%-35.2%) (eTable 6 in Supplement 2). The top 2 cancer types with MBRT matching scores greater than 50 were gastric (n = 5) and breast (n = 5) cancers. The background genomic alterations between the 2 groups of matching scores were similar (eFigure 5 in Supplement 1). At the time of prespecified interim analysis with a median follow-up of 7.9 months (range, 0.5-13.2 months), 34 patients (19.8%; 95% CI, 14.1%-26.5%) received MBRT (Table; Figure 4A; eFigure 6A, B in Supplement 1). Among 49 patients with options of MBRT included in the CDx list of FoundationOne CDx in a tumor-agnostic setting, 30 patients (61.2%) received the corresponding MBRT. Among 56 patients with options of MBRT not in the CDx list, 4 patients received the corresponding MBRT (Figure 4A). Twenty-six patients received evidence level A MBRT, 2 received evidence level B MBRT, and 6 received evidence level C MBRT (Table; eFigure 6A in Supplement 1). Eleven patients could receive the corresponding MBRT in clinical trials. Patients who had not received MBRT at the time of interim analysis had potentially effective treatment regimens (eFigure 6C, D in Supplement 1). In particular, 23 patients had an option of evidence level A MBRT, including immune checkpoint inhibitors for MSI-high or TMB-high tumors. At the time of interim analysis for prognosis, 28 patients had died (the median overall survival had not been reached) and the 1-year survival rate was 79.7% (95% CI, 71.3%-86.0%) (eFigure 7 in Supplement 1) in the per-protocol set of patients (n = 172). Table. Summary of Patients Who Received MBRT Patient Cancer type Histologic details Biomarker MBRT Evidence level Genomic alterationsa Access Type Annotation 012 Stomach Adenocarcinoma MSI-high Others Immune checkpoint inhibitor A Yes Clinical trial 017 Uterus Adenocarcinoma ERBB2 Amplification Trastuzumab C Yes Clinical trial 025 Stomach Adenocarcinoma ERBB2 Amplification Anti-HER2 agents A Yes Approved 026 Lung Small cell carcinoma TMB-high (21 mutations/Mb) Others Immune checkpoint inhibitor A Yes Approved 028 Breast Adenocarcinoma BRCA1 Others PARP inhibitor A Yes Clinical trial 030 Stomach Adenocarcinoma ERBB2 Amplification Trastuzumab, T-DXd A Yes Approved 031 Stomach Adenocarcinoma ERBB2 Amplification Anti-HER2 agents A Yes Approved 032 Ovary Adenocarcinoma ERBB2 Amplification Anti-HER2 agents C Yes Clinical trial 038 Lung Adenocarcinoma ALK fusion Fusion ALK inhibitor A Yes Approved 045 Lung Adenocarcinoma EGFR L858R Missense mutation EGFR-TKI A Yes Approved 047 Pancreas Adenocarcinoma ERBB2 Amplification Anti-HER2 agents C Yes Clinical trial 059 Colon Adenocarcinoma BRAF V600E Missense mutation Cetuximab + encorafenib ± binimetinib A Yes Approved 062 Ovary Adenocarcinoma LOH score-high Others PARP inhibitor A No Approved 067 Colon Others RANBP2-ALK fusion Fusion ALK inhibitor B Yes Clinical trial 074 Skin melanoma Others BRAF V600E Missense mutation Dabrafenib + trametinib, encorafenib + binimetinib A Yes Approved 075 Colon Others BRAF V600E Missense mutation Cetuximab + encorafenib ± binimetinib A Yes Approved 087 Lung Adenocarcinoma TMB-high (35 mutations/Mb) Adenocarcinoma Immune checkpoint inhibitor A Yes Approved 093 Breast Adenocarcinoma BRCA1 Truncation PARP inhibitor A Yes Approved 096 Biliary tract Adenocarcinoma ERBB2 Amplification Anti-HER2 agents C Yes Clinical trial 108 Colon Adenocarcinoma BRAF V600E Missense mutation Cetuximab + encorafenib ± binimetinib A Yes Approved 112 Stomach Adenocarcinoma ERBB2 Amplification Anti-HER2 agents A Yes Approved 118 Colon Adenocarcinoma BRAF V600E Missense mutation Cetuximab + encorafenib ± binimetinib A Yes Approved CDK12 E659* Truncation Platinum, PARP inhibitor C No Approved 122 Colon Small cell carcinoma ERBB2 S310Y Missense mutation pan-HER TKI, T-DXd C No Clinical trial 125 Ovary Adenocarcinoma LOH Score-high Others PARP inhibitor C No Approved 134 Lung Adenocarcinoma EML4-ALK fusion Fusion ALK inhibitor A Yes Approved 135 Stomach Adenocarcinoma MSI-high Others Immune checkpoint inhibitor A Yes Clinical trial 145 Biliary tract Adenocarcinoma TMB-high (20 mutations/Mb) Others Immune checkpoint inhibitor A Yes Approved 155 Skin melanoma Others BRAF V600R Missense mutation Dabrafenib + trametinib, encorafenib + binimetinib B No Clinical trial 165 Biliary tract Adenocarcinoma FGFR2-KIAA1598 fusion Fusion FGFR inhibitor A Yes Approved 166 Stomach Adenocarcinoma MSI-high Others Immune checkpoint inhibitor A Yes Approved 167 Breast Adenocarcinoma ERBB2 Amplification Anti-HER2 agents A Yes Approved 179 Lung Adenocarcinoma TMB-high (10 mutations/Mb) Others Immune checkpoint inhibitor A Yes Approved 181 Stomach Adenocarcinoma MSI-high Others Immune checkpoint inhibitor A Yes Approved 183 Stomach Adenocarcinoma MSI-high Others Immune checkpoint inhibitor A Yes Clinical trial Abbreviations: MBRT, molecular-based recommended therapy; MSI, microsatellite instability; PARP, poly(adenosine diphosphate-ribose) polymerase; TMB, tumor mutational burden. a Alterations included in the companion diagnostics list in the tumor-agnostic setting. Discussion To our knowledge, this study is the first comprehensive analysis to prospectively evaluate the clinical utility of the next-generation CGP (FoundationOne CDx [F1CDx]) test in patients with chemotherapy-naive cancer. Actionable cancer genomic alterations were found in 100.0% of the patients, druggable cancer genomic alterations in 63.4%, and MBRT in 61.0%. These findings are clinically important because more than 50% of patients with advanced solid tumors had druggable cancer genomic alterations and potentially had the opportunity to access molecularly matched treatments based on CGP before starting the SOC. In the present study, 34 patients (19.8%) received MBRT at the time of interim analysis. Among them, 26 patients received evidence-level A MBRT. Moreover, because as many as 23 patients had options of evidence-level A MBRT among 71 patients who had not received MBRT, we expected that more patients would benefit from CGP before the SOC. The proportions of MBRT evidence levels observed in this study were similar to those in previous reports,12,17,33 except a few more patients with evidence-level A MBRT were present in our study than the others. Therefore, the present findings suggest that CGP before the SOC could provide an opportunity for receiving potentially effective treatments. We need continuous efforts to standardize and update the molecular tumor board discussion as reported previously.34 Molecular-based recommended therapy targeting genes included in the CDx list in a tumor-agnostic setting were recommended to 49 patients, and 30 (61.2%) of them received the MBRT early in their treatment journey. Molecularly targeted drugs accompanied by CDx generally exhibit high efficacy in the tumor-agnostic setting, including tropomyosin receptor kinase (TRK) inhibitors for TRK fusion-positive cancers35,36 and pembrolizumab for MSI-high or TMB-high tumors.37,38 Thus, CGP before the SOC might present patients with an opportunity to receive effective treatments in the subsequent lines of chemotherapy. Limitations This study has limitations. Because the median follow-up period was short (7.9 months), overall survival could not be determined accurately. To investigate the survival benefit of CGP before the SOC, we are currently performing a prospective observational study to follow up the patients in the FIRST-Dx study. Second, access to drugs was limited because off-label use was not accepted in Japan. That means, even if a patient has a recommended evidence level C or D treatment with rare driver gene alterations or treatment with more than a 50% matching score, the patient cannot be treated with off-label use drugs. To introduce precision oncology effectively in clinical practice, an extended option for patients with cancer to gain access to an investigational drug outside of clinical trials, such as a compassionate use program, might improve the accessibility of anticancer drugs in Japan. Conclusions The findings of this prospective observational cohort study suggest that CGP testing for patients with previously untreated metastatic or recurrent tumors of gastrointestinal, pancreatic, biliary tract, lung, breast, gynecologic, and melanoma origin might have a notable clinical benefit for individual patients by providing them an opportunity to receive highly effective MBRT early in the disease course. These findings may support the modification of the insurance coverage in Japan of CGP from the last line to prior-to-initiation of the primary treatment in patients with metastatic or recurrent solid tumors. The genomics-guided oncology using CGP testing before SOC for patients with advanced solid tumors may be the first step for future advances in precision oncology. Supplement 1. eFigure 1. Events in FIRST-Dx Study According to the Timeline eFigure 2. List of Actionable Genomic Alterations eFigure 3. Actionable/Druggable Cancer Genomic Alterations and Molecular-Based Recommended Therapies eFigure 4. Tumor Mutational Burden and Microsatellite Instability eFigure 5. Background Genomic Alterations According to Matching Score eFigure 6. Biological Targets of MBRT in Tumor-Agnostic Setting and Evidence Levels of the MBRT eFigure 7. Kaplan-Meier Plot of Overall Survival Click here for additional data file. Supplement 2. eTable 1. Patient Characteristics eTable 2. Success Rates of Comprehensive Genomic Profiling eTable 3. Summary of CGP Test Results and Molecular-Based Recommended Therapies eTable 4. Number of Molecular-Based Recommended Therapy Per Patient eTable 5. Companion Diagnostics Indications of F1CDx in Japan eTable 6. Summary of Matching Score Click here for additional data file. Supplement 3. Data Sharing Statement Click here for additional data file. ==== Refs References 1 Haendel MA, Chute CG, Robinson PN. Classification, ontology, and precision medicine. N Engl J Med. 2018;379 (15 ):1452-1462. doi:10.1056/NEJMra1615014 30304648 2 Sunami K, Naito Y, Komine K, . Chronological improvement in precision oncology implementation in Japan. Cancer Sci. 2022;113 (11 ):3995-4000. doi:10.1111/cas.15517 35976133 3 FDA Fact Sheet CDRH’s approach to tumor profiling next generation sequencing tests. 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Molecular screening program to select molecular-based recommended therapies for metastatic cancer patients: analysis from the ProfiLER trial. Ann Oncol. 2019;30 (5 ):757-765. doi:10.1093/annonc/mdz080 30865223 17 Kondo T, Matsubara J, Quy PN, . Comprehensive genomic profiling for patients with chemotherapy-naïve advanced cancer. Cancer Sci. 2021;112 (1 ):296-304. doi:10.1111/cas.14674 33007138 18 Dienstmann R, Lonardi S. Is upfront full molecular profiling needed in all patients with colorectal cancer in daily practice? Lancet Oncol. 2022;23 (9 ):1129-1131. doi:10.1016/S1470-2045(22)00035-3 36055302 19 World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013;310 (20 ):2191-2194. doi:10.1001/jama.2013.281053 24141714 20 Sicklick JK, Kato S, Okamura R, . Molecular profiling of cancer patients enables personalized combination therapy: the I-PREDICT study. Nat Med. 2019;25 (5 ):744-750. doi:10.1038/s41591-019-0407-5 31011206 21 Naito Y, Aburatani H, Amano T, ; Japanese Society of Medical Oncology; Japan Society of Clinical Oncology; Japanese Cancer Association. Clinical practice guidance for next-generation sequencing in cancer diagnosis and treatment (edition 2.1). Int J Clin Oncol. 2021;26 (2 ):233-283. doi:10.1007/s10147-020-01831-6 33249514 22 Kato S, Adashek JJ, Shaya J, . Concomitant MEK and cyclin gene alterations: implications for response to targeted therapeutics. Clin Cancer Res. 2021;27 (10 ):2792-2797. doi:10.1158/1078-0432.CCR-20-3761 33472910 23 Meric-Bernstam F, Hurwitz H, Raghav KPS, . Pertuzumab plus trastuzumab for HER2-amplified metastatic colorectal cancer (MyPathway): an updated report from a multicentre, open-label, phase 2a, multiple basket study. Lancet Oncol. 2019;20 (4 ):518-530. doi:10.1016/S1470-2045(18)30904-5 30857956 24 Jhaveri KL, Wang XV, Makker V, . Ado-trastuzumab emtansine (T-DM1) in patients with HER2-amplified tumors excluding breast and gastric/gastroesophageal junction (GEJ) adenocarcinomas: results from the NCI-MATCH trial (EAY131) subprotocol Q. Ann Oncol. 2019;30 (11 ):1821-1830. doi:10.1093/annonc/mdz291 31504139 25 André F, Ciruelos E, Rubovszky G, ; SOLAR-1 Study Group. Alpelisib for PIK3CA-mutated, hormone receptor–positive advanced breast cancer. N Engl J Med. 2019;380 (20 ):1929-1940. doi:10.1056/NEJMoa1813904 31091374 26 Subbiah V, Iannotti NO, Gutierrez M, . FIGHT-101, a first-in-human study of potent and selective FGFR 1-3 inhibitor pemigatinib in pan-cancer patients with FGF/FGFR alterations and advanced malignancies. Ann Oncol. 2022;33 (5 ):522-533. doi:10.1016/j.annonc.2022.02.001 35176457 27 Dumbrava EI, Alfattal R, Miller VA, Tsimberidou AM. Complete response to a fibroblast growth factor receptor inhibitor in a patient with head and neck squamous cell carcinoma harboring FGF amplifications. JCO Precis Oncol. 2018;2 :PO.18.00100. doi:10.1200/PO.18.00100 31123723 28 Salzmann M, Pawlowski J, Loquai C, . MEK inhibitors for pre-treated, NRAS-mutated metastatic melanoma: a multi-centre, retrospective study. Eur J Cancer. 2022;166 :24-32. doi:10.1016/j.ejca.2022.02.008 35272084 29 Schoenfeld AJ, Bandlamudi C, Lavery JA, . The genomic landscape of SMARCA4 alterations and associations with outcomes in patients with lung cancer. Clin Cancer Res. 2020;26 (21 ):5701-5708. doi:10.1158/1078-0432.CCR-20-1825 32709715 30 Gounder M, Schöffski P, Jones RL, . Tazemetostat in advanced epithelioid sarcoma with loss of INI1/SMARCB1: an international, open-label, phase 2 basket study. Lancet Oncol. 2020;21 (11 ):1423-1432. doi:10.1016/S1470-2045(20)30451-4 33035459 31 LoRusso P, Gounder MM, Patel MR, . A phase I dose-escalation study of the MDM2-p53 antagonist BI 907828 in patients (pts) with advanced solid tumors. J Clin Oncol. 2021;39 (15 )(suppl ):3016. doi:10.1200/JCO.2021.39.15_suppl.3016 32 de Bono J, Mateo J, Fizazi K, . Olaparib for metastatic castration-resistant prostate cancer. N Engl J Med. 2020;382 (22 ):2091-2102. doi:10.1056/NEJMoa1911440 32343890 33 Aoyagi Y, Kano Y, Tohyama K, . Clinical utility of comprehensive genomic profiling in Japan: result of PROFILE-F study. PLoS One. 2022;17 (3 ):e0266112. doi:10.1371/journal.pone.0266112 35358259 34 Tamborero D, Dienstmann R, Rachid MH, ; Cancer Core Europe consortium. Support systems to guide clinical decision-making in precision oncology: the Cancer Core Europe Molecular Tumor Board Portal. Nat Med. 2020;26 (7 ):992-994. doi:10.1038/s41591-020-0969-2 32632195 35 Drilon A, Laetsch TW, Kummar S, . Efficacy of larotrectinib in TRK fusion-positive cancers in adults and children. N Engl J Med. 2018;378 (8 ):731-739. doi:10.1056/NEJMoa1714448 29466156 36 Doebele RC, Drilon A, Paz-Ares L, ; trial investigators. 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==== Front Neurol Genet Neurol Genet nng NNG Neurology: Genetics 2376-7839 Wolters Kluwer Baltimore NXG-2023-000027 10.1212/NXG.0000000000200087 3 120 176 177 181 91 Research Article Phenotype Presentation and Molecular Diagnostic Yield in Non-5q Spinal Muscular Atrophy https://orcid.org/0000-0003-1112-7464 Fernández-Eulate Gorka MD Theuriet Julian MD https://orcid.org/0000-0002-9802-2683 Record Christopher J. MD Querin Giorgia MD, PhD Masingue Marion MD https://orcid.org/0000-0002-2365-7932 Leonard-Louis Sarah MD Behin Anthony MD Le Forestier Nadine MD https://orcid.org/0000-0003-1563-9432 Pegat Antoine MD Michaud Maud MD Chanson Jean-Baptiste MD Nadaj-Pakleza Aleksandra MD Tard Celine MD, PhD Bedat-Millet Anne-Laure MD https://orcid.org/0000-0001-9599-6573 Sole Guilhem MD Spinazzi Marco MD https://orcid.org/0000-0002-1846-3017 Salort-Campana Emmanuelle MD Echaniz-Laguna Andoni MD, PhD https://orcid.org/0000-0002-4945-5431 Poinsignon Vianney PharmD Latour Philippe PhD Reilly Mary M. MD Bouhour Francoise MD https://orcid.org/0000-0002-4054-2838 Stojkovic Tanya MD From the Nord/Est/Ile-de-France Neuromuscular Reference Center (G.F.-E., G.Q., M. Masingue, S.L.-L., A.B., T.S.), Institut de Myologie, Pitié-Salpêtrière Hospital, Paris; Electromyography and Neuromuscular Department (J.T., A.P., F.B.), Hospices Civils de Lyon; Centre for Neuromuscular Diseases (C.J.R., M.M.R.), UCL Queen Square Institute of Neurology, London, United Kingdom; Neurology Department (N.L.F.), Pitié-Salpêtrière Hospital, Paris; Nord/Est/Ile-de-France Neuromuscular Reference Center (M. Michaud), Central Nancy University Hospital; Nord/Est/Ile-de-France Neuromuscular Reference Center (J.-B.C., A.N.-P.), Strasbourg University Hospitals; Nord/Est/Ile-de-France Neuromuscular Reference Center (C.T.), U1172, Lille University Hospital; Nord/Est/Ile-de-France Neuromuscular Reference Center (A.-L.B.-M.), Rouen University Hospital; Neuromuscular Reference Center ‘AOC’ (G.S.), Bordeaux University Hospitals (Pellegrin Hospital); Neuromuscular Reference Center (M.S.), Angers University Hospital; Neuromuscular and ALS Reference Center (E.S.-C.), La Timone University Hospital, Marseille; French National Center for Rare Neuropathies (A.E.-L.), Neurology Department, Bicêtre University Hospital, INSERM U1195, Paris-Saclay University; Molecular Genetics Lab (V.P.), Bicêtre University Hospital, Le Kremlin Bicêtre; and Center for Biology - East (P.L.), Neurological Hereditary Disorders Unit, Hospices Civils de Lyon, France. Correspondence Dr. Stojkovic tanya.stojkovic@aphp.fr Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the authors. Submitted and externally peer reviewed. The handling editor was Associate Editor Margherita Milone, MD, PhD. 8 2023 17 7 2023 17 7 2023 9 4 e20008716 12 2022 26 5 2023 Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 2023 American Academy of Neurology https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Background and Objectives Spinal muscular atrophy (SMA) is mainly caused by homozygous SMN1 gene deletions on 5q13. Non-5q SMA patients’ series are lacking, and the diagnostic yield of next-generation sequencing (NGS) is largely unknown. The aim of this study was to describe the clinical and genetic landscape of non-5q SMA and evaluate the performance of neuropathy gene panels in these disorders. Methods Description of patients with non-5q SMA followed in the different neuromuscular reference centers in France as well as in London, United Kingdom. Patients without a genetic diagnosis had undergone at least a neuropathy or large neuromuscular gene panel. Results Seventy-one patients from 65 different families were included, mostly sporadic cases (60.6%). At presentation, 21 patients (29.6%) showed exclusive proximal weakness (P-SMA), 35 (49.3%) showed associated distal weakness (PD-SMA), and 15 (21.1%) a scapuloperoneal phenotype (SP-SMA). Thirty-two patients (45.1%) had a genetic diagnosis: BICD2 (n = 9), DYNC1H1 (n = 7), TRPV4 (n = 4), VCP, HSBP1, AR (n = 2), VRK1, DNAJB2, MORC2, ASAH1, HEXB, and unexpectedly, COL6A3 (n = 1). The genetic diagnostic yield was lowest in P-SMA (6/21, 28.6%) compared with PD-SMA (16/35, 45.7%) and SP-SMA (10/15, 66.7%). An earlier disease onset and a family history of the disease or consanguinity were independent predictors of a positive genetic diagnosis. Neuropathy gene panels were performed in 59 patients with a 32.2% diagnostic yield (19/59). In 13 additional patients, a genetic diagnosis was achieved through individual gene sequencing or an alternative neuromuscular NGS. Discussion Non-5q SMA is genetically heterogeneous, and neuropathy gene panels achieve a molecular diagnosis in one-third of the patients. The diagnostic yield can be increased by sequencing of other neuromuscular and neurometabolic genes. Nevertheless, there is an unmet need to cluster these patients to aid in the identification of new genes. OPEN-ACCESSTRUE ==== Body pmcHereditary motor neuropathies (HMN) are a heterogeneous group of diseases characterized by peripheral motor neuron weakness with minimal or absent clinical and neurophysiologic sensory impairment, which differentiates them from Charcot-Marie-Tooth (CMT) disease and the hereditary sensory neuropathies.1 Distal HMN (dHMN) represent the most common clinical presentation of HMN.2,3 The term spinal muscular atrophy (SMA) is mainly used when referring to HMNs with proximal weakness at onset, either isolated (P-SMA) or concomitant with distal (PD-SMA) or scapuloperoneal (SP-SMA) motor weakness.4 A homozygous exon 7 and 8 deletion in the SMN1 gene represents the main cause of SMA,5 ranging from 50-94% of patients depending on the series.6,7 In the absence of this deletion (non-5q SMA), several genes have been described, such as TRPV4, DYNC1H1, BICD2, GLE1, UBA1, ASAH1, VRK1, EXOSC3, SLC52A3 and SLC52A2, VAPB, HEXB, SETX, LMNA, and TFG with different clinical phenotypes and ages at onset of disease.4,6,8-12 These and other genes may overlap with other nosological entities such as dHMN, CMT type 2, amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia (HSP), or even mitochondrial disease and myopathy.4,13 Nevertheless, around 70% or more of non-5q SMA patients do not have a definitive molecular diagnosis.6 Unfortunately, although some reviews of non-5q SMA patients have been published,4,14 clinical series of patients are lacking and the value of routine neuropathy next-generation sequencing (NGS) gene panels is largely unknown. The aim of this study was to describe the clinical and genetic landscape of a large multicentric series of patients with non-5q SMA and evaluate the performance of neuropathy gene panels in these patients. Methods Inclusion and Exclusion Criteria Between April and May 2021, we retrospectively reassessed the clinical records of adult patients (18 years or older) with an HMN and predominant proximal weakness (SMA) from 10 neuromuscular reference centers in France. Sixty-one patients were included based on the following criteria: (1) Patients had a motor neuropathy or motor neuronopathy on electroneuromyography (ENMG), defined by the presence of large-size (>2 mV in amplitude) simple-shaped motor unit potentials and reduced recruitment. In addition, reductions of compound motor action potential (CMAP) amplitudes and less than 30% reduction of normal sensory nerve action potential (SNAP) amplitudes (reference values for adult patients: >10 µV in lower limbs, >8 µV for ulnar, and >15 µV for median and radial nerves) were accepted. (2) Patients were negative for the SMN1 deletion and (3) had a confirmed genetic diagnosis or at least undergone a neuropathy NGS gene panel. Patients with clinical or neurophysiologic disease progression at 6 months were excluded. Furthermore, clustered data from 10 adult patients followed in the Centre for Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London (United Kingdom) were obtained following the same inclusion and exclusion criteria. Based on the clinical presentation, patients were classified in 3 groups: (1) Patients with P-SMA had pure proximal motor weakness at first visit, (2) patients with PD-SMA had proximal and distal weakness but pure proximal involvement at disease onset or proximal-predominant weakness at first visit, and (3) patients with SP-SMA had scapuloperoneal weakness. Demographic (date of birth, sex) and clinical information (age at onset, family history of neuropathy, consanguinity, neurologic, orthopedic, and respiratory manifestations) were collected. Additional data such as ENMG findings, creatine kinase (CK) levels, the need of noninvasive ventilation (NIV), and molecular biology results were collected when available, as well as loss of ambulation as functional outcome. Molecular Biology Investigations The molecular biology investigations performed in each patient are shown in eTable 1 (links.lww.com/NXG/A614). Neuropathy Gene Panels Fifty-nine neuropathy gene panels were performed in total. Forty-eight were performed at the molecular genetic laboratory of Hospices Civils de Lyon. DNA was extracted from blood samples using Nucleospin Blood L (Macherey-Nagel) on automat Microlab STARlet (Hamilton). Capture was performed with Roche KAPA Hypercap v3.0. Sequencing was performed using Illumina NextSeq 500. Copy number variations were detected using DeCovA_1.6.0. Sequencing depth at 30x was over 99,95%. One hundred two genes are included in this panel (eAppendix 1, links.lww.com/NXG/A613). Eleven additional neuropathy gene panels were sequenced by the National Hospital for Neurology and Neurosurgery (NHNN) in London (n = 4, 15–32 genes) and the molecular genetic laboratories from Kremlin-Bicêtre (n = 4, 127 genes), Limoges (n = 2, 124 genes), and Angers (n = 1, 148 genes). Alternative Neuromuscular Gene Panels and Individual Gene Sequencing Eight patients underwent an alternative large neuromuscular gene panel: whole genome sequencing (WGS) with a ∼180 gene virtual panel performed by the NHNN in London (n = 3), a whole exome sequencing (WES) focused on known neuromuscular genes (n = 1), and large neuromuscular gene panels in Paris (n = 2), Strasbourg (n = 1), and Nancy (n = 1). In 8 patients, a genetic diagnosis was confirmed through individual sequencing of other clinically suspected neuromuscular or neurometabolic genes. Whole Exome Sequencing Furthermore, in 20 patients without a molecular diagnosis after a neuropathy NGS panel, WES was performed at Hospices Civils de Lyon. Exome enrichment was performed with the SeqCap EZ MedExome Target Enrichment Kit (Roche Diagnostics). Sequencing was performed on a NextSeq 500 (Illumina) platform, aligned using Borrows-Weeler Aligner to a hg19 reference genome; variants were called using Genome Analysis Tool Kit Unified genotyper software, and filtering of data were performed based on an in-house pipeline: 1000 genomes frequency <0.01, maximum ExAC occurrences heterozygotes 250, maximum ExaC occurrences homozygotes 2, minimal read depth >7, mosaïcism >0.15, coding exons ± 10. Variants were classified according to the 2015 American College of Medical Genetics Standards and Guidelines for the interpretation of sequence variants,15 and patients with pathogenic or likely pathogenic variants were considered as genetically confirmed. Data Analysis and Statistics Data analysis was performed using JASP version 0.16.2. Mean and median values, SD, and range of quantitative variables and absolute or relative frequencies of categorical variables were reported. Association was studied through the Student t test (continuous dependent variables) and χ2 test (categorical dependent variables). A multivariate logistic regression was performed to assess the association of relevant clinical variables with a definitive molecular diagnosis. Normality was verified through the Shapiro-Wilk test. Statistical significance was established at p ≤ 0.05. Ethics Approval In accordance with French legislation, the study was registered and is accessible to the French data protection authority (CNIL, Commission nationale de l'informatique et des libertés). This study was approved by the ethics committee of Île-de-France VI on 01/04/2021. Data Availability Anonymized data from this study will be shared by request from any qualified investigator. Results Clinical Description of Patients and Ancillary Testing Seventy-one patients from 65 different families were included. Thirty-four patients (47.9%) were female, and most patients had no family history of neuromuscular disease or consanguinity and were therefore classified as sporadic (60.6%). Onset of the disease ranged from 0 to 58 years, and 36 patients (50.7%) had an early onset (younger than 18 years). At presentation (38.2 ± 14.9 years), 21 patients (29.6%) showed exclusive proximal weakness (P-SMA), 35 (49.3%) associated distal weakness (PD-SMA), and 15 patients (21.1%) a scapuloperoneal phenotype (SP-SMA). Clinical and ancillary data are shown in Table 1. Differences in clinical variables between the P-SMA, PD-SMA, and SP-SMA groups are shown in eTable 2 (links.lww.com/NXG/A615). Table 1 Clinical Features and Ancillary Examinations of Non-5q Spinal Muscular Atrophy (SMA) Patients On ENMG, all patients had neurogenic motor unit potentials as per inclusion criteria, with 27 (38%) showing reduction of CMAP amplitude and 6 (8.5%) a minimal reduction of SNAPs associated with the peripheral motor neuron involvement. For 60 patients, follow-up data were available and 5 patients (8.3%) had lost ambulation at a median of 5 years (range 4–54 years) from symptom's onset. Genetic Diagnosis and Predictive Variables In 32 patients (45.1%), a genetic diagnosis was achieved. The molecular diagnostic yield was lowest in the P-SMA (6/21, 28.6%) vs the PD-SMA (16/35, 45.7%) and SP-SMA (10/15, 66.7%) clinical groups. The different genes found in each non-5q SMA clinical group can be seen in Figure 1. Figure 1 Distribution of Patients With a Positive Genetic Diagnosis According to the Spinal Muscular Atrophy (SMA) Subtype Thirty-two patients with a genetic diagnosis achieved through neuropathy gene panel (n = 19: DYNC1H1 x6, BICD2 x4, TRPV4 x4, HSPB1, VCP, VRK1, DNAJB2, MORC2), individual gene sequencing (n = 8: BICD2 x3, AR x2, HSPB1, HEXB, and COL6A3), and other neuromuscular NGS (n = 5: BICD2 x2, DYNC1H1, VCP, and ASAH1). Neg: negative; NGS: next-generation sequencing; P-SMA: proximal SMA; PD-SMA; proximo-distal SMA; SP-SMA: scapuloperoneal SMA. Fifty-nine neuropathy NGS gene panels were performed, with a positive genetic diagnosis in 19/59 (32.2%). There was no significant difference in the performance of the neuropathy gene panel from Hospice Civils de Lyon (n = 48) vs other neuropathy gene panels in this series (n = 11) (eTable 3, links.lww.com/NXG/A616). In 8 additional patients, a genetic diagnosis was achieved through individual gene sequencing: BICD2 (3 patients from a previously reported family with 6 affected individuals),16 AR (n = 2), HSPB1, HEXB, and COL6A3. Reasons for this were genetic testing before routine gene panel sequencing in family members were an index case was found to carry a variant in the gene through exome sequencing (BICD2),16 genetic testing before routine gene panel sequencing and a plausible clinical picture (HSPB1), a particular or noncorresponding pattern on muscle MRI (HEXB and COL6A3), gynecomastia and elevated CK levels (patient with Kennedy disease), and a negative neuropathy gene panel and elevated CK levels (second patient with Kennedy disease). Finally, 5 patients were diagnosed through an alternative neuromuscular NGS: BICD2 (n = 2), DYNC1H1, VCP, and ASAH1. Furthermore, 20 patients underwent WES, which did not confirm any additional pathogenic or likely pathogenic variants in a known neurologic disease-causing gene. Patients who received a definitive genetic diagnosis were more likely to have an earlier age at onset, a family history of the disease or consanguinity, and contractures and/or arthrogryposis. In a multivariate logistic regression, an earlier onset and a family history of the disease or consanguinity retained statistical significance (Table 2). Table 2 Predictors of a Positive Genetic Diagnosis Clinical Features of the Most Common Non-5q SMA–Associated Genes The most common genes in non-5q SMA patients were BICD2 (n = 9) and DYNC1H1 (n = 7), associated with SMA with lower extremity predominance (SMA-LED), and TRPV4 (n = 4), implicated in SP-SMA. These 3 genes are dominantly inherited; however, only 13/20 patients had a clear AD family history of the disease, the rest were therefore categorized as sporadic cases. We summarize below the features of these 3 most common genes.BICD2 (9 patients from 7 different families). Variants: c.1502G>C (n = 3) from the previously reported family,16 c.2042C>T (n = 2), c.2108C>T, c.2080C>T, c.1617C>G, and c.380A>G. The latter 2 had never been reported: The c.1617C>G variant segregated correctly within the family and predicted as likely pathogenic in silico; the c.380A>G variant was predicted as a likely pathogenic in silico (because it is absent from population databases, well conserved, and situated in a mutational hotspot, with a clinical and muscle MRI phenotype compatible with BICD2 SMA-LED).17 However, given that SMA-LED is not a disease with a single genetic etiology and that segregation was not performed, we retain this variant as a variant of unknown significance (VUS) for now. Globally, although all types of phenotypical presentations were possible between and within families [PD-SMA (n = 5), SP-SMA (n = 3) or P-SMA (n = 1)], BICD2 patients frequently showed some degree of distal weakness on presentation (7/9) and a disease onset younger than 10 years (8/9). Normal or brisk deep tendon reflexes (DTR) (6/9) with 1 patient presenting some degree of spasticity, scapula alata (4/9), and axial weakness (3/9) were relatively frequent. Two patients had mild respiratory insufficiency, 1 needing overnight NIV. No patient had lost ambulation at follow-up. All patients had a motor neuronopathy on ENMG, one with mild sensory involvement. DYNC1H1 (7 patients from 7 families). Variants: c.2327C>T (n = 2), c.752G> A, c.1792C>T, c.752G>T, c.1427T>C, c.596A>C. The latter 2 variants have never been reported and were predicted as likely pathogenic in silico. Segregation for the c.596A>C confirmed a de novo origin; however, segregation for the c.1427T>C variant could not be performed because the patient was adopted. Clinically, DYNC1H1 patients presented most frequently a PD-SMA (5/7). Disease onset was always in younger than 5 years, 3/7 patients had neonatal arthrogryposis, and 3/7 learning difficulties since childhood. DTRs were mostly abolished (6/7). Only 1 patient had moderate respiratory insufficiency in the context of obesity. Of the 4 patients who were followed up, none had lost ambulation. All patients had a pure motor neuronopathy on ENMG. TRPV4 (4 patients from 3 families). Variants: c.557G>A (n = 2) and c.694C>T (n = 2), all previously reported. These patients showed predominantly a SP-SMA (3/4), although 1 patient presented with a PD-SMA without scapula alata. Although onset of the first progressive symptom was in adult age (18–53 years), 2 patients had a personal history of arthrogryposis and one of scoliosis and pes cavus. DTRs were always abolished. Three patients had some degree of respiratory insufficiency, none requiring ventilation. Three of these patients also had vocal cord paralysis with hypophonia. One patient showed a minimal reduction of normal SNAP on ENMG. All but 1 patient remain ambulant, the latter losing ambulation at age 5 years (before the onset of weakness) because of arthrogryposis. Other Non-5q SMA–Associated Genes Other variants in genes implicated in HMNs were found. In 2 SP-SMA patients with lower-limb distal involvement, scapula alata, and a disease onset in adult life, HSPB1 variants were found (AD transmission). Two patients (SP-SMA and P-SMA) carried variants in the VCP and one (SP-SMA) in the VRK1 gene (both AD), all 3 with an adult disease onset. A previously reported patient with an adult-onset PD-SMA carried a variant in the MORC2 gene.18 One patient with an infantile-onset PD-SMA carried a homozygous pathogenic variant in the DNAJB2 gene (AR). The specific genetic variants can be seen in eTable 1 (links.lww.com/NXG/A614). It is important that 2 patients with a PD-SMA phenotype without clinical nor ENMG sensory involvement and an onset of symptoms in their late 20s carried CAG repeat expansions in the AR gene (AD transmission). None of these patients had bulbar symptoms, and only one had very high CK levels (2000U/L) and gynecomastia. Furthermore, 2 patients carried biallelic variants (AR transmission) in genes implicated in lysosomal storage disorders (LSD). One patient carries 2 new compound heterozygous pathogenic variants in the HEXB gene (c.881A>G and c.1082+2T>C), compatible with a diagnosis of Sandhoff disease. This patient presented with a pure P-SMA, an onset of symptoms at 11 years, and no other neurologic manifestation of the disease (cerebellar, extrapyramidal, cognitive, or psychiatric); DTRs were normal, and there was no upper limb tremor. ENMG was compatible with a motor neuronopathy. Importantly, the patient was found to carry a VUS in the DYNC1H1 gene, but the muscle MRI pattern did not correspond to the one previously described in DYNC1H1-associated SMA (Figure 2),19 so a leukocyte hexosaminidase analysis was performed, finding deficient A and B enzymatic activities. A second patient which we have previously reported20 carries compound heterozygous variants in the ASAH1 gene (c.77C>G and c.125+1G>A) and has a deficient leukocyte ceramidase activity. This patient presented with a pure P-SMA, an onset of symptoms at 19 years and again no other neurologic manifestation of the disease (epilepsy). The patient had scoliosis and club feet, upper limb tremor, and retained DTRs. ENMG was compatible with a motor neuronopathy. Figure 2 Differing MRI Muscle Involvement in Non-5q Spinal Muscular Atrophy (SMA) Patients T1-weighted MRI sequences of 5 patients with variants in the 3 most frequent non-5q SMA genes (DYNC1H1, BICD2, and TRPV4) as well as in Sandhoff disease (HEXB) and COL6A3. Muscles with yellow arrows had marked fat replacement; muscles with white arrows were completely or partially spared. (A) Quadriceps, semitendinosus, and adductor magnus muscle degeneration in a patient with Sandhoff disease presenting as P-SMA. (B) The previously described quadriceps muscle degeneration with adductor longus and semitendinosus sparing in a patient with DYNC1H1 PD-SMA.19 In this case, sartorius and gracillis muscle fat replacement is also observed. (C) Quadriceps and sartorious muscle degeneration with more important lower limb involvement in a patient with BICD2 PD-SMA. Notice some degree of paraspinal muscle atrophy and important scoliosis. (D) Tibialis anterior and gastrocnemius medialis involvement with sparing of muscles of the thigh and paraspinal and deltoid muscle degeneration in a patient with TRPV4 SP-SMA. (E) Finally, the characteristic muscle fat replacement in a “sandwich patter” of soleus and quadriceps muscles (yellow arrowheads) in a patient with COL6A3 mutations and a pure SMA phenotype. AL = adductor longus; AM = adductor magnus; De = deltoid; GM = gastrocnemius medialis; Gr = gracillis; PS = paraspinal; Quad = quadriceps; Sa = sartorious; ST = semitendinosus; TA = tibialis anterior. Finally, in 1 patient with a PD-SMA, retained DTRs, contractures in all 4 limbs since early childhood, and a neurogenic ENMG and muscle biopsy with no evidence of myopathy, who had no genetic diagnosis (after a 102 gene neuropathy panel, SMN1 deletion, and hexosaminidase analysis); a characteristic “sandwich” sign on muscle MRI, typical of collagenopathies, was observed (Figure 2).21 Direct sequencing of the COL6A3 gene found a previously unreported likely pathogenic homozygous c.5867A>G variant as well as a previously described heterozygous missense variant (c.7447A>G), which probably acts as a modulator of the disease.22 A sibling, who presents a similar clinical phenotype and a predominant neurogenic ENMG and muscle biopsy, carried the same variants. Their mother was heterozygous for the c.5867A>G variant, and the father was deceased. Discussion We have described the clinical and genetic spectrum of non-5q SMA, whether P-SMA, PD-SMA or SP-SMA, in a large series of patients without the SMN1 deletion. Furthermore, this study provides evidence that the diagnostic yield of neuropathy NGS gene panels in non-5q SMA is still low (almost one-third of patients were diagnosed) but differs across the different clinical presentations. This study first confirms that variants in 3 genes, BICD2, DYNC1H1, and TRPV4, account for almost two-thirds of genetically confirmed non-5q SMA patients. Nevertheless, each gene shows a distinctive clinical presentation, and while both BICD2 and DYNC1H1 patients had an early disease onset and some degree of distal weakness, BICD2 patients had more frequently scapula alata and preserved or brisk DTRs, while DYNC1H1 patients had more frequently orthopedic manifestations and learning difficulties. TRPV4 patients present most frequently with SP-SMA and even though orthopedic manifestations since childhood are frequent, onset of motor weakness occurs in adult life. Variants in 8 additional genes were found and account for a significant genetic heterogeneity in non-5q SMA, with multiple possible pathophysiologic mechanisms. These genes can overlap with other nosological entities such as CMT and dHMN (HSBP1, DNAJB2, MORC2),2,23 Kennedy disease (AR),24 myopathies (VCP, COL6A3), ALS (VCP),25,26 LSDs (ASAH1 and HEXB),27 and other developmental disorders (VRK1, MORC2).28-31 Non-5q SMA is also a clinically heterogenous disorder, with different ages at onset of symptoms, patterns of muscle weakness, and associated signs such as abnormal DTRs or orthopedic manifestations. It is important that these clinical features affect the performance of genetic studies. While an earlier onset and a family history of the disease or consanguinity are independent predictors of a positive genetic diagnosis, contractures and/or arthrogryposis as well as SP-SMA phenotype also seem to increase the likelihood of a definitive genetic diagnosis. A previous genetic-focused study testing the diagnostic yield of a panel of 62 non-5q SMA genes vs a broad 479 neuromuscular gene panel6 reported a diagnostic yield of 13% for the non-5q SMA panel vs a 33% for the broad neuromuscular panel; the latter comparable with the performance of neuropathy gene panels in our study. Therefore, neuropathy gene panels (coupled to in-depth phenotyping) seem an adequate strategy in the genetic diagnosis of non-5q SMA patients. With the increase throughput capability of NGS technologies, comes an increase in the number of VUS, which require an important amount of time and expertise to interpret the variants and avoid misclassification. An example of this is the patient with Sandhoff disease, who was previously found to carry a VUS in the DYNC1H1 gene. In this case, careful analysis of the muscle MRI prompted the search for other alternative diagnosis. This further shows that ancillary testing, and in particular muscle MRI, is crucial to increase the proportion of correctly diagnosed non-5q SMA patients in the advent of NGS techniques. Muscle MRI can not only help discriminate between neuropathy and myopathy32 but also show particular patterns of muscle fat replacement described in DYNC1H1 and BICD2 patients17,19 or characteristic MRI signs, like the “sandwich” sign in collagenopathies.21 Nevertheless, a significant proportion of non-5q SMA patients do not have a definitive genetic diagnosis. Strategies that may increase the number of genetically diagnosed patients can be extracted from the results of this study. First, Kennedy disease should be more frequently searched for, even in the absence of other features such as gynecomastia or sensory involvement, which are not systematic.33 Second, the performance of NGS panels in non-5q SMA could be increased if other neurometabolic disease–associated genes were to be included. This is especially critical because some motor neuron mimics such as SLC52A2 and SLC52A3 genes show a dramatic clinical response to riboflavin34 and that therapeutic trials are ongoing in LSDs such as Sandhoff disease (NCT04221451). Third, the frontier between neuropathies and myopathies is increasingly porous. A well-known example of this is the VCP gene, with varied neurologic presentations (myopathy, neuropathy, or ALS) for most genetic variants.35 Furthermore, almost half of the patients showed moderate elevations of CK levels, a feature which is fairly common in diseases involving motor nerve damage.36 Unexpectedly, 1 patient with a pure SMA (based on repeated ENMG and muscle biopsy findings) but contractures and a muscle MRI compatible with a collagenopathy was found to carry likely pathogenic variants in the COL6A3 gene. Indeed, collagen VI animal models show alterations in myelination and axonal growth,37 and in humans, a previous COL6A3 patient with myopathic and neuropathic changes on ENMG has been described.22 However, this is a report of a patient with pure SMA probably because of mutations in the COL6A3 gene. Finally, WGS, and particularly long-read WGS, may be a more suitable approach than WES in genetically unconfirmed non-5q SMA to search for intronic variants as well as structural variants (SV) because these may account for significant part of the missing heritability in non-5q SMA, as is the case in other neuromuscular diseases.38-40 In Figure 3, the reader can find our recommended diagnostic workflow for an adult patient with a non-5q SMA. Figure 3 Flow Diagram With the Recommended Diagnostic Approach in Non-5q Spinal Muscular Atrophy (SMA) Patients All 3 different sections (genetic, clinical, and ancillary findings) are critical for a correct final diagnosis. Findings in each section should be confronted to findings in the other 2 sections before establishing a definitive diagnosis. Genes underscored show an autosomal dominant (AD) transmission while the rest show an autosomal recessive (AR) transmission, with the exception of the COL6 genes (COL6A1, COL6A2, COL6A3), which may be both AD and AR. The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. CK = creatine kinase; NCS = nerve conduction studies; NGS = next-generation sequencing; SNAP = sensory nerve action potential. In summary, non-5q SMA is a clinically and genetically heterogenous disorder, and neuropathy NGS gene panels achieve a genetic diagnosis in about one-third of patients followed in specialized neuromuscular reference centers, with an earlier onset and a family history of the disease being an independent predictor of a positive genetic diagnosis. Two-thirds of these patients carry pathogenic variants in 3 genes (BICD2, DYNC1H1, and TRPV4); however, non-5q SMA may also be caused by other overlapping genes, whose identification is laborious if not included in the NGS gene panel. Unfortunately, more than half of the patients do not have a precise genetic diagnosis despite gene panels and WES. Thus, there is an unmet need to cluster these rare patients in view of the identification of new common genes through higher throughput NGS techniques. Study Funding The authors report no targeted funding. Disclosure The authors report no relevant disclosures. Go to Neurology.org/NG for full disclosures. Appendix Authors Glossary ALS amyotrophic lateral sclerosis CK creatine kinase CMAP compound motor action potentials CMT Charcot-Marie-Tooth DTR deep tendon reflexes ENMG electroneuromyography HMN hereditary motor neuropathies HSP hereditary spastic paraplegia NGS next-generation sequencing NHNN National Hospital for Neurology and Neurosurgery NIV noninvasive ventilation SMA spinal muscular atrophy SNAP sensory nerve action potentials VUS variant of unknown significance WES whole exome sequencing WGS whole genome sequencing ==== Refs References 1. Pipis M, Rossor AM, Laura M, Reilly MM. Next-generation sequencing in Charcot–Marie–Tooth disease: opportunities and challenges. Nat Rev Neurol. 2019;15 (11 ):644-656.31582811 2. Rossor AM, Kalmar B, Greensmith L, Reilly MM. The distal hereditary motor neuropathies. J Neurol Neurosurg Psychiatry. 2012;83 (1 ):6-14.22028385 3. Beijer D, Baets J. The expanding genetic landscape of hereditary motor neuropathies. 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Sevilla T, Lupo V, Martínez-Rubio D, et al. Mutations in the MORC2 gene cause axonal Charcot-Marie-Tooth disease. Brain. 2016;139 (Pt 1 ):62-72.26497905 24. Spada ARL, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature. 1991;352 (6330 ):77-79.2062380 25. Weihl CC, Pestronk A, Kimonis VE. Valosin-containing protein disease: inclusion body myopathy with Paget's disease of the bone and fronto-temporal dementia. Neuromuscul Disord. 2009;19 (5 ):308.19380227 26. Johnson JO, Mandrioli J, Benatar M, et al. Exome sequencing reveals VCP mutations as a cause of familial ALS. Neuron. 2010;68 (5 ):857-864.21145000 27. Platt FM, d'Azzo A, Davidson BL, Neufeld EF, Tifft CJ. Lysosomal storage diseases. Nat Rev Dis Prim. 2018;4 (1 ):27.30275469 28. Renbaum P, Kellerman E, Jaron R, et al. Spinal muscular atrophy with pontocerebellar hypoplasia is caused by a mutation in the VRK1 gene. 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==== Front Case Rep Oncol Case Rep Oncol CRO CRO Case Reports in Oncology 1662-6575 S. Karger AG Basel, Switzerland 530965 10.1159/000530965 Case Report Complete Response to Immunotherapy in a Patient with MUTYH-Associated Polyposis and Gastric Cancer: A Case Report Complete Response to Immunotherapy in MAP and Gastric Cancer 2696829 Mathias-Machado Maria Cecilia a 2696828 Peixoto Renata D. a 2696830 Ashton-Prolla Patricia b 2696831 Da Silva Leonard Medeiros a c 2696832 Dienstmann Rodrigo a c a Oncolínicas Brasil, São Paulo, Brazil b Department of Genetics of the Federal University of Rio Grande Do Sul, Porto Alegre, Brazil c Oncoclinicas Precision Medicine, São Paulo, Brazil Correspondence to: Maria Cecília Mathias-Machado, maria.mathias@medicos.oncoclinicas.com 10 7 2023 Jan-Dec 2023 10 7 2023 16 1 504510 5 12 2022 2 5 2023 2023 © 2023 The Author(s). Published by S. Karger AG, Basel 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission. Abstract MUTYH-associated polyposis syndrome is an uncommon, autosomal recessive colorectal polyposis syndrome caused by biallelic inactivation of MUTYH. Most patients present with multiple colorectal polyps. However, other primary tumor sites have been described as less frequent. In this report, we describe the case of a young patient with a germline biallelic pathogenic MUTYH mutation with three different primary tumors. We focused on a metastatic gastric adenocarcinoma that presented with complete bowel obstruction secondary to extensive peritoneal carcinomatosis and achieved complete response upon treatment with immunotherapy. The patient’s tumor presented with a high tumor mutational burden and a 100% combined positive score, which certainly contributed to the complete response to immunotherapy. To date, no studies have described the association of MUTYH-related tumors with high PD-L1 expression, but we hypothesized that it may be linked to the increased antigenicity of these cancers. Keywords Gastric cancer MUTYH Immunotherapy Case report No funding was used for this manuscript. ==== Body pmcIntroduction MUTYH-associated polyposis (MAP) is an autosomal recessive colorectal polyposis syndrome caused by biallelic inactivation of MUTYH. The main clinical manifestations of affected patients are colorectal polyposis and colorectal cancer (CRC). In patients with multiple colorectal adenomas and no identifiable APC mutations, biallelic MUTYH inactivation can be found in 7–13% of those with >100 adenomas and 14–40% of those with 10–99 adenomas [1–4]. Usually, MAP patients develop 10–100 colorectal polyps by the fifth or sixth decade [5, 6] and have an increased lifetime risk for CRC (43%–63% at age 60 years, which may reach 80%–90% in the absence of proper surveillance) [7, 8]. Approximately 60% of MAP patients are diagnosed with CRC at presentation [7–13]. In a few MAP patients, CRC may develop in the absence of colorectal polyposis. Germline monoallelic MUTYH alterations are common in the general population, reaching 1%–2% prevalence in northern European, Australian, and US populations. Biallelic alterations are much less common and account for <1% of individuals with the diagnosis of CRC [14, 15] but increases to 2% in young-onset CRC patients and to 10%–25% in CRC patients whose tumors harbor somatic KRAS c.34G>T (G12C) [13]. This association is linked to a distinct mutational signature in MUTYH-associated tumors, with prominent G:C>T:A transversions in NpCpA or NpCpT contexts [16]. Extracolonic manifestations of MAP are less frequently described. However, patients appear to have an increased risk of gastric and duodenal polyps [17, 18]. In one study that included 276 patients with MAP from 181 families, gastric and duodenal polyps were noted in 11 and 17 percent of patients, respectively. Higher risk for gastric cancer (GC) has been observed, although the trend was not significant [17]. In another cohort of 226 carriers of MUTYH germline alterations, increased risk for GC was identified only in monoallelic but not in biallelic mutation carriers [11]. Interestingly, patients with GC exhibiting low MUTYH expression seem to have a poor outcome when compared to those expressing high levels of MUTYH [19]. Furthermore, in a large European multicenter cohort of MAP patients, the incidence of extraintestinal malignancies was almost twice that observed in the general population (standardized incidence ratio 1.9; 95% CI: 1.4–2.5) with an overall lifetime risk of developing cancer of 38%. The estimated lifetime risk of cancers of the duodenum, ovaries, bladder, and skin was significantly higher as compared with the general population (4, 10, 6, and 17%, respectively) [17], and there is some evidence of an increased risk for breast and endometrial cancer. In addition, MAP patients may also present with thyroid nodules and benign adrenal lesions [20, 21], osteomas, congenital hypertrophy of the retinal pigment epithelium, dental cysts, desmoid tumors, sebaceous hyperplasia, and Muir-Torre phenotype with sebaceous gland tumors [22]. Here, we report a case (with the consent of the patient) of a young female patient with germline biallelic pathogenic MUTYH variants and diagnosis of advanced GC, who achieved complete clinical response to anti-PD1 therapy. Case Report Here, we report the case of a 21-year-old female patient with past medical history of depression and hypothyroidism with no family history of cancer. In July 2014, the patients were submitted to total colectomy due to a diagnosis of stage 3 CRC. During adjuvant oxaliplatin-based therapy for 6 months, the patient presented with a pulmonary nodule finding on routine thoracic imaging, which was biopsied, confirming an early-stage primary lung adenocarcinoma. The lung cancer was treated with surgical resection and adjuvant cisplatin and pemetrexed doublet. At that time, the patient declined genetic counseling and germline testing. In October 2020, the patient presented with an intestinal sub-occlusion and was submitted to a segmental enterectomy due to an intestinal intussusception. The pathology report was consistent with an intramucosal adenocarcinoma of a hamartomatous dysplastic lesion. Two months later, the patient presented with yet another intestinal sub-occlusion. She underwent an upper endoscopy with a biopsy finding of a poorly cohesive gastric adenocarcinoma and strong immune infiltration, as shown in Figure 1. She was subsequently submitted to an exploratory laparoscopy with findings consistent with diffuse peritoneal involvement precluding surgical resection. Pathology reported a poorly cohesive, poorly differentiated carcinoma with a preserved expression of mismatch repair proteins on immunohistochemistry, as shown in Figure 2. Immunohistochemistry was performed on Autostainer Link 48 (Dako) using PT-Link Dako for antigenic retrieval and EnVision FLEX for visualization. Antibody (clones) included were MLH1 (ES05), PMS2 (EP51), MSH2 (FE11), MSH6 (EP49). Also, PD-L1 staining (DAKO 22C3 clone) showed combined positive score and tumor proportion score of 100% and 60%, respectively. Due to the intestinal sub-occlusion, the patient remained hospitalized with total parental nutrition due to incapacity of any oral ingestion. Fig. 1. Poorly cohesive adenocarcinoma (yellow arrows) admixed with abundant tumor-infiltrating lymphocytes (red arrows) in subperitoneal connective tissue (scale 50 μm). Fig. 2. Preserved expression of mismatch repair proteins (MLH1, PMS2, MSH2, MSH6) on immunohistochemistry. MHL1 (a); PMS2 (b); MSH2 (c), and MSH6 (d) (scale 50 μm). Given the strong immune infiltration with high PD-L1 expression and considering patient's denial for any cytotoxic chemotherapy, the medical team decided to offer first-line therapy with pembrolizumab. A few weeks after initiating treatment in January 2021, the sub-occlusion was resolved, and parenteral nutrition was discontinued. The patient achieved complete clinical response in January 2023 and remains in complete remission until the present day (more than 1 year and 6 months), as shown in Figure 3. Fig. 3. Comparison of the patients’ imaging studies (computerized topographies) from December 2020 prior to immunotherapy and January 2023 with immunotherapy (created with BioRender.com). To further characterize this spectacular response, the patient’s gastric tumor samples were submitted to next-generation sequencing (NGS) with a broad panel of 180 genes (customized ArcherDX VariantPlex and FusionPlex, Oncoclínicas Precision Medicine) and revealed somatic mutations in KRAS (G12C), BRAF (G464V), PIK3CA (Q546K), SMAD4 (S474*), and MAP2K1 (K57 N). Based on NGS, tumor was classified as microsatellite stable (MSS) but had moderate to high mutational tumor burden (26.5 mutations per Mb). We also referred the patient to genetic counseling, and a germline NGS panel detected biallelic pathogenic MUTYH mutation (Y179C). Discussion The case reported here demonstrates for the first-time major response to immunotherapy in advanced GC of a patient harboring germline biallelic mutation in MUTYH. The MUTYH gene encodes for DNA glycosylase, a key enzyme in DNA base excision repair (BER) [23]. BER is responsible for correcting DNA alterations arising spontaneously from normal metabolic processes, lesions induced by chemical carcinogens, and abasic sites. MUTYH loss of function alterations leads to defective BER and an increased incidence of G:C to T:A transversions throughout the genome. The most commonly described germline loss of function MUTYH variants are Y179C (rs34612342) and G396D (rs36053993), which account for 75% of the reported mutations and are most prevalent in Caucasians [24–26]. MAP increases individual risks not only of CRC but also of extracolonic tumors such as ovarian, duodenal, and bladder cancers, which are usually diagnosed at younger ages as their sporadic counterparts [13]. The association of biallelic loss of function germline MUTYH alterations with GC remains still controversial. Although promising results of immunotherapy in MAP patients with CRC have been published, little is known about the response in those with non-CRC tumors. MUTYH-associated tumors are characterized by a moderate mutator phenotype when compared to proficient mismatch repair CRC, with an average of 5.3 mutations per Mb. Despite being MSS, the higher mutation burden is associated with increased lymphocyte infiltration and frequent loss of HLA I class expression, suggestive of immune-driven selective pressure, resembling cancers with high-level microsatellite instability (MSI-H) [27–29]. High mutation burden and lymphocyte infiltration are known predictive markers for tumor response to immune checkpoint inhibitors, as seen in MSI-H CRC [30, 31]. Therefore, it has been hypothesized that tumors harboring MUTYH gene inactivation could be sensitive to immunotherapy, and 1 case report of patient with CRC harboring two mutations in MUTYH documented pronounced response to nivolumab therapy [29]. The mechanism to explain increased immunogenicity of high tumor mutational burden (TMB) cancers is believed to be related to the accumulation of so-called mutation-associated neoantigens, mutant proteins resulting from nonsynonymous mutations, which are strongly immunogenic. As previously stated, MUTYH-associated CRCs, although MSS, show a distinct mutational signature, with frequent G:C>T transversions and increased TMB combined with prominent lymphocyte infiltration [27, 32] which could render these tumors sensitive to therapy with immune checkpoint inhibitors [33, 34]. Indeed, our patient’s poorly cohesive gastric tumor had moderate to high TMB and abundant lymphocyte infiltration. So far, no studies have described the association of MUTYH-related tumors with high PD-L1 expression, but we hypothesized that it may be linked to the increased antigenicity of these cancers, as in our case. Interestingly, MAP patients with CRC may present a KRAS G12C transversion in up to 60% of the cases [27, 35, 36], which is most likely due to the intrinsic BER defect resulting in an accumulation of DNA transversions throughout the genome but especially in APC and KRAS [37]. Therefore, considering the low prevalence of KRAS G12C mutations in unselected CRC population (<4%) [38], it seems reasonable to consider screening these cases for germline MUTYH alterations. The same may be true for GC, as in our patient’s case, given the extreme rarity of this genomic alteration in general (<1%) [39]. The present case contributes to the description of the MAP-associated phenotype with description of a very young patient diagnosed with three primary tumors. It contributes to the growing evidence for an association of MAP with GC and demonstrates that the impressive response to immunotherapy seen in patients with MUTYH-associated CRCs may also be achieved in other primaries, such as GCs. Due to the rarity of MUTYH cancers and the strong association with KRAS G12C somatic mutations, case reports like the one described here will increase awareness of the medical community on emerging genomic biomarkers of immunotherapy response beyond MSI and high TMB. The CARE Checklist has been completed by the authors for this case report, attached as online supplementary material (for all online suppl. material, see https://doi.org/10.1159/000530965). Statement of Ethics This retrospective review of patient data did not require ethical approval in accordance with local/national guidelines. The patient described in the case report has given consent to publish this case, and written informed consent was obtained from the patient for publication of the details of their medical case and any accompanying images. Conflict of Interest Statement The authors report no conflict of interest. Funding Sources No funding was used for this manuscript. Author Contributions Maria Cecilia Mathias-Machado, Renata D. Peixoto, Patricia Ashton-Prolla, Leonard Medeiros Da Silva, and Rodrigo Dienstmann contributed to conception, research, and manuscript writing. Data Availability Statement The data that support the findings of this study are openly available in PubMed at https://pubmed.ncbi.nlm.nih.gov. Supplementary Material ==== Refs References 1. Jones S , EmmersonP, MaynardJ, BestJM, JordanS, WilliamsGT, . Biallelic germline mutations in MYH predispose to multiple colorectal adenoma and somatic G:C-->T:A mutations. 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==== Front J Hand Surg Glob Online J Hand Surg Glob Online Journal of Hand Surgery Global Online 2589-5141 Elsevier S2589-5141(19)30113-6 10.1016/j.jhsg.2019.09.008 Original Research Effect of Bupivacaine Liposome Injectable Suspension on Sensory Blockade and Analgesia for Dupuytren Contracture Release Vandepitte Catherine F. MD, PhD ∗ Van Boxstael Sam MD ∗ Duerinckx Joris F. MD † Leunen Ine MD ∗ Kuroda Maxine M. PhD, MPH ∗ Mesotten Dieter MD, PhD ∗ Van De Velde Marc MD, PhD ‡ Hadzic Admir MD, PhD admir@nysora.com ∗‡∗ ∗ Department of Anesthesiology, Ziekenhuis Oost-Limburg, Genk, Belgium † Department of Orthopedic Surgery, Ziekenhuis Oost-Limburg, Genk, Belgium ‡ Department of Anesthesiology, University Hospitals Leuven, K.U.L, Leuven, Belgium ∗ Corresponding author: Admir Hadzic, MD, PhD, Department of Anesthesiology, Schiepse Bos 6, Ziekenhuis Oost-Limburg, 3600 Genk, Belgium. admir@nysora.com 1 10 2019 10 2019 1 4 191197 16 4 2019 6 9 2019 © 2019 THE AUTHORS. Published by Elsevier Inc. on behalf of The American Society for Surgery of the Hand. 2019 https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Purpose To study the efficacy of bupivacaine liposome injectable suspension in prolonging sensory blocks of the median and ulnar nerves for subjects with Dupuytren contracture release by collagenase injection. We hypothesized that combining liposome bupivacaine and bupivacaine hydrochloride would extend the duration of blocks without added complications. Methods We randomized 32 subjects scheduled for Dupuytren contracture release with collagenase Clostridium histolyticum injections to receive forearm blocks of the median and ulnar nerves with a mixture of 5 mL liposome bupivacaine 1.33% plus 2.5 mL bupivacaine hydrochloride 0.5% per nerve (n = 16) or 7.5 mL bupivacaine hydrochloride 0.5% alone per nerve (n = 16). Sensory block and analgesia were assessed through the first posttreatment week. Results Sensory block was nearly 4 times longer in subjects who received the liposome bupivacaine mixture compared with subjects who received bupivacaine hydrochloride alone. Most subjects (13 of 16) who received the liposome bupivacaine mixture had adequate analgesia for finger manipulation to rupture the cords, whereas most subjects (15 of 16) who received bupivacaine hydrochloride alone required additional anesthesia. Subjects in the liposome mixture group reported lower pain scores through the first 3 days after treatment. There were no serious side effects. Conclusions Addition of liposome bupivacaine to forearm blocks for Dupuytren contracture release prolonged sensory block and improved pain scores without increasing side effects or impairing hand function. Supplemental lidocaine injections for the painful phases of Dupuytren contracture release with collagenase C histolyticum injections were not required by most subjects who received liposome bupivacaine. Type of study/level of evidence Therapeutic I. Key words bupivacaine liposome injectable suspension collagenase Clostridium histolyticum Dupuytren contracture release forearm block ==== Body pmcDupuytren contracture is a common disease of the connective tissue of the hand in which the formation of subcutaneous nodules and cords causes disabling flexion contracture of one or more fingers.1,2 The cords of these contractures can be enzymatically weakened after injection of collagenase Clostridium histolyticum (CCH), allowing the fingers to be manipulated to break up the cords.3 Pain is caused by multiple injections of collagenase into the cords (phase 1 of treatment), during manipulation of the fingers to break up the cords (phase 2, 24 to 72 hours after injection), and from the inflammatory response, which can last several days.4 Ideal analgesia would provide adequate pain relief for all phases of treatment (Fig. 1). Although forearm (median and ulnar) nerve blocks can provide adequate analgesia for phase 1 of treatment, they have a relatively short duration.5,6Figure 1 Phases of treatment in Dupuytren contracture release with CCH injections. A recent study4 reported that 43% of patients had severe pain during finger manipulation even when analgesia was provided with a wrist block (10 mL of 2% mepivacaine); 53% of patients had pain after injection of collagenase despite treatment with an oral analgesic combination of acetaminophen 650 mg, ibuprofen 600 mg, or metamizole 575 mg every 8 hours. Bupivacaine liposome injectable suspension (EXPAREL, Pacira Pharmaceuticals, Inc, Parsippany, NJ) is an extended-release formulation of a local anesthetic that has been approved by the Food and Drug Administration for infiltration into the surgical wound, and recently (April, 2018) for use in interscalene brachial plexus blocks. Although the efficacy of liposome bupivacaine for postoperative pain has been studied with respect to soft tissue infiltration,7 it has not been examined in the median and ulnar nerve blocks of the forearm. Thus, although local blocks can be used for CCH treatment of Dupuytren contracture, we chose forearm nerve blocks to study the effects of liposome bupivacaine in the small peripheral nerves. The objective of this study was to evaluate the analgesic benefit of liposome bupivacaine in prolonging forearm blocks of the median and ulnar nerves and analgesia for subjects with Dupuytren contracture release with injections of CCH into the affected cords. We primarily tested the hypothesis that adding liposome bupivacaine in forearm blocks would sufficiently prolong the sensory block to provide adequate pain relief for Dupuytren contracture release with CCH. We secondarily tested the hypothesis that adding liposome bupivacaine in forearm blocks would improve pain scores in the first posttreatment week. Materials and Methods This double-blind, randomized, controlled trial was approved by the Ethics Committee of Ziekenhuis Oost-Limburg, Genk, Belgium (16/011), and by FAGG, the Belgian federal agency for medicines and health products (2016-001656-22). The trial was registered at www.clinicaltrials.gov (NCT03106519) in March, 2017. Patients scheduled for Dupuytren contracture release who were aged 18 to 85 years, had an American Society of Anesthesiologists physical status of I to III, and were able to understand the purpose and risks of the study were eligible to participate. Patients were excluded if they were pregnant, had a history of allergic or adverse reaction to local anesthetics, used pain medications within 28 hours before treatment, had a suspected or known recent history (less than 3 months) of drug or alcohol abuse, had an infection at the planned block site, had a body mass index greater than 44 kg/m2, or had any chronic condition or psychiatric disorder that could compromise neurological or study assessments. Consenting subjects were randomized in a computer-generated 1:1 ratio to receive a mixture of 5 mL bupivacaine liposome injectable suspension 1.33% plus 2.5 mL bupivacaine HCl 0.5% or 7.5 mL bupivacaine HCl 0.5% alone to the median and ulnar nerves before CCH injection (Xiapex, Swedish Orphan Biovitrum, AB, Stockholm, Sweden). Each subject received a total drug volume of 15 mL (7.5 mL/nerve). The traditional bupivacaine HCl used in common clinical practice was mixed with the extended-release formulation of liposome bupivacaine (EXPAREL). This model has been used to facilitate early interscalene brachial plexus block onset and prolong block duration. After treatment, all subjects received the standardized multimodal regimen for pain control with acetaminophen 1 g every 6 hours and diclofenac 75 mg 2 times daily. Transmucosal tramadol 50 mg (every 6 hours as needed) was used for breakthrough pain. In addition, subjects received rescue medication upon request for breakthrough pain or as necessary upon discharge home. Anesthesiology staff members performing the nerve blocks were not blinded to drug treatment but did not participate in subject assessments. Surgeons and research personnel were blinded to drug treatment because blocks were performed in a separate procedure room outside the operating theater. Subjects were not told their allocated treatment. A strict blind was maintained so that the surgeon (who injected CCH and then manipulated the fingers) and the research personnel (who conducted the follow-up assessments of the subjects) would not know the assigned drug arms. The use of supplementary local anesthesia was based on subject request for pain relief and not the surgeon’s perception of need. Subjects left the hospital facility on the same day of the CCH injection. Nerve blocks Study medication (bupivacaine liposome injectable suspension mixed with bupivacaine HCl or bupivacaine HCl alone) was administered at least 30 minutes before CCH injection and was deposited in the tissue plane around the median and ulnar nerves at the level of the mid-forearm. All blocks were administered under ultrasound guidance. The injectate was deemed adequately distributed when it encircled the nerve, as documented by ultrasound. The forearm blocks were the sole anesthetic modality for the CCH injections and were administered without premedication. We chose to use a more proximal approach to median and ulnar nerve blocks at the level of the forearm because a more distal approach (wrist blocks) failed to provide analgesia during finger manipulation in 43% of patients in a study by Sanjuan-Cerveró et al.4 Time to onset and offset of sensory blockade was tested in 5-minute intervals from injection of the study medication up to 30 minutes, and hourly thereafter until discharge. Sensory block onset was defined as time from end of each block procedure to no sensation in the median and ulnar nerve distributions. Sensory block duration was defined as time from block onset to time to return of complete sensation. Block success or failure was defined as absence or presence of full sensation in the areas and muscles supplied by the median and ulnar nerves as assessed by pinprick.8 Inadequate analgesia for phase 2 (finger manipulation to break up the cords) was defined as the need for additional injections of lidocaine 1% into the affected tissues of the hand to allow for effective manipulation and cord breakage. Vital signs (blood pressure, heart rate, and oxygen saturation) were monitored every 3 minutes for the first 30 minutes after the block, every 5 minutes up to 1 hour after the block, and every 15 minutes until discharge. We obtained 12-lead electrocardiograms before block and approximately 2 hours after nerve blocks. Posttreatment assessments Worst pain (modified Brief Pain Inventory) was reported as a numeric rating scale (NRS) score ranging from 0 (no pain) to 10 (most extreme pain).9 The NRS was recorded before and after block. Before discharge home, subjects were given clear instructions regarding scheduling and questions that would be asked during the posttreatment telephone interviews. Specifically, subjects were trained to assess sensation and distinguish among complete sensation, light touch, and no sensation. They were also trained to assess the presence or absence of weakness in the blocked hand. Subjects were given a daily diary in which to record the NRS score before taking each dose of rescue medication (tramadol). The blinded research staff used standardized scripts to collect pain scores via phone interviews at 24 hours (D1 am), 36 hours (D1 pm), 48 hours (D2 am), 60 hours (D2 pm), 72 hours (D3 am), and 84 hours (D3 pm), and at D4, D5, D6, and D7. The worst pain was asked as question 1 from the modified Brief Pain Inventory: “Please rate your pain at its worst in the last 24 hours from 0 (no pain) to 10 (pain as bad as you can imagine).” This item, based on the NRS, was already familiar to subjects because it was asked during screening and in phase 1 of the study. The phone interview inquired about numbness and weakness or dysesthesias. Polystyrene foam cups were given to subjects to take home upon discharge; subjects were instructed that use of these cups referred to use with the operated hand. The functionality of the hand was assessed by asking subjects, “Are you able to use a polystyrene foam cup?” Training in motor assessments using the polystyrene foam cup was done before the research intervention and repeated before discharge home. In addition, we assessed the presence or absence of the sensory and motor block by asking the patient to report numbness and/or weakness in the operated hand every 12 hours. At the 48-hour visit, the presence or absence of sensory block and hand weakness of the intrinsic muscles were evaluated, as well as the ability to use the polystyrene foam cup. Subjects returned for finger manipulation and cord rupture 48 hours after the CCH injections. Lidocaine 1% was injected if the subject reported pain during manipulation of the fingers. Side effects were recorded through day 7, including nausea, vomiting, fever, constipation, severe itching of the skin, dizziness, sleepless nights, excessive sweating, urinary retention, headache, and heart palpitations. Statistical analysis The sample size calculation, estimated on duration of sensory block, assuming a minimum difference important to detect at 48 hours (SD, 32 hours), α = .01, and power of .90, yielded 15 subjects/group. This was increased to 16 subjects/group to accommodate block failures and losses to follow-up. If bupivacaine liposome injectable suspension is capable of prolonging nerve block, its pharmacokinetic data suggest that the formulation should have a conduction block at least 48 hours longer than non-encapsulated bupivacaine HCl, the active component of liposome bupivacaine.10 We selected the SD of 32 hours because there is substantial variability in duration of nerve blocks with the currently available local anesthetics. Because liposome bupivacaine is an encapsulated formulation that releases an active substance, over 72 hours, an even wider variability in block duration might be anticipated. Continuous variables are presented as means (SD) and categorical (nominal and ordinal) variables as n (%) or as a ratio as in gender and treated limb laterality. Duration of sensory and motor block was compared between groups by Student t test or Mann-Whitney U test, as appropriate. The proportions of subjects requiring additional anesthesia for phase 2 of treatment were compared between groups by chi-square test. Efficacy analyses followed intent-to-treat principles. Worst pain was analyzed by generalized estimating equations (GEE) to examine group differences over time. The GEE method is flexible with respect to the type of outcome variable (including possibly skewed continuous distributions and ordinal measures) and to observations that are unequally spaced over time. For instance, worst pain is reported at discharge (D0 pm), at D1 am and pm, D2 am and pm, and D3 am and pm, and then on D4, D5, D6, and D7. In the GEE analyses, a conservative unstructured correlation structure was used so as not to assume the relative magnitude of correlation between any 2 pairs of observations (although GEE is robust against choosing an incorrect correlation structure). Link function was identity for these continuous outcome measures. Tests of differences between groups for reported side effects were not planned, because the number of each side effect was anticipated to be small, and subjects could report more than one side effect. Instead, the relative risk (with 95% confidence interval) for at least one side effect through day 7 was reported. P < .05 was deemed statistically different. Results We assessed 55 patients for eligibility; 32 were randomly assigned (16/group) (Fig. 2). The groups did not differ in sociodemographic characteristics or severity of disease (Table 1).Figure 2 Consolidated Standards of Reporting Trials diagram for 2-arm study of bupivacaine liposome injectable suspension mixed with bupivacaine HCl or bupivacaine HCl alone. Table 1 Sociodemographic Characteristics and Clinical Features of 32 Patients Undergoing Forearm Blocks of Median and Ulnar Nerves for Dupuytren Contracture Release Demographics, pain, and arm functionality before surgery Mixture of 5 mL Liposome Bupivacaine 1.3% Plus 2.5 mL Standard Bupivacaine 0.5% (per Median and Ulnar Nerve) (n =16) 7.5 mL Standard Bupivacaine 0.5% per Median and Ulnar Nerve) (n = 16) Gender (M : F) 14 : 2 14 : 2 Age, y (range) 66.2 (48–76) 63.5 (35–82) Body mass index, kg/m2∗ 25.7 (0.05) 26.5 (0.04) Race (%)  American Indian/Alaska native 0 0  Asian 0 0  Black/African American 0 0  Native Hawaiian/Pacific Islander 0 0  White 16 (100) 16 (100)  Other 0 0 American Society of Anesthesiologists physical status (%)  I 4 (25) 7 (44)  II 10 (63) 8 (50)  III 2 (12) 1 (6) Surgical side (R : L)† 3 : 13 8 : 8 Functionality of surgical hand (before surgery) (%)  Unable to use 0 0  Only light activity 0 0  Able to do some activities 0 0  Able to do most activities 0 1 (6)  Slight restrictions only 8 (50) 6 (38)  Normal 8 (50) 9 (56) NRS (0–10)  At rest 0.6 (2.0) 0.2 (1.0)  During movement 1.5 (2.4) 0.4 (0.9) Data are shown as means (SD) or mean (range) for continuous variables and n (%), ratio, or median (range) for discrete (nominal, ordinal) variables. ∗ Body mass index was missing for one subject (active comparator group). † Pearson chi-square (2-sided) P = .063; Fisher exact (2-sided) P = .14. Duration of sensory block was significantly longer in the liposome bupivacaine mixture group compared with the bupivacaine HCl–alone group (3.8 [1.1] vs 1.0 [0.3] days, respectively; P < .001). Additional anesthetic intervention to complete phase 2 of treatment was required by 15 of 16 subjects (94%) who received bupivacaine HCl alone, compared with 3 of the 16 subjects (20%) who received the liposome bupivacaine mixture (P < .001). Hence, no additional anesthesia was required in 80% of subjects who received the liposome bupivacaine mixture. Previous pharmacokinetic analyses in femoral nerve block demonstrated that the peak concentration of liposome bupivacaine occurs during the first 72-h interval.10,11 In the current study, subject-reported worst pain (modified Brief Pain Inventory question 1) over the first 3 posttreatment days was analyzed. Generalized estimating equations (GEE) showed that pain over this interval was significantly lower in subjects who received the liposome bupivacaine mixture compared with those who received bupivacaine HCl alone (GEE P = .010) (Fig. 3).Figure 3 Mean worst (NRS) pain scores with 95% confidence interval from discharge after phase 2 through first posttreatment week in 32 subjects undergoing forearm blocks for Dupuytren contracture release (GEE P = .01 over the first 3 days). All subjects underwent a multimodal oral pain regimen throughout the study period. Three subjects took tramadol through day 7 for breakthrough pain: 2 in the liposome bupivacaine mixture group (total dose of 400 and 150 mg, respectively) and one in the bupivacaine HCl–alone group (100 mg). Among subjects who received bupivacaine HCl alone, the proportion who reported numbness decreased rapidly within the first 48 hours. In contrast, numbness appeared to persist through days 3 and 4 in at least 7 subjects (greater than 40%) who received the liposome bupivacaine mixture (GEE P = .008) (Fig. 4). Among subjects who received bupivacaine HCl alone, no weakness was reported within 24 hours after injection. In contrast, subjects who received the liposome bupivacaine mixture experienced weakness, which subsided by the end of the fourth treatment day (GEE P = .03). Assessment of subjects’ sensorimotor block and ability to adduct and abduct the fingers (intrinsic hand muscles) by the research staff just before finger manipulation at 48 hours revealed that no subject who received bupivacaine HCl alone had residual sensory or motor block. In contrast, all subjects who received liposome bupivacaine had some degree of both sensory and motor block and weakness in adduction-abduction of the fingers (P < .001). This indicated that the duration of the blockade with liposome bupivacaine mixture exceeded that of bupivacaine HCl alone. Prolonged numbness and subjectively reported weakness did not appear to affect hand function, as assessed by subjects’ ability to use a polystyrene foam cup throughout the first posttreatment week.Figure 4 Sensory block (numbness) from discharge after phase 2 through first posttreatment week in 32 subjects undergoing forearm blocks for Dupuytren contracture release. Among subjects who received bupivacaine HCl alone, the percentage who reported numbness decreased rapidly within the first 48 hours; in contrast, numbness appeared to persist through days 3 and 4 in at least 40% of subjects who received the liposome bupivacaine mixture (GEE P = .008). Skin tears frequently occur during finger manipulation for Dupuytren contracture release. In this study, almost all subjects had skin tears that were successfully treated by bandaging the hand after the cords were released. No subject reported symptoms consistent with local anesthetic systemic toxicity, including bradycardia, hypotension, arrhythmia, and seizure. There were no differences in occurrence of reported side effects between treatment arms (relative risk for at least one side effect = 1.33; 95% confidence interval, 0.35–5.03) (Table 2).Table 2 Frequency of Side Effects Among 32 subjects Undergoing Forearm Blocks of Median and Ulnar Nerves for Dupuytren Contracture Release Side Effect Mixture of 5 mL Liposome Bupivacaine 1.3% Plus 2.5 mL Bupivacaine HCl 0.5% (per Median and Ulnar Nerve) (n = 16) 7.5 mL Bupivacaine HCl 0.5% per Median and Ulnar Nerve) (n = 16) None 12 (75) 13 (81) Unique subjects with side effects, n 4 (25) 3 (19) Total side effects, n∗ 6 (38) 5 (31) Specific side effect∗  Bleeding wound 1 (6) 0  Dizziness 0 1 (6)  Headache 2 (12) 1 (6)  Itching of skin 1 (6) 1 (6)  Nausea 0 1 (6)  Sleepless night 2† (12) 1 (6) Data are shown as n (%). Percentages of specific side effects are based on n = 16 in each group. Relative risk for at least one side effect = 1.33 (95% confidence interval, 0.35–5.03). ∗ Percentages for total number of side effects and specific side effects do not sum to 100% because some subjects reported more than one side effect. † Sleepless nights in the same subject on 2 separate dates. Discussion The addition of liposome bupivacaine 1.33% to bupivacaine HCl 0.5% in median and ulnar nerve blocks prolonged the duration of sensory block and analgesia compared with bupivacaine HCl alone in subjects with Dupuytren contracture release with injections of CCH. Injections of collagenase resulted in inflammatory response, ecchymosis, and swelling of the treated hand in all research subjects, requiring adequate analgesia. As opposed to subjects treated with the liposome bupivacaine mixture, nearly all in the active comparator group required lidocaine injections for additional analgesia. Overall, pain intensity was relatively low in both groups, probably because all subjects received multimodal analgesia. Regardless, worst pain in the first 72 hours was lower among subjects who received the liposome bupivacaine mixture than in those who received bupivacaine HCl alone. This extended analgesic effect likely resulted from the prolongation of sensory blockade by sustained release of free bupivacaine released from liposomes and was longer by approximately threefold compared with bupivacaine HCl alone (3.8 vs 1.1 days, respectively; P < .001). The prolonged sensory block did not prevent any subject from using a polystyrene foam cup. Liposome bupivacaine is a novel formulation of bupivacaine HCl, and no dosing recommendations or dose–response studies in the distal peripheral nerves of the upper extremity were available from the literature. Nonetheless, limited dosing information is available from several studies in which the drug was used for the brachial plexus block and the larger nerve blocks. For instance, injection of 133 mg (10 mL) of liposome bupivacaine resulted in successful femoral block.12 Moreover, 5 mL of 0.25% bupivacaine HCl immediately followed by 10 mL of liposome bupivacaine 133 mg in the interscalene brachial plexus block prolonged sensory block and analgesia with the same volume (15 mL) of bupivacaine 0.25%.6 Because the median and ulnar nerve surface areas to be blocked in the current study are 30% to 50% that of the femoral nerve, we empirically chose to mix 5 mL of liposome bupivacaine and 2.5 mL of bupivacaine HCl 0.5%.13,14 We relied on the relative difference in anatomical size between the femoral nerve and the smaller peripheral nerves to approximate the dosing for the current study. Although this may not be an ideal dose or mixture, there was no guidance in the literature suggesting a different dose. Bupivacaine HCl 0.5% was added to liposome bupivacaine to speed the onset of anesthesia for the CCH injection procedure because the liposome bupivacaine suspension contains only 3% of free drug available for immediate blockade, which would not be adequate to result in fast onset of the blockade. The liposome bupivacaine group received a larger total dose of bupivacaine: 79 mg of bupivacaine HCl (a combination of 66.5 mg bupivacaine HCl in liposome bupivacaine 5 mL plus 12.5 mg bupivacaine HCl) compared with the bupivacaine HCl–alone group (37.5 mg bupivacaine HCl). Nonetheless, the actual dose of free bupivacaine available for nerve blockade after injection was larger in the bupivacaine HCl–alone group than in the liposome bupivacaine group (37.5 versus 14.5 mg, respectively). The liposome bupivacaine suspension contains only 3% of free bupivacaine (66.5 mg × 3% ∼ 2 mg + 12.5 mg = 14.5 mg), and pharmacokinetic studies showed that free bupivacaine is gradually released from the liposomes over 72 hours or more, which is distinctly different from an injection of bupivacaine HCl alone, in which the entire dose (7.5 mL bupivacaine 0.5% = 37.5 mg) is immediately available for nerve blockade. The duration of blockade by free bupivacaine from the liposome bupivacaine suspension is limited because only a small amount of free drug is quickly absorbed. Hence, any duration of analgesia beyond 36 hours is likely caused by the extended release of free bupivacaine from liposome bupivacaine, rather than a function of the larger total mass of bupivacaine HCl in the liposome bupivacaine group.11,15,16 A limitation of this study is that surgical dressings made motor assessments difficult. Nonetheless, analgesic benefit, rather than hand function, was the primary purpose of the study. We therefore opted to ask the subjects whether they could use a polystyrene foam cup, and about their perception of numbness or weakness in the surgical hand. Indirect assessment over the phone of their ability to hold a polystyrene foam cup and their perception of numbness and/or weakness of the surgical hand suggested that any motor block was minor or nonexistent. This could be because liposome bupivacaine releases small amounts of bupivacaine HCl over 72 hours after injection, which may be adequate for autonomic and sensory blockade but not for motor blockade. The study did not include plasma pharmacokinetic determination; however, no subjects had signs or symptoms of systemic toxicity or electrocardiogram abnormality suggestive of local anesthetic systemic toxicity. Another limitation of the study is that a comparison with local infiltration was not performed, although it is a common practice among hand surgeons. Nerve block and infiltration models require different methods of drug administration. We believe that a more direct comparison of the efficacy of liposome bupivacaine versus bupivacaine HCl was examined by using the nerve block model alone. Nonetheless, because local infiltration is a common practice among hand surgeons, future studies that include an infiltration arm of the trial should be conducted. The addition of liposome bupivacaine prolonged sensory block for both phases of Dupuytren contracture release and improved pain scores in the first posttreatment week. Future studies of the efficacy of liposome bupivacaine in more extensive and reconstructive hand surgeries are indicated. Studies should also be conducted to determine the dose–response and best anatomical sites of application of liposome bupivacaine that optimize nerve exposure to small amounts of active drug released in a sustained fashion from the liposomes, detailed examination of the hand function after forearm blocks with liposome bupivacaine, and cost-efficacy of the treatments. Acknowledgments This study was supported by a research grant from 10.13039/100013738 Pacira Pharmaceuticals , Inc (Parsippany, NJ). Pacira Pharmaceuticals was not involved in study design; in data collection, analysis, or interpretation; in writing of this report; or in the decision to submit this article for publication. The authors would like to acknowledge the orthopedic surgical and nursing team at Ziekenhuis Oost-Limburg who facilitated patient care (Joëlle Caretta, RN, Sonja Moonen, RN, Birgit Lohmar, RN, and Ine Vanweert, RN) and the research team at Ziekenhuis Oost-Limburg who collected the subject assessments and followed the subjects by phone after discharge (Ingrid Meex, PhD, Marijke Cipers, RN, and Gülhan Özyürek, MSc). Declaration of interests: Admir Hadzic serves a consultant for Pacira Pharmaceuticals and has received consulting honoraria and educational and research grants. No benefits in any form have been received or will be received by the other authors related directly or indirectly to the subject of this article. ==== Refs References 1 DiBenedetti D.B. Nguyen D. Zografos L. Ziemiecki R. Zhou X. Prevalence, incidence, and treatments of Dupuytren’s disease in the United States: results from a population-based study Hand (N Y) 6 2 2011 149 158 21776198 2 Hindocha S. McGrouther D.A. Bayat A. Epidemiological evaluation of Dupuytren’s disease incidence and prevalence rates in relation to etiology Hand (N Y) 4 3 2009 256 269 19145463 3 Desai S.S. Hentz V.R. The treatment of Dupuytren disease J Hand Surg Am 36 5 2011 936 942 21527148 4 Sanjuan-Cerveró R. Carrera-Hueso F.J. Vazquez-Ferreiro P. Pain associated with treatment of Dupuytren contracture with collagenase Clostridium histolyticum J Hand Surg Am 42 2 2017 e104 e114 5 Dasta J. Ramamoorthy S. Patou G. Sinatra R. Bupivacaine liposome injectable suspension compared with bupivacaine HCl for the reduction of opioid burden in the postsurgical setting Curr Med Res Opin 28 10 2012 1609 1615 22900785 6 Vandepitte C. Kuroda M. Witvrouw R. Addition of liposome bupivacaine to bupivacaine HCl versus bupivacaine HCl alone for interscalene brachial plexus block in patients having major shoulder surgery Reg Anesth Pain Med 42 3 2017 334 341 28157791 7 Hamilton T.W. Athanassoglou V. Mellon S. Infiltration at the surgical site for the management of postoperative pain Cochrane Database Syst Rev 2 2017 CD011419 28146271 8 Bigley G.K. Sensation Walker H.K. Hall W.D. Hurst J.W. Clinical Methods: The History, Physical, and Laboratory Examinations 3rd ed. 1990 Butterworths Boston, MA 9 Atkinson T.M. Mendoza T.R. Sit L. The Brief Pain Inventory and its “pain at its worst in the last 24 hours” item: clinical trial endpoint considerations Pain Med 11 3 2010 337 346 20030743 10 Ilfeld B.M. Viscusi E.R. Hadzic A. Safety and side effect profile of liposome bupivacaine (Exparel) in peripheral nerve blocks Reg Anesth Pain Med 40 5 2015 572 582 26204387 11 Butterworth J. IV Clinical pharmacology of local anesthetics Hadzic’s Textbook of Regional Anesthesia and Acute Pain Management 2nd ed. 2017 McGraw-Hill New York, NY 12 Hadzic A. Minkowitz H.S. Melson T.I. Liposome bupivacaine femoral nerve block for postsurgical analgesia after total knee arthroplasty Anesthesiology 124 6 2016 1372 1383 27035853 13 Eichenberger U. Stöckli S. Marhofer P. Minimal local anesthetic volume for peripheral nerve block Reg Anesth Pain Med 34 3 2009 242 246 19587623 14 Keplinger M. Marhofer P. Marhofer D. Effective local anaesthetic volumes for sciatic nerve blockade: a clinical evaluation of the ED99 Anaesthesia 70 5 2015 585 590 25644578 15 Hadzic A. Abikhaled J. Harmon W. Impact of volume expansion on the efficacy and pharmacokinetics of liposome bupivacaine Local Reg Anesth 8 2015 105 111 26673040 16 Hu D. Onel E. Singla N. Kramer W.G. Hadzic A. Pharmacokinetic profile of liposome bupivacaine injection following a single administration at the surgical site Clin Drug Investig 33 2 2013 109 115

PMC010xxxxxx/PMC10352963.txt

==== Front RSC Adv RSC Adv RA RSCACL RSC Advances 2046-2069 The Royal Society of Chemistry d3ra01533e 10.1039/d3ra01533e Chemistry The key role of pretreatment for the one-step and multi-step conversions of European lignocellulosic materials into furan compounds Kammoun Maroua a https://orcid.org/0000-0002-3184-7656 Margellou Antigoni b Toteva Vesislava B. c Aladjadjiyan Anna d https://orcid.org/0000-0003-3044-3016 C-9080-2014 Sousa Andreai F. ef Luis Santiago V. g https://orcid.org/0000-0001-6867-6240 Garcia-Verdugo Eduardo g https://orcid.org/0000-0001-8658-8500 Triantafyllidis Konstantinos S. b https://orcid.org/0000-0002-4352-6542 Richel Aurore a a Laboratory of Biomass and Green Technologies, University of Liege Belgium a.richel@uliege.be b Department of Chemistry, Aristotle University of Thessaloniki 54124 Thessaloniki Greece c Department of Textile, Leather and Fuels, University of Chemical Technology and Metallurgy Sofia Bulgaria d National Biomass Association Plovdiv Bulgaria e CICECO—Aveiro Institute of Materials, Department of Chemistry, University of Aveiro 3810-193 Aveiro Portugal f Centre for Mechanical Engineering, Materials and Processes, Department of Chemical Engineering, University of Coimbra Rua Sílvio Lima—Polo II 3030-790 Coimbra Portugal g Dpt. of Inorganic and Organic Chemistry, Supramolecular and Sustainable Chemistry Group, University Jaume I Avda Sos Baynat s/n E-12071-Castellon Spain 18 7 2023 12 7 2023 18 7 2023 13 31 2139521420 8 3 2023 4 7 2023 This journal is © The Royal Society of Chemistry 2023 The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/ Nowadays, an increased interest from the chemical industry towards the furanic compounds production, renewable molecules alternatives to fossil molecules, which can be transformed into a wide range of chemicals and biopolymers. These molecules are produced following hexose and pentose dehydration. In this context, lignocellulosic biomass, owing to its richness in carbohydrates, notably cellulose and hemicellulose, can be the starting material for monosaccharide supply to be converted into bio-based products. Nevertheless, processing biomass is essential to overcome the recalcitrance of biomass, cellulose crystallinity, and lignin crosslinked structure. The previous reports describe only the furanic compound production from monosaccharides, without considering the starting raw material from which they would be extracted, and without paying attention to raw material pretreatment for the furan production pathway, nor the mass balance of the whole process. Taking account of these shortcomings, this review focuses, firstly, on the conversion potential of different European abundant lignocellulosic matrices into 5-hydroxymethyl furfural and 2-furfural based on their chemical composition. The second line of discussion is focused on the many technological approaches reported so far for the conversion of feedstocks into furan intermediates for polymer technology but highlighting those adopting the minimum possible steps and with the lowest possible environmental impact. The focus of this review is to providing an updated discussion of the important issues relevant to bringing chemically furan derivatives into a market context within a green European context. Currently, an awareness of the chemical industry towards the furanic compounds production, renewable molecules alternatives to fossil molecules, which can be transformed into a wide range of chemicals and biopolymers. European Cooperation in Science and Technology 10.13039/501100000921 FUR4SUSTAIN CA18220 Fundação para a Ciência e a Tecnologia 10.13039/501100009091 LA/P/0006/2020 CEECIND/02322/2020 UID/EMS/00285/2020 Centro de Investigação em Materiais Cerâmicos e Compósitos 10.13039/100007889 UIDB/50011/2020 UIDP/50011/2020 Federación Española de Enfermedades Raras 10.13039/100007889 Unassigned pubstatusPaginated Article ==== Body pmc1. Introduction The transition from petroleum-based refineries to novel biorefineries is a key to maintaining sustainable global development and preserving the future of the planet. To adapt this context in an action plan, natural resources other than petrol should be involved as input to generate different value-added products, such as chemicals, materials, and fuels. Featured by guaranteed supply, large availability, constant production, and reasonable cost, renewable sources constitute a key element in facing challenges reduce and fossil resource use. Vegetal biomass implication represents an advanced conception of the emerged existing model of food and paper biorefineries while producing a wide range of different platform molecules fulfilling the different societal requirements. Moreover, renewable biomass introduction has a beneficial impact on the balance of complex trade-offs among the socioeconomic and environmental points of view by promoting value-added product generation, creating new opportunities for employment, and reducing fossil resource use.1 The furan compound-driven biorefinery approach, based on lignocellulosic biomass as a renewable resource, has lately evolved. The carbohydrate part of LCB (free sugars such as hexoses or pentoses, or from oligo- or polysaccharides) can be derived into versatile furan-based molecules, such as 5-HMF (5-hydroxymethylfurfural) or 2-F (2-furfural), which are promising bridges between biomass and a panel of possible chemicals and biopolymers.2 In fact, 5-HMF and 2-F were identified as platform molecules since the early 2000s and both were obtained by the selective dehydration of C5 or C6 monosaccharides (including representative C5 sugars, such as d-xylose or C6 entities, such as d-glucose, d-fructose, or d-mannose).3 Macromolecules in LCB have different susceptibilities to pretreatment, and the most common situation is hemicellulose removal as it consists of short side chains, and has a lower degree of polymerization, and inferior crystallinity compared to cellulose. During hydrothermal pretreatment, hemicellulose is easily extracted into oligo or polysaccharides or monomers as free sugars. In contrast, a low yield of cellulose is extracted into cellobiose and glucose.4–6 The acid medium attacks hemicellulose and promotes its removal, and during LHW, autohydrolysis is induced through hydronium catalyzed reactions, leading to hemicellulose extraction.7,8 Ionic liquids (IL) can dissolve both hemicellulose and cellulose by dint of the polarity of N–O bonds, which breaks cellulose hydrogen bond, forming new hydrogen bonds with the solute.9,10 Cellulose has low solubility in deep eutectic solvents (DESs) compared to lignin and hemicellulose. DESs especially remove lignin and hemicellulose, due to their intrinsic ionic nature, which breaks the supramolecular H-bonds stabilizing the hemicellulose–lignin complex, allowing easily the breaking of the covalent bonds and leaving a cellulose rich-solid.11,12 Supercritical fluids reduce cellulose crystallinity, promote its decomposition into α- and β-glucosides and deal with direct carbohydrate hydrolysis.13 The recovered sugars from cellulose and hemicellulose can be directly dehydrated to furanic compounds or undergo some added hydrolysis to be transformed. Their conversion depends on pretreatment parameters and their structural changes. Many technical papers have been published on the only production routes involving “simple” monosaccharides such as xylose,14 glucose,15 and fructose.16 However, this review pays excessive attention to the whole biorefinery schema starting from raw materials from which carbohydrates would be extracted and the mass balance on the whole value chain. It is an unquestionable fact that from an industrial perspective, the issue of exploiting lignocellulosic resources, which are more strategic as sources of (C6 and C5) carbohydrates that can be converted into furan-based compounds of interest for the polymer materials sector. Therefore, within a green European context, the present review intends to put forward two particular lines of thought. On the one hand, the choice of the most suitable lignocellulosic biomasses as the raw material to be converted into furan intermediates. Thirty-three European lignocellulosic biomasses emerging from agricultural residues, agri-food byproducts, energy crops, forest matrices, plant grasses, and non-food sectors are discussed. On the other hand, this review describes in detail all the technological approaches allowing us to convert these matrices into furan compounds of interest. Focusing on the green processes, particularly under hydrothermal conditions and using neoteric solvents, potential carbohydrate extraction and furanic compound formation in the fewest possible steps and with the lowest possible environmental impact are investigated. 2. Furanic compounds' potential towards new bio-polymers Furanic compounds, notably 5-HMF and 2-furfural, are promising “starting point” molecules derived from LCB, to form new platform molecules. Fig. 1 provides an overview of the molecular structures of the main furan entities under investigation to date. The economic and environmental stakes related to 5-HMF and 2-furfural are high and supported by multiple research initiatives on the subject, which results in abundant scientific literature.17 The state of the art describes extensively the production of furans from hexoses and pentoses, which have often been considered as the “model case” studies (Fig. 2). A lot of fundamental knowledge has been gained on the role of the catalyst, the solvent (whether it is an aqueous solution, an organic solvent, or an ionic liquid),18 and the reaction mechanism;19 more specifically, d-glucose can be converted via isomerization to d-fructose, which is further dehydrated to 5-HMF, with homogeneous (inorganic acids and bases and metal chlorides) or heterogeneous (metal oxides, metal-doped zeolites, etc.) catalysts exhibiting basicity or Lewis acidity (for d-glucose isomerization) and Brønsted acidity (for d-fructose dehydration to 5-HMF).3,20–23 Different organic solvents, mixtures of water-organic solvents, ionic liquids, and even pure water have been used as reaction media.24,25 Attention should be paid to the selective production of 5-HMF instead of organic acids (levulinic acid and formic acid) and humin by-products, stability of catalysts, and improvement of current separation methods of products, e.g., use of biphasic systems.22,23,26 Similarly, 2-furfural is produced from d-xylose via dehydration. Since the early 20s, the industrial production of 2-F is based on the acidic transformation of biomass derived C5 sugars, but the low yield (50%) obtained, due to undesired side-reactions, led to further research on this process.27d-Xylose conversion to 2-furfural is catalyzed either by homogeneous organic and inorganic acids (HCl, H2SO4, metal chlorides, etc.) or heterogeneous catalysts, such as metal oxides and zeolites.28,29 Regarding the solvents, water, organic solvents, or mixtures of water with organic solvents are frequently used.28–30 Fig. 1 Main molecules derived from 5-HMF or 2-F exploited for the design of new biobased polymers and additives (non-exhaustive list).17 Fig. 2 Hydrothermal conversion of d-glucose (A) and d-fructose (B) into 5-HMF and derivatives. Reaction intermediates and/or subsequent decomposition (or rearrangement) products are not mentioned.18 The most reported case study in the scientific literature, and the most studied by many private actors, is undoubtedly the case of 5-HMF-based furan-2,5-dicarboxylic acid (FDCA) allowing obtaining poly(ethylene 2,5-furanoate) (PEF), a possible alternative to the traditional poly(ethylene terephthalate) (PET),31 but also suitable for a myriad of other biopolymers and technical additives.32 Given the chemical reactivity of furan rings and the presence of C <svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata> Created by potrace 1.16, written by Peter Selinger 2001-2019 </metadata><g transform="translate(1.000000,15.000000) scale(0.017500,-0.017500)" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg> O and C–O functional groups on whether 5-HMF or 2-F, numerous studies have been reported on the development of catalytic tools allowing their functionalization into a panel of derived molecules, among which we find 2,2′-bifuran-5,5′-dicarboxylic acid (BFDCA), which offers an alternative to bisphenol A for the design of dimethacrylate resins with thermomechanical properties comparable with those of traditional BisGMA (dimethacrylate derivative of bisphenol A).33,34 The potential of 5-HMF among others is underlined through various works reported in the state of the art, with the alternative synthesis of “symmetrical” 2,5-functional derivatives such as 2,5-bis(hydroxymethyl)furan (BHF), or diformylfuran (DFF), and the “unsymmetrical” monomers such as 5-ethoxymethylfurfural (EMF), furfuryl alcohol (FA), 5-hydroxymethylfuranoic acid, 5-(chloromethyl)furfural (CMF), and diamines and diepoxy analogs.35 Nevertheless, the exploitation of specific chemical tools also allows obtaining other derivatives of interest for the plastics sector, with a loss of the typical furan ring structure, since it is possible to synthesize medium chain diols such as 1,6-hexanediol or even anhydrides (maleic anhydride).36 It is commonly accepted that, in general, all aforementioned furan derivatives offer unique molecular architectures and notable physicochemical properties, opening new prospects in polymer science and technology. However, the great question is not on their value but rather on how to prepare them, starting from lignocellulosic matrices available to explore in the European context. 3. Lignocellulosic matrices as potential inflows for the production of furanic entities Considering the natural composition of lignocellulosic biomass, highly enriched in carbohydrate polymers namely cellulose (source of d-glucose via hydrolysis) and hemicellulose (mostly rich in C5 and C6 carbohydrates),37 and the attractive perspectives for 5-HMF or 2-F production therefrom, if suitable pretreatment steps are set up for its isolation, several authors' assessments for 2020–2030 have identified four strategic primary sources of lignocellulosic biomass that could support the development of biobased industrial activities, at the European level. These are (1) forest biomass, (2) agricultural residues, (3) non-food (or energy) crops, and (4) food or non-food side-products.38 However, one should have in consideration the fact that these four primary sources of lignocellulosic materials are heterogeneously distributed within the EU, both in terms of quantities and territorial repartition. This is due, among other things, to differences in climate (and soil type) between the EU member states, differences in “production tradition”, or differences in the management of the end of life of the by-products. Although it is rather complex to quantify rigorously the available biomass flows in Europe, it can be said that agricultural residues represent the most important sector with a (dry) mass of available lignocellulosic materials of 342 million tons (on an annual basis), followed by the forestry sector with 325 million tons and the forestry by-product sector accounting for 185 million tons. The food waste sector could contribute nearly 89 million tons. However, as already stated before these raw material flows are unevenly distributed in the European zone. Forestry production is massively concentrated in the Nordic countries such as Finland and Sweden, whereas France, Spain, and Slovenia are also producers but to a lesser extent. Agricultural production (mainly cereals) is strongly concentrated in France and Spain. The other European countries contribute in an equivalent way, with specificities of production. This is particularly true for sugar beet (which is produced mainly in Belgium, Germany and the Netherlands) in addition to northern France, and for olive (produced mainly in the southern countries such as Greece, Spain and Portugal). Table 1 presents the four primary sectors of raw lignocellulosic materials as well as representative examples of interest for Europe. Table 1 also proposes the chemical composition of these lignocellulosic matrices, besides cellulose and hemicellulose previously mentioned, also the aromatic biopolymer lignin together with minor amounts of other organic components (proteins, secondary metabolites, etc.) and minerals.37 As can be seen, this chemical composition is found specific, with cellulose contents fluctuating between 10 and 75% of the dry weight of the starting material, and hemicellulose contents oscillating between 5 and 35%. If we estimate (by simple calculation based on C6 contents) the theoretical yields of 5-hydroxymethylfurfural and 2-furfural that can be obtained, we deduce that all these raw materials are not equivalent in their strict potentiality to offer important yields of 5-HMF. The same conclusion can also be formulated if we consider the conversion of pentoses from hemicellulose to 2-F. Chemical composition of several lignocellulosic matrices and theoretical yields for 5-HMF and 2-F productions Matrices Chemical composition (%, referred to dry matter) 5-HMF yielda (kg) 2-F yielda (kg) Cellulose Hemicelluloses Lignin Proteins Inorganics Forest biomass and grasses Spruce 45.0 18.4 27.6 1.1 1.0 350.0 53.8 Beechwood 41.9 18.2 21.6 0.6 0.4 325.9 24.1 Hybrid poplar 39.0 17.0 25.0 3.3 2.0 303.3 103.9 Eucalyptus 46.7 17.3 28.0 0.6 0.6 363.2 65.4 Pine 38.6 20.5 29.2 0.6 4.7 300.2 36.8 Spruce barks 24.1 12.9 36.8 1.8 5.7 187.4 51.5 Willow 34.6 17.8 18.8 3.2 0.1 269.1 115.2 Birchwood 42.7 26.4 16.9 3.0 0.1 332.1 144.7 Black locust 45.0 19.0 29.0 3.6 0.8 350.0 119.7 Fresh grass 18.6 27.7 4.4 26.7 12.8 144.7 36.8   Agricultural and agri-food residues Wheat straw 40.8 35.3 8.2 2.9 7.4 317.3 152.8 Corn stovers 37.5 18.7 13.4 4.7 5.7 291.7 119.7 Sugarcane bagasse 42.1 21.5 24.0 0.9 1.6 327.4 124.2 Sugar beet pulp 28.5 28.1 9.7 9.4 5.7 221.7 135.0 Ryegrass 22.1 25.6 4.7 8.6 8.5 171.9 130.6 Olive kernels 37.7 22.8 29.1 4.4 2.7 293.2 129.9 Oat hulls 30.0 34.0 13.2 3.6 6.1 233.3 137.0 Oat straw 37.6 23.3 12.9 5.3 2.2 292.4 135.0 Fescue 31.3 15.2 19.4 7.3 11.5 243.4 97.3 Peach kernels 21.1 22.1 40.5 1.6 2.9 164.1 123.5 Apple pomace (cider industry) 23.3 24.9 15.4 3.5 1.6 181.2 51.8 Grape pomace (wine industry) 15.8 7.9 20.3 11.2 13.4 122.5 87.7 Orange peels (juice industry) 11.1 12.6 1.0 7.8 4.2 86.3 25.5 Destarched wheat bran 14.8 47.7 8.1 12.2 1.7 115.1 209.9 Spent coffee grounds 12.4 39.1 23.9 17.4 1.3 96.4 28   Energy crops Miscanthus 48.5 20.1 22.4 1.4 4.3 377.2 111 Hemp (woody core) 48.0 12.0 28.0 3.0 2.0 373.3 31.2 Switchgrass 33.5 26.5 18.1 5.3 6.4 260.6 90.2   Residues from non-food sectors Sawdust 45.1 28.1 24.2 1.3 1.2 350.8 45.2 Oak barks 24.0 13.0 25.0 1.1 9.0 186.7 55.1 Organic household waste 14.8 9.8 3.7 15.8 11.8 115.1 1.2 Garden waste 20.3 16.9 17.5 9.6 31.3 157.9 74.9 Mowing residues 38.3 31.0 6.4 4.7 7.7 297.9 36.2 a Theoretical yield (kg based on the conversion of 1 ton of dry biomass). The analysis in Table 1 suggests that wood and its by-products as well as specific crops (such as hemp or miscanthus) or agricultural residues (cereal straws or sugarcane bagasse) offer timely potential to produce furan compounds in significant quantities. Either softwoods or hardwoods can offer (theoretically) up to one third of their weight in 5-HMF, while some by-products of the food industry seem, however, less suitable. While the total hemicellulose content within a lignocellulosic material is a key factor to consider, it is worth mentioning that the very specific chemical nature of these hemicelluloses is also variable between the different materials presented in Table 1. The hemicellulose structure can be “homogeneous” or “heterogeneous” depending on the diversity (or not) of the “monomers” (oses) which constitute them. Amongst these “monomers”, the most representative are pentoses (xylose and arabinose), hexoses (glucose, mannose, and galactose), hexuronic acids (4-O-methyl-d-glucuronic acid, d-glucuronic acid, and d-galacturonic acid) and acetyl groups.39 Based on the combination of the above building blocks, hemicellulose can be classified as xylan-based (homoxylans, glucuronoxylans, arabinoxylans, and glucuronoarabinoxylans/arabinoglucuronoxylans), mannan-based (homomannans, galactomannans, glucomannan, and galactoglucomannan), xyloglucan and galactans.39 As evidenced in Table 1, hemicellulose contents in wood could reach 18 to 30 wt%. Hardwoods are usually composed of xylans, while hemicelluloses in softwoods are mostly mannan and galactoglucomannan-type. Agricultural biomasses (cereal straws) and fresh grasses exhibit the highest amounts of hemicelluloses, mostly xylans and arabinoxylans.40 Based on these theoretical results, variable yields of 5-HMF were observed, ranging from 86 kg/ton dry raw biomass to more than 350 kg/ton dry raw biomass. These high yields were detected in rich cellulose matrices such as spruce, eucalyptus, miscanthus, and hemp sawdust. Concerning 2-F formation from hemicellulose, only pentoses were considered to calculate the theoretical yield, which oscillated between 1.2 and 209 kg/ton dry raw biomass. The most interesting lignocellulosic feedstocks to produce 2-F are pentose rich hemicellulose matrices such as destarched wheat bran, hybrid poplar, willow, birchwood, and black locust. In addition to this purely quantitative perspective, two additional points are equally important. On the one hand, they concern the availability of the lignocellulosic resource and its ability to be mobilized, and on the other hand the “technical” accessibility of the carbohydrate components within these lignocellulosic matrices. If the first point is related to economic, political and often territorial strategies, the second point is related to purely technical aspects linked to the efficiency of the pretreatment phases, which we will discuss below. 4. Role of the pretreatment in the “lignocellulose-to-furan” pathway In order to transform lignocellulosic matrices into 5-HMF and/or 2-F, the prerequisite remains the pretreatment step whose purpose is to separate and individualize the main lignocellulosic components. Once the structural polysaccharides are isolated, subsequent hydrolysis steps (chemical or enzymatic) are then implemented to obtain the hexoses and/or pentoses that will be converted into furan derivatives (Fig. 3). Numerous pretreatment strategies are referenced in the state of the art and include chemical (acids or bases under aqueous conditions, and/or more or less innovative solvents including ionic liquids (ILs)), mechanical, and biological methods or a combination of several of these approaches. Each pretreatment method can be specific to the raw material and/or the targeted applications.41 The formation of simple furanic compounds such as 5-HMF or 2-F during the pretreatment phases has been demonstrated for decades. This formation has often been avoided, especially because 5-HMF (and 2-F) is a known inhibitor of alcoholic fermentation and its presence decreases bioethanol yields.42 It is recognized that pretreatment methods involving acids, whether solvent (or ionic liquid) methods with acid catalysts or hydrothermal methods, are the most likely to generate these furan species. Therefore, it is these particular pretreatment methods that are currently being used to produce specifically 5-HMF and 2-F. Fig. 3 Multi-step conversion of lignocellulosic resources into furanic compounds. ILs stands for ionic liquids. 4.1. Hydrothermal pretreatments Pretreatment methods that take place in the aqueous phase (with or without the addition of acid catalysts) are known under the scientific term of “hydrothermal pretreatments” (HT). They are classified as “physicochemical pretreatment” and encompass two distinct processes, namely, liquid hot water (LHW) and steam explosion (SE).43 HT is recognized as a “green” process due to the absence (or minimum use) of toxic chemicals or added catalysts and was initially developed for the second-generation bioethanol production, with the aim to remove hemicellulose and partially degrade the robust lignocellulosic structure, leading ultimately to enhanced enzyme hydrolysis of cellulose to glucose. Considering the operational conditions of these processes, LHW utilizes water as the only solvent at elevated temperatures, in the range of 130–270 °C, for a time period of ca. 15 to 180 min depending on temperature.44–49 Similar to LHW, SE uses steam instead of water, under pressure (<50 bar) for relatively short reaction times (ca. up to 1–10 min) in the temperature range 160–240 °C, followed by a fast explosive decompression of the high-pressure steam.43,50–52 HT pretreatment of lignocellulosic biomass has been applied on the pilot and industrial scale mainly for the production of second generation bioethanol considering also the “side” streams of hemicellulose and lignin, with Inbicon in Denmark and SEKAB in Sweden being two representative examples.53,54 4.1.1. Hydrothermal pretreatment in neat water HT pretreatment has the great advantages, from a green perspective, of using only water and without addition of any acid catalyst, and hemicellulose is the main biomass component which is solubilized and partially hydrolyzed, and recovered in the liquid product stream. As discussed previously, hemicelluloses are heterogeneous polysaccharides and published results have evidenced that, generally, hardwoods are preferred for HT pretreatment due to their acetyl groups, which induce self-catalyzed mild acid hydrolysis of hemicellulose.47,55 Cellulose and lignin are less affected during HT pretreatment and remain in the solid fraction, while small amounts of acid soluble lignin and extractives, such as non-structural sugars, proteins, terpenes, waxes and phenolic compounds, depending on the type of biomass, are removed to the liquid fraction.56 Hemicellulose deconstruction during LHW is carried out via first-order reactions, catalyzed by hydronium ions, produced due to the subcritical state of water (T < 374 °C, P < 221 bar).57 Hemicellulose polymer (H) is initially depolymerised into oligomers (O), which with further breakdown are converted to monomers (M) and degradation products (DP). The reaction pathways and reaction steps for solubilization and hydrolysis of hemicellulose can be described by eqn (1a) and (1b).581a 1b1 Xylan → XOSH → XOSL → xylose → furfural → DP 1b2 Arabinan → AOS → arabinose → furfural → DP where XOSH denotes the high molecular weight xylo-oligosaccharides, XOSL the lower molecular weight xylo-oligosaccharides and AOS the arabino-oligosaccharides. More specifically, hemicellulose in biomass is partially acetylated and, under hydrothermal conditions, acetic acid is formed via hydrolysis of ester bonds.59 The in situ produced acetic acid promotes hydrolytic reactions of hemicellulose and to a lesser extent of cellulose into xylan and glucan oligomers, which can be further hydrolyzed to form xylose and glucose, respectively (Fig. 4, step 1). Under the mildly acidic hydrothermal conditions (notably for pH ∼ 2.5–3.5), C5 sugars (xylose and arabinose) can be dehydrated towards furfural formation, which can be further converted to formic acid via hydrolytic fission of the aldehyde group or to 2-furoic acid via oxidation (Fig. 4, step 2).47,60,61 Fig. 4 Reaction pathways in the hydrothermal treatment of lignocellulosic biomass towards sugars and degradation products. Similarly, 5-HMF can be produced via dehydration of C6 sugars (glucose, galactose, and arabinose) and can be further converted into levulinic and formic acids (Fig. 4, step 2).18 The extent of these reactions, as well as the biomass solubilization degree and hemicellulose removal, is controlled by the residence time and temperature of the pretreatment. Their combined effect can be expressed by the severity factor Ro (eqn (2)).62–64 During HT pretreatment, water auto-ionizes into hydronium ions, leading to a pH drop, increasing the severity factor leading to promoting acetyl and uronic group cleavage, and thus improving hydrolysis reactions.65 When small amounts of acid catalysts (i.e., H2SO4 and HCl) are used to facilitate hemicellulose hydrolysis, the modified severity factor is expressed by eqn (3), where t is the residence time (min) and T is the temperature (°C).66–682 Ro = t·e(T−100)/14.75 3 In addition to temperature, time and use of small amounts of acids, the liquid-to-solid (biomass) ratio (LSR) may have a relatively small effect on biomass solubilization and a more pronounced impact on liquid product selectivity and yields, as discussed below.69,70 Furthermore, considering the biomass particle size effects in LHW, they are correlated with heat and mass transfer limitations and it was shown that the particle size affects only sugar extraction and hemicellulose removal from biomass and not their thermal degradation in the liquid fraction.71,72 It should be highlighted that biomass particle size is a less important parameter than temperature and time and its effect is more profound in combination with that of LSR. Considering the effect of temperature and time, under mild treatment conditions (log Ro ≤ 2.76, corresponding to ≤150 °C and short time of 15 min), low solubilization of beechwood sawdust was observed (≤6 wt%), while under harsher conditions (log Ro ≥ 4.14, 170 °C, 120–180 min) the solubilization was increased substantially (to about 40 wt%) and complete removal of hemicellulose occurred at log Ro = 4.69, corresponding to 220 °C and 15 min.47 Regarding product yields, a maximum xylan recovery (60 wt%) in the liquid fraction was determined at 80 wt% hemicellulose removal, which was obtained at moderate severities (log Ro = 3.8–4.1). Higher severities led to xylose degradation product formation, mainly furfural and formic acid.47 Differentiations have been observed in the LHW pretreatment between softwood and hardwood biomass. More specifically, by increasing log Ro from 3.3 to 4.7, the hardwoods, grapevine prunings and poplar branches, showed higher solubilization ranging from 28 to 48% and 23.5 to 40%, respectively, while the softwood pine sawdust was more resistant to hydrothermal pretreatment with solubilization ranging from 19 to 34%.59 The recovery of xylan from poplar and grapevine and of mannan/galactan from pine in the liquid products showed a maximum of about 60% at moderate severities (log Ro 3.8–4.1), corresponding to 70–85% hemicellulose removal. By comparing the above results of the two relevant works, it can be suggested that the solubilization/removal of hemicellulose is similar for different types of hardwood biomass (forest and agricultural) and higher than that in softwood biomass (pine), while the recovery of hemicellulose in the liquids reaches a similar maximum and in the same severity region.47,59 A wide variety of forestry and agricultural residues have been hydrothermally treated in neat water: olive tree prunings with 37.6–45.3% biomass solubilization at log Ro = 3.06–4.83 (ref. 73) and 34.4% solubilization at 3.79,68 cotton stalks with 33% biomass solubilization and 82% xylan removal at 4.29 (ref. 74) bagasse with biomass solubilization of 14.8–27.6% at 2.8–4.8 (ref. 75) and xylan removal of 34.5% and 80% at 3.83 and 4.42,76 eucalyptus sawdust with 0–48.2% biomass solubilization at 2.07–5.60 and 77.7% xylan removal at 3.3,77 maple wood with 80% xylan removal at 4.25,78 corn cobs with 10–40% biomass solubilization at 3.06–4.43 (ref. 79) and 74.1% xylan solubilization at 4.13,80 poplar with 1.9–23% biomass solubilization and 6.8–62.8% xylan removal at 3.06–3.76 (ref. 81) and 50–77% xylan solubilization at 3.6–4.2,46 hazelnut shells with 35.4% biomass solubilization at 4.44,82 hazelnut tree prunings with 14–36% biomass solubilization and 43.2–98.5% hemicellulose removal at 3.24–4.89,83 brewer's spent grains with 64.06% xylan solubilization at 4.13,80 corn husks with 42% xylan solubilization at 4.13,80 wheat straws with 16.5–45.7% biomass solubilization at 3.36–5.14 (ref. 84) and 39.26% xylan solubilization at 4.13 (ref. 80) and almond shells with 39.3% biomass solubilization at 3.79 (ref. 68). A correlation of pretreatment conditions (temperature and time) with biomass solubilization and hemicellulose/xylan removal, based on the above previously reported works, can be observed in Fig. 5. Fig. 5 Correlation of xylan solubilization/removal and biomass solubilization with hydrothermal (LHW) pretreatment temperature and time. Data are from ref. 46, 47, 59, 68, 73–75, 77–85. Liquid to solid ratio (LSR) in LHW pretreatment can be varied in the range of 1–20 but usually is around 10.70 An increase of solid concentration can raise acetic acid concentration and thus enhance the autohydrolysis mechanism. In general, biomass solubilization is slightly affected by LSR, while product distribution in the pretreatment liquor seems to exhibit differentiations. Under mild pretreatment conditions, at 160 °C for 45 min (log Ro = 3.42), an increase of LSR from 1 to 11 resulted in a decrease of xylose and xylan concentrations but an increase of xylose and acetic acid weight percentages (on biomass) in the liquor.70 Garrote et al. studied the mild autohydrolysis of eucalyptus wood at 175 °C for 60 min (log Ro = 3.99) and found that an increase of LSR from 6 to 10 resulted in a decrease of xylose and xylan concentration, as well as a decrease of acetic acid, furfural and 5-HMF concentration in the liquors.69 Fujimoto et al. studied the effect of solid concentration of the same wood under two pretreatment conditions.86 At log Ro = 4.22, an increase of solid concentration (decrease of LSR) resulted in a decrease of xylose yield but an increase of acetic acid, furfural and 5-HMF, while at log Ro = 4.52, an increase of solid concentration resulted in a decrease of xylose and furfural yields but almost in similar acetic acid and 5-HMF.86 A correlation of biomass solubilization and hemicellulose/xylan removal with log Ro and LSR, based on the above previously reported works, can be observed in Fig. 6. Fig. 6 Correlation of xylan solubilization/removal and biomass solubilization with hydrothermal pretreatment conditions, expressed by log Ro and LSR. Data are from ref. 46, 47, 59, 68, 73–75, 77–85. Steam explosion (SE) is also one of the most studied pretreatment processes of lignocellulosic biomass feedstocks. The parameters affecting the SE process are the biomass particle size, residence time and temperature. Although hemicellulose removal is indeed enhanced at elevated temperatures, it can simultaneously lead to sugar degradation.87 Higher severities achieved at 270 °C for 1 min (log Ro = 5.01) resulted in optimal hemicellulose solubilization towards degradation products, while lower temperature and longer residence time (190 °C, 10 min) avoided their formation.50 Similar to LHW, the SE process has been applied to a vast variety of lignocellulosic feedstocks. In the SE pretreatment of banana rachis, an increase of the severity factor from 2.97 to 3.78 resulted in hemicellulose and amorphous cellulose solubilization towards monosaccharides.88 Similar results were obtained for tall fescue grass, which was pretreated in a wide range of severities log Ro = 1.4–4.2. A gradual increase of hemicellulose removal was observed and at the highest severity, all the hemicellulose has been extracted in the liquid fraction.89 SE of olive tree prunings led to 34–51% biomass solubilization at log Ro = 3.83–4.71, attributed to hemicellulose and extractives, while a higher hemicellulose sugar recovery of 35.6% was obtained at a severity of 4.12.90 4.1.2. Dilute acid hydrothermal pretreatment The efficiency of hydrothermal pretreatment towards increased solubilization and hemicellulose removal in the liquor can be enhanced by the use of dilute acid solutions. Alkaline conditions (NaOH or NaCO3) are not preferred due to the simultaneous lignin removal in the liquid fraction even under mild conditions.64 Similar results were obtained in the HT pretreatment of sugarcane bagasse, where an increasing concentration of NH3 was used, which did not significantly affect hemicellulose removal but dramatically increased lignin removal.91 Sulfuric acid (H2SO4) is the most widely used inorganic acid in the HT pretreatment, in various low concentrations, usually in the range of 0.1 to 2 wt%. Hydrochloric (HCl), phosphoric (H3PO4) and acetic acids (CH3COOH) are less frequently used.92 In a comparative study, HCl resulted in low lignin removal and high furanic formation, whereas CH3COOH provided low furanic production but high lignin removal and H2SO4 showed an intermediate performance.64 Intense conditions (either high temperatures or prolonged pretreatment times) combined with relatively high acid concentrations (>2 wt%) led to higher solubilization but also induced partial degradation of sugars towards furanics and organic acids, as well as to the formation of low molecular weight phenolic compounds from lignin. Generally, addition of dilute sulfuric acid may increase the solubilization of lignocellulosic biomass feedstocks by more than 10 times, reaching 40.5 wt% solubilization, while hemicellulose removal can reach up to 100%,75,83,93 as can be observed in Fig. 7. Hemicellulose sugar recovery reached 98% in HT pretreatment of brewer's spent grains at log Ro = 2.28 combined with 1% w/v H2SO4.94 Careful selection of pretreatment temperature, time and acid concentration can tune the composition of the liquid fraction. Indicatively, in sulfuric acid catalyzed HT pretreatment of poplar, an increase of temperature from 130 to 180 °C increased xylan degradation from 70 to 91%, initially towards xylose (T < 140 °C) and further towards furanics (T > 140 °C) and acids (T > 200 °C).95 Similarly, in acetic acid catalyzed HT pretreatment of the same wood, the higher acetic acid concentration (10% v/v) and the higher pretreatment time (50 min) resulted in 87.8% xylan removal in liquor, mainly in the form of xylose (46.4%), while at lower acid concentration (5% v/v) and shorter time (30 min), xylan removal was equal to 71.3% toward xylo-oligosaccharides (55.8%) with lower xylose yield (32.9%).81 Fig. 7 Correlation of biomass solubilization with log Ro in dilute acid (H2SO4, wt%) HT pretreatment. Data are collected from ref. 68, 75, 83, 93–95. Redox environment was also pointed out as an important parameter in auto-hydrolysis and dilute-acid pretreatment of biomass. Addition of O2 improved xylan removal in the liquid whose composition proved to be affected by the severity of the conditions. Under the most severe conditions in the presence of O2, xylan was removed towards the formation of furanics (furfural, 2-furoic acid, and 5-HMF) and organic acids (acetic acid, formic acid and levulinic acid), resulting also in lower glucan recovery in the solid fraction.75 Alternatively to the classic heating, microwave heating has been applied in the acetic acid catalyzed corn stover leading to higher heating rate and high glucose yield in the solid fraction.96 In SE pretreatment the addition of acid catalysts or the impregnation of wood chips with acids prior to pretreatment is more often, allowing for significantly decreased residence temperature and time, and inducing improved hydrolysis rate, decreased degradation products and increased removal of hemicellulose and lignin.87 Addition of 1 wt% H2SO4 in the pretreatment of almond shells, olive tree prunings and grapevine prunings resulted in similar biomass solubilization (53.5%, 50.4% and 49.9%, respectively) but with different hemicellulose recovery percentages (14.19%, 27.68% and 43.31%, respectively) in the liquid fraction.68 Immersion of wheat straw in 0.2% H2SO4 prior to SE resulted in 36–48% biomass solubilization at log Ro = 2.95–4.24 with increased recovery of hemicellulose in the liquid fraction, while glucose remains in the solid.97 Apart from sulfuric acid, other inorganic acids have been also utilized in the catalyzed SE pretreatment and H3PO4 resulted in slightly higher sugarcane bagasse solubilization compared to H2SO4.66 4.1.3. Effect of HT pretreatment on biomass/cellulose enzymatic digestibility The majority of pretreatment studies target the efficient removal of hemicellulose and/or part of lignin, in order to limit the inhibition phenomena in the subsequent biochemical processes towards 2G-bioethanol production, i.e., the enzymatic hydrolysis of cellulose and hemicellulose into monomeric sugars and their subsequent fermentation. The pretreated solids are more susceptible to glucose production by hydrolysis with cellulases (exo/endo-glucanases, etc.) and in the last years, to xylose production in the presence of xylanases.98 In a following step, the produced sugars are converted to ethanol via microbial fermentation. The above processes can be combined in one step and the process is known as simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and co-fermentation (SSCF).99 As a consequence, optimization of biomass pretreatment is crucial for the efficient valorization towards ethanol production. Both LHW and SE may induce structural and physicochemical alterations in biomass particles, such as the disruption of the lignocellulose complex and rigid structure, increase of particle external surface area and accessibility of enzymes, hydration and crystallinity decrease of cellulose, as well as removal of hemicellulose and part of lignin via depolymerization into low molecular weight fragments.48,100,101 Generally, HT pretreatment enhances biochemical processes with moderate to intense HT conditions, resulting in maximum yields of glucose or ethanol.47,73,86 HT pretreatment also proved to facilitate microbial activity during anaerobic digestion for methane production.102 Furthermore, delignification of HT solids may further facilitate enzymatic hydrolysis. Removal of “surface” lignin, i.e., partially solubilized lignin during HT pretreatment that has been recondensed on the biomass surface, can be achieved by mild extraction with “green” and easily recoverable solvents, e.g., acetone or ethanol, resulting in a 25% increase in enzymatic digestibility.103 The complete delignification of HT solids showed controversial effects on enzymatic hydrolysis. Alkaline post-treatment of HT-hazelnut shells, with NaOH either in water or in ethanol/aqueous solutions, as well as through organosolv and acid-catalyzed organosolv post-treatments did not improve the enzymatic hydrolysis of cellulose to glucose compared to the direct acid catalyzed organosolv pretreatment of raw biomass.82 On the other hand, sequential organosolv-hydrothermal pretreatment of cotton stalks resulted in higher ethanol production.74 Similarly, delignification of HT vine shoots with NaClO2 in aqueous solution of acetic acid led to enhanced ethanol production.104 Ball milling of HT solids prior to the hydrolysis was proved to enhance the enzymatic digestibility of cellulose to glucose.77,105 4.1.4. Production of furanics (furfural and 5-HMF): cascade (two-step) and in situ (one-step) processes during HT pretreatment Instead of studying the conversion of “purified” monosaccharides into furanics as described in Fig. 1, process modelling and optimization studies have been performed utilizing “real” streams of hydrothermally pretreated biomass and down-stream catalytic hydrolysis and dehydration steps, in order to evaluate the potential industrial scale production of 5-HMF where heat integration, and mass and energy balance are also critical.106 The one-step (in situ) aimed production of furanics from raw biomass feedstocks during HT pretreatment in neat water is rarely referred in the literature, despite its evident usefulness. The HT pretreatment of lignocellulosic feedstocks results in relatively low yields of furfural and 5-HMF, in the range of 0.05–2 wt% on initial biomass, which are usually not considered as the primary target but as degradation by-products.64,73,107 As can be observed in Fig. 8, the concentration of furfural in the pretreatment liquor may reach up to 5 mg ml−1 in a wide range of log Ro = 2.5–4.7, thus indicating that other parameters in addition to temperature and time, such as biomass type, may have an important effect. The concentration of 5-HMF is even lower, not exceeding 1 mg ml−1 in most cases. The addition of homogeneous catalysts in the hydrothermal pretreatment process can improve the in situ production of furanics. Dilute inorganic acids under severe conditions of LHW pretreatment resulted in higher furanic production.94,95,108 Fig. 8 Furanic production via LHW pretreatment. Numbers correspond to acid concentrations: 1 : 0.5% v/v H2SO4, 2 : 1%v/v H2SO4, 3 : 1.6 w/v H2SO4, 4 : 400 g kg−1 CH3COOH, 5 : 3 wt% H2SO4, 6 : 0.5 wt% H2SO4, and 7 : 3% w/v H2SO4. Purple numbers correspond to furfural and blue numbers to 5-HMF. Data are from ref. 47, 59, 75, 76, 78–80, 85, 92, 94, 95, 108, 114 and 115. However, intense hydrothermal conditions in the presence or absence of dilute acids, at ca. log Ro > 4, usually lead to enhanced “loss” of biomass carbon as humins or even gaseous products.47,48,109 Aiming to avoid mineral acids, metal chlorides can be utilized as catalysts in the HT pretreatment, which under optimal reaction conditions can convert almost 98% of corn stover hemicellulose towards furfural production (25% wt% of liquid product) without significantly affecting cellulose and lignin.110 In broader research, poppy stalk carbohydrates were converted to furfural and 5-HMF in neat water over a wide range of metal chlorides and among them, CuCl2 was the most active, resulting in 79.9% furfural and 12.2% 5-HMF production yields (based on xylose and glucose of raw biomass) under optimized conditions.111 In general, hydrothermal pretreatment under intense conditions, low LSR and addition of inorganic acids may enhance the production of furfural and 5-HMF, but care of biomass degradation to humins should be taken. To this end, direct furfural and 5-HMF production from lignocellulosic biomass can be further enhanced using co-solvents, which can also minimize the humin formation. The most promising co-solvents are γ-valerolactone (GVL), tetrahydrofuran (THF) and dioxane, which can partially replace water in the hydrothermal pretreatment and enhance the furanic production in the presence or absence of catalysts.25,29,112,113 Another important parameter considering the viable production of furanic compounds from lignocellulosic biomass is their separation and recovery from aqueous pretreatment solutions. Generally, adsorption, distillation, precipitation or solid/liquid phase extraction is applied to selectively recover 5-HMF and furfural. High costs derived by large amounts of solvent consumption and scaling issues led to most suitable processes. 5-HMF recovery from aqueous solutions via extraction has been studied with many solvents, but methyl isobutyl ketone (MIBK) and 2-methyltetrahydrofuran (2-MTHF) gained great interest due to the high recovery yields of 5-HMF.116,117 Similarly, furfural can also be recovered from aqueous solutions either via classic extraction separation or CO2 assisted phase separation.118–120 Considering a low cost and environmentally friendly solvent, 2-pentanone is an alternative for furfural separation, providing low separation load in case of downstream conversion of furfural to fuels, because 2-pentanone can also be converted to hydrocarbon fuels.121 Another interesting approach is the recovery of both furfural, 5-HMF and acetic acid from aqueous solutions with polyethylenimine, a soluble polyelectrolyte, which was successfully applied in biomass pretreatment slurries to remove the fermentation inhibitors.122 Similarly, furanics and acetic acid can be removed from the hydrolysate via sorption on activated charcoal and anion exchange resins.108 4.2. Neoteric solvents as reaction media for pretreatment and conversion of lignocellulosic matrices Neoteric solvents have gained interest in the last decade and have been deeply investigated as alternative media for the pretreatment and the conversion of the lignocellulosic feedstock into furanic derivatives. In particular, the use of ionic liquids (ILs), deep eutectic solvents (DES) and supercritical fluids is discussed, highlighting the advantages, strengths, and opportunities of their use in this field. These neoteric solvents present unique properties in comparison with traditional solvents, allowing not only the development of more efficient processes in terms of diversity of materials and catalytic efficiency but also the reduction of the environmental impact for the key treatments and synthetic transformation required to transform lignocellulose materials into high-added value products. 4.2.1. Pretreatment of biomass feedstock in ILs Ionic liquids (ILs), which are very particular solvents composed exclusively of ions and generally having negligible vapour pressure, high thermal and chemical stability, and outstanding solvation potential,123,124 have emerged as an attractive alternative for the production of furan-derived compounds from biomass.125 The specific properties of the ILs can be fine-tuned by the right design and combination of anion and cation components. It is estimated that combination of the possible cations and anions would result in the formulation of more than (1012) potential ILs,126 so one can infer the huge flexibility of this class of solvents. Importantly, the structure of the ILs can be adjusted to be used not only as a solvent for an efficient biomass pretreatment, but also to enable its catalytic transformation into valuable chemicals and biofuel.127–133 The pretreatment of the biomass is a key step for its further conversion into chemical products. In this regard, ILs are among the most efficient solvents for the dissolution of lignocellulosic biomass (i.e., wood chips, straw, cornhusks, etc.).134 Hence, ILs offer a simple alternative to conventional pretreatment methods. Imidazolium based ILs represent the most common type of ILs but pyridinium, phosphonium, morpholinium and ammonium derived ILs have also been evaluated for biomass pretreatment.135,136 While they are generally considered to be ecofriendly compounds due to their non-volatility and non-flammability, the green calcification of the ILs should be carefully considered as some of them present potential toxicity and biodegradability issues.137 Nevertheless, ILs are efficient media for the fractionation of biomass into its components, some of them being a suitable feedstock (i.e., polysaccharides and sugars) for the production of 5-HMF. The typical initial step is the heating of the biomass in the presence of the ILs. The generated non-covalent interactions with the structural elements of the involved biomass help to disrupt the non-covalent interactions between lignocellulose components, leading to its dissolution without significant degradation.138 Different solvents (i.e., acetone–water mixtures, alcohol, or dilute acid) are added to the mixture or removed from it (i.e., by evaporation) in order to induce the precipitation of the different components of the lignocellulosic biomass.139 This type of process requires, for every step, a well-designed separation and purification of the product from the IL-phase, as well as an energy-efficient recycling of the IL, if a potential industrial application of ILs for biomass pretreatment is envisaged (Fig. 9) as well as in the perspective of a green chemistry approach. Fig. 9 General scheme for the preparation of furanic derivatives in IL-based processes. The initial reports of biomass dissolution dealt with cellulose. Although the first report on the dissolution of cellulose in a mixture of 1-ethylpyridinium chloride and a nitrogen-containing base dates back to 1934,140 it was not until the seminal work of Rogers et al. that the potential of room temperature ILs for cellulose dissolution was fully revealed.141 The IL-dissolved cellulose could be regenerated without severe degradation by addition of water or other precipitating solvents (e.g., ethanol or acetone). In general, regarding the structure of the cation, decreasing the length of the alkyl chains (decreasing their hydrophobic character) or introducing allyl chains improved the dissolution power of the ILs.142 On the other hand, anions like Cl−, HCOO− and OAc− provided good dissolution capacity. These anions are relatively small and good hydrogen bond acceptors, favouring the formation of hydrogen bonds with the acidic hydrogen atoms of the hydroxyl groups of the polysaccharides. In imidazolium cations, the proton on the C2 position of the ring is relatively acidic and can also form hydrogen bonds with the oxygen atoms in cellulose (Fig. 10).143,144 Possible additional interactions (i.e., van der Waals interactions and hydrophobic effects) have been also claimed to occur between the cation and the cellulose.145,146 The use of an additional organic solvent with the ILs can induce co-solvency and anti-solvency effects.35 Fig. 10 Schematic representation of the main interactions involved in the dissolution and activation for hydrolysis of polysaccharide fiber in ILs. Later studies have demonstrated that ILs can be used to process different types of lignocellulosic biomass, such as agricultural residues, dedicated energy crops and forest biomass.147,148 As for cellulose, ILs' structure and properties can be fine-tuned to improve the capacity for dissolving lignocellulosic biomass from different sources (Fig. 11).32,149,150 The most commonly used ILs for lignocellulosic pretreatment are [EMIM][OAc] (1-ethyl-3-methylimidazolium acetate), [AMIM][Cl] (1-allyl-3-methylimidazolium chloride) and [BMIM][Cl] (1-butyl-3-methylimidazolium chloride).10 The appropriate design of the IL can lead to a reduction of the required dissolution temperature, price, ecotoxicological profile and viscosity, which are key factors to enable their industrial implementation. A simple to prepare, biocompatible and low cost (∼$1 per kg) protic IL such as ethanolamine acetate has been reported for the direct production of biofuels via one-pot synthesis.151 Protic ionic liquids are a subset of ILs produced through the combination of a Brønsted acid and a Brønsted base.152 In this example, after pretreatment of switch grass, the whole slurry is directly used for simultaneous saccharification and fermentation with commercial enzyme cocktails and wild type yeast strains, generating 70% of the theoretical ethanol yield. On the other hand, the so called ionoSolv process based on the use of triethylammonium hydrogen sulfate ([TEA][HSO4]) as a protic ionic liquid has been implemented for the fractionation of even highly recalcitrant feedstocks and for separation of cellulose-rich pulp and lignin fractions.153,154 In addition, the easy recycling, high yields, and low pretreatment times facilitate potential commercialization of this process.155 Fig. 11 Some examples of ILs used for the processing and transformation of lignocellulose. Apart from the fractionation of the biomass, ILs can also be used as solvents for the depolymerisation of polysaccharides like cellulose, hemicellulose, inulin or starch into the corresponding sugars.32 As mentioned above, the basic anion from the IL disrupts the crystallinity of the polysaccharide that is transformed into an amorphous structure by interfering with the hydrogen bonding network, enhancing its dissolution but also facilitating its modification. On the other hand, the interaction between the cation and the glycosidic moieties of the polysaccharide makes the carbohydrates more susceptible to hydrolysis. Thus, the addition of an acid catalyst, either homogeneous (i.e., sulfuric, acetic, oxalic acids, etc.) or heterogeneous (Amberlyst, zeolite, MOF, etc.), to the polysaccharide dissolved in the IL facilitates the catalytic hydrolysis of the glycosidic bonds linking the sugar monomers in the polysaccharide, leading to its depolymerisation into the corresponding monosaccharide.156 Alternatively, different task-specific ILs displaying a built-in acid function in the cation (e.g., –SO3H and –COOH) or/and in the anion (e.g., HSO4−) have been designed to play a dual role as a solvent for the dissolution of the polysaccharide and as an acid catalyst for its hydrolysis into sugars.128,157 4.2.2. Converting biomass feedstock into 5-HMF ILs in the presence of an additional catalyst or playing by themselves a catalytic role can also provide a pathway for the transformation of monosaccharides into 5-HMF.16,158 It has been observed that ILs minimise decomposition of the produced HMF in comparison with other conventional solvents, which is a step forward within the green chemistry principle of waste-prevention. This stabilization of 5-HMF from decomposition provides a great potential for the use of imidazolium ILs in highly selective and efficient biomass-derived 5-HMF synthesis, even though imidazolium-based ILs are associated with toxicity issues. Lansalot-Matras et al. demonstrated that the presence of ILs in IL/DMSO mixtures had a positive effect for the acid-catalyzed dehydration of fructose to 5-HMF.159 Thus, under catalyst-free conditions a 27% 5-HMF yield was reported after 15 h by adding 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]) to DMSO, while in pure DMSO only trace amounts of 5-HMF were obtained. The yield could be further improved using a polymer-supported acid (Amberlyst-15) as a co-catalyst, reaching an 87% yield in 24 h. These results indicate the non-innocent nature of the ILs in the conversion of fructose to 5-HMF. Lewis acidic cations such as those in metal chlorides are also suitable catalysts for the dehydration of fructose to 5-HMF.160 Zhang and co-workers demonstrated that metal chlorides (i.e., CrCl2) dissolved in 1-alkyl-3-methylimidazolium chloride can efficiently catalyse the synthesis of 5-HMF from fructose, while other less potentially harmful metal chlorides in [EMim]Cl showed poor activity or very low selectivity to 5-HMF.161 From the tested imidazolium ILs displaying different N-alkyl chain lengths (octyl, butyl and ethyl) the one with the shorter chain ([EMIM][Cl]) provided the higher yield (73% 5-HMF yield at 120 °C). 5-HMF can also be obtained from other sugars, although the efficiency of the reaction is significantly affected by the nature of the feedstock. When sucrose is used (a disaccharide of glucose and fructose) the sugar is nearly quantitatively transformed into 5-HMF and unreacted glucose.87 Indeed, to convert glucose to 5-HMF, longer reaction times and higher reaction temperatures are required. Two different reactions are needed to convert glucose into 5-HMF: (i) isomerization of glucose into fructose, and (ii) dehydration of fructose to 5-HMF. The coordination of the ILs with a metal salt enables the proton transfer at the isomerization required to convert glucose into fructose.162 The nature of the ILs defines again the reaction conditions (temperature and time) and the yield obtained. In general, ILs containing imidazolium cations combined with nucleophilic anions such as halides (e.g., Cl− or Br−) led to more efficient conversions of sugars into 5-HMF.32 Zhao and co-workers achieved a significant conversion of cellulose and glucose, with ca. 60% and 90% isolated yields, respectively, mediated by CrCl3/[BMIM][Cl] under microwave irradiation (MW).163 Microwave heating allowed for a remarkable reduction in the time required (from hours to minutes). Thus, the combined use of MW and ILs can lead to a fast and more cost-effective process for the conversion to 5-HMF in comparison with conventional heating.164,165 Because of the high dielectric constant and the ionic nature of the ILs, the reaction is heated up quickly, volumetrically and simultaneously leading to a more efficient heating.166 For instance, up to 97% yield of 5-HMF can be obtained from fructose in [BMIM][Cl], in the absence of catalyst, in only three minutes under microwave irradiation.167 The combination of [BMIM][Cl]/CrCl3 yielded ca. 70% of 5-HMF from glucose in only thirty seconds, which represents a yield significantly higher than the one obtained under conventional heating (ca. 48% 5-HMF yield). With this system, a 5-HMF yield of 54% was obtained for cellulose conversion at 150 °C during 10 min of reaction time, with the [BMIM][Cl]/CrCl3 being recyclable for six cycles of use.168 J. P. Hallett and co-workers have reported the efficient use of [BMIM][OTf] for the synthesis of 5-HMF from fructose. The water content plays an important role in stabilising the obtained 5-HMF and in suppressing side reactions. Thus, high yields (>80% 5-HMF yield) from high substrate loadings (14% w) can be obtained in a short reaction time (<10 min), even with the use of non-coordinative ILs and halide anion free ILs.169 The use of ILs as reaction media can also decrease the operational temperature required for the dehydration of sugars to 5-HMF with a reduction in the energy requirements and operational costs. In general, under conventional conditions temperatures as high as 100 to 300 °C are needed. However, when ILs are used, much lower temperatures, in the range of 80–140 °C or lower, can be usually applied, which constitutes a major advantage from the energetic point of view. Indeed, Smith, Qi and co-workers have reported an efficient catalytic system for the conversion of fructose into 5-hydroxymethylfurfural at room temperature.170 Reactions proceeded smoothly and efficiently with 78–82% 5-HMF yields at 25 °C after pre-dissolving the fructose into [BMIM][Cl] and subsequently adding a small amount of a co-solvent (i.e., acetone, DMSO, ethanol, etc.) and using Amberlyst-15 as the catalyst. The transformation of fructose to 5-HMF can also be achieved with a high yield (>90% HMF yield) at room temperature using multicatalytic systems such as [BMIM][Cl]/WCl6,171 or a mixture of [BMIM][Cl] and an acid IL like [HNMP][CH3SO3] (N-methyl-2-pyrrolidonium methyl sulfonate).172 This reduction of the temperature can allow for the continuous extraction of 5-HMF by using organic solvents with low boiling points. This can simplify the implementation of large-scale biphasic continuous systems enabling the 5-HMF separation and recovery by solvent distillation and the recycling of the catalytic IL-phase for subsequent catalytic cycles. Despite being more challenging than the conversion of fructose, other complex carbohydrates such disaccharides (i.e., sucrose, maltose, lactose and cellobiose)173 and polysaccharides (i.e., inulin, cellulose, chitosan and starch)87 can also be transformed to 5-HMF using ILs. The nature of the ILs, the co-catalyst and the reaction conditions should be properly adjusted depending on the feedstock, as the increase in the degree of polymerization of the feedstock gradually decreases the efficiency of the catalytic system. ILs, being compatible with different catalytic units, also open the possibility to integrate different steps required for the synthesis of furanic derivatives (i.e., dissolution, hydrolysis, isomerization, dehydration, etc.) in a multistep cascade process, where the product from one step becomes the feedstock of the next one without any need of purification processes between them. This integration can save time and energy in pretreatment and isolation steps, increasing profitability and enabling its industrial implementation. For instance, the combination of two metal chlorides (CuCl2/CrCl2) dissolved in [EMIM][Cl] (1-ethyl-3-methylimidazolium chloride) catalyzes the single-step conversion of cellulose to 5-HMF with an unrefined 96% purity among recoverable products (at 55% 5-HMF yield at 80–120 °C).174 The catalytic performance of the CuCl2/CrCl2/[EMIM][Cl] catalytic system can be preserved after the isolation of 5-HMF, keeping the activity for at least three consecutives runs. Some other paired metal catalytic systems have been studied, such as CrCl2/CuCl2, CrCl2/CrCl3, CrCl3/FeCl3, CrCl3/FeCl2, CrCl3/AlCl3, CrCl3/SnCl4, CrCl3/MnCl4, and CrCl3/CuCl2, in this context.175,176 In the search of low-temperature, one-pot processes, Raines and co-workers used ortho-carboxyl-substituted phenylboronic acids as organocatalysts, together with MgCl2·6H2O/H2SO4, to convert cellulose and cellulose-rich municipal waste (i.e., cotton, paper towels, and newspaper) to 5-HMF in [EMIM][Cl] at 105 °C. 5-HMF yields as high as 41% could be achieved with a catalytic system that is devoid of heavy metals.177 A variety of related catalytic systems have been exploited for lignocellulosic biomass conversion to 5-HMF. In these systems, the ILs assist the dissolution and fractionation of the biomass, and as a source of active sites for subsequent transformation of sugars to 5-HMF.17,32,53,72,75 For instance, corn stalk, rice straw and pine wood treated with CrCl3·6H2O/[BMIM][Cl] produced 5-HMF and furfural in 45–52% and 23–31% yields, respectively, after 3 min.178 In summary, IL solvent systems have shown a huge potential for the preparation of 5-HMF from biomass. They can be used for the pretreatment and conditioning of different lignocellulosic materials and for the saccharification of the polysaccharides into the sugars needed for the synthesis of furanic compounds. They also allow the integration of the pretreatment and transformation of different types of natural feedstocks into 5-HMF in a single process. However, there are still some challenges for the acceptance of this technology as a feasible alternative to conventional processes. Among them, we can highlight the reduction of the IL price, the decrease in viscosity, which is an important technical parameter, the improvement in cellulose dissolving capacity, the selection of ILs not providing additional environmental concerns, and the reduction of the conditions required to carry out the corresponding processes at ambient temperature. There is also a need to develop simple strategies for an efficient separation/recovery of all the elements (i.e., products, catalysts, ILs, and co-solvents) used in the process. A simple and efficient separation and reuse of the IL-phase can mitigate the concerns on their cost.179 Although different approaches have been proposed for the isolation of 5-HMF and the recovery of ILs, such as the use of supercritical CO2 or high-vacuum entrained distillation, or the use of membranes, these approaches are still far away from being suitable for large scale 5-HMF production. 4.2.3. Use of deep eutectic solvents (DESs) Deep eutectic solvents can also be used for the pretreatment of biomass (i.e., delignification and solubilization of cellulose) and as reaction media for its further conversion to 5-HMF.180,181 Deep eutectic solvents (DESs) are special liquid solvents resulting from the mixture of two components: a hydrogen-bond donor (HBD) and a hydrogen-bond acceptor (HBA) (Fig. 12).182,183 The mixture of these components, forming strong hydrogen-bonding interactions, presents an eutectic point temperature below that of an ideal liquid mixture. DESs share some of the properties of ILs (i.e., low volatility and nonflammability) but they are often easier to prepare, and typically display low cost, low toxicity for properly selected components, and can be prepared from highly biodegradable raw materials.184 Research into the use of DESs is still in the early stages of development especially compared to IL-based technologies, but they have already been successfully applied for biomass pretreatment, carbohydrate conversion, and lignin valorization. For instance, the pretreatment of eucalyptus wood with the DES prepared from choline chloride and lactic acid (ChCl–lactic acid) afforded >80% of delignification and 94% yield of glucose.185 The ChCl–citric acid DES was proposed as both a solvent and catalyst for the dehydration of fructose to 5-HMF and resulted in 78% yield of 5-HMF at 80 °C after 1 h.186 Fig. 12 Some examples of DESs used for the processing and transformation of lignocellulose. ChCL: choline chloride, LA: lactic acid, CA: citric acid, N2222: tetraethylammonium, and EG: ethylene glycol. Su and co-workers have developed the dehydration of glucose to 5-HMF under continuous flow conditions using CrCl3−[N2222][Cl]/EG as the catalytic system. A 42% 5-HMF yield was obtained at 150 °C using 10% glucose in only 3.64 min residence time, while under batch conditions only a 10% yield was reached in a much longer period (30 min).187 After the reaction, the products were extracted with ethyl acetate as the solubility of the DES and CrCl3 in ethyl acetate was negligible. The treated catalytic system could be efficiently recycled for four runs. Catalytic valorization of native biomass has been systematically evaluated in the choline chloride/oxalic acid DES system.188 This acidic DES promotes transformations of structurally branched glucans, fructans, or xylans into glucose (yields up to 68%), fructose (yields up to 60%), xylose (yields up to 73%), 5-HMF (yields up to 14%), or furfural (yields up to 72%). A choline chloride–formic acid–SnCl4·5H2O (ChCl–Fa–SnCl4) catalytic system has been reported for the transformation of herbal residues into furanic derivatives.189 Up to 67% total furfural and xylose yield from herbal residues of Heteropogon contortus (L.) Beauv was reached at 140 °C in 10 min. The same catalytic mixture led to a total yield of 93.8% of 5-HMF and glucose from herbal residues of Anemarrhena asphodeloides Bunge at 120 °C. A better understanding of the mechanistic aspects for both pretreatment and conversion of the biomass in DES media is required to be able to design more efficient systems. There is also a need to increase the thermal stability of the DES as well as developing efficient ways to isolate/recover the products and for the recycling of the DES-based systems. 4.2.4. Application of supercritical fluids The use of supercritical fluids (scFs) constitutes another promising alternative for the pretreatment, fractionation and conversion of biomass into furanic derivatives. ScFs present a series of properties (i.e., gas-like mass transfer with liquid-like solvation), which can be exploited to achieve a simpler fractionation/pretreatment, depolymerisation and conversion of the reducing saccharides into furanic derivatives. The use of high temperatures and pressures is an economic bottleneck for the widespread utilization of supercritical pretreatment techniques on a large scale. However, supercritical fluids are a mature technology currently industrially applied for a wide range of processes dealing with biomass (i.e., supercritical extraction). In the same cases, the additional cost can be compensated by not requiring the addition of any catalyst or solvent and the high quality of the building blocks obtained free of impurities that may hamper further valorization of the biomass (i.e., fermentation). In this context, sub- and supercritical CO2 have been evaluated to develop the concept of green biorefineries.190 Supercritical CO2 (scCO2) presents a low critical pressure and temperature (7.4 MPa and 31.0 °C) in comparison with other supercritical fluids (i.e., 22.1 MPa and 374.2 °C for scH2O). Furthermore, CO2 can be considered as a low cost, nontoxic, non-flammable, readily available solvent and can be easily removed from the final product by simple de-pressurisation.191 scCO2 has proved as a suitable tool to perform the treatment of lignocellulosic biomass prior to biological processing.192,193 Thus, scCO2 has been used for cellulose pretreatment, reducing its crystallinity by ca. 50% and enabling its enzymatic hydrolysis to glucose.194 An enhancement of up to 18–51% in the yield of reducing sugars can be achieved from dilute acid hydrolysis of scCO2-pretreated corncob, cornstalk, and rice straw.195 Other systems included H2SO4-catalyzed cascade hydrolysis–dehydration of rice husk enabled by CO2 extraction, which directly gave furfural in a high yield of around 90%.196 High-pressure CO2 in contact with water produces, in a reversible way, carbonic acid.197–199 This has opened the way to the direct use of this carbonic acid as an environmental friendly catalytic system for the dehydration of carbohydrates to 5-HMF.200 Yields of 5-HMF from fructose, glucose, sucrose, and inulin of ca. 67%, 36%, 50%, and 47%, respectively, at 190 °C and 2 MPa of CO2 in a mixture of 2-propanol/water have been reported.201,202 This evidences the capacity of CO2 to provide an acid catalyst for an environmentally friendly synthesis of 5-HMF from carbohydrates, achieving efficiencies comparable to those obtained with a typical homogeneous or heterogeneous acid catalyst. Supercritical water (scH2O) is another scF used for processing and transforming lignocellulosic materials.203 Subcritical and supercritical water exhibit unique characteristics, including increased diffusivity and decreased hydrogen bonding and dielectric constant. By varying the temperature and pressure of the water, its dielectric constant (ε) decreases, from ε ∼ 78 under room conditions to ∼6 at the vapor–liquid critical point, increasing dramatically, at this point, the solubility of organic compounds.204 On the other hand, the ion product of supercritical water is two orders of magnitude higher than that at ambient temperature. High concentrations of H+ and OH− can be obtained in SCW, and it is possible to provide a perfect environment for acid- or base-catalysed reactions.205,206 These properties can be exploited to develop hydrothermal pretreatment processes, under sub-critical and supercritical conditions, in the absence or presence of an additional acid catalyst, for the selective production of reducing sugars. A novel technology for the continuous and selective hydrolysis of cellulose and biomass to sugars has been reported based on the use of scH2O in a fast heating and fast cooling reactor. This reactor set-up together with the unique properties of the scH2O allows for the dissolution and hydrolysis of cellulose in a very short reaction time.207,208 At 400 °C and 25 MPa, a total recovery of C-5 sugars was achieved in 0.19 s, while the highest yield of C-6 sugars (65% w/w) was achieved at 0.22 s reaction time. The main hydrolysis product of C-6 and C-5 sugars was glycolaldehyde (20% w/w), while 5-HMF production was highly inhibited (yields lower than 0.5% w/w). The process for the hydrolysis of biomass in scH2O has been scaled-up from laboratory to pilot plant scale. Sugar beet pulp and wheat bran were used to validate the scaling up. The larger particle size (250 μm vs. ≤150 μm) used for the pilot plant scale slows down the hydrolysis. This reduction increased the selectivity and reduced sugar degradation (degradation yield < 15%). For sugar beet pulp, the yields for sugars were 55% and 66%, respectively for pilot plant and lab experiments, respectively. However, the selectivity reached values around 90% for both sugar beet pulp and wheat bran in the pilot plant.209 Low yields of 5-HMF have been reported for the treatment of fructose in supercritical water in the presence of 10 mM H2SO4 (35% selectivity, 80% conversion, 25 MPa, 180 °C, 600 s residence time). The substitution of scH2O by supercritical acetone in the presence of water (acetone : water 90 : 10) resulted in higher yields of 5-HMF (77% selectivity, 98% conversion, 20 MPa, 180 °C, 120 s residence time).210 Alternatively, when the reaction was carried out in scMeOH, the corresponding furfural ether was obtained (methoxymethylfurfural, MMF, 79% selectivity, 99% conversion, 20 MPa, 240 °C, 2 s residence time), while the reaction in subcritical acetic acid (scAcOH) afforded the corresponding furfural-ester although in lower yields (5-acetoxymethylfurfural, AMF, 38% selectivity, 98% conversion, 20 MPa, 180 °C, 120 s residence time).211 4.3. Critical assessment of potential of pretreatment technologies for carbohydrate extraction and furanic compound formation A comparison between the different pretreatment approaches based on the carbohydrate recovery and furan yield is summarized in Table 2. The hydrothermal pretreatment is cheap and effective for hemicellulose extraction by selective hydrolysis of ether bonds, resulting in a high sugar recovery. LHW and steam explosion do not require any chemical recovery after the process, but low product concentrations are recuperated without catalysts. Regulating different parameters (temperature, time, LSR, and dilute acid catalyst), the formation of 2-furfural can be promoted. This treatment can be applied in a wide range of biomass types, such as hardwood and softwood forestry and agricultural residues as well as food industries and food wastes. Main effects of lignocellulosic structure and furanic compound yield related to pretreatment technology Pretreatment Main effects on biomass structure Carbohydrate removal/recovery Key furanic compounds yields Hydrothermal LHW -Complete hemicellulose removal -Hemicellulose removal up to (100 wt%)47,59,83,84 -Low yield of 5-HMF and furfural in catalyst-free conditions (0.05–2 wt%)64,72 -Structural changes on cellulose Steam -Fiber separation -Xylan recovery (50–82 wt%)47,59 -Auto-hydrolysis and partial. Solubilization of the hemicelluloses -With catalysts conditions, CuCl2, up to 79.9% furfural and 12.2% 5-HMF production yields111 -Reduction of cellulose crystallinity Dilute acid -High recovery of sugars from hemicellulose -Hemicellulose recovery up to (98%)94 Furfural yield up to (11.09%)212 -Low recovery of sugar from cellulose -Xylan removal up to (87.8%)81 -Enhance cellulose accessibility to enzymes -Conversion of soluble hemicelluloses into fermentable sugars Neoteric Ionic liquids -Dissolution of cellulose and hemicellulose -Carbohydrate's removal (60–90%)163 -Catalyst-free conditions: 27% 5-HMF yield159 -With catalyst conditions: 54–80% 5-HMF yields159,161,168,169 DES -Liquid phase is rich in hemicellulose sugar -Glucose yields (32–94%)185,213 -5-HMF yields (42–78%)187,214 -Cellulose is in pretreated solid phase -Xylose yield up to (73%)188 -Furfural yield up to (70%)188,189 Supercritical fluids -Promote hemicellulose decomposition and decrease cellulose crystallinity -Glucose yields (14–51%)192 -5-HMF yields (18–67%)201,202 Supercritical fluid pretreatment is attractive because it is non-toxic and nonflammable to extract carbohydrates and transform them into furanic compounds. This method is effective for high cellulose and hemicellulose content biomass such as wheat straw, sugar beet pulp, oat hulls, apple pomace, and garden waste. However, similarly to hydrothermal treatment, it requires high temperature and pressure. In ionic liquid media, depending on the based solvent, complex polysaccharides can be solubilized by breaking β-O 4-bonds, thus enabling their application to a wide range of lignocellulosic biomass feedstocks, by fine tuning the treatment parameters and catalysis to achieve optimum lignocellulosic fractionation and conversion. One major advantage of ILs is the fact that they do not require high pressure, can be recyclable, and when adequately selected they can have low toxicity. However, this solvent has severe drawbacks that may hamper upscaling, the fact that they are not easy to prepare and some of them are considered toxic. With a similar mechanism, deep eutectic solvents have the same advantages over ILs since they tend to be cheaper and easier to formulate. Furthermore, DESs can degrade the physical structure of the biomass with minimal energy consumption during pretreatment, comparable with the chemical and physical methods in terms of energy consumption and conversion efficiency. This treatment type can be applied on biomass enriched in cellulose, such as spruce, beechwood, hybrid poplar, birchwood, black locust, fescue, miscanthus, hemp, and sawdust. The drawback of their high viscosity requires a high consideration on a pilot scale together with cost effective strategies for the recovery and reuse of the DESs in multiple cycles without compromising their performance. 5. Circular biorefinery as a transitional object of the bioeconomy Integrating lignocellulosic biomass as an alternative non-fossil resource is an incentive for developing a bio-based economy. Biorefineries are based on renewable feedstocks that can be assembled from several supply categories, available at low cost in a short time throughout the year. From an environmental point of view, the oil refinery is a main contributor to carbon dioxide and air pollution. Conversely, biomass-based biorefineries utilize a wide range of feedstocks: (i) cultivated biomass, whose growth contributes to carbon capture from the atmosphere and storage for varying periods of time,215,216 and (ii) exploited biomass which was earlier left untouched or burned for energy needs, causing a significant amount of greenhouse emission, and therefore its exploitation contributes to waste management and reduction in polluting gas emissions.217 Unlike petroleum-based refineries, where the process is costly due to the needed oxidation step to synthesize hydrocarbons, in biorefineries, renewable feedstocks are naturally rich in oxygen and converted into functional intermediates with lower energy.217 Shifting toward green and sustainable biorefineries is a prospect for the future circular bioeconomy. It can start with integrating biomass-based refineries with current petrochemical refineries by exploiting the current infrastructure, thus minimizing the startup cost of the biorefinery. In addition to that, several processes, notably chemically catalyzed reactions already used in petroleum refineries, can be adapted for biorefineries. Technoeconomic assessment is a needed tool to design novel circular biorefinery schemes. This analysis starts with an economic analysis of process scalability from the laboratory to pilot and industrial scales. Based on the laboratory study, the equipment cost can be estimated with the scaling exponent for each equipment (eqn (4))218 and adjusted to the base year using the Chemical Engineering Plant Cost Index (CEPCI) expressed by (eqn (5)).2194 where CAE: calculated actual equipment, CBE: base cost equipment, EC: equipment capacity, EBC: equipment base capacity, and f: empirical scaling exponent.5 where AC = adjusted cost, Co: reference cost, CEPCI: studied equipment price, and CEPCIo: equipment base price. The pretreatment method cost is directly associated with capital expenditure (CAPEX) and operational expenditures (OPEX). CAPEX consists of start-up and fixed costs (FC), which englobes equipment. Regarding hydrothermal and supercritical fluid treatment, the reactor vessel is the most expensive in capital cost.220 For example, in LHW pretreatment (14.33 × 106 L, at 180 °C, 10 min, and 20% solid : water ratio), a fixed capital investment of (US$ 102 × 106) represents 28.17%.219,221 In subcritical conditions, the pretreatment of lignocellulosic biomass for industrial processes of the order of 310 tons of feedstock daily represents US$ 27.4 × 106 of the FC, and 35% (US$ 9.6 × 106) is associated with the reactor.219,222 However, in the case of the ILs and DESs, the capital cost of the reactor is expected to be lower than that of supercritical fluids and hydrothermal treatment, which require special instrumentation for high temperatures and excessive pressure.155 OPEX are subcategorized into direct production costs, including raw material, waste steam charges, and fixed operating costs, including labor and general expenses.223 In the case of supercritical fluid and hydrothermal pretreatment, the energy demand dominates as the major operating cost and represents around 70% of OPEX.224 For example, the needed heat required for lignocellulosic biomass treatment under these conditions varied between 610 kJ kg−1225 and 1096 × 106 kcal day−1.222 However, the hydrothermal process is carried out without using chemicals or catalysts.219 And among supercritical fluids, scCO2 presents the lowest cost compared to other supercritical fluids.139 Unlike HT and ScFs, OPEX in IL and DES-based biorefineries concerns mainly chemicals. ILs suffer from high costs ranging from $20 to 101 kg−1. A bulk-scale of 1-methylimidazolium hydrogen sulfate was studied in order to decrease the cost index ratio to 1.47 and an exchange rate of 1.1 $ €−1.155 However, DESs are cheaper than ILs and easily recyclable, where operating costs dropped significantly from $1573.56 million to $1060.03 million when DES recycling time increased from 5 to 20.226 Optimizing the total cost in biorefineries is recommended depending on the base treatment type. For example, in hydrothermal conditions using higher solid loading can improve energy efficiency,221 and implementation of renewable energy, like wind farms, can contribute to achieving lower prices of electricity.227 Concerning DES and IL-based pretreatment, some improvements can be made by reducing costs and loading, and increasing recycling.226,228 6. Conclusions and outlook It is an unquestionable fact that fossil resources available for the world population are facing environmental, economic, and social challenges. As an alternative, biomass constitutes an inexhaustive source for platform molecules such as 5-hydroxymethylfurfural and 2-furfural. Both of them can be derived from lignocellulosic carbohydrates to form new chemicals and bio-based polymers. Therefore, a screening of chemical identification and quantification of major molecules of four categories of LCB, including forest biomass and grass, agricultural and agri-food residue, energy crops, and residues from non-food sectors, showed that these renewable resources revealed a considerable potential within the European context to be exploited for furanic compound formation aiming at their use in the bioplastic industry. In a green chemistry context, an updated overview of ecofriendly pretreatment technologies was comparatively reported to efficiently convert them into furans. Regarding the hydrothermal pretreatment in neat water, hemicellulose is completely solubilized and recovered in the liquid fraction. Solubility of biomass, hemicellulose removal and liquid fraction composition are strongly related to the severity and the process parameters, mainly temperature and time. Furfural and 5-HMF yields are increased under more severe conditions, but attention should be paid to avoid carbon loss due to humin formation. Alternatively, the formed humins can be upgraded by recovering them followed by added-value material production. Aiming to increase the isolation of hemicellulose as xylose monomers in the liquid fraction, dilute acid pretreatment at relatively lower temperature is preferred, coupled with controlled downstream dehydration of xylose to furfural. Contrarily, neoteric solvents solubilize carbohydrates and result in high furanic compound yield. Lignocellulosic biomass is an attractive renewable source to be exploited for bio-based molecule production for new polymers and additives. However, there are challenges for the wide-scale commercialization of these value-added products to satisfy the community demands. As a starting premise, it is recommended that future investigations for further industrial development take into account the following gaps: • Tackle the whole value chain system including (i) feedstock cultivation, harvesting, collection, transport, and storage; (ii) processes concerning biomass pretreatment technologies and monosaccharide extraction; (iii) intermediate molecule production notably 5HMF and 2-F, (iv) and finally desired products such as chemicals, biomaterials, and biofuels. • Ensure sustainable biomass supply regarding geolocation, storage, and transport logistics. • Manage development practices of standardized process techniques to achieve the most adequate process conversion. • Establish different combined approaches using efficient green processes to fulfil sustainable production. • Provide an action plan to adapt commercialization and eventually application of produced furanic products. Abbreviations 2-F 2-Furfural 2-MTHF 2-Methyltetrahydrofuran 5-HMF 5-Hydroxymethylfurfural AOS Arabino-oligosaccharides BFDCA 2,2′-Bifuran-5,5′-dicarboxylic acid BHF 2,5-Bis(hydroxymethyl)furan ChCl Choline chloride CMF 5-(Chloromethyl)furfural DES Deep eutectic solvents DFF Diformylfuran EMF 5-Ethoxymethylfurfural FA Furfuryl alcohol FDCA Furan-2,5-dicarboxylic acid GVL γ-Valerolactone HBA Hydrogen-bond acceptor HBD Hydrogen-bond donor HT Hydrothermal pretreatments ILs Ionic liquids LCB Lignocellulosic biomass LHW Liquid hot water LSR Liquid-to-solid ratio MIBK Methyl isobutyl ketone PEF Poly(ethylene 2,5-furanoate) PET Poly(ethylene terephthalate) scCO2 Supercritical CO2 ScFs Supercritical fluids SE Steam explosion THF Tetrahydrofuran XOSH Xylo-oligosaccharides XOSL Xylo-oligosaccharides Conflicts of interest There are no conflicts to declare. Supplementary Material This publication is supported by COST Action FUR4Sustain—European network of FURan based chemicals and materials FOR a Sustainable development, CA18220, supported by COST (European Cooperation in Science and Technology). This work was developed within the scope of CICECO—Aveiro Institute of Materials (UIDB/50011/2020 & UIDP/50011/2020) & LA/P/0006/2020, financed by national funds through the FCT—Fundação para a Ciência e a Tecnologia/MEC (PIDDAC). This research is also sponsored by FEDER funds through the program COMPETE—Programa Operacional Factores de Competitivid-ade—and by national funds through the FCT under the project UID/EMS/00285/2020. The FCT is also acknowledged for the re-search contract under Scientific Employment Stimulus to A. F. S. (CEECIND/02322/2020). Our final words go to our dearly remembered college and friend Anna Aladjadjiyan. ==== Refs References Manzanares P. 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==== Front Environ Sci Technol Environ Sci Technol es esthag Environmental Science & Technology 0013-936X 1520-5851 American Chemical Society 37014786 10.1021/acs.est.3c00267 Article Molecular Dynamics Simulation Prediction of the Partitioning Constants (KH, Kiw, Kia) of 82 Legacy and Emerging Organic Contaminants at the Water–Air Interface https://orcid.org/0000-0002-4734-8667 Lemay Amélie C. *†∥ Sontarp Ethan J. ‡∥ Martinez Daniela † Maruri Philip † Mohammed Raneem † Neapole Ryan † Wiese Morgan † https://orcid.org/0000-0002-4018-6466 Willemsen Jennifer A. R. † https://orcid.org/0000-0002-5265-7229 Bourg Ian C. †§ † Department of Civil and Environmental Engineering, Princeton University, Princeton, New Jersey 08544, United States ‡ Department of Geosciences, Princeton University, Princeton, New Jersey 08544, United States § High Meadows Environmental Institute, Princeton University, Princeton, New Jersey 08544, United States * Email: alemay@princeton.edu. 04 04 2023 18 04 2023 57 15 62966308 10 01 2023 23 03 2023 22 03 2023 © 2023 American Chemical Society 2023 American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). The tendency of organic contaminants (OCs) to partition between different phases is a key set of properties that underlie their human and ecological health impacts and the success of remediation efforts. A significant challenge associated with these efforts is the need for accurate partitioning data for an ever-expanding list of OCs and breakdown products. All-atom molecular dynamics (MD) simulations have the potential to help generate these data, but existing studies have applied these techniques only to a limited variety of OCs. Here, we use established MD simulation approaches to examine the partitioning of 82 OCs, including many compounds of critical concern, at the water–air interface. Our predictions of the Henry’s law constant (KH) and interfacial adsorption coefficients (Kiw, Kia) correlate strongly with experimental results, indicating that MD simulations can be used to predict KH, Kiw, and Kia values with mean absolute deviations of 1.1, 0.3, and 0.3 logarithmic units after correcting for systematic bias, respectively. A library of MD simulation input files for the examined OCs is provided to facilitate future investigations of the partitioning of these compounds in the presence of other phases. All-atom molecular dynamics simulations predict the adsorption and partitioning of 82 legacy and emerging organic contaminants at the water−air interface. adsorption PFAS phthalate PCB PAH paraben micropollutant aerosol National Science Foundation 10.13039/100000001 CBET 1931611 Princeton University 10.13039/100006734 NA U.S. Department of Energy 10.13039/100000015 DE-AC02-05CH11231 document-id-old-9es3c00267 document-id-new-14es3c00267 ccc-price ==== Body pmcIntroduction A key hindrance to the mitigation of organic contaminants (OCs) is insufficient knowledge of the thermodynamic constants that reflect their affinity for different phases and interfaces in natural and engineered systems.1−9 This limited availability of fundamental thermodynamic data reflects a widening gap between, on the one hand, the ability of regulatory agencies to carry out experimental characterizations of substances that can be highly hazardous or challenging to synthesize and, on the other hand, the rapidly increasing diversity of OCs illustrated by the nearly 70 000 chemicals or mixtures registered globally in the past decade10,11 or by the nearly 4000 applications for regulatory approval submitted to the US Environmental Protection Agency (EPA) between 2016 and 2021.12 Efforts to predict the partitioning of OCs in natural and engineered systems have focused primarily on the development of approaches that rely on correlations against various properties of individual OC molecules (including chemical formula, functional groups, size, conformation, polarity, and other properties predicted using various levels of refinement up to quantum chemical calculations) trained using pre-existing partitioning data.3,13−18 Alternatively, the well-established COSMOtherm approach relies on quantum mechanical simulations of individual organic solutes carried out using a dielectric continuum approximation for the solvent.19,20 Efforts to cross-validate and benchmark existing approaches such as SPARC, COSMOtherm, SIMPOL.1, and poly-parameter linear free energy relationships (ppLFERs) indicate that, for water–air partitioning and related properties (e.g., saturation vapor pressure), these approaches differ from each other and from experimental results by ∼1 order of magnitude for sparsely functionalized, weakly soluble compounds.3,21,22 For more soluble, highly functionalized compounds (properties common to many contaminants of emerging concern),23 however, different well-established approaches deviate by up to 10 orders of magnitude, and their predictive utility is therefore highly uncertain.3 All-atom molecular dynamics (MD) and density functional theory (DFT) simulations provide a potential alternative approach to filling the knowledge gap outlined above by enabling predictions of the partitioning behavior of environmental contaminants in explicitly simulated phases and multiphase systems. The promise of this approach is illustrated by its use in drug discovery and toxicology.24−28 In the environmental sciences, increasing numbers of studies over the last two decades have used all-atom simulations to gain insight into the local coordination, energetics, and dynamics of OCs in natural systems.29−35 However, few of these studies have generated information on the equilibrium constants controlling partitioning between phases or adsorption at interfaces.36−42 Here, we evaluate the feasibility of using all-atom MD simulations to generate thermodynamic data for OC partitioning in natural systems. We focus on systems containing water, air, and a water–air interface as a simple environmental multiphase system with important implications in contaminant fate and transport.43−45 To evaluate OC partitioning, we create and test an open library of input files for a diverse set of 82 OCs. The compounds examined span those produced and released with minimal regulation prior to 1970 and categorized as legacy contaminants (benzene, formaldehyde, polychlorinated biphenyls, tetrachloroethylene, polycyclic aromatic hydrocarbons, etc.) to more recently produced and less regulated contaminants of emerging concern (nonionic surfactants, per- and polyfluoroalkyl substances, phthalates, pyrethroid pesticides, etc.) as well as ubiquitous pharmaceutical products (acetylsalicylic acid, ibuprofen, naproxen) and compounds naturally present at the water–air interface (acetic acid, glyoxal, isoprene). For each compound, we determine its Henry’s law constant, KH, and its interfacial adsorption coefficients from water and air, Kiw and Kia. We compare our results with published experimental data to quantify the accuracy and precision of our predicted thermodynamic constants. Finally, we examine our simulation results to gain insight into the relation between these constants and the molecular structure of the organic compounds. Simulation Methodology Database of Organic Contaminants The list of 82 compounds examined here is presented in Table SI1. This list contains similar numbers of legacy contaminants, for which experimental data exist to verify partitioning and adsorption at the water–air interface for many compounds, and contaminants of emerging concern, which the US EPA and National Science Foundation (NSF) have identified as key research priorities, for which fewer experimental data exist. The contaminants span those classified as per- and polyfluoroalkyl substances (PFAS), bioaccumulative endocrine disruptors detected in the blood of 98% of Americans;46,47 nonsteroidal anti-inflammatory drugs (NSAIDs), ubiquitous pharmaceutical chemicals detected in drinking and groundwater that can cause adverse ecotoxicological effects;48,49 phthalates, plasticizers widely used in personal care products with known epigenetic and endocrine-disrupting toxicological effects;50−52 polycyclic aromatic hydrocarbons (PAHs), a key category of carcinogenic and/or mutagenic compounds associated with combustion processes;53−55 polychlorinated biphenyls (PCBs), a class of industrial persistent organic pollutants (POPs) that remains one of the predominant threats to water and air quality worldwide;53−58 and multiple other compounds of concern in environmental or human health.23,59−63 The list also includes seven compounds that are naturally present at or near the water–air interface, including biogenic semivolatile organic compounds that play important roles in aerosol chemistry and indoor air quality.64,65 Input files for the compounds in Table SI1 were developed using the OPLS-AA model, a general-purpose interatomic potential model widely used for MD simulations of systems containing organic compounds and liquid water.66,67 The OPLS-AA model represents the potential energy of organic-water systems as a sum of pairwise van der Waals and Coulomb interactions (represented, respectively, using the Lennard-Jones 6–12 model and a fixed partial charge for each atom) as well as contributions associated with intramolecular bond lengths, angles, and torsions. For compounds that carry ionizable groups, the protonation state was selected to represent the predominant species at pH 7. For compounds that contain uncommon atom linkages for which bond, angle, or torsion parameters do not exist in the OPLS-AA model, the parameters for the most similar linkages were used as noted in the comments of the input file for each compound. The program Moltemplate68 was used to develop the compound input files. A database of input files for carrying out MD simulations of the compounds in Table SI1 using the open-source program LAMMPS69 is available at [https://github.com/amelielemay/MD-Contaminants]. Molecular Dynamics Simulations Simulations were carried out using the code LAMMPS on the Cori supercomputer at the National Energy Research Scientific Computing Center (NERSC) and on the Tiger CPU cluster at Princeton University. The simulation cell size was 32 × 120 × 32 Å3 with periodic boundary conditions in all directions. Simulated systems contained a 60-Å-thick slab of liquid water (5700 water molecules) adjacent to a 60-Å-thick slab of void space, with a single organic molecule located near one of the water–air interfaces (Figure 1A). For charged compounds, charge balance was ensured by the addition of Na+ or Cl– counterions. To prevent the location of the water–air interface from drifting during the simulations, the overall center of mass of the water O atoms was fixed in the y direction. In addition, a 10-Å-thick reflective wall was placed in the void space at each end of the simulation cell to prevent evaporated water molecules from drifting to the neighboring periodic image of the simulation cell in the y direction. Simulations were carried out in the canonical (NVT) ensemble at a temperature of 298 K using a Verlet algorithm with a timestep of 1 fs. Interatomic interactions were resolved in real space up to a cutoff of 15 Å. Coulomb interactions beyond this cutoff were resolved in reciprocal space using the particle–particle, particle mesh (PPPM) method70 with a precision of 99.9%. Water molecules were kept rigid using the SHAKE algorithm.71 Figure 1 Illustration of our MD simulation methodology: (A) snapshot of a simulated system showing an organic molecule at the water–air interface (C, O, and H atoms in cyan, red, and white, respectively); (B) density profiles of water O atoms and organic atoms (blue and red lines) as a function of distance normal to the water–air interface (y) in a standard (“unbiased”) MD simulation, averaged over 40 ns of simulation; (C) free energy profiles of the organic contaminant as a function of y determined either from the density profile of the organic molecule center of mass in an unbiased simulation (black line) or using the umbrella sampling (US) methodology (gray line). Molecular dynamics simulations solve Newton’s equations of motion for a many-particle system using semiempirical interatomic potential parameters.72 As such, simulation predictions are sensitive to the choice of interatomic potential model. Here, we used the OPLS-AA model for organic molecules,66 the Dang–Smith model for Na+ and Cl–,73 and the simple point charge (SPC) model for water.74 These models have been extensively used to predict the conformation and coordination of organic compounds in water.28,75,76 The few existing comparative studies on the relative ability of different interatomic potential models to predict organic compound partitioning at the water–air interface suggest that the combination of the SPC and OPLS-AA models is among the most accurate.36,76−78 To gain insight into the sensitivity of the simulation predictions to the choice of interatomic potential models, a subset of the simulations was repeated using the extended simple point charge (SPC/E) water model (Figure SI4).79 Future investigations should more extensively characterize the sensitivity of simulation predictions to the choice of interatomic potential models, including other general-purpose organic models such as AMBER,80 CHARMM,81 and GROMOS82 and other water models such as TIP4P2005.83 Calculation of the Free Energy Profile Two methods were used to determine the free energy (FE) profile of OCs as a function of distance from the interface. In the first method, applied for a subset of the simulated compounds, standard (“unbiased”) MD simulations were carried out with the OC initially located near one of the interfaces. Simulations were equilibrated for 423 ps in the NVT ensemble, after which production runs were carried out for 40 ns in the same ensemble. Average density profiles of water O atoms (ρOW) and of the center of mass of the organic molecule (ρOM) were determined as a function of y (Figure 1B). Finally, the FE of the organic compound as a function of y, relative to an arbitrary reference, was calculated as ΔF ∝ −RT ln ρOM. The resulting ΔF values, obtained in the NVT ensemble, are Helmholtz free energies, but they are essentially identical to Gibbs free energies as Py ≈ 0 in our simulated systems. As illustrated by the black line in Figure 1C, a limitation of the resulting FE profiles is their limited extent in the y direction, as the unbiased method only yields precise estimates of FE in the narrow range of y values that is extensively sampled by the organic molecule. To characterize the FE profiles of organic compounds in a wider range of y coordinates spanning the two bulk fluids, we used the umbrella sampling (US) method, a “biased” MD simulation approach.37,38,77,84 In the US method, a harmonic biasing potential is used to tether the compound to a certain reaction coordinate. The resulting analysis yields the FE profile in the vicinity of the tethering location. The method is repeated for different tethering locations. Finally, the results of the different biased simulations are combined using an iterative calculation, the weighted histogram analysis method (WHAM), that determines the optimal offsets between the FE profile sections obtained with different tethering locations.85 In this study, each compound was tethered to a range of y coordinates that spanned from bulk air to bulk water. The tethering locations were separated by intervals of 2 Å. Each simulation was preceded by 9.2 ps of equilibration with no biasing potential, 13 ps of equilibrium during which the harmonic force constant was increased to 0.1 eV Å–2, and 400 ps of equilibration with a fixed harmonic force constant of 0.1 eV Å–2. Finally, production runs were carried out for 40 ns with a minimum of 11 tethering locations, such that each FE profile required at least 440 ns of simulation. Results obtained with different tethering locations were combined using 104 iterations of the WHAM algorithm. A representative example of the resulting free energy profiles is shown as a gray line in Figure 1C. Results and Discussion Free Energy Profiles Predicted FE profiles for the 82 compounds examined here are presented in Figure SI1. Profiles are reported as average values calculated over 40 ns of simulation at each tethering location, with error bands indicating the precision (95% confidence intervals) evaluated by dividing each 40 ns simulation into four 10 ns intervals. Reported FE profiles were normalized relative to the average value in bulk water, defined as the FE plateau in the 1 Å range of y coordinates immediately below the maximum tethering location. Profiles are also reported for a subset of the compounds in Figure SI2 based on unbiased simulations. For all compounds for which both US and unbiased simulations were carried out, good agreement was observed between the shapes of the FE minima determined by both methods. Several notable trends emerge from the collection of ΔF profiles in Figure SI1. First, almost all compounds examined here exhibit surfactant-like properties with a FE minimum in the vicinity of the water–air interface. This minimum had an average depth of 15.0 ± 7.6 kJ mol–1 relative to the value in bulk water (where the range reflects ±σ, where σ is the standard deviation (SD) of the 75 values with FE minima at the interface) and was located 0.3 ± 0.8 Å above the water surface. This minimum indicates that most compounds examined here exhibit a strong preference (relative to both bulk water and air) for being located at the interface, where they can form favorable interactions with water molecules without strongly disrupting the water hydrogen bond network. Second, the FE profiles of charged compounds do not stabilize in the vapor phase, as shown in Figure 2, indicating that exsolution of ionic species is highly unfavorable. The three zwitterions in the database (glyphosate, glufosinate, PFAS-5-3-FtB) also did not reach a stable ΔF value in the air phase. Although we are not aware of any previous reports of the free energy profiles of organic ions or zwitterions across the water–air interface, such a profile was reported recently for Cl– at a flat water–air interface86 and previously for Cl– at water–organic liquid interfaces87,88 These results indicated that the free energy profile of Cl– only begins to stabilize at distances >15 Å beyond the water surface and that accurate sampling of the free energy is complicated by hysteresis in the formation of water fingers and the residual hydration of the ion in air. For this reason, the Henry’s law constant (KH) and the adsorption constant relative to air (Kia) were not determined for the charged compounds, and only the adsorption constant relative to water (Kiw) is reported for these compounds. This finding is consistent with the expectation that, for compounds that exist predominantly in ionized form in liquid water, measured KH values reflect the exsolution of the less predominant uncharged form.6 Prediction of such KH values using the methodology used here (where molecules cannot spontaneously gain or lose protons) would require simulating the uncharged form of these compounds and adjusting the predicted FE of evaporation to account for their FE of protonation or deprotonation. For compounds that have pKa values near 7, our predicted Kiw and Kia values should be treated with caution as the protonation state of organic compounds may vary in the vicinity of the water–air interface.89,90 Figure 2 Representative subset of FE profiles obtained for neutral and charged compounds (upper and lower panels, respectively). The uncharged compounds display an FE plateau in both phases at distances about 10 Å above or below the water surface (located at y = 36.45 Å). For the charged compounds, a plateau is not observed in the vapor phase on the length scales examined by our simulations. Third, 32% (26 of 82) of the profiles exhibit a ΔF barrier of at least 0.25 kJ mol–1 located about 5 Å below the water surface, as reported in some previous MD simulation studies.37,91 This barrier indicates the existence of an organic-depleted region on the water side of the interface, near the location of the second monolayer of water molecules at the interface,92 with possible implications for mass transfer and chemical transformation kinetics of organic compounds at the water–air interface.93 A full list of the compounds exhibiting such a barrier is given in Table SI2, and a subset is shown in Figure 3. Figure 3 Representative subset of FE profiles exhibiting an energy barrier between the FE minimum at the interface (near the Gibbs dividing surface of water at y = 36.45 Å, indicated as a vertical dashed line) and the FE plateau in bulk liquid water (around 45 Å). Partitioning between Water and Air The Henry’s law solubility constant, KH, is the quotient of the concentration of a dissolved compound in water (mol m–3) and its partial pressure in air (Pa) in the limit of infinite dilution. We determined the value of this constant for each of the compounds in our library from its FE of evaporation, ΔFevap = ΔFa – ΔFw. The FE in bulk air, ΔFa, was calculated as the average value in the 1-Å-wide range of y values directly above the minimum tethering location, while the FE in bulk water, ΔFw, was calculated as the average value in the 1-Å-wide range of y values directly below the maximum tethering location. The Henry’s law solubility constant was then calculated as KH = (1/RT) × exp(ΔFevap/RT), where T is the absolute temperature (298 K) and R is the ideal gas constant. Our predictions of KH are reported as log(KH), with 95% confidence intervals calculated as 2σ/√n, where σ is the SD of predicted log(KH) values estimated from n = 4 distinct FE profiles calculated from the four 10 ns intervals of each simulation. Our predicted values of log(KH) are compiled in Table SI1. To evaluate the potential existence of a systematic bias in our MD simulation predictions of log(KH), predicted values are presented in Figure 4A as a function of the extensive database of log(KH) values compiled by Sander.94 The values from the Sander compilation used here were those listed as being most reliable, designated in the compilation with an M, L, or V. Confidence intervals were assigned to the Sander values as 2σ/√n, where n is the number of reliable KH values listed for each compound and σ = 0.190 is the estimated SD of experimental log(KH) values, computed as the average SD of log(KH) for the 10 compounds with the most reported KH values in Sander’s database. Compounds with no highly reliable experimental KH values in Sander’s database are omitted from Figure 4. Compounds for which approximations in atoms linkages or charges were made because there were no existing parameters in the OPLS-AA database (as indicated in Table SI1) were omitted from Figure 4A and the calculation of the regression line (they are included in Figure 4B). Finally, decabromodiphenyl ethane (DBDE) was excluded from both plots because it is an outlier that differs from the experimental value by 6.36 log units. This molecule also differs from others in our dataset by its high abundance of aromatic C–Br linkages, suggesting that the OPLS-AA parameters for this functional group may require further evaluation. Figure 4 Predicted vs average measured log(KH) values (with KH in mol m–3 Pa–1) based on (A) MD simulation results and (B) bias-corrected MD simulation results. The error bars indicate 95% confidence intervals on the predicted and average measured values. As rough scales for the polarity and size of simulated OCs, we use the sum of the magnitudes of the partial charges and van der Waals radii, respectively, of the atoms in the OPLS-AA model of each organic molecule. Linear regression of the results presented in Figure 4A reveals a systematic bias between the values predicted by our simulations and the measured values characterized by the relation log(KH,predicted) = 0.6885 × log(KH,measured) – 1.5136. In short, MD simulations carried out using the SPC and OPLS-AA interatomic potential models tend to underestimate the affinity of organic compounds for water vs air, particularly for more hydrophilic compounds. Most of this bias likely reflects the approximate nature of the interatomic potential models. However, part of it could possibly be associated with the experiments; for example, measurements using the inert gas stripping method have been found to overestimate KH, in some cases by several orders of magnitude, if their interpretation does not account for adsorption at the water–air interface.8,17,95,96 Results presented in Figure 4A suggest that MD simulation predictions of log(KH) obtained with the OPLS-AA and SPC models can be empirically corrected for their systematic bias using the relation log(KH,corrected) = [log(KH,predicted) + 1.5136]/0.6885. The resulting bias-corrected simulation results, presented in Figure 4B, match the average measured log(KH) values with a mean absolute difference (MAD) of 1.1 logarithmic units. The accuracy and precision of our simulation predictions (reflected by the systematic bias and by the MAD after bias correction) are commensurate with those reported for established methods used to predict log(KH). For example, a comparison of log(KH) values predicted using SPARC, COSMOtherm, and ppLFER approaches for natural organic compounds with 0 to 3 functional groups (consistent with most compounds examined here) yielded an average MAD of 1.5 log units between different approaches,3 implying an MAD of at least 0.8 log units between the predictive models and experimental results. The outliers for which our bias-corrected simulation predictions deviate from the experimental values by three or more logarithmic units are hexachlorobenzene (−1.73, 3.23), 1,3-dichlorobenzene (−2.47, 1.08), and clofenotane (DDT) (−0.325, −3.54). DDT is a compound for which charge adjustments and atom type substitutions were made as it contains unusual combinations of functional groups for which OPLS-AA model parameters are not available. Our results suggest a need to develop OPLS-AA parameters for this compound. The mismatch observed for hexachlorobenzene and 1,3-dichlorobenzene is unexpected as no adjustments were made for these compounds. The other benzene derivatives (benzene, ethylbenzene, 1,2- and 1,4-dichlorobenzene, toluene, 4-chlorophenyl, and 4-bromophenyl phenyl ether) match the experimental data well; therefore, we cannot conclusively state why two of the benzene-derived compounds deviate from the experimental values. Adsorption at the Water–Air Interface The interfacial adsorption coefficients, Kiw and Kia, are the quotients of the surface excess of a compound at the water–air interface, Γi (mol m–2), and its concentration in bulk water or air, Cw or Ca (mol m–3), respectively. The interfacial surface excess Γi is conventionally defined relative to the Gibbs surface of water using the relation1 where ρOM(y) is the density profile of the organic compound as a function of distance in the direction normal to the interface, yi is the location of the Gibbs surface of water (i.e., the interface location imposed such the surface excess equals zero for water), and ymin and ymax are coordinates in bulk air and bulk water. Based on eq 1 and the Boltzmann relation ρOM ∝ exp(−ΔF/RT), the interfacial adsorption coefficient Kiw = Γi/Cw is obtained from the FE profile through the relation2 The interfacial adsorption coefficient Kia = Γi/Ca is determined in an analogous manner. The integrals in eqs 1 and 2 were evaluated from predicted FE profiles using a small-rectangle approximation with a bin width of 0.1 Å. Results are presented in Table SI1. Experimental values of Kiw and Kia are far less commonly reported in the literature than KH. A literature search for experimental Kiw and Kia values of the compounds in our database resulted in 11 and 10 values, respectively. We chose not to convert between published Kiw and Kia values using the relationship KHRT = Kiw/Kia due to the uncertainty associated with KH values for compounds for which there are few experimental data. Additionally, we excluded 1,3-dichlorobenzene from our comparison and regression computation, as it is one of the three outliers in Figure 4B. Published log(Kiw) values for six compounds (benzene, ethylbenzene, toluene, 1,1,1-trichloroethane, tetrachloroethylene, trichloroethylene) as reported by Hoff97 (based on a gas chromatography-based method that measures Kiw and Kia simultaneously) and for four PFAS (PFOA, PFOS, PFBS, PFHxS) as compiled by Brusseau98 (based on surface tension measurements) are compared with our simulation predictions in Figure 5A. Experimental log(Kia) values for nine compounds (benzene, ethylbenzene, 1,2-dichlorobenzene, 1,4-dichlorobenzene, toluene, tetrachloroethylene, 1,1,1-trichloroethane, 1,4-dioxane, trichloroethylene) reported by Hoff et al.97 and Kelly et al.99 are compared with our simulation predictions in Figure 5B. Figure 5 Predicted vs measured log(Ki) values (with Ki in cm) based on (A) interfacial-water coefficients (Kiw) and (B) interfacial-air coefficients (Kia). Results shown in Figure 5A reveal that our predicted log(Kiw) values are consistent with experimental results, with a MAD of 0.5 log units prior to bias correction. A regression line plotted through both PFAS and legacy compounds is statistically consistent with a 1:1 relationship (slope of 1.3 ± 0.6, intercept of 1.3 ± 2.6, reflecting the t0.975, n–2 confidence intervals). If PFAS and legacy compounds are considered separately, our MD simulation results tend to overestimate reported log(Kiw) values for the former and underestimate log(Kiw) for the latter. This may reflect a bias in our simulations associated with the OPLS-AA model parameterizations of C–F, C–H, and C–Cl bonds. Alternatively, it also may reflect a bias in the experimental database due to the different methodologies used to measure log(Kiw) values for PFAS vs legacy contaminants in Figure 5A. Results shown in Figure 5B indicate that predicted log(Kia) values generally underestimate the experimental values, with a mean difference of −1.4 logarithmic units. Linear regression of the results in Figure 5B yields a slope of 0.5 ± 0.2 (intercept of −3.4 ± 0.8), which indicates that the simulations increasingly underestimate log(Kia) with increasing compound hydrophilicity. In short, our simulations underestimate the propensity of OCs to adsorb from the air phase onto the surface of liquid water, particularly for the more hydrophilic compounds. Overall, our results shown in Figure 5 suggest that the systematic bias in predicted log(KH) values in Figure 4A results from an underestimation of the tendency of OCs (and particularly of hydrophilic OCs) to adsorb from air onto the water surface, whereas the simulations correctly predict the partitioning of OCs between the interface and bulk water. This finding is consistent with the fact that the OPLS-AA model was developed to represent organic compounds in condensed phases (such as water, hydrocarbon fluids, biological matrices) rather than in gas phases.100 We note, in passing, that our log(Kiw) results for PFAS shown in Figure 5A may help resolve a debate regarding the suitability of the Langmuir and Freundlich isotherms for PFAS adsorption at the water–air interface.98,101−104 The consistency of our predictions with the low-salinity, low-concentration results compiled by Brusseau98 suggests the existence of a finite log(Kiw) value at infinitely low loading, in agreement with the Langmuir model. Sensitivity to the Interatomic Potential Models As a preliminary evaluation of the sensitivity of our predictions to the choice of interatomic potential model, seven of the compounds in our library were simulated using the SPC/E water model in addition to the SPC model. The only difference between the SPC and SPC/E water models is that the latter assigns 3% larger partial charges to water O and H atoms, resulting in a larger dipole moment and stronger hydrogen bonds. Free energy profiles predicted with the two water models are compared in Figure SI2. Our results show that the SPC model yields higher log(KH) values and lower or similar log(Kiw) values, with mean differences of 0.4 and −0.5 logarithmic units, respectively. In other words, the SPC/E model predicts a smaller affinity of OCs for bulk water. Log(KH) values predicted using the SPC model more closely match experimental values for five of the six compounds with reliable published values. Implications for Environmental Partitioning The environmental implications of our KH and Kiw values are illustrated in Figure 6, a partitioning space plot indicating the phase or interface that holds the largest quantity of each compound in a generic warm cloud at 298 K. Dashed lines delineate regions in log(KH)–log(Kiw) space where the associated compounds have greater total abundance in water, air, or at the interface, which depend on the relative values of water volume Vwater, air volume Vair, and water–air interfacial area Ainterface in the system of interest. A representative warm cloud was assumed to have an aqueous phase consisting of spherical droplets with a radius of 10–5 m and a volume fraction of water of 3 × 10–7 m3/m3.105 The charged compounds, for which exsolution is so unfavorable that KH could not be quantified, are drawn as red symbols near the right edge of the diagram. Figure 6 Partitioning space plot15,105 showing the expected phase in which each compound would primarily be located in a cloud at 298 K (with Vwater/Vair = 10–7 and Vwater/Ainterface = 3.3 × 10–6 m). Values noted on the left and lower axes indicate the log(Kiw) values and bias-corrected log(KH) values of each compound as predicted using our MD simulations. Values noted on the right and upper axes indicate the locations in log(KH)–log(Kiw) space where the three dashed boundaries would intersect in systems with different Vwater/Ainterface and Vwater/Vair ratios. Specifically, the Vwater/Ainterface ranges indicated along the right axis correspond to different water droplet diameters, with approximate ranges shown for ultrafine aerosols (∼10–9 to 10–8 m), fog droplets (∼10–6 to 10–5 m), and rain droplets (∼10–4 to 10–3 m). The Vwater/Vair ranges along the upper axis correspond to different water saturation levels, with approximate ranges shown for the soil vadose zone (∼10 to 90%), industrial foams (∼1 to 40%),112−114 and clouds (<1%).105 In a warm cloud, most neutral compounds are predicted to be located predominantly in air due to the large abundance of this phase. Seven neutral compounds are located predominantly in water (bisphenol-A, benzidine, 2,4,6-trinitrotoluene, N-methylpyrrolidone, methylparaben, simazine, carbaryl, hexachlorobenzene), although we note that the last compound shows an unexpectedly large log(KH) value according to Figure 4B. Two neutral compounds are predicted to be located predominantly at the water–air interface (4-nonylphenol diethoxylate, cyfluthrin). The charged compounds, conversely, are all predominantly located in bulk water or at the interface, with different adsorbed fractions depending on their log(Kiw) values. Among the charged compounds, the seven highest log(Kiw) values belong to the PFAS investigated in this study. Thus, our results confirm that remediation efforts based on interfacial processes, such as foam fractionation or reactive micro- and nanobubbles, should be particularly effective for PFAS compounds, in agreement with experimental observations.106−111 The boundaries drawn in Figure 6 are sensitive to the water-to-air and water-to-interface ratios of different environmental systems. For example, in an unsaturated soil system with Vwater/Vair = 3 × 10–1, in contrast with the generic cloud with Vwater/Vair = 3 × 10–7 reflected in Figure 6, all three boundaries move to the left by six logarithmic units, placing most compounds predominantly in water. In a system containing ultrafine aqueous aerosol droplets with a 10 nm radius (Vwater/Ainterface = 3.3 nm), the boundaries shift downward by three logarithmic units, placing many compounds predominantly at the interface. The finding that many compounds in our study are located within only a few logarithmic units of the horizontal or diagonal boundaries in Figure 6 even in a warm cloud (with low Vwater/Vair and relatively high Vwater/Ainterface) is consistent with observations that adsorption at water–air interfaces can impact OC behavior in many engineered and natural systems, including during gas stripping,95 sea spray aerosol emission,115 and wet deposition.45 The configuration used in our MD simulations had Vwater/Vair ≈ 0.5 (a value typical of unsaturated soils) and Vwater/Ainterface ≈ 3 nm (a value typical of ultrafine aerosol particles116,117 and adsorbed water films on mineral surfaces).118,119 The corresponding partitioning space plot, presented in Figure SI5, is consistent with our observations that, in unbiased MD simulations of our studied system, most OCs predominantly sample the interfacial region (Figures 1 and SI3). Implications for Contaminant Fate and Transport The nearly ubiquitous existence of a FE minimum at the water–air interface indicates that most OCs exhibit higher concentrations at the interface than in either fluid, with potential implications for OC fate, transport, and remediation5,45,115,120 and for the environmental transformations of natural organic compounds.90,91 This concentrating effect of the interface is most pronounced for compounds with well-established surfactant properties, such as PFAS,98,104,115 but remains significant for most other OCs examined here, in agreement with previous studies.38,45,91,95 The affinity of most OCs for the interface implies that water–air interfaces in most natural systems carry a significant loading of adsorbed organic molecules. This observation suggests a need to examine how OC adsorption is modulated by pre-existing adsorbed organic substances. Previous studies indicate that cooperative or competitive adsorption at the interface can give rise to relatively complex adsorption trends.121,122 For instance, PFAS present in mixtures or with other surfactants exhibit sorption isotherms that differ from their monocomponent behavior,119 with significant impacts on their fate and transport.123 Our results also have implications for the adsorption of OCs from air onto solid particles. In this context, relative humidity (RH) has been identified as a significant factor due to the formation of adsorbed water films that compete with OCs for the particle surface.124−127 Measurements have shown that the adsorption of certain OCs to solid surfaces, such as 2-propanol to activated black carbon124 and di-n-butyl phthalate (DnBP) to indoor dust,127 decreases with RH. The relationship does not hold for all OCs, however, as shown by the minimal impact of RH on the adsorption of 1,2,4-trimethylbenzene (1,2,4-TMB) to carbon124 and di(2-ethylhexyl)phthalate (DEHP) to dust.127 A potential explanation for the lack of a consistent trend is that adsorbed water may both compete with OCs for adsorption on the dry surface but also create a water–air interface on which OCs can adsorb. Regarding water treatment, our findings have implications, for example, for the use of micro- and nanobubbles in water purification. These bubbles exhibit an enhancement of reactive oxygen species at the water–air interface that can oxidize OCs.5,128 Our results suggest that the accumulation of OCs at the interface may enhance the efficiency of associated remediation technologies, as already noted for PFAS.106,110,111 Future Research Opportunities The use of MD simulations to determine partitioning coefficients will likely complement, rather than supplant, existing predictive tools due to the significant time, computational resources, and expertise in molecular modeling required to implement the approach. However, MD simulations provide the notable advantages of providing detailed molecular-scale views of OCs and their coordination in systems in interest, and future establishment of standard simulation routines could greatly reduce the time required. Although the list of compounds examined here remains limited, extension to additional compounds or to different conditions (e.g., temperature6 or salinity)129 should be straightforward. To facilitate such studies, we provide a library of MD simulation input files for the compounds examined here [https://github.com/amelielemay/MD-Contaminants]. We encourage efforts to expand this database; to refine it in the case of compounds identified as outliers, for example, using DFT simulations;16,130 to use it to evaluate the affinity of OCs for other phases and interfaces;26,57,131 to evaluate its performance against quantum mechanical approaches;62 to evaluate the importance of solvent effects in molecular docking simulations;27,28,132,133 and to apply machine learning approaches to identify clusters and trends within our compound library.134,135 Supporting Information Available The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.3c00267.All FE profiles, SPC vs SPC/E model comparison, unbiased vs US simulation comparison, comparison of simulations with and without a charge-balancing ion, all Henry’s Law and interfacial adsorption coefficients (KH, Kiw, Kia), and quantitative characteristics of FE profiles (PDF) Supplementary Material es3c00267_si_001.pdf Author Contributions ∥ A.C.L. and E.J.S. contributed equally. All authors developed the database of MD simulation input files. A.C.L. and E.J.S. carried out the umbrella sampling simulations and analyzed the results. A.C.L., E.J.S., and I.C.B. wrote the initial manuscript. All authors provided input on the manuscript. The authors declare no competing financial interest. Acknowledgments This research was supported by the US National Science Foundation under Grant No. CBET 1931611. A.C.L., E.J.S., D.M., P.M., R.M., R.N., and M.W. acknowledge support from the Summer Internship Program of the High Meadows Environmental Institute (HMEI) at Princeton University. A.C.L. and E.J.S. were additionally supported by the Grand Challenges program of the HMEI at Princeton University. 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==== Front ACS Infect Dis ACS Infect Dis id aidcbc ACS Infectious Diseases 2373-8227 American Chemical Society 37347230 10.1021/acsinfecdis.2c00649 Review CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance https://orcid.org/0000-0003-0220-4990 Mayorga-Ramos Arianna † Zúñiga-Miranda Johana † Carrera-Pacheco Saskya E. † Barba-Ostria Carlos ‡* Guamán Linda P. *† † Centro de Investigación Biomédica (CENBIO), Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito 170527, Ecuador ‡ Escuela de Medicina, Colegio de Ciencias de la Salud Quito, Universidad San Francisco de Quito USFQ, Quito 170902, Ecuador * linda.guaman@ute.edu.ec * cbarbao@usfq.edu.ec 22 06 2023 14 07 2023 9 7 12831302 31 12 2022 © 2023 The Authors. Published by American Chemical Society 2023 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). The emergence of antibiotic-resistant bacterial strains is a source of public health concern across the globe. As the discovery of new conventional antibiotics has stalled significantly over the past decade, there is an urgency to develop novel approaches to address drug resistance in infectious diseases. The use of a CRISPR-Cas-based system for the precise elimination of targeted bacterial populations holds promise as an innovative approach for new antimicrobial agent design. The CRISPR-Cas targeting system is celebrated for its high versatility and specificity, offering an excellent opportunity to fight antibiotic resistance in pathogens by selectively inactivating genes involved in antibiotic resistance, biofilm formation, pathogenicity, virulence, or bacterial viability. The CRISPR-Cas strategy can enact antimicrobial effects by two approaches: inactivation of chromosomal genes or curing of plasmids encoding antibiotic resistance. In this Review, we provide an overview of the main CRISPR-Cas systems utilized for the creation of these antimicrobials, as well as highlighting promising studies in the field. We also offer a detailed discussion about the most commonly used mechanisms for CRISPR-Cas delivery: bacteriophages, nanoparticles, and conjugative plasmids. Lastly, we address possible mechanisms of interference that should be considered during the intelligent design of these novel approaches. CRISPR-Cas antimicrobial design antibiotic resistance document-id-old-9id2c00649 document-id-new-14id2c00649 ccc-price Special Issue Published as part of the ACS Infectious Diseases virtual special issue “Drug Resistance in Infectious Diseases and Beyond”. ==== Body pmcIntroduction The spread of antibiotic-resistant bacteria is a source of major global concern as it is a rising cause of fatal therapeutic failures, and this is anticipated to continue increasing in the future.1 On the other hand, the discovery of new antibiotics has stalled significantly over the past decade, and the development of novel approaches to address the spread of antimicrobial resistance genes demands substantial efforts.2,3 This dichotomy emphasizes the need to develop innovative antimicrobial strategies as a viable alternative to existing antibiotics.4 One major disadvantage of conventional antibiotics is the lack of species specificity, as they can induce metabolic and structural damage into large bacterial communities of both beneficial and pathogenic strains.5 Additionally, the literature has linked antibiotic selection pressure to the dramatic enrichment of drug-resistant strains in all areas of the biosphere.6−8 Using a CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)-Cas system for the precise elimination of targeted bacterial populations holds promise as an innovative approach for new antimicrobial agent design.9 The specificity of the CRISPR-Cas approach relies upon the interaction between the Cas protein and a guide RNA (gRNA) sequence designed for targeting unique DNA sequences within the pathogenic target strain.10 The Cas nuclease/gRNA complex can then induce double-strand breaks (DSBs) in the selected sequence, which can lead to cell death or the loss of the targeted plasmid (plasmid curing).11 Under this premise, it is crucial to develop appropriate strategies to deliver the CRISPR-Cas complex into all bacteria of the target population and to foresee possible mechanisms of interference or resistance. CRISPR-Cas Antimicrobials: Origin and Design CRISPR-Cas: The Adaptive Bacterial Immune System Bacteria have shown several strategies to prevent their damage or death by antibiotics, including the use of efflux pumps to deplete the antibiotic from the cell, the use of specific enzymes to degrade or inactivate the antibiotic, and the modification of essential cellular functions to prevent inhibition or binding by an antibiotic.12 These antibiotic resistance mechanisms are commonly acquired by horizontal transfer of plasmids and other mobile elements, which have obtained resistance genes from antibiotic-producing soil organisms.12,13 However, they can also be gained by mutation, often in genes related to the antibiotic mode of action.13 Horizontal gene transfer occurs via conjugation (plasmids), via transduction (bacteriophages), and via transformation (spontaneous uptake of bacterial DNA released by dying organisms followed by integration into the acceptor genome).13,14 Plasmids harbor resistance genes and distinctive origins of replication, which allow them to initiate the replication process separately from the host genomic DNA. Multiple plasmids can be present in a given bacterium.12 Bacteriophages can transfer genetic material containing resistance genes during their bacterial infection life cycle. Studies demonstrated that non-human-associated viromes are a vast reservoir of antibiotic-resistance genes (ARGs).15 On the other hand, conjugative transposons, integrative and conjugative elements (ICE), and integrons (all containing resistance genes) can be acquired through conjugation or by transduction. Transposons can integrate and move to different genomic sites within a cell by means of transposases or recombinase enzymes.16,17,16 As a consequence, one of the promising approaches that cleaves bacterial DNA or inhibits expression of critical genes is the use of the CRISPR-Cas systems. The CRISPR-Cas system was first described in 2007 as a complex bacterial defense mechanism, or immune system, against viruses and plasmids.18 Subsequently, substantial efforts have been made to adapt this system into a powerful gene editing tool with broad application for several organisms.19−21 In general terms, this bacterial immune system makes copies of the invader pathogen genome and incorporates them into the CRISPR loci as spacer sequences with the help of specific Cas proteins (e.g., Cas1 and Cas2).19,22 These spacer sequences are separated by repetitive sequences, forming an array of spacers and repetitive sequences. Short CRISPR RNAs (crRNAs) are then transcribed containing the full or partial spacer sequence.22 Finally, both the Cas nucleases and crRNA targeting sequence associate into a cleavage complex and induce a specific lethal cut in the invader’s genome, thus eliminating the invader and preventing bacterial death.23 Depending on the molecules involved and the cas operon distribution, the CRISPR-mediated immune mechanism can be classified into three main types: I, II, and III.20,22 Nevertheless, other types and subtypes of CRISPR-Cas systems are constantly described in the literature as new studies broaden the field. Here we describe CRISPR-Cas type I, II, III, and VI as these have been used in the development of CRISPR-based antimicrobials (Table 1). Table 1 Current CRISPR-Cas-Based Antimicrobials in Developmenta CRISPR-Cas system (type) Pathogen Delivery system Target location Gene target Refs In Vivo Study CRISPR-Cas9 (Type II) E. coli Cri-nanocomplex (carbon quantum dots) Chromosome (pap gene cluster) papG (53) E. coli Conjugative plasmid (TP114 plasmid) Chromosome (AMR cassette inserted in glmS terminator) cat (54, 55) E. faecalis Conjugative plasmid (PRP pPD1) Plasmid (pAM771, pCF10) ermB (56) tetM E. faecalis Conjugative plasmid (PRP pPD1) Plasmid (pAM714) repB (57, 58) S. aureus Bacteriophage (temperate phage) Chromosome (thermonuclease encoding region) nuc (59, 60) S. aureus MGEs (staphylococcal pathogenicity islands [SaPIs]) Chromosome (agr and listeriolysin encoding regions) agrA (61) hly S. aureus Bacteriophage (phiNM1 phage) Chromosome (kanamycin resistance encoding regions) Aph-3 (62) mecA   CRISPR-Cas3 (Type I) C. difficile Bacteriophage (temperate phage) Chromosome (CR11 array) ND (63)   In Vitro Study CRISPRi (derived from CRISPR-Cas9) E. coli Conjugative plasmid (recombinant plasmids) AcrAB-TolC-associated mRNAs acrA, acrB (44) tolC M. abscessus Conjugative plasmid (pLJR962) Peptidoglycan biosynthesis-associated mRNAs pbpB (64) cwIM   CRISPR-Cas9 (Type II) E. coli Conjugative plasmid (pSC101) Plasmid (pNDM-5) blaNDM-5 (65) E. coli Conjugative plasmid (suicide plasmid) Plasmid (multiple AMR-bearing plasmids) mcr-1 (66) blaKPC-2 blaNDM-5 E. coli Conjugative plasmid (transposon-associated suicide plasmid) Chromosome and plasmid (colistin resistance encoding regions) mcr-1 (67) Enterobacteriaceae spp. Conjugative plasmid (pSB1C3) Plasmid (pSB1A2) blaTEM-1 (68) E. coli MGEs (pro-active genetic system) Plasmid (bla harboring pET) bla (69) E. faecium Conjugative plasmid (pVDM1001) Chromosome (lac operon encoding region) lacL (70) S. aureus Nonconjugative plasmid (Apa I-cut pLI50) Chromosome (WTA biosynthesis encoding regions) tarO (71) tarH tarG S. aureus Cri-nanocomplex (polymer-derivatized SpCAS9) Chromosome (PBP2a encoding regions) mecA (72)   CRISPR-Cas3 (Type I) E. coli Bacteriophage (λ phage, T7 phage) Plasmid (pNDM, pCTX) ndm-1 (73) ctx-M-15   CRISPR-Cas13a (Type IV) E. coli Phagemids (Cas13a encapsulated with bacteriophage capsid) Chromosome and plasmid (multiple AMR-encoding regions) blaIMP-1 (47) blaOXA-48 blaVIM-2 blaNDM-1 blaKPC-2 mcr-1 mcr-2 a Abbreviations: AMR, antimicrobial resistance; Cri-nanocomplex, nanosized CRISPR complex; MGE, mobile genetic element; ND, nondisclosed; PBP2a, penicillin-binding protein 2a; PRP, pheromone-responsive plasmid; WTA, wall teichoic acid. The Type I CRISPR-Cas system identified in Escherichia coli includes the CRISPR-associated complex for antiviral defense (Cascade) and Cas3.19,24 The cascade complex subunit Cas6 recognizes an eight-nucleotide sequence upstream of the spacer sequence and releases a crRNA. crRNA associates with Cascade and directs the complex to its target DNA cleavage site19,25 (Figure 1-A). On the other hand, the Type II CRISPR-Cas system, derived from Streptococcus pyogenes, is based on the recognition of a genome’s specific dsDNA sequence by a gRNA, which recruits a Cas9 nuclease (to form a ribonucleoprotein complex) that will introduce a DSB.25,26 This gRNA is formed by a crRNA and a trans-encoded crRNA (tracrRNA), which is complementary to a repetitive sequence. Subsequently, this RNA duplex is cleaved by RNase III, leaving the Cas9 in a complex with both tracrRNA and crRNA23,25 (Figure 1-B). Efficient cleavage by type I and II systems require complementarity of the first eight (type I), six to eight (type II) nucleotides of the target DNA and the crRNA. Also, a protospacer-adjacent motif (PAM) next to the target sequence must be present.12 Given the importance of these regions, mutations could result in a pathogen escaping the CRISPR-mediated immunity.23 The type III CRISPR-Cas system can be divided into two subtypes,type III-A (Staphylococcus epidermidis) or III-B (Pyrococcus furiosus), which recruit Cas6 and, similarly to type I CRISPR-Cas, give origin to crRNA.22,25 This crRNA is further processed at the 3′ end by Cmr or Csm effector complexes. In contrast to the other systems, type III requires transcription of the target sequence and cleavage of the DNA (type III-A) and RNA (type III-B) target27−29 (Figure 1-C). Finally, type VI CRISPR-Cas contains Cas13 enzymes and targets single-stranded RNA (ssRNA) instead of DNA. The particularity of these Cas13 enzymes is the ability to process pre-crRNA to its mature form and to perform the degradation of a target ssRNA.30 However, the degradation only occurs when the Cas13-crRNA complex binds to the target ssRNA31 (Figure 1-D). Interestingly, evidence suggests that the pre-crRNA can be enough to recognize the target ssRNA, and therefore, crRNA is not a key player in this type of system.32 Figure 1 CRISPR-Cas systems employed for developing next-generation antimicrobials. Left panel shows the molecular mechanisms of CRISPR-Cas systems present in bacteria that have been used to develop CRISPR-based antimicrobials (A, B, C, and D). Right panel shows a strategy based on the type II system with a mutant Cas9 nuclease (dCas9) also used to develop CRISPR-based antimicrobials (E). dCas9 refers to a catalytically dead Cas9. *Nonfunctional nuclease domains RuvC and HNH. RNA polymerase (RNAP). CRISPR-Cas: Designing Novel Antimicrobials CRISPR-Cas-based technology has transformed genome engineering, previously limited to the use of specialized custom DNA-binding proteins, by benefiting from the specificity of the interaction between the Cas protein and an easily malleable target-specific gRNA complex.33 As a result, the CRISPR-Cas system is now celebrated as a highly revolutionary genome-editing tool with several potential applications.34 Remarkably, the discovery of the type II CRISPR-Cas9 systems and the development of related easy-to-use toolsets have opened the door to a wide range of applications for both fundamental scientific research and future clinical therapeutics.35−37 These target-specific dual gRNA complexes rely on the interplay between two well-characterized types of RNA: the crRNA (CRISPR RNA) and the tracrRNA (Trans-activating CRISPR RNA).31 Moreover, Streptococcus pyogenes-derived Cas9 (SpCas9) is not only the most commonly used nuclease for genome editing at the moment, but it was also the first nuclease to be applied outside of prokaryotic cells and repurposed for genome editing in mammalian cells.10,38,39 In the past few years, the CRISPR-Cas system has also emerged as a promising strategy for designing new-generation antimicrobials that can target specific bacterial populations.9,40 This offers an excellent opportunity to fight antibiotic resistant pathogens by cleaving their DNA or by selectively inhibiting genes involved in resistance, pathogenicity, or virulence.41 As seen in Table 1, some of the most commonly targeted genes are related to antibiotic-inactivation enzymes like beta-lactamases (bla genes)42 as well as genes related to membrane structure and cell permeability (cmr-1 gene).43 The simultaneous use of multiple gRNAs has also been implemented to target complex antibiotic resistance mechanisms like the AcrAB-TolC efflux pump system (AcrA, AcrB, and TolC genes) in Escherichia coli.44 The existing body of literature has described three primary mechanisms of how CRISPR-Cas can mediate antimicrobial activity, these include double-strand chromosomal cleavage, global RNA degradation, and inhibition of essential gene expression in the target bacteria.45−47 One of the first attempts to create a CRISPR-Cas antibacterial agent used the well-known CRISPR-Cas9 nuclease system (Type I) to induce specific chromosomal DSBs and reported potent bacterial killing by targeting diverse locations throughout the genome.48 It is important to note that the induction of plasmid DNA cleavage does not lead to host bacteria death but can be applied deliberately to eliminate resistance within the target population.49 A different strategy proposes the use of CRISPR-Cas13 systems (Type VI) due to its promiscuous ssRNA cleavage activity and subsequent bacterial RNA degradation which can eventually halt bacterial growth.47,50 Additionally, CRISPR-Cas antimicrobial studies have also shown an inclination toward the use of optimized CRISPR interference (CRISPRi) approaches (Figure 1-E) as opposed to traditional gene inactivation routes. The CRISPRi system was first derived from a catalytically inactive Cas9 protein, which causes transcription blockage.51 This approach can be repurposed to repress gene expression of important genes involved in antimicrobial resistance (AMR) and pathogenicity.44,52 Ultimately, most of these studies have still favored the CRISPR-Cas9 mechanisms over others to design their antimicrobial system in vitro and in vivo (Table 1). The literature has demonstrated that CRISPR-Cas-based strategies can enact antimicrobial effects by two approaches: induce bacterial death or cure antibiotic-resistance-expressing plasmid.9 The outcome of the system is determined by the location of the target DNA as CRISPR-Cas cleavage of chromosomal sequences can lead to the death of the target bacteria while targeting a plasmid-bound sequence leads to plasmid loss from the host cell (Figure 2).48,74,75 Ultimately, it is fundamental to consider the delivery mechanisms involved in the construction of CRISPR-Cas antimicrobials as they should be able to reach virtually all bacteria within the target population.76 The three main CRISPR-Cas vehicles explored in recent research to combat AMR bacteria are bacteriophages, nanoparticles, and conjugative plasmids (Figure 2). Figure 2 CRISPR-Cas system antimicrobials: mechanisms of action and delivery. CRISPR-Cas System Delivery Bacteriophages The use of bacteriophages for infectious diseases treatment has been considered almost as soon as they were first described in 1915.77,78 However, the initial exploration and application of phage therapy were quickly overtaken by the discovery of antibiotics after they were successfully introduced during World War II.79 Nevertheless, research in phage therapy did not fully recede as a number of research groups from institutions like the Eliava Institute and the Hirszfeld Institute continued research on the therapeutic use of phage cocktails.80 The natural life cycle of bacteriophages requires replication inside a bacterial host, and they possess inherent bacteria-specific targeting and killing machinery which pose an attractive tool for the treatment of bacterial infection.81 Bacteriophages are by far the most abundant microorganisms on the planet, and they can be found in a variety of environments where bacteria are present.82 Bacteriophages can also be classified into two main groups according to their establishment and release mechanisms: virulent (host cell lysis) and temperate phages (stay latent inside the host).81 Remarkably, bacteriophages also had great importance in the discovery of the CRISPR system and its function as a primitive bacterial adaptive immune system via CRISPR-Cas nucleases.79,83 In general, viral therapy has been extensively explored in medical research, and several fields like oncology have made promising advances regarding oncolytic virus treatments.84 Excitingly, in 2015 the first oncolytic virotherapy was approved by the U.S. Food and Drug Administration (FDA) for melanoma treatment, and several other candidates are undergoing the latest stages of clinical trials.85,86 The safe use of bacteriophage therapy in human patients may not be inconceivable although the literature still highlights several considerations regarding phage design and possible limitations for future applications.87 CRISPR-Cas Delivery Using Bacteriophages As explained previously, bacteriophages can be classified into virulent and temperate groups depending on their life cycle. Virulent phages inject their genome into the host, hijack the host machinery for replication, and release their progeny by host cell lysis.88 This process involves the action of two types of proteins, holins and lysins, to perforate the bacterial cytoplasmic membrane and the bacterial cell wall, respectively.89 Temperate phages employ a lysogenic cycle where the viral genetic material gets inserted into the host genome as a prophage and stays dormant while being replicated along with the host genome.90,91 This type of dormant genome insertion has been associated with the acquisition of antibiotic-resistance genes and virulence factors, which has prompted researchers to suggest lytic phages a more suitable vehicle for bacteriophage therapy.92 Classic bacteriophage therapies often take advantage of previously existing bacteriophages which can be isolated from the same environment where the target pathogen resides naturally.93 The literature suggests that bacteriophages capable of targeting clinically associated bacteria could be isolated from hospital wastewater containers or sewage.94 Selection for suitable phages then involves a sterilization step where the sample gets cleared of all external microorganisms and then the solution gets plated into a culture of the targeted bacteria in vitro to assess the formation of plaques.95 After initial isolation and selective assays, these phages are characterized and genetically sequenced to enter the library of prospective therapeutic applications.95 Remarkably, it is also important to characterize their interactions and possible inflammatory effects on human cells and tissues.96 Recently, the incorporation of antimicrobial payloads, such as CRISPR-Cas nucleases, has become a staple approach to enhance traditional phage-based therapies.97 The literature has extensively illustrated how both lytic and temperate phages can be used for CRISPR-Cas delivery in target bacteria (Figure 3).98 Nevertheless, the complexity related to phage genome manipulation should not be underestimated as it demands suitable engineering tools as well as knowledge about secondary morphological and functional repercussions of such changes.99−101 The main challenge for phage genetic engineering is related to space limitations inside the capsid which can hinder the addition of large DNA fragments like a CRISPR-Cas system without disrupting the packaging and delivery of the genome.98 Notably, in 2015, an innovative dual system was designed to increase the efficiency of AMR E. coli treatment by employing both temperate and lytic bacteriophages coupled with CRISPR-Cas targeting.73 The selected CRISPR-Cas3 system was paired with resistance to lytic phages and successfully introduced into temperate bacteriophages by removing nonfundamental accessory genes as a way to overcome the capsid space limitation. Hence, the CRISPR-Cas system only acted as a resensitization tool against conventional AMR while conferring a selective advantage to the strains that accepted resensitization. Bacteria that fail to accept the CRISPR-Cas resensitization were immediately killed by the engineered lytic phages introduced as a second part of this two-step phage treatment.73 Figure 3 Bacteriophages for CRISPR-Cas9 delivery. In time, improved selection of phage models allowed the election of better-suited phages like fSABov for the introduction of a CRISPR-Cas9 system targeting methicillin-resistant Staphylococcus aureus (MRSA) infections in vivo.59 The success of this optimization relies on the removal of major virulence genes from the phage genome which significantly reduced cytotoxicity in human peripheral blood mononuclear cells (PBMCs). In addition, this study demonstrated that the host range of their fSABov vector can be expanded by enhancing the phage tail fiber protein.59 Additionally, some studies have also tested the suitability of using bacterial genetic mobile elements such as staphylococcal pathogenicity islands (SaPIs) packaged inside helper delivery phage capsids.61 Two antimicrobial CRISPR-Cas9 modules (CRISPR-Cas9 bactericidal and CRISPR-dCas9 virulence-blocking modules) were introduced in exchange for SAPIs toxigenic genes. Moreover, the deletion of the SaPI capsid morphogenesis genes provided an additional 30 Kb packaging space which enabled the prospective insertion of additional killing/inhibition modules to counteract potential bacterial resistance. Subsequent in vivo assays yielded promising results in mice infected with S. aureus during a murine subcutaneous abscess model and a peritoneal lethal infectious model.61 Ultimately, CRISPR-Cas3 bacteriophage antimicrobials also found success in a study targeting Clostridium difficile both in vitro and in vivo.63 Limitations The major limitation of the use of bacteriophages as delivery vehicles is the variability in infection susceptibility between different bacterial strains.102 This means that one phage vehicle can only target a reduced range of bacteria because their affinity depends on specific receptors on the bacterial membrane surface.103 A proposed solution is the amplification of the host range by using “phage cocktails” where different bacteriophages are combined to increase the delivery rate of the antibacterial system.104−106 Another limitation can be a reduced phage survival rate at the administration site (in situ) as many bacteriophages isolated from the environment are not equipped to survive inside a human host.107 To avoid this, bacteriophages should undergo a rigorous selection process and can also be genetically modified to survive the immune response of the human host.104 Phages are also susceptible to other environmental factors like temperature variations, pH, organic substances, and mechanical stress among others.108,109 Phage encapsulation is one common protection mechanism employed for prospective therapeutic phages (it provides a platform for phage storage and increases the phage survival rate until it reaches its target location).110 The literature has demonstrated that encapsulation enhances the adsorption and distribution of phages by enabling them to evade immune responses, withstand stomach acidity, and resist enzymes and free radicals found in tissue fluids, thereby increasing their circulation time.111−113 Enteral administration is the most common, but some other administration routes involve topical administration, inhalation, or injections.114 Safety-of-use concerns have also been raised as replicative phages could also acquire host mobile genetic elements (MGEs) which could lead to the subsequent spread of virulence genes.115 In order to prevent contamination, some studies have opted for virulence-factor gene deletion from the host strains used in bacteriophage amplification.59 Ultimately, all these are questions that require further research before bacteriophage can enter a clinical application phase. Conjugative Plasmids Several limitations regarding the use of bacteriophages and other synthetic vehicles have prompted researchers to seek more bacteria-compatible delivery routes. Research studies have assessed the use of conjugative plasmids as engineered mobile elements to transport genetic information and deploy desired functionalities within a bacterial population.116 Plasmid sequences are highly programmable as there is a wide range of molecular biology tools which can turn them into relatively simple platforms for bacterial genetic engineering in situ.117 Conjugative plasmids are circular genetic sequences that are capable of transferring from one bacterial cell to another through a process called conjugation.118 Conjugative plasmids contain the necessary genetic information for their own transfer and can also cotransfer nonconjugative plasmids with appropriate oriT sites.119,120 Briefly, the bacterial conjugation mechanism generally comprises the involvement of a type IV secretion system (T4SS) for the mobilization of DNA between a donor and a recipient cell in close physical proximity with a variety of transfer efficiencies.55,118 Notably, several studies have demonstrated that transfer of CRISPR-Cas systems between different species of bacteria can be achieved using this transfer mechanism (Figure 4).116,121,122 Therefore, it is not surprising that conjugation is proposed as a natural delivery mechanism for CRISPR-Cas-based antimicrobials, which can benefit from the transfer efficiency of native conjugative plasmids.75 Figure 4 Plasmid conjugation for CRISPR-Cas9 delivery. CRISPR-Cas Delivery Using Conjugative Plasmids Genetically distinguishable plasmids cannot coexist stably inside the same host cell, which has prompted researchers to classify them into Incompatibility (Inc.) groups.123 Some studies have also uncovered the underlying mechanisms responsible for the entry exclusions that prevent the access of Inc. plasmid groups.124 These inherent incompatibility properties suggest that plasmids utilized in synthetic biology with prospective clinical applications should be uncommon within the targeted microbial population.125 However, plasmid screening within heterogeneous populations can be very demanding and time-consuming. One of the first attempts to develop a CRISPR-Cas antimicrobial conjugative plasmid used an Inc.P plasmid, one of the rarest type of plasmids conferring resistance in Extended Spectrum Beta-Lactamase (ESBL) strains, for targeting blaTEM and blaCTX-M beta-lactamase genes within an E. coli population in vitro.116,126 Further studies have also effectively targeted blaTEM-related genes in laboratory E. coli strains and Enterobacteriaceae clinical isolates.68 An important question to consider for plasmid-driven delivery methods is that the CRISPR-Cas conjugative plasmid requires a host cell that can act as a donor toward target strains. Therefore, some research groups have suggested the delivery of this system inside well-known probiotic strains.54 In 2021, a study achieved high transfer efficiency and treatment effectiveness using a TP114 plasmid carrying a CRISPR antimicrobial system delivered by the probiotic E. coli Nissle 1917.54,127 In this model, a previously known TP114 plasmid was optimized via accelerated laboratory evolution to target AMR E. coli and Citrobacter rodentium with impressive results after 4 consecutive days of oral treatment in mice.54,55 The use of narrow host range plasmids such as pheromone-responsive plasmids (PRPs) has also been explored for the delivery of CRISPR-Cas antimicrobials within their specific target population: Enterococcus faecalis.56,57 PRPs exhibit a naturally high conjugation frequency, the ability to infiltrate and propagate within intestinal E. faecalis without antibiotic selection, and a competitive advantage via bacteriocin-mediated activity.57,128−130 In 2019, a study used PRP pPD1 to target multi-drug-resistant E. faecalis both in vivo and in vitro and uncovered the striking difference in the effectiveness of the in vitro model versus the in vivo intestinal mouse assay.57 The CRISPR-Cas system was able to effectively block the target plasmid dissemination within the intestinal mouse model while displaying a lower antiplasmid activity during in vitro assays. These differences have been attributed to several variables affecting the CRISPR-Cas system activity during in vivo settings such as planktonic vs biofilm lifestyles, donor-to-recipient ratio, and assay sampling time points.57 The same year, an associated research group found promising results by selectively depleting the erythromycin-resistant enterococcal population of a murine intestine model by several orders of magnitude.56 These results also demonstrated that a plasmid-born CRISPR-Cas antimicrobial can protect the donor strains from the uptake of the targeted antibiotic resistance. However, this study also detected perturbations in the effectiveness of the CRISPR-Cas antimicrobial after it was translated into gnotobiotic mice. Similarly to the previous study, these findings suggest that the CRISPR-Cas system benefits from the presence of microbial complexity and other external factors found in natural conditions.56 Research has also been directed toward the development of community-wide genetic modification tools in situ by engineering the mobilome. One study demonstrated that a range of replicative (mobile plasmids) and integrative (Himar transposon) vectors can exert in situ genetic modifications of the gut mouse microbiome with precise genetic payload.131 Results obtained in this study also uncovered the challenges faced by probiotic donor strains to establish long-term stability within complex microbial communities. Consequently, the optimization and use of native gut bacteria as plasmid donors have been proposed to ensure the persistence of the donor strain in situ without the aid of antibiotic-mediated microbiota clearance.131 More recently, the combination of CRISPR-Cas systems and transposon-mediated DNA delivery has been proposed to conduct species- and site-specific genome modifications.132 This novel arrangement demonstrated that MGEs can also be optimized to improve the specificity and dissemination of CRISPR-Cas systems within microbial communities.132 On the other hand, a number of studies have also strived to enhance the efficiency of current CRISPR-Cas antibiotic mechanisms by developing an active self-copying mechanism denominated pro-active genetics (ProAG).69 In this way, the gRNA cassette is flanked with homologous sequences to the target cleavage sequence of a high copy number plasmid. The ProAG gRNA will get copied into the high copy number plasmid leading to two scenarios: amplification of gRNA expression and inactivation of the AMR sequence. The results from this study show that this ProAG system improves the inactivation of the target plasmid by ∼100-fold in comparison to the classic CRISPR-Cas-based antiplasmid methods.69 Overall, the use of conjugative plasmids as delivery vehicles holds great promise for a variety of engineering purposes. During conjugation, the donor strain shares the plasmid as a single-stranded DNA (ssDNA) which is subsequently complemented within the recipient bacterium with compatible host-specific modifications.133 This could potentially facilitate the transfer and acceptance of conjugative plasmids into diverse bacterial species without prior knowledge of the specific features of those bacteria. Another advantage of conjugative plasmids is their compatibility with a wide range of bacteria, not limited by receptor recognition unlike bacteriophages.54 Ultimately, the co-joint use of CRISPR conjugative antimicrobials and bioengineered donor strains can help circumvent the disadvantages that bacteriophages face in vivo when the target bacteria resides inside harsh environmental conditions (low pH, gastric fluid, proteases, etc.) such as the gut.54 Limitations The first straightforward limitation toward the clinical application of conjugative plasmids is their dependence on a donor vector (often a probiotic bacterial strain) that needs to be introduced into the patient to replicate, transport and conjugate the plasmid module. Therefore, conjugative plasmids are limited by targeted population traits (planktonic vs biofilm formation) as well as the robustness of the donor strain to thrive and establish long-term colonization in different environments like skin (external) or intestinal (internal) sites of infection.121,134 Additionally, the application of living donor vectors is limited to the gastrointestinal tract and external locations as it can be detrimental to inoculate these microorganisms into noncolonized tissues. Another very important consideration is the risk of acquisition of AMR-related genes into the plasmid vector system as the literature has extensively described the role of plasmids in the horizontal gene transfer of AMR genes among unrelated bacteria.135 At the moment, a couple of studies have shown that the presence of the CRISPR-Cas system can immunize the donor bacteria to the acquisition of the target AMR gene, but this pressing issue requires further investigation.56 The use of conjugative plasmids as delivery vehicles is also limited by their naturally low transfer rate, prompting numerous investigations aimed at enhancing the transfer efficacy of their selected plasmid vectors.54 In addition, it is well documented that plasmid acquisition and maintenance enforce a high metabolic cost to the donor cell which can hinder their metabolism, growth, and reproduction.136 Bacterial hosts are often prompted to lose nonadvantageous plasmids after exposure to nutrient scarcity, harsh environmental conditions, or competition.137 This opens the question about how disadvantageous harboring an entire CRISPR-Cas system can be for the probiotic donor strain and how it can affect its survival rate under natural conditions. It could add a layer of complexity in the development of these vehicles as the donor strain will also be required to undergo an optimization process before it can be suitable for clinical applications. The efficiency of plasmid transfer within bacterial biofilms has also been strongly debated in the literature.138 It is known that close proximity of bacterial communities can be beneficial for horizontal gene transfer and the spread of MGEs like plasmids.139,140 Nevertheless, several studies have brought attention to a disparity of plasmid distribution among different sections of a biofilm population.141,142 The highest rate of plasmid transfer appears to be localized in the biofilm interfaces where bacteria are still undergoing replication and high metabolic activities.143,144 Considering the mechanism of action for CRISPR-Cas antimicrobials, these unequal distributions may impair the reach of their antimicrobial effects and severely limit their use. Lastly, the target bacteria can still harbor a plasmid from the same Inc. group as the conjugative plasmid vehicle. This can trigger what is known as plasmid competition between the resident plasmid and the CRISPR-Cas module.145 In addition, it is important to consider that bacteria possess several mechanisms (detailed in the following sections) that could inactivate the CRISPR-Cas sequence within the genetic module. Consequently, this can lead to the creation of a defective, but still viable and replicating, CRISPR-Cas plasmid which could hinder the entrance of nonimpaired peers into the target cell. Nanoparticles Nanotechnology, through nanomaterials, offers an alternative to improve antimicrobial therapy and gene delivery.146 Nanoparticles are delivery enhancers for antimicrobial agents and novel antimicrobial material distinct from conventional drugs.147 Generally, promising nanoparticle-based antimicrobials operate under two main mechanisms against bacteria: (i) alteration of membrane potential and integrity and (ii) induction of oxidative stress through the generation of reactive oxygen species (ROS) catalyzed by nanoparticles.148 Nanoparticles have a large specific surface area due to their small size, which enhances their reactivity, contributes to their distinctive physical and chemical properties, and renders them highly efficient in eradicating bacteria.149 Furthermore, their nanosize-related properties allow them to penetrate deep into bacterial cells and damage their internal components, contributing to their antimicrobial activity.149 Several attempts have been made to improve nanoparticle design by incorporating surface recognition elements such as ligands or phage-derived receptor binding proteins which enable them to selectively identify and bind epitopes on the bacterial membrane.150 In addition, a frequent approach in the field of nanoparticle biorepatterning involves the customization of nanoparticles using aptamers or specific antibodies that exhibit high affinity toward diverse bacterial elements.150,151 Ultimately, the interaction between nanoparticles and bacteria can also be influenced by electrostatic forces.152 As demonstrated in recent studies, nanoparticle-based antimicrobials can be tailored with biodegradable cationic polycarbonates, enhancing the electrostatic interaction between the nanomaterials and bacterial membranes and considerably improving antimicrobial activity.153 In addition to their antibacterial potential, nanoparticles can fight infections by carrying drugs to deliver antimicrobial agents. Antibiotics alone may present limitations such as poor penetration into bacterial infection sites, low drug bioavailability, antibiotic-related side effects, and antibiotic resistance.154,155 In this sense, polymeric nanoparticles have been considered a valuable tool to improve the efficacy and safety of classic antibiotics. Wrapping antibiotics in polymeric nanoparticles can enhance the solubility of hydrophobic drugs and increase the antibiotic concentration at the infection site.155 Additionally, polymeric nanoparticles can provide a controlled release of antibiotics, which may help reduce the frequency of dosing needed and improve the safety of treatment.156,157 CRISPR-Cas Delivery Using Nanomaterials Nanoparticles can be used not only to deliver drugs but also to deliver genes.146 Genetic elements face several difficulties during target cell entrance due to serum protein adsorption, rapid clearance into the bloodstream, phagocyte uptake, inability to escape the endosome, lack of targeting ability, and the toxicity induced by the immune system, limiting their use in medical treatments.155 To overcome this drawback, delivery systems such as metallic nanoparticles, lipid nanoparticles, and polymeric nanoparticles are under study. They have generated significant interest among researchers as protective envelopes from degradation in biological fluids by nucleases and proteases for fragile sequences like Cas9, mRNA, and sgRNA.158−160 Although CRISPR-Cas9 has the potential to knock out antibiotic resistance genes, the delivery system is inefficient.161 For this reason, a number of strategies for nanomaterial-mediated delivery of CRISPR-Cas9 systems have been developed, which include (i) Cas protein and sgRNA encoding plasmid, (ii) Cas9 mRNA and sgRNA, and (iii) complex of Cas9 protein and sgRNA (Figure 5).148 In addition, the physicochemical characteristics of the nanoformulation are essential for its targeting and release. For example, nanoparticles can be chemically modified to add functional groups that stabilize Cas9 and mRNA, protecting it from degradation and granting high target specificity.159,162 The nanoparticle surface can also be conjugated with cell-penetrating peptides (enhancing cellular uptake) and/or nuclear localization signal peptides (for internal delivery).155 Branched polyethylenimine has been used to form nanocomplexes with spCas9 and facilitate the delivery and binding between the nanocomplex and the cell wall of AMR bacteria.72 Figure 5 Nanoparticles for CRISPR-Cas9 delivery. Lipid nanoparticles are one of the most explored systems for CRISPR delivery. Lipid nanoparticles can be used to encapsulate negatively charged CRISPR plasmid DNA and mRNA, guiding and protecting the mRNA during its cell membrane diffusion.163,164 Polymeric nanoparticles have been favored for CRISPR delivery due to their low immunogenicity and their high compatibility with living tissues.165,166 Furthermore, metal-based nanoparticles have also been considered suitable for delivering ribonucleoprotein (RNP) complexes.155 For example, the design of a polymer-derivatized Cas9 and a gRNA nanocomplex (Cri-nanocomplex) displayed significant inhibition of MRSA growth in the presence of methicillin by altering the mecA gene.72 In another study, carbon quantum dots covalently conjugated to Cas9 and papG-targeted gRNA were employed to form a Cri-dot-papG nanocomplex for the delivery of a CRISPR-Cas antibiotic in Uropathogenic E. coli (UPEC). This system targeted the gene associated with the fimbrial adhesion virulence factor (papG gene) and registered a significant decrease in papG expression, thereby reducing the pathogenicity of UPEC.53 Limitations For proper Cas mechanism and protein expression, accurate delivery of the CRISPR-Cas system is critical. However, there are several challenges at the in vivo level, such as the immunogenicity caused by the vector160 or the efficient packaging and localization of the CRISPR-Cas components using nanoparticles. The risk of immunogenicity associated with CRISPR-Cas9 gene editing highly depends on the delivery route used.160 However, some CRISPR-Cas-based therapies incorporate the protein within biocompatible nanoparticles to reduce immunogenesis.167,168 For example, vectors based on lipid nanoparticles may offer a viable alternative for in vivo applications since lipids are less immunogenic than other vectors, such as viruses.169 However, there needs to be more knowledge of the interaction of nanomaterials with cells and tissues. Another limitation is the efficient packaging and localization of system components, for which nanoparticles can be tailored in various ways, for example, by modifying cell-penetrating peptides and specific cell receptors to enhance protein uptake by cells and its interaction with target cells.155 Another strategy is a surface modification with biocompatible polymers such as polyethylene glycol to reduce the elimination mediated by reticuloendothelial cells and increase the lifetime in the blood.160 This strategy will contribute to reducing the toxicity of metal nanoparticles for drug delivery which, at low doses, are generally not toxic, but their prolonged administration can cause long-term aggregation and potential toxicity. There are still studies on the CRISPR-Cas-nanoparticle delivery system before they can be applied as delivery systems for antibacterial activity. Further investigation with in vivo subjects is required to determine short- and long-term toxicity. Bacterial Mechanisms of Resistance CRISPR-Cas systems have been developed as a new form of antimicrobial defense to counteract bacterial infections. However, different studies have shown that bacteria can develop mechanisms to evade the CRISPR-Cas system’s action with an overall frequency of approximately 10–4,170 potentially rendering CRISPR-Cas systems ineffective as a stand-alone antimicrobial agent. Research has shown that bacteria can acquire several mutations during the natural process of CRISPR adaptive immune responses. Occasionally a mutation may grant resistance to phages or antibiotics.171,172 Currently, the role of CRISPR as a precision genome editing tool is widely celebrated; however, studies have shown that many newly discovered CRISPR systems are not quite precise. Type III CRISPR mechanisms demonstrated indiscriminate targeting for DNA cleavage, unlike the Type II CRISPR system, which relies on the Cas9 nuclease to cut specific DNA sequences.173 Therefore, the rational design of CRISPR-based tools like novel antibiotics has shown strong inclinations toward using Cas9 nucleases.174 However, the heavy reliance on these specific machinery raises concerns about future resistance mechanisms. The literature has described several risk factors, such as the spontaneous mutations in the Cas genes or the target sequences and the presence of the anti-CRISPR (Acr) genes in the target host genomes175 (Figure 6-A). Here we describe the main mechanisms involved in CRISPR-Cas resistance by bacteria. Figure 6 (A) Mechanisms of resistance toward CRISPR-Cas. (B) Bacterial community responses interfering with CRISPR-Cas antimicrobials. Escapers through Mutations Mutations in either the CRISPR-Cas complex or the targeted genetic region can enable bacteria to evade the effects of the CRISPR-Cas system and survive. If a mutation in the first CRISPR-Cas complex to enter the bacteria occurs, the bacteria would still be sensitive to a functional system (Figure 6-A). Still, if the target region undergoes large deletions, the bacteria would effectively evade the system and become resistant.74,176 Research has recorded some examples of bacteria using mutations to evade the CRISPR-Cas system. In a 2018 study, E. coli was exposed to a phage carrying a protospacer with a specific PAM sequence and developed mutations in the PAM sequence of the target gene, rendering it unrecognizable to the CRISPR-Cas system.177 As a result, the bacteria were able to survive and continue to replicate, while the phage was unable to infect the cells.177 Another example of bacteria using mutations to evade the CRISPR-Cas system was observed in Streptococcus thermophilus, which was exposed to a plasmid that carried a protospacer with a specific PAM sequence. The bacteria were able to develop mutations in the PAM sequence of the target gene, preventing the CRISPR-Cas system from recognizing and destroying the plasmid. As a result, the bacteria were able to maintain the plasmid and continue to replicate.176 In addition to mutations in the PAM sequence, mutations can also occur in the spacer sequence, which is the small fragment of foreign DNA incorporated into the bacterial genome. If the spacer sequence is mutated, it may no longer match the target DNA, rendering the CRISPR-Cas system ineffective. Mutations in the spacer sequence have been observed in Vibrio parahaemolyticus, which was exposed to a phage that carried a protospacer with a specific spacer sequence. The bacteria were able to develop mutations in the spacer sequence, preventing the CRISPR-Cas system from recognizing and destroying the phage.178 A few scenarios have been described after the loss of large genetic sequences. The first can lead to positive outcomes, resulting in the inactivation of antibiotic resistance or virulence traits related to those genes.155 Nonetheless, point mutations where the gene function is preserved can enable bacteria to escape recognition by the Cas nucleases while maintaining their pathogenic traits.179 This issue can be solved by programming the CRISPR-Cas system to simultaneously target several positions at the undesired locus. Fittingly, the natural mechanism of CRISPR immunity encompasses multiple targeting sequences, enabling easy and convenient multiplexing for antimicrobial sequence targeting.9 Some studies, however, showed no correlation between the number of target sites and killing efficiency by assessing 10 different gRNAs with 1 to 25 target sites in an E. coli genome and obtaining similar results.180 Other studies indicate that a high number of target sites can still limit the evolution of resistant strains with chromosomal mutations.181 In addition to the previous resistance response, it was found that the most common site of mutations was localized in the plasmid-encoded SpCas9 used to introduce the system into the target strain. Some studies described recombination and deletions that resulted in the inactivation of the CRISPR loci and the elimination of Cas genes and target sequences, to name a few.48,63 A proposed solution was reintroducing an intact copy of the plasmid to circumvent this resistance mechanism.182 Reduction of the CRISPR array to a single spacer repeat could prevent recombination between repeats and subsequent spacer deletion.40 One strategy for overcoming mutations in the targeted DNA sequences is to use multiple gRNAs to target different sites within the bacterial genome. This approach increases the likelihood of destroying the target DNA, even if it has mutated in one or more of the gRNA binding sites. Multiple gRNAs can be delivered simultaneously using a variety of methods, including plasmids, viral vectors, or nanoparticles.183 Another strategy is to use CRISPR-Cas systems that target different parts of the bacterial genome. For example, CRISPR-Cas9 and CRISPR-Cas13 systems target DNA and RNA, respectively. Targeting different parts of the genome can increase the likelihood of destroying the target, even if it has mutated in one part of the genome. Additionally, using different CRISPR-Cas systems can be effective against bacteria that have developed resistance to one particular system.184,185 A third strategy is to engineer the CRISPR-Cas system to target regions of the genome that are less likely to mutate. For example, the CRISPR-Cas system can be designed to target conserved regions of the genome, which are less likely to undergo mutations such as the gyrA gene in E. coli. The system was effective in killing antibiotic-resistant E. coli strains, which had mutations in other regions of the gyrA gene.186 A fourth strategy is to use high-throughput screening to identify gRNAs that are effective against bacteria with specific mutations. In this approach, large libraries of gRNAs are synthesized and screened for their ability to target mutated sequences. A study successfully utilized a library of 1.4 million gRNAs to identify all gRNAs effective against antibiotic-resistant E. coli strains.187,188 Inactivation via Anti-CRISPR (Acr) Proteins Anti-CRISPR proteins (Acrs) interact with essential components of the diverse CRISPR-Cas systems inhibiting the cleavage activities of the protein.189 It has been reported that most CRISPR-Cas systems can undergo inactivation, especially in prokaryotes where Acrs are highly diverse.190 For example, phylogenetic studies in a pool of around 600 genes belonging to a Pseudomonas aeruginosa strain collection found acr-related genes in at least 30% of them,191 restricting CRISPR-Cas applications. The first ACR protein to be discovered was AcrF1, which was identified in a study of the Type I-F CRISPR-Cas system in Pseudomonas aeruginosa. Since then, many other ACR proteins have been identified in various bacterial species, including Type II-A, Type II-C, and Type V-A CRISPR-Cas systems.191 ACR proteins function by binding to various components of the CRISPR-Cas system, such as the Cas proteins or the gRNA, and preventing them from binding to the target DNA. This inhibition can occur at different stages of the CRISPR-Cas process, including DNA binding, RNA-guided complex formation, and target cleavage.192 One of the most well-studied ACR proteins is AcrIIA4, which inhibits the activity of the Type II-A CRISPR-Cas system found in Streptococcus pyogenes. AcrIIA4 binds to the Cas9 protein and prevents it from binding to the target DNA. This inhibition is reversible and can be overcome by increasing the concentration of Cas9, suggesting that AcrIIA4 is a competitive inhibitor.193,194 The discovery of ACR proteins has important implications for the use of CRISPR-Cas systems in genome editing and other applications. The presence of ACR proteins can reduce the efficiency and specificity of the CRISPR-Cas system, leading to off-target effects and potentially reducing the therapeutic potential of this technology.189,195 Anti-anti-CRISPR strategies refer to the development of tools and techniques to overcome the inhibitory effects of anti-CRISPR (ACR) proteins on CRISPR-Cas systems. These strategies are important for improving the efficiency and specificity of CRISPR-Cas systems in genome editing and other applications. One possible solution has been described as an “anti-anti-CRISPR” strategy where anti-CRISPR-associated (aca) genes, generally encoded at the 3′-end of acr gene regions, are included in the antimicrobial design as they play a role in the repression of Acr genes.192,196 One approach to anti-anti-CRISPR strategies involves the development of small molecule inhibitors that can bind to and inhibit the activity of ACR proteins. These inhibitors could be used to prevent ACR proteins from binding to the CRISPR-Cas system and inhibiting its activity, thereby improving the efficiency of genome editing and other applications.197,198 Another approach is the use of engineered CRISPR-Cas systems that are resistant to the inhibitory effects of ACR proteins. For example, mutations can be introduced into the Cas protein or gRNA to prevent binding by ACR proteins, or alternative nucleases can be used that are not affected by ACR proteins.199 Other Forms of Escape Bacteria can also resist the CRISPR-Cas system by modifying their own DNA. One such modification is methylation, which involves adding a methyl group to the DNA sequence. This modification can prevent the CRISPR-Cas system from recognizing and destroying the target DNA by masking the PAM sequence required for recognition. A study found that some bacteria can methylate their DNA in the PAM sequence, preventing the CRISPR-Cas system from recognizing and destroying the target DNA.200 Some authors also hypothesize that E. coli “escapers” survive the DSBs within the chromosome when there is a low expression of SpCas9 to overcome the RecA-mediated repair.201 In recent research, researchers choose the promoters J23116, J23111, and J23100 for weak, intermediate, and strong expression of SpCas9, respectively. The expression strength was linked to the killing efficiency of the system (Figure 6-A). They demonstrate that the level of SpCas9 is an important parameter to consider to reduce the number of cells surviving DSBs.180 Impact of Environmental Conditions and Lifestyle on the Ability of Bacteria to Survive CRISPR-Based Antimicrobials Population Variations Microbial communities, composed of a diverse array of genera, species, and strains of microorganisms coexisting in the same environment, are characterized by complex networks of interactions where each member performs specific functions while simultaneously competing for limited resources.202,203 Competition for resources is a fundamental driving force behind the evolution of microbial communities. In environments where resources are limited, such as in soil or in the human gut, microbial communities must compete for nutrients and other essential factors in order to survive and reproduce.202,204 This competition can trigger a selective pressure for all the microbes suited for efficient acquisition and utilization of scarce resources, as well as those that are able to outcompete other microbes for these resources.205 Mechanisms, such as horizontal gene transfer (HGT), which promotes genetic diversity, are critical for microbial adaptation and evolution under these conditions.206 It is important to discuss how some of these mechanisms may restrict the use of CRISPR-based antimicrobials or limit their effectiveness against certain groups of bacteria. Interference of Different Plasmids and Other Mobile Genetic Elements MGEs, including plasmids, transposons, bacteriophages, and integrative conjugative elements, are important drivers of bacterial evolution and can significantly impact the effectiveness of CRISPR-based antimicrobials.76,175,207 As mentioned previously, microbial evolution can be shaped by competition for resources, prompted by the acquisition of beneficial genes or genetic elements (plasmids and MGEs) and enabling microbes to utilize resources effectively or protect themselves against external hazards.208,209 The ability of MGEs to facilitate the transfer of genetic information between different bacterial species allows for the evolution of novel genetic combinations and the emergence of new bacterial lineages.210 In highly competitive environments, MGEs provide an advantage for bacteria,209,211,212 and as a result, bacteria have evolved adaptations to restrict or reduce the action of genomic defense systems.208 Currently, CRISPR-based antimicrobials are not widely utilized, therefore no reports of resistance to this type of antimicrobial have been documented in clinically significant bacteria. Nevertheless, a critical concern for this type of antimicrobial is the possibility of bacteria escaping CRISPR-Cas dsDNA breakage due to mutations in chromosomal regions encoding for CRISPR-Cas effector protein, gRNA, or the target sequence,49,62,213−215 thereby inducing resistance. Along with CRISPR resistance resulting from mutations in chromosomal genes, increasing evidence suggests that several proteins possessing anti-CRISPR activity are encoded in plasmids and other MGEs, which can promote resistance to CRISPR-Cas systems, thereby limiting their use as antimicrobial agents. In a recent study, it was identified that anti-CRISPR proteins are up to 15 times overrepresented in the MGEs of ESKAPE pathogens when compared to the “nonmobile” genome.216 Furthermore, loci that code for anti-CRISPR proteins have been detected in MGEs of various Gram-negative bacteria,217 some of the clinical relevance.218 Considering the high frequency of horizontal transfer events between bacteria and the selective pressure that CRISPR-based antimicrobials could impose, it is expected that the transfer of plasmids that confer resistance to cutting by Cas nucleases will pose a challenge for the use of this type of antimicrobial therapy alternatives. Microbial Biofilms An adaptation mechanism employed to survive in diverse environments is the growth of biofilms, which are communities of microorganisms that form on various surfaces and are surrounded by an extracellular matrix of biopolymers.219 These structures have several characteristics that contribute to their survival, including surface adherence protection from desiccation, chemical perturbation, invasion by other bacteria, and killing by immune cells.220 Biofilm formation is also one of the main sources of antibiotic resistance in bacterial communities due to the poor penetration of these molecules into the biofilm matrix. Cells within biofilms are significantly more resistant to antimicrobials than their free-living counterparts as demonstrated by the literature with some studies showing up to 1000-fold increased resistance.221 Even though the resistance of biofilms to antimicrobials is often considered a passive or nonspecific mechanism, a combination of acquired and adaptive mechanisms contributes to antibiotic resistance in these structures.222 Genetic adaptation within biofilms allows cells to adapt to their surroundings and increase their resistance to antibiotics as shown by the expression of multi-drug resistance proteins by cells within biofilms.223 Some antibiotics can also induce the expression of resistance-related enzymes in these cells.224 External factors such as the environment and lifestyle of the target bacteria can significantly impact the effectiveness of CRISPR-Cas-based antimicrobials,180 potentially affecting the treatment of AMR-born infections. The environment where bacteria are localized can affect their growth rate, metabolic activity, and gene expression, which can further impact their antimicrobial susceptibility. Similarly, it has been extensively described that bacterial lifestyles significantly affect their ability to survive and replicate in the presence of antimicrobials.220,225 CRISPR-based antimicrobials may face the same limitation as conventional antibiotics if they target bacteria inside biofilms as the presence of the extracellular matrix can hinder the antimicrobial penetration,220,223 making it more difficult for CRISPR-Cas systems to reach their targets and cleave targeted DNA sequences (Figure 6-B). Additionally, bacteria within a biofilm tend to have a high degree of heterogeneity in metabolic states and gene expression,224,226 leading to individual cells exhibiting resistance due to reduced permeability, low metabolic activity, and the production of persister cells.220,227 As a result, current biofilm treatments often require higher doses of conventional antimicrobials administered for longer periods of time compared to the doses and duration needed to treat planktonically grown cells.221,223 As discussed in this section, some features of the lifestyle of microbial communities have been identified as a potential obstacle to the effectiveness of CRISPR-Cas-based systems as antimicrobial agents. Nonetheless, certain lifestyle changes could also provide opportunities for the successful deployment of this type of antimicrobial. A study demonstrated that biofilm formation significantly enhanced the conjugative transfer proficiency of a CRISPR-bearing plasmid.121 This effect can be attributed to the proximity of cells within a biofilm community which can be advantageous to enhance CRISPR-based antibacterial killing. Despite the antimicrobial potential of CRISPR-based systems, certain bacterial strains still exhibit a natural resistance to CRISPR-Cas and represent a significant challenge for CRISPR application. In light of these limitations, CRISPR-Cas antimicrobials may not be effective as standalone treatments, but instead, they could be employed as a supplementary approach to address microbial resistance. Concluding Remarks and Future Prospects Due to the proliferation of antibiotic-resistant bacteria, the management of infectious diseases is becoming increasingly challenging. The high specificity and reprogrammability of CRISPR-Cas constructs have piqued the interest of the research community for the development of a plethora of novel applications. In vitro and in vivo studies have shown that CRISPR-Cas systems have the potential to serve as next-generation antimicrobials. Although the regulatory and ethical aspects of CRISPR-Cas-based antimicrobials and their clinical application have not been widely discussed in the literature, the general use of the CRISPR-Cas technology has raised several concerns.228 A significant issue to consider is the risk of off-target mutations with consequent deleterious effects observed in large genome sequences such as human cell lines.229,230 Bearing in mind that CRISPR-CAS-based antimicrobials will be in contact with the patient tissue, it is essential to anticipate and resolve these hazards during the early stages of antimicrobial design by optimizing the guide RNA target system231 and upgrading the delivery vehicle specificity. Furthermore, the risks related to the potential release of the CRISPR-Cas system into the environment need to be assessed, and strict control regulations for therapeutic handling and application should be enforced.231 It is imperative to engage and educate the medical and general populations regarding all distinct aspects of this new technology as well as to update legislative guidelines related to the use of gene-editing tools to ensure their well-informed and safe application.232 Prospective applications of CRISPR-Cas-based antimicrobials include clearance of the AMR pathogenic microorganisms or restoring their susceptibility to conventional antibiotics. However, the main challenge of using CRISPR-Cas systems as therapeutic solutions is the development of effective vectors that can deliver exogenous DNA. Currently, the most commonly employed methods for CRISPR-Cas delivery include bacteriophages, nanoparticles, and conjugative plasmids. There are several challenges and limitations that can be foreseen in the development of CRISPR-based antimicrobials. The most concerning one is the generation of resistance mechanisms by targeted bacteria. Further research is necessary to assess the true potential of CRISPR-Cas systems in more realistic microbial communities and to fully understand the risks associated with this technology. The authors declare no competing financial interest. Acknowledgments We thank Dr. Alexander Mueller for technical support. All the figures in this Review were created with Biorender.com. ==== Refs References Tacconelli E. ; Carrara E. ; Savoldi A. ; Harbarth S. ; Mendelson M. ; Monnet D. L. ; Pulcini C. ; Kahlmeter G. ; Kluytmans J. ; Carmeli Y. ; Ouellette M. ; Outterson K. ; Patel J. ; Cavaleri M. ; Cox E. M. ; Houchens C. R. ; Grayson M. L. ; Hansen P. ; Singh N. ; Theuretzbacher U. ; et al. 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==== Front 101313252 34584 J Vis Exp J Vis Exp Journal of visualized experiments : JoVE 1940-087X 36912520 10.3791/64886 nihpa1914263 Article Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice Harvey Kyra 1 Madsen Peter J. 23 Smith Tiffany 2 Griffin Crystal 12 Patterson Luke 12 Vitanza Nicholas A. 45 Storm Phillip B. 23 Resnick Adam C. 23 Foster Jessica B. 12 1 Division of Oncology, Children’s Hospital of Philadelphia 2 Center for Data Driven Discovery in Biomedicine, Children’s Hospital of Philadelphia 3 Division of Neurosurgery, Children’s Hospital of Philadelphia 4 Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute 5 Department of Pediatrics, Seattle Children’s Hospital, University of Washington Corresponding Author: Jessica B. Foster, fosterjb@chop.edu 13 7 2023 24 2 2023 24 2 2023 18 7 2023 192 10.3791/64886https://creativecommons.org/licenses/by-nc-nd/3.0/ JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors, raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors. ==== Body pmcIntroduction Despite improvements in chemotherapy, radiotherapy, and surgery, central nervous system (CNS) tumors are the deadliest malignancy in pediatrics1, underscoring an important need for new approaches with more successful outcomes. With significant advances in the field of immunotherapy, adoptive cellular therapy (ACT) approaches have shown promising results in various cancers, especially hematological malignancies2. Chimeric antigen receptor (CAR) T cell therapy, a specific type of ACT, takes advantage of the immune system’s natural ability to recognize and kill harmful cells by redirecting the specificity of T cells to generate tumor-targeting T cells3. CAR T cell therapy has demonstrated substantial success in the treatment of leukemias and lymphomas4, making it a promising immunotherapeutic approach and encouraging its investigation in solid tumors. However, thus far, CAR T cell therapy in solid tumors has achieved little clinical success and faces many challenges, such as inefficient tumor penetration, limited targetable antigens, and the suppressive tumor microenvironment5. Recent clinical trials have begun evaluating CAR T cell therapy for pediatric CNS tumors, providing proof of concept and early evidence of T cell activity in preliminary reports6,7,8. While most initial preclinical data focused on intravenous delivery of the CAR T cells, recent preclinical evidence has suggested the superiority of locoregional delivery in the CNS9,10, which has also been utilized successfully in several clinical trials6,7,8,11. Preclinical studies to date that have incorporated the locoregional delivery of CAR T cells in the CNS have relied on a single intracranial dose of CAR T cells delivered stereotactically9,10. However, clinical trials in humans have required repeated infusions of CAR T cells in the CNS6,7,8,11, underscoring a need to evaluate multiple repeated infusions in preclinical development. The goal of this procedure is to successfully test serial CAR T cell infusions using a catheter in orthotopic murine models of pediatric brain tumors. The advantage of this technique is the avoidance of multiple surgical procedures to provide repeated intra-CNS treatments. Cannulas have primarily been used for microdialysis sampling of neurotransmitters and the delivery of neuroactive substances in neuroscience and behavioral research in rodents12, with limited reports of their use for the delivery of anti-cancer therapeutics. Building on the prior reports, this protocol uses a stereotactically placed indwelling cannula system to deliver CAR T cells in xenograft murine models of CNS tumors. The protocol can be utilized to test additional therapeutics in murine models of neurological or neuro-oncologic disorders, and may be helpful to test novel therapeutics where bypassing the blood-brain barrier is critical for efficacy. Protocol All protocol procedures were approved by the Institutional Animal Care and Use Committee of the Children’s Hospital of Philadelphia (IAC 19-000907), which is accredited by the AAALAC. This study used 6-12-week-old NOD scid gamma (NSG) mice with orthotopic xenograft tumors; however, the protocol can be utilized on any mouse strain. NSG mice were housed in sterile barrier conditions and surgery performed under sterile biosafety cabinets. When human material such as tumor cells or T cells are being used, procedures and handling must be completed in ABSL-2 biosafety cabinets. 1. Preparing the mouse for surgery Anesthetize the mouse in an induction chamber with isoflurane (2-4%) at an oxygen flow rate of 1 L/min until an adequate plane of anesthesia is reached (approximately 5 min). Weigh the mouse using a scale to the nearest 0.1 g and administer subcutaneous slow-release (SR) buprenorphine (1 mg/kg) or other analgesic. NOTE: SR buprenorphine provides analgesia for 72 h. Shave the fur on the top of the mouse’s head using electric clippers or depilatory agents. Use a spatula to gently open the bottom of the stereotactic arm and insert the guide cannula using forceps. Tighten the screw on the arm to secure the cannula so that approximately 1/2 to 2/3 of the white plastic portion of the cannula is protruding from the bottom of the opening, along with the entire 5 mm metal length of the cannula. Insert and secure the mouse’s top teeth in the bite bar of the stereotaxic apparatus. Pull the nose cone forward and tighten it, ensuring the mouse is inhaling isoflurane. Mount the mouse on the warmed stereotaxic apparatus using ear cuffs or ear bars, avoiding excessive pressure. NOTE: Warmed tray should have a rectal thermometer inserted, and the warming tray should adjust to maintain a normal body temperature of the mouse during the procedure. Apply sterile ophthalmic ointment to both eyes using a cotton-tipped applicator. Wipe the surgical site with povidone-iodine on a pad or applicator, followed by an alcohol pad. Perform this step three times in total. Before beginning the procedure, perform a toe pinch with forceps to assess for adequate sedation. 2. Surgical procedure NOTE: All aspects of the surgical procedure utilize sterilized instruments and aseptic techniques. The mice continue under anesthesia with isoflurane (2-4%) throughout the duration of the procedure, approximately 10-20 min. Gently pick up the scalp between the ears with forceps. Using sterile scissors, cut the lifted scalp parallel to the skull and remove an oval flap of skin (0.75-1 cm in length) to expose the skull. NOTE: Scissors are preferred over a scalpel to provide a clean, oval-shaped opening and to prevent unnecessary damage to surrounding skin and tissue. Push away fascia using a scalpel or cotton-tipped swabs and a hemostatic cotton pellet to help slow excessive bleeding as needed. NOTE: Using the wooden side of a sterile cotton tip can also push away fascia and help avoid excessive bleeding. Identify the landmarks bregma and lambda, the respective anterior and posterior marks on the skull where the cranial plates meet13. NOTE: Identification can be augmented by wiping the top of the exposed skull with hydrogen peroxide. Gently score the skull using a scalpel to create a surface for the acrylic to attach. Scoring should include multiple linear lines approximately 0.5-1 cm in length at 90° angles to each other. Using the stereotaxic arm, localize the cannula to the landmark of interest (bregma or lambda). Once localized, raise the cannula tip 1-2 mm above the skull surface and move to the desired coordinates. For intratumoral injections, this uses the same A/P and M/L coordinates as the tumor placement. On the exposed skull, away from the area where the cannula will enter, make two screw holes with an 18 G needle or a surgical drill. Ensure that the holes are spaced out to include enough room for the cannula. Using a drill bit, twist through the screw holes until they catch on the skull. Insert and fasten two screws into the holes using a flat-tipped screwdriver. Then, gently pull the screws up to ensure they are secured. NOTE: Do not insert the screws until they are flush with the skull, or they may damage the mouse brain underneath. Leave at least a 1-2 mm gap between the screw and the skull. Using an 18 G needle or surgical drill, drill through the skull at the identified coordinates to create a hole for the cannula to be inserted. Using the stereotactic arm, lower the cannula to the desired D/V coordinate. NOTE: The D/V coordinate of cannula implantation needs to account for the projection length of the dummy and treatment cannulas, and may be more superficial than the orthotopic injection of tumor cells (Figure 1). In a porcelain 12-well plate, fill one well with acrylic resin powder (approximately 0.3 g) and 10-15 drops (approximately 0.5-0.75 mL) of acrylic resin liquid. This produces a viscous white-colored material. Draw up the mixture into a 1 mL syringe and use it to coat and cover the skull, filling in the spaces around the cannula and screws. NOTE: The viscous material hardens into cement over time, so this step should be completed promptly after mixing. While the cement is still pliable, loosen the screw on the stereotactic arm and use a spatula in the opening at the bottom to gently release the cannula from the holder and slowly retract the stereotactic arm upward away from the mouse. Once the cement is completely dry, insert the dummy cannula into the guide cannula and tightly secure it by turning clockwise. Once the procedure is complete, place the mouse back in its warmed home cage to carefully monitor, ensuring adequate recovery and recording any post-procedure observations, including that the mouse has fully regained consciousness, before returning to the colony. NOTE: It is generally recommended to place only half of the cage on a heating pad to allow for the animal to move to the cooler side to avoid overheating. Administer additional analgesics as needed if the mice display behaviors indicative of pain post-operatively, such as meloxicam 5 mg/kg subcutaneously delivered once daily for up to 3 days. 3. Preparing CAR T cells Measure the pre-transfected CAR T cell concentration using a cell counter. Centrifuge pre-transfected T cells at 200 x g for 5 min at room temperature (RT). Aspirate the supernatant using a sterile Pasteur pipet on a vacuum aspiration system and resuspend the pellet in phosphatae buffered saline (PBS) to a desired concentration. Typical delivery volumes are 2-5 μL. Typical cell doses are 0.5-5 x 106 cells. 4. CAR T cell infusions Prepare the treatment cannula by feeding the top through a small piece of PKG tubing. Fill the treatment syringe with the CAR T cell suspension and insert it through the other end of the PKG tubing, enough to cover the top of the treatment cannula. Anesthetize the mouse with isoflurane (2-4%) at an oxygen flow rate of 1 L/min. Stabilize the guide cannula using forceps at the base, and then carefully unscrew and remove the dummy cannula, allowing for access into the guide cannula. NOTE: A stereotaxic setup is not necessary, but can be used to stabilize the head for treatment. Infuse the CAR T cells for 1 min and hold the treatment cannula in place for an additional 1 min following the cessation of infusion. Remove the treatment cannula and screw the dummy cannula tightly back in place. Administer subcutaneous meloxicam (5 mg/kg) for optional pain control. Representative Results Successful cannula implantation into the mouse CNS Mice with successful cannulation have a secure guide cannula in place, that does not interfere with activities of daily living (Figure 2A) and that can easily pass a treatment cannula and deliver fluid without resistance (Figure 2B). In our experience, the majority of cannulas remain in place for over 4 weeks, although 0-25% can be dislodged over time. Verification of correct placement can be confirmed with Evans blue dye injected into the cannula. For example, a cannula inserted into the lateral ventricle shows Evans blue dye throughout the ventricular system as it travels through the cerebral spinal fluid, confirming correct placement (Figure 2C). Cannulas inserted into the tumor show Evans blue dye at the site of tumor implantation. Effective delivery of CAR T cells into the CNS for treatment of xenograft tumors. The efficacy of the intracranial cannula system and the therapeutic efficacy of CAR T cells in murine models can be measured through a variety of mechanisms, including bioluminescent imaging and overall survival. GPC2-directed CAR T cells were tested against murine medulloblastoma and diffuse midline glioma models, 7316-4509 and 7316-3058, respectively, using repeated CAR T cell dosing via the stated intracranial cannula system14. Orthotopic tumor placement and engraftment were confirmed by bioluminescent imaging, and cannulas were placed into the tumor bed using the same coordinates as the orthotopic tumor placement. The treatments consisted of GPC2 CAR T cell infusions once to twice a week for 2-4 weeks, totaling four to six doses. Following treatment, GPC2-directed CAR T cell therapy induced significant tumor regression in the medulloblastoma model 7316-4509 (p < 0.01) (Figure 3A) and significantly prolonged survival in thalamic diffuse midline glioma 7316-3058 (p < 0.05) (Figure 3B)14. Discussion CAR T cell therapy has revolutionized the treatment of hematological cancers and shows promising value in treating solid brain tumors6,7,8. This protocol was designed to enable the preclinical evaluation of locoregional CAR T cell delivery for the treatment of pediatric CNS tumors. The cannula system replicates an Ommaya or Rickham reservoir, an intraventricular catheter system currently being used in ongoing clinical trials of CAR T cell therapy in pediatric CNS tumors6,7,8, underscoring the relevance and translational potential of these methods. This system allows for the repeated delivery of CAR T cells that bypass the blood-brain barrier, again similar to methods being employed in ongoing clinical trials. Locoregional delivery can provide maximal efficacy in the CNS9 and may also reduce the risk of systemic toxicities associated with trafficking from circulation15. While stereotactic delivery can provide a single dose into the CNS, the advantage of this system is the opportunity to provide multiple repeated doses into a specified location in the CNS without the need for multiple surgeries. The limitations of this procedure include a fixed delivery site without the ability to change location or make adjustments once the cannula is in place, and the potential for dislodgement of the cannula. A critical step in this protocol is the implantation of the fixed guide cannula at a D/V coordinate that takes into consideration the projection of the treatment cannulas. The treatment cannula will protrude beyond the tip of the guide cannula, and so care must be taken to ensure the placement will result in the delivery of CAR T cells to the region of interest. Projection lengths of the treatment cannula can be customized, and in the author’s experience, 0.5 mm is a useful projection length. This length ensures that the therapeutic does not remain in the guide cannula upon dispensing, but also does not require significant adjustment of the D/V coordinates for the guide cannula to the region of interest. An additional important step in this protocol is the time the treatment cannula is left in place following CAR T cell infusion. The treatment cannula should be held in place for at least 1 min following the end of infusion, to prevent leaks and the loss of locoregional delivery of CAR T cell therapy. Troubleshooting this method is straightforward, with most complications involving difficulty removing the dummy cannula or inserting the treatment cannula into the fixed guide cannula, likely due to dried blood on the interior of the guide cannula. This can be easily resolved by gently passing the dummy cannula through the guide cannula until there is less resistance and the debris has been cleared. The acrylic resin can occasionally dislodge from the skull, resulting in the loss of the cannula system. In our experience, this is generally limited by scoring the skull with a scalpel and the placement of two screws. In addition, any items from the cage that may accidentally apply force to the cannula while the mouse is moving around are removed, such as particular mouse enrichment huts with small openings. In conclusion, described here is a protocol for the insertion of a cannula system in murine models of CNS tumors for the repeated delivery of CAR T cells. Cannula placement can be adjusted to multiple locoregional delivery locations, testing the efficacy of different delivery sites. In addition, this system can be used for additional therapeutics beyond CAR T cells to evaluate efficacy when bypassing the blood-brain barrier, and may also be useful for the evaluation of therapeutics in murine models of non-oncologic disorders. Acknowledgments Funding for this work was provided by the Matthew Larson Foundation, Grayson Saves Foundation, Hyundai Hope on Wheels Young Investigator Award, Kortney Rose Foundation, National Institutes of Health NCI K12 CA076931-19 and 1K08CA263179-01, and the Department of Defense W81XWH-21-1-0221. Figure 1: Guide cannula with projection dummy and treatment cannulas. (A) Guide cannula with 0.5 mm projection and 2 mm projection dummy cannulas in place. (B) Guide cannula with 0.5 mm projection and 2 mm projection treatment cannulas in place. Please click here to view a larger version of this figure. Figure 2: Treatment cannula delivery system for repeated doses of CAR T cells. (A) Cannula implanted in a mouse skull with a cap in place when not in use. (B) Infusion of CAR T cells into a pontine tumor via the treatment cannula while the mouse is under anesthesia. (C) Verification of cannula placement in the lateral ventricle using Evans blue dye. Dye was inserted through the cannula, followed by euthanasia and brain excision, with dye present throughout the ventricles. Please click here to view a larger version of this figure. Figure 3: GPC2-directed CAR T cells mediate antitumor responses and prolong survival in pediatric brain tumors in vivo. (A) Quantification of bioluminescence of the orthotopic group 4 medulloblastoma xenograft 7316-4509 treated with either GPC2- or CD19-directed mRNA CAR T cells. Doses are indicated by arrows on the graph. Data displayed as mean with SD, n = 9-11 mice per arm. (B) Overall survival of mice implanted with thalamic DMG xenograft 7316-3058 treated with six repeated doses of 2 x 106 CAR T cells. Doses are indicated by arrows on the graph. n = 7 mice per arm. **p < 0.01; *p < 05; ns = not significant. This figure has been reproduced with permission from Foster et al.14. Please click here to view a larger version of this figure. A complete version of this article that includes the video component is available at http://dx.doi.org/10.3791/64886. Disclosures JBF holds a patent related to glypican 2 (GPC2)-directed immunotherapies. All other authors have nothing to disclose. ==== Refs References 1. Curtin SC , Minino AM , Anderson RN Declines in cancer death rates among children and adolescents in the United States, 1999-2014. NCHS Data Brief. 257 ,1–8 (2016). 2. Rohaan MW , Wilgenhof S , Haanen JBAG Adoptive cellular therapies: the current landscape. Virchows Archiv. 474 (4 ), 449–461 (2019).30470934 3. Sadelain M , Brentjens R , Riviere I The basic principles of chimeric antigen receptor design. Cancer Discovery. 3 (4 ), 388–398 (2013).23550147 4. Maude SL Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. The New England Journal of Medicine. 378 (5 ), 439–448 (2018).29385370 5. Wagner J , Wickman E , DeRenzo C , Gottschalk S CAR T cell therapy for solid tumors: Bright future or dark reality? Molecular Therapy. 28 (11 ), 2320–2339 (2020).32979309 6. Vitanza NA Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis. Nature Medicine. 27 (9 ), 1544–1552 (2021). 7. Majzner RG GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature. 603 (7903 ), 934–941 (2022).35130560 8. Vitanza NA Intraventricular B7-H3 CAR T cells for diffuse intrinsic pontine glioma: preliminary first-in-human bioactivity and safety. Cancer Discovery. 13 (1 ), 114–131 (2023).36259971 9. Theruvath J Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors. Nature Medicine. 26 (5 ), 712–719 (2020). 10. Donovan LK Locoregional delivery of CAR T cells to the cerebrospinal fluid for treatment of metastatic medulloblastoma and ependymoma. Nature Medicine. 26 (5 ), 720–731 (2020). 11. Brown CE Regression of glioblastoma after chimeric antigen receptor T-Cell therapy. The New England Journal of Medicine. 375 (26 ), 2561–2569 (2016).28029927 12. Bourne JA Intracerebral microdialysis: 30 years as a tool for the neuroscientist. Clinical and Experimental Pharmacology and Physiology. 30 (1–2 ), 16–24 (2003).12542448 13. Zhou P Automatically detecting bregma and lambda points in rodent skull anatomy images. PLoS One. 15 (12 ), e0244378 (2020).33373400 14. Foster JB Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors. Journal for Immunotherapy of Cancer. 10 (9 ), e004450 (2022).36167467 15. Akhavan D CAR T cells for brain tumors: Lessons learned and road ahead. 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==== Front J Patient Exp J Patient Exp JPX spjpx Journal of Patient Experience 2374-3735 2374-3743 SAGE Publications Sage CA: Los Angeles, CA 10.1177/23743735231179040 10.1177_23743735231179040 Research Article The Association of Emergency Severity Index Score and Patient and Family Experience in a Pediatric Emergency Department https://orcid.org/0000-0003-3499-1642 Vukovic Adam A MD, MEd 12 https://orcid.org/0000-0003-3986-8731 Krentz Callie MD 1 Gauthier Abigail MD 1 Harun Nusrat PhD 3 Porter Stephen C MD, MSc 12 1 Department of Pediatrics, 12303 University of Cincinnati College of Medicine , Cincinnati, OH, USA 2 Division of Emergency Medicine, 2518 Cincinnati Children's Hospital Medical Center , Cincinnati, OH, USA 3 Division of Biostatistics and Epidemiology, 2518 Cincinnati Children's Hospital Medical Center , Cincinnati, OH, USA Adam A Vukovic, Division of Emergency Medicine, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, MLC 2008, Cincinnati, OH 45229, USA. Email: adam.vukovic@cchmc.org 16 7 2023 2023 10 23743735231179040© The Author(s) 2023 2023 Cleveland Clinic https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage). The study aim was to determine the relationship between a patient's Emergency Severity Index (ESI) score and their or their family's response to the key performance indicator (KPI) question on the post-visit patient and family experience (PFE) survey. Retrospective review of patients presenting to the Pediatric Emergency Department between July 1, 2021, and June 30, 2022, who completed the KPI question on an associated post-visit survey. We performed univariate analyses on all candidate variables; multivariable linear regression identified independent predictors of KPI on the PFE survey. A total of 8136 patients were included in the study. Although ESI score was significantly associated with PFE in univariate analysis, this association was lost in the multivariable model. Independent associations were appreciated with race/ethnicity, time to provider, length of stay, and procedure performance during the visit. Although ESI is not independently associated with PFE in this study, its interaction with factors such as time to provider, length of stay, and procedure performance may be important for emergency department providers creating interventions to impact experience during low acuity visits. pediatric emergency experience triage typesetterts19 cover-dateJanuary-December 2023 ==== Body pmcIntroduction Patients and their families seek care in Pediatric Emergency Departments (PEDs) for emergencies real and perceived at all hours of the day. Multiple studies have investigated the motives of families seeking care in the PED.1–5 Commonly, caregivers view their child's illness or injury with higher perceived urgency than providers.2,5 Caregivers also seek empathy for a child's suffering and maintain expectations around being a responsible parent for their ill child. 1 Overnight, an emergency department may be the only place to seek care.2,3 The PED may be the closest site of care for the family and, in the event of a perceived emergency, viewed as the quickest way to have their child seen.4,5 The PED offers a perceived immediate fix to a problem and an opportunity to have a sick child feel better versus having to delay treatment while awaiting an ambulatory appointment.1,3 When a patient arrives at the PED, they are triaged using the Emergency Severity Index (ESI) system, which is a validated 5-level triage scale that applies a clinical assessment to the patient and assigns a triage level 1 to 5, with 1 denoting a patient who requires immediate emergency lifesaving medical care and 5 identifying the least severe patients with less urgent needs. The ESI considers the patient's illness, vital signs, and potential need for PED resources when assigning a triage level. The American College of Emergency Physicians and the Emergency Nurses Association support the use of the ESI as a tool in emergency care. 6 Most patients in the United States who seek emergency care are triaged via the ESI system. 7 Specifically in pediatrics, the current version of ESI has been shown to be a reliable tool in predicting hospitalization, PED length of stay, and resource utilization. 8 Overall, ESI is a crucial tool in the PED to determine which patients need expedited care. However, while prioritizing those patients with lower ESI scores, those with higher scores may wait longer for definitive care. Wait times have been associated with overall patient and parent satisfaction in PED visits.9–12 Wait times were identified as the second strongest predictor in overall patient satisfaction only behind how well physicians and nurses work together. 9 In a survey of patients and parents in a PED, long wait times were highlighted as an aspect of their care that was described as poor. 12 Notably, children being treated had poorer experiences with wait time when compared to their parents. 12 Making waiting a positive experience was noted to be uniquely important contributor to the patient and family experience (PFE) in the PED.13,14 One survey of PED families noted that longer wait times were identified as a problem more often in the night or overnight shifts. Of note, most respondents in this study who completed the survey overnight were completed by parents of patients with lower acuity triage scores. 14 PFE surveys are frequently given after PED visits asking families to rate their experience. PFE scores have been shown to be inversely related to wait times and door-to-room times. 11 Abbreviated wait times and optimized waiting room experiences can improve the PED experience for patients and families.12,15 Given the connection between ESI and wait times, ESI scores may be correlated with PFE survey results. The purpose of this study is to identify any association between a patient's ESI level and their or their family's experience with that encounter, as identified through the key performance indicator (KPI) on our post-visit PFE survey. Methods Study Design and Setting This is a retrospective cohort study of patient encounters resulting in discharge from either of 2 freestanding PEDs between July 1, 2021, and June 30, 2022, in which a PFE survey was completed after the visit. This single-center study was performed across 2 campuses in which freestanding PEDs are present. Our institution has >650 inpatient beds and sees approximately 100 000 PED visits annually, resulting in approximately 35 000 admissions annually. Our institution uses the National Research Corporation (NRC) Health survey platform for soliciting feedback from patients and families, with results accessible through the NRC Health Portal, which has been used for the last 3 years. Around 8000 survey responses are received annually following PED visits. Our institutional review board deemed this study exempt from review. Given this exemption and the minimal risk to included patients in this retrospective study, consent was not obtained for those included in this study. Subject Selection Inclusion Criteria. All encounters for patients <21 years of age who were treated in and discharged from either of our PEDs with a completed PFE survey after the visit were eligible for inclusion. This includes patients of all genders, races, and ethnicities, regardless of presenting complaint. Patient encounters were included if, in the post-visit PFE survey, there was a response provided to the KPI question: “How would you rate our facility?” Exclusion Criteria. Encounters for patients 21 years of age or older, those who left prior to provider assessment and standard discharge, those admitted to the hospital, and those transferred to another institution for definitive care were excluded from this study. Patients were excluded if there was no corresponding PFE survey to their PED visit, or if there is no response to the KPI “rate facility” question. Lastly, a patient who visits one of our PEDs within 7 days of a prior PED visit and is discharged on both occasions will not be issued a PFE survey on their second visit and thus is not eligible for this study. Data Collection Preexisting data from 2 sources were used to complete this study. Data were collected on all patients who completed a PFE survey during the defined study period. Single data extraction from the NRC health portal generated a list of all patients eligible for inclusion in the study. The NRC health portal is not directly connected to our institutional electronic health record (EHR), and thus the contact serial number (CSN) from each PFE survey was used for a second data extraction from our EHR. The CSN is a unique identifier that ties data to the specific visit and is consistent across both platforms. We queried EPIC (Verona, WI, 2022) to pull clinical data related to each encounter to generate a final database, which included experience-level data cross-referenced with clinical elements from the EHR. Data Collected Data collected from the NRC Health Portal included: patient name, medical record number (MRN), CSN, encounter date, PFE survey response date, location of encounter, and responses to the following prompts: How would you rate our facility (KPI)? How likely would you be to recommend our emergency department to friends or family? I/my child was seen in a timely manner. Data collected from our EHR included: patient name, MRN, CSN, date of birth, encounter date, race, ethnicity, and ESI score. We also extracted whether a prescription was written at the time of discharge, if any lab work was ordered during the encounter, if any imaging studies were ordered during the encounter, or if a procedure had been performed during the visit. We chose these a priori based on the theory that the occurrence of any of these interventions during a patient encounter might impact PFE. We identified procedures in our EHR review as any intravascular catheter placement, ketamine administration, or laceration repair/abscess drainage documentation. We chose this specifically to align with our definition of procedure in the NRC health portal and felt that this identified our most common procedures and sedative used in nearly all other procedures requiring sedation. Lastly, we collected time to provider and length of stay. We defined time to a provider as the time of patient arrival to the time they were first seen by a provider (defined as a medical student, resident, Pediatric Emergency Medicine fellow or attending, staff pediatrician, or advanced nurse practitioner); we defined length of stay as the time of patient arrival to the time of patient discharge. Data Analysis We generated frequencies and proportions for all categorical variables and means and standard deviations for all continuous variables. Statistical associations with the continuous facility rating were assessed using the Wilcoxon rank-sum or Kruskal-Wallis tests for categorical variables and the Pearson correlation coefficient for continuous variables in the univariate analyses. For our primary outcome, we used multivariable linear regression to identify independent predictors of KPI on the PFE survey adjusting for other factors. Candidate variables included: sex, race, ethnicity, ESI score, prescription written, labs ordered, imaging ordered, procedure performed, time to provider, and length of stay. We did not include responses to the following prompts: How likely would you be to recommend our emergency department to friends or family; I/my child was seen in a timely manner. Our multivariable model included all objective clinical variables included in univariate analysis. SAS® version 9.4 was used for all analyses (SAS Institute Inc). Results There were 9795 PFE surveys completed during the study period, of which 8136 survey respondents answered the KPI question and were included in our analysis (Table 1). The mean (SD) age of patients included in our analysis was 7.7 (5.9) years, and approximately half were male (51%). Most respondents were White (66%), with Black (16%) respondents comprising the second highest race. Three-quarters of patients were triaged as ESI 3 or 4 (74%), and most did not receive prescriptions (62%), have labs or imaging ordered (64%), or undergo a procedure (84%). Table 1. Demographic Data for Patients Discharged From the Pediatric Emergency Department During the Study Period Who Completed the Post-Visit PFE Survey. a Characteristic Answered KPI, n = 8136 Did Not Answer KPI, n = 1659 Age (years, SD) 7.7 (5.9) 7.3 (6.0) Sex (N, %)  Male 4130 (51) 863 (52)  Female 4006 (49) 796 (48) Race (N, %)  Asian 245 (3) 112 (7)  Black 1317 (16) 407 (25)  Middle Eastern 42 (1) 19 (1)  Multiple 329 (4) 75 (5)  Other 96 (1) 53 (3)  Preferred category not available 496 (6) 192 (12)  Unknown 227 (3) 59 (4)  White 5383 (66) 742 (45) Ethnicity (N, %)  Hispanic 895 (11) 344 (21)  Non-Hispanic 7153 (88) 1300 (78)  Refused/Unknown 87 (1) 15 (1) ESI (N, %)  1 6 (0) 1 (0)  2 1732 (21) 293 (18)  3 3505 (43) 655 (40)  4 2508 (31) 605 (36)  5 381 (5) 104 (6) Prescription written (N, %)  Yes 3068 (38) 706 (43)  No 5068 (62) 953 (57) Labs ordered (N, %)  Yes 2935 (36) 639 (39)  No 5201 (64) 1020 (61) Imaging ordered (N, %)  Yes 2716 (33) 472 (28)  No 5420 (67) 1187 (72) Procedure performed (N, %)  Yes 1324 (16) 198 (12)  No 6812 (84) 1461 (88) Abbreviations: KPI, Key Performance Indicator; PFE, patient family experience; ESI, Emergency Severity Index. a Value assigned at triage to indicate anticipated resource needs as well as illness or injury severity; Prescription written, labs ordered, imaging ordered, and procedure performed refer to any of those elements occurring during the patient encounter. Table 2 presents the univariate analysis comparing PFE survey responses or specific clinical aspects of the encounter with responses to the KPI question on the post-visit PFE survey. Except for prescriptions, imaging, and procedure performance, all variables were statistically significant in univariate analysis (P < .05). ESI levels were significantly associated with the KPI response, with higher acuity patients (ESI 1 and 2) providing more positive responses than lower acuity patients (ESI 3 and 4). Notably, ESI level 5 patients provided scores similar to ESI level 2 patients; however, there were far less responses in this triage category than in ESI 2, 3, or 4. Both time to provider and length of stay demonstrated statistically significant negative associations with the KPI question on our PFE survey. This suggests that with longer time to providers and length of stays, responses to the KPI are less positive. Table 2. Univariate Association of PFE Survey Responses and Specific Aspects of Clinical Care With Facility Rating on PFE Survey. a PFE or clinical aspect How would you rate our facility? (mean [SD], N) P Sex  Male 8.5 (2.5), 4130 .035  Female 8.4 (2.6), 4006 Race  Asian 8.2 (2.7), 245 <.0001  Black 8.4 (2.6), 1317  Middle Eastern 8.8 (2.6), 42  Multiple 8.5 (2.6), 329  Other 9.1 (1.8), 96  Preferred category not available 9.3 (1.7), 496  Unknown 8.8 (2.3), 227  White 8.4 (2.5), 5383 Ethnicity  Hispanic 9.1 (2.0), 895 <.0001  Non-Hispanic 8.4 (2.6), 7153  Refused/Unknown 8.4 (2.6), 87 ESI  1 9.5 (0.8), 6 <.0001  2 8.8 (2.2), 1732  3 8.3 (2.7), 3505  4 8.5 (2.5), 2508  5 8.8 (2.3), 381 Prescription written  Yes 8.5 (2.4), 3068 .83  No 8.4 (2.6), 5068 Labs ordered  Yes 8.4 (2.6), 2935 .043  No 8.5 (2.5), 5201 Imaging ordered  Yes 8.5 (2.5), 2716 .84  No 8.4 (2.5), 5420 Procedure performed  Yes 8.5 (2.4), 1324 .55  No 8.5 (2.5), 6812 How likely would you be to recommend our emergency department to friends or family?  0-6 4.3 (2.8), 1526 <.0001  7-8 8.0 (1.1), 992  9-10 9.7 (0.8), 5618 I/my child was seen in a timely manner.  Yes 9.1 (1.8), 6605 <.0001  No 5.7 (3.2), 1529 Time to provider b -0.3 <.0001 Length of stay b -0.2 <.001 Abbreviations: PFE, patient family experience; ESI, Emergency Severity Index. a Facility rating is on a scale of 0 to 10. Value assigned at triage to indicate anticipated resource needs as well as illness or injury severity; prescription written, labs ordered, imaging ordered, and procedure performed refer to any of those elements occurring during the patient encounter. b Measure represents Pearson correlation coefficient between measure and question, “How Would you Rate our Facility?.” Results from our multivariate analysis are displayed in Table 3. The point estimate represents the effect of the clinical aspect on KPI response in a linear regression model. There was no independent association between ESI score and the patient or family response to the KPI question on our post-visit PFE survey. There is a statistically significant association between race and response, driven by the “preferred category not available” group. Similarly, patients and families identifying as Hispanic race responded more positively to the KPI question on the post-visit PFE survey. Lastly, patients with procedures performed had an independent positive association with the KPI question response, while length of stay and time to provider had independent negative associations. Table 3. Multivariable Regression Model of Specific Aspects of Clinical Care Predicting KPI Response on PFE Survey. a Clinical aspect Estimate P Sex .6  Male  Female −0.03 Race .02  Asian −0.2 .1  Black −0.04 .6  Middle Eastern 0.3 .4  Multiple 0.05 .7  Other 0.3 .3  Preferred category not available 0.5 .0006  Unknown 0.3 .09  White 0 Ethnicity .0005  Hispanic 0.5 .0002  Refused/Unknown −0.1 .6  Non-Hispanic 0 ESI .4  1 0.5 .6  2 0.07 .6  3 −0.05 .7  4 −0.07 .6  5 0 Prescription written 0.08 .1 Labs ordered −0.1 .1 Imaging ordered 0.1 .06 Procedure performed 0.3 <.0001 Time to provider −0.008 <.0001 Length of stay −0.002 <.0001 Abbreviations: KPI, Key Performance Indicator; PFE, patient family experience; ESI, Emergency Severity Index. a Value assigned at triage to indicate anticipated resource needs as well as illness or injury severity; prescription written, labs ordered, imaging ordered, and procedure performed refer to any of those elements occurring during the patient encounter. Time to provider is the time of arrival to the first provider assessment; Length of stay is the time between patient arrival and discharge. Discussion In this study, we hypothesized that ESI triage levels might serve as an independent predictor for PFE during PED visits. Although there is no independent association between these variables, the results of this study demonstrated several key findings. In our multivariable model, clinical factors, including time to provider, total length of stay, and procedure performance are associated with the KPI score, with abbreviated time to provider, shorter length of stay, and any procedure performance positively impacting experience. This is consistent with other studies, which have demonstrated that increased door-to-room time is associated with lower patient satisfaction, supporting the concept of waiting room interventions to improve overall patient experience.9–13 In the wake of a global pandemic, as institutions continue to endure increased stress on existing healthcare and staffing resources, experience-driven efforts should primarily focus on these metrics.16,17 We did find univariate associations between ESI scores and KPI, an association that was lost in the multivariate model. It is likely that this association is lost because of the inherent interaction between ESI and time to provider and total length of stay. A more ill or injured child may also be more likely to have a procedure performed. However, recognizing these factors, providers in the front of house, such as waiting room or triage, might focus experience-driven interventions on patients with higher ESI triage scores or in whom procedures and interventions are unlikely to be performed to help optimize their experience when times to providers are prolonged. Considerations here could include accurate and visible updates on expected wait times, positive distractions during the waiting room experience, and liberalization of diet in low-risk patients. In one quality improvement study, family experiences in the ED were improved through communication around wait times and waiting room rounds. 15 This study did not address the illness and injury spectrum captured through ESI levels, which might have promoted targeted expectation setting for families of patients with higher ESI levels, where longer waits and times to providers are more likely to be endured. Although prescribing medications or performing labs and imaging would not likely lead to higher KPI scores, procedure performance has an independent association with PFE. More research into the relationship between procedures and PFE is needed as there is a relative lack of literature in this area. Although we did not directly study the interaction between procedure performance and ESI, it can be postulated that more ill or injured children (with resultant lower ESI scores) may be more likely to have a procedure performed. This interaction may have also contributed to the lack of association between ESI and PFE scores in our multivariate model. In this study, we found that patients and families who identified as Hispanic race responded more positively to the KPI question in the PFE survey. This is consistent with a similar study from 2018 that looked at differences in patient experience between Hispanic and non-Hispanic white patients, which found that Hispanics tended to be more satisfied with their care than their non-Hispanic counterparts 17 . Similar results have been noted in other settings.18,19 This interaction deserves further exploration, especially given the frequently reported disparate care for Hispanic patients in the PED.20–23 Limitations As this study is retrospective, it is limited by factors inherent to this study design. Based on its retrospective nature and data collection method, there may be independent predictors of PFE that were not assessed in this study. Also, due to the voluntary nature of the PFE post-visit response survey, our results are based only on families who filled out the survey after their visit. This may bias our results toward those families who had a particularly positive or negative experience. Similarly, our department only receives post-visit surveys from families discharged, and the experience of admitted patients is not considered in this study. Although admission might suggest a higher likelihood of certain PED tasks (imaging, labs, procedures, etc) and lower ESI levels (higher illness or injury severity), the experience of these patients is not included in this study. We believe this is mitigated by the sheer number of patients in this study. This study was performed in a tertiarily care pediatric ED with a high focus on PFE, and its results may not be generalizable to all care centers. Conclusion In the PED, patients and families experiencing shorter wait times, time to providers, and those in whom a procedure was performed are likely to have a more positive experience during their visit. Although ESI triage score is not an independent predictor of experience, it is associated with variation in wait time and can be used as a proxy for these measures to design interventions aimed at improving the experience of patients and their families presenting to the PED. Author Contributions: AAV conceived the study and designed the trial. AAV, CK, and AG performed data collection. NH performed statistical analysis. CK, AG, and AAV drafted the manuscript, while NH and SCP contributed substantially to its revision. AAV takes responsibility for the paper as a whole. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. ORCID iDs: Adam A Vukovic https://orcid.org/0000-0003-3499-1642 Callie Krentz https://orcid.org/0000-0003-3986-8731 ==== Refs References 1 Costet Wong A Claudet I Sorum P Mullet E . Why do parents bring their children to the emergency department? A systematic inventory of motives. Int J Family Med. 2015;2015 :978412. doi:10.1155/2015/978412 26618002 2 Burokienė S Raistenskis J Burokaitė E Čerkauskienė R Usonis V . Factors determining parents’ decisions to bring their children to the pediatric emergency department for a minor illness. Med Sci Monit. 2017;23 :4141-8. doi:10.12659/msm.902639 28845042 3 Haasz M Ostro D Scolnik D . Examining the appropriateness and motivations behind low-acuity pediatric emergency department visits. Pediatr Emerg Care. 2018;34 :647-9. doi:10.1097/pec.0000000000001598 30180100 4 Smith V Mustafa M Grafstein E Doan Q . Factors influencing the decision to attend a pediatric emergency department for nonemergent complaints. Pediatr Emerg Care. 2015;31 :640-4. doi:10.1097/pec.0000000000000392 25822234 5 Nicholson E McDonnell T De Brún A , et al. Factors that influence family and parental preferences and decision making for unscheduled paediatric healthcare - systematic review. BMC Health Serv Res. 2020;20 :663. doi:10.1186/s12913-020-05527-5 32680518 6 Work EPS. Triage scale standardization: joint statement by the American College of Emergency Physicians and the Emergency Nurses Association. Ann Emerg Med. 2010;56 :451.20868927 7 McHugh M Tanabe P McClelland M Khare RK . More patients are triaged using the Emergency Severity Index than any other triage acuity system in the United States. Acad Emerg Med. 2012;19 :106-9. doi:10.1111/j.1553-2712.2011.01240.x 22211429 8 Green NA Durani Y Brecher D DePiero A Loiselle J Attia M . Emergency Severity Index version 4: a valid and reliable tool in pediatric emergency department triage. Pediatr Emerg Care. 2012;28 :753-7. doi:10.1097/PEC.0b013e3182621813 22858740 9 Byczkowski TL Fitzgerald M Kennebeck S , et al. A comprehensive view of parental satisfaction with pediatric emergency department visits. Ann Emerg Med. 2013;62 :340-50. doi:10.1016/j.annemergmed.2013.04.025 23787210 10 Magaret ND Clark TA Warden CR Magnusson AR Hedges JR . Patient satisfaction in the emergency department—a survey of pediatric patients and their parents. Acad Emerg Med. 2002;9 :1379-88. doi:10.1197/aemj.9.12.1379 12460841 11 Nichol JR Fu R French K Momberger J Handel DA . Association between patient and emergency department operational characteristics and patient satisfaction scores in a pediatric population. Pediatr Emerg Care. 2016;32 :139-41. doi:10.1097/pec.0000000000000723 26928092 12 Parra C Vidiella N Marin I Trenchs V Luaces C . Patient experience in the pediatric emergency department: do parents and children feel the same? Eur J Pediatr. 2017;176 :1263-7. doi:10.1007/s00431-017-2954-4 28669089 13 Barbarian M Bishop A Alfaro P , et al. Patient-reported experience in the pediatric emergency department: what matters most? J Patient Saf. 2021;17 :e1166-70. doi:10.1097/pts.0000000000000472 29432338 14 Bal C AlNajjar M Thull-Freedman J Pols E McFetridge A Stang AS . Patient reported experience in a pediatric emergency department. J Patient Exp. 2020;7 :116-23. doi:10.1177/2374373519826560 32128380 15 Emerson BL Setzer E Bechtel K Grossman M . Improving patient experience scores in a pediatric emergency department. Pediatr Qual Saf. 2021;6 :e417. doi:10.1097/pq9.0000000000000417 34235347 16 El Desoky S Mashat S Bana S , et al. Efficiency of using pediatrics emergency services and triage evaluation. Pediatr Emerg Care. 2018;34 :417-21. doi:10.1097/pec.0000000000000754 29851918 17 Deng J Heybati K Garcia C Huang E Zhou F . The COVID-19 endemic: calm before the storm of paediatric viral respiratory illnesses. QJM. 2022;116 (5 ):329–331. 10.1093/qjmed/hcac264 18 Figueroa JF Reimold KE Zheng J Orav EJ . Differences in patient experience between hispanic and non-hispanic white patients across U.S. Hospitals. J Healthc Qual. 2018;40 :292-300. doi:10.1097/jhq.0000000000000113 29252871 19 Wong MS Steers WN Hoggatt KJ Ziaeian B Washington DL . Race differences in patient experience by hispanic ethnicity among veteran health administration users. J Gen Intern Med. 2021;36 :1821-4. doi:10.1007/s11606-020-06023-6 32654088 20 Hartford EA Blume H Barry D Hauser Chatterjee J Law E . Disparities in the emergency department management of pediatric migraine by race, ethnicity, and language preference. Acad Emerg Med. 2022;29 :1057-66. doi:10.1111/acem.14550 35726699 21 Congdon M Schnell SA Londoño Gentile T , et al. Impact of patient race/ethnicity on emergency department management of pediatric gastroenteritis in the setting of a clinical pathway. 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==== Front Digit Health Digit Health DHJ spdhj Digital Health 2055-2076 SAGE Publications Sage UK: London, England 10.1177/20552076231187474 10.1177_20552076231187474 Original Research How did Chinese public health authorities promote COVID-19 vaccination on social media? A content analysis of the vaccination promotion posts Luo Chen 1 https://orcid.org/0000-0003-1842-3717 Dai Runtao 2 https://orcid.org/0000-0002-4080-9125 Deng Yuying 3 Chen Anfan 45* 1 School of Journalism and Communication, 12390 Wuhan University , Wuhan, Hubei, China 2 School of Journalism and Communication, 12442 Tsinghua University , Beijing, China 3 Department of Sociology and Communication, Faculty of Social Sciences, 16779 University of Salamanca , Salamanca, Spain 4 School of Humanities and Social Science, 12652 University of Science and Technology of China , Hefei, Anhui, China 5 School of Journalism and Communication, 26451 The Chinese University of Hong Kong , Shatin, Hong Kong SAR, China * Current address: School of Communication, The Hong Kong Baptist University, Hong Kong, China Yuying Deng, Department of Sociology and Communication, Faculty of Social Sciences, University of Salamanca, Salamanca 37007, Spain. Email: yuyingdeng_irene@usal.es 16 7 2023 Jan-Dec 2023 9 2055207623118747410 2 2023 26 6 2023 © The Author(s) 2023 2023 SAGE Publications Ltd, unless otherwise noted. Manuscript content on this site is licensed under Creative Commons Licenses https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage). Objective Drawing upon the health belief model, this study aims to analyze the message characteristics of coronavirus disease 2019 (COVID-19) vaccination promotion messages posted by influential Chinese public health institutions and how those characteristics affect audiences’ participative engagement on Weibo, which is a popular social media site in China. Methods Two Chinese phrases for the COVID-19 vaccine were adopted as search terms to retrieve qualified posts on Weibo from 1 December 2019 to 18 March 2023. A total of 2546 posts by the top nine most impactful public health institutions were retained for quantitative content analysis. Message characteristics derived from the health belief model and participative engagement indicators were coded by the authors. Results Among health belief model constructs, the collective-oriented constructs (i.e., benefits, cues to action, and susceptibility) appeared in almost half of the posts, while the individual-oriented constructs (i.e., barriers, self-efficacy, and severity) were mentioned less. Moreover, negative binomial regression models revealed that collective-oriented constructs and self-efficacy facilitated engagement, while other constructs played impeding roles. Conclusions Appearances and functions of the health belief model's constructs in the COVID-19 vaccination promotion context are closely associated with China's collectivistic culture. Furthermore, constructs conforming to people's psychological traits are likely to promote public engagement and may facilitate vaccination behavior. COVID-19 vaccination health belief model public health authorities participative engagement content analysis typesetterts19 cover-dateJanuary-December 2023 ==== Body pmcIntroduction Although an increasing number of countries have loosened the regulations (e.g., travel bans, social distancing, and regular nucleic acid tests) regarding the COVID-19 pandemic, 1 promoting COVID-19 vaccination remains indispensable to curb the pandemic's resurgence. 2 As of January 2023, nearly 70% of the world population had received at least one dose of a COVID-19 vaccine. 3 However, vaccine allocation is unbalanced between developed and developing countries, and the coverage rate in specific at-risk groups (e.g., people aged over 60 and children) is suboptimal.2,4 China, as one of the most populous countries in the world, achieves the large-scale vaccination objective but also experiences slow vaccination promotion among certain groups. 5 For instance, the COVID-19 vaccine coverage in China before October 2022 has already exceeded 90%, but promoting vaccination among the elderly is still on the way. 5 Yet as the Chinese government is branded with its collectivistic culture and embracement of collectivization in political and economic policies, getting all people vaccinated is the most emphasized health-promoting goal during the pandemic. 6 The burgeoning social media propels public health institutions and professionals to leverage social media platforms to conduct health promotion and education.7,8 During the pandemic, the Chinese government strived to embrace social media as a cost-effective channel to promote COVID-19 vaccination. 9 Also, it is prevalent for the public to seek health information online. 10 Thus, understanding the content characteristics of COVID-19 vaccination promotion messages released by public health authorities is crucial for evaluating past efforts and formulating proper strategies for future public health emergencies. Furthermore, since social media enables health information generation, acquisition, sharing, and endorsing, mirroring audience engagement and response to persuasive information,10,11 it is also imperative to learn how health content characteristics affect participative engagement to inform social media-based health promotion campaigns. 12 To fulfill the preceding objectives, the health belief model (HBM) was adopted as the overarching framework. HBM comprises six components—perceived susceptibility, perceived severity, perceived benefits, perceived barriers, cues to action, and self-efficacy. 13 Perceived susceptibility refers to a subjective evaluation of the likelihood of contracting a disease. 14 Perceived severity refers to an assessment of the disease's seriousness and the magnitude of its ramifications. 14 Perceived benefits denote an individual's beliefs about the effectiveness of an action to alleviate the disease threat.13,14 Contrary to perceived benefits, perceived barriers represent an evaluation of factors that discourage one from practicing preventive behavior (e.g., fear of side effects). 13 Regarding the last two components, cues to action stand for stimuli that trigger the desire to adopt the preventive behavior, and self-efficacy is an individual's self-confidence in their ability to execute the behavior. 13 Since its inception, HBM has been widely adopted to understand why people succeed or fail to take preventive health measures. 15 Noticeable efforts also confirmed HBM's explanatory power in analyzing, designing, and delivering health campaign messages. For instance, Madden and associates’ content analysis of websites related to the HPV vaccine based on HBM found that more than half of the websites reported benefits, severity, and susceptibility. 16 Another content analysis of HPV vaccine coverage on YouTube by Briones and associates found that fewer than half of the videos indicated the high benefits of the vaccine and susceptibility to HPV, while more than half of them reported barriers and severity. 17 Wang and Lu's 18 analysis of COVID-19 vaccine-related tweets from three international news agencies disclosed that the most mentioned HBM construct was barriers, followed by benefits, susceptibility, cues to action, severity, and self-efficacy. Although studies have shown how HBM enlightens health-related content analysis and design,19,20 scant efforts have been paid to bridging HBM constructs and participative engagement. However, participative engagement is critical in the social media context because social media affords more flexible content-based interaction than traditional communication patterns. 21 Some scholars also interpret the intensity of participative engagement as the effect of online health promotion. 22 Previous studies commonly took three social media metrics (the number of retweets, likes, and comments) to capture three conceptual dimensions (viral reach, affective evaluation, and message deliberation) of participative engagement.12,21,22 Taking the above rationales into account, this study aims to investigate the message characteristics of COVID-19 vaccination promotion messages posted by representative social media accounts of Chinese public health institutions under the guidance of HBM. Furthermore, we intend to examine how the message attributes affected participative engagement on Chinese social media. Besides the primary constructs of HBM, mentioning target groups (e.g. children and the elderly) or not is incorporated because boosting vaccination coverage in specific groups has been repeatedly underscored in China's vaccination promotion strategies. 23 Therefore, the two research questions of this study are as follows. RQ1: To what extent did COVID-19 vaccination promotion posts posted by Chinese public health institutions convey information related to susceptibility to COVID-19, the severity of COVID-19, benefits of vaccination, barriers of vaccination, cues to vaccination, self-efficacy, and target groups? RQ2: How did the seven message characteristics (as enumerated in RQ1) influence social media users’ participative engagement, including sharing, commenting, and liking? Method This is an observational study based on textual data (i.e. posts) collected from one of the most prevalent Chinese social media platforms, Sina Weibo (https://weibo.com/, hereafter Weibo). To answer our research questions, quantitative content analysis was adopted. Manual coding and formal analysis were performed by two native Chinese speaker researchers through online collaboration in March 2023. Methodological details are elucidated in the following sections. Informed consent is not necessary for this study because it does not involve animal or human subjects. All data were collected from open-accessed posts released by public health institutions intended for public health education and promotion. Data We retrieved posts from Chinese public health institutions’ official accounts on Weibo, the biggest microblogging site in China, 24 with 252 million daily active users by the first quarter of 2022. 25 The central government of China required affiliated public institutions to create and maintain Weibo accounts to enhance government responsiveness. 26 As a result, numerous government agencies in China communicate with the public on Weibo, including agencies in the public health sector. 27 To ensure representativeness, the ten most influential public health authorities’ Weibo accounts were included based on the latest report released in January 2022 by the Weibo Data Center and the Public Opinion Research Center of the People's Net. 28 It should be noted that the account of the Health Commission of Guangxi Province closed public access to its historical posts, leaving nine accounts in the following analyses, including China's national health commission and eight provincial health commissions (accounts’ detailed information is listed in Table 1). According to the report, an account's impact is a composite index comprising dissemination scope, online service quality, interaction degree, and identity level. 28 The higher the score, the higher the impact. Table 1. Accounts’ detailed information. ID Account Account's affiliation Impact factor 1. @Jiankang Zhongguo The National Health Commission 74.88 2. @Jiankang Chengdu Guanwei The Health Commission of Chengdu 74.61 3. @Shenzhen Weijianwei The Health Commission of Shenzhen 68.04 4. @Jiankang Jiangsu Weibo The Health Commission of Jiangsu 67.78 5. @Shoudu Jiankang The Health Commission of Beijing 67.27 6. @Jiankang Guangzhou The Health Commission of Guangzhou 65.82 7. @Jiankang Shandong The Health Commission of Shandong 61.54 8. @Jilinsheng Weisheng Jiankangwei The Health Commission of Jilin 58.06 9. @Jiankang Hebei Guanwei The Health Commission of Hebei 57.27 We set the data collection period from 1 December 2019 to 18 March 2023. Huang and associates indicated that 1 December 2019 was the symptom onset date of the first identified COVID-19 patient in China. The ending date, 18 March 2023, is the day before our formal analysis. 29 The combinations of the Chinese words for “COVID” and “vaccine” were adopted as the search terms. Through the built-in search function of Weibo, we were able to retrieve relevant posts containing any of the search terms under each account in the specified time range. Since our study focuses on how Chinese public health authorities promoted COVID-19 vaccination on social media, posts without an explicit purpose of promoting COVID-19 vaccination were eliminated, resulting in a final sample consisting of 2546 posts. Here, we provide a sample post not explicitly promoting COVID-19 vaccination to better illustrate our selection criteria: “Recently, the Beijing Municipal Center for Disease Control and Prevention issued guidelines for preventing environmental pollution in COVID-19 vaccination sites. The guidelines indicate that after the injection, the cotton swab pressed on the inoculation site should be uniformly discarded in a designated recycling container and should not be taken away from the COVID-19 vaccination site.” Analysis Content analysis, “a research technique for making replicable and valid inferences from texts to the contexts of their use,” 30 was selected as the primary method. The unit of analysis in this study is every single post. We constructed a codebook (as shown in Table 2) according to the HBM framework to better quantify the posts of interest. For HBM's core constructs, if one post met the definition of a construct in the coding book, then the corresponding value was coded as one; otherwise, zero. For other constructs, such as the three indicators of participative engagement, we coded the numbers as displayed on Weibo. We also considered other constructs that may affect participative engagement, including content (e.g. availability of supplementary information) 31 and account (e.g. number of followers) 32 characteristics. Table 2. The codebook of this study. Construct Definition Example a Attribute(s) HBM constructs Susceptibility Whether the post contains the likelihood of contracting COVID-19 (16, 18) “The Omicron variant is highly infectious and everyone is susceptible to it!” 0 = Absent, 1 = Present Severity Whether the post contains the seriousness of contracting COVID-19 (18) “The elderly are more likely to develop severe and critical illnesses after contracting COVID-19.” 0 = Absent, 1 = Present Benefits Whether the post contains the effectiveness of COVID-19 vaccination in deterring the COVID-19 threat (16,19) “Receiving the COVID-19 vaccine is the most economical and effective approach to contain COVID-19.” 0 = Absent, 1 = Present Barriers Whether the post contains obstacles to taking the COVID-19 vaccine, including physical health risks and psychological risks (15,16) “Some adverse reactions after taking the COVID-19 vaccine include headache, fever, feeling feeble, and swelling at the injection site.” 0 = Absent, 1 = Present Cues to action Whether the post contains elements to activate COVID-19 vaccine uptake (e.g., expert testimony, role models) (19) “An Olympic athlete invites you to get the COVID-19 vaccine.” 0 = Absent, 1 = Present Self-efficacy Whether the post contains guidance on getting the COVID-19 vaccine (15, 18) “If you want to make a vaccination appointment, you just need to visit the nearest community health service center and register.” 0 = Absent, 1 = Present Target groups Whether the post contains specific groups that are at risk of COVID-19 or taking the COVID-19 vaccine “Pregnant women should defer the COVID-19 vaccination plan.” 0 = Absent, 1 = Present Participative engagement constructs Viral reach The number of retweets the post received As displayed Continuous variable Message deliberation The number of comments the post received As displayed Continuous variable Affective evaluation The number of likes the post received As displayed Continuous variable Controls Supplementary information Whether the post includes an URL As displayed 0 = Absent, 1 = Present Original post or not Whether the post is originally created As displayed 0 = Not original, 1 = Original Multimodality Whether the post contains other media formats than text The post contains a video or an image 0 = Absent, 1 = Present Posting date The posting date of the post As displayed Date format Followers The number of fans the account has As displayed Continuous variable Number of posts The total number of posts the account has sent As displayed Continuous variable Note. HBM: health belief model; COVID-19: coronavirus disease 2019. a Examples here are sentences selected from qualified posts. We conducted pilot coding to examine the agreement between the coders before formal analysis. Two authors first coded 280 (11.00%) randomly selected posts to calculate the intercoder reliability after training. Cohen's Kappa coefficient was adopted, 22 and the average value was 0.60 (ranging from 0.40 to 0.90) in the first round, which denotes moderate agreement. 33 In the second round, we discussed the incongruencies and settled comprehension divergence. Another 100 posts were randomly sampled to retest the intercoder reliability. The average value of Cohen's Kappa coefficient reached 0.82 (ranging from 0.66 to 0.95), indicating a satisfactory agreement between the coders. 33 The remaining posts (N = 2166) were split into two halves for independent coding. Following the previous experience of predicting social media participative engagement indicators,32,34 two competing count models (i.e. the Poisson model and the negative binomial model) were compared to answer RQ2 due to the count variable nature of the outcomes. We also follow previous practices to adopt the multivariate analysis of variance (MANOVA) for robustness check,11,32 which helps cross-validate the count model's findings. Results Descriptive statistics of core constructs are listed in Table 3. Regarding RQ1, Table 3 shows that among HBM constructs, benefits were most frequently mentioned (68.54%), followed by cues to action (63.83%), susceptibility (43.17%), barriers (25.77%), self-efficacy (21.96%), and severity (13.16%). It can be inferred that the primary vaccination promotion strategy adopted by Chinese public health authorities was to accentuate the benefits and provide action stimuli. An additional analysis was performed to examine the internal relations within the HBM constructs (see Appendix A). Pearson chi-square tests ( Appendix Table A2) show that benefits, cues to action, and susceptibility were likely to co-occur in a post, implying that the public health authorities mainly advocated vaccination by demonstrating the COVID-19 vaccine's power and encouraging the public to get vaccinated to mitigate the spread of the virus. More than one-third of the posts (36.84%) had clear target groups, such as people over 60, teenagers, and pregnant women. Table 3. Descriptive statistics of categorical variables (N = 2546). Variable Frequency of occurrence (%) Susceptibility 1099 (43.17%) Severity 335 (13.16%) Benefits 1745 (68.54%) Barriers 656 (25.77%) Cues to action 1625 (63.83%) Self-efficacy 559 (21.96%) Target groups 938 (36.84%) Regarding RQ2, we first compared the two competing models with the three participative engagement metrics as outcome variables and other variables as predictors. Besides, the posting date was controlled on a monthly basis (i.e. 39 month-based dummy variables were put into the model) to parse out potential confounding effects. For example, the number of infected cases is constantly changing, and controlling the time factor partly contributes to ruling out the influences from such time-varying variables. Additionally, the “abrupt policy shift” timepoint (i.e. 7 December 2022) in China's domestic pandemic control was considered and controlled to minimize the confounding effect brought by the policy change. 35 The “New 10-point Plan” was issued on 7 December 2022 to ease the impact of longstanding control measures. 35 With the initiation of the “New 10-point Plan” large-scale nucleic acid testing, health code checking, and centralized quarantine were no longer required for most cases. 36 Hence, the rolling out of the new plan denotes a relaxation of China's COVID-19 control. The comparison results (as shown in Appendix B) indicate that the negative binomial model can always produce smaller residuals than the Poisson model in predicting the three metrics. Therefore, we chose the negative binomial model to predict the three participative engagement indicators. The multicollinearity test revealed that there were no multicollinearity problems (the maximum VIF value was 1.64). As displayed in Table 4, the three negative binomial regression models demonstrate that mentioning susceptibility (for retweets: B = 0.74, p < .001; for comments: B = 0.36, p < .001; for likes: B = 0.51, p < .001) and benefits (for retweets: B = 0.63, p < .001; for comments: B = 0.27, p < .01; for likes: B = 0.24, p < .05) was positively associated with three kinds of participative engagement while mentioning severity (for retweets: B = −0.56, p < .001; for comments: B = −0.93, p < .001; for likes: B = −1.12, p < .001), barriers (for retweets: B = −0.51, p < .001; for comments: B = −0.57, p < .001; for likes: B = −0.89, p < .001), and target groups (for retweets: B = −0.59, p < .001; for comments: B = −0.75, p < .001; for likes: B = −0.80, p < .001) was negatively associated with them. Cues to action (B = 0.24, p < .05) and self-efficacy (B = 0.42, p < .001) were only positively associated with commenting. Among the remaining variables, it can be inferred that an original post featuring media richness (i.e. multimodality) and supplementary information free and sent by an account with more fans is more likely to trigger participative engagement. The robustness check results are summarized in Appendix C, which were highly similar to the findings of negative binomial regressions, confirming the robustness of our results. Table 4. Results of negative binomial regression models on participative engagement (N = 2546). Number of retweets Number of comments Number of likes Variable B (SE) Exp(B) B (SE) Exp(B) B (SE) Exp(B) Constant −12.76*** (1.32) 0.00 −6.43*** (1.63) 0.00 −3.37* (1.60) 0.03 Susceptibility 0.74*** (0.09) 2.09 0.36*** (0.10) 1.43 0.51*** (0.11) 1.67 Severity −0.56*** (0.12) 0.57 −0.93*** (0.14) 0.40 −1.12*** (0.15) 0.33 Benefits 0.63*** (0.09) 1.88 0.27** (0.10) 1.31 0.24* (0.11) 1.27 Barriers −0.51*** (0.10) 0.60 −0.57*** (0.11) 0.57 −0.89*** (0.13) 0.41 Cues to action 0.15 (0.10) 1.16 0.24* (0.11) 1.27 −0.01 (0.12) 0.99 Self-efficacy 0.03 (0.10) 1.03 0.42*** (0.11) 1.51 0.13 (0.12) 1.13 Target groups −0.59*** (0.09) 0.55 −0.75*** (0.11) 0.47 −0.80*** (0.13) 0.45 Control variables Followers (log-transformed) 0.52*** (0.03) 1.69 0.76*** (0.05) 2.14 0.71*** (0.05) 2.03 Number of posts (log-transformed) 0.30* (0.12) 1.36 −0.52** (0.15) 0.60 −0.52** (0.16) 0.60 Original post 2.19*** (0.15) 8.95 2.00*** (0.17) 7.41 2.42*** (0.17) 11.26 Multimodality 1.22*** (0.09) 3.40 0.26* (0.10) 1.29 0.29** (0.11) 1.33 Supplementary information −0.78*** (0.11) 0.46 −1.49*** (0.12) 0.22 −1.74*** (0.13) 0.18 Control policy change 0.48 (0.27) 1.62 −1.71*** (0.31) 0.18 −1.50*** (0.33) 0.22 Posting date Controlled Controlled Controlled Log-likelihood −8664.11 −8269.96 −10712.82 Pseudo R2 0.06 0.07 0.08 AIC 17422.23 16633.92 21519.64 BIC 17696.82 16908.51 21794.23 Note. AIC: Akaike information criterion; BIC: Bayesian information criterion. The posting date was controlled on a monthly basis, we omitted the exact coefficients for brevity. *p < .05; **p < .01; ***p < .001. Discussion This study analyzes the presence of HBM constructs in Chinese health authorities’ vaccination promotion posts and examines how those constructs affect engagement indicators. Some intriguing findings merit further discussion. Similar to a previous study by Li et al., 20 cues to action and benefits were mentioned in most posts. Besides, as a concept referring to the chance of being infected, susceptibility is widely adopted and appeared in almost half of the posts. 14 Other constructs were mentioned less, including barriers, self-efficacy, and severity. The “fragmented” presentation of HBM constructs echoes previous findings, which may be partly due to the long-established collectivistic culture in China. 19 The collectivistic culture sets group aims as priorities and emphasizes the roles of interdependence and external social norms in combating the COVID-19 risk. 37 Thus, highlighting group safety and mass mobilization are frequently employed strategies in promoting COVID-19 vaccination in collectivistic cultures-oriented societies. 37 Accordingly, benefits and cues to action are mainly about the vaccine's effectiveness in building the colony immune defense, containing the virus contagion, and protecting the whole society. On the other hand, susceptibility also reminds audiences that COVID-19 is highly contagious and endangers public health. Contrarily, barriers, self-efficacy, and severity are mainly individual-oriented aspects, such as whether the vaccination causes adverse reactions (i.e. barriers) or whether one can get the vaccine conveniently (i.e. self-efficacy). The negative binomial regressions show that frequently appeared constructs in posts (i.e. benefits, cues to action, and susceptibility) held positive associations with at least one of the participative engagement indicators. These HBM components are rooted in collectivism, which may arouse a high degree of collective identity and encourage Weibo users’ active engagement. For instance, to protect the safety of the neighborhood or the broader community, users may be alert to the highly infectious virus and retweet the message to exhibit the function of countermeasures and encourage others to practice the recommended behaviors. In addition, self-efficacy, which indicates one has the ability to perform the recommended response, helps strengthen people's confidence in COVID-19 vaccination. 38 The negative effects of severity, barriers, and target groups on participative engagement may be ascribed to the impact of loss frames. Compared with benefits and self-efficacy, which underscore the COVID-19 vaccine's strengths and individual competence in performing the behavior, severity, barriers, and target groups are more relevant to personal losses. The prospect theory suggests that people are risk-seeking when precautions are framed in losses but risk-averse when precautions are framed in gains. 39 Consequently, loss frames are deemed more effective for encouraging detection behaviors, while gain frames are more effective for facilitating prevention behaviors. 40 As a typical preventive behavior, vaccination framed in losses would be less persuasive and more likely to inhibit engagement than those framed in gains. However, it should be noted that we merely provide a tenable explanation (i.e. collective-oriented constructs vs. individual-oriented constructs) for the differential effects of message characteristics but do not rule out other possible explanations. For instance, some fear appeal theories (e.g. the extended parallel process model) suggest that perceived threat is composed of perceived severity and perceived susceptibility, in which perceived susceptibility represents an individual's beliefs about his or her likelihood of experiencing the health threat.38,41,42 Thus, susceptibility can also be treated as an individual-level factor derived from the personal appraisal of external threats. According to Tang et al., 8 posts containing susceptibility help the audiences understand the risk of COVID-19, contributing to participative engagement amidst uncertainty. In summary, susceptibility as a content characteristic focused more on COVID-19's impacts on public health, while susceptibility as a psychological appraisal leans toward individual perception. The individual-level perception of susceptibility may also arouse fear and stimulate engagement. These complementary explanations require fine-grained evidence from surveys or experiments to examine. Similarly, regarding the promoting roles of benefits, cues to action, and self-efficacy in participative engagement, an alternative explanation could be that the positively valenced constructs conform to the Chinese government's commitment to building a harmonious society and advocating positive energy in the whole society. According to Chen and Wang, 43 the positive energy discourse underscores maintaining a positive attitude in daily life, even in the face of public crises. Therefore, adopting benefits, cues to action, and self-efficacy in vaccination promotion posts strengthens hope and one's determination to overcome difficulties, which complies with the tenets of positive energy and boosts morale in combating COVID-19. Those constructs may further stimulate social media users’ engagement. It is worth noting that some inconsistent findings exist in other studies. For example, a computer-assisted content analysis study by Tang et al. based on HBM found that severity significantly promoted public engagement on Twitter while benefits’ predicting effect on public engagement was not significant. 8 Two reasons may account for this. For one thing, the Western context highlights independence and self-determination when facing health risks.37,44 Emphasizing severity might intensify their COVID-19 risk perception, which in turn motivates online engagement, such as retweeting or endorsing the severity-laden tweets. Contrarily, interdependence is highly valued in a collectivist society such as China, leading to reduced individual capacity to take preventive measures and nurturing a risk-averse mentality. 44 Hence, accentuating severity may trigger a boomerang effect among Chinese netizens and induce information avoidance since it largely heightens the concern of personal loss. These cultural differences could partly explain the opposite effects of severity on participative engagement in the two studies or two divergent cultural settings. For another, the study by Tang et al. 8 was performed in the first six months of 2020, when COVID-19 vaccines were still under development. Against that background, the benefits of the COVID-19 vaccine as an effective defense for preventing COVID-19 were not fully recognized then. However, our study wraps an extended period, enabling a sufficient demonstration of the COVID-19 vaccine's effectiveness. All the disparate findings underscore the significance of cross-context comparisons, especially utilizing longitudinal and comparative evidence to warrant a more nuanced understanding. Practical implications This study bears some practical implications for vaccination campaigns on social media. First of all, as an integral part of the public health campaign, vaccination promotion should adhere to the context-sensitive or culture-sensitive principle by designing tailored persuasive messages conforming to the cultural characteristics. In China, emphasizing vaccination's benefits in lowering group contagion's possibility and presenting explicit cues about vaccination motivate people to follow social norms and vaccinate for the public good. Second, potential losses at the individual level should not be overemphasized. On social media, people may already encounter numerous posts (especially personal narratives and help-seeking messages) elucidating COVID-19's severity. 32 Of great importance is to strengthen self-efficacy by providing detailed instructions on getting the vaccine, which would entice deeper levels of information diffusion, deliberation, and nurturing positive attitudes toward the national vaccination program. Third, although enhancing vaccination coverage among specific groups aligns with the policies, public health practitioners must balance the threat and efficacy. Since an overdose of threats on certain groups compromises the messages’ persuasive power, a better strategy may be providing direct guidance or encouragement to the whole population and decreasing the occurrence frequency of specific groups. Fourth, the roles of control variables also inform Chinese public health authorities on maximizing the influence of vaccination promotion posts. It is advisable to create original posts using multiple media types (e.g. infographics and videos) to present detailed persuasive messages to facilitate online public participation, which may translate into vaccination behavior in the offline setting. Limitations The findings of this study should be interpreted with caution. Only the top nine public health authorities’ posts were analyzed, which overlooked other less influential accounts. Future attempts covering more public health authorities could disclose a comprehensive landscape of China's COVID-19 vaccination promotion. Besides, critical time points can be further explored, such as whether the promotion strategies differed before and after milestones (e.g. government approval of certain vaccines) or how content characteristics were associated with participative engagement at different phases of COVID-19 infection (e.g. waxing or waning). 20 Moreover, participative engagement differs from actual vaccination behavior. Whether posts containing HBM constructs activate actual vaccination necessitates further testimony. Lastly, the retrospective nature of this study restrains our ability to check data integrity before the formal analysis. Future research may pursue a real-time data collection approach to better curate the social media corpus and retrieve a complete dataset. Conclusions Drawing on the HBM framework, this theory-driven content analysis scrutinizes how prominent Chinese health authorities promoted COVID-19 vaccination on social media. We found that collective-oriented constructs were more employed than individual-oriented ones, which is consistent with the features of collectivistic culture. The relationships between HBM constructs and participative engagement were discussed, demonstrating the differential effects of health concepts. The function of a newly added context-specific variable—target groups, was also disentangled. Our findings not only summarize past efforts in social media-based public health campaigns during the pandemic but also provide reference to coping with future public health emergencies. Acknowledgements The authors would like to thank the anonymous reviewers who provided valuable feedback which helped improve the quality of the paper. Appendix A Pearson chi-square tests of internal relations among health belief model (HBM) constructs (N = 2546). Table A1. The results of Pearson chi-square values. HBM constructs 1 2 3 4 5 1. Susceptibility – 2. Severity 67.47*** – 3. Benefits 250.71*** 15.71*** – 4. Barriers 75.81*** 182.18*** 23.44*** – 5. Cues to action 176.53*** 0.15 234.06*** 212.84*** – 6. Self-efficacy 99.69*** 0.40 100.29*** 41.67*** 164.66*** Note. HBM: health belief model. Asymptotic significance level (two-sided) at *p < .05; **p < .01; ***p < .001. Table A2. The results of phi values. HBM constructs 1 2 3 4 5 1. Susceptibility – 2. Severity 0.16*** – 3. Benefits 0.31*** 0.08*** – 4. Barriers −0.17*** 0.27*** −0.10*** – 5. Cues to action 0.26*** 0.01 0.30*** −0.29*** – 6. Self-efficacy −0.20*** 0.01 −0.20*** 0.13*** −0.25*** Note. HBM: health belief model. *p < .05; **p < .01; ***p < .001. Appendix B The comparison between the Poisson model and the negative binomial model. Figure B1. Model comparison of predicting the number of retweets. Figure B2. Model comparison of predicting the number of comments. Figure B3. Model comparison of predicting the number of likes. Table B1. Statistical indicators of model comparison on predicting the number of retweets. The Poisson model The negative binomial model The sum of the absolute residual 0.40 0.19 The sum of the Pearson value 2298.85 231.52 AIC 150110.49 17422.23 BIC 150379.24 17696.82 AIC: Akaike information criterion; BIC: Bayesian information criterion. Table B2. Statistical indicators of model comparison on predicting the number of comments. The Poisson model The negative binomial model The sum of the absolute residual 0.38 0.20 The sum of the Pearson value 2104.73 198.15 AIC 278678.13 16633.92 BIC 278946.87 16908.51 AIC: Akaike information criterion; BIC: Bayesian information criterion. Table B3. Statistical indicators of model comparison on predicting the number of likes. The Poisson model The negative binomial model The sum of the absolute residual 0.39 0.35 The sum of the Pearson value 2040.60 851.63 AIC 354038.50 22313.38 BIC 354549.06 22401.01 AIC: Akaike information criterion; BIC: Bayesian information criterion. Appendix C Results of the robustness check (multivariate analysis of variance [MANOVA]). Table C1. The results of MANOVA (N = 2546). Variable Number of retweets B(SE) Number of comments B(SE) Number of likes B(SE) Constant −5.31*** (0.89) −5.85*** (0.95) −5.16*** (1.09) Susceptibility 0.70*** (0.06) 0.07 (0.07) 0.12 (0.07) Severity −0.52*** (0.09) −0.44*** (0.09) −0.56*** (0.11) Benefits 0.52*** (0.06) 0.27*** (0.07) 0.32*** (0.08) Barriers −0.32*** (0.07) −0.14* (0.07) −0.31*** (0.08) Cues to action 0.22*** (0.06) 0.25*** (0.07) 0.33*** (0.08) Self-efficacy 0.18** (0.07) 0.36*** (0.07) 0.28** (0.08) Target groups −0.18** (0.07) −0.36*** (0.07) −0.46*** (0.08) Control variables Followers (log-transformed) 0.28*** (0.02) 0.32*** (0.03) 0.43*** (0.03) Number of posts (log-transformed) 0.05 (0.08) 0.19* (0.08) 0.05 (0.10) Original post 1.12*** (0.10) 0.90*** (0.11) 1.21*** (0.12) Multimodality 0.88*** (0.06) −0.09 (0.06) −0.16* (0.07) Supplementary information −0.45*** (0.08) −0.80*** (0.08) −0.95*** (0.10) Control policy change 0.24 (0.21) −0.78*** (0.22) −0.93*** (0.26) Posting date Controlled Controlled Controlled F-value 30.76*** 27.29*** 31.61*** R 2 0.36 0.33 0.36 Note. MANOVA: multivariate analysis of variance. Outcome variables were log-transformed before entering into the model because of the non-normal distributions. The posting date was controlled on a monthly basis, we omitted the exact coefficients for brevity. *p < .05; **p < .01; ***p < .001. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Ethical approval: This study did not require approval by the institutional review board because it involved the use of public access data only. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. Informed consent: Informed consent is not necessary for this study because it does not involve animal or human subjects. All data were collected from open-accessed posts released by public health institutions intended for public health education and promotion. 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==== Front Digit Health Digit Health DHJ spdhj Digital Health 2055-2076 SAGE Publications Sage UK: London, England 10.1177/20552076231187608 10.1177_20552076231187608 Original Research Enhanced electrocardiogram machine learning-based classification with emphasis on fusion and unknown heartbeat classes https://orcid.org/0000-0002-6427-1008 Al-mousa Amjed Baniissa Joud Hashem Tala Ibraheem Tala Computer Engineering Department, 113338 Princess Sumaya University for Technology , Amman, Jordan Amjed Al-mousa, Computer Engineering Department, Princess Sumaya University for Technology (PSUT), Amman 11941, Jordan. Email: a.almousa@psut.edu.jo 16 7 2023 Jan-Dec 2023 9 2055207623118760813 3 2023 23 6 2023 © The Author(s) 2023 2023 SAGE Publications Ltd, unless otherwise noted. Manuscript content on this site is licensed under Creative Commons Licenses https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage). Building an electrocardiogram (ECG) heartbeat classification model is essential for early arrhythmia detection. This research aims to build a reliable model that can classify heartbeats into five heartbeat types: normal beat (N), supraventricular ectopic beat (SVEB), ventricular ectopic beat (VEB), fusion beat (F), and unknown beat (Q), with a focus on enhancing the predictions of the uncommon Q and F heartbeats. The base dataset used is the MIT-BIH SupraVentricular Database, which was used to train and compare the performance of five machine learning models: logistic regression, Random Forest (RF), K-nearest neighbor, linear support vector machine, and linear discriminant analysis. In addition to using the synthetic minority oversampling technique, data extracted from multiple databases for the F and Q classes were combined with the original base dataset. These methods resulted in significant improvement in the recall for the rare F and Q classes when compared to the literature. The RF algorithm produced the best performance with an accuracy of 97% and recall values equal to 97%, 93%, 95%, 95%, and 30% for N, SVEB, VEB, F, and Q, respectively. ECG classification machine learning fusion beat unknown beat random forest typesetterts19 cover-dateJanuary-December 2023 ==== Body pmcIntroduction According to data from the World Health Organization (WHO), the leading causes of death globally in the past few years have been cardiovascular diseases, including coronary artery disease, stroke, and heart failure. 1 Arrhythmia, or abnormal heart rhythms, can be a symptom of cardiovascular disease or a risk factor for developing one. For example, arrhythmia can result from damage to the heart muscle due to a heart attack or other forms of heart damage. According to Mayo Clinic, arrhythmia can be defined as irregular heartbeats when the electrical signals that coordinate the heart's beats do not function properly.2,3 Early arrhythmia detection is essential because it can help to identify and treat abnormal heart rhythms at an early stage, which can help to prevent or manage serious complications. When arrhythmia is not detected and treated early, it can cause various symptoms, including palpitations, dizziness, shortness of breath, and fainting. It can also increase the risk of stroke and other serious complications. For example, myocardial infarction (MI), also known as a heart attack, is a silent and fatal cardiovascular disease that can cause sudden death without warning. Thus an accurate and automated ECG classification is critical in the diagnosis and treatment of heart diseases, as reported by Rai and Chatterjee 4 in addition to being effective in the detection of cardiac arrhythmias, as they also reported in Rai and Chatterjee.5,6 Several methods can be used to classify heartbeats, including manual analysis of electrocardiogram (ECG) tracings by trained healthcare providers or automated analysis employing machine learning algorithms. Both methods identify heartbeat features, such as the shape and duration of the various waves and complexes present in ECG tracing. Then, use these features to classify heartbeats into different categories as defined by the Association for the Advancement of Medical Instrumentation EC57 standard, 7 shown in Table 1. Classifiers aim to build reliable models that can classify heartbeats into the five heartbeat super-classes: normal beat (N), supraventricular ectopic beat (SVEB), ventricular ectopic beat (VEB), fusion beat (F), and unknown beat (Q). Table 1. Heartbeat categories according to AAMI. Heartbeat superclass Heartbeat annotation N (Normal) N (Normal) L (Left bundle branch block beat) R (Right bundle branch block beat) e (Atrial escape beat) j (Nodal (junctional) escape beat) SVEB (Supraventricular ectopic beat) A (Atrial premature beat) a (Aberrated atrial premature beat) J (Nodal (junctional) premature beat) S (Supraventricular premature beat) VEB (Ventricular ectopic beat) V (Premature ventricular contraction) E (Ventricular escape beat) F (Fusion beat) F (Fusion of ventricular and normal beats) Q (Unknown beat) Q (Unclassifiable beat)/(Paced beat) f (Fusion of paced and normal beat) Figure 1 shows a diagram of a typical ECG signal. The signal consists of three major parts: The P wave, the QRS complex, and the T wave. The diagram shows critical parameters like the PQ, ST, QRS, and QT intervals. These parameters constitute a key part of the features that will be used later in constructing the ECG classification model. Figure 1. ECG diagram. ECG: electrocardiogram. While numerous studies in ECG classification have used machine learning techniques, most of them targeted achieving higher overall accuracy. Something that can be easily biased if you have an unbalanced distribution of classes. Overall accuracy can be easily manipulated by increasing the number of samples of an easy-to-detect class. This is the case for ECG datasets, as most contain a large percentage of the normal (N) class, which has a very high recall rate. In such cases, considering the per-class recall numbers would be a more accurate measure of the classifier's performance than the overall accuracy. The fact that the F and Q classes had the fewest instances in these datasets contributed to the poor performance of classification models in correctly identifying these classes. When a class has very few instances, it is more difficult for the model to learn its distinctive features and patterns. As a result, the model may not be able to generalize well and may struggle to classify new instances of that class correctly. This work adds value to the existing literature on ECG classification using machine learning by explicitly focusing on improving the recall of the Q and F classes, which are often associated with severe cardiac conditions and often have the fewest number of instances in the datasets. This is a significant contribution, as many previous studies did not address the issue of the performance of these specific classes. Tackling the problem of uncommon heartbeats is done using two approaches. The first is deploying the synthetic minority oversampling technique (SMOTE), which oversamples the minority classes to create additional synthetic data. While the second relies on combining data for the minority classes from multiple datasets. Using these approaches together has shown tremendous improvement in the recall of the Q and F heartbeats. It is worth noting that the base dataset used in this work is the MIT-BIH Supraventricular Arrhythmia Database created by Greenwald. 8 The remaining parts of the paper are organized as follows: the Related Work section presents the related literature regarding ECG heartbeat classification. The Dataset and Experimental Setup section will detail the primary datasets used and the experimental setup. The Machine Learning Algorithms section describes the machine algorithms used in this article. Meanwhile, the Results and Analysis section presents the results of the three cases: the original dataset, after using SMOTE, and when combining multiple datasets. Finally, the conclusion presents a summary of findings and future research direction. Related work Machine learning has been proven to be helpful in the medicinal field. For example, in the prediction of multiple diseases like diabetes, 9 heart disease,10,11 and strokes. 12 As for ECG heartbeat classification, several approaches have been taken regarding signal preprocessing and various models and implementations. Wang et al. 13 have used the Easy Ensemble technique and global heartbeat information for an imbalanced heartbeat classification on the MIT-BIH arrhythmia dataset. 14 The MIT-BIH arrhythmia dataset is similar to the MIT-BIH supraventricular arrhythmia dataset used in this research as it comes from the same holster records and thus has the same features but fewer instances. Wang et al. tested their database using the interpatient scheme. Their experimental results showed that the global heartbeat information is helpful for heartbeat classification and achieved an average accuracy of 95.6% and a recall of 91.7%, 89.9%, 87.8%, and 55.4% for types N, SVEB, VEB, and F, respectively. Zhang et al. 15 proposed a method that selects effective feature subsets for distinguishing a class from classes by performing a One-versus-One comparison on a support vector machine (SVM) binary classifier on the MIT-BIH Arrhythmia Database. 14 Zhang et al. achieved recall values of 88.94%, 79.06%, 85.48%, and 93.81% for classes N, SVEB, VEB, and F. Meanwhile, Diker used a different dataset and compared the results of three different models that used artificial neural networks, SVM, and k-nearest neighbor (KNN) and used a 10-fold crossvalidation method for better performance. 16 They achieved the best results with the SVM model, achieving an accuracy of 85.1%. Alarsan and Younes 17 combined the MIT-BIH Supraventricular Arrhythmia Database 8 and the MIT-BIH Arrhythmia Database 14 to get a total of 205,146 instances. In addition, it was implemented using three models, Decision Trees, Random Forest (RF), and Gradient-Boosted Trees, and achieved the highest accuracy of 98.03% with RF for multiclassification. Bhattacharyya et al. 18 studied the effect of extracting 61 features using the time-series feature extraction library (TSFEL) and then applying a weighted majority ensemble of RF and SVM on the MIT-BIH arrhythmia dataset. The purpose of incorporating the TSFEL during feature extraction and the SMOTE is to create a more balanced dataset. The paper achieved an accuracy of 98.21% and recall values of 99.5%, 74.2%, 94.22%, 73.21%, and 0% for N, SVEB, VEB, F, and Q classes, respectively. In addition, L. Wang 19 proposed a new approach for arrhythmia classification using a three-heartbeat multi-lead (THML) ECG data, in which each fragment contained three complete heartbeat processes of multiple ECG leads. The THML ECG data preprocessing method is formulated, and four arrhythmia classification models are constructed using 1D-CNN and a priority model with an integrated voting method. The classification of the dual ECG lead with THML data achieved a recall of 96.37%, 96.99%, 80.47%, 22.75%, and 8.33% for N, VEB, SVEB, F, and Q classes. Table 2 shows a summary of the four key research papers, showing the pros and cons of each one of them. Table 2. Summary of the main related literature. Literature Pros Cons Z. Zhang et al. (2014) Proposed automatic heartbeat classification using a disease-specific feature selection approach. Two independent training procedures are executed for the data acquired from each lead for evaluation. Training data was not balanced. Class Q was discarded. S. Bhattacharyya et al. (2021) Implemented an Ensemble of RF and SVM using a weighted majority algorithm (WMA). Performed 10-fold crossvalidation on the training set. Although high accuracy was achieved, the recall for the minority classes was relatively low, mapping to 74.2%, 73.21%, and 0% for SVEB, F, and Q, respectively. T. Wang et al. (2022) Adopt multiple features from the heartbeat (three types of local and global features). Used Easy Ensemble balancing technique and compared against other balancing methods like SMOTE. The adopted features have different distributions, and direct concatenation may affect classification performance. Class Q was discarded. L. Wang et al. (2022) Proposed a novel method that uses a three-heartbeat multi-lead (THML). The majority voting method was used on four models. The THML ECG data relies on the accuracy of the QRS wave detection algorithm. Dataset and experimental setup The work presented in this article creates a heartbeat classification model and studies the impact of adding multiple datasets on enhancing its performance in predicting the Q and F classes. The primary dataset used is the MIT-BIH supra-ventricular dataset, 8 which will be referred to as the base dataset throughout the text. The other supplementary datasets used are: The MIT-BIH arrhythmia dataset. 14 The IN-CART 12-lead Arrhythmia Database. 20 The Sudden Cardiac Death Holter Database. 21 All these datasets are available in the public domain and share similar features. Thus, data from these datasets can be merged simply by appending their records. The data merge process is focused on adding the Q and F classes to the primary dataset, whereas other instances are dropped. Datasets The MIT-BIH supraventricular dataset is a dataset that consists of 78 ECG recordings, each lasting about 30 minutes. The recordings were obtained from patients with various clinical conditions, including healthy individuals and those with structural heart disease, and were collected using the standard 12-lead ECG equipment. A 12-lead ECG is named as such because it records the heart's electrical activity from 12 different viewpoints or leads. The test uses ten electrodes, one attached to each of the four limbs and six to the chest. The four attached to the limbs create six views of the heart in the vertical plane (I, II, III, VL, VF, and VR). Meanwhile, the remaining six electrodes are attached to the chest and are responsible for the six views V1 to V6 that record the activity in the horizontal plane. Thus, the 12-lead ECG as a diagnostic test provides a complete picture of the heart's electrical activity, recording information from six limb leads and six chest leads. 22 In the referenced dataset, only leads II and V5 were used. The dataset consists of 184,428 instances, where each instance is a single ECG signal representing a single heartbeat. The dataset consists of 34 features, including one for the patient record and another for the heartbeat classification type (Label). The remaining 32 features are divided into 16 features representing the signals of lead II and 16 features representing the features of lead V5. The features of both leads are identical. The only difference is the placement of the electrodes on the body. Table 3 describes the features list. Table 3. Feature description. Attribute Description Feature type Record A number assigned to the patient Numeric Type Classification of heartbeat Object pre-RR The period between the end of one R-R interval and the beginning of the next. Numeric post-RR The period following the R-R interval. Numeric pPeak The peak of the P wave. Numeric tPeak The peak of the T wave. Numeric rPeak The peak of the R wave. Numeric sPeak The peak of the S wave. Numeric qPeak Small, positive deflection in the QRS complex. Numeric qrs interval Duration of the QRS complex. Numeric pq interval The time between the P wave's start and the QRS complex's start. Numeric qt interval The time between the start of the QRS complex, and the end of the T wave. Numeric st interval The time between the S wave's end and the T wave's start. Numeric qrs morphologies For the morphology features, five samples are taken in the QRS complex (between the on-set and off-set points of the QRS complex 23 ) Numeric Data attribute information Figure 2 reveals the distribution of values for some features provided within the dataset. Figure 2(a) shows the sPeak distribution, where it can be seen that the number of instances grows increasingly from −4 mv toward zero, followed by a sharp drop in the number of instances toward the positive values. This indicates the normal range of “sPeak” varies within negative millivolts. Moreover, all intervallic features measured by millisecond are distributed between positive 1 ms up to 150 ms. For example, Figure 2(b) refers to qt-interval, where most instances are centered around 13 ms, while the pq-interval, shown in Figure 2(c), has the most instances at 2 ms. Figure 2. Features distribution (a) histogram of the peak of the S wave (b) histogram of the QT interval (c) histogram of the PQ interval (d) histogram of the post-RR (e) histogram of the pre-RR (f) histogram of the peak of the T wave. It is important to note that the normal range for the pre-RR and post-RR intervals can vary depending on many factors, including a person's age, gender, and underlying health conditions. That is why they have a more widespread normal distribution, as shown in Figure 2(d) and (e). However, other attributes like the tPeak seem more concentrated within a smaller range, as shown in Figure 2(f). Figure 3 illustrates the distribution of each class within the base dataset. It can be seen that the classes of the dataset are imbalanced. For example, the F and Q classes constitute 0.012% and 0.043% of the data, respectively. This is significantly lower than the number of instances from other classes, such as the N class, contributing to around 87.913% of the dataset. Such imbalance can lead to poor performance in the less frequently occurring classes since the model may have difficulty learning the characteristics of these minority classes. Moreover, the model's accuracy could be misleading when applied to an imbalanced dataset because it does not consider the recall of rare classes. Figure 3. Distribution of classes. Data pipeline Figure 4 shows the data pipeline for this work. Once the data is cleaned, it is split into training and testing sets, and each is scaled separately. Since the dataset is unbalanced, the SMOTE balancing technique is used to compensate for the minority classes in the dataset. Afterward, the model is trained and undergoes a series of iterations for hyperparameters turning. Once the model training and optimization are complete, its performance is evaluated on the test dataset. Such a process is a standard practice in any machine learning project. Figure 4. Data pipeline. Data preprocessing is essential in data analysis because rows are often incomplete, noisy, or inconsistent and may need to be cleaned and transformed before they can be used effectively. For example, the feature “record” was dropped since it only refers to the patient number and does not help predict the type of ECG signal, so it was discarded. The base dataset is free from noisy rows. However, the values must be scaled for better performance, efficiency, and accuracy. The normalization technique which results in the most significant improvement is the standardization scaling technique. Standardization is often used to scale variables to a common scale because it preserves the shape of the distribution of the data. It can be helpful if the data is not normally distributed and is less sensitive to outliers. The standardization equation is referred to in Equation (1). (1) XStand=x−mean(x)StandardDeviation(x) Machine learning algorithms This work will examine several machine learning algorithms to evaluate their performance in the ECG classification problem. As shown in Figure 5, the machine learning models are RF, logistic regression (LR), KNN, linear SVM (LSVM), and linear discriminant analysis (LDA). Any of these models will have hyperparameters that need to be tuned. However, all of them will have the same input features, which are 16 inputs per lead. These are the (p, q, r, s, t) peaks, (pq, qt, st, qrs) intervals, the Pre-RR and post-RR, and the five qrs morphologies. The model will classify any input ECG into one of the five classes N, SVEB, VEB, Q, and F. Figure 5. Detailed classification model diagram. Logistic regression LR is a type of statistical model that is used for classification tasks. It is called “logistic regression” because it uses a logistic function to predict the probability of an event occurring. It predicts the probability that an ECG signal belongs to a particular class. 24 Random Forest RF is one of the machine learning algorithms used for classification. The basic concept of this algorithm is that it is made of many individual decision trees that work together to make predictions. Each decision tree in an RF is trained on a random subset of the data and makes predictions based on the features of that tree. The predictions from the individual decision trees are then combined to make the final prediction of the RF. 25 K-nearest neighbor The KNN algorithm applies proximity to classify or predict how a single data point will be grouped. It is a nonparametric, supervised learning classifier. It operates under the concept that related points can be discovered close to one another. A class label is chosen by a majority vote, meaning that it chooses the one that is most frequently used in the region of a particular data point.26,27 Linear support vector machine LSVM works by finding a hyperplane in a high-dimensional space that maximally separates classes, where the hyperplane is a line that separates the data. Finding a line that perfectly separates linearly separable classes is possible. For nonlinear classes, finding a good separation line is possible by adding additional dimensions and then projecting the data into a higher dimensional space. 28 Linear discriminant analysis LDA is a dimensionality reduction technique. It finds the linear combination of features that maximizes the separation between the different classes. This is done by finding the projection of the data onto a lower-dimensional space. LDA is a supervised method that considers class labels when finding the data projection. 29 Results and analysis This section presents the results on the MIT-BIH supraventricular dataset (the base dataset), then shows the SMOTE technique's impact on enhancing the results. Then shows how combining data from multiple sources enhances the results. Finally, the best results are compared against the latest in the literature. Base dataset results To compare the performance of different models, recall, precision, and F1-scores were used alongside accuracy. Precision is defined as the model's ability to correctly predict the positive class, whereas recall is the ability of the model to identify all positive instances correctly. The primary performance measure used in the comparison in this work is the recall. One would want to detect all types of classes and not miss any. Equations for the precision, recall, F1-score, and specificity are presented in Equations (2)–(5). (2) Precision=TPTP+FP (3) Recall=TPTP+FN (4) f1-Score=2*Precision*RecallPrecision+Recall (5) Specificity=TNTN+FP Logistic regression Figure 6(a) shows the confusion matrix of the LR model. No instances are detected as Q or F based on the values provided. Although the test data includes 24 instances labeled as Q and seven instances labeled as F, the model predicts them as N, SVEB, Q, and VEB instead. This shows the model's weakness in detecting Q and F classes, causing the recall to be zero. The accuracy given by this model equals 93%. However, accuracy is not enough indicator of performance for classification models since it does not consider data imbalance. Table 4 shows the overall results of LR. Figure 6. Confusion matrix of all algorithms (a) confusion matrix for LR (b) confusion matrix for RF (c) confusion matrix for KNN (d) confusion matrix for LSVM (e) confusion matrix for LDA. LR: logistic regression; RF: Random Forest; KNN: K-nearest neighbor; LSVM: linear support vector machine; LDA: linear discriminant analysis. Table 4. Analysis of LR classifier on original data. Type Precision Recall F1-score Specificity N 95% 98% 97% 59.6% SVEB 65% 42% 51% 98.4% VEB 77% 63% 70% 99% F 0% 0% 0% 100% Q 0% 0% 0% 100% SVEB: supraventricular ectopic beat; VEB: ventricular ectopic beat; LR: logistic regression. Random Forest RF yields a total accuracy of 98%. As shown in the confusion matrix in Figure 6(b) and Table 5, the model performs better for the F class since it detects two out of seven instances as F, and the recall is equal to 29%. However, the Q class does not improve, with its recall still equal to zero. Table 5. Analysis of RF classifier on original data. Type Precision Recall F1-score Specificity N 98% 99% 99% 87.8% SVEB 91% 80% 85% 99.4% VEB 95% 92% 93% 99.7% F 50% 29% 36% 99.9% Q 0% 0% 0% 100% SVEB: supraventricular ectopic beat; VEB: ventricular ectopic beat; RF: Random Forest. K-nearest neighbor KNN provides an accuracy of 97%. Table 6 provides the recall, precision, F1 score, and specificity of both F and Q classes, which shows better readings than previous algorithms. Moreover, Figure 6(c) shows that two out of seven instances are correctly predicted as F, and only one is detected correctly as Q. Table 6. Analysis of KNN classifier on original data. Type Precision Recall F1-score Specificity N 98% 99% 99% 87.3% SVEB 87% 80% 83% 99.1% VEB 95% 91% 93% 99.7% F 67% 29% 40% 99.9% Q 50% 4% 8% 99.9% SVEB: supraventricular ectopic beat; VEB: ventricular ectopic beat; KNN: K-nearest neighbor. Linear support vector machine The measured accuracy is 92%. Like LR and as shown in Figure 6(d), not a single instance of Q or F is detected. LR produces slightly better results than Linear SVM in terms of accuracy, F1-score, recall, precision, and specificity, as shown in Table 7. Table 7. Analysis of LSVM classifier on original data. Type Precision Recall F1-score Specificity N 94% 99% 96% 51% SVEB 63% 30% 41% 98.7% VEB 77% 60% 68% 99% F 0% 0% 0% 100% Q 0% 0% 0% 100% SVEB: supraventricular ectopic beat; VEB: ventricular ectopic beat; LSVM: linear support vector machine. Linear discriminant analysis The LDA measured accuracy is 92%. As shown in Figure 6(e), all instances of F are predicted as either N or VEB. As for Q, only four instances are correctly predicted. Table 8 illustrates the precision, recall, F1-score, and specificity values. Table 8. Analysis of LDA classifier on original data. Type Precision Recall F1-score Specificity N 95% 98% 96% 58.3% SVEB 60% 39% 47% 98.1% VEB 74% 62% 67% 98.8% F 0% 0% 0% 99.9% Q 4% 17% 6% 99.8% SVEB: supraventricular ectopic beat; VEB: ventricular ectopic beat; LDA: linear discriminant analysis. Results using SMOTE technique The charts in Figure 7 compare the recall of the different models before and after applying data augmentation using SMOTE. Figure 7(a) shows the impact of applying SMOTE on the LR model. While the Q and F classes experienced huge growth, the recall of the N class dropped to 74%. As for the SVEB and VEB, an increase in recall values is detected. Figure 7. Recall values before and after SMOTE (a) LR (b) RF (c) KNN (d) LSVM (e) LDA. SMOTE: synthetic minority oversampling technique; LR: logistic regression; RF: Random Forest; KNN: K-nearest neighbor; LSVM: linear support vector machine; LDA: linear discriminant analysis. Figure 7(b) refers to the RF model. After applying SMOTE, there are upward trends in recall in all classes except the N class, which dropped only by 1%, and the F class, which did not change. Overall, the performance of SVEB and VEB is outstanding. However, F and Q did not mark any considerable improvements. The analysis of the KNN model is illustrated in Figure 7(c), where SMOTE clearly shows enhancement in the F class, which increases to 71%, whereas Q increases to 21%. Also, SVEB and VEB classes slightly rise in recall values, yet N decreases by 3% after implementing SMOTE. Figure 7(d) compares the Linear SVM model. SMOTE causes F and Q classes to rise to 71%. However, this causes a significant fall in the results for the N class, dropping from 99% to 69%. This model is considered weak since all classes have a low recall compared to the previous algorithms. Finally, Figure 7(e) shows information about the LDA model results. Q and F recall increases to more than 70% after SMOTE. Similarly, the SVEB and VEB recall increases slightly to 63% and 66%, respectively. However, the recall of the N class becomes worse than its value before SMOTE. In summary, SMOTE efficiently improves the recall of the F and Q classes in LR, LSVM, and LDA models. However, the overall performance of all classes is not at its best, as the accuracy drops to 72%, 69%, and 66%, respectively. Results of using the combined dataset More F and Q instances are added from different datasets and merged into the base dataset to improve the results further. The new data is cleaned and prepared before merging with the base dataset. This guarantees accurate analysis to compare the results and benchmark the improvements fairly. Starting with The Sudden Cardiac Death Holter Database, 21 many instances have features of lead V2 only and instances with features of lead V5 only. For data consistency, instances with missing values from either lead are dropped. The cleaned data is then merged with the clean version of the MIT-BIH arrhythmia dataset 14 and the IN-CART 12-lead Arrhythmia Database. 20 However, since the main target is increasing the unique instances for both Q and F classes, instances with class N, VEB, and SVEB are dropped from the merged dataset to diminish the unbalancing that would arise. Finally, the merged dataset is added to the base MIT-BIH supraventricular dataset. Figure 8 shows the number of the Q and F classes before and after adding the instances from the supplementary datasets. The final distribution of the classes after adding the new instances are added is shown in Figure 9. Then, this new combined dataset is processed using the data pipeline presented earlier, shown in Figure 4, including data splitting, scaling, balancing, and modeling. Figure 8. Number of instances of types F and Q before and after adding multiple datasets. Figure 9. Percentage of instances after adding multiple datasets. Figure 10 compares the recall values between those generated using the base dataset and those generated using the combined dataset. It is worth noting that both cases utilized the SMOTE technique to oversample minority classes. Figure 10(a) compares the results for the LR model; it clearly shows that the F class gained the most benefit after merging the datasets as its recall increased to 92%. In contrast, the Q class performed worse after the merge, dropping from 75% to 48%. As for the remaining classes, N and VEB classes showed slight enhancement, while SVEB remained unchanged. Figure 10. Recall values of base dataset versus combined dataset both after SMOTE (a) LR (b) RF (c) KNN (d) LSVM (e) LDA. SMOTE: synthetic minority oversampling technique; LR: logistic regression; RF: Random Forest; KNN: K-nearest neighbor; LSVM: linear support vector machine; LDA: linear discriminant analysis. Figure 10(b) shows a similar comparison for the RF model. The F class shows massive growth, increasing from 29% to 94%. Also, the Q class has improved from 4% to 19%. Moreover, this model did not affect the remaining classes significantly, as they performed well after merging the datasets. Similar results in Figure 10(c) show the comparison for the KNN model, where it shows that recall of F and Q increased to 95% and 26%, respectively, without negatively affecting the remaining classes. As for Linear SVM, its results are presented in Figure 10(d). Adding more instances for F and Q classes positively affected the recall of the F class. However, Q class recall dropped from 71% to 37%. Generally, this model did not achieve good results for both the base and the merged datasets. Finally, Figure 10(e) illustrates the results for LDA before and after combining the multiple datasets. Merging the datasets resulted in worse results for all classes except for the N class. To further analyze the details of these algorithms, the confusion matrices for these algorithms are shown in Figure 11. Among these algorithms, RF and KNN proved to be the most efficient models capable of predicting all classes with the most negligible errors, as shown in Figure 11(b) and (c). They achieved the best recall values for all classes and improved the predictions of the Q and F classes. RF achieved an accuracy of 97%, making it the most reliable classifier that optimized the predictions of the F and Q classes and raised their recall values to 94.4% and 18.5%, respectively. Meanwhile, LSVM, LR, and LDA provided good recall values for the Q and F classes; however, they were excluded from further consideration due to the apparent drop in results for the other classes, as shown in Figure 11(a), (d) and (e). KNN provided good recall values, but after comparing it with RF, it can be seen that RF provides better recall values for N, SVEB, and VEB. Figure 11. Confusion matrices of all algorithms using the combined dataset and SMOTE (a) confusion matrix for LR (b) confusion matrix for RF (c) confusion matrix for KNN (d) confusion matrix for LSVM (e) confusion matrix for LDA. SMOTE: synthetic minority oversampling technique; LR: logistic regression; RF: Random Forest; KNN: K-nearest neighbor; LSVM: linear support vector machine; LDA: linear discriminant analysis. These results demonstrate that the dataset preprocessing using SMOTE and the data-merging techniques have significantly improved the classification models’ performance. And that the RF can provide better classification accuracy for the given dataset. Finally, the RF model was fine-tuned by altering two hyper-parameters, “n estimators” and “min samples leaf.” 30 Figure 12 shows the normalized confusion matrix of the RF classifier, which exceeds the previously reported KNN classifier performance for the Q and F classes. The final performance metrics of the fine-tuned RF model across all classes are provided in Table 9 and Figure 13. Figure 12. Confusion matrix of RF model after parameter optimization. RF: Random Forest. Figure 13. Analysis of the RF classifier after fine-tuning. RF: Random Forest. Table 9. Analysis of the RF classifier after parameter optimization. Type Precision Recall F1-score Specificity N 100% 97% 98% 96.7% SVEB 71% 93% 80% 97.3% VEB 92% 95% 93% 99.5% F 98% 95% 96% 100% Q 19% 30% 23% 100% Accuracy 97% SVEB: supraventricular ectopic beat; VEB: ventricular ectopic beat; RF: Random Forest. In conclusion, considering all results, the most efficient models that proved their capability of predicting all classes with the least errors were RF and KNN after applying SMOTE on the merged dataset since they achieved the best recall values for all classes and improved the predictions of Q and F classes. Looking for the best accuracy, RF achieved an accuracy equal to 97%. However, KNN recorded 96%. Thus, RF is considered the most reliable classifier, which optimized the predictions of F and Q classes and raised their recall values to 95% and 30%, respectively. Thus, it will be selected for comparison against other works in the literature. Results comparison with literature To compare the obtained results with previous related work, Table 10 shows the recall values of the five classes for this work and four leading research papers. Considering the recall of the F class, it can be seen that in this work, it is 95% exceeding the 93.8% reported by Zhang et al. 15 Table 10. Comparison of recall and accuracy with other papers. Type This work Zhang et al. 15 * Bhattacharyya et al. 18 T. Wang et al. 13 * L. Wang et al. 19 N 97% 88.9% 99.5% 91.7% 96.37% SVEB 93% 79% 74.2% 89.9% 80.47% VEB 95% 85.4% 94.2% 87.8% 96.99% F 95% 93.8% 73.2% 55.4% 22.75% Q 30% 0% 0% 0% 8.33% Accuracy 97% 86.6% 98.2% 95.6% 94.4% Moreover, the Q class has the highest percentage at 30% in this work, while others reported 0% in this category, and 19 has the highest percentage among the four individuals at 8.33%. Notably, works denoted with (*) chose to drop all Q instances from their datasets. But in this work, adding multiple datasets and implementing data augmentation could improve the recall to 30%, as shown in Figure 14, without affecting the remaining classes. Besides that, better results were achieved even for SVEB class. While it is true that the performance of work 19 outperformed our results by around 2% in the specific VEB category, it is essential to note that the performance of the remaining classes was worse than what this work achieved. Taking accuracy into consideration, work in Bhattacharyya et al. 18 achieved better accuracy. However, accuracy was sacrificed to enhance the predictions of F and Q that were neglected in most papers. Figure 14. Comparison of recall values with other papers. Conclusion In summary, this article aimed to build an ECG heartbeat classification model with an enhancement to the Recall values of types F and Q without affecting the recall of the other types. After comparing five different supervised machine-learning algorithms on the base dataset, SMOTE technique was implemented on the five models, and their results were compared. It was noticeable that for most models, the recall for types N, SVEB, and VEB was much higher than for types F and Q. As such, instances of types F and Q from three other datasets were combined with the base dataset. SMOTE was then implemented to balance the data as a further enhancement, and the results were reevaluated. It was concluded that the RF model after SMOTE was implemented had the best overall results considering that the accuracy remained high and the recall for all types increased. This final model provided an accuracy of 97% and a recall of 97%, 93%, 95%, 95%, and 30% for types N, SVEB, VEB, F, and Q, respectively. The clinical application of such a model would be having physicians in remote areas analyze the ECGs quickly without needing a cardiologist for every ECG analysis. A practical deployment of this machine-learning model can be done by adding a processing layer to capture a single heartbeat, extracting the required parameters from the heartbeat, then feeding them into the machine-learning model for classification. The model can be installed on IoT devices and provide preliminary classification (diagnosis), saving time and lives. While the scope of this work focused on the machine learning part, building a fully functional prototype could be considered in future work. Acknowledgements Not Applicable. Contributorship: Amjed Al-Mousa provided a continuous review of the means and methods used, revised the manuscript several times, and prepared the final version, which was approved by all authors. Joud Baniissa, Tala Hashem, and Tala Ibraheem contributed to conceptualization, conducted the literature review, and contributed to data preparation and code implementation. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. Ethical approval: This work did not involve any human or animal trials. The datasets used in this work are anonymous and available in the public domain. Consent statement: Patient consent is not applicable in this research, as there has been no human or animal testing involved. Guarantor: Amjed Al-Mousa. ORCID iD: Amjed Al-mousa https://orcid.org/0000-0002-6427-1008 ==== Refs References 1 WHO. WHO reveals leading causes of death and disability worldwide: 2000–2019. 12 2020. [Online], https://www.who.int/news/item/09-12-2020-who-reveals-leading-causes-of-death-and-disability-worldwide-2000-2019. 2 Mayo Clinic. Heart arrhythmia. 2023. [Online], https://www.mayoclinic.org/diseases-conditions/heart-arrhythmia/symptoms-causes/syc-20350668. 3 Mayo Clinic. Cardiac Abalation. 2023. [Online], https://www.mayoclinic.org/tests-procedures/cardiac-ablation/about/pac-20384993. 4 Rai HM Chatterjee K . Hybrid CNN-LSTM deep learning model and ensemble technique for automatic detection of myocardial infarction using big ECG data. Appl Intell 2022; 52 : 5366–5384. 5 Rai HM Chatterjee K . A unique feature extraction using MRDWT for automatic classification of abnormal heartbeat from ECG big data with multilayered probabilistic neural network classifier. 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==== Front Am J Sports Med Am J Sports Med AJS amjsports The American Journal of Sports Medicine 0363-5465 1552-3365 SAGE Publications Sage CA: Los Angeles, CA 37318086 10.1177/03635465231179693 10.1177_03635465231179693 Articles Shoulder Physiological Tensioning During Lower Trapezius Transfer for Irreparable Posterosuperior Rotator Cuff Tears May Be Important for Improvement in Shoulder Kinematics https://orcid.org/0000-0001-7393-1891 Muench Lukas N. MD *†‡ https://orcid.org/0000-0001-7899-6299 Rupp Marco-Christopher MD † Obopilwe Elifho MS ‡ Mehl Julian MD † Scheiderer Bastian MD † Siebenlist Sebastian MD † Elhassan Bassem T. MD § Mazzocca Augustus D. MS, MD § Berthold Daniel P. MD †‡ † Department of Sports Orthopaedics, Technical University of Munich, Munich, Germany ‡ Department of Orthopedics & Sports Medicine, University of Connecticut, Farmington, Connecticut, USA § Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA Investigation performed at the Department of Orthopedics & Sports Medicine, University of Connecticut, Farmington, Connecticut, USA * Lukas N. Muench, MD, Department of Sports Orthopaedics, Technical University of Munich, Ismaninger Strasse 22, Munich, 81675, Germany (email: lukas.muench@tum.de). 15 6 2023 7 2023 51 9 24222430 18 8 2022 28 4 2023 © 2023 The Author(s) 2023 American Orthopaedic Society for Sports Medicine https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Background: Lower trapezius transfer (LTT) has been proposed for restoring the anteroposterior muscular force couple in the setting of an irreparable posterosuperior rotator cuff tear (PSRCT). Adequate graft tensioning during surgery may be a factor critical for sufficient restoration of shoulder kinematics and functional improvement. Purpose/Hypothesis: The purpose was to evaluate the effect of tensioning during LTT on glenohumeral kinematics using a dynamic shoulder model. It was hypothesized that LTT, while maintaining physiological tension on the lower trapezius muscle, would improve glenohumeral kinematics more effectively than undertensioned or overtensioned LTT. Study Design: Controlled laboratory study. Methods: A total of 10 fresh-frozen cadaveric shoulders were tested using a validated shoulder simulator. Glenohumeral abduction angle, superior migration of the humeral head, and cumulative deltoid force were compared across 5 conditions: (1) native, (2) irreparable PSRCT, (3) LTT with a 12-N load (undertensioned), (4) LTT with a 24-N load (physiologically tensioned according to the cross-sectional area ratio of the lower trapezius muscle), and (5) LTT with a 36-N load (overtensioned). Glenohumeral abduction angle and superior migration of the humeral head were measured using 3-dimensional motion tracking. Cumulative deltoid force was recorded in real time throughout dynamic abduction motion by load cells connected to actuators. Results: Physiologically tensioned (Δ13.1°), undertensioned (Δ7.3°), and overtensioned (Δ9.9°) LTT each significantly increased the glenohumeral abduction angle compared with the irreparable PSRCT (P < .001 for all). Physiologically tensioned LTT achieved a significantly greater glenohumeral abduction angle than undertensioned LTT (Δ5.9°; P < .001) or overtensioned LTT (Δ3.2°; P = .038). Superior migration of the humeral head was significantly decreased with LTT compared with the PSRCT, regardless of tensioning. Physiologically tensioned LTT resulted in significantly less superior migration of the humeral head compared with undertensioned LTT (Δ5.3 mm; P = .004). A significant decrease in cumulative deltoid force was only observed with physiologically tensioned LTT compared with the PSRCT (Δ–19.2 N; P = .044). However, compared with the native state, LTT did not completely restore glenohumeral kinematics, regardless of tensioning. Conclusion: LTT was most effective in improving glenohumeral kinematics after an irreparable PSRCT when maintaining physiological tension on the lower trapezius muscle at time zero. However, LTT did not completely restore native glenohumeral kinematics, regardless of tensioning. Clinical Relevance: Tensioning during LTT for an irreparable PSRCT may be important to sufficiently improve glenohumeral kinematics and may be an intraoperatively modifiable key variable to ensure postoperative functional success. lower trapezius transfer shoulder kinematics tendon transfer posterosuperior rotator cuff tear graft tensioning typesetterts1 ==== Body pmcRecently, lower trapezius transfer (LTT) has come to the fore as a promising joint-preserving treatment option for young and active patients with irreparable posterosuperior rotator cuff tears (PSRCTs).8,13,31,33,36 Alternative surgical approaches include debridement, biceps tenotomy or tenodesis, partial repair, latissimus dorsi transfer, superior capsular reconstruction, subacromial balloon spacer, and reverse total shoulder arthroplasty, without clear evidence-based guidelines described in the literature. 20 As the line of pull of LTT is almost parallel to that of the native infraspinatus muscle, it holds the ideal anatomic position for restoring an insufficient anteroposterior muscular force couple.8,16,24,26,28 When attached to the supraspinatus and infraspinatus insertion sites, LTT has also been shown to generate abduction moment arms throughout shoulder motion, consequently mimicking the native supraspinatus muscle. 28 While the potential for physical therapy may be limited in this patient population, initial clinical results after LTT for irreparable PSRCTs have been encouraging, including significant improvements in range of shoulder motion, patient-reported outcome scores, and pain relief.8,13,31,33 However, the fact that the lower trapezius is a relatively weak muscle with only a short excursion poses a clinical challenge, as it requires the interposition of a graft to bridge the gap to the insertion site.15,18,28 Current options for interposition involve the use of an Achilles tendon allograft13,14,31 or hamstring tendon autograft33,36 to prevent excessive strain on the transferred trapezius muscle. Regardless of the graft choice, adequate tensioning during LTT may be a critical factor for the sufficient restoration of shoulder kinematics and postoperative functional improvement. A biomechanical study by Omid et al 26 recently found that LTT tensioned according to the physiological cross-sectional area ratio of the lower trapezius muscle restored native glenohumeral joint reaction forces better compared with overtensioned or undertensioned LTT. However, that investigation was performed using a static model, which limits the transferability to clinical practice. Further, the effects of tensioning during LTT on range of abduction motion, superior humeral head migration, and compensatory deltoid force during dynamic testing remain unknown. Thus, the purpose of the present study was to evaluate the effect of tensioning during LTT on the glenohumeral abduction angle, superior migration of the humeral head, and cumulative deltoid force using a dynamic shoulder model. It was hypothesized that LTT, while maintaining physiological tension on the lower trapezius muscle, would improve glenohumeral kinematics more effectively than undertensioned or overtensioned LTT. Methods A total of 10 fresh-frozen cadaveric shoulders (mean donor age, 56.5 ± 17.2 years; 5 male and 5 female; 4 left and 6 right) were obtained from MedCure and used for the study. All specimens underwent visual and radiographic examinations to detect and exclude those with tears of the rotator cuff tendons and capsule, joint contracture, moderate to severe osteoarthritis, or bony defects. The study was reviewed via a Human Research Determination Form by the institutional review board of the University of Connecticut, and it was concluded that no institutional review board approval was required. Specimen Preparation Specimen preparation was performed according to a previously described method.1,11,23 After specimens were thawed overnight at room temperature, dissection of the skin, subcutaneous tissue, and muscles was performed. Care was taken to leave the rotator cuff muscles and the coracoacromial ligament preserved. The anterior, middle, and posterior portions of the deltoid tendon were detached from the muscle belly at the deltoid tuberosity and preserved with anchor loops sutured to the tendinous insertions using a locking running stitch (No. 2 FiberWire; Arthrex), allowing for attachment of each of the 3 deltoid heads to an individual shoulder simulator actuator.1,11,29 Similarly, the humeral tendinous insertions of the latissimus dorsi and pectoralis major muscles were carefully preserved. Suture loops were placed so that they covered the whole insertional footprint to ensure physiological load distribution, which were then each attached to an individual actuator. The rotator cuff muscles (supraspinatus, subscapularis, infraspinatus, and teres minor) were sharply released from the scapula and separated from the underlying capsule. Again, care was taken to meticulously prevent any disruption of tissue. As previously described, the infraspinatus and teres minor muscles were simulated as a single unit. The supraspinatus, subscapularis, and infraspinatus/teres minor muscles were sutured to pulley straps (No. 5 FiberWire; Arthrex) to avoid pull-through during load application.1,11,19,29 A steel rod was cemented into the distal humerus and loaded with 1.7 kg, 30 cm distal from the center of the humeral head, representing a constant moment arm for each tested shoulder.19,35 To prevent changes during testing, the glenohumeral joint capsule was vented by opening the rotator interval.1,11,29 Subsequently, the scapular body was placed in a custom rectangular box with the medial border aligned perpendicular to the ground and the glenoid tilted 10° superiorly, while bone cement was poured into the box to ensure proper fixation.1,11,17,29,35 Testing Setup For biomechanical testing, a validated dynamic cadaveric shoulder model was utilized (Figure 1). ‖ As previously described, the shoulder simulator consisted of up to 6 linear screw-driven actuators (Bimba) connected to 444-N load cells (Futek). A universal strain gauge signal conditioner (Model CSG110; Futek) was linked to a panel mount display (Model IMP650; Futek), and test and measurement software (Sensit Version 2.5.1.0; Futek) was used for load cell data acquisition in real time.1,11,29 Figure 1. Schematic drawing of the testing setup. Respective pulleys for the anterior (AD), middle (MD), and posterior deltoid (PD) tendons as well as for the supraspinatus (SSP) tendon were placed according to anatomic landmarks to re-create native force vectors. The subscapularis (SSC) and infraspinatus/teres minor unit (ISP/TM) were loaded statically with a hanging weight. The lines of pull of the latissimus dorsi (LD) and pectoralis major (PM) muscles were placed according to their anatomic positions and loaded with a hanging weight. Starting at their humeral insertions, the suture loops of each muscle were routed over a guide pulley, which was placed on a perfectly level slide rail, allowing for medial and lateral (relative to the mounted shoulder specimen) motion of the guide pulley during dynamic abduction. The potted scapular body was mounted to the shoulder simulator on a 6 degrees of freedom jig with the scapula in 10° of anteflexion and a 10° superior tilt of the glenoid, resulting in a 110° angle between the scapular spine and vertical axis. 35 The anatomic lines of action of the 3 portions of the deltoid, the subscapularis, and the infraspinatus/teres minor unit were routed using custom 7 mm–diameter frictionless pulleys, while the cable attached to the supraspinatus tendon was aligned with a tilt of 10° to the horizontal plane. 35 The pulley for the anterior deltoid was placed over the tip of the coracoid process, approximately 5 mm anterior to the anterolateral corner of the acromion. The middle deltoid pulley was routed over a point 5 mm posterior to the anterolateral corner of the acromion. The posterior deltoid pulley was placed at the posterolateral edge of the acromion, in line with the scapular spine, to re-create native force vectors.1,11,29,35 The lines of pull of the latissimus dorsi and pectoralis major muscles were placed according to their anatomic positions.21,26 Starting at the respective humeral insertions, the suture loops of each muscle were routed over a guide pulley, which was placed on a perfectly level slide rail, allowing for medial and lateral (relative to the mounted shoulder specimen) motion of the guide pulley during dynamic abduction (Figure 1). 21 Motion Analysis and Dynamic Biomechanical Testing As previously described, 4 infrared cameras (Vero Version 1.3; Vicon Motion Systems) were mounted around the shoulder simulator to cover a 180° field of view. 23 A stationary triad, consisting of 3 optical markers, was placed on the acromion, with its center in line with the pulley of the middle deltoid, 23 while a second moving triad was mounted to the humeral shaft with its longitudinal axis in line with the center of the stationary triad placed on the acromion. In a displacement-controlled setting, computer software (SiNet Hub Programmer Version 1.29; Applied Motion Products) was utilized to generate custom motion profiles (for each native specimen) for the individual actuator of the supraspinatus and the anterior, middle, and posterior deltoid muscles separately.1,11,23,29 To ensure the re-creation of physiological positional changes in latissimus dorsi and pectoralis major vectors during abduction motion, the distance from the starting position of the guide pulley on the slide rail (0° of abduction) to its position at 60° of abduction was measured. 21 Subsequently, the guide pulleys of the latissimus dorsi and pectoralis major muscles were each attached to an individual actuator. For each specimen, the measured distance was used to calculate the velocity for the actuators of the latissimus dorsi and pectoralis major guide pulleys. This allowed the guide pulleys to move along the slide rail at the calculated velocity during dynamic abduction, while re-creating the physiological vectors of the latissimus dorsi and pectoralis major muscles at each abduction angle. 21 A 3-dimensional (3D) motion tracking system (Nexus Version 2.8; Vicon Motion Systems) and 4 infrared cameras (Vero Version 1.3) with a frame rate of 250 Hz and a position accuracy of 0.01 mm and 0.1° recorded each motion profile. 23 The shoulder was abducted in neutral rotation from 0° to 60° in the scapular plane with the scapula fixed, corresponding to approximately 90° of total shoulder abduction.1,11,23,29 The subscapularis and infraspinatus/teres minor unit were loaded statically with a 1.36-kg hanging weight, allowing for balanced abduction motion. 27 Based on previously determined cross-sectional area ratios, the latissimus dorsi and pectoralis major muscles were each statically loaded with a 24-N (2.45-kg) hanging weight.26,34 Each motion cycle was repeated 3 times to generate reliable data of applied forces.1,11,29 To maintain centering of the glenohumeral joint at the resting position, 10 N was applied to the supraspinatus as well as to the anterior, middle, and posterior deltoid muscles.1,11,23,29 Every testing cycle started with the specimen in its resting position of 0° of abduction and neutral rotation. Individual tendon excursion was measured, and velocity (0.1 inch/s for the middle deltoid) was calculated to reach 60° of glenohumeral abduction as previously described.9,29 Force in each muscle was specified to increase linearly.23,29 For each specimen, an individual motion profile was generated in the native state and maintained throughout the following testing conditions. 23 Testing Conditions Specimens remained in the shoulder simulator during all testing and surgical repair. To avoid performance bias, all surgical procedures were performed by the same surgeon (L.N.M.). Each specimen underwent the 5 following conditions, with each specimen being its own control: (1) native, (2) irreparable PSRCT, (3) LTT with a 12-N load (undertensioned), (4) LTT with a 24-N load (physiologically tensioned according to the cross-sectional area ratio of the lower trapezius muscle), and (5) LTT with a 36-N load (overtensioned).2,26,34 After being tested in the native state, an irreparable PSRCT was created by sharply dissecting the footprint of the supraspinatus muscle and cranial part of the infraspinatus muscle on the greater humeral tuberosity.4-6,21 The supraspinatus muscle belly was detached from the supraspinous fossa to create an irreparably retracted tear (Figure 2A). Subsequently, LTT was performed using an Achilles tendon allograft, as previously described by Elhassan et al 12 (Figure 2B). Briefly, the osseous portion of the calcaneus was removed, and 2 sutures (No. 2 FiberWire) were placed at the thick end of the allograft using a Krackow stitch configuration. Next, 2 suture anchors (5.5-mm SwiveLock anchor; Arthrex) were placed at the greater tuberosity in an anteromedial and anterolateral position, with each being preloaded with one of the sutures placed in the allograft. Subsequently, a second row was added posterior to the first row using 2 double-loaded suture anchors (5.5-mm Corkscrew anchor; Arthrex), while ensuring appropriate tension of the graft. The thin distal side of the allograft was then tagged with another suture, which was used to route the line of pull of LTT by using the dorsal scapular spinal tubercle as an anatomic landmark.21,25 The LTT was then loaded with a 12-N (undertensioned), 24-N (physiological tension), and 36-N (overtensioned) hanging weight in a randomized order (conditions 3-5).2,26,34 Figure 2. (A) An irreparable posterosuperior rotator cuff tear was created by sharply dissecting the footprint of the supraspinatus muscle and cranial part of the infraspinatus muscle on the greater humeral tuberosity. (B) Lower trapezius transfer was performed using an Achilles tendon allograft, which was then loaded with a 12-N (undertensioned), 24-N (physiological tension), and 36-N (overtensioned) hanging weight in a randomized order. Outcome Measures Outcome variables included (1) glenohumeral abduction angle (in degrees), (2) superior migration of the humeral head (in millimeters) relative to the native state, and (3) cumulative deltoid force (in Newtons).1,11,29 Glenohumeral abduction angle and superior migration of the humeral head were recorded using a 3D motion tracking system (Nexus Version 2.8) with 4 infrared cameras (Vero Version 1.3). Motion analysis software (ProCalc; Vicon Motion Systems) was used to analyze recorded 3D motion video. 23 Superior migration of the humeral head was calculated as the change in the distance between the 2 tripods relative to the native state. Cumulative deltoid force was recorded in real time throughout the range of motion by load cells (Futek) connected to the actuators.1,11,23,29 Cumulative deltoid force was calculated as the sum of anterior, middle, and posterior deltoid forces.1,11,23,29 Specimens underwent 3 testing cycles for each condition.1,23,29 Statistical Analysis A priori power analysis was performed to determine detectable differences in the dependent variables given the estimated standard deviations. 29 For the glenohumeral abduction angle, an error variance of 1° across all conditions with a correlation of 0.3 between measurements was assumed. A sample size of 6 specimens was determined to provide 80% power to detect a 1° difference in the shoulder angle at an alpha level of .05. Descriptive statistics including means and standard deviations were calculated to characterize the specimens. Repeated-measures analysis of variance was performed to examine differences in the glenohumeral abduction angle, superior migration of the humeral head, and cumulative deltoid force among the various testing conditions. The distribution of model residuals was examined to ensure that large deviations from normality were not present. When significant, post hoc paired t tests with a corrected alpha using the Holm-Bonferroni sequential correction method were performed to determine which pairwise comparisons were statistically significant. The alpha level for all analyses was set at .05. All statistical analyses were performed using commercial software (Stata 15.2; StataCorp). Results Glenohumeral Abduction Angle The PSRCT significantly decreased the glenohumeral abduction angle compared with the native state (Δ–24.0°; P < .001). Physiologically tensioned (Δ13.1°), undertensioned (Δ7.3°), and overtensioned (Δ9.9°) LTT each significantly increased the glenohumeral abduction angle compared with the PSRCT (P < .001 for all). LTT with physiological tension achieved a significantly greater glenohumeral abduction angle than undertensioned LTT (Δ5.9°; P < .001) or overtensioned LTT (Δ3.2°; P = .038). However, LTT did not restore the native glenohumeral abduction angle, regardless of tensioning (Tables 1 and 2). Table 1 Outcome Variables for Each Testing Condition a Glenohumeral Abduction Angle, deg Superior Migration of Humeral Head, mm Cumulative Deltoid Force, N Native 55.3 ± 2.3 — 250.8 ± 33.7 PSRCT 31.2 ± 5.6 21.0 ± 5.9 303.8 ± 33.6 LTT with 12-N load 38.5 ± 3.5 17.0 ± 7.5 298.7 ± 26.9 LTT with 24-N load 44.3 ± 3.0 11.7 ± 7.2 284.6 ± 22.4 LTT with 36-N load 41.1 ± 4.1 14.4 ± 8.8 289.0 ± 25.8 a Data are presented as mean ± SD. LTT, lower trapezius transfer; PSRCT, posterosuperior rotator cuff tear. Dash indicates no data available. Table 2 Comparison of Glenohumeral Abduction Angle Among Testing Conditions a Difference (95% CI) P Value PSRCT vs native −24.0 (−26.8 to −21.3) <.001 b LTT with 12-N load vs native −16.8 (−19.5 to −14.1) <.001 b LTT with 24-N load vs native −10.9 (−13.6 to −8.2) <.001 b LTT with 36-N load vs native −14.2 (−16.9 to −11.5) <.001 b LTT with 12-N load vs PSRCT 7.3 (4.5 to 10.0) <.001 b LTT with 24-N load vs PSRCT 13.1 (10.4 to 15.8) <.001 b LTT with 36-N load vs PSRCT 9.9 (7.2 to 12.6) <.001 b LTT with 24-N load vs LTT with 12-N load 5.9 (3.2 to 8.6) <.001 b LTT with 36-N load vs LTT with 12-N load 2.6 (−0.1 to 5.3) .059 LTT with 36-N load vs LTT with 24-N load −3.2 (−6.0 to −0.5) .038 b a P values were adjusted for multiple comparisons using the Holm-Bonferroni sequential correction method. LTT, lower trapezius transfer; PSRCT, posterosuperior rotator cuff tear. b Statistical significance. Superior Migration of Humeral Head The PSRCT as well as each of the LTT tensioning conditions showed a significant increase in superior migration of the humeral head compared with the native state (P < .001 for all) (Tables 1 and 3). However, LTT resulted in a significant decrease in superior migration of the humeral head compared with the PSRCT, regardless of tensioning. Physiologically tensioned LTT achieved significantly less superior migration of the humeral head compared with undertensioned LTT (Δ5.3 mm; P = .004). Table 3 Comparison of Superior Migration of Humeral Head Among Testing Conditions a Difference (95% CI) P Value PSRCT vs native −21.1 (−24.3 to −17.8) <.001 b LTT with 12-N load vs native −17.0 (−20.2 to −13.8) <.001 b LTT with 24-N load vs native −11.7 (−14.9 to −8.4) <.001 b LTT with 36-N load vs native −14.4 (−17.6 to −11.1) <.001 b LTT with 12-N load vs PSRCT 4.1 (0.8 to 7.3) .042 b LTT with 24-N load vs PSRCT 9.4 (6.1 to 12.6) <.001 b LTT with 36-N load vs PSRCT 6.7 (3.4 to 9.9) <.001 b LTT with 24-N load vs LTT with 12-N load 5.3 (2.1 to 8.6) .004 b LTT with 36-N load vs LTT with 12-N load 2.6 (−0.6 to 5.9) .204 LTT with 36-N load vs LTT with 24-N load −2.7 (−6.0 to 0.5) .204 a P values were adjusted for multiple comparisons using the Holm-Bonferroni sequential correction method. LTT, lower trapezius transfer; PSRCT, posterosuperior rotator cuff tear. b Statistical significance. Cumulative Deltoid Force The PSRCT as well as each of the LTT tensioning conditions showed a significant increase in the cumulative deltoid force compared with the native state (P < .001 for all) (Tables 1 and 4). Only physiologically tensioned LTT resulted in a significant decrease in the cumulative deltoid force compared with the PSRCT (Δ–19.2 N; P = .044). However, there were no significant differences between physiologically tensioned, undertensioned, and overtensioned LTT. Table 4 Comparison of Cumulative Deltoid Force Among Testing Conditions a Difference (95% CI) P Value PSRCT vs native 52.9 (38.3 to 67.6) <.001 b LTT with 12-N load vs native 47.9 (33.2 to 62.5) <.001 b LTT with 24-N load vs native 33.7 (19.1 to 48.4) <.001 b LTT with 36-N load vs native 38.2 (23.5 to 52.8) <.001 b LTT with 12-N load vs PSRCT −5.1 (−19.7 to 9.6) .990 LTT with 24-N load vs PSRCT −19.2 (−33.9 to −4.6) .044 b LTT with 36-N load vs PSRCT −14.8 (−29.4 to −0.1) .240 LTT with 24-N load vs LTT with 12-N load −14.2 (−28.8 to 0.5) .290 LTT with 36-N load vs LTT with 12-N load −9.7 (−24.4 to 4.9) .970 LTT with 36-N load vs LTT with 24-N load 4.4 (−10.2 to 19.1) .990 a P values were adjusted for multiple comparisons using the Holm-Bonferroni sequential correction method. LTT, lower trapezius transfer; PSRCT, posterosuperior rotator cuff tear. b Statistical significance. Discussion The most important finding of this study was that LTT with physiological tension was most effective in improving glenohumeral kinematics after an irreparable PSRCT. Physiologically tensioned LTT resulted in a significant increase in the glenohumeral abduction angle along with significantly less superior migration of the humeral head and a lower cumulative deltoid force compared with the PSRCT. However, LTT did not completely restore native glenohumeral kinematics, regardless of tensioning. These biomechanical observations indicate that physiological tensioning during LTT may be an intraoperatively modifiable key variable to ensure improvement in postoperative glenohumeral kinematics and functional success. Restoration of the anteroposterior muscular force couple has been shown to be essential for ensuring centered abduction motion and the prevention of superior decentralization of the humeral head.3,7,11,32 Based on the advantageous anatomic location of the lower trapezius muscle, the force vector after LTT closely resembles that of the native infraspinatus muscle, thus allowing for sufficient balancing of the anteroposterior muscular force couple in the transverse plane during abduction motion.7,8,16,24-26,28 Consistently, the present study found a significant improvement in the glenohumeral abduction angle along with a reduction in superior migration of the humeral head after LTT compared with the PSRCT, regardless of tensioning. However, one of the main drawbacks of LTT is the need for interposition of a graft to bridge the gap to the insertion site. In an anatomic feasibility study by Gracitelli et al, 15 direct isolated LTT did not allow for reaching the insertion site of the infraspinatus muscle at the greater tuberosity in any case, and sutures were not viable for this purpose. A recent study using a biomechanical computed tomography model further showed that LTT resulted in a muscle elongation of 113.1 mm compared with its resting length when attached to the supraspinatus insertion, highlighting the requirement of graft interposition. 28 Although the general notion exists that an autograft may show faster incorporation along with a lower inflammatory response compared with an allograft, the use of an Achilles tendon allograft is currently the most commonly established technique. 8 Further, tensioning during LTT should be considered as an intraoperatively modifiable key variable, with probably immediate effects on biological healing, remaining creep within the graft, and the ability to restore native shoulder kinematics.8,26 Using a static biomechanical shoulder model, Omid et al 26 investigated the effect of 3 different tensioning conditions of the Achilles tendon allograft for LTT in the setting of an irreparable PSRCT. The authors found that adequately tensioned LTT with a load proportional to the physiological cross-sectional area ratio of the lower trapezius muscle most sufficiently recentered the humeral head in the anteroposterior plane and restored glenohumeral compression forces to that of the native rotator cuff. 26 Interestingly, undertensioned LTT, simulating the result of excessive creep of the graft, was not able to generate enough glenohumeral compression forces, whereas overtensioned LTT led to overcorrection. 26 Similarly, the present study found that physiologically tensioned LTT resulted in significantly greater abduction motion compared with both undertensioned and overtensioned LTT, while at the same time being most effective in preventing superior decentralization of the humeral head. Based on the observations of Omid et al, 26 this improvement may be a result of the restoration of native joint compression forces and recentering of the humeral head, allowing for balanced abduction motion. In the case of undertensioned LTT, the anteroposterior muscular force couple is not adequately balanced with a more internally rotated position of the humeral head, whereas overtensioning leads to increased external rotation, with both scenarios impeding centered abduction motion and thus not completely restoring native kinematics. Previous biomechanical investigations showed that insufficiency of the posterosuperior rotator cuff leads to a disruption of the synergy between the rotator cuff and deltoid muscles, with a subsequent increase in compensatory deltoid forces to prevent the loss of abduction motion.1,4-6,10,11,29 This is consistent with the present results, as an irreparable PSRCT resulted in a significant increase in the cumulative deltoid force. Physiologically tensioned LTT was the only tensioning condition that achieved a significant decrease in the cumulative deltoid force compared with the PSRCT, which may be a result of recentering of the humeral head combined with the restoration of adequate joint compression forces. 26 Reducing compensatory deltoid forces may be clinically important to protect patients from developing postoperative deltoid fatigue. However, none of the LTT tensioning conditions restored the native cumulative deltoid force. This may be because the deltoid muscle needs to compensate for the remaining insufficiency of the posterosuperior rotator cuff. During initial abduction motion, the line of pull of the deltoid muscle is almost vertical 30 ; thus, LTT may not be able to completely resist the vertically directed, increased deltoid force, consequently allowing for some amount of superior humeral head translation and subsequent limitations in abduction motion. Furthermore, the ability to reduce the cumulative deltoid force may be limited by the testing setup, as LTT was only statically loaded in this model using a hanging weight. As LTT has also been shown to generate abduction moment arms throughout shoulder motion when attached to the supraspinatus and infraspinatus insertion sites, dynamic loading of LTT would probably show a considerably better ability to reduce deltoid forces, while also more closely reflecting in vivo shoulder kinematics. 28 This fact may in part explain why a complete restoration of native glenohumeral kinematics could not be achieved in the present study. Certainly, transferability of the present findings to clinical practice is difficult, as tensioning during LTT is challenging to assess intraoperatively. In conjunction with already existing biomechanical data, 26 physiological tensioning during LTT seems to most sufficiently restore joint compression forces, humeral head centering, abduction motion, and compensatory deltoid forces. However, there is an unmet clinical need to develop better intraoperative methods to assess adequate tensioning during tendon transfer accurately and reproducibly, potential changes over time, and its effect on biological healing. Although graft interposition is generally seen as one of the main disadvantages of the LTT technique, the intraoperative modification of graft tension also carries the opportunity to optimize functional outcomes by addressing interindividual anatomic variations. Surgical options to adjust graft tension intraoperatively may include changing the graft length as well as fixing the graft in varying positions of shoulder abduction and rotation. However, there will always remain the issue that it only represents the preload at time zero. The stretching and contracting ability of the transferred muscle and its adaptations over time are very challenging to assess. In addition, biological healing of the allograft to the lower trapezius muscle may further affect shoulder biomechanics; however, this is impossible to assess in this model. There were several limitations to the study. With this being a biomechanical cadaveric study, only time-zero data are reported without an evaluation of biological healing or changes in graft tension over time. Second, as the same specimen was used for consecutively testing the different conditions, there may be some potential for creeping of tissue, especially of the joint capsule, with increased laxity over time. This was accounted for by randomizing the testing order. Third, the high age of the donors of the cadaveric shoulders may not always reflect clinical practice, as patients indicated for tendon transfer usually comprise a younger population. In addition, an assessment of shoulder kinematics was only performed during dynamic glenohumeral abduction in the scapular plane, leaving the effects of LTT tension during other functional tasks unknown. Further, LTT was only loaded statically based on previously determined physiological cross-sectional area ratios,2,34 limiting the transferability to clinical practice. In addition, the necessity of securely mounting the specimen to the shoulder simulator with a fixed scapula eliminated any scapulothoracic motion, precluding an assessment of the potential disruption of scapulothoracic motion caused by LTT. Thus, full torso specimens could not be used, which limits the replication of the true dynamic lines of pull of LTT, the pectoralis major muscle, and the latissimus dorsi muscle. However, every attempt was made to place the respective force vectors in a physiological orientation by using several anatomic landmarks such as the inferior scapular angle and scapular spine. Finally, for all the limitations stated here, the findings of the present study have to be tempered when translating the data to the in vivo setting. Conclusion LTT was the most effective in improving glenohumeral kinematics after an irreparable PSRCT when maintaining physiological tension on the lower trapezius muscle at time zero. Kinematic effects included an increase in the glenohumeral abduction angle, less superior migration of the humeral head, and a lower cumulative deltoid force compared with undertensioned and overtensioned LTT. However, LTT did not completely restore native glenohumeral kinematics, regardless of tensioning. Submitted August 18, 2022; accepted April 28, 2023. One or more of the authors has declared the following potential conflict of interest or source of funding: The UConn Musculoskeletal Institute, UConn Health, has received direct funding and material support from Arthrex; the company and the institute had no influence on the study design, data collection, or interpretation of the results or the final article. B.T.E. has received consulting fees from DJO, Arthrex, and Integra LifeSciences. A.D.M. has received consulting fees and research support from Arthrex, consulting fees from Astellas Pharma, and speaking fees from Kairos Surgical. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto. ORCID iDs: Lukas N. Muench https://orcid.org/0000-0001-7393-1891 Marco-Christopher Rupp https://orcid.org/0000-0001-7899-6299 ‖ References 1, 9, 11, 17, 19, 21-23, 29, 35. ==== Refs References 1 Adams CR Comer B Scheiderer B , et al . The effect of glenohumeral fixation angle on deltoid function during superior capsule reconstruction: a biomechanical investigation. Arthroscopy. 2020;36 (2 ):400-408.31902546 2 Akagi R Kanehisa H Kawakami Y Fukunaga T . Establishing a new index of muscle cross-sectional area and its relationship with isometric muscle strength. J Strength Cond Res. 2008;22 (1 ):82-87.18296959 3 Apreleva M Parsons IM 4th Warner JJ Fu FH Woo SL . Experimental investigation of reaction forces at the glenohumeral joint during active abduction. J Shoulder Elbow Surg. 2000;9 (5 ):409-417.11075325 4 Berthold DP Bell R Muench LN , et al . 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PMC010xxxxxx/PMC10353028.txt

==== Front Am J Sports Med Am J Sports Med AJS amjsports The American Journal of Sports Medicine 0363-5465 1552-3365 SAGE Publications Sage CA: Los Angeles, CA 37306059 10.1177/03635465231178292 10.1177_03635465231178292 Articles Knee Tibial Slope on Conventional Lateral Radiographs in Anterior Cruciate Ligament–Injured and Intact Knees: Mean Value and Outliers Weiler Andreas MD, PhD * Berndt Rosa MS † Wagner Michael MD * Scheffler Sven MD, PhD * Schatka Imke MD ‡ Gwinner Clemens MD †§ * Sporthopaedicum Berlin, Berlin, Germany † Center for Musculoskeletal Surgery, Charité–University Medicine Berlin, Berlin, Germany ‡ Center for Radiology and Nuclear Medicine, Charité–University Medicine Berlin, Berlin, Germany Investigation performed at Charité–University Medicine Berlin, Berlin, Germany § Clemens Gwinner, MD, Center for Musculoskeletal Surgery, Charité–University Medicine Berlin, Augustenburger Platz 1, 13353 Berlin, Germany (email: clemens.gwinner@charite.de). 12 6 2023 7 2023 51 9 22852290 31 8 2022 19 4 2023 © 2023 The Author(s) 2023 American Orthopaedic Society for Sports Medicine https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Background: An increased tibial slope (TS) has been identified as a risk factor for anterior cruciate ligament (ACL) injury and graft failure after ACL reconstruction. However, different imaging modalities are used to determine the TS, resulting in divergent values. Consequently, no reference values and no consensus on thresholds can be reached, which in turn is mandatory for indicating correction osteotomies when facing outlier TS. Purpose: To determine the mean values of the TS and the incidence of their outliers in large cohorts of patients with ACL-injured and noninjured knees and to determine the feasibility of measuring TS on conventional lateral radiographs (CLRs). Study Design: Cross-sectional study; Level of evidence 3. Methods: TS of ACL-injured knees (n = 1000, group A) and ACL-intact knees (n = 1000, group B) was measured by 3 experienced examiners. Medial TS was measured on CLRs using the technique of Dejour and Bonnin. Patients with radiographs with poor image quality, osteoarthritis, previous osteotomies, or nondigital radiographs were excluded. The intra- and interrater reliability was calculated using the intraclass correlation coefficient. Results: The mean TS was significantly higher in group A than in group B (10.04°± 3° [range, 2°-22°] vs 9.02°± 2.9° [range, 1°-18°], respectively; P < .001). Significantly more participants in group A had TS larger than 12° (≥12°, 32.2% vs 19.8%, P < .001; ≥13°, 20.9% vs 11.1%, P < .001; ≥14°, 13.5% vs 5.7%, P < .001; ≥15°, 8% vs 2.7%, P < .001; ≥16°, 3.7% vs 1.4%, P = .0005), respectively. In contrast, significantly more participatns in group B had TS 8° or less (≤8°, 32.1% vs 42.7%, P < .001; ≤7°, 20% vs 30.9%, P < .001; ≤6°, 12.4% vs 19.8%, P < .001; ≤5°, 6.6% vs 12%, P = .0003; ≤4°, 2.8% vs 5.3%, P = .0045). The intraclass correlation coefficient revealed a good to excellent reliability throughout measurements. Conclusion: Median values for the TS were 9° for uninjured and 10° for ACL-injured knees on CLRs. Notwithstanding its statistical significance, this finding might be negligible in clinical practice. However, a significantly larger number of outliers were found in the ACL-injured group exceeding a TS of 12° and demonstrating an incremental proportion with increasing TS, serving as a potential threshold for correction osteotomy. Furthermore, CLRs in the largest cohort to date exhibited high reproducibility, proving the feasibility of CLRs as a routine measurement for TS. anterior cruciate ligament tibial slope mean values outliers conventional lateral radiographs typesetterts1 ==== Body pmcIncreasing evidence shows that the configuration of the dorsal inclination of the proximal tibia—namely, the tibial slope (TS)—has a strong effect on anterior cruciate ligament (ACL) strain, ACL injury rates, and failure rates after ACL reconstruction (ACLR).16,25,29,30,32 It has been shown that an outlier TS (≥12°) is associated with an increased number of ACL graft failures and produces an odds ratio of 11.6 for repeated ACL graft insufficiency after ACLR. 13 On the basis of these findings, some authors advocate measuring the TS in every case of ACL injury to identify outliers as a neglected risk factor for ACL graft failure.10,13 Particularly in the revision situation, assessment of the TS seems to be mandatory to decide whether a correction osteotomy might be indicated.1,7,28,31 A paucity of studies have used a uniform TS assessment, reporting on a threshold for an increased failure rate after ACLR or slope-correcting osteotomies. To date, different acquisition techniques have been introduced to measure the TS, such as magnetic resonance imaging (MRI), computed tomography (CT), and conventional lateral radiographs (CLRs). Every modality allows various measurements using different anatomic references. 6 This results in inconsistent data, obstructs comparison between studies with difference acquisition techniques, and impedes the ability to reach a consensus on a standardized measurement for the clinical routine. CLRs are commonly available in the clinical setting, and potential confounders have been elucidated. 12 Concerning measurement of the TS, the technique described by Dejour and Bonnin 8 is widely used. However, the number of patients with ACL injury in the available studies is limited, ACL-intact controls are usually not included, and few studies report on intra- and interrater reliability.4,18,21,29 Thus, little consensus can be reached regarding mean values of TS and its outliers in patients with and without ACL injury. However, this information is mandatory to judge the clinical feasibility of CLRs in this regard. The aim of the current study was, first, to determine mean values of TS in a large patient cohort and the incidence of outliers on CLRs in a standardized fashion. Second, we compared ACL-injured and noninjured knees, and third, we determined the intra- and interrater reliability. It was hypothesized that the TS and its outliers in ACL-injured knees would be significantly higher compared with those in the control group. Furthermore, we hypothesized that measurements using CLRs in a standardized fashion would result in high intra- and interrater reliability. Methods The institutional database was retrospectively reviewed for CLRs of patients with an ACL injury (group A). ACL insufficiency was confirmed via MRI and clinical examination. Patients with concomitant injuries of the posterior cruciate ligament, combined lower extremity fractures, osteoarthritis higher than grade II according to Kellgren and Lawrence, or previous osteotomies of the ipsilateral knee were excluded. In addition, we excluded patients whose radiographs did not include a true lateral view of the tibia as determined by proper superimposition of the femoral condyles (<5 mm). 12 We excluded radiographs with poor quality, such as a short tibial shaft (<12.5 cm), low resolution, or poor contrast, and nondigital radiographs. The inclusion process in the study group (group A) was set at N = 1000 CLRs. The control group (group B) consisted of 1000 CLRs of patients after exclusion of an ACL insufficiency. These numbers were based on the sample size calculation. CLRs in group B were performed to exclude degenerative changes or fractures adjacent to the knee, and special care was taken to exclude patients with possible ACL injury. The same exclusion and inclusion criteria were applied as in group A. The inclusion process is illustrated in Figure 1. Figure 1. Flowchart depicting the selection of study participants. CLR, conventional lateral radiograph. Anthropometric and descriptive measures including age and sex were documented. The study protocol was approved by the local ethics committee (EA2/016/21), and the study was carried out in accordance with the Declaration of Helsinki and all participants provided informed consent. Radiological Assessment TS was measured on CLRs by 3 experienced observers (A.W., R.B., C.G.) using a picture archiving and communication system workstation (Centricity RIS-I 4.2 Plus; GE Healthcare). All observers were blinded to all other parameters. The TS is regarded as the angle between the posterior inclination of the medial tibial plateau and a line perpendicular to the tibial shaft axis. The tibial shaft axis was determined according to Dejour and Bonnin 8 using 2 midpoints between the anterior and posterior tibial cortex: one below the tibial tuberosity (approximately 5 cm below the joint line) and the other one 15 cm below the tibial joint line. Subsequently, the TS was determined between the tangent line to the medial tibial plateau, which is commonly superimposed on lateral radiographs, and to a line 90° to the diaphyseal axis. Although the best available radiographs were chosen, not all included enough tibial shaft to determine the width of the shaft at a distance of 15 cm distal to the joint line. In these instances, the most distal width (if ≥12.5 cm) was measured based on the aforementioned conditions (Figure 2).12-15 Figure 2. Measurement of the tibial slope, which is defined as the angle (*) between the tangent line of the medial tibial plateau and a line perpendicular to the tibial shaft axis. Statistical Analysis Statistical analysis was performed using SPSS 22 (IBM Corporation) and Prism Version 6 (GraphPad Software). Continuous data are expressed as mean ± SD and or median according to their distribution. D’Agostino and Person omnibus normality tests were used to test Gaussian distribution. Parametric data were compared using a t test, and nonparametric data were assessed using a Mann-Whitney test. Chi-square test was used to determine statistically significant differences between categorical variables. Multivariate analysis was performed to account for demographic differences between groups. P < .05 was considered statistically significant. A power analysis was performed to calculate the sample size, using G*Power software (Heinrich-Heine-Universität). Intra- and interrater reliability was rated using the intraclass correlation coefficient according to Shrout and Fleiss 26 (2,1; absolute agreement). Intraclass correlation coefficient was categorized as slight, 0 to 0.2; fair, 0.21 to 0.4; moderate, 0.41 to 0.6; good, 0.61 to 0.8; and excellent, >0.8. 19 To test interrater reliability, we compared 100 measurements between each observer. To test intrarater reliability, we performed 50 measurements twice—at least 14 days apart—and consequently compared them between each individual observer. Results Inclusion of 1000 patients per group resulted in a power >0.9 to protect against undue rejection of the null hypothesis, thus achieving the anticipated level of statistical power and validation. Group Characteristics The mean age of patients was 36.6 years (range, 18-66 years) in group A and 41.2 years (range, 18-65 years) in group B (P < .001). Group A contained significantly more male patients (63.5%) compared with group B (48.3%) (P < .001). Multivariate analysis revealed no dependence between sex and TS but a statistically significant dependence between age and TS (P < .001; regression coefficient, –0.03). With respect to the affected side, group A had 50.8% right knees, and 53.4% of the cases in group B involved the right side (not significant). Analysis of the Tibial Slope The mean TS was 10.04°± 3° (median, 10°; range, 2°-22°) in group A and 9.02°± 2.9° (median, 9°; range, 1°-18°) in group B. This difference was statistically significant (P < .001). The numbers of patients with a TS of 2° to 16° are depicted in Figure 3. Figure 3. Distribution and trend lines of the tibial slope (TS) between 2° and 16° within the patient cohort. A significant divide is seen in favor of patients with intact anterior cruciate ligament (ACL) (light gray) at TS ≤ 8°. This is also evident for patients with ACL injury (dark gray) at TS ≥12°. Outlier analysis revealed a significant divide in favor of group A marking the beginning of a TS of 12°. More specifically, in group A compared with group B, there was a significantly larger number of patients with TS ≥12° (32.2% vs 19.8%; P < .001), TS ≥13° (20.9% vs 11.1%; P < .001), TS ≥14° (13.5% vs 5.7%; P < .001), TS ≥15° (8% vs 2.7%; P < .001), and TS ≥16° (3.7% vs 1.4%; P = .0005), respectively. In contrast, there was a significant divide for group B with a TS ≤8° (Group A: 32.1% vs Group B: 42.7%; P < .001), TS ≤7° (20% vs 30.9%; P < .001), TS ≤6° (12.4% vs 19.8%; P < .001), TS ≤5° (6.6% vs 12%; P = .0003), and TS ≤4° (2.8% vs 5.3%; P = .0045), respectively. The outlier ratio (percentage of group A divided by percentage of group B) between a TS of 2° and 16° is presented in Figure 4. Figure 4. Distribution and trend lines of the outlier ratio (percentage of group A divided by percentage of group B) between a tibial slope of 2° and 16°. Values >1 are depicted in light gray, >1 in dark gray. Inter- and Intrarater Reliability Inter- and intrarater reliability was assessed for all 3 observers (observer 1, A.W.; observer 2, R.B.; observer 3, C.G.) (Table 1). Table 1 Inter- and Intrarater Reliability Rated Intraclass Correlation Coefficient (ICC) Observer ICC (95% CI) Agreement 1 vs 2 0.78 (0.69-0.84) Good 1 vs 3 0.93 (0.86-0.96) Excellent 2 vs 3 0.95 (0.93-0.97) Excellent 1 vs 1 0.92 (0.86-0.96) Excellent 2 vs 2 0.88 (0.79-0.94) Excellent 3 vs 3 0.95 (0.89-0.98) Excellent Discussion The working hypothesis, that the mean TS on CLRs in ACL-injured knees would be significantly higher compared with that in ACL-intact knees, was supported by the current results. Notwithstanding its statistical significance, there was only a minor absolute difference of 1°, with both groups demonstrating considerable ranges. Consequently, one must consider this discrepancy to be of small clinical relevance. Nonetheless, ACL-injured knees exhibited a significantly higher portion of outliers above a TS of 12°, becoming even more pronounced with further increase of TS. In contrast, ACL-intact knees showed a significantly larger portion below a TS of 8°. This was also evident in the outlier ratio. In addition, assessment of the TS on CLRs using the technique by Dejour and Bonnin 8 in a standardized fashion resulted in high inter- as well as intraobserver reliability, underlining the clinical feasibility of this modality for measurement of TS. Micicoi et al 23 advocated that approximately 50% of healthy individuals present bony risk factors for sustaining ACL injuries, including 54% with an increased medial TS and 15% with an increased lateral TS. However, this connection between bony risk factors and actual ACL injury might be oversimplified, as shown by the results of the study and current clinical understanding for ACL injuries. Both groups in the present study revealed only a negligible mean difference, indicating that most patients who have ACL injury will present within normal TS ranges. Differences between groups were especially distinguished at both ends of the TS spectrum, as approximately one-third of ACL-injured and only one-fifth of ACL-intact knees showed a TS ≥12°. This finding is in line with several other studies that proposed this threshold as a risk factor for failure after ACLR.13,20,25,30 Notably, the divide of TS became even more pronounced with increasing TS. Consequently, this subset of patients could be categorized as outliers, as shown in the outlier ratio. Future studies need to delineate whether these patients qualify for TS correction osteotomy, as already advocated.1,7,27,28 Meric et al 22 reported the TS of >13,000 patients measured on CT scans before knee arthroplasty; the investigators defined their outliers to be <4° and >10° and found approximately 30% of cases lying outside this range. Given that the nominal values found on MRI and CT scans are usually 4° to 5° lower than those found on CLRs, this might explain the difference with the current results.17,24 It again becomes evident that the TS describes an acquisition-dependent tendency of the dorsal inclination of the tibial plateau within an equally measured cohort and not a nominal figure. Thus, comparisons between cohorts that are not uniformly measured will introduce considerable inaccuracy and will ultimately prevent firm conclusions. Because conventional radiography is the most common imaging technique used in clinical practice and is conducted in almost every case of suspected ACL injury, the current study was performed to delineate feasibility for assessment of the TS. In contrast to CLRs, cross-sectional imaging allows for discrimination of the medial and lateral TS yet requires specialized software and is more time-consuming. 12 The current results, which we obtained in the largest patient cohort to date using CLRs and the technique by Dejour and Bonnin, 8 showed a high inter- and intraobserver reliability, indicating a high reproducibility for clinical routine. Moreover, mean values for ACL-intact knees in the present study concur with those in the literature, which reports a mean TS of 8.4° to 9.9° for CLRs.4,18,21,29 With respect to the literature, the Dejour and Bonnin technique shows wide application, high reproducibility, and moderate to strong correlation with MRI assessment of the TS.12-15,17,24 Taking the aforementioned into account, it might be reasonable to propose this technique as the standard measurement of TS in the clinical setting. Our study has a few limitations that should be noted. Because of its retrospective design, information on patients in the ACL-intact group was based on the clinical history of our institutional database. Thus, MRI scan with the presence of an intact ACL was not available in all patients. However, patients with a medical history or examination suggestive for ACL injury were excluded. The characteristic differences between groups A and B should be noted, as well as the fact that we could not account for potential disparity regarding body mass index and ethnicity.2,3 In group A, patient age was significantly lower and significantly more male patients were included compared with group B. This might be attributable to the fact that group A represents a younger and more active group of patients who are prone to ACL injury compared with group B, whose radiographs were taken because of undefined trauma or exclusion of degenerative changes. Of note, conflicting results have been reported about the effect of aging (after skeletal maturity up to the presence of severe osteoarthritis) and TS.2,5,11 Patient sex did not have a significant effect on the TS, yet multivariate analysis showed a significant dependence between age and TS. Even if the regression coefficient might indicate that this is clinically negligible, it still must be noted when interpreting the results. As already discussed, another drawback of the current study is the measurement of TS on standard radiographs, thus potentially ignoring a possible asymmetry between the medial and lateral TS. 9 Nevertheless, radiographs have been reported to provide an accurate and objective quantification of the TS as well as high correlation with MRI assessment.12,17 In addition, the clinical use of radiography might be higher than that of MRI. Conclusion The TS was significantly higher in 1000 patients with an ACL-injured knee compared with 1000 patients with an ACL-intact knee. The mean values differed by only 1°, which might be negligible in clinical practice. However, a significantly larger number of outliers were noted in the ACL-injured group exceeding a TS of 12°, which became more distinct with increasing TS and thus qualified as a potential threshold for correction osteotomies. Submitted August 31, 2022; accepted April 19, 2023. The authors declared that they have no conflicts of interest in the authorship and publication of this contribution. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto. ==== Refs References 1 Akoto R Alm L Drenck TC Frings J Krause M Frosch KH . Slope-correction osteotomy with lateral extra-articular tenodesis and revision anterior cruciate ligament reconstruction is highly effective in treating high-grade anterior knee laxity. Am J Sports Med. 2020;48 (14 ):3478-3485.33135908 2 Aljuhani WS Qasim SS Alrasheed A Altwalah J Alsalman MJ . The effect of gender, age, and body mass index on the medial and lateral posterior tibial slopes: a magnetic resonance imaging study. Knee Surg Relat Res. 2021;33 (1 ):12.33832540 3 Bisicchia S Scordo GM Prins J Tudisco C . Do ethnicity and gender influence posterior tibial slope? 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Tibial slope is highly variable in patients undergoing primary total knee arthroplasty: analysis of 13,546 computed tomography scans. J Arthroplasty. 2015;30 (7 ):1228-1232.25795234 23 Micicoi G Jacquet C Khakha R , et al . Femoral and tibial bony risk factors for anterior cruciate ligament injuries are present in more than 50% of healthy individuals. Am J Sports Med. 2021;49 (14 ):3816-3824.34710345 24 Naendrup JH Drouven SF Shaikh HS , et al . High variability of tibial slope measurement methods in daily clinical practice: comparisons between measurements on lateral radiograph, magnetic resonance imaging, and computed tomography. Knee. 2020;27 (3 ):923-929.32061503 25 Salmon LJ Heath E Akrawi H , et al . 20-year outcomes of anterior cruciate ligament reconstruction with hamstring tendon autograft: the catastrophic effect of age and posterior tibial slope. Am J Sports Med. 2018;46 (3 ):531-543.29244525 26 Shrout PE Fleiss JL . 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PMC010xxxxxx/PMC10353029.txt

==== Front Am J Sports Med Am J Sports Med AJS amjsports The American Journal of Sports Medicine 0363-5465 1552-3365 SAGE Publications Sage CA: Los Angeles, CA 35971803 10.1177/03635465221095563 10.1177_03635465221095563 Current Concepts The Use of Intra-articular Platelet-Rich Plasma as a Therapeutic Intervention for Hip Osteoarthritis: A Systematic Review and Meta-analysis Lim Anthony BA * Zhu John B. BA * https://orcid.org/0000-0001-9454-3978 Khanduja Vikas MA (Cantab), MSc, FRCS (Tr&Orth), PhD †‡ * University of Cambridge School of Clinical Medicine, Cambridge, UK † Addenbrooke’s–Cambridge University Hospital, Cambridge, UK Investigation performed at the University of Cambridge School of Clinical Medicine, Cambridge, UK ‡ Vikas Khanduja, MA (Cantab), MSc, FRCS (Tr & Orth), PhD, Young Adult Hip Service, Department of Trauma & Orthopaedics, Addenbrooke’s–Cambridge University Hospital, Box 37, Hills Road, Cambridge, CB2 0QQ, UK (email: vk279@cam.ac.uk). 7 6 2022 7 2023 51 9 24872497 7 11 2021 10 3 2022 © 2022 The Author(s) 2022 American Orthopaedic Society for Sports Medicine https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License (https://creativecommons.org/licenses/by-nc-nd/4.0/) which permits non-commercial use, reproduction and distribution of the work as published without adaptation or alteration, without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage). Background: There is a cohort of patients in whom hip preservation surgery is not indicated, because they have developed signs of early osteoarthritis (OA), and nor can they have a hip replacement, as they are too early in the disease process. Management of this cohort of patients is not standardised and both pharmacological and nonpharmacological measures are utilised to reduce pain. Interventions available for early OA include intra-articular injections of steroids, viscosupplementation and more recently platelet-rich plasma (PRP). However, the use of PRP in hip OA has not yet been studied systematically. Purpose: To assess intra-articular PRP as a therapeutic intervention for hip OA, including the duration of efficacy, influence of dose and composition of PRP, and the incidence of adverse effects. Study Design: A systematic review and meta-analysis; Level of evidence, 4. Methods: We performed literature searches on the MEDLINE, EMBASE, CINAHL, WEB OF SCIENCE, COCHRANE, and SCOPUS databases, and the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines were followed. Data were pooled using random-effects meta-analysis. We assessed the quality of the included studies using the methodological index for non-randomized studies instrument, with an additional assessment for randomized controlled trials with the revised Cochrane risk of bias tool for randomized trials. This is the first study to concisely collate the available data on the use of PRP in hip OA. Results: Eight studies were included in the analysis, with data from a total of 331 patients. PRP significantly reduced pain compared with the baseline at multiple time points, with the greatest effect at the 1- to 2-month follow-up, but PRP significantly improved function only at the 1- to 2-month follow-up. A significantly larger reduction in pain was achieved with a single injection of PRP compared with multiple injections, a total injected dose of PRP <15 mL compared with ≥15 mL, and use of a leukocyte-poor PRP preparation compared with leukocyte-rich PRP. There were no lasting adverse effects. Conclusion: Low- and moderate-quality evidence suggests that PRP reduces pain and improves function at the end-point follow-up of studies compared with the baseline. Moderate-quality evidence suggests that a larger reduction in pain is achieved with a single injection of PRP compared with multiple injections, and low-quality evidence attributes a larger reduction of pain with a total injected dose of PRP <15 mL compared with ≥15 mL and using leukocyte-poor PRP compared with leukocyte-rich PRP. hip joint injection intra-articular osteoarthritis platelet-rich plasma typesetterts1 ==== Body pmcHip osteoarthritis (OA) is a progressive degenerative disease involving the articular cartilage and surrounding structures of the hip, 17 resulting in pain and dysfunction, often beyond the confines of the hip. 13 It is one of the most prevalent disabling musculoskeletal problems, affecting 11% of adults in England. 23 While end-stage hip OA is successfully treated by total hip arthroplasty, 8 the National Institute for Health and Care Excellence (NICE) guidelines include nonpharmacological and pharmacological management for earlier states of OA. 21 One such option is an intra-articular injection of corticosteroid, considered an adjunct to the core treatments and providing immediate, albeit short-term, pain relief. 39 This has led to research into other injectable therapies for hip OA, 12 although these have not yet appeared in routine use and have not been recommended by the NICE guidelines in the United Kingdom. One of the interventions that has gained significant popularity over the past decade is platelet-rich plasma (PRP). PRP is an autologous product derived from whole blood that contains elevated platelet levels, 16 as well as higher concentrations of growth factors, including platelet-derived growth factor (PDGF), transforming growth factor β, and vascular endothelial growth factor. 22 There are several PRP collection protocols and preparation characteristics available from many commercial systems. 9 The main steps involved in the production of PRP are shown in Figure 1. Generally, the production of PRP requires the collection of whole venous blood, which is then mixed with an anticoagulant prior to centrifugation. 18 A single or double centrifugation process is then performed to separate the erythrocytes and concentrate the platelets. The concentrated platelets are found with leukocytes in the “buffy coat,” 5 from which various methods can be used to isolate the platelets with or without leukocytes. The platelets can then be activated with calcium chloride or applied directly without activation. Figure 1. Flowchart of the main steps involved in the production of platelet-rich plasma. PRP can be categorized into several different types based on differences in platelet isolation and activation method, centrifugation speed, and collection systems —and multiple classification systems exist. 25 One important categorization of PRP is into leukocyte-rich or leukocyte-poor PRP, defined as having a leukocyte concentration above or below the baseline, respectively. 15 The presence of leukocytes has been associated with elevated catabolic cytokines, which may partially antagonize the anabolic cytokines contained within platelets. 32 Regardless of the preparation system, PRP universally contains supraphysiological amounts of platelets and growth factors and has been shown to have an overall anti-inflammatory effect and a positive effect on chondrogenesis, 33 indicating its use as a therapeutic intervention for OA. While multiple studies have been performed to look into the use of PRP to treat knee OA, 34 there have been fewer looking specifically at hip OA, recommended to be kept separate from knee OA by the European League Against Rheumatism because of differences in anatomy, development, and treatment applicability. 38 Studies that do focus on hip OA have varying conclusions, with controlled studies typically using a comparator of hyaluronic acid (HA)—itself an emerging injectable. Therefore, the aims of this systematic review and meta-analysis were as follows: (1) Assess the efficacy of intra-articular PRP on patient-reported outcomes for hip OA; (2) determine the duration of efficacy after PRP injections; (3) assess the influence of composition and dosage of PRP on efficacy; and (4) review the incidence of adverse effects from PRP therapy. Methods This systematic review and meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols checklist 27 (see Appendix 1, available in the online version of this article), following advice from the Cochrane Handbook. 11 The study was registered on the PROSPERO database (ID: CRD42021245553). Literature Search We conducted a literature search using MEDLINE (via PUBMED), EMBASE (via OVID), CINAHL (via EBSCOhost), WEB OF SCIENCE, COCHRANE, and SCOPUS databases from database inception to December 2, 2020. No language restrictions were applied, and our search terms were a combination of Medical Subject Headings terms and keywords relating to PRP, OA, and the hip joint (see Appendix 2, available online). The reference lists of included studies were also searched to find additional studies that were not identified by our initial search. We attempted to contact authors of conference abstracts and clinical trials found in the search to include further studies. Study Selection After deduplication, 2 authors (A.L., J.B.Z.) independently screened all potentially eligible studies. We included studies that assessed the use of intra-articular injections of PRP as a stand-alone treatment in the treatment of hip OA in adults that reported a patient-reported outcome at any time point as well as the dosage of PRP used and any treatment complications. We excluded studies that used PRP in augmentation to arthroscopy or as an intraosseous injection, as well as any animal studies or those not published in English. Disagreements were resolved through discussion, and a third reviewer (V.K.) made the final decision in the event no consensus was reached. Data Extraction The following data were extracted from eligible studies: study details—title, authors, publication year, study design, study setting, and inclusion/exclusion criteria; participant information—sample size, mean age, sex ratio, and hip OA severity; intervention—dose, frequency of administration of PRP injections, and information regarding the preparation methods of PRP; the effect size and P values of both primary and secondary outcome measures for patients treated with PRP from baseline to follow-ups, and adverse effects. Where data were not reported, authors were contacted to obtain the relevant information. Quality Assessment The quality of the included studies was assessed independently by the 2 authors using the methodological index for non-randomized studies (MINORS) instrument. 30 Studies were assessed through 12 items (8 items for noncomparative studies), each of which was given a score out of 2 based on whether the item was reported and adequate. In addition, randomized controlled trials (RCTs) were assessed with the revised Cochrane risk of bias tool for randomized trials. 31 This tool included 5 domains, each of which was assessed to determine an item as low (+), high (−), or some concerns (?) over the risk of bias. The overall risk of bias was judged to be low if each domain was assessed as (+), some concerns if at least 1 domain was assessed as (?) but no domains were assessed as (−), and high risk if 2 or more domains were marked as (?), or if 1 or more domains were marked as (−). Risk of bias plots were made using the robvis web app. 19 Any disagreement was discussed and a third reviewer (V.K.) resolved any outstanding conflicts. Data Analysis The results of the studies were analyzed in RStudio Version 1.3.1093 (RStudioTeam, 2020). For continuous outcomes, data were preprocessed to obtain mean ± SD using the estimation by Wan et al, 36 if not already recorded within the original papers. Outcomes were expressed as the mean difference (MD) or the standardized mean difference (SMD) depending on the similarity of the used scales. We used the I2 statistic to measure heterogeneity between studies, with a value <50% representing low heterogeneity and a value >75% indicating high heterogeneity. The outcomes were pooled using a random-effects model. A subgroup analysis was performed to identify any sources of heterogeneity. Results Literature Search A flow diagram of studies found is presented in Figure 2. After reading the full text, 8 studies1,3,4,6,7,26,29,35 were included for the final analysis. Five studies were reported to be RCTs.3,4,6,7,35 All 5 studies compared PRP with HA, and 1 study additionally compared a combined PRP + HA treatment. 4 Three studies were nonrandomized clinical trials. Two studies evaluated PRP in a single patient cohort26,29 and 1 study compared PRP with combination treatments of PRP + cortisone and PRP + cortisone + recombinant IL1R-antagonist 1 as a retrospective nonrandomized intervention study. Authors of 3 studies were contacted to request further information regarding the composition of PRP used, 1 of which responded. Figure 2. Flowchart of the selection process according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement. OA, osteoarthritis; PRP, platelet-rich plasma. Participants Of participants who received PRP treatment alone, a total of 331 patients were analyzed. There were 50.5% men and 49.5% women, with a mean age of 59.8 ± 11.5 years. Patients were included based on both the clinical criteria and a radiological grading system, with 7 of the 8 studies using the Kellgren and Lawrence (K-L) classification system. Of the patients, 19% were grade I, 35% grade II, 28% grade III, and 18% grade IV. Sánchez et al 26 classified hips with the Tönnis scale, with 30% grade 2 and 70% grade 3. Intervention PRP was prepared using a single spin technique in 3 of the 8 studies,1,26,35 with the rest employing a double spin procedure. Most studies involved a total of 3 intra-articular injections at 1-week intervals, with 2 studies29,35 using a single injection, and Baltzer et al 1 reporting 6 injections. A range of 2 to 8 mL of PRP was injected per injection, giving a total dosage ranging from 6 to 24 mL of PRP. Five studies1,6,26,29,35 prepared leukocyte-poor PRP, with the other 33,4,7 using leukocyte-rich PRP. Outcomes All studies assessed pain reduction using a 10-cm visual analog scale (VAS). In addition, 5 studies3,4,7,26,35 recorded the Harris Hip Score (HHS), 54,6,7,26,35 recorded the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and 1 29 recorded patient outcomes using the Hip disability and Osteoarthritis Outcome Score (HOOS). Five studies1,4,26,29,35 reported proportions of responders to treatment, defining responders as those experiencing a reduction in pain intensity >20% to 50%, varying between studies. The total length of follow-ups ranged from 4 to 14 months after the last PRP injection. Quality Assessment The MINORS scores of the included studies are shown in Appendix 3 (available online). Seven of the 8 studies3,4,6,7,26,29,35 included clear aims and outcomes, and the design was prospective in 6 studies.3,4,6,7,26,35 A summary of the risk of bias of the included RCTs is shown in Appendix 4 (available online). Two RCTs7,35 were judged to have some concerns overall, with the other 3 RCTs3,4,6 judged to be at high risk of bias overall. Pain Assessment (VAS) All studies reported VAS scores, with a significant reduction of VAS scores at the endpoint follow-up compared with baseline scores (MD, −1.599 [95% CI, −2.326 to −0.872]; P =.001) (Figure 3). There was significant heterogeneity (χ2 = 28.96 [df = 7]; I2 = 75.8%). Reported VAS scores were grouped into 5 time points after treatment: immediate (1-2 weeks), early (1-2 months), midterm (3-4 months), late (6 months), and long term (12+ months). A statistically significant VAS reduction was seen at early, late, and long-term follow-ups, with significant heterogeneity at all time points except for the early follow-up (χ2 = 4.23 [df = 4]; I2 = 5.4%) (Figure 4). There was a varying effect of OA severity on VAS scores between studies. Three studies4,29,35 found greater improvement with early OA (K-L grades I-II) over severe OA (K-L grades III-IV), with 2 further studies6,7 demonstrating that pain effects were maintained long term in a cohort mainly with early OA, 7 but they disappeared at the 12-month follow-up in a cohort with mainly severe OA. 6 Sánchez et al 26 used the Tönnis scale and found that 10 of the 11 patients who did not respond to treatment (27.5% of patients overall) had Tönnis grade 3 hips. However, 2 studies1,3 found no correlation between the VAS and the K-L grade, with Battaglia et al 3 showing a more profound short-term reduction in the VAS with K-L grade IV OA compared with K-L grade III OA, although these differences were not present by the 12-month follow-up. A short-term effect with severe OA was also noticed by Singh et al, 29 where there was no difference between patients with early or severe OA in terms of the proportion of responders at 12 weeks. Figure 3. A forest plot for the visual analog scale (VAS) scores at the endpoint. IV, inverse variance; PRP, platelet-rich-plasma. Figure 4. A forest plot for the visual analog scale (VAS) scores at immediate (1-2 wk), early (1-2 mo), midterm (3-4 mo), late (6 mo), and long-term (12+mo) follow-up. IV, inverse variance; PRP, platelet-rich-plasma. Function Assessment Seven of the 8 studies3,4,6,7,26,29,35 reported an outcome measure that wholly or partially covered function. SMDs were calculated for WOMAC–function, HOOS–Activities of Daily Living, and HHS scores. There was no significant difference in function scores at the endpoint compared with the baseline (SMD, −1.755 [95% CI, −3.953 to 0.442]; P = .098) (Figure 5). When pooling effects at different time points, a statistically significant improvement in function was only seen at the early follow-up (SMD, −0.784 [95% CI, −1.118 to −0.450]; P = .003), with no significant heterogeneity at this timepoint (χ2 = 4.90 [df = 4]; I2 = 18.4%) (Figure 6). There was limited reporting on function outcomes stratified by initial OA severity, with conflicting findings from studies. Similar to VAS scores, Singh et al 29 found an improvement in function only with K-L grades I-II, with no improvement at any time interval in patients with K-L grades III-IV. However, Battaglia et al 3 found no difference in the temporal variation of the HHS between any K-L grade OA. Figure 5. A forest plot for the function scores at the endpoint. HHS, Harris Hip Score; HOOS–ADL, Hip disability and Osteoarthritis Outcome Score–Activities of Daily Living; IV, inverse variance; PRP, platelet-rich-plasma; SMD, standardized mean difference; WOMAC, Western Ontario and McMaster Universities Osteoarthritis Index. Figure 6. A forest plot for the function scores at immediate (1-2 wk), early (1-2 mo), mid (3-4 mo), late (6 mo), and long-term (12+mo) follow-ups. HHS, Harris Hip Score; HOOS–ADL, Hip disability and Osteoarthritis Outcome Score–Activities of Daily Living; IV, inverse variance; PRP, platelet-rich-plasma; SMD, standardized mean difference; WOMAC, Western Ontario and McMaster Universities Osteoarthritis Index. Effect of PRP Dose on Efficacy Subgroup analyses were performed to investigate the effect of injection number and total dose PRP injected on endpoint VAS scores compared with the baseline. Subgroups were formed based on whether single or multiple PRP injections were administered and whether <15 mL or ≥15 mL of PRP was injected in total, designated as low and high doses, respectively. The test for injection number differences showed a statistically significant quantitative subgroup effect (P = .006), with a greater reduction in the VAS score seen after a single injection (MD, –2.496 [95% CI, –5.377 to 0.384) compared with multiple injections (MD, –1.359 [95% CI, –2.243 to 0.475]) (Figure 7A). In addition, studies that gave a single injection were homogeneous (χ2 = 0.49 [df = 1]; I 2 = 0%), while significant heterogeneity remained among the studies giving multiple injections (χ2 = 18.96 [df = 5]; I2 = 74%). Figure 7. (A) A subgroup analysis for endpoint VAS scores with a single versus multiple injections of PRP. (B) A subgroup analysis for endpoint VAS scores with a high versus low total dose of PRP. IV, inverse variance; PRP, platelet-rich-plasma; VAS, visual analog scale. The results of the subgroup analysis for total dose differences also showed a significant quantitative subgroup effect (P = .029), with a greater reduction in the VAS score after a low total dose (MD, −2.166 [95% CI, −3.693 to −0.639]) compared with a high total dose (MD, −1.081 [95% CI, −1.487 to −0.676]) (Figure 7B). Heterogeneity remained among low dose studies (χ2 = 9.39 [df = 3]; I2 = 68%) but disappeared among high dose studies (χ2 = 1.05 [df = 3]; I2 = 0%). Effect of PRP Composition on Efficacy A subgroup analysis was performed to investigate the effect of PRP composition on endpoint VAS scores compared with the baseline. Subgroup analyses were formed based on whether leukocyte-poor or leukocyte-rich PRP was injected. The test for subgroup differences showed a significant subgroup effect (P = .022), with a greater reduction in VAS scores after leukocyte-poor PRP was administered (MD = −1.959 [95% CI, −3.098 to −0.819]) compared with leukocyte-rich PRP (MD = −0.958 [95% CI, −1.591 to −0.325]) (Figure 8). Heterogeneity disappeared among the 3 studies3,4,7 that used leukocyte-rich PRP (χ2 = 0.55 [df = 2]; I2 = 0%) but remained among studies that used leukocyte-poor PRP (χ2 = 17.52 [df = 4]; I2 = 77%). Figure 8. A subgroup analysis for endpoint visual analog scale (VAS) scores with a leukocyte-rich versus leukocyte-poor platelet-rich-plasma (PRP) preparation. IV, inverse variance. Adverse Effects Seven of the 8 studies1,3,4,6,7,26,35 reported the presence or absence of adverse effects, 4 of which1,4,6,35 did not observe any adverse effects among patients treated with PRP. In the other 3 studies,3,7,26 postinjection pain was reported in patients, which universally was transient and self-limiting. In addition, 2 individual events—1 mild rash and 1 superficial hematoma—were reported, both of which spontaneously resolved without long-term complications. Discussion This systematic review and meta-analysis aimed to assess the efficacy of PRP as a treatment for hip OA, including any effects of posttreatment duration, dosage, and composition on efficacy. The use of stand-alone PRP injections in the hip OA-specific context is an emerging concept, with the present study suggesting that PRP is beneficial and safe for patients, with demonstratable reductions in pain over a few months.20,37 The present study demonstrates the beneficial effect of PRP in a larger group (N = 331 patients), including an RCT conducted after the previous reviews had been published. 35 It utilized a comprehensive, systematic search strategy to identify all the available evidence in the main electronic databases. There was a significant improvement in pain outcomes as measured by the VAS scale at the endpoint compared with the baseline, with a peak improvement at the midterm (3-4 mo) follow-up, although this was not significant. While there was also a trend for functional scores to improve at the endpoint compared with the baseline, this was not significant, with a significant improvement only seen at the early (1-2 mo) follow-up. This is in contrast to the literature on the use of PRP for knee OA, 28 where the function may be significantly improved even at the 12-month follow-up. However, PRP metrics may be crucial in providing this sustained improvement in function, with a potential critical dose needed with knee OA 2 yet to be achieved with hip OA. In addition, patients with hip OA tend to be younger than those with knee OA, 10 and while joint stability matters more in the knee, range of motion is more crucial in the hip. 24 Therefore, there is a need to focus on hip OA as a stand-alone disease when considering the use of PRP, rather than extrapolating based on results with knee OA. Although data reporting was too limited to perform a subgroup analysis stratified by OA severity, findings between studies provided conflicting indications about the effect of OA severity on outcomes. While 2 studies1,3 found no correlation between posttreatment outcomes and initial OA severity, most studies found that patients with early OA (K-L grades I-II or Tönnis grade 2) had a greater beneficial effect on outcomes, which was maintained long term at the 12-month follow-up. Nonetheless, there may still be a short-term benefit of PRP even in patients with severe OA, with 2 studies3,29 finding a relatively greater reduction in pain at short-term follow-up in hips with severe OA compared with those with early OA. Future trials should report outcomes stratified by OA grade to establish whether the efficacy of PRP does differ between OA severity and whether there is a short-term benefit when severe OA is treated with PRP. This study is novel in that it used outcome measures at baseline as the comparator with posttreatment outcomes, unlike previous reviews that use a control group with injected HA. Intra-articular HA injections are a separate emerging treatment for hip OA and do not provide a true gold standard against which PRP can be compared. Few clinical trials have compared PRP injections with control treatments—such as acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroid injections—combined with physical therapy, which form the mainstay of nonoperative hip OA management according to current recommendations. 21 In addition, this is the first study to assess the effect of PRP dosage, injection number, and leukocyte concentration in the management of hip OA. When PRP was given as a single injection, a low total dose (<15 mL), or a leukocyte-poor preparation of PRP, there was a trend for a greater improvement of pain outcomes. Adverse effects in all studies were temporary and self-limiting and did not affect reported outcomes. There are several limitations to this study. This review included 8 articles analyzing 331 patients treated with PRP, reflecting the very small number of trials that exist. Only 2 studies adopted a double-blind approach, which when combined with the use of baseline scores in the meta-analysis, could present potential bias in outcome measurement. In addition, there may be a bias arising from selective reporting of data, as we cannot exclude the possibility of studies failing to find an effect of PRP treatment and thus remaining unpublished. Hip OA characteristically involves inflammation and subsequent degeneration of articular cartilage, 17 with the aim of therapeutic intra-articular injections being to directly suppress the inflammation in the joint space. Trials assessing the efficacy of injected PRP therapy should therefore include an objective outcome measure assessing the direct effect of PRP on the articular cartilage using biomarkers or radiographic OA scoring. None of the included studies reported such a measure during the follow-up period, although 1 study 4 did measure growth factor concentration in the PRP from a subsample of patients. In addition, studies differed on therapeutic management during the follow-up period, varying between prohibiting all anti-inflammatory drug consumption throughout 6 to allowing NSAID consumption from 48 hours after treatment. 3 Reported outcomes could therefore have been influenced by concurrent therapy rather than have been attributed to the PRP injection alone. There was also a large variation in the preparation methods, and reporting thereof, used in producing PRP. This may have contributed to the considerable heterogeneity found, as shown by the removal of heterogeneity in the subgroup analysis of studies where a high total dose of PRP (≥15 mL) was injected, studies that gave a single injection of PRP, or studies that prepared PRP to be leukocyte rich. Future trials should adopt a standard coding system and follow “minimum reporting requirements” 14 when describing PRP preparation to limit this issue. The scarcity of data available on the use of PRP to treat hip OA may have also contributed to the heterogeneity existing between studies. There was significant heterogeneity between studies at the endpoint and all follow-up points apart from the early 1- to 2-month follow-up in both pain and function scores. Therefore, we can only suggest with any confidence a short-term effect of PRP on improvement of outcomes in hip OA, with only weak evidence for a sustained improvement in pain scores. In each subgroup analysis, heterogeneity remained in 1 of the 2 subgroups, suggesting larger numbers of patients are needed to provide stronger evidence of any differential effect of PRP injection number, dosage, or leukocyte presence. The future trial design needs to incorporate the aforementioned limitations into a randomized double-blind controlled trial. A large patient cohort with clinically and radiographically diagnosed unilateral hip OA of differing severity treated with a single injection of a low dose of PRP prepared to a leukocyte-poor protocol should be studied. The control group should use the current gold standard therapy of analgesia and physical therapy alongside a single corticosteroid injection. Total follow-up should be extended beyond 12 months, with assessments at 1 to 2 months, 6 months, 12 months, and 24 months to provide a clearer picture of how long any PRP effects may last. At the baseline and each follow-up appointment, radiographic appearance and inflammatory markers should be assessed alongside patient-reported pain and function scores to ascertain whether efficacy from PRP injections is clinically significant. Conclusion Low- and moderate-quality evidence suggests that PRP reduces pain and improves function in patients with hip OA compared with baseline, with the strongest evidence for an effect at the 1- to 2-month follow-up. Moderate quality evidence suggests that a larger reduction in pain is achieved with a single injection of PRP compared with multiple injections, and low-quality evidence attributes a larger reduction of pain with a total injected dose of PRP <15 mL compared with ≥15 mL, or using a leukocyte-poor PRP preparation compared with leukocyte-rich PRP. Finally, there were no lasting adverse effects associated with PRP injections. Large, methodologically rigorous trials using gold standard control groups should be conducted to test whether PRP injections have a benefit for patients with hip OA over other currently used forms of nonsurgical management. Supplemental Material sj-pdf-1-ajs-10.1177_03635465221095563 – Supplemental material for The Use of Intra-articular Platelet-Rich Plasma as a Therapeutic Intervention for Hip Osteoarthritis: A Systematic Review and Meta-analysis Click here for additional data file. Supplemental material, sj-pdf-1-ajs-10.1177_03635465221095563 for The Use of Intra-articular Platelet-Rich Plasma as a Therapeutic Intervention for Hip Osteoarthritis: A Systematic Review and Meta-analysis by Anthony Lim, John B. Zhu and Vikas Khanduja in The American Journal of Sports Medicine The authors thank Matthew Pettit for his advice regarding the meta-analysis. Submitted November 7, 2021; accepted March 10, 2022. One or more of the authors has declared the following potential conflict of interest or source of funding: V.K. is an educational consultant for Smith & Nephew and Arthrex. V.K. is also on the Executive Board for BHS as President and ESSKA as Chair of EHPA. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto. An online CME course associated with this article is available for 1 AMA PRA Category 1 Credit™ at http://www.sportsmed.org/aossmimis/Members/Education/AJSM_Current_Concepts_Store.aspx. In accordance with the standards of the Accreditation Council for Continuing Medical Education (ACCME), it is the policy of The American Orthopaedic Society for Sports Medicine that authors, editors, and planners disclose to the learners all financial relationships during the past 12 months with any commercial interest (A ‘commercial interest’ is any entity producing, marketing, re-selling, or distributing health care goods or services consumed by, or used on, patients). Any and all disclosures are provided in the online journal CME area which is provided to all participants before they actually take the CME activity. In accordance with AOSSM policy, authors, editors, and planners’ participation in this educational activity will be predicated upon timely submission and review of AOSSM disclosure. Noncompliance will result in an author/editor or planner to be stricken from participating in this CME activity. ORCID iD: Vikas Khanduja https://orcid.org/0000-0001-9454-3978 ==== Refs References 1 Baltzer AWA Ostapczuk MS Stosch D Seidel F Granrath M . A new treatment for hip osteoarthritis: clinical evidence for the efficacy of autologous conditioned serum. Orthop Rev (Pavia). 2013;5 (2 ):59-64. doi:10.4081/or.2013.e13 23888203 2 Bansal H Leon J Pont JL , et al . Platelet-rich plasma (PRP) in osteoarthritis (OA) knee: correct dose critical for long term clinical efficacy. 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==== Front Gerontologist Gerontologist geront The Gerontologist 0016-9013 1758-5341 Oxford University Press US 35705108 10.1093/geront/gnac083 gnac083 Forum AcademicSubjects/SOC02600 RISE: A Conceptual Model of Integrated and Restorative Elder Abuse Intervention Burnes David PhD Factor-Inwentash Faculty of Social Work, University of Toronto, Toronto, Ontario, Canada Connolly Marie-Therese JD Elder Justice Collaborative, Washington, District of Columbia, USA Salvo Erin JD Office of Aging and Disability Services, Maine Department of Health and Human Services, Augusta, Maine, USA Kimball Patricia F MS Elder Abuse Institute of Maine, Brunswick, Maine, USA Rogers Geoff BA Silberman School of Social Work, Hunter College, New York, New York, USA Lewis Stuart MD, FACP Division of Geriatrics and Primary Care, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA Meeks Suzanne PhD, FGSA Decision Editor Address correspondence to: David Burnes, PhD, Factor-Inwentash Faculty of Social Work, University of Toronto, Toronto, Ontario, 246 Bloor Street West, M5S 1V4, Canada. E-mail: david.burnes@utoronto.ca 8 2023 15 6 2022 15 6 2022 63 6 966973 17 2 2022 24 5 2022 14 7 2022 © The Author(s) 2022. Published by Oxford University Press on behalf of The Gerontological Society of America. 2022 https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Abstract Despite a growing number of elder abuse (EA) cases nationwide, response programs such as adult protective services (APS) lack a defined, prolonged intervention phase to address these complex situations. This article presents RISE, a model of EA intervention that works alongside APS or other systems that interact with at-risk older adults. Informed by an ecological-systems perspective and adapting evidence-based modalities from other fields (including motivational interviewing, teaming, restorative justice, and goal attainment scaling), the RISE model intervenes at levels of the individual older adult victim, individual harmer, their relationship, and community to address EA risk and strengthen systems of support surrounding the victim-harmer dyad. The RISE model addresses an intervention gap in existing systems to better meet the needs of EA victims and others in their lives, leading to more sustainable outcomes. Ecological-systems Intervention Mistreatment Model U.S. Department of Health and Human Services 10.13039/100000016 90EJSG0031-01-00 ==== Body pmcElder abuse (EA) is recognized by clinicians, policymakers, and researchers as a pervasive problem with serious consequences. EA refers to an intentional act or lack of action by a person in a relationship of trust that causes harm or risk of harm to an older adult; its subtypes include financial, emotional/psychological, physical, sexual abuse, and neglect by others (National Research Council, 2003). Approximately 10% of older adults in the United States aged 60 years and older living in the community experience some form of EA each year (Pillemer et al., 2016). Another 30 million “concerned others” suffer significant distress simply because they know or are trying to help an older adult being mistreated (Breckman et al., 2018). EA is associated with detrimental consequences, including premature mortality, poor physical and mental health, and increased health care utilization (Yunus et al., 2019). In the absence of effective prevention strategies, EA cases are projected to grow as the population ages. Little evidence exists to inform initiatives to prevent EA, intervene effectively, and better the lives of those who are mistreated (Pillemer et al., 2016). Adult protective services (APS), the primary public agency responsible for receiving/responding to reports of alleged EA, consists of state and county agencies, governed by state-specific laws and procedures. In 2019, APS received over 1.3 million reports of alleged maltreatment of which it investigated and substantiated approximately 260,000 allegations and provided some type of service in nearly 130,000 cases (National Adult Maltreatment Reporting System [NAMRS], 2019). Trends suggest that reports to APS and APS caseloads are rising (NAMRS, 2019). Yet, outcomes of modern APS programs have not been examined in rigorous client-centered ways, exposing EA victims to a system that lacks evidence of effectiveness (Ernst et al., 2014). Several factors complicate EA intervention research. Interventions lack clear definitions of success and rarely identify or measure metrics of success, particularly from the perspective of older adults themselves (Burnes et al., 2018). EA often arises in the context of complex familial relationships, yet most interventions focus only on victims, despite victims’ wishes to also obtain support for the alleged harmer or address harms within the victim-harmer relationship (Burnes et al., 2021). Most APS interventions are, by terms set forth in law/protocol, limited and brief, usually lasting only the time it takes to complete an investigation or implement a crisis response. When services are offered, many clients are reluctant or decline to engage them (Burnes, 2017). Some EA victims live with cognitive impairment, but interventions often lack sufficient methods to discern their wishes and support their decisions. Finally, social service agencies may lack the infrastructure and/or resources to do high-quality intervention research. The current APS practice model typically involves three phases: intake, investigation, and substantiation. Nationwide, cases remain open, on average, for 65 days with the investigation phase lasting 53 days (NAMRS, 2019). Though some APS programs provide services during and/or after the investigation to meet the immediate safety needs of clients, the current APS system generally lacks a dedicated, defined, conceptually driven, and evidenced based intervention phase to support EA cases. Most substantiated cases are closed after APS provides brief crisis intervention supports/services and/or referrals to external entities (Liu et al., 2022; e.g., home care, visiting nurse, criminal justice, and aging network services). Guidelines from the Administration for Community Living (ACL; 2016) and National APS Association Education Committee (NAPSA-EC, 2013) recommend that an effective APS model should include distinct service planning and intervention phases for substantiated cases. The “RISE” model, described here, is a novel EA response intervention and the cornerstone of an ongoing state-wide study of a partnership between APS and a community-based organization. The name, RISE, was chosen because the intervention model operates at Relational, Individual, Social, and Environmental levels. While there is not a direct correspondence, it also reflects the overall goals and core components of the model: Repair Harm (restorative approaches), Inspire Change (motivational interviewing), Support Connection (teaming), and Empower Choice (supported decision-making). RISE was built from the ground up as a stakeholder-driven, conceptually based, advocacy-framed model of community-based EA intervention to address a broader range of victims’ needs and wishes and existing systems’ structural limitations. RISE was designed to focus on an older adult victim/client’s expressed goals and needs, including support for the harmer and/or the victim-harmer relationship if the client wishes. RISE complements existing APS services, working simultaneously or sequentially, depending on the case. RISE emphasizes a restorative not retributive approach to individuals and relationships that integrates four modalities (described later) with evidence in other contexts that have been adapted to EA. Critically, RISE allows for a longer intervention phase than most existing systems to build relationships and trust with clients and is, therefore, hypothesized to result in more sustainable outcomes. Conceptual Framework Guiding RISE The ecological-systems framework, a dominant perspective to understand EA victimization risk, captures the complex etiology of EA by recognizing that risk is a function of factors situated at different levels of ecological-systemic organization. Drawing on this perspective, the seminal EA Theoretical Risk Framework proposed by the National Research Council (2003) conceptualizes indicators of EA risk as attached to several eco-systemic levels, including the individual victim, the individual harmer, the relationship between them, the formal and informal social support for the victim and harmer, the surrounding systems, and socio-cultural context. Similarly, the abuse intervention model (AIM) conceptualizes risk factors as attached to the individual victim, the harmer, as well as the surrounding context (e.g., victim-harmer relationship, social, and culture; Mosqueda et al., 2016). This eco-systemic-informed intervention theory suggests that effective EA intervention models should have the capacity to work with both older adult victims and alleged harmers, as well as to strengthen relevant relationships and social supports surrounding them individually and as a dyad to address the full scope of risk factors characterizing a given case (Burnes, 2017; Liu et al., 2019; Mosqueda et al., 2016). EA intervention theory and APS best practices also recommend that intervention models be person-centered (ACL, 2016; Burnes, 2017; NAPSA-EC, 2013). Service plan development should involve collaboration with clients to understand their unique concerns and circumstances, their understanding of the problem, and to construct a person-centered, self-determined intervention. Cultural sensitivity is essential to person-centeredness as victims may have differing beliefs and experiences of what constitutes EA and appropriate intervention based on their intersection with various socio-cultural identities, including culture, race, ethnicity, indigeneity, economic and citizenship status, age, ability, neurodiversity, gender identity and expression, sexuality, geographical context, and religion/faith. A culturally sensitive, person-centered approach is particularly important in working with communities that have been systemically marginalized. EA victimization risk is also highly contextualized and uniquely constellated across eco-systemic levels; each EA case requires a tailored combination of service plan interventions to address clients’ needs and achieve their preferred resolution outcomes (Burnes et al., 2016). EA interventions must recognize older adults’ right to make their own choices; that they may have numerous other concerns; and that “success” of outcomes should be primarily defined by victims, not professionals (ACL, 2016). Indeed, working to support older adults’ self-determination is often crucial to building relationships with EA victims and, for many, is a threshold for accepting help. RISE Model Development The need for an enhanced model of EA response intervention became evident in a pilot study that investigated the feasibility and implementation of goal attainment scaling (GAS) with EA cases handled by Maine’s APS program (Burnes et al., 2018). GAS is a collaborative, client-centered treatment planning and measurement strategy that tracks changes in goal attainment over the course of an intervention (Turner-Stokes, 2009). During this study, some clients’ goals included getting support for alleged harmers, often family members (e.g., mental health, substance use, and housing). Consultations with APS caseworkers and supervisors involved in the study suggested that responsive systems could be strengthened if the intervention phase was less time-limited and allowed for direct work not only with victims but also with alleged harmers (Burnes et al., 2018). The earler findings emerging from the GAS pilot study prompted a meeting in 2017 with key stakeholders in Maine, as well as EA researchers and policy experts nationwide, to explore and solicit input about a potential enhanced EA response intervention model. The stakeholder meeting included Maine’s Department of Health and Human Services (DHHS) Commissioner, representatives from numerous DHHS offices, APS director, supervisors, caseworkers, and experts working with other governmental, academic, or nonprofit entities. That meeting was followed by additional meetings with a broad range of stakeholders whose knowledge intersects with EA intervention or whose expertise could inform model development, for example, mental health, substance use, employment counseling, caregiver support, individual and family therapy, financial services, legal services, aging network services, health care, long-term care, ombudsman, law enforcement, prosecution, homecare, child welfare, domestic violence, sexual assault, transitional housing, and community-based victim advocacy organizations. Those stakeholders’ input was instrumental to the development of RISE and reinforced the need for a longer, and more holistic EA intervention phase than the current APS structure and response system allows for. That input also informed numerous discussions about how best to approach model development and pilot testing. The RISE Model RISE (Figure 1) is a client-centered model of EA intervention that integrates and adapts intervention components from other relevant fields. “RISE Advocates” implement the model, engaging and building relationships with clients and working collaboratively with them using the key processes and model core components below to achieve client-driven goals. RISE Advocates typically enter the role with an educational background in social work, which provides clinical training to engage with RISE model processes/components and practice with vulnerable populations around the issue of family/interpersonal violence. Figure 1. A conceptual model of integrated and restorative elder abuse intervention. Key Model Processes Engagement and goal setting are constantly in action throughout a case, representing critical, fluid dimensions of RISE, and hypothesized as contributing to positive case outcomes. Engagement Client engagement represents a challenge in any EA intervention. Only 15% of EA victims living in the community report their mistreatment to formal systems (Burnes, Acierno et al., 2019). Many who do interface with formal systems decline supports/services offered (Ernst et al., 2014; Rizzo et al., 2015). Reluctance to engage with APS may be due to stigma, distrust of government, marginalization, fears about what might happen to themselves (e.g., loss of autonomy or caregivers and nursing home placement), fears about consequences for harmers (e.g., prosecution and incarceration), or fears of severing (family) relationships (Burnes, 2017). Offering an engagement model that clients believe will respond to their needs is central to developing a successful EA intervention model. The broader social services literature demonstrates that the quality of the client/practitioner relationship predicts retention and successful case outcomes when working with reluctant or vulnerable populations (Dearing et al., 2005; Yatchmenoff, 2005). Advocates receive training around developing a client-centered practice philosophy/orientation and on a specific practice technique called creative engagement. This technique emphasizes enhanced listening, observation, and improvisation/adaptation skills (Fritsch et al., 2009) to foster skills of engagement and relationship building with clients. Given some clients’ concerns and fears of engaging with a government-based service system, the RISE Advocate role is intentionally housed within and administered through a community-based organization. This placement also gives RISE Advocates more flexibility than APS, lengthening engagement with clients where indicated, and expanding the tools available to them to engage clients and develop trusting, collaborative working relationships that, in turn, facilitate clients’ openness to setting goals and accepting support. The RISE model’s core components described later (e.g., motivational interviewing) were selected specifically and in large part, because they are known to facilitate ingredients to promote client engagement and to develop strong client-practitioner relationships, including collaboration and coconstruction of meaningful goals, expectations, and tasks (Horvath & Greenberg, 1989). Goal-setting Formal goal setting is an effective way of facilitating behavioral change (Locke & Latham, 2002) and has the potential to facilitate increased self-efficacy through enactive attainment or self-witnessed progress along with clear, measurable goal attainment (Bandura, 1986). GAS is a feasible goal-setting and measurement strategy to use in the context of EA intervention (Burnes et al., 2018). GAS is a client-centered goal-setting procedure that tracks changes in goal attainment over the course of an intervention and has been used successfully with older adults experiencing complex, heterogeneous problems other than EA (Turner-Stokes, 2009). GAS is a formal and systematic goal-setting process that requires the client and practitioner to collaboratively identify mutually understood goals, tasks, and expectations. RISE Advocates use GAS to track progress toward person-centered case goals. GAS guides the advocate-client dyad through a process to understand varying expectations of success on each goal. As the RISE intervention moves through the various core components described later, goals can be set that are attached to the victim, harmer, victim-harmer relationship, as well the informal and formal supports in the surrounding social support systems. Model Core Components The RISE model core components in this section are described in the order that they most commonly play out in practice and that broadly follow the eco-systemic perspective conceptualizing the work (e.g., starting with individual-level practice). As a note, however, the core components may not operate in a linear sequence, but rather in an iterative/recursive manner, separately or concurrently depending on client preference and case progression. RISE Advocates receive specialized training from experts on each of these four core components and in adapting them in EA cases. Motivational interviewing Motivational interviewing is a collaborative, person-centered, goal-oriented modality designed to elicit change with clients who are experiencing ambivalence in relation to a specific problem. It acknowledges that clients enter an intervention with varying levels of receptiveness for change (Rollnick & Miller, 1995). Through raising awareness about the challenges caused by, consequences of, and risks they face with an ongoing problem, clients will consider the benefits of a change in their situation (Cummings et al., 2009). For example, motivational interviewing can mitigate the impact of trauma that makes change difficult, thereby facilitating an older adult’s readiness to access medical care, transition to new environments, or accept in-home care. Advocates help clients (and sometimes harmers) explore their own motivation for change. Throughout a motivational interviewing process, the client-practitioner dyad builds on a mutual understanding of treatment goals and tasks that are desired by the client to reach those goals (Miller & Rollnick, 2002). A motivational interviewing approach aligns with RISE’s person-centered orientation and targets older victims’ ambivalence about making decisions to address their mistreatment. Motivational interviewing has also shown evidence to reduce EA revictimization risk and advance intervention goals (Mariam et al., 2015). Supported decision-making Person-centered goal setting with clients who have cognitive decline follows a three-tiered protocol in the RISE model depending on the client’s cognitive ability to participate in goal setting and the availability of a trusted third-party substitute decision-maker. Tier 1 involves clients who continue to have the cognitive ability to express needs, goals, and service plan preferences. In these cases, clients are directly involved in goal setting using supported decision-making principles. APS guidelines suggest that supported decision-making should be used when clients in cognitive decline maintain decision-making abilities around their preferences (ACL, 2016; NAPSA-EC, 2013). Although there is no data about using supported decision-making in the context of APS or EA, from a practice perspective, supported decision-making entails a process through which older adults receive assistance from RISE Advocates to understand the mistreatment situation and the choices to be made to make their own service planning decisions without a substitute decision-maker. A supported decision-making strategy is intended to discern older people’s wishes, respect their right to self-determination, and align with the person-centered practice orientation central to RISE (National Council on Disability, 2018). Tier 2 involves clients who, in general, lack the cognitive ability to participate in a goal-setting process. Attempts will be made to work directly with the older adult. However, the RISE Advocate will consult with trusted others and/or authorized substitute decision-makers, such as powers of attorney or guardians, to set goals if further support is needed. Tier 3 involves clients who lack both the ability to participate in goal setting and a substitute decision-maker. The handling of these situations differs depending on state law and practice. In states such as Maine, where APS serves as the guardian of last resort (i.e., public guardian), goal-setting occurs collaboratively between the RISE Advocate and APS guardianship caseworker with input from appropriate others. Teaming Teaming is a collaborative model proposing that more favorable and sustainable outcomes occur when interventions engage the shared commitment and responsibility of a collective of formal and informal supporters (Snyder et al., 2012). Social support is consistently associated with lower rates of EA (Pillemer et al., 2016), thus, RISE’s teaming component aims to activate this protective factor. Teaming can strengthen family, concerned other, and social support networks surrounding the victim and, if victims desire, the accused harmer, and integrate the harmer into the intervention phase. Facilitated by the RISE Advocate, the teaming process identifies and gathers relevant informal (e.g., family members, friends, and community members) and formal (e.g., APS caseworkers, law enforcement, aging network services, and health professionals) stakeholders who agree to share responsibility in supporting the victim (and/or accused harmer) toward sustained revictimization risk reduction, even after the case has closed (Epstein et al., 2003). The informal/formal teaming members are assigned specific tasks to support the victim (e.g., weekly check-ins, transportation, and service referral) or accused harmer (e.g., counseling, anger management, check-ins, and caregiver respite), addressing specific risk factors present in the case (Epstein et al., 2003). In turn, the teaming process generates teaming-level goals that can be integrated into the client’s service plan. The client’s team meets regularly to address challenges as they arise and to hold teaming members accountable. Teaming is designed to facilitate a comprehensive mode of intervention and build victim/harmer support systems toward longer-lasting outcomes. The teaming approach aligns with EA research findings that stronger victim social support networks facilitate help-seeking behavior (Burnes, Breckman et al., 2019) and favorable long-term health and psychosocial outcomes (Acierno et al., 2017). Restorative justice approaches In RISE, EA victims can choose to include the accused harmer in the intervention process. Interventions involving accused harmers integrate restorative justice principles to help repair the victim-harmer relationship and initiate harmer-specific goals in the service plan. Restorative justice principles focus on repairing and making amends for harm caused in a way that is meaningful to and agreed upon by the victim, and that focuses on transformation, healing, and reintegration of the parties into their community. EA victims are given the opportunity to talk about how they were affected and share their needs and wishes, at least with the Advocate, and if the client wishes, more broadly. Another goal of restorative justice is to hold harmers accountable in ways more likely to produce positive results for both parties (Beck et al., 2015). Restorative justice approaches often result in agreed upon restorative action plans, including actions harmers will take to repair harms and prevent further mistreatment (Beck et al., 2015; Groh, 2010). Sometimes restorative approaches involve working with the victim and the harmer separately. Other times, they provide a process to intervene at the level of the victim–harmer relationship. The restorative process can also integrate services not just for the victim but also for the accused harmer. Certain cases, such as where the accused harmer is unwilling to engage, may not be appropriate for restorative justice. Also, certain conventional restorative justice elements, such as a “circle” or “conference,” may not be appropriate/feasible in some cases. For example, harmful victim-perpetrator power dynamics or changing life circumstances (i.e., health/cognitive stratus and instabilities) may preclude participation. Prevention The RISE model was designed to work with both substantiated EA cases and to serve a preventive role in at-risk or subthreshold (or “unsubstantiated”) cases. We recognize the importance of working with people in cases that do not necessarily meet threshold substantiation criteria yet may present indications of risk with the potential for escalation without proper supports/services in place. While service system resource limitations usually preclude a focus on such prevention, and little prevention research exists to inform such efforts, the RISE model has the capacity to work with subthreshold cases using the key processes and core components described earlier. RISE/APS Partnership Structurally, the APS/RISE arrangement is viewed as a complementary, cross-sector partnership between a state-government agency and a community-based nonprofit organization. As explained earlier, RISE Advocates are intentionally housed within a community-based organization external to APS to facilitate greater client engagement and to allow more freedom in providing longer, more flexible support. The community-based organization should have a focus on aging issues, a service/practice-oriented mission, and be embedded within a system/network that understands the resources available to support older adults. Both parties are involved with the EA case and benefit from one another. The APS worker completes the investigation and substantiation phases of case work and may facilitate certain state-sanctioned processes (e.g., guardianship, application for certain benefits, and medical evaluations), while the Advocate is responsible for implementing the direct practice key processes and core components of the RISE intervention model. Depending on the case, the APS worker makes a referral to the RISE Advocate during or following the APS investigation phase and shares pertinent information with the RISE Advocate. Conclusion RISE represents an innovative, reproducible, and testable framework for EA intervention that could make a difference to individuals and families. It is grounded in theory, advocacy, and relationship building, guided by clients’ wishes, and responsive to clients’ needs. It was designed with the flexibility to work with both the victim and accused harmer and to strengthen support surrounding them as individuals and as a dyad. The integrated adaptation of modalities demonstrating evidence in other fields―engagement, GAS, motivational interviewing, teaming, and restorative justice―to EA represents a new, hopeful approach. The modalities, combined with longer intervention phases than those routinely offered by existing systems, are hypothesized to result in more favorable and sustainable outcomes. Aligning with the conceptual orientation of the RISE model and the objectives of each core component, several key outcomes can be targeted by the model. These outcomes include revictimization risk reduction (across various levels of eco-systemic influence), self-determination and empowerment (client-centeredness, supported decision-making), self-efficacy and goal attainment (GAS), ambivalence and readiness for change (motivational interviewing), social support and connectedness (teaming), and relationship quality/repair and perpetrator accountability (restorative justice). Broader outcomes relate to client safety, well-being, and mistreatment severity, which are targeted by the model as a whole. APS is a natural partner for RISE given the central role APS plays responding to EA nationwide. However, RISE could also be adapted to augment other systems that interact with at-risk or victimized older adults, such as health care, legal or aging network services. RISE also has the potential to augment or provide alternative options to EA cases in the legal system. This could be accomplished by giving law enforcement, prosecutors, judges, financial enforcement and/or those administering guardianships the option to refer cases to RISE, in lieu of or in addition to legal intervention. RISE may serve as a useful stand-alone, community-based EA intervention model, including in countries without an APS system. Finally, although RISE has been conceptualized to fit within a community-based organization, it may be possible to house RISE within other systems, including health care or aging network settings that work with older adults. Further research is needed to understand the feasibility and implementation of RISE in working with real-world EA scenarios alongside APS or other systems. Rigorous intervention research is required to determine the effect of RISE on key case outcomes, including those outlined earlier, the intervention mechanisms of change, and for whom and under what circumstances RISE demonstrates effects. The research process also requires reflexivity in response to stakeholder feedback and evaluation data to identify RISE model limitations or gaps. Potential gaps to consider at this juncture include a more explicit integration of evidence-based trauma-informed practice and a greater understanding of adapting RISE with clients across various intersecting socio-cultural identities. In summary, the RISE model could be a critical and needed addition to society’s response to EA if, as hypothesized, it helps fill gaps in existing systems and better meets the needs and wishes of EA victims and others in their lives. Acknowledgments We wish to thank all of the investigators, supervisors, staff, and leadership at Adult Protective Services in Maine, as well as the Advocates, staff, and leadership at the Elder Abuse Institute of Maine, who dedicated so much time and effort to making this project possible. We would like to thank the U.S. Department of Health & Human Services, Administration for Community Living for funding and supporting this project. Thank you to the Elder Justice Foundation for supporting the initial GAS work precursing the current article. Thank you, Andie MacNeil, for your ongoing research efforts on this RISE project. We also would like to thank members of the national research and practice advisory groups whose input helped to inform the work. Please note that the current manuscript is a Forum piece without data and, therefore, the replicability, availability, and accessibility of data, analytic methods and other materials is not relevant. Similarly, the information reported in this manuscript was not preregistered. Funding This work was supported by the U.S. Department of Health and Human Services, Administration for Community Living (90EJSG0031-01-00). Conflict of Interest None declared. ==== Refs References Acierno, R., Hernandez-Tejada, M. A., Anetzberger, G. J., Loew, D., & Muzzy, W. (2017). The National Elder Mistreatment Study: An 8-year longitudinal study of outcomes. 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==== Front Gerontologist Gerontologist geront The Gerontologist 0016-9013 1758-5341 Oxford University Press US 36544399 10.1093/geront/gnac179 gnac179 Diversity and Equity in ADRD Research AcademicSubjects/SOC02600 Mechanisms by Which Cultural-Centric Narrative Influences Interest in ADRD Research Among African American Adults Lingler Jennifer H PhD, MA, CRNP, FAAN School of Nursing, University of Pittsburgh, Pittsburgh, Pennsylvania, USA University of Pittsburgh Alzheimer’s Disease Research Center, Pittsburgh, Pennsylvania, USA Ren Dianxu PhD School of Nursing, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Tamres Lisa K MS School of Nursing, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Knox Melissa L BS School of Nursing, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Mbawuike Uchenna BS, MPPM ZemiTek, LLC, Bethesda, Maryland, USA https://orcid.org/0000-0002-8329-9225 Williams Ishan C BA, PhD School of Nursing, University of Virginia, Charlottesville, Virginia, USA Robinson Renã A S BS, PhD Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA Department of Neurology, Vanderbilt University Medical Center, Nashville, Tennessee, USA Cameron Judy L PhD Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Terry Melita H BS University of Pittsburgh Alzheimer’s Disease Research Center, Pittsburgh, Pennsylvania, USA Garrett Marita BS, MA Civically, Inc., Pittsburgh, Pennsylvania, USA Graduate School of Public & International Affairs, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Meeks Suzanne PhD, FGSA Decision Editor Address correspondence to: Jennifer H. Lingler, PhD, MA, CRNP, FAAN, Department of Health and Community Systems, School of Nursing, University of Pittsburgh, Pittsburgh, PA 15261, USA. E-mail: linglerj@pitt.edu 8 2023 21 12 2022 21 12 2022 63 6 10601066 20 9 2022 09 11 2022 09 2 2023 © The Author(s) 2022. Published by Oxford University Press on behalf of The Gerontological Society of America. 2022 https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Abstract Background and Objectives Insufficient ethnoracial diversity is a pervasive challenge in Alzheimer’s disease (AD) research. The Recruitment Innovations for Diversity Enhancement (RIDE) is grounded in the premise that culturally informed narratives of research participation can inspire individuals from a given culture-sharing group to consider research enrollment. This study examines factors associated with interest in AD research among Black or African American adults following exposure to RIDE narrative campaign materials. Research Design and Methods A community-based sample of 500 Black or African American adults viewed RIDE narrative materials online and completed a survey of perceptions about research, AD risk, and likelihood of enrolling in AD research. Logistic regression examined predictors and mediators of self-reported likelihood of participating in AD research. Results Most (72%) participants reported interest in being contacted for AD research opportunities. After controlling for key variables, prior experience with clinical research and trust in medical researchers emerged as independent predictors of likelihood of enrolling in AD research. Perceived burden of AD research partially mediated the effects of prior research experience and trust on likelihood of enrollment. Perceived benefits of AD research also played a mediating role, accounting for over one third of the effect of trust on likelihood of enrollment. Discussion and Implications This study advances the field’s understanding of how narrative may function to enhance diversity in AD research. Findings suggest that participant narratives should address experiences regarding the burdens and potential benefits of AD research participation as these factors may influence decisions leading to subsequent research enrollment. Alzheimer’s disease Health equity Narrative medicine Recruitment National Institutes of Health 10.13039/100000002 R01AG054518 P30AG066468 UL1 TR001857 ==== Body pmcInsufficient enrollment of persons from diverse racial and ethnic backgrounds is a pervasive challenge in clinical research on Alzheimer’s disease and related disorders (ADRD), with the overwhelming majority of participants in both observational and intervention research identifying as non-Hispanic White (Canevelli et al., 2019, Gilmore-Bykovskyi et al., 2019). The underrepresentation of individuals from communities of color in ADRD research is problematic at two broad levels. First, there is a fundamental need for racially and ethnically representative research samples to ensure that findings from studies are applicable to the general population of persons affected by ADRD (Barnes 2019; Portacolone et al., 2020). Second, adequate enrollment of persons from historically underrepresented and minoritized groups is essential for understanding and ultimately combating the cognitive health disparities, including those noted between African American and non-Hispanic White older adults (Lin et al., 2022; Sluder 2020; Suran 2022; Zhou et al., 2016). Reasons underlying such underrepresentation are complex. Indeed, empirical evidence demonstrates that while persons from racial and ethnic minority communities experience less awareness of and access to research, and face multilevel barriers and limited facilitators to participating, overall, they are no less willing to participate in clinical research than nonminority groups, even in the face of well-documented historical mistrust of the clinical research institution (Bardach et al., 2021). Calls to increase representation in ADRD research have grown dramatically in recent years, beginning with the prioritization of diversity and inclusion in the National Alzheimer’s Project Act agenda (Nye et al., 2022). These calls were renewed through a series of advocacy initiatives that followed George Floyd’s killing and the ensuing spotlight on how systemic racism extends to virtually every dimension of American life, including clinical research (Parker et al., 2022). Most recently, advocates for diversity in ADRD research have pointed to the gross underrepresentation of persons of color in the clinical trials leading to the Food and Drug Administration approval of aducanumab, the field’s first disease-modifying treatment for Alzheimer’s disease (AD). Less than 1% of over 3,000 combined participants in the aducanumab trials identified as Black or African American and only 3% identified as Hispanic (including those of European Spanish descent; Manly & Glymour, 2021). Recognizing the need for data on the efficacy of disease-modifying therapies for AD in a representative population, the Centers for Medicare and Medicaid Services (CMS) has taken the unprecedented action of imposing a requirement that any studies supplying aducanumab under the CMS coverage with evidence development provision must, by protocol, include “A study population whose diversity of patients are representative of the national population with MCI due to AD or mild AD dementia” (Medicare Coverage Database, 2022). Meeting such a requirement and responding to similar charges going forward will require substantial resources and innovative approaches to addressing the wide range of barriers to recruiting participants of color into ADRD research (Pugh et al., 2022). As current efforts to identify and disseminate effective strategies for community engagement and recruitment are accelerated and new efforts begin, there will be a concomitant need to understand relationships among key variables influencing research enrollment and to deepen the field’s understanding of why, how, and under what circumstances various strategies for enhancing diversity in research are effective. The current report represents an early step in this direction. Our objective was to examine pathways by which a newly developed story-telling approach may promote enrollment in ADRD research among African American adults. The Recruitment Innovations for Diversity Enhancement (RIDE) in AD Research study is grounded in the premise that culturally informed narratives of research participation can inspire other individuals from a given culture-sharing group (in this case, African American adults) to consider enrolling in research (Robinson et al., 2020). RIDE involved developing and disseminating narrative materials to share the research participation experiences of African American adults who are enrolled in ADRD research with those who are not. As previously described (Robinson et al., 2020), RIDE is guided by the model put forth by Larkey and Hecht (2010), which posits that sharing personally engaging and culturally congruent stories of engagement in health behaviors can positively influence health-related attitudes, beliefs, and behaviors among individuals within a culture-sharing group or community. In the context of clinical research, such attitudes and behaviors can be operationalized, respectively, as receptiveness to and participation in research studies. When applying this model to the research context, there is also a critical need to account for the role of known correlates of study enrollment, including trust (Corbie-Smith et al., 2002; Li et al., 2022; Scharff et al., 2010), and individuals’ perceptions of the risk to benefit ratio of research participation (Cox et al., 2019; Hughes et al., 2017). The current analysis aims to examine how such factors are associated with interest in ADRD research following exposure to RIDE narrative campaign materials. Method Design, Sample, and Setting This prospective survey study was part of a larger investigation of RIDE in AD Research (AG054518). Five hundred community members were recruited using web-based advertising on social media, community partner e-mail lists (including contacts at traditionally African American churches, sororities, fraternities, and community groups), through directed mailing service of individuals in the local community, and through Pitt+Me, an online database managed by the University of Pittsburgh Clinical and Translational Science Institute (UL1 TR001857). Inclusion criteria were: (a) self-identification as Black or African American; (b) residence within one of the seven counties nearest to the University; (c) age 18 years or older; and (d) able to read English. Exclusion criteria were: (a) current or previous participation at an Alzheimer’s Disease Research Center (ADRC), and (b) residence in a long-term care facility or group care setting. Procedures This study received Institutional Review Board approval (IRB #2003004). All participants provided electronic informed consent via the Qualtrics platform. Upon provision of e-consent, participants were sent a link to a separate Qualtrics survey battery. As depicted in Figure 1, the survey battery consisted of assessments to be completed both before and following the viewing of an ~2-min video telling the research participation story of an African American adult. Each participant viewed one of two videos featuring either a male or female participant. The male and female versions of the video were shared with equal frequency based on a randomization scheme that was programmed into Qualtrics. Because the videos were produced before the coronavirus disease 2019 (COVID-19) pandemic, but shown during the pandemic, a disclaimer was added stating that videos were recorded prepandemic and do not depict the COVID mitigation procedures (e.g., masking and social distancing) that have been adopted in research settings. Figure 1. Procedure for survey data collection. AD = Alzheimer’s disease. Prior to viewing the video, participants completed a basic demographic questionnaire (Sereika & Engberg, 2006), subjective memory assessment (Zelinski & Gilewski, 2004), rating of perceived risk of developing AD (Chung et al., 2009), and the Trust in Medical Researchers questionnaire (Hall et al., 2006). Participants also rated, on a scale of 1–5, how worried they currently were that they or their loved one would get COVID-19 (or, if previously infected, worried about getting it again). After viewing the video, participants rated their level of interest in learning about AD research (5-point Likert scale from Strongly Agree to Strongly Disagree), how likely they are to agree to participate in an AD research study (4-point Likert scale from Not Likely to Extremely Likely; Lingler et al., 2010), and indicated whether or not they would like to be contacted by someone from the ADRC to learn more about participating in AD research (yes/no). Lastly, participants completed an 11-item assessment of motivations for participation in AD research (Agarwal et al., 2007) and the 21-item Perceived Research Burden Assessment (Lingler et al., 2014). Participants were also asked whether their interest in AD research participation is for themselves or for a loved one. Those endorsing the latter were provided with assessment versions including items that were adapted to apply to AD research study partners. Data Analysis Descriptive analyses were conducted including frequency and percentages for categorical variables, mean and standard deviation for continuous variables. Mediation analyses were performed to explicate the association of prior experience with clinical research and trust in medical researchers with intention to enroll as potentially mediated by the variables of perceived benefits/burden of research participation (Figure 2). A parametric regression approach (PROC CAUSALMED procedure in SAS version 9.4) was used to estimate the total effect, the natural indirect effects (NIE), and natural direct effects (NDE) of prior experience with clinical research/trust in medical researchers on intention to enroll (Yung et al., 2018). Two models were estimated: a multivariate linear regression model for perceived burden/benefits (mediator) conditional on prior experience with clinical research/trust in medical researchers (exposure) and all study confounders and a multivariate logistic regression model for the likelihood of enrolling (outcome) conditional on prior experience with clinical research/trust in medical researchers, perceived burden/benefits, and all study confounders. The NDE represented the effect of prior experience with clinical research/trust in medical researchers on likelihood of enrolling that was independent of perceived burden/benefits. An NIE represented the proportion of prior experience with clinical research/trust in medical researchers that could be explained by its association with perceived burden/benefits. To quantify the magnitude of mediation, the study estimated the proportion of the association mediated by perceived burden/benefits (NIE/[NDE + NIE]). All p values were two-tailed, and statistical significance was set at alpha level of 0.05. Figure 2. Mediating pathway of the association of prior experience with clinical research/trust in medical researchers with self-reported likelihood of enrolling. AD = Alzheimer’s disease. Results Sample characteristics are detailed in Table 1. Most participants ranged from 18 to 79 years old with nearly half of the sample falling between 40 and 59 years of age. Of note, most (77.2%) were female and nearly half (45.6%) were college-educated. When asked how likely they would be to enroll in a study such as the one portrayed in the video, the mean response was 2.97 on a 5-point Likert scale where 5 represents very likely and most (70.1%) participants clicked “yes” when asked if they would like to be contacted by a staff member from the ADRC. Table 1. Participant Characteristics (N = 500) Characteristic n % M SD Age  18–39 106 21.2%  40–59 237 47.4%  60–79 157 31.4% Sex  Female 386 77.2%  Male 114 22.8% Highest educational level attained  Less than HS or HS degree 49 9.8%  Some college or technical school 223 44.6%  University or postgraduate degree 228 45.6% Income meets basic needs  Yes 437 87.4%  No 63 12.6% Prior experience with clinical research  Yes 344 69.1%  No 154 30.9% Agree to be contacted to learn more about participating  Yes 298 70.1%  No 127 29.9% Worry about getting COVID-19  Not worried at all 126 25.2%  A little worried 165 33.0%  Somewhat worried 139 27.8%  Very worried 44 8.8%  Extremely worried 26 5.2% Trust in medical researchers 39.3 7.0 Perceived AD risk 35.3 26.4 Perceived benefit of research participation 31.2 7.3 Perceived burden of research participation 45.0 15.5 Notes: AD = Alzheimer’s disease; COVID-19 = coronavirus disease 2019; HS = high school. Over two thirds of the sample had prior experience with clinical research. The mean rating on the Trust in Medical Researchers Scale was above the midpoint at 39.3 on a 60-point scale where higher scores indicate more trust. Consistent with the relationships delineated in Figure 2, in models adjusting for demographic factors and perceived risk of AD, both prior experience with clinical research and degree of trust in medical researchers were positively associated with self-reported intention to enroll in AD research (Table 2). Also shown in Table 2, we found that more trust leads to less perceived burden and less perceived burden leads to more likely enrollment (by self-report). This finding is consistent with Table 2, where the analysis revealed that more trust leads to more perceived benefit and more perceived benefit leads to more likely enrollment (by self-report). Table 2. Mediation Analyses: Adjusted Direct and Indirect Association for Likelihood of Enrollment Variable OR (95% CI) p With prior clinical research experience via perceived burden of research participation Effect  Total 2.96 (1.28, 4.63) .022  NIE 1.27 (1.04, 1.50) .020  NDE 2.32 (1.11, 3.53) .03 Mediated proportion (%) 32.2 (12.7, 51.8) .0012 With Trust in Medical Researchers via perceived burden of research participation Effect  Total 1.07 (1.03, 1.11) .001  NIE 1.02 (1.01, 1.03) .0027  NDE 1.05 (1.01, 1.09) .012 Mediated proportion (%) 25.3 (5.38, 45.2) .013 With Trust in Medical Researchers via perceived benefit of research participation Effect  Total 1.09 (1.02, 1.16) .009  NIE 1.03 (1.01, 1.05) <.001  NDE 1.06 (1.00, 1.12) .06 Mediated proportion (%) 35.3 (10.3, 60.4) .006 Notes: AD = Alzheimer’s disease; CI = confidence interval; NIE = natural indirect effect; NDE = natural direct effect; OR = odds ratio. The above mediation analyses each controlled for age, sex, education, income, and AD risk. In addition to these significant direct effects, indirect effects were also identified. Specifically, ratings of the perceived burden of research participation partially mediated the relationship of prior research experience (32%) and trust in medical researchers (25%) with self-reported likelihood of enrolling in AD research. An indirect effect was also detected for perceived benefits of AD research, with regression analysis showing that 35% of the relationship of trust in medical researchers to likelihood of enrolling in AD research was accounted for by perceptions of the benefits of such research. Discussion The current analysis builds on a well-established literature showing that both having trust in medical researchers and perceiving research participation to be beneficial are positively associated with study enrollment decisions, particularly among adults identifying as Black or African American (Bardach et al., 2021; Bonevski et al., 2014; Unger et al., 2021). Upon examining the interplay between these key variables, we demonstrated that perceptions of benefit play a partial mediating role in the overarching relationship of trust to likelihood of study enrollment. That is, one way in which trust in medical researchers influences enrollment decisions is by affecting how individuals appraise the potential benefits of a research opportunity that is being presented or considered (Pugh et al., 2022). This finding is important because, while perceptions of trust are likely formed through experiences occurring over the course of many years preceding a given research enrollment opportunity, perceptions of a given study’s potential benefit are formed as one learns about that study through outreach activities and during the processes of recruitment and informed consent. Without question, investigators and research staff members must work to earn and maintain the trust of prospective research participants and the communities to which they belong (Portacolone et al., 2020). Fundamental to this process is transparency about what research study entails and why it is being conducted. However, research teams, including team members who are engaging in outreach and engagement, can take careful steps to ensure that messaging regarding a study’s potential benefits, whether to the individual or for future generations, is clear and compelling. When applying Larkey and Hecht’s (2010) framework for using culturally centric narrative to promote research enrollment, this can be accomplished by communicating information about potential benefits in a way that emphasizes those benefits identified as most important to, and is framed from the perspective of, members from a given culture-sharing community. This approach is especially promising given research showing that gain-framed messages about research procedures yield higher rates of study agreement than loss-framed messages (Witbracht et al., 2020) as it is reasonable to speculate that consulting community members on the content and framing of such messages may enhance their impact. Indeed, weaving the narratives of persons from underrepresented communities into research recruitment materials is a major goal of the ongoing RIDE study. Of equal importance to infusing community members’ voices into recruitment messaging around study benefits, investigators can design studies to directly maximize the potential for benefit. For example, in a clinical trial, teams can work to minimize the proportion of participants randomized to placebo; or adopt a pragmatic design that affords participants or their treating clinicians some degree of choice at various points in the protocol. While some commentators note that pragmatic trial designs might have lower internal validity than traditional trials, others have pointed to the advantage of greater external validity (Ford & Norrie, 2016). Balancing perceptions of a study’s benefit are perceptions of the burdens that may be associated with participation. Studies quantifying prospective and enrolled participants’ perceptions of research burden are fewer, and in the context of ADRD research are only beginning to emerge. This is especially important in ADRD research given the potential invasiveness of common research procedures like lumbar puncture. One recent study of 443 participants in longitudinal research on ADRD showed that higher perceived participant burden was associated with lower rates of attendance at follow-up visits and higher rates of dropout (Gabel et al., 2022). The current study adds to this evidence base by linking perceived participant burden to the potential for research enrollment. Specifically, the current analysis showed that appraisals of the level of burden that one expects to experience upon study participation act, in part, to mediate the effect of trust in medical research on self-reported likelihood of enrolling. We also found that the positive effect of prior experience on likelihood of enrollment was partially mediated by perceptions of the burdens of participating in a given study. Taken together, these findings underscore the importance of making research participation as low-risk and hassle-free as possible. While the risks of a given study depend on the nature of its procedures or interventions which have been described as “direct” burdens, an emerging literature points to opportunities to minimize the hassle factor involved with research participation. Strategies such as remote research visits, reimbursement for travel and parking, and flexibility in the requirement of a study partner have all been credited with having the potential to minimize the logistical burdens of study participation, including in the context of ADRD research (Grill et al., 2019). Limitations Despite efforts to recruit a community-based sample, we were limited to remote recruitment during the COVID-19 pandemic and the majority of the sample enrolled through the Pitt+Me database. While we excluded individuals who reported current or past participation in ADRD research, we nevertheless attracted a relatively research-friendly sample with the vast majority expressing interest in ADRD research after viewing a video depicting the research experiences of a healthy control or individual with mild cognitive complaints. To that end, because all participants viewed a RIDE video, we could not examine how exposure to storytelling interacts with other variables in our model. Mirroring other samples, women were overrepresented in this survey as were financially secure individuals with relatively high levels of education. There is a great need to extend this work in samples that have far less familiarity with and/or trust in clinical research by perhaps using a recruitment approach that focuses exclusively on engaging individuals through community partners. Conclusion This study demonstrates that when potential participants begin evaluating the potential merits of a given study, those who are less trusting may view the research opportunity with more skepticism when forming an appraisal of the potential benefits and burdens of participation, which may in turn affect enrollment decisions. Conversely, higher levels of trust may favorably affect perceptions of a study’s benefits and burdens. Taken together, these findings suggest that trust is necessary, but not sufficient to promote ADRD research enrollment, and underscores the need for research teams to simultaneous work to build and maintain trusting relationships with prospective participants and to be intentional in efforts to optimize the ratio of benefits to burdens when designing study protocols. Data Availability This study drew on survey data collected and managed in REDCap. 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Journal of the American Medical Association, 327 (8 ), 709. doi:10.1001/jama.2022.0979 Unger, J. M., Hershman, D. L., Till, C., Minasian, L. M., Osarogiagbon, R. U., Fleury, M. E., & Vaidya, R. (2021). “When offered to participate”: A systematic review and meta-analysis of patient agreement to participate in cancer clinical trials. Journal of the National Cancer Institute, 113 (3 ), 244–257. doi:10.1093/jnci/djaa155 33022716 Witbracht, M. G., Bernstein, O. M., Lin, V., Salazart, C. R., Sajjadi, S. A., Hoang, D., Cox, C. G., Gilen, D. L., Grill, J. D. (2020). Education and message framing increase willingness to undergo research lumbar puncture: A randomized controlled trial. Frontiers in Medicine, 7 :493. doi:10.3389/fmed.2020.00493 33043024 Yung, Y. F., Lamm, M., & Zhang, W. (2018). Causal mediation analysis with the CAUSALMED procedure. In Proceedings of the SAS Global Forum 2018 Conference. SAS Institute Inc. Zelinski, E. M., & Gilewski, M. J. (2004). 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==== Front Eur Heart J Case Rep Eur Heart J Case Rep ehjcr European Heart Journal: Case Reports 2514-2119 Oxford University Press US 10.1093/ehjcr/ytad301 ytad301 Case Report Coronary Intervention AcademicSubjects/MED00200 Ehjcr/6 Ehjcr/7 Ehjcr/9 Ehjcr/11 Ehjcr/35 Rotational atherectomy of left main stem immediately after transcatheter aortic valve implantation in a patient with symptomatic severe aortic stenosis and an impaired left ventricular systolic function: a case report https://orcid.org/0000-0002-0703-7805 Satti Zahir Cardiothoracic Department, Freeman Hospital, Newcastle University, Freeman Rd, Newcastle upon Tyne, NE7 7DN, UK https://orcid.org/0000-0003-0811-7679 Farag Mohamed Cardiothoracic Department, Freeman Hospital, Newcastle University, Freeman Rd, Newcastle upon Tyne, NE7 7DN, UK https://orcid.org/0000-0003-3642-318X Egred Mohaned Cardiothoracic Department, Freeman Hospital, Newcastle University, Freeman Rd, Newcastle upon Tyne, NE7 7DN, UK https://orcid.org/0000-0002-3088-8878 Alkhalil Mohammad Cardiothoracic Department, Freeman Hospital, Newcastle University, Freeman Rd, Newcastle upon Tyne, NE7 7DN, UK Russo Giulio Handling Editor Abd ElSamad Sherif Mohammad Editor Cvijic Marta Editor Bahaa Hesham Editor Mohan Siddhartha Editor Kadioglu Hikmet Editor Corresponding author. Tel: +44 191 233 6161, Email: mohammad.alkhalil@nhs.net Conflict of interest: None declared. 7 2023 08 7 2023 08 7 2023 7 7 ytad30103 10 2022 09 4 2023 06 7 2023 18 7 2023 © The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Abstract Background Severe aortic stenosis (AS) and coronary artery disease (CAD) often coexist since they both share the same risk factors and pathophysiology. Patients with severe AS with prohibitive surgical risk are often treated with transcatheter aortic valve implantation (TAVI) and percutaneous coronary intervention (PCI) as a staged or concurrent procedure. Significant calcified CAD and left ventricular (LV) systolic impairment in such patients would add more challenges to the management. A clear consensus on the timing of revascularization of such patients in relation to the TAVI procedure is lacking. Case summary Herein, we present an 86-year-old male who presented to a local district hospital with non-ST-segment elevation myocardial infarction (N-STEMI) and decompensated heart failure. His transthoracic echocardiography showed moderate LV systolic impairment with low-flow severe AS. He was initially treated with dual anti-platelet and diuretic therapy and subsequently underwent coronary angiography that revealed severe calcified shelf-like left main stem (LMS) and moderate left anterior descending (LAD) disease. He was successfully treated with TAVI and rotational atherectomy (RA)-assisted PCI to LMS and LAD in the same setting. Conclusion There is limited evidence on effective strategies to tackle high-risk angioplasty with concurrent TAVI in patients with impaired LV function. We performed TAVI and RA to LMS and LAD in the same setting using no mechanical circulatory support (MCS). Management strategies should be individualized to highly selected patients taking into account LMS involvement, calcium modulation strategies, haemodynamic instability, or cardiogenic shock and whether MCS is needed. Left main stem Percutaneous coronary intervention Rotational atherectomy Severe aortic stenosis Transcatheter aortic valve implantation Impaired left ventricular function Case report ==== Body pmcLearning points Concurrent transcatheter aortic valve implantation (TAVI) and rotational atherectomy (RA)-assisted percutaneous coronary intervention (PCI) to left main stem in a relatively stable high-risk patient can be performed safely without mechanical circulatory support. Transcatheter aortic valve implantation prior to RA-assisted PCI can minimize the risk of haemodynamic compromise resulting from slow coronary flow. Coronary revascularization in patients undergoing TAVI needs to be individualized based on current available evidence. Introduction There is limited evidence to support rotational atherectomy (RA) of left main stem (LMS) disease during transcatheter aortic valve implantation (TAVI). Transcatheter aortic valve implantation has expanded as an effective treatment for patients who pose risk of mortality from conventional surgical aortic valve replacement (SAVR).1 Patients with severe aortic stenosis (AS) frequently have coexistent coronary artery disease (CAD); the optimal management remains an area of ambiguity in the literature and warrants further research.2, 3 Timeline Time Events 25 December 2021 Admission with chest pain and shortness of breath. Echocardiogram 13 April 2021: moderate-to-severe AS. Coronary angiography: severe calcified LMS and LAD disease. 31 December 2021 Transferred to a tertiary centre. 4 January 2022 Heart Team (HT) decision: TAVI + percutaneous coronary intervention (PCI) to LMS/LAD. Predicted mortality by EuroSCORE II: 11.29%. SYNTAX Score II: PCI SYNTAX Score II: 42.3. PCI 4-year mortality: 17.9%. CABG SYNTAX Score II: 49.2. CABG 4-year mortality: 29.9%. 14 January 2022 12:38 TAVI performed. 12:45 RA of LMS and LAD. 15:04 Electrocardiograph (ECG): sinus rhythm (SR), left bundle branch block (LBBB), QRS duration 130 ms. 15 and 16 January 2022 ECG: SR, first degree heart block, LBBB, QRS Duration 128 ms. Discharged. Case presentation An 86-year-old male admitted to a district hospital with chest pain and shortness of breath. He was managed as acute coronary syndrome with decompensated heart failure. Coronary angiography demonstrated heavily calcified shelf-like 70–80% LMS stenosis, with 60−70% stenosis in mid-left anterior descending (LAD) (see Supplementary material online, Videos S1–S3). He was initially referred for consideration of SAVR plus surgical revascularization. He has a history significant of asthma, hypertension, hypercholesterolemia, prostate cancer with previous radiotherapy, and chronic kidney disease (creatinine of 132 µmol/L with an estimated glomerular filtration rate of 42 mL/min/1.73 m2). Clinical examination revealed haemodynamically stable patient with a heart rate of 82 b.p.m., blood pressure of 107/57 mmHg, respiratory rate of 18 breaths per minute, and oxygen saturation of 94%. He had a quiet second heart sound and estimated central venous pressure of 12–13 mmHg. There was reduced air entry on the right lung with evidence of bilateral peripheral oedema. Echocardiogram showed a calcified tri-leaflet aortic valve (AV) with restricted mobility, peak gradient of 50 mmHg, mean gradient of 31 mmHg, and AV area of 0.9 cm2 with left ventricular (LV) ejection fraction of 40% (Figure 1 and Supplementary material online, Video S4). The anterior wall was hypokinetic with normal left ventricle cavity size [diastolic LV internal diameter (LVIDD) of 4.57 cm] and moderately concentric LV hypertrophy (septum thickness of 1.44 cm and posterior wall of 1.39 cm). He had low stroke volume index (SVi) of 28.4 mL/m2. Aortic valve calcium score was >3000 confirming severe AS. Computed tomography (CT) of the aorta confirmed suitability for transfemoral TAVI. The right coronary height was 19 mm, and the left coronary height was 15.6 mm. Figure 1 Long-axis transthoracic echocardiography image and continuous wave Doppler image highlighting severe aortic stenosis. The HT concluded that the severity of AS was secured without the need to perform dobutamine stress echocardiography (DSE) based on low indexed valve area of 0.47 cm2/m2 in the context of low flow alongside the degree of calcified AV leaflets on CT. The consensus was in favour of inpatient TAVI plus PCI to LMS as high surgical risk [The calculated Society of Thoracic Surgeons (STS) score of 8.44%] and patient’s preference for a less invasive approach. Transcatheter aortic valve implantation was performed under conscious sedation using a 14 French (F) eSheath inserted into the right femoral artery. The LV was pre-conditioned by performing very short burst of incremental rapid pacing at 120, 140, 160, and 180 b.p.m. Following each pacing episode, we ensured both blood pressure and ECG changes return to baseline. A 26 mm Sapien 3 Ultra (Edwards Lifesciences Inc., Irvine, CA, USA) valve was successfully deployed under rapid pacing via the LV wire with no significant paravalvular leak (see Supplementary material online, Video S5). Subsequently, the left coronary artery was selectively engaged using 7F EBU 4.0 guide catheter via the eSheath. A Fielder FC guidewire crossed the lesions and was exchanged to rota extra support guidewire using Turnpike LP microcatheter. A Rota Pro 1.5 burr was used to debulk coronary artery calcification (CAC) in LMS and mid LAD (see Supplementary material online, Video S6). A total of 3 runs per lesion were used and each run lasting for less than 20 s at speed of no more than 170 g. The LAD and LMS lesions were pre-dilated using 3.0 × 12 mm Accuforce Non-Compliant Balloon (NCB) and 3.5 × 12 mm Quantum Apex NCB, respectively. A 3.0 × 18 mm Xience Sierra drug-eluting stent (DES) was deployed in mid-LAD and post-dilated with a 3.5 × 12 NCB. The LMS lesion was treated with a 4.0 × 15 mm Xience Sierra DES and optimized with a 5.0 × 8 mm NCB with excellent angiographic result (see Supplementary material online, Videos S7 and S8). The procedure was performed with total contrast volume of 190 mL and activated clotting time (ACT) between 250 and 300 s. Patient developed LBBB with marked first-degree AV block and PR interval of >300 ms (Figure 2). He was discharged on aspirin and clopidogrel 2 days later. At 4-month follow-up, the patient reported no chest pain or shortness of breath and noted significant improvement in his exercise capacity. His peak and mean gradients across the valve were 19 and 12 mmHg, respectively, with effective orifice area of 1.78 cm2. There was an improvement in his LV dysfunction from moderate to mild LV dysfunction with an estimated ejection fraction of 50%. His LVIDD was 4.4 cm with septum and posterior wall thickness of 1.4 and 1.36 cm, respectively. Figure 2 (A) Twelve-lead electrocardiograph on admission to our centre and prior to transcatheter aortic valve implantation and percutaneous coronary intervention to left main stem and left anterior descending. (B) Twelve-lead electrocardiograph post-transcatheter aortic valve implantation showing left bundle branch block. Discussion This case demonstrates that RA in patients with severely calcified LMS and concomitant severe AS and LV impairment maybe safe. Transcatheter aortic valve implantation could potentially act as a mechanical support device to facilitate high-risk coronary intervention. This is important since the prevalence of degenerative severe AS and CAD increases with age and both conditions coexist in 50% of patients aged ≥ 70 years old and in 65% in those aged ≥ 80 years old.4 Previous studies have shown that 40–75% of patients undergoing TAVI also have significant CAD (defined as from >50% to 70% narrowing in an epicardial artery). Coronary artery disease and severe AS often share a similar pathophysiology and risk factors.5 Severe AS is associated with abnormally diminished coronary flow reserve (CFR) that may be secondary to LV hypertrophy and higher resting flow. This phenomenon can contribute to the development of symptoms, LV systolic and diastolic dysfunction, and adverse outcomes. Adverse outcomes can be further exaggerated in the presence of CAC during angioplasty. There is lack of robust data and large randomized trials to guide optimal management and the impact of significant CAD in TAVI population.6 The TAVI-LM registry has shown that TAVI plus LMS PCI can be performed safely in patients with high surgical risk.7 Abdel-Wahab et al.8 demonstrated that PCI before TAVI using CoreValve (Medtronic, Minneapolis, MN, USA) was not associated with increased adverse events and was safe at 30 days and 6 months compared with TAVI alone. Several anatomical and procedural factors need to be considered when performing PCI in patients undergoing TAVI. Overhanging stents following revascularization of ostial coronary lesions can be crushed with expanded transcatheter heart valves. This needs to be considered when performing PCI prior to TAVI. The use of balloon valvuloplasty (BAV) prior to PCI may be associated with reduction in procedural risk. The risk of converting severe AS to aortic regurgitation has limited the use of this strategy. On the other hand, performing PCI after TAVI may add technical challenges, particularly when trying to engage guiding catheters.9 The presence of severe coronary calcification combined with impaired LV function has added further procedural risks. Both factors can synergistically increase the risk of no reflow and a require tailored approach to patients undergoing PCI and TAVI. It is well established that severe CAC is associated with worse procedural and long-term outcomes. This includes difficult stent delivery, stent under expansion, stent thrombosis, vessel dissection, coronary perforations, myocardial infarction, repeat vascularization, and even increased risk of death.10 Therefore, RA was used in our case to adequately prepare the LMS lesion by debulking CAC and allowing for optimal stent expansion. Alternative calcium modifications strategies such as the use of intra-vascular lithotripsy (IVL) would have also been an option. The discrepancy in the size of the targeted arteries would have prompted the use of two shockwave balloons to manage the coronary lesions. Whilst IVL is less likely to cause no reflow compared with RA, the stable blood pressure post-TAVI and meticulous attention to the burr speed and duration enabled us to complete the PCI procedure without complications. The ACTIVATION (PercutAneous Coronary inTervention prIor to transcatheter aortic VAlve implantaTION) trial concluded that death rates and re-hospitalization events were comparable among patients who underwent combined PCI and TAVI vs. TAVI-alone.11 Nonetheless, the presence of unprotected LMS disease was an exclusion criterion from this study, and therefore its results cannot be applied to our case. Although performing PCI prior to TAVI is a relatively common strategy to obviate the challenges associated with coronary access post-TAVI, the combination of PCI and TAVI in a single setting is safe and cost-effective.12 The use of same vascular access, reducing ischaemic burden during TAVI, possible decrease in the risk of contrast nephropathy, and reduction in radiation exposure to both patient and operators provide an advantage over performing PCI and TAVI in two different settings.5 The other challenge in this case was the presence of LV systolic impairment. High-risk angioplasty in this setting carries additional risk of intra-procedural haemodynamic instability.13,14 The presence of untreated severe AS would inevitably exacerbate the risk. Therefore, we elected to perform TAVI to improve the haemodynamic status of the patient. Accessing the coronary ostia following TAVI may be challenging, particularly with a self-expanding valve. This may less be of an issue when using balloon expandable valve, particularly given the large top cell size that would allow easy access for future coronary intervention. By treating AS first, we obviate the need to use mechanical circulatory support (MCS) device. Overcoming pressure overload using TAVI prior to high-risk PCI would simplify the procedure and allow early discharge for patients. In conclusion, high-risk LMS PCI using RA may be safe in patients undergoing TAVI with concomitant LV impairment. The decision to perform TAVI first would potentially avoid the need to use MCS in these settings. Supplementary Material ytad301_Supplementary_Data Click here for additional data file. Lead author biography Dr Mohammad Alkhalil is Consultant Cardiologist and Structural Heart Interventionist at the Freeman Hospital. He graduated from Damascus University and completed his interventional cardiology training at the Royal Victoria Hospital in Belfast, UK and Toronto General Hospital in Canada. He obtained MSc in Cardiology at Trinity College Dublin, Ireland and DPhil degree in Cardiovascular Medicine at University of Oxford, UK. His research interests include atherosclerosis, myocardial infarction, transcatheter aortic valve implantation (TAVI), and cardiac magnetic resonance imaging. Supplementary material Supplementary material is available at European Heart Journal – Case Reports. Slide set: A fully edited slide set detailing this case is available online as Supplementary data. Consent: In accordance with COPE guidelines, patient-informed consent was obtained. Funding: None declared. Data availability The authors confirm that the data supporting the findings of this case report are available within the manuscript and its supplementary materials. Further details can be requested by contacting the corresponding author (M.A.). ==== Refs References 1 Reardon MJ , Van MieghemNM, PopmaJJ, KleimanNS, SøndergaardL, MumtazM, et al Surgical or transcatheter aortic-valve replacement in intermediate-risk patients. N Engl J Med 2017;376 :1321–1331.28304219 2 Paradis JM , FriedJ, NazifT, KirtaneA, HarjaiK, KhaliqueO, et al Aortic stenosis and coronary artery disease: what do we know? What don’t we know? A comprehensive review of the literature with proposed treatment algorithms. Eur Heart J 2014;35 :2069–2082.24970334 3 Alkhalil M , JabriA, PuriR, KalraA. Revascularization in the transcatheter aortic valve replacement population. Interv Cardiol Clin 2021;10 :553–563.34593117 4 Iung B . Interface between valve disease and ischaemic heart disease. 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Am J Cardiol 2012;109 :581–586.22133754 9 Ramee S , AnwaruddinS, KumarG, PianaRN, BabaliarosV, RabT, et al The rationale for performance of coronary angiography and stenting before transcatheter aortic valve replacement: from the interventional section leadership council of the American College of Cardiology. JACC Cardiovasc Interv 2016;9 :2371–2375.27931592 10 Lee MS , GordinJS, StoneGW, SharmaSK, SaitoS, MahmudE, et al Orbital and rotational atherectomy during percutaneous coronary intervention for coronary artery calcification. Catheter Cardiovasc Interv 2018;92 :61–67.29045041 11 Patterson T , ClaytonT, DoddM, KhawajaZ, MoriceMC, WilsonK, et al ACTIVATION (Percutaneous Coronary inTervention prIor to transcatheter aortic VAlve implantaTION): a randomized clinical trial. 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==== Front J Endocr Soc J Endocr Soc jes Journal of the Endocrine Society 2472-1972 Oxford University Press US 10.1210/jendso/bvad089 bvad089 Clinical Research Article AcademicSubjects/MED00250 Clinical and Biochemical Characteristics of Untreated Adult Patients With Resistance to Thyroid Hormone Alpha https://orcid.org/0000-0002-3022-0755 Dahll Louise Koren Hormone Laboratory, Department of Medical Biochemistry and Biochemical Endocrinology and Metabolism Work Group, Oslo University Hospital, Oslo 0424, Norway lokoda@ous-hf.no https://orcid.org/0000-0003-2619-4192 Westbye Alexander Bauer Hormone Laboratory, Department of Medical Biochemistry and Biochemical Endocrinology and Metabolism Work Group, Oslo University Hospital, Oslo 0424, Norway Vinorum Kristin Department of Medical Genetics, Oslo University Hospital, Oslo 0424, Norway Sejersted Yngve Department of Medical Genetics, Oslo University Hospital, Oslo 0424, Norway Barøy Tuva Department of Medical Genetics, Oslo University Hospital, Oslo 0424, Norway https://orcid.org/0000-0002-9615-1035 Thorsby Per Medbøe Hormone Laboratory, Department of Medical Biochemistry and Biochemical Endocrinology and Metabolism Work Group, Oslo University Hospital, Oslo 0424, Norway Institute of Clinical Medicine, University of Oslo, Oslo 0316, Norway https://orcid.org/0000-0001-7619-8701 Hammerstad Sara Salehi Institute of Clinical Medicine, University of Oslo, Oslo 0316, Norway Department of Endocrinology, Oslo University Hospital, Oslo 0424, Norway Department of Pediatric Medicine, Oslo University Hospital, Oslo 0424, Norway Correspondence: Louise K. Dahll, MD, The Hormone Laboratory, Department of Medical Biochemistry, Oslo University Hospital, Postboks 4950, Aker sykehus, Nydalen, Oslo 0424, Norway. Email: lokoda@ous-hf.no. 03 7 2023 04 7 2023 04 7 2023 7 8 bvad08908 2 2023 23 6 2023 18 7 2023 © The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. 2023 https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Abstract Background Thyroid hormone resistance due to pathogenic variants in thyroid hormone receptor alpha (THRA) is rare and descriptions of patients are sparse. The disorder is probably underdiagnosed as patients may have normal thyroid function tests. Treatment with thyroxine in childhood improves clinical symptoms. However, it is not clear if treatment has beneficial effects if started in adulthood. Cases We investigated 4 previously untreated Caucasian adult first-degree-related patients with the THRA c.788C > T, p.(Ala263Val) variant identified by a gene panel for intellectual disability in the index patient. Clinical data and previous investigations were obtained from medical reports. Results During childhood and adolescence, short stature, short limbs, metacarpals, and phalanges, and delayed bone age maturation were observed. Delayed motor and language development and decreased intellectual and learning abilities were described. Abdominal adiposity, round face, and increased head circumference were common features. All individuals complained of tiredness, constipation, and low mood. While thyrotropin (TSH) and free thyroxine (FT4) were within the reference range, free triiodothyronine (FT3) was high. FT4/FT3 ratio and reverse T3 were low. Other main features were low hemoglobin and high LDL/HDL ratio. Conclusion Investigation of 4 first-degree-related adult patients with untreated resistance to thyroid hormone alpha (RTHα) revealed more pronounced phenotype features and hypothyroid symptoms than previously described in patients treated with levothyroxine from childhood or adolescence. The delay in diagnosis is probably due to normal thyroid function tests. We suggest that THRA analysis should be performed in patients with specific clinical features, as treatment in early childhood may improve outcomes. thyroid hormone thyroid hormone receptor THRA short stature obesity ==== Body pmcThyroid hormones are important regulators of growth and development, metabolic rate, energy homeostasis, heart and bowel function, central nervous system maturation and cholesterol conversion. The THRA and THRB genes encode thyroid hormone receptors (TR), both producing multiple isoforms through alternative splicing. The isoforms TRβ1, TRβ2 and TRα1 bind triiodothyronine (T3) while TRα2 does not bind T3 but acts as an inhibitor by competing for DNA [1, 2]. These nuclear receptors, found in most cells, form monomers, homodimers, or heterodimers with retinoid X receptors at DNA binding sites on target genes (thyroid hormone-responsive elements) and mediate gene regulation by binding of T3 [3, 4]. Pathogenic variants in the receptors are rare but can cause disease due to cellular resistance to T3. The clinical symptoms are receptor specific, because TRα and TRβ are unevenly distributed in the tissues. TRα is the predominant receptor in the central nervous system (CNS), intestines, bone, and cardiac and skeletal muscle, while TRβ is predominant in the hypothalamus, pituitary, retina, cochlea, thyroid gland, liver, and kidneys [5, 6]. Pathogenic variants in THRB causing resistance to thyroid hormone beta (RTHβ) are suspected if the patient has normal or slightly elevated thyroid stimulating hormone (TSH) and elevated tetraiodothyronine (T4) and freeT4 (FT4). Heterozygous pathogenic variants in THRA are associated with resistance to thyroid hormone alpha (RTHα), causing autosomal dominant nongoitrous congenital hypothyroidism. The receptor translated from the alternative allele causes thyroid hormone resistance by inhibiting wild-type receptor action in a dominant negative manner. The pathogenic THRA variants described so far are most frequently located in the C-terminal ligand-binding domain (LBD) [2, 7]. Nonsense, frame shift, and missense variants close to the C-terminus appear to lead to more severe phenotypic features than missense variants located in the more N-terminal gene segment common to the TRα1 and TRα2 isoforms. A key molecular pathogenic defect may be persistent co-repressor binding, rather than coactivator recruitment per se. The variants associated with the most severe outcomes do not only prevent T3-binding but also coactivator recruitment [8-11]. The p.(Ala263Val) variant is located in the N-terminal segment of the LBD common to the TRα1 and TRα2 isoforms. This variant is found to cause reduced affinity for T3 which in vitro is overcome by high doses of T3 [12]. Phenotypic variation between patients, and also between family members with the same pathogenic variant in THRA has been reported [13]. Here we report available clinical and laboratory characteristics from childhood and onwards of 4 previously untreated adult patients with RTHα. These descriptions may aid pediatricians to identify the disease in patients at an early age. Material and Methods A father and his 3 adult children, 2 daughters and a son, were referred from the genetic department to the outpatient thyroid clinic at Oslo University Hospital in 2018 for assessment. All 4 had the THRA c.788C > T, p.(Ala263Val) variant and had not previously received treatment. The index patient was referred to the genetic department at the age of 15 due to clinical features and was diagnosed with RTHα at the age of 18. The children’s mother did not present the gene variant and volunteered as a control. This study was approved by the Institutional Review Board and the Norwegian Regional Committee for Medical and Health Research Ethics (REK). Written informed consent was obtained from all family members. Medical Records, Clinical Features Information regarding clinical descriptions, developmental tests, milestones, symptoms of hypothyroidism, and a family history for preparing a pedigree are based on medical records and investigations at admission to the outpatient thyroid clinic. Genetic Investigation Molecular genetic analyses using genomic DNA from the index patient was initiated at age 13 and included a chromosomal microarray (SurePrint G3 Human Exon 4 × 180k Microarray Kit, Agilent Technologies), Sanger sequencing of NSD1, SHOX, PTEN, and PTEN promotor and multiplex ligation dependent probe amplification of SHOX and PTEN (MRC Holland P026 and P018). At age 18, a diagnostic in silico gene panel for intellectual disability containing 849 genes (Supplementary Table S1) [14] was analyzed (Gene sequences were obtained using SureSelect Human All Exon v5 exome kit (Agilent Technologies), and a HiSeq2500 sequencer (Illumina). Variants with global allele frequency >1% in ExAC [15] or 1000 genomes [16], or >5% in local in-house databases, were removed. Of the remaining variants, only a heterozygous variant in THRA (NM_199334.3): c.788C > T, p.(Ala263Val) (chr17(GRCh37):g.38244559C > T) was suspected to be disease-causing. Biochemical Investigation: Thyroid Function Tests Morning blood samples after an overnight fast were collected, centrifuged, and analyzed shortly after retrieval at the Hormone laboratory, Oslo University Hospital using available commercial kits. TSH (reference range, 0.5-3.6 mU/L) was measured with noncompetitive immunofluorometric analysis by Autodelfia (Wallac Oy, Turku, Finland- RRID:AB_2877703). FT4 (8.0-21.0 pmol/L) was measured with solid-phase time-delayed fluoro-immunoassay with back-titration by Autodelfia (Wallac Oy, Turku, Finland- RRID:AB_2801661). FT3 (2.8-7.0 pmol/L) was measured with competitive electrochemiluminescence immunoassay by Cobas e601 (Roche Diagnostics, Indianapolis, IN, USA AB_2827368). Reverse T3 was analyzed using a competitive RIA (DIAsource ImmunoAssays S.A, Belgium). Sex hormone–binding globulin (SHBG), thyroxine binding globulin (TBG), and insulin-like growth factor 1 (IGF-1) using a chemiluminescent immunometric assay (Siemens IMMULITE 2000, Siemens). Androstenedione was analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS Agilent, 1290 UHPLC coupled to a 6490 triple quadrupole MS) by a method developed at the Hormone laboratory. Cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured as routine analyses at Oslo University Hospital. Developmental Tests Information regarding clinical development is based on medical records and investigations on developmental assessments performed with Leiter-R, Wisc-R, and ITPA-test [17-19]. Results Characteristics The family pedigree is shown (Fig. 1A). The participants’ characteristics and photographic images are presented (Table 1 and Fig. 2, respectively). Heart rate and blood pressure were normal for the 3 siblings, while the father had elevated systolic and diastolic blood pressure. All 4 patients were overweight, were of short stature and had short limbs. Growth curves for patient 4 are presented in Supplementary Fig. S1A and S1B [20] (percentiles from [21, 22]). Figure 1. (A) Pedigree of the affected family. Molecular analysis of the index patient (arrow), her parents, siblings, and nephew. Black squares/circles indicate individuals confirmed to have the nucleotide substitution (c.788C > T) corresponding to an alanine to valine substitution at codon 236. Family members with confirmed normal THRA variants are indicated WT/WT. Pedigree created with Biorender.com. (B) Sanger sequencing of THRA. Sanger sequencing chromatogram of THRA (index patient). The heterozygous c.788C > T, p.(Ala236Val) variant is shown. Figure 2. Photographs of patients. (A) Patient 3:10 years old, (B) Patient 4: 7 years old, (C) Index patient: 3 years old, From left to right (D) Mother (unaffected), patient 3, patient 4, index-patient, patient 2 (father). Photo taken 4 years after admission. Note: elbows reach the middle of the ribcage. Table 1. Clinical and auxological characteristics Index patient Patient 2 Patient 3 Patient 4 *Mother Age (years) 19 64 26 25 60 Gender F M F M F Weight kg (ref. range)a 119 (48-77) 97 (NA) 155 (48-77) 111 (57-101) 81.7 (NA) Height cm (ref. range)a,b 165 (154-179)a 161 (177)b 163 (154-179) 170 (167-194) 165 (NA) Arm span cm (ref. range)c 160 (166) 157 (162) 158.5 (164) 170 (171) 169 (166) Sitting height cm (ref. range)d 88.6 (82-91) 88.6 (NA) 90.1 (81-90) 91.8 (81-92) 82.8 (NA) BMI kg/m2 (ref. range)e 43.8 (18-28) 37.4 (27) 58.0 (18-28) 38.0 (17-30) 30.0 (26) Head circumference cm (ref. range)f 60.1 (52-58) 63.0 (53-59) 60.5 (52-57) 61.0 (54-59) 54.5 (52-58) Blood pressure mmHg (ref. range)g 122/83 (94-136/47-80) 170/114 (101-175/58-100) 118/79 (94-136/47-80) 127/82 (103-148/47-83) 126/83 (98-175/51-95) Heart rate bpm (ref. range)g 57 (51-95) 72 (44-91) 65 (51-95) 68 (47-91) 83 (46-94) Shoe size (continental) (predicted size based on height)h 42 (38) 42 (<39) 42 (38) 44 (41) 39 (38) Values outside the reference ranges are marked in bold. Abbreviations: BMI, body mass index; bpm, beats per minute; F, female; M, male. a Reference ranges are extracted from available literature. Patient 3 and 4: Height, weight or BMI and range (± 2 SD) from Norwegian 19-year-olds [23]. b Patient 2 and mother: Mean height of Norwegian military conscripts (by year of birth) (Statistics Norway, table SY 108). c Predicted arm span from height [24]. d Sitting height range (mean ± 2 SD) predicted from patient height [25]. Patient 3 and 4 predicted using ratios for age 21 years. Dutch population. e BMI [26]. f Head circumference range (3rd to 97th percentile) of adult females and males [27]. British population. g Systolic and diastolic blood pressure and heart rate range (calculated from mean ± 2SD) for the Norwegian HUNT3 (2006-2008) cohort [28]. Index patient compared to age group 20-29 years. h Estimated shoe size by height [29] in parenthesis. Conversion of US Women's size to continental size based on a Nike size chart. *Mother served as control. Laboratory Assessment The variant was confirmed by Sanger sequencing in the index patient (Fig. 1B) and found to co-segregate with the disease in 3 affected and 4 unaffected family members. The variant altered an evolutionary conserved amino acid, and no amino acid substitutions affecting codon 263 were present in the Genome Aggregation Database (v2.1.1 and v3.1.2). Thyroid function tests showed normal TSH and FT4 (Table 2). FT3 levels were just below the upper reference range or slightly elevated in all the patients. In all 4 patients, FT4/FT3 ratio was lower compared with both healthy controls without any thyroid diseases and anonymized reference values from our laboratory (negative thyroid peroxidase antibodies TPO-Ab] and thyrotropin-receptor antibodies [TRAb], and TSH within the reference range), regardless of age (Fig. 3 and Table 2). Similarly, rT3 levels were lower than the reference range (Table 2). Figure 3. Distribution of FT4/FT3 ratio. Median calculated FT4/FT3 ratio by age group comparing patients to controls. Percentile distribution of FT4/FT3 ratios in presumed healthy patients was obtained by exporting data from 4.5 years of clinical routine production results. Anonymized data was filtered to remove likely pathologic results; patients with at least one positive TPO-Abs or TRAS, or a TSH outside the reference range, and patients that were analyzed more than once were removed. Results were grouped in 5-year age categories and percentiles calculated for each category. The healthy controls group consisted of 47 healthy individuals (mean age 40 years) (FT4/FT3 ratio range, 2.2-3.7). Table 2. Thyroid function test results Index patient Patient 2 Patient 3 Patient 4 Mothera Reference range TSH 2.9 0.55 2 1.9 0.82 0.5-3.6 (mU/L) FT4 15 13 9.6 9.6 19 8.0-21.0 (pmol/L) FT3 7.8 6.2 6.8 7.2 3.8 2.8-7.0 (pmol/L) FT4/FT3 1.9 2.1 1.4 1.3 5 See Fig. 3 rT3 0.18 0.21 0.11 0.12 0.28 0.26-0.69 (nmol/L) TT4 125 118 86 84 92 60-150 (nmol/L) TT3 2.9 2.4 2.8 3 1.3 1.2-2.8 (nmol/L) TBG 22.6 15.6 17.5 20.3 19 14-31 (mg/L) Results in bold show low FT4/FT3 ratio and high FT3. Abbreviations: TSH, thyroid stimulating hormone; T3, triiodothyronine; T4, thyroxine; TBG, thyroxine binding globulin. a The mother served as a control. Reference ranges (RR) for the mother at the local hospital were TSH: 0.27-4.2 mIU/L, FT4: 12-22 pmol/L, FT3: 2.6-5.7 pmol/L. Hemoglobin below or in the low normal reference range was a common feature in all participants regardless of age and sex. LDL levels were in the upper reference range and the LDL/HDL ratio was high. Fasting C-peptide was above the reference range in the 2 patients with the highest body mass index (BMI) (Table 3). Table 3. Biochemical characteristics Index Patient Patient Patient Mothera Reference ranges Patient 2 3 4 Hemoglobin 11.4 14.1 11.6 13.8 12.3 age ≥ 12 F: 11.7-15.3, M: 13.4-17 g/dL EVF 0.32 0.42 0.36 0.42 0.38 age ≥ 12 F: 0.35-0.46, M: 0.40-0.50 MCV 88 94 94 90 98 80-100 fL HbA1C 33 39 44 39 58 20-42 mmol/mol C-peptide fasting 1749 898 1690 1105 947 300-1480 pmol/L Total cholesterol 4.7 6.1 5.5 5.2 3.8 age 18-29: 2.9-6.1 age ≥ 50 3.9-7.8 mmol/L LDL cholesterol 3.25 4.75 4.3 3.55 1.63 age 18-29: 1.5-4.2, age 50-79: 2.1-4.9 mmol/L HDL-cholesterol 0.97 1.09 0.97 1.33 1.7 F: 1.0-2.7, M: 0.8-2.1 mmol/L LDL/HDL 3.4 4.4 4.4 2.7 0.96 Recommended ratio: 1-3.5 Lipoprotein (a) <20 <20 114 131.9 238 >75 nmol/L increase risk of atherosclerosis Haptoglobin 1.8 2.5 2.2 2.8 1.5 0.4-2.1 g/L Lactate dehydrogenase 181 290 206 182 121 age 18-69: 105-205 U/L Total CK (>99% CK-MM) 125 345 173 197 79 F ≥ age 18: 35-210, M: age 18-49:50-400, M: age ≥ age 50: 40-280 U/L CK-MB 2 4 2 2 3 ≤5 µg/L Androstenedione 3.5 2.9 6.9 5 4.6 F: 0.9-7.9, F > 50 years: 0.5-2.9, M: 1.2-4.7 nmol/L IGF-1 25 16.4 13.1 18.5 9.2 age 19-24: 10-51, age 25-29: 9-34, age ≥ 50: 4-22 nmol/L SHBG 36 50 28 25 124 F: 23-100, M age < 60: 8-60, age > 60: 15-90 (nmol/L) Values outside the reference ranges are marked in bold. Abbreviations: CK-MM/CK-MB, skeletal muscle/cardiac muscle isoenzyme of creatinine kinase; EVF, erythrocyte volume fraction; HbA1C, glycated hemoglobin A1C; IGF-1, insulin-like growth factor-1; MCV, mean corpuscular volume; SHBG, sex hormone–binding globulin. a The mother served as a control. RR for the mother: SHBG (23-150 nmol/L). Clinical Description The index patient A female, the youngest of 5 siblings, was nearly 20 years old on admission to the thyroid outpatient clinic. She was born 2 weeks overdue. At birth, her mother immediately recognized the facial features and body composition of her husband and 2 of her children—later diagnosed with RTHα. Birth weight (BW) was 4.96 kg and birth height (BH) was 53 cm (median BW for girls in Norway is 3.6 kg [reference range, 95%: 2.7-4.6], and median BH 50.4 cm [reference range, 95%: 46.5-54.4]) [23]. Her mother had gestational diabetes mellitus. At delivery she presented with hypoglycemia and was treated with glucose intravenously. Lactose intolerance was detected when she was a toddler. Recurrent otitis media led to bilateral paracentesis at the age of 1 and adeno-tonsillectomy at age 6. She started walking at 13 to 14 months. She was short and had stout thighs and calves. Delayed language development was observed from 20 months. She was tested with the Illinois Test of Psycholinguistic Abilities (ITPA) test at the age of 5 years and 8 months. Her average score was lower than the expected. She scored above the standard deviation in 2 of the visual tasks and below the standard deviation in 3 of the auditory tasks. She could pronounce “r” and learned to ride a bicycle when she was 8 years old. She had general learning difficulties, with a cognitive capacity in the lower normal range. From the age of 10 she received teaching support. She achieved normal grades at school and completed upper secondary vocational education. During childhood and adolescence, she was often tired and had little energy to meet friends after school. She went to bed between 9 and 10 in the evenings and slept for 10 to 11 hours. At the age of 12 her skeletal age was normal, but micromelia (short metacarpals and phalanges) was recognized by the radiologist. When she and her 2 affected siblings were teenagers, they had to wear shoes that were 2 sizes too large because of abnormally wide feet. At that time she had normal thyroid function tests: TSH, 1.6 mmol/L (reference range [RR] 0.5-4.9), and FT4, 13 pmol/L (9-16). Repeated measurements after 2 years showed stable TSH and low normal FT4, 10 pmol/L. Due to short stature with short limbs, macrocephaly, and developmental delay, the NSDI, SHOX, and PTEN gene were analyzed with normal results. When she was 15 years old, she was referred to Oslo University Hospital for genetic counseling. By then she had developed pronounced truncal obesity with a BMI of 40 kg/m2. She had a large round head, broad face, flat nasal bridge, a narrow palpebral fissure length, and short limbs. Her voice was nasal, and she spoke with a slight lisp. At the age of 18, analysis of a gene panel for intellectual disability disclosed the c.788C > T p.(Ala263Val) variant in THRA. Upon admission to the thyroid outpatient clinic, she had a nasal voice and complained of tiredness and constipation, passing stool every third to fourth day. Compared to Norwegian 19-year-old females she was short and severely obese (Table 1) [30, 31]. Her head circumference was increased [23]. In contrast to a normal population [24], her limbs were short for her height (sitting height in the upper range) (Fig. 2D) and her arm span was shorter than her body height. She had broad hands, large feet and wore shoes 4 sizes larger than estimated from her body height [29]. Patient 2 The father was 64 years old at admission to the outpatient clinic. Based on information in the patient record, he was delivered 3 weeks overdue, with BW 3.2 kg and BH 49 cm (reference mean BW 3.5 kg [SD 0.57] and mean BH 50.9 cm [SD 2.7]) [32]. The father started to walk at 16 months old, he said “mummy” when he was 1 year old but did not speak any other words until he was 3 years old. He was referred to the oral surgery department at the nearest University Hospital when he was 7 years old for extraction of 2 worn down (caries decayed) deciduous teeth. He was at this time described as a short, stout, nervous and anxious boy who had suffered from eczema, frequent colds, bronchitis, and otitis media. A big tongue and short thighs were observed. His speech was indistinct, he stuttered and could not pronounce “k” and “g” and was referred to a speech therapist. He received teaching support for 3 years from the age of 8. At age 11, he was referred to the pediatric outpatient clinic at the nearest University Hospital for investigation of short stature and suspected hypothyroidism. At that time, he had anemia with hemoglobin 10.2 g/dL, and bone age maturation corresponding to 6 to 7 years. Insulin-glucose load, ACTH and metyrapone test, air encephalogram, hormonal pituitary examination, and other biochemical status were normal, and the investigation concluded with constitutional growth delay. Protein-bound iodine was in the low normal range and primary hypothyroidism was considered less likely. At this time (11 years) his height was 124.5 cm (8 cm below the 2.5 percentile), and he weighed 30.9 kg (about 2 kg above the 97.5 percentile in relation to height) [32]. At admission to the outpatient thyroid clinic, his appearance was stout with a round face, broad hands and feet, and short fingers. He had a large chest with numerous moles on his chest and back and some skin tags in the neck, abdominal obesity, and short extremities (Fig. 2D). Despite being physically active, his weight had gradually increased by 34 kg from when he was 23 years old. Cortisone injections were given each spring to treat hay fever and he took nasal spray for allergic rhinitis. He complained of tiredness, exhaustion, and low mood. He was shorter and more overweight than the average for Norwegian men of the same age (Table 1) [31, 33] and his head circumference was increased [23]. Patient 3 The older sister, (7 years older than the index patient) was first evaluated in the thyroid outpatient clinic when she was 26 years old. She was born after a normal pregnancy and had normal weight and height at delivery, BW 3.57 kg and BH 50 cm. She learned to walk without support when she was 19.5 months. Her language development was delayed and she could only utter some two-word sentences when she was 2 years old. As an infant and toddler, she was treated with hydrocortisone cream for atopic eczema. Due to obstructive episodes related to several upper respiratory tract infections she received inhalation therapy with salbutamol and budesonide. At 5 years and 4 months she was referred to the local Educational and Psychological Counselling Service (“Pedagogisk psykologisk tjeneste”). The evaluation described a delay of 1.5 to 2 years in mental and intellectual function. Special education assistance was recommended in daycare and at school. From the age of 5, she was described as significantly overweight and was therefore examined in primary care when she was 9 years old. Blood tests showed TSH, 1.8 mIU/L (reference range, 0.35-5); FT4, 9 pmol/L (11-23); and FT3, 7.6 pmol/L (3.5-6.5). She had an iron sufficient microcytic anemia with an red blood cell count slightly below normal and hemoglobin of 11.2 g/100 mL (11.5-14,5). At age 11 her BMI was 34 kg/m2 and she weighed 66.5 kg, 22.5 kg above the 97.5 percentile for height [23]. She attended a standard public school but had an individual education program. The third year of lower secondary school she attended a school specifically tailored to children with disabilities. She completed a vocational education at upper secondary level, but her school performance was in the lower half of the grading system. She was often tired and could fall asleep during daytime. Heavy sleeping forced her to use 5 alarm clocks to wake herself in the morning. When tested, her apnea-hypopnea index did not meet the criteria for obstructive sleep apnea. Borderline high level glycated hemoglobin (HbA1c) was detected when she was 21 years old, and 2 years later she presented with gestational diabetes. Patient 3, along with her mother (who weighed 125 kg) and the index patient, were referred to a nutritionist and were evaluated by an institution specializing in treatment of patients with morbid obesity. A change in diet and lifestyle was successful and they achieved significant weight loss. However, both patient 3 and the index patient soon regained weight to pretreatment levels in contrast to their mother, who sustained her weight loss of more than 40 kg. On evaluation, patient 3 was obese, class 3. The investigation revealed numerous moles on her back, her thyroid was slightly enlarged with a firm nodule in the right lobe, about 10 mm in diameter. She complained of tiredness and periodically a lack of energy to meet friends, and had only capacity to perform housework 20 minutes a day. She had abdominal pain related to constipation and hard stool every third to fourth day. She used desogestrel contraceptives, and a beta 2 agonist (terbutalinsulfat) and budesonide for inhalation when obstructive. Patient 4 The older brother (6 years older than the index patient) was 25 years old at admission. He was born after an uncomplicated pregnancy and delivery, BW 3.75 kg and BH 52 cm (median BW for boys in Norway is 3.7 kg [reference range, 95%: 2.8-4.6], median BH 50.7 cm [reference range, 95%: 46.5-54.9]) [23]. As a toddler, he was treated for atopic eczema and upper airway infections with hydrocortisone cream, beta 2 agonist (terbutalinsulfat), and budesonide. He had recurrent lung and ear infections and was treated with adenoidectomy and bilateral myringotomy when he was 4 years old. At age 6 he was referred to the local pediatric ward for investigation of short stature. His height was 106.3 cm, 3 cm below the 2.5 percentile. Weight 22 kg, equivalent to the 97.5 percentile. His growth curve was followed regularly (Supplementary Fig. S1) [20] until he was 17 years of age at the local pediatric ward. His height was consistently below the 2.5 percentile and his weight gradually increased. At age 7, his upper front teeth were worn down completely; he had speech impediments and hoarseness and was treated by a speech therapist. At the same age he was referred to an ophthalmologist because of stumbling over uneven terrain at the daycare center. He also complained of visual difficulties when solving puzzles. Visual examination showed only mild hyperopia, which was corrected with glasses for a few years until retesting of his vision revealed short-sightedness. At age 8, he was investigated by neurologists at Oslo University Hospital, who concluded that he had delayed development of speech, delayed growth, and delayed psychomotor development. He was described as having narrow eye slits, a short philtrum, a thin upper lip, worn teeth, and stout calve muscles. At age 9, intellectual tests [17, 18] were not conclusive of mental retardation. The inquirer stated that his concentration problems probably negatively biased the test results. At age 13 years and 7 months, x-ray investigation revealed mesomelia (shortening of the forearms and lower legs) corresponding to the lower normal reference range for a child aged 11.5 years, while the humerus and femur were in the lower normal range for chronological age. Short metacarpals and phalanges were also observed. Blood tests performed at age 14 showed elevated FT3, 7.9 pmol/L (3.0-5.8) but normal TSH, 1.7 mIU/L (0.5-4.9); FT4, 13 pmol/L (9-16); and IGF-1, 20 nmol/L (19-130). He started school one year delayed with special educational facilitation. From lower secondary school he attended a school providing adapted education and completed a vocational education at upper secondary level. At admission to the outpatient thyroid clinic he had abdominal obesity, increased head circumference [23, 30], a broad face and chest, broad hands and feet with short fingers and toes, a pigmented area on his back and upper abdomen, numerous moles and a few skin tags on his back and face (Table 1). His thyroid was barely palpable and without nodules. The patient complained of tiredness, he usually went to sleep at 11 Pm, woke up at noon the next day, and had excessive daytime sleepiness. He could fall asleep for hours during the daytime when sitting down without anything to occupy him. He had problems with concentration, and information was retained only if repeated several times. He had always suffered from constipation, with hard stool every second day and straining when passing a stool. He used antihistamine for hay fever as needed and aripiprazole (Abilify) 400 mg every fourth week from 20 years of age for paranoid delusions. The Mother (nonaffected family member) was 60 years old at admission. She has a history of gestational diabetes, cardiovascular disease, and fibromyalgia. She presented normal clinical characteristics, normal thyroid function, and normal biochemical findings except for lipoprotein(a) and HbA1c (Tables 1-3). All her children were born approximately 14 days overdue. Discussion In this paper we present the clinical and laboratory data of 4 first-degree-related patients with RTHα. RTHα is a rare genetic disease and this is the first family in Norway reported to have a pathogenic THRA variant. Thyroid hormone resistance can be caused by variants in THRB or THRA. The incidence of RTHβ has been estimated to be 1:40 000 [34]; symptoms vary from asymptomatic to debilitating. Although RTHβ is rare, the condition is readily recognized due to elevated thyroid function tests with nonsuppressed TSH. The incidence of RTHα is unknown, as pathogenic variants in THRA are reported much less frequently than in THRB. The condition is rarely suspected due to normal levels of TSH and FT4. Symptoms of RTHα are caused by T3-resistance in tissues where TRα is the predominant receptor. The phenotype of RTHα is not univocal and patients present with a varying degree of delayed development of skeletal, motor, and neuropsychological function [35]. The 3 siblings in our study and their father presented similar features; short stature, short limbs, increased head circumference, and numerous moles. When the index patient was born, the mother immediately recognized common facial features and body compositions of the affected father and siblings. We therefore hypothesize that there may be visible signs present already at birth that may aid early identification of RTHα. Furthermore, clinical features were characterized by delayed language development, motor dyscoordination, fatigue, constipation, and obesity. Delayed language development, obesity, low height, and learning disabilities were the main reasons for early assessment in the specialist health care. All 4 patients were tested for thyroid function during childhood or adolescence. Due to normal TSH and FT4, hypothyroidism was considered less likely although the patients presented typical symptoms. Attempts to reveal a genetic etiology were performed over a timespan of 5 years until the disease-causing THRA etiology was disclosed by analysis of a gene panel for intellectual disability of the index patient at age 18 years. Thereafter, extensive thyroid hormone testing at admission to the outpatient thyroid clinic revealed decreased FT4/FT3 ratio (Fig. 3) and rT3 compared with healthy controls. Similar results are reported by others [12, 36]. Previously, the same variant was reported in 4 patients with hypothyroidism from 2 separate families, and functional studies showed that p.(Ala263Val) resulted in reduced T3-binding and inhibited the transcriptional activity of the wild-type receptor in a dominant negative manner [12, 36]. Furthermore, a different substitution of the same amino acid, p.(Ala263Ser), was reported in 7 patients from a family with mild symptoms of hypothyroidism [13]. Based on this, the c.788C > T, p.(Ala263Val) variant was classified as pathogenic according to ACMG-AMP guidelines [37]. This is the third reported family with the missense variant c.788C > T, p.(Ala263Val) in THRA. In the first family, 3 patients, a mother and 2 sons, were treated with levothyroxine from early childhood [12]. The mother had tentatively been treated as a toddler with levothyroxine which improved her growth and constipation. When 2 of her sons at age 3 years presented similar features and symptoms as their mother, they were also offered levothyroxine treatment, although blood tests could not confirm hypothyroidism. In the second family, levothyroxine treatment was started in a 17-year-old relatively asymptomatic boy in whom the mutation was diagnosed during investigation for possible delayed puberty [36]. We note that the index patient scored below 1 SD from the mean for 3 auditory tasks when evaluated as a child. Recently, THRA was implicated in the normal development of hearing in mice [38], and the authors suggested THRA patients may present with mild hearing loss. According to the reported RTHα cases listed in the Human Gene Mutation database professional (HGMD Pro), accessed September 21, 2022, treatment with thyroxine has been tried in 21 patients with 14 different THRA mutations. Thyroxine treatment was started before the age of 3 years in 6 patients, 3 patients started treatment as adults, and 15 patients in childhood or adolescence [7, 8, 11, 12, 36, 39-46]. Treatment of patients with mutations in the C-terminal segment of the ligand-binding domain or in patients with nonsense or frameshift variants gave limited or no benefit [39-41]. Treatment seems to give better response when started at an early age and in patients with missense variants located in the LBD of THRA common for the TRα1 and TRα2 isoforms [11, 42]. Treatment with levothyroxine (LT4) in early childhood is reported to have a beneficial effect on growth and development, improvement of motor dyscoordination, constipation, and increase basal metabolism. To date, effects of LT4 have not been clearly demonstrated on heart rate, and only a limited effect has been shown on cardiac function measured by echocardiography [39, 40]. The previously untreated adult patients presented in this study have deficits caused by pathogenic variants in the LBD of THRA common for the TRα1 and TRα2 isoforms. Thyroxine may have less benefit in untreated adult patients with such RTH mutations and to which extent they will benefit from thyroxine treatment remains to be seen. Both bone and the central nervous system develop most rapidly before 3 years of age, and for patients with RTHα it may be crucial to start high dosage levothyroxine treatment as infants. The effects of LT4 treatment depend on the severity and the location of the mutation. For those who have responded favorably, the treatment has increased growth and basal metabolism, ameliorated constipation, improved concentration, alertness, muscle coordination, and lipid profile and reduced weight. It is unclear whether the treatment improves cognitive function. There is limited knowledge of the effect of liothyronine (LT3) treatment in this patient group [8]. It remains to further test the effect of treatment with LT4 or LT3 in adults with RTHα. Results from patient-related outcome measures have not been published. The assumption that treatment with LT4 from childhood in patients with RTHα may ameliorate patients’ phenotype is supported by this study. The 4 previously described patients with the same THRA variant seem to have had milder symptoms than the 4 untreated adult patients assessed in the present study, who all presented varying degrees of constipation, tiredness, fatigue, concentration problems, social dysfunction, low mood, and obesity. More pronounced symptoms might also reflect different phenotypes in patients with the same variant in THRA. Conclusion Patients with RTHα typically present normal thyroid function tests when testing only TSH and FT4. However, typical clinical features together with symptoms of hypothyroidism and specific laboratory findings (low FT4/FT3 ratio, high FT3) should raise suspicion of RTHα and be followed by genetic testing (sequencing of the THRA gene). The importance of early diagnosis is crucial as treatment in early childhood might improve growth and neuropsychological development. Further studies are needed to assess potential effects of levothyroxine treatment when started in adulthood. Acknowledgments The authors thank the patients and their family for participating in this study. The authors would like to thank Robin Holtedahl for English editing of the manuscript. Funding This study was funded by the author's institution and a small grant from the Norwegian patient organization for thyroid disorders (Stoffskifteforbundet). S.S.H. has previously received a grant from South-Eastern Norway Regional Health Authority. Author Contributions All authors contributed to the design, collation of data and manuscript preparation. Disclosures The authors declare no conflicts of interest. Data Availability All data are presented in the article. Abbreviations BH birth height BMI body mass index BW birth weight FT3 free triiodothyronine FT4 free thyroxine HDL high-density lipoprotein IGF-1 insulin-like growth factor 1 LBD ligand-binding domain LDL low-density lipoprotein RTHα/β resistance to thyroid hormone alpha/beta T3 triiodothyronine T4 thyroxine THRA thyroid hormone receptor alpha TR thyroid hormone receptor TSH thyrotropin (thyroid stimulating hormone) ==== Refs References 1 Wejaphikul K , GroenewegS, Hilhorst-HofsteeY, et al Insight into molecular determinants of T3 vs T4 recognition from mutations in thyroid hormone receptor α and β. J Clin Endocrinol Metab. 2019;104 (8 ):3491‐3500.30817817 2 Paisdzior S , KnierimE, KleinauG, et al A new mechanism in THRA resistance: the first disease-associated variant leading to an increased inhibitory function of THRA2. Int J Mol Sci. 2021;22 (10 ):5338.34069457 3 Cheng S-Y , LeonardJL, DavisPJ. Molecular aspects of thyroid hormone actions. Endocr Rev. 2010;31 (2 ):139‐170.20051527 4 Mendoza A , HollenbergAN. New insights into thyroid hormone action. Pharmacol Ther. 2017;173 :135‐145. 10.1016/j.pharmthera.2017.02.012 28174093 5 Moran C , ChatterjeeK. Resistance to thyroid hormone due to defective thyroid receptor alpha. Best Pract Res Clin Endocrinol Metab. 2015;29 (4 ):647‐657.26303090 6 Minakhina S , BansalS, ZhangA, et al A direct comparison of thyroid hormone receptor protein levels in mice provides unexpected insights into thyroid hormone action. Thyroid. 2020;30 (8 ):1193‐1204.32122258 7 Furman AE , DumitrescuAM, RefetoffS, WeissRE. Early diagnosis and treatment of an infant with a novel thyroid hormone receptor α gene (pC380SfsX9) mutation. Thyroid. 2021;31 (6 ):1003‐1005.33198587 8 Espiard S , SavagnerF, FlamantF, et al A novel mutation in THRA gene associated with an atypical phenotype of resistance to thyroid hormone. J Clin Endocrinol Metab. 2015;100 (8 ):2841‐2848.26037512 9 Markossian S , GuyotR, RichardS, et al CRISPR/Cas9 editing of the mouse THRA gene produces models with variable resistance to thyroid hormone. Thyroid. 2018;28 (1 ):139‐150.29205102 10 Sun H , WuH, XieR, et al New case of thyroid hormone resistance α caused by a mutation of THRA/TRα1. J Endocr Soc. 2019;3 (3 ):665‐669.30842990 11 le Maire A , Bouhours-NouetN, SoamalalaJ, et al Two novel cases of resistance to thyroid hormone due to THRA mutation. Thyroid. 2020;30 (8 ):1217‐1221.32204686 12 Moran C , AgostiniM, VisserWE, et al Resistance to thyroid hormone caused by a mutation in thyroid hormone receptor (TR)α1 and TRα2: clinical, biochemical, and genetic analyses of three related patients. Lancet Diabetes Endocrinol. 2014;2 (8 ):619‐626.24969835 13 Demir K , van GuchtAL, BuyukinanM, et al Diverse genotypes and phenotypes of three novel thyroid hormone receptor-alpha mutations. J Clin Endocrinol Metab. 2016;101 (8 ):2945‐2954.27144938 14 Dahll LK . Supplementary material. Dataset. List of genes included in the gene panel for intellectual disability. figshare. Posted May 18, 2023. 10.6084/m9.figshare.22931303.v1 15 Lek M , KarczewskiKJ, MinikelEV, et al Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016;536 (7616 ):285‐291.27535533 16 Genomes Project C , AutonA, BrooksLD, et al A global reference for human genetic variation. Nature. 2015;526 (7571 ):68‐74.26432245 17 Roid GH , MillerLJ. Leiter International Performance Scale-Revised (Leiter-R). Stoelting; 1997:10 18 Wechsler D . Wechsler Intelligence Scale for Children (3rd ed.). (WISC-III): Manual. San Antonio, TX: The Psychological Corporation; 1991. 19 Kirk SA , KirkWD, McCarthyJJ. Illinois Test of Psycholinguistic Abilities. University of Illinois Press; 1968. 20 Hammerstad, Sara Salehi (2023). Supplemantary Figure 1: Growth Curve. figshare. Figure. 10.6084/m9.figshare.23623569.v2 21 Knudtzon J , WaalerPE, SkjaervenR, SolbergLK, SteenJ. [New Norwegian percentage charts for height, weight and head circumference for age groups 0–17 years]. Tidsskr Nor Laegeforen. 1988;108 (26 ):2125‐2135.3206414 22 Waaler PE . Anthropometric studies in Norwegian children. Acta Paediatr Scand Suppl. 1983;308 :1‐41.6581680 23 Juliusson PB , RoelantsM, NordalE, et al Growth references for 0–19 year-old Norwegian children for length/height, weight, body mass index and head circumference. Ann Hum Biol. 2013;40 (3 ):220‐227.23414181 24 Jarzem PF , GledhillRB. Predicting height from arm measurements. J Pediatr Orthop. 1993;13 (6 ):761‐765.8245203 25 Fredriks AM , van BuurenS, van HeelWJ, Dijkman-NeerincxRH, Verloove-VanhorickSP, WitJM. Nationwide age references for sitting height, leg length, and sitting height/height ratio, and their diagnostic value for disproportionate growth disorders. Arch Dis Child. 2005;90 (8 ):807‐812.15863466 26 Meyer HE , TverdalA. Development of body weight in the Norwegian population. Prostaglandins Leukot Essent Fatty Acids. 2005;73 (1 ):3‐7.15916890 27 Bushby KM , ColeT, MatthewsJN, GoodshipJA. Centiles for adult head circumference. Arch Dis Child. 1992;67 (10 ):1286‐1287.1444530 28 Holmen J , HolmenTL, TverdalA, HolmenOL, SundER, MidthjellK. Blood pressure changes during 22-year of follow-up in large general population - the HUNT study, Norway. BMC Cardiovasc Disord. 2016;16 (1 ):94.27176717 29 Giles E , VallandighamPH. Height estimation from foot and shoeprint length. J Forensic Sci. 1991;36 (4 ):1134‐1151.1919473 30 Statistics Norway, Yearbook 2013, Table 1 Conscripts, by height and weight (SY 108); 2013. 31 Rybak A , BentsD, KrugerJ, GrothD. The end of the secular trend in Norway: spatial trends in body height of Norwegian conscripts in the 19(th), 20(th) and 21(st) century. Anthropol Anz. 2020;77 (5 ):415‐421.32588018 32 Sundal A . The Norms for Height (Length) and Weight in Healthy Norwegian Children from Birth to 15 Years of Age. Universitetet i Bergen Årbok 1957 Medisinsk rekke Nr 1. A.S. John Griegs Boktrykkeri Bergen; 1957. 33 Brandkvist M , BjorngaardJH, OdegardRA, et al Genetic associations with temporal shifts in obesity and severe obesity during the obesity epidemic in Norway: a longitudinal population-based cohort (the HUNT study). PLoS Med. 2020;17 (12 ):e1003452. 34 Lafranchi SH , SnyderDB, SesserDE, et al Follow-up of newborns with elevated screening T4 concentrations. 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Sci Signal. 2022;15 (738 ):eabj4583. 39 Bochukova E , SchoenmakersN, AgostiniM, et al A mutation in the thyroid hormone receptor alpha gene. N Engl J Med. 2012;366 (3 ):243‐249.22168587 40 Moran C , SchoenmakersN, AgostiniM, et al An adult female with resistance to thyroid hormone mediated by defective thyroid hormone receptor alpha. J Clin Endocrinol Metab. 2013;98 (11 ):4254‐4261.23940126 41 Tylki-Szymańska A , Acuna-HidalgoR, Krajewska-WalasekM, et al Thyroid hormone resistance syndrome due to mutations in the thyroid hormone receptor α gene (THRA). J Med Genet. 2015;52 (5 ):312‐316.25670821 42 van Gucht AL , MeimaME, Zwaveling-SoonawalaN, et al Resistance to thyroid hormone alpha in an 18-month-old girl: clinical, therapeutic, and molecular characteristics. Thyroid. 2016;26 (3 ):338‐346.26782358 43 van Mullem A , van HeerebeekR, ChrysisD, et al Clinical phenotype and mutant TRα1. 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==== Front Eur Heart J Case Rep Eur Heart J Case Rep ehjcr European Heart Journal: Case Reports 2514-2119 Oxford University Press US 10.1093/ehjcr/ytad283 ytad283 Case Report Valvular Heart Disease AcademicSubjects/MED00200 Ehjcr/2 Ehjcr/12 Ehjcr/45 Ehjcr/36 Ehjcr/14 Mitral valve repair with papillary muscle repositioning for functional mitral regurgitation (Type IIIb) with metal allergies: a case report https://orcid.org/0000-0001-5851-2736 Mitsuishi Atsuyuki Department of Cardiovascular Surgery, Kochi Medical School Hospital, 185-1, Kohasu, Okohcho, Nankoku-shi, Kochi Prefecture 783-8505, Japan Miura Yujiro Department of Cardiovascular Surgery, Kochi Medical School Hospital, 185-1, Kohasu, Okohcho, Nankoku-shi, Kochi Prefecture 783-8505, Japan https://orcid.org/0000-0001-5681-8854 Kubo Toru Department of Cardiology and Geriatrics, Kochi Medical School Hospital, 185-1, Kohasu, Okohcho, Nankoku-shi, Kochi Prefecture 783-8505, Japan Sinning Christoph Handling Editor EL-Andari Ryaan Editor Lorusso Roberto Editor Gobolos Laszlo Editor Zhu Michael Z L Editor Athithan Lavnya Editor Waight Michael Editor Corresponding author. Tel +81-88-880-2375 (Office), +81-88-866-5811 (Hospital), Email: atmitsu@kochi-u.ac.jp Conflict of interest: None declared. 7 2023 26 6 2023 26 6 2023 7 7 ytad28328 12 2022 01 1 2023 22 6 2023 18 7 2023 © The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Abstract Background Prosthetic heart valves, rings, and clips commonly used in heart surgery may contain metals, such as nickel and cobalt, that can cause severe hypersensitivity reactions in allergic patients. These reactions can cause paravalvular leakage and valve dysfunction. Similarly, stainless steel sternal wires can cause contact dermatitis. We should select rings, valves, and wires that do not contain any metals known to cause allergies in patients undergoing cardiac surgery. Case summary We report the case of a 79-year-old man with severe functional mitral regurgitation (Type IIIb) and a history of nickel and cobalt allergies. We safely performed mitral valve repair with papillary muscle repositioning with nickel- and cobalt-free rings in this patient. He was discharged from the hospital on the 26th postoperative day without dialysis intervention. Two years after surgery, mitral and tricuspid regurgitation had not worsened. Discussion According to the 2020 American Heart Association guidelines, surgery for severe functional mitral valve insufficiency (Type IIIb) is considered class IIb. Meanwhile, transcatheter edge-to-edge repair is class IIa. Long-term regurgitation is difficult to control with valve replacement and annuloplasty alone; recurrence has been observed. Therefore, additional techniques were considered. Papillary muscle repositioning has been reported and shown good results. The method used in the present case made intervening in the subvalvular tissue easy and demonstrated technical feasibility, safety, and effectiveness. Functional mitral regurgitation Metal allergies Papillary muscle repositioning Spiral suspension Mitral valve repair Case report ==== Body pmcLearning points Patients with metal allergies and requiring valvular repair/replacement surgery pose a unique clinical challenge. We safely performed mitral valve repair with papillary muscle repositioning with nickel- and cobalt-free rings to treat a patient with functional mitral regurgitation (Type IIIb) and nickel and cobalt allergies. Introduction Prosthetic heart valves, rings, and clips commonly used in heart surgery may contain metals, like nickel and cobalt, that cause severe hypersensitivity reactions in allergic patients. These reactions can result in paravalvular leakage, leading to valve dysfunction.1 Similarly, contact dermatitis, including pruritus or pain in the sternum, can develop in reaction to monofilament stainless steel sternum wire. Therefore, in patients with known metal allergies, the use of artificial valves, clips, and sutures containing any metal allergens should be avoided. According to the 2020 American Heart Association (AHA) guidelines,2 surgery for severe functional mitral valve insufficiency (Type IIIb) is considered class IIb; transcatheter edge-to-edge repair (TEER) is class IIa. Long-term regurgitation is difficult to control with valve replacement and annuloplasty alone,3,4 and recurrence has been observed. Therefore, additional techniques were considered. Papillary muscle repositioning has been reported and shown good results. Herein, we safely performed mitral valve repair with papillary muscle repositioning using nickel- and cobalt-free rings to treat a patient with functional mitral regurgitation (Type IIIb) and nickel and cobalt allergies. Summary Figure Twelve years before admission to our hospital Echocardiography: left ventricular end-diastolic (LVEDD) and end-systolic diameters (LVESD) were 63 and 45 mm, respectively. Ejection fraction (EF) of 49%, mild mitral regurgitation (MR), and cardiac dysfunction were observed. Medical treatment was initiated. Six years before admission to our hospital Echocardiography: LVEDD and LVESD had improved to 57 and 45 mm, respectively. EF was approximately 60%; however, taking oral medication became difficult due to traumatic subdural haematoma, and MR gradually worsened. One year before admission to our hospital MR became severe, and the patient was hospitalized with symptoms of heart failure. Heart failure improved with medical treatment. Five months before admission to our hospital Dyspnoea on exertion and pulmonary hypertension developed, which were improved with medical treatment. One month before admission to our hospital Dyspnoea on exertion worsened, and the patient was readmitted to the hospital. Due to recurrent heart failure, an elective surgery was planned after the previous hospital admission, following the successful management of heart failure. Postoperative day (POD) 0: The patient was extubated. POD 13: LVEDD and LVESD had improved to 62 and 53 mm, respectively. EF was approximately 40%, and MR was trivial. POD 25: The patient was discharged from the hospital. Case presentation The patient was a 79-year-old man with a history of methicillin-resistant Staphylococcus aureus infection (acquired during brain surgery), mitral regurgitation (MR), chronic atrial fibrillation for 9 years, and decreased left ventricular contractility. He had eight long-term hospitalizations during which he underwent multiple operations. Metal allergies to nickel (Ni) and cobalt (Co) were detected 8 years prior. The MR gradually worsened and became more severe. Dyspnoea on exertion appeared approximately 2 years prior and gradually worsened. One year prior, he was hospitalized for heart failure. Soon after discharge, he gained weight and experienced dyspnoea on exertion, accompanied by lower-leg oedema. Echocardiography showed severe MR, reduced ejection fraction (EF, 50%), and deterioration of pulmonary hypertension (pulmonary pressure: 37/16/25 mmHg). Because of recurrent heart failure, an elective surgery was planned after the previous hospital admission, following the successful management of heart failure. Preoperative echocardiography showed the following: left ventricular internal end-diastolic (LVEDD) and end-systolic diameters (LVESD) of 68 and 48 mm, respectively—EF of 45%, severe MR with central jet (tenting height, 12 mm) (Figures 1 and 2A), moderate tricuspid regurgitation (TR) (tenting height, 8 mm) (Figure 2B), a left atrial diameter of 66 mm, and progressive MR with increased diuretics. Using coronary angiogram and technetium-99m, cardiologists ruled out ischaemia and amyloidosis as the cause of LV dysfunction. Although the LV size was large, the LVEF was over 40% and the wall thickness was approximately 13 mm. Therefore, it was not a typical dilated cardiomyopathy (DCM); yet DCM could not be excluded as the cause of LV dysfunction. Figure 1 Mitral regurgitation with central jet. Figure 2 (A) Mitral valve; tenting height 11.1 mm and annulus 39 mm. (B)Tricuspid valve; tenting height 9.3 mm, tenting area 2.83 mm2, and annulus 49 mm. Prosthetic valves, rings, and TEER containing Ni and Co were contraindicated. Additionally, the sternal closure required a Ni- and Co-free sternal wire. We selected Ni- and Co-free rings for mitral and tricuspid annuloplasty. We used titanium wire for sternal closure as stainless steel can contain traces of nickel. Cardiopulmonary bypass was established through median sternotomy using standard aortic and bicaval cannulation. Antegrade blood cardioplegia was administered, and the left atrium was accessed via a transseptal approach. No chord rupture was observed. Left atrial ablation was performed using a modified maze procedure with cryoablation. The left atrial appendage was then resected. A 28-mm Ni- and Co-free Séguin Ring (Séguin Semi-Rigid Annuloplasty Ring; Abbot Medical, Austin, TX, USA) was chosen for mitral valve repair. After the annular mattress sutures were tied, they were passed around the annuloplasty ring again, taking additional bites of atrial tissue and tied again (the double-suture technique). The tricuspid valve was examined after the closure of the interatrial septum. The diameter of the annulus was 55 mm. In addition to annulus enlargement, tethering was also contributing to regurgitation. Subsequently, a pledgeted CV4 suture (GORE-TEX® Suture; W. L. Gore & Associates Inc., Newark, DE, USA) was placed at the base of the anterior papillary muscle (PM) and continuously sutured clockwise to the base of the secondary and then inferior PM. The CV4 suture crossed the interventricular septum and externalized the septal ring of the right atrium. After tricuspid annuloplasty using a 27-mm Ni- and Co-free ring (Duran Flexible Partial Ring; Medtronic Inc., Minneapolis, MN, USA) the CV4 suture was passed through the ring. Suture length adjustments were performed under a water test, and the sutures were tied (spiral suspension technique). Next, a pledgeted CV4 suture was passed through the head of the anterior and posterior PM via an aortotomy. It was passed from the left ventricular cavity through the mid-septal fibrous annulus and exteriorized through the aortic wall beneath the commissure between the non-coronary and left coronary aortic cusps (PM repositioning) (Figures 3A and B). Aortotomy was closed with two layers of continuous sutures, and the sternum was closed with a pure titanium wire (Yokozuna wire; USCI Holdings, Tokyo, Japan). Transoesophageal echocardiography confirmed that the MR and TR had disappeared. Postoperative transthoracic echography revealed a trivial MR (tenting height, 8 mm) and disappearance of TR. Figure 3 (A, B) Papillary muscle repositioning; a pair of CV-4 sutures was passed through the anterior and posterior papillary muscles (APM and PPM, respectively), and papillary muscle refixation and ligation were added through the aorto-mitral continuity and aortic valve. After undergoing treatment for heart failure in the cardiology department, the patient was discharged from the hospital on the 26th postoperative day without dialysis intervention. Two years after surgery, MR and TR had not worsened on echocardiography (Figure 4A, B). Figure 4 Mitral valve echocardiography (A) preoperative image. (B) Postoperative image. Discussion Prosthetic heart valves, rings, and clips commonly used in heart surgery may contain metals that can cause severe hypersensitivity reactions in allergic patients. These reactions can cause paravalvular leakage, leading to valve dysfunction.1 Herein, we report the case of a patient with functional MR (Carpentier Type IIIb) and metal allergies. Guidelines recommend TEER (American College of Cardiology (ACC)/AHA Guideline class IIa) and mitral valve surgery (ACC/AHA Guideline class IIb) for patients with functional MR.2 However, TEER (MitraClip™; Abbot Medical) could not be used due to the patient’s allergy. Patients with severe nickel allergies have been reported to be discharged from the hospital without serious complications following aortic valve replacement with On-X Aortic Valve (CryoLife, Kennesaw, GA, USA).5 Valve replacement can produce a short pump run and has a low risk of a re-run. However, because of the patient’s history of subdural haematoma, we avoided using a mechanical valve which would involve taking warfarin. Also, all biological valves (Table 1) available in Japan contain Ni, Co, or alloys, excluding them from consideration for this patient. Table 1 Metal components of surgical materials in Japan Mitral bioprosthetic valve MITRIS RESILIA Ni, Co, Cr, Mo, Mn, C, Be, Fe EPIC Ni, Co, Mo, Mn Mosaic alloy (Co, Cr, W, Ni, Fe, Mn, Si, C) Mitral mechanical valve SJM Regent/Masters Ni/Co alloy, Cr, Mo, W SJM Standard No metal ATS AP 360 C, Ti alloy (N, C, Fe, Al, Ba, Ti), Ni/Co/Cr alloy ATS Standard Ti alloy (N, C, Fe, Al, Ba, Ti) ON-X Ti alloy (Ti, Al, V, N, C, H, Fe, O) Ring or band for mitral valve Profile 3D Ring Ti alloy CGFuture Ring & Band Ni, Co, Cr, Mo alloy Duran ring No metal Simu Form Ni/Co/Cr/Mo alloy Simu Plus ring W PhysioII (5200) Ni, Co, Cr PhysioFlex (5300) Ni/Ti alloy Rigid Saddle Ring Metal Free Seguin Metal Free Ring or band for tricuspid valve Contour-3D Ti alloy Tri-ad2.0 ring Ni/Co/Cr/Mo alloy MC3(4900) Ti alloy, Al, Ba PhysioTricuspid (6300) Ti alloy, Al, Ba MEMO 3D/MEMO 4D Ni, Co, Cr, Cu. C, Fe, Ti, Nb Ring or band for mitral & tricuspid valve Cosgrove (4600) Metal free Tailor Ring & Band Metal free Duran Ring & Band Metal free Clip MitraClip Co/Cr alloy, Ni/Ti alloy Wire Cosmo wire  Ni, Cr, Mo, Fe alloy Yokozuna  Ti Ni, nickel; Co, cobalt; Cr, chromium; Mo, molybdenum; Mn, manganese; Be, beryllium; Fe, iron; W, tungsten; Ti, titanium; Ni, nickel; Si, silicon; C, carbon; Al, aluminium; Ba, barium; V, vanadium; N, nitrogen; H, hydrogen; O, oxygen; Cu, copper; Nb, niobium. As the metal components of alloys are often unclear, we selected rings without alloys, Ni, or Co and those we had used before. However, it is difficult to control regurgitation during the long-term period and recurrence has been reported.3,4 Therefore, techniques other than annuloplasty are being considered in cases where valves cannot be used. PM repositioning has shown good results,6,7 although neo-chord implantation in combination with a stabilizing annular ring can be quicker and less extensive. Thus, PM repositioning was added in this case to prevent long-term regurgitation recurrence. Posterior PM repositioning alone may be sufficient for ischaemic functional MR.8 However, in this case, due to LV systolic dysfunction of unknown aetiology, the possibility of DCM was considered and both anterior and posterior PM repositioning were performed in accordance with Langer et al.9 Additionally, in TR with severely tethered leaflets, repair with ring annuloplasty alone cannot prevent residual or recurrent TR. Rigid ring annuloplasty showed a lesser incidence of late TR recurrence than other valve repairs, including ring-spare techniques such as De Vega.10 Eishi et al.11 reported a spiral suspension technique that approximates the anterior PM, accessory PM for the inferior leaflet (inferior accessory PM), and inferior PM. We used this technique and rigid ring annuloplasty to treat TR with severely tethered leaflets. Additionally, attention was paid to the sternal closure in this patient, which required a Ni- and Co-free sternal wire. A few cases with metal allergies where alternative sutures were used instead of monofilament stainless steel in cardiac surgery have been reported.12,13 However, these alternative materials have problems with strength. Considering the price in Japan, we used a wire made of pure titanium instead of a sternal plate. Titanium wire (Yokozuna wire; USCI Holdings) has a tensile strength of ≥160% (strength after fastening the thoracic bone model) compared to the stainless wire that was selected. In conclusion, we performed mitral valve repair, with additional subvalvular surgical techniques for long-term regurgitation control, to treat functional MR in a patient with Ni and Co allergies. Follow-up with medium- to long-term evaluations, assessing for MR exacerbation and left ventricular re-expansion, is important. The methods used in this case made intervening in the subvalvular tissue easy and demonstrated technical feasibility, safety, and effectiveness. Acknowledgements We would like to thank Elsevier Language (https://webshop.elsevier.com) for the English language editing. Consent: The authors confirm that written consent for submission and publication of this case report including image(s) and associated text has been obtained from the patients in line with COPE guidance. Funding: This study received no funding from private, public, or not-for-profit agencies. Lead author biography I’ve graduated from Niigata university, Niigata, Japan. I’m working at Kochi Medical School Hospital, Kochi, Japan. My main area of interest includes cardiovascular surgery. Data availability No new data were generated or analysed in support of this research. ==== Refs References 1 Lyell A , BainWH, ThomsonRM. Repeated failure of nickel-containing prosthetic heart valves in a patient allergic to nickel. Lancet 1978;2 :657–659.80580 2 Otto CM , NishimuraRA, BonowRO, CarabelloBA, ErwinJPIII, GentileF, et al 2020 ACC/AHA guideline for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 2021;143 :e72–e227.33332150 3 Goldstein D , MoskowitzAJ, GelijnsAC, AilawadiG, ParidesMK, PerraultLP, et al Two-year outcomes of surgical treatment of severe ischemic mitral regurgitation. N Engl J Med 2016;374 :344–353.26550689 4 Warwick R , PullanM, PoullisM. Mathematical modelling to identify patients who should not undergo left ventricle remodelling surgery. Interact Cardiovasc Thorac Surg 2010;10 :661–665.20093268 5 Lusini M , BarbatoR, SpadaccioC, ChelloM. Aortic valve replacement in a patient with severe nickel allergy. J Card Surg 2011;26 :618–620.21929741 6 Langer F , SchäfersHJ. RING Plus STRING: papillary muscle repositioning as an adjunctive repair technique for ischemic mitral regurgitation. J Thorac Cardiovasc Surg 2007;133 :247–249.17198821 7 Langer F , KuniharaT, HellK, SchrammR, SchmidtKI, AicherD, et al RING+STRING: successful repair technique for ischemic mitral regurgitation with severe leaflet tethering. Circulation 2009;120 :S85–S91.19752391 8 Levine RA , SchwammenthalE. Ischemic mitral regurgitation on the threshold of a solution from paradoxes to unifying concepts. Circulation 2005;112 :745–758.16061756 9 Langer F , KuniharaT, MiyaharaS, FahrigL, BlümelM, KlärA, et al Bilateral papillary muscle repositioning: successful repair of functional mitral regurgitation in dilative cardiomyopathy. Eur J Cardiothorac Surg 2020;57 :285–292.31364693 10 Sohn SH , KimKH, LeeY, ChoiJW, HwangHY. Long-term outcomes of rigid ring versus De Vega annuloplasty for functional tricuspid regurgitation: a propensity score-matching analysis. J Thorac Cardiovasc Surg 2021;161 :1788–1798.e5.31948739 11 Eishi K , MiuraT, MatsumaruI, TanigawaK, ObaseK. Spiral suspension, a novel repair technique for severe functional tricuspid regurgitation. J Thorac Cardiovasc Surg 2018;156 :649–652.29530578 12 Barber FA , HerbertMA, RichardsDP. Sutures and suture anchors: update 2003. Arthroscopy 2003;19 :985–990.14608318 13 Keçeligil HT , KolbakırF, AkarH, KonuralpC, DemirZ, DemirağMK. Sternal closure with resorbable synthetic loop suture material in children. J Pediatr Surg 2000;35 :1309–1311.10999685

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==== Front Gerontologist Gerontologist geront The Gerontologist 0016-9013 1758-5341 Oxford University Press US 36652299 10.1093/geront/gnac190 gnac190 Workforce Issues AcademicSubjects/SOC02600 Developing the Occupational Communion Scale: Belonging-Based Social Connections Are Vital for Work Engagement, Self-Efficacy, and Positive Affect in Aged Care Workforces https://orcid.org/0000-0002-4726-5685 Elliott Kate-Ellen J PhD Wicking Dementia Research and Education Centre, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia School of Psychological Sciences, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia Quinn Michael G PhD School of Psychological Sciences, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia Stirling Christine M PhD School of Nursing, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia Sanderson Kristy PhD School of Health Sciences, University of East Anglia, Norwich, Norfolk, UK Robinson Andrew L PhD Wicking Dementia Research and Education Centre, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia Martin Angela J PhD Menzies Institute for Medical Research and Tasmanian School of Business and Economics, University of Tasmania, Hobart, Tasmania, Australia Scott Jennifer L PhD School of Psychological Sciences, College of Health and Medicine, University of Tasmania, Hobart, Tasmania, Australia Meeks Suzanne PhD, FGSA Decision Editor Address correspondence to: Kate-Ellen J. Elliott, PhD, University of Tasmania, School of Psychological Sciences, Private Bag 30, Hobart, Tasmania 7001, Australia. E-mail: KateEllen.Elliott@utas.edu.au 8 2023 18 1 2023 18 1 2023 63 6 10281038 27 1 2022 01 12 2022 18 3 2023 © The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Abstract Background and Objectives A multifaceted construct called occupational communion (OC), defined as a sense of belonging based on social interaction at work, has been proposed to understand why care workers were positively engaged in their jobs over time, even though they were very demanding. Rich qualitative data on the multiple aspects of OC in care work exist, but a valid measure does not. Research Design and Methods We applied a mixed-method systematic scale development process to measure OC. Aged and dementia care workers in Australia (76%) and other countries participated in a focus group and online surveys (N = 2,451). We also used interview data from our prior study. The study involved 3 components: (1) scale development and design; (2) pilot test validation with exploratory factor analysis; and (3) confirmatory validation via confirmatory factor analysis. The third component assessed convergent and discriminant validity using measures of communion, self-efficacy, work engagement, job and life satisfaction, intention to leave, positive and negative affect, and mood. Results We developed a 28-item Occupational Communion Scale (OCS) with good internal consistency (composite reliability = 0.75–0.91) across 6 factors: (1) “natural” carer, (2) psychological need to care, (3) connection with clients, (4) connection with coworkers, (5) desire for more connection, and (6) blurred boundaries. All validity measures correlated with OC and work engagement, self-efficacy, and positive affect showed the strongest association. Discussion and Implications The OCS can be used to design and evaluate interventions addressing aged care workforce engagement, social connections and well-being, and care outcomes. Caregiving—formal Measurement Psychometrics Well-being Workforce issues National Health and Medical Research Council 10.13039/501100000925 E0023237 Australian Research Council 10.13039/501100000923 University of Tasmania Research Enhancement E0021909 Wicking Dementia Research and Education Centre University of Tasmania 10.13039/501100001249 045135 ==== Body pmcResearch and development of care workforces is a current priority to manage the aging population and age-related conditions such as dementia (ACSWT, 2018; Pickett et al., 2018; Prince et al., 2016). Instability associated with high staff turnover is a problem for care services, with care failures linked to inadequate skilled labor supply in Australia, the United Kingdom, and the United States (Atkinson et al., 2018; Howe et al., 2012; Pagone & Briggs, 2021; Spetz et al., 2015). The coronavirus 2019 (COVID-19) pandemic has further exacerbated care workforce issues and strengthened the need to reinvest in care sectors (Meeks & Degenholtz, 2021). Localized solutions are highly contextual and consider the wide ecosystem of care is necessary to resolve the workforce crisis, particularly in the absence of substantive investment to redress the longstanding problems with job quality, remuneration, and low status of aged care work (Scales, 2021). Finding evidence-based ways to build capacity, knowledge, skills, coping, and interest in caring workforces is essential (Colombo et al., 2011; Elliott et al., 2012; Prince et al., 2016). Research that considers the psychosocial nature of the care work environment has much potential, especially given the evident emotional and interpersonal requirements of aged care work (Brenenbaum et al., 2017; King, 2012). Social and organizational psychological theories, such as communion (Bakan, 1966), and job demands–resources (Demerouti et al., 2001) that seek to understand interpersonal relationships and perceptions about social connections in the work context may offer insights for assessment and the design of future interventions. The term communion was coined alongside agency more than 50 years ago (Bakan, 1966), each reflecting fundamental modes of human existence corresponding to “getting along” or “getting ahead” (Able & Wojciszke, 2018; Helgeson, 1994). Communion (warmth, being focused on others) is a trait-like construct where individual fulfillment is experienced through close relationships and a sense of belonging by forming bonds (Able & Wojciszke, 2018; Guisinger & Blatt, 1994). Mutually rewarding interactions in close relationships can result in positive well-being when caring for others (measured by positive and negative affect and life satisfaction; Le et al., 2018). When the desire to care for others outweighs caring for the self, the consequences can be poor psychological health such as depressive symptoms (Helgeson & Fritz, 1998; called unmitigated communion). Similar findings may exist when caring relationships are bounded by psychological contracts of employment (O’Donohue & Nelson, 2009). Social connectedness with colleagues (de Oliveira Maciel & Camargo, 2016; van der Borg et al., 2017) and potential clients may have implications for workforce well-being and engagement in the job. Applying the concept of communion to work contexts is not new. Several studies have applied communion across a range of occupational arenas such as career development, motivation, success, and corporate social responsibility (Abele & Spurk, 2011; Chiaburu & Carpenter, 2013; Van Marrewijk, 2003), female careers and hierarchical structures (Ellery-Brown, 2011; Pringle & McCulloch-Dixon, 2003), and bullying and turnover in care organizations (Kim & Glomb, 2010; Regts & Molleman, 2013). Despite some exploration of communion in work contexts, no studies have adapted measures to suit work-specific relationships. Instead, these studies have mostly adopted self-report questionnaires with a personality trait approach, which was sometimes assessed as a subscale alongside other factors. For example, communion personality traits are often associated with relating to others, cooperation, empathy, agreeableness, global feelings of “we,” togetherness, and/or close social connection (Barrick et al., 2002; Spence & Helmreich, 1978). Communion values include compassion, humility, harmony, equality, and trust (Trapnell & Paulhus, 2012), as well as behaviors such as helping others (Buchanan & Bardi, 2015). Rarely have these self-reports focused on social connection with clientele, and the communion concept has not been tailored to the context of the psychosocial work environment. Traditionally, communion has been explored within familial, friendship, and intimate partner contexts rather than the occupational arena. The concept of communion has more recently been expanded to care work, by adding the perspective of employees with caring roles. The term communion was used instead of social connection or social support, because in comparison within the psychological literature, there was less consensus as to the precise definition of these terms (Kim et al., 2008; see Elliott et al., 2013, for further discussion). In an earlier study, we identified occupational communion (OC) as a multifaceted concept and defined OC “as a sense of belonging based on social interaction at work that can assist adaptive coping” (Elliott et al., 2013, p. 771). This research builds on our earlier qualitative study of capacity building in Australian aged and dementia care workers (Elliott et al., 2013) by quantifying OC on a scale. Close relationships, being well suited to the job, and caring for the self as well as clients, were reported characteristics in the aged care workforce and were found to influence well-being, self-efficacy (e.g., confidence in abilities), job attraction, and satisfaction (Elliott et al., 2016; Peng & Mao, 2015; Piercy, 2000; Sheridan & Agim, 2014; Xiao et al., 2021). These aspects of care work relate to communion because they represent a care worker’s intrinsic focus on helping others and forming social bonds, which can be personally rewarding and motivating. Communion in the context of aged care work appears to have a nuanced expression of the purpose behind this orientation toward others. When we described OC in our qualitative study (Elliott et al., 2013), we highlighted that those social interactions, and togetherness with coworkers and clients, were considered as psychological needs that were met at work but not in personal lives. Positivity was experienced when relationships with clients were rewarding and workers could build trust, and advocate for and meet their clients’ needs, but opportunities for social connection with colleagues were lacking (Elliott et al., 2013). The application of communion to care work has, therefore, meant the concept is changing to account for additional aspects specific to professional caring relationships, which may not be mutual, or reciprocal (i.e., when workers feel that they cannot burden clients with their own personal worries or concerns; Elliott et al., 2013). Elliott et al. (2013) proposed a conceptual model of OC (see Supplementary Figure 1) that explains possible relationships with other constructs relevant for occupational health and well-being, such as job demands–resources and capacity, and resilience indicators in care work. In this model, aspects of OC, such as social connections with colleagues and clients, can buffer against the job demands of grief and isolation, resulting in adaptive capacity such as confidence in abilities and positive emotions. There may also be implications for job resources such as work engagement. We detail, in this study, how we used our OC model to inform hypotheses and choose validation measures to explore the assessment of OC. The objective of this study was to develop and validate a measure of OC. We first hypothesized that a measure of OC would have multiple domains as displayed in the circle at the center of Supplementary Figure 1. These were positive, rewarding, and trusting close relationships with clients and coworkers; care and compassion for clients; being suited to the job; psychological need for social interaction; desire for coworker social connection and support; and motivation to help and advocate for clients, sometimes above one’s own needs. We expected that the multiple aspects of OC would correlate with a measure of general communion. Validation measures based on the conceptual model for job demands–resources variables included job satisfaction and work engagement (see Supplementary Figure 1). Capacity and resilience indicators were included as intent to leave, self-efficacy, mood and affect, and satisfaction with life. The measures were chosen to correlate with OC in aged care across some or all of the multiple aspects of the concept. The definition of OC includes an association between social interaction and positive adjustment at work. This is informed by literature on the influence of the psychosocial work environment on worker health and well-being (Finne et al., 2014) and communion and well-being from the perspectives of close partners and family (Helgeson & Fritz, 1998; Le et al., 2018). Therefore, psychometric testing of convergent and discriminant validity between OC and organizational health and well-being outcomes was examined. The relationships between OC, job demands and resources, and resilience may all have relevance for building capacity in care workforces (Elliott et al., 2018). Secondly, we hypothesized that indicators of resilience would be positively related to aspects of OC such as connectedness with coworkers and clients, employees’ strong sense of being suited to the job, a psychological need for social interaction, and togetherness, whereas resilience indicators would be negatively related to aspects of OC that reflect putting others’ needs above their own and when social connection with colleagues was lacking. We expected that measures of negative mood and or affect correlate better with aspects of OC that represent isolation from coworkers, more so than aspects of OC that relate to interactions with clients. Instead, parts of OC that represent social interactions with clients and advocating for their needs would be more likely to correlate with measures of work engagement, self-efficacy, and positive affect. This may represent that employees want more control over their jobs via changes in the psychosocial work environment (Elliott et al., 2017; Gleason & Miller, 2021) so that better opportunities are available to interact with colleagues, and that connections with clients are meaningful and meet a psychological need not fulfilled elsewhere. The aim of the research was to develop a scale to measure OC. This will enable the conceptual model of capacity and resilience in care work to be empirically tested and inform the development and evaluation of workforce interventions. The study involved three components. The first component was the scale development and design and applied a triangulated qualitative approach with data from review of the literature, interviews (from previously collected data on OC; Elliott et al., 2013), and a focus group. The second component involved an exploratory factor analysis (EFA) used to examine the scale factor structure. The third component was a confirmatory validation via confirmatory factor analysis (CFA) of the model developed using EFA and included convergent and discriminant validity testing. Method Participants In the first component, qualitative data were collected prior to this study by Elliott et al. (2013) from a convenience sample of community-based aged and dementia care workers (N = 25 interviews); mean age 53 years (SD = 9.6); majority (N = 22) female. Only the qualitative interview data that formed the theme called OC were used in the first component of the study. Face validity of OC was tested on another convenience sample of aged and dementia care workers (N = 7 focus group) based in a residential aged care facility. For the second component using EFA, the OC scale was piloted on Australian aged and dementia care workers (N = 329) recruited via a university-led Massive Open Online Course (MOOC) on Understanding Dementia from October 2014 to March 2015. Participants were invited through a link to a project webpage to complete an online survey about psychological adjustment to job roles and workplace supports. Additional sample characteristics are outlined in Table 1. Table 1. Sample Characteristics for the EFA and CFA Samples in Initial Validation of the Occupational Communion Scale. Variable EFA sample (N = 329) CFA samplea (N = 2,115) % Mean (SD) Range % Mean (SD) Range Age (in years) 49.3 (9.8) 22–67 47.7 (11.5) 18–76 Sex 95.7% 91.5%  Female  Male 4.3% 8.5% Highest education level  Year 10 or below 8% 7%  Year 11 or 12 7% 9%  Vocational certificate/diploma 54% 40%  University degree (incl. postgraduate) 27% 45%  Other/not specified 2% <1% Country of residence  Australia 100% 76%  New Zealand — 9%  Canada — 6%  United Kingdom and Ireland — 6%  Other Europe, Asia, Africa, South and Central America — <1% Length of time working in sector 8.3 (8.6) 8.7 (9.5) Length of time in current workplace 4.8 (5.6) 5.2 (6.4) Notes: CFA = confirmatory factor analysis; EFA = exploratory factor analysis; SD = standard deviation. aWhere percentages do not equal 100%, remainder is not specified. For the third component, the EFA factor structure was tested using CFA from a global sample of N = 2,115, recruited via the same university-led MOOC on Understanding Dementia, this time from August to December 2015, in which paid care workers were invited to complete an online survey about organizational health and well-being. Although most participants worked in Australia, 24% were from other countries. Additional sample characteristics are outlined in Table 1. Ethics approval was granted from the Tasmanian Social Sciences Human Research Ethics Network (H0013800) prior to beginning the studies and all participants gave their informed consent before participating. Materials For the second component involving EFA, the only questionnaire was the OC measure. The initial OC measure was 52 items rated on a 6-point Likert scale (0 = strongly disagree to 5 = strongly agree). For the third component applying CFA, participants completed the OC measure and other measures on organizational health and well-being. These measures included the assessment of convergent and discriminant validity (see Table 5 for names and descriptions of each measure). Procedure Item development occurred following a review of literature on communion applied to the work setting, close re-examination of qualitative data from semi-structured interviews describing OC, and a focus group. The first author developed an item pool from the interview data in the OC theme (see Elliott et al., 2013) and was then cross-referenced with existing items from measures of communion in non-work contexts. A total of 52 items were generated and a 4-point Likert scale was used (1 = strongly disagree to 4 = strongly agree) to rate statements relating to the past month. Items were then reviewed by five interdisciplinary experts (aged care nursing, community nursing, management, clinical psychology, and psychiatry) and the face validity of OC was tested in a focus group comprising a convenience sample of aged and dementia care workers (N = 7). The focus group discussed the definition of OC, then completed the scale and reflected on the item wording, comprehensibility, and ease of use. Based on the focus group qualitative comments, the response set was changed to a 6-point Likert rating, and respondents were asked to consider the previous month. The initial item pool of 52 items is available from the first author. For both the EFA and CFA samples, participants completed an online survey about adjustment to their job roles and workplace supports. Analyses Exploratory factor analysis A factor model for the Occupational Communion Scale (OCS) was developed from the initial 52 items using EFA using Mplus version 7 (Muthen & Muthen, 2012). Items were screened for normality and skew, and intercorrelations between items were calculated. Items showing multicollinearity (r > 0.85) or items that did not correlate with other items above r = 0.3 were removed. Items where there was minimal or no endorsement of one end of the Likert scale were removed. Following this, an EFA was conducted using robust weighted least squares (WLSMV) method of estimation and oblimin rotation. WLSMV is appropriate for categorical data such as Likert scales (Brown, 2015), and an oblique oblimin rotation was appropriate given OCS factors were expected to correlate. Initially, three- to seven-factor solutions were extracted and analyzed for statistical and theoretical fit. The appropriateness of EFA models was determined by the fit indices derived from Mplus. Mplus calculates χ2 likelihood ratios; however, χ2 can reject models with relatively small residual variance when there is a large sample size (see Brown, 2015, for an outline of the limitations of χ2). In view of this, additional fit indices were used to evaluate model fit. Mplus provides approximate (or practical) fit indexes for the Tucker–Lewis Index (TLI), the comparative fit index (CFI), and the root-mean-squared error of approximation (RMSEA). For the CFI and TLI, values of 0.95 or above were taken as indicating good model-data fit, with values above 0.90 indicative of acceptable fit (Brown, 2015; Hu & Bentler, 1999). Based on guidelines suggested by Hu and Bentler (1998), RMSEA values close to 0.06 or below were taken as good fit, with values from 0.06 to 0.08 inferred as moderate fit, and 0.08–0.10 as marginal fit as per the guidelines of Browne and Cudeck (1993). Following initial solutions, for each model that showed reasonable fit, an EFA was repeated such that items were removed if they did not substantively load on factors, or those that cross-loaded on factors were subsequently removed, to ensure a clear factor solution. Confirmatory factor analysis Factor solutions taken forward from the EFA were then evaluated using CFA with the larger global sample. The robust WLSMV estimator was used, and model evaluation used the same criteria as specified for the EFA. Once a final factor structure had been determined from CFA, internal consistency of the measure was considered using composite reliability (CR; Fornell & Larcker, 1981; Raykov, 1997). CR is an alternative to Cronbach’s alpha, and uses factor loadings to calculate internal consistency, and thus is often done in conjunction with CFA and related techniques (Peterson & Kim, 2013). Further analyses To assess the convergent and discriminant validity of the OCS with other constructs, each acceptable CFA model was carried forward to a subsequent analysis where each of the six OCS factors was correlated (using a freely estimated covariance path) with the observed total score of a range of measures. This was again undertaken in Mplus with a WLSMV estimator. Model fit was not a criterion in this analysis; rather the significance and size of the correlation (covariance) path between the measures and the OCS factors were assessed. Results Exploratory Factor Analysis From the initial screening of the 52-scale items, 16 items were excluded for reasons including low correlations with other items (seven items correlated r > 0.3 with 1 or no other items in the data set, indicative of orphan items); minimal or no endorsement of one end of the Likert scale (six items: in all cases minimal/no endorsement of the strongly disagree or disagree options); or for both of these reasons (three items). Subsequently, EFA was conducted with the remaining 36 items. Two- to seven-factor solutions were extracted and fit indices for these EFAs are shown in Table 2. Initial screening indicated that the two-, three-, and four-factor solutions were not acceptable statistically, with fit indices outside desired values, as shown in Table 2. Inspection of each of these models also identified that each model had as many cross-loading items as items loading on a single factor, and showed residual correlations >0.2, further indicative of poor fit (Gorsuch, 1983). Table 2. Fit Indices for the Extracted Exploratory Factor Analysis (EFA) Solutions EFA model χ2 CFI TLI RMSEA Value df Two-factor solution 2,601.01* 559 0.77 0.74 0.11 Three-factor solution 1,954.53* 525 0.84 0.81 0.09 Four-factor solution 1,455.10* 492 0.89 0.86 0.08 Five-factor solution 1,107.48* 460 0.93 0.90 0.07 Five-factor revised 33 item 912.09* 373 0.94 0.91 0.07 Six-factor solution 927.51* 429 0.94 0.92 0.06 Six-factor revised 28 item 553.04* 225 0.96 0.93 0.07 Seven-factor solution 755.55* 399 0.94 0.96 0.05 Notes: CFI = comparative fit index; TLI = Tucker–Lewis index; RMSEA = root-mean-square error of approximation. *p < .0001. As can be seen, the five-factor solution showed marginal fit though with only one residual correlation >0.1, this model was carried further to CFA testing, as once items within each factor were interpreted, there were items clustered around common themes. The six-factor solution showed acceptable fit with the items within each factor interpreted, there were items clustered around common themes, and this model was carried further to CFA testing. The seven-factor solution, although statistically acceptable, produced a solution that did not provide a more theoretically or practically distinct structure than the six-factor model described later, and included a factor with only two items loading >0.4. Although in special cases, two items can be acceptable for a subscale, it is generally considered preferable to have at least three items per factor (see Brown, 2015; Kline, 2016), and given the seven-factor model did not provide a distinct solution, in the interests of parsimony, it was not considered further. To create more coherent and interpretable factors in the models carried forward, both the five- and six-factor solutions were further refined by eliminating items that cross-loaded across multiple factors, or which did not load substantively (>0.4) on any factor. In the case of the five-factor solution, four items did not load on any item, and a single item cross-loaded on two factors; the subsequent 33-item, five-factor EFA showed acceptable fit, as seen in Table 2. In the case of the six-factor solution, six items did not load on any item, two items cross-loaded on two factors; the subsequent 28-item EFA acceptable-to-good statistical fit is seen in Table 2. Confirmatory Factor Analysis The 33-item five-factor model identified in the EFA was tested with CFA in the larger sample, but showed poor fit by all indices, χ2(485, N = 2,115) = 9,900.92, p < .001, CFI = 0.85, TLI = 0.84, RMSEA = 0.096. The five-factor model was thus not considered further. The 28-item six-factor model identified in the EFA and subjected to CFA using the larger sample showed acceptable fit by the fit indices χ2(335, N = 2,115) = 4,829.93, p < .001, CFI = 0.92, TLI = 0.91, RMSEA = 0.08 [90% CI: 0.078, 0.082], even though χ2 was significant. Table 3 shows the CFA factor loadings for the six-factor model and internal consistency (CR) for the six-factor CFA model. Internal reliability for the measure ranged from 0.74 to 0.91, which is well within acceptable limits (Fornell & Larcker, 1981; Raykov, 1997). Factor intercorrelations, shown in Table 4, were all 0.6 or below. Thus, although some factors were moderately intercorrelated, each can be considered a statistically distinct construct. Table 3. Standardized (STDYX) Factor Loadings and Internal Reliability (Composite Reliability) Statistics for the Six-Factor Confirmatory Factor Analysis Item number Question Factor loading Factor CR “Natural” carer factor 0.81  5 My job comes easily and naturally to me 0.78  3 I feel like I belong in my job 0.76  11 I know I’m good at what I do 0.75 Psychological need to care factor 0.74  12 My job matches my personal needs 0.74  6 My job fills a void in my life 0.71  10 If it wasn’t for my job I would feel somewhat empty inside 0.63 Connection with clients factor 0.91  34 I feel connected to my clients 0.87  35 I have a powerful emotional connection to the people I care for 0.81  26 I generally have a strong connection with all the people I care for at work 0.77  32 Getting to know the person I’m caring for is the best part of my job 0.77  25 Sometimes the people I care for are like my extended family 0.73  33 My clients get the best care because I know them well 0.73  30 It is important that I remain caring for the people I care for as long as I can 0.70  31 The best part of work is when I can share a cup of tea with my clients 0.64  29 Having balanced relationships with the people I care for are an important part of my work 0.63  36 It is important for me to fix problems for the people I support in my work 0.61 Connection with coworker factor 0.87  50 The quality of my relationships at work are of a high standard 0.85  51 I feel respected in most of my relationships at work 0.81  47 I feel connected to my coworkers 0.75  49 I have the opportunity to make relationships at work with others doing similar work as me 0.71  52 I speak up at work for the needs of my coworkers 0.67 Desire for more connection factor 0.84  42 I want more interaction with other workers who care for people with dementia 0.87  43 I want more opportunities to share the ups and downs of my work with other workers 0.85  44 My job would be better if I could share and learn more about the “tricks of the trade” 0.72  45 When I care for a person with dementia I wish for more support from work 0.52 Blurred boundaries factor 0.83  38 I have to blur the professional boundaries to provide the best care for people I provide care for 0.93  48 I blur the professional boundaries with my colleagues now and again so I can do my job well 0.74  39 It’s hard to keep my personal life and work life separate 0.67 Notes: CR = composite reliability. All-factor loadings significant at p < .001 level. Table 4. Latent Factor Intercorrelation (Covariance) Matrix for the Six-Factor Occupational Communion Scale Factor Psychological need to care Connection with clients Connection with coworkers Desire for more connection Blurred boundaries “Natural” carer 0.59** 0.49** 0.59** 0.18** −0.16** Psychological need to care 0.49** 0.36** 0.32** 0.28** Connection with clients 0.44** 0.51** 0.35** Connection with coworkers 0.23** 0.07* Desire for more connection 0.35** *p < .01. **p < .001. Following confirmation of the fit of the six-factor model, three authors reviewed and interpreted (K.-E. J., Elliott, M. J. Quinn, and J. L. Scott) items comprising each factor, and described the following domains: “natural” carer; psychological need to care; connection with clients; connection with coworkers; desire for more connection; blurred boundaries. Further Analyses The six-factor model was carried forward, and each OCS factor was correlated with observed scores of measures. The direction and strength of the relationships shown in Table 5 were as expected. Overall, the “natural” carer and desire for more connection with coworker factors had the highest number of significant moderate relationships across occupation and well-being measures, particularly for work engagement, self-efficacy, and positive affect. Table 5. Standardized Correlations (STDYX) Between the OCS Latent Factors, With Observed Scores From Other Measures (With Measure Details) Construct Measure N of items (aCronbach α) Likert response High score indicates… Correlations with OCS factors “Natural” carer Psychological need to care Connection with clients Connection with coworkers Desire for more connection Blurred boundaries Communion Communion, Personal Attribution Questionnaire (Spence & Helmreich, 1978) 6 (0.86) 5-pt High levels of communion 0.34** 0.11** 0.19** 0.21** 0.08** −0.14** Work engagement Utrecht Work Engagement Scale (Schaufeli et al., 2006) 3 (0.82) 6-pt High engagement in work 0.50** 0.35** 0.32** 0.37** 0.13** −0.03 Job satisfaction Job Satisfaction Scale (Spector, 1985) 36 (0.91) 6-pt High job satisfaction 0.29** 0.21** 0.07* 0.36** −0.19** −0.17** Intent to leave Intention to leave (Cho et al., 2009) 1 (n/a) 7-pt High intention to leave. −0.21** −0.21** −0.09** −0.23** 0.06 0.05 General self-efficacy General Self-Efficacy Scale (Schwarzer & Jerusalem, 1995) 10 (0.89) 4-pt Good self-efficacy 0.50** 0.18** 0.25** 0.31** 0.10** −0.12** Psychological distress Kessler 10 (K-10: Kessler et al., 2003) 10 (0.89) 5-pt High psych. distress −0.25** 0.07* 0.05 −0.19** 0.11** 0.22** Depression Centre for Epidemiological Studies—Depression scale (Radloff, 1977) 20 (0.72) 4-pt High levels of depressive symptomatology −0.32** 0.01 −0.01 −0.28** 0.10** 0.23** Positive affect Positive and Negative Affect Schedule (PANAS; Watson et al., 1988) 10 (0.94) 5-pt High positive affectivity 0.43** 0.20** 0.26** 0.37** 0.13** −0.07* Negative affect As previously 10 (0.92) 5-pt High negative affectivity −0.25** >0.01 0.03 −0.20** 0.10** 0.23** Satisfaction with life Satisfaction with Life Scale (Diener et al., 1985) 5 (0.91) 7-pt Highly satisfied with life. 0.29** 0.09** 0.13** 0.32** 0.03 −0.05 Notes: CFA = confirmatory factor analysis; OCS = Occupational Communion Scale. aCronbach’s alpha derived from CFA sample. *p <.01. **p <.001. Discussion and Implications Research on social connection is expanding to include new aspects, such as OC, which is relevant for caring professionals’ resilience and engagement in their work. To our knowledge, this is the first study to develop and test a measure of OC, which advances the literature on the assessment of social psychological concepts that appear vital to engage and develop aged and dementia care workforces. The valid and reliable 28-item OCS appears easy for care workers to understand and complete, and was theory driven and informed by evidence. It is intended for all direct care workers and health professionals who may work to partner in care with older people including people living with dementia. The first hypothesis was supported to show OC is a multifaceted construct represented by six dimensions: (1) “natural” carer, (2) psychological need to care, (3) connection with clients, (4) connection with coworkers, (5) desire for more connection, and (6) blurred boundaries (see Supplementary Figure 2). We also found substantive moderate associations between OC and work engagement, self-efficacy, positive affect, job satisfaction, and intention to leave the job, which suggests this construct may be useful to inform interventions aimed to engage employees in their jobs and enhance well-being at work, with potential to prevent high staff turnover. OC is redefined as a sense of belonging based on social interactions in care work that includes multiple domains of being suited to the job, a psychological need to care (e.g., “fills a void”), and strong and rewarding connections with clients and coworkers, which must be balanced alongside navigating professional boundaries and seeking better connections to assist with adaptive coping among care workers. Although OC means care workers can get a sense of positivity from connections with others, there is a flip side that can be negative. Values-based recruitment and retention strategies may focus on candidates’ predisposition for caring (i.e., their sense of being a “natural” carer, belonging, and being suited to the job). Our results showed that the “natural” carer dimension of OC strongly aligned with self-efficacy, work engagement, and positive affect. This is supported by previous research indicating a significant relationship between person–job fit (a match between the employees’ characteristics and their job roles) and self-efficacy (Peng & Mao, 2015). Being suited to the job has also been reported as a reason for entering aged care with a passion for the job (Xiao et al., 2021). Significant moderate negative relationships were also found between “natural carer” and mood indicators, which suggest the potential for poor health when workers are unsuited to their caring roles, which is similar to past reports of the connection between depression and job misfit (Ford, 2012). Meaningful work may be an important factor to consider for engaging aged and dementia care workers in their job roles over time. In our study, the psychological need to care dimension of OC was characterized by an alignment between personal needs and the caring duties of the job, as though the work fills a missing part of the self. This dimension was significantly and moderately positively related to work engagement, with substantive correlations also found with job satisfaction and positive affect, and negatively with intent to leave. It was, however, quite unrelated to mood indicators. It represents what some care workers have described as “empty nest syndrome” (Elliott et al., 2013) and may be important for work motivation and self-fulfilling work (Allan et al., 2016). The psychological need dimension of OC has a high potential to inform marketing strategies to attract people into the care workforce. Assessment of social connections in care work can be broadened to include relationships with clients. We found a connection with clients dimension was characterized by powerful emotional ties. This is when employees prioritize getting to know the client and enjoy sharing socially, being dependable, and advocating to fix clients’ problems. Such interactions by employees may lead to positive levels of engagement, satisfaction, positive affect, and self-efficacy, as indicated by our covariance results. These findings support the benefits espoused by the move toward relational care models in aged care services, and when caring for those with dementia (Nolan et al., 2004). Understanding relationships in the care work environment, and how workers interact, may be an essential part of increasing staff numbers to relieve high demands of care (Cooke & Baumbusch, 2021). Connection with coworkers appeared to be more influential across well-being outcomes such as satisfaction with life. This confirms previous findings that indicate the positive associations between good relationships with coworkers and quality of care, self-belief, social support, and positive work engagement (García-Sierra et al., 2016; Halbesleben, 2010; Xiao et al., 2021). They also suggest that positive and enhanced relationships between managers and coworkers may build an engaged and motivated care workforce, with implications for training and career advancement opportunities (Scales, 2021). Relationships with coworkers (van der Borg et al., 2017) may also underpin success in team-based skills training (i.e., occupational adaption framework, McKay et al., 2021). Poor health outcomes can result when personal and professional boundaries are blurred. In care work settings, this may occur when people place others’ needs above their own to extreme levels. This has been found for other carer contexts such as with unmitigated communion in those caring for patients with cardiovascular disease (Helegson & Frits, 1998). Qualitative studies have described a similar blurred boundary phenomenon (Elliott et al., 2013; Bailey et al., 2015; Mears, 2009; Piercy, 2000). The associations between blurred boundaries and mood indicators shown in our study highlight that employers need to be aware of the possible psychosocial risks for their employees combined with “friend and family-like” relationships with clients. Further, there can be implications for compliance with regulations (Sheridan & Agim, 2014) that underpin good-quality care. The concept of OC has a potential to inform workplace development programs. Prior to quantifying OC, it was applied in a training manual for interprofessional dementia care (Rudd et al., 2012). The new measure extends this potential application, and we suggest that the OCS can be used to assess staff and potentially the workplace, throughout the career pathway (from entry to exit) to inform these initiatives. Strengths-based programs that promote the positives and address unmet social interaction needs may suit care workers. Strategies may aim to develop social support mechanisms to reduce isolation such as providing more opportunities for good-quality peer, supervisor, or team interactions. This could follow other professions where workers are required by regulatory standards to undertake peer consultation which includes a critically reflective focus on practice (World Federation of Medical Education, 2015). Addressing this issue may mean there is potential to change stigma and the negative public perception of care work (Machha et al., 2021) by improving job attractiveness. The strengths of our research include the multiple independent samples, confirmatory analysis, and a unique focus on care work that contributes to existing social and organizational psychological theory. A limitation of our study is the specialized nature of the employee sample, as results may not generalize beyond care workers, which is a predominantly female workforce. Although the samples are narrow in scope, their characteristics are similar to available workforce demographics (Mavromaras et al., 2017). Future research may investigate the utility of the OCS in general healthcare workers, or in roles where employees have repetitive social interactions with people other than their colleagues, particularly when helping others is the aim of the job. Consideration of workforce subgroups and how they may differ on the OCS may be useful in more clearly understanding the construct of OC, and ways to improve capacity and resilience in the care workforce (e.g., latent class/profile analysis). The OCS may be relevant for other regions and cultures; however, further cross-cultural research is necessary to comment thoroughly on the generalizability of the measure. Future research should focus on further validation methods (e.g., test–retesting, concept analysis) and testing mediation models. In summary, OC has a theoretically coherent multidimensional structure that is associated with established organizational health and well-being variables. The application of OC to human resourcing decisions, such as job selection, recruitment, orientation to and engagement in job roles, as well as retention efforts, appear promising. Supplementary Material gnac190_suppl_Supplementary_Material Click here for additional data file. Funding Funding was from National Health and Medical Research Council, and Australian Research Council (E0023237), University of Tasmania Research Enhancement Grant Scheme (E0021909), an Early Career Seeding Grant from University of Tasmania, Wicking Dementia Research and Education Centre, and a PhD scholarship jointly provided by TIME for Dementia (Tasmania and Victoria Dementia Training Study Centre, Australia), the Wicking Dementia Research and Education Centre and the University of Tasmania, Australia (045135, awarded to J. L. Scott and A. L. Robinson; K.-E. J. Elliott stipend). Conflict of Interest None declared. Author Contributions K.-E. J. Elliott led and formulated the research questions, designed, and carried out the study in collaboration and with advice from C. M. Stirling, J. L. Scott, A. L. Robinson, K. Sanderson, A. J. Martin, and M. G. Quinn. M. G. Quinn analyzed the data and wrote the results section and assisted with writing the full article. All authors assisted with writing the article. Data Availability Data are not stored in a public repository as participants did not provide their informed consent to share their deidentified data. This study was not preregistered. ==== Refs References Abele, A. E., & Spurk, D. (2011). The dual impact of gender and the influence of timing of parenthood on men’s and women’s career development: Longitudinal findings. International Journal of Behavioral Development, 35 (3 ), 225–232. doi:10.1177/0165025411398181 Abele, A. E., & Wojciszke, B. (Eds). (2018). 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==== Front Fungal Syst Evol Fungal Syst Evol FUSE Fungal Systematics and Evolution 2589-3823 2589-3831 Fungal Systematics and Evolution 10.3114/fuse.2023.11.01 Articles Cylindromonium dirinariae sp. nov. (Ascomycota, Hypocreales), a new nectrioid lichenicolous species on Dirinaria applanata in Japan Ohmaki A. 1 Okane I. 2 * Crous P.W. 3 Verkley G.J.M. 3 1 Agro-Bioresources Science and Technology, Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan 2 Faculty of Life and Environmental Sciences, Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan 3 Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, Netherlands *Corresponding author: okane.izumi.fw@u.tsukuba.ac.jp Corresponding editor: A.J.L. Phillips 23 1 2023 1 2023 11 110 8 11 2022 2 1 2023 © 2023 Westerdijk Fungal Biodiversity Institute 2023 https://creativecommons.org/licenses/by-nc-nd/4.0/ Fungal Systematics and Evolution is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License A nectrioid fungus forming a pinkish colony with mainly solitary phialides producing ellipsoid, aseptate conidia in mucoid packets was isolated from Dirinaria applanata. Our taxonomic study based on morphology and phylogenetic analysis using ITS rDNA sequences revealed that the isolates represented a member of the genus Cylindromonium. Based on further morphological examination, nucleotide sequence comparison, and phylogenetic analysis based on LSU rDNA, tef1, and rpb2 in addition to the phylogenetic analysis using the ITS rDNA sequences, the fungus from Dirinaria represents a new species, which is described here as Cylindromonium dirinariae sp. nov. Furthermore, inoculation experiments revealed that this species can also produce perithecia when inoculated on the host lichen in laboratory environments. Citation: Ohmaki A, Okane I, Crous PW, Verkley GJM (2023). Cylindromonium dirinariae sp. nov. (Ascomycota, Hypocreales), a new nectrioid lichenicolous species on Dirinaria applanata in Japan. Fungal Systematics and Evolution 11: 1–10. doi: 10.3114/fuse.2023.11.01 culture inoculation lichenicolous fungi Nectriaceae new taxon phylogeny ==== Body pmcINTRODUCTION Lichenicolous fungi is a term used to circumscribe fungi that grow on lichens. They can interact with their lichen hosts as saprophytes, parasites and commensalistic parasymbionts. Lichenicolous fungi usually establish a symbiotic relationship with a single species or genus of lichens, while some species have a wide host range (Diederich et al. 2018). Approximately 2 300 species of lichenicolous fungi have been described on lichens globally and they are classified into ca. 400 genera, ca. 100 families, 55 orders and 10 classes. Ninety-six percent of the total number of lichenicolous fungi are ascomycetes and four percent of the fungi are basidiomycetes (Diederich et al. 2018). About 166 lichenicolous fungi have been reported from Japan to date (Frisch et al. 2018, Ohmura & Kashiwadani 2018, Tadome et al. 2018, Zhurbenko & Ohmura 2018a, b, Zhurbenko et al. 2018, Zhurbenko & Ohmura 2019, 2020, Frisch et al. 2020, Tadome & Ohmura 2021, 2022, Tadome et al. 2022). In spite of these reports, there has been relatively little research conducted on this fungal group in Japan. Therefore, many species remain to be discovered and described. The genus Cylindromonium, with the type species C. eugeniicola, was segregated from Acremonium based on analyses of ITS and LSU rDNA sequence data (Summerbell et al. 2011, Crous et al. 2019b). Cylindromonium species are known to be lichenicolous, mycophilic, or saprophytic (Gams 1971, Crous et al. 2019b, 2020, 2021). Cylindromonium was established as a genus to accommodate acremonium-like taxa with unbranched, hyaline, phialidic conidiophores, and cylindrical 1-septate conidia (Crous et al. 2019b). A total of five asexual species have been assigned to Cylindromonium, for which no sexual morph has thus far been reported (Crous et al. 2019b, 2020, 2021). During our research on the diversity of lichenicolous fungi in Japan, a fungus colonising the Physciaceae lichen Dirinaria applanata was found. The purpose of this study is to describe the morphological, physiological, and ecological features of this species, clarify the link to its sexual morph, and discuss its taxonomic placement. MATERIALS AND METHODS Collection materials Field investigations were performed from September 2020 to March 2021 in Tsukuba city, Ibaraki prefecture, Japan. Specimens of the fungus growing on the lichen host D. applanata were found on the bark of Zelkova serrata. A voucher specimen was deposited in the National Museum of Nature and Science (TNS), Tsukuba, Japan. A living ex-type culture was deposited in the Biological Resource Center of the National Institute for Technology and Evaluation (NBRC). Cylindromonium lichenicola strains (CBS 188.70 and CBS 415.70A) were also examined for comparison purposes. Morphological observations Samples were observed using a dissecting microscope [M165 C (Leica, Wetzlar, Germany)] and a differential interference contrast compound microscope [BX53 (Olympus, Tokyo, Japan)]. Anatomical examination was performed using hand-cut sections mounted in a drop of water or clear lactophenol. Photographs were taken using a microscope digital camera [Flexacam C3 (Leica, Wetzlar, Germany) or DP23 (Olympus, Tokyo, Japan)]. Dimensions of ascospores, conidia, conidial mass, phialide and hyphal width are given as (minimum–) range of mean ± standard deviation (–maximum) (n = number of measurements). Chemical reactions of the perithecia were observed by using 10 % KOH. To determine if there is a significant difference between each dimension of the present fungus and C. lichenicola, the t-test was performed using Microsoft Excel. Isolation of fungal cultures Fungal cultures were isolated from freshly collected material. Mycelium or single ascospores were picked up using a flamed needle and plated on 1 % malt extract agar (MEA). To confirm differences in colony characteristics on each agar medium, mycelial plugs were subcultured on 1 % MEA, potato dextrose agar (PDA) (Nissui Pharmaceutical, Tokyo, Japan), oatmeal agar (OA) (Becton Dickinson and Co, New Jersey, USA), Sabouraud maltose agar (SMA) (Thermo Fisher Scientific, Massachusetts, USA), malt yeast extract agar (MYA) (Ahmadjian 1961) and Sabouraud glucose agar (SGA) (Stocker-Wörgötter 2002), confirming the recipes of these media according to Crous et al. (2019a). Colour of colonies were determined based on Kornerup & Wanscher (1978). DNA extraction, PCR amplification and sequencing Perithecia were sampled from specimen TNS-L-131533, mycelium from specimen TNS-L-131534, and mycelia from a culture derived from specimen TNS-L-131535. For DNA extraction, fungal tissues were suspended in 20 μL of DNA extraction buffer [10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01 % sodium dodecyl sulfate (SDS), 0.01 % Proteinase K], incubated at 37 °C for 60 min, and denatured 90 °C for 10 min; 30 μL of sterile distilled water (SDW) added to the tubes, and stored in a freezer at -20 °C. Partial sequences of the nuc rDNA ITS1-5.8S-ITS2 (ITS), large subunit (LSU) nuc rDNA regions, elongation factor 1-alpha (tef1), and RNA polymerase II second largest subunit (rpb2) were amplified as these regions are frequently used for phylogenetic analyses of Nectriaceae (Lombard et al. 2015, Crous et al. 2019b, 2020, 2021). The ITS region was amplified using the primers ITS5 and ITS4 (White et al. 1990), LSU rDNA using primers LIC24 (Miadlikowska & Lutzoni 2000) and LR7 (Vilgalys & Hester 1990) or LR0R (Rehner & Samuels 1994) and LR6 (Vilgalys & Hester 1990), tef1 using EF1-983F and EF1-1567R (Rehner & Buckley 2005), and rpb2 using RPB2-5F2 and RPB2-7cR (O’Donnell et al. 2007). PCR was performed in a 15 μL reaction volume containing 1 μL DNA template, 7.5 μL GenRED PCR Mix Plus (Nippon Gene, Tokyo, Japan), 1.5 μL each primer (2 pmol/μL), and 3.5 μL distilled water. The PCR was performed in a TaKaRa PCR Thermal Cycler Dice® Touch (TaKaRa, Shiga, Japan) as follows for the ITS region; 5 min at 95 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 53 °C, 1 min at 72 °C, and a final step of 8 min at 72 °C. PCR conditions for LSU, tef1 and rpb2 were set according to Frisch et al. (2020), Rehner & Buckley (2005) and O’Donnell et al. (2007), respectively. PCR products were checked by electrophoresis on a 1.5 % agarose gel stained with Midori Green Direct DNA Stain (Nippon Genetics, Tokyo, Japan) and visualised using WSE-5200 Printgraph 2 M (ATTO, Tokyo, Japan). The PCR products were purified using a FastGeneTM Gel/PCR extraction kit (Nippon Genetics, Tokyo, Japan) and ExoSAP-ITTM (Thermo Fisher Scientific, Massachusetts, USA) following the manufacturer’s instructions. Sequences were obtained via a DNA sequencing service using Applied Biosystems 3730xl DNA analyzer (Thermo Fisher Scientific, Massachusetts, USA) (Eurofins Genomics, Tokyo, Japan). Data and accession number of the voucher specimen, and the obtained sequences from the International Nucleotide Sequence Database (INSD) are shown in Table 1. Phylogenetic analysis and comparison of sequence data Both newly generated ITS sequences and reference sequences were used in the phylogenetic analysis. Sequence data of loci other than ITS do not exist for some isolates of the genus Cylindromonium. Therefore, only ITS, LSU and rpb2 of four Cylindromonium species were used for the phylogenetic analysis (Table 1). ITS sequence data of species with relatively high identity (> 85 %) in the BLAST search and other hypocrealean fungi in the Nectriaceae were included in the analysis to infer the taxonomic position of the targeted fungi (Table 1). In these analyses, Stachybotrys chartarum (KM231858) was chosen as the outgroup (Lombard et al. 2015). Sequences of each locus (ITS, LSU, tef1 and rpb2) were compared with those of C. lichenicola. All sequences analysed in this study were deposited in the DNA Data Bank of Japan (DDBJ), a member of International Nucleotide Sequence Database Collaboration (INSDC). Assembling forward and reverse strands of the sequenced loci were carried out with MUSCLE v. 3.6 (Edgar 2004) in MEGA v. 7 (Kumar et al. 2016) to obtain consensus sequences. DNA sequences were aligned using the online version MAFFT v. 7 (Katoh et al. 2019) (https://mafft.cbrc.jp/alignment/server/) with default settings. MEGA v. 7 (Kumar et al. 2016) was used to truncate sequences up to the determined edge of the dataset. Phylogenetic analyses were performed with Maximum likelihood (ML) using an online version W-IQ-Tree v. 1.6.12 (Trifinopoulos et al. 2016) (http://iqtree.cibiv.univie.ac.at/). All characters were equally weighted, and gaps were treated as missing data. The ML analysis for the ITS region alignment using the TIM2+F+I+G4 model and for a combined alignment of the three loci, ITS, LSU, rpb2 using the TN93+G (for ITS and LSU) and TN93+I (for rpb2) were performed with 1 000 bootstrap replicates. FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and MEGA X were used for plotting the phylogenetic trees. Sequence alignments were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S30024). Inoculation experiments Symptomless thalli of D. applanata, the host lichen of C. dirinariae, and those of the non-host lichen Parmotrema tinctorum were collected in Tsukuba city, Ibaraki prefecture, Japan and confirmed as non-infected via microscopy. Following this step, the lichen surface was cleaned using an ultrasonic cleaner with 0.005 % Aerosol® OT (a surface-active agent) for 1 min. This step was repeated five times. After this process, samples were rinsed with distilled water. Agar pieces including hyphae of isolates of C. dirinariae and C. lichenicola were inoculated onto the lichen thalli and maintained in 90-mm-diam glass Petri dishes with a sheet of wet filter paper to retain a high humidity at room temperature. Lichens inoculated were misted from above and moistened when they began to dry, every 2 to 3 d. In addition, some lichens were not inoculated and maintained under the above conditions as a control. RESULTS Taxonomy Classification: Nectriaceae, Hypocreales, Sordariomycetes. Cylindromonium dirinariaeOhmaki & Okane, sp. nov. MycoBank MB 846061. Fig. 1. Etymology: Name refers to the host genus Dirinaria from which it was isolated. Diagnosis: Ascomata perithecial, globose, orange; phialides short; conidia aggregated in mucoid packets in the apex of phialides, ellipsoid, aseptate; the species differs from all other Cylindromonium species by its characteristic DNA sequences (ITS, LSU, tef1, rpb2) and from its closest relative C. lichenicola also by its shorter phialides. Description: Ascomata occur on the upper surface or soralia of the host lichen thallus; perithecial, scattered, globose, 80–100 μm diam, poculiform when dry, pale orange when young but later becoming dark, KOH negative, ascomatal wall layers of textura globosa, 14–18 μm thick. Asci broadly cylindrical to clavate, non-stipitate, (21.2–)24.5 ± 2.3(–29.1) × (3.9–)5.0 ± 0.8(–6.5) μm (n = 12), unitunicate, apex simple, 8-spored. Ascospores biseriate, ellipsoid, hyaline, smooth, medially 1-septate, (5.0–)7.8 ± 1.1(–10.1) × (1.8–)2.6 ± 0.4(–3.7) μm, length/breadth (l/b) = (2.1–)2.6 ± 0.4(–3.9) (n = 40). Mycelium consisting of hyaline, smooth, septate, branched, 2 μm diam hyphae. Conidiogenous cells arising directly from aerial hyphae, hyaline, smooth, subcylindrical, 20–35 μm tall, 2 μm wide at the base, tapering to 1 μm at the apex, phialidic, with non-flared collarette. Conidia solitary, adhering in a slimy mass, hyaline, smooth, aseptate, ellipsoid with obtuse ends, (4.1–)5.9 ± 0.9(–10.3) × (1.5–)2.4 ± 0.4(–3.5) μm, l/b = (1.5–)2.55 ± 0.5(–3.9) (n = 75). Culture characteristics: Colonies flat, circular or irregular, with moderate aerial mycelium and smooth, lobate margin, reaching 20 mm diam on MEA, PDA, OA, SMA and MYA, 30 mm diam on SGA after 3 wk at 23 °C in darkness. On MEA, PDA, SMA, SGA and MYA surface and reverse strong brownish orange; on OA surface grayish orange. Host: Dirinaria applanata. Distribution: Japan. Typus: Japan, Ibaraki, Tsukuba, Tennodai, Univ. of Tsukuba, 36°06’08”N, 140°06’24”E, lichenicolous on Dirinaria applanata on bark of Zelkova serrata, 1 Aug. 2020, A. Ohmaki, I. Okane, K. Ohmachi, K. Miyazawa & K. Gibu, FAO 005 (holotype TNS-L-131533, culture ex-type NBRC 115852); DDBJ: ITS = LC731273; LSU = LC731274; tef1 = LC731275; rpb2 = LC744391. Additional materials examined: Japan, Ibaraki, Tsukuba, Tennodai, Univ. of Tsukuba, 36°06’08”N, 140°06’24”E, from Dirinaria applanata on the bark of Zelkova serrata, 1 Aug. 2020, A. Ohmaki, I. Okane, K. Ohmachi, K. Miyazawa & K. Gibu, NBRC 115851 = TNS-L-131534 (FAO 004), DDBJ: ITS = LC731276; LSU = LC744402; tef1 =LC744396; rpb2 =LC744390; 6 Nov. 2020, A. Ohmaki, TNS-L-131536 (FAO 090), in an inoculation experiment with isolates on D. applanata (Ibaraki: Tsukuba, 1 Aug. 2020, A. Ohmaki, I. Okane, K. Ohmachi, K. Miyazawa & K. Gibu, TNS-L-131533); Tsukuba, Tennodai, Univ. of Tsukuba, 36°06’38”N, 140°06’16”E, from Dirinaria applanata on the bark of Zelkova serrata, 15 Mar. 2021, A. Ohmaki, NBRC 115853 = TNS-L-131535 (FAO 006), DDBJ: ITS = LC731277; LSU = LC744401; tef1 = LC744395; rpb2 = LC744392. Notes: The ex-type strain of C. lichenicola (CBS 425.66) was not available for study, so we conducted morphological observations on other available strains of C. lichenicola (CBS 188.70 and CBS 415.70A) that are similar in terms of their collection sites and hosts. In addition, these strains were also studied when the genus Cylindromonium was established in Crous et al. (2019b). Morphological features of the C. lichenicola strains (CBS 188.70 and CBS 415.70A) shown in Table 2 correlated well with the original description provided by Gams (1971) [conidia size 5.5–9.8 × 1.5–2.5 μm (l/b = 3.0–4.4), phialide length 30–60 μm, phialide width base 2.0–3.0 μm, apex 0.7–1.5 μm]. As a result of the t-test, there were significant differences between C. dirinariae and C. lichenicola in conidia, phialide length and width (Table 2). As for phialide length, phialides of C. dirinariae were about half as long as those of C. lichenicola. Materials examined of Cylindromonium lichenicola: Germany, Probsteierhagen, Schüttbrehm, from unnamed apothecia of lichen on tree bark, Oct. 1996 (living strain CBS 188.70), GenBank: LSU = LC744399; tef1 = LC744398; rpb2 = LC744393. Netherlands, Utrecht, Amelisweerd, from aerial algae on tree bark, Oct. 1968 (living strain CBS 415.70A), GenBank: LSU = LC744400; tef1 = LC744397; rpb2 = LC744394. Phylogeny The ITS sequences derived from DNA extracted from a perithecium (specimen TNS-L-131533) and that from mycelia from another specimen (TNS-L-131534) of C. dirinariae were identical. The Phylogenetic analysis based on the ITS region revealed that all three sequences, adding that from mycelia (TNS-L-131535) to the above two, were grouped in a fully supported clade (Fig. 2). The clade was sister to C. lichenicola and linked with five Cylindromonium species including the type species of the genus Cylindromonium, C. everniae. The clade including the Cylindromonium species also included species of Trichonectria (Bionectriaceae) and Phialoseptomonium (Nectriaceae). Sequences of LSU, tef1, and rpb2 derived from specimen TNS-L-131533, TNS-L-131534 and TNS-L-131535 of C. dirinariae were identical. The ITS sequence from TNS-L-131535 differed from the other two sequences from TNS-L-131533 and TNS-L-131534 in one site. While the sequences of ITS, LSU, and tef1 of C. lichenicola strains CBS 188.70 and CBS 415.70A were identical, those of rpb2 of the two stains were different in 18 sites. Comparison of C. dirinariae with C. lichenicola (CBS 188.70 and CBS 415.70A) showed that there were 19 gap sites (96.7 % in nucleotide identity) in ITS, 11 gap sites including 2 bp deletions (98.8 %) in LSU, 26 gap sites in tef1 (93.7 %), 153 gap sites (CBS 415.70A vs. C. dirinariae) or 155 gap sites (CBS 188.70 vs C. dirinariae) including 44 bp insertions in rpb2 (84.9–85.1 %). Phylogenetic analysis based on the concatenated sequences of ITS, LSU and rpb2 showed that three sequences of C. dirinariae grouped together with full bootstrap support and clearly segregated from C. lichenicola and other species (Fig. 3). Inoculation experiments The two Cylindromonium spp. studied were able to colonise the inoculated lichens, except for failures due to contamination, extreme dryness or moisture (Fig. 4; Table 3). Cylindromonium dirinariae colonised and produced perithecia on the thalli of D. applanata. Colonies reached 5 mm diam about 1 wk post inoculation. Pinkish discolouration was observed in the part of lichen’s thalli covered with hyphae of C. dirinariae. The asexual morph also developed on inoculated thalli. Perithecia developed 2 wk post inoculation. Morphological features of C. dirinariae on the inoculated lichens coincided well with those of C. dirinariae observed in the field. On the other hand, although C. lichenicola also colonised and produced the asexual morph on D. applanata, no perithecia were produced. Cylindromonium dirinariae and C. lichenicola colonised and sporulated asexually, but no perithecia were produced on P. tinctorum. The lichen’s thalli were covered with hyphae of the lichenicolous fungi, and discoloured brownish around the point of inoculation. Colonies reached 5 mm diam after 1 wk and 1.5–2 cm diam after 2 wk post inoculation. Lichens used for controls remained healthy. In addition, three single ascospore cultures were obtained of C. dirinariae, and each culture was inoculated onto thalli of D. applanata. As a result, they colonised the lichen and produced perithecia. DISCUSSION In the molecular phylogenetic analysis based on ITS sequence data and concatenated sequences of three loci, the three sequences of C. dirinariae clustered in a single clade and positioned as sister to C. lichenicola, which is the most closely related species in morphology and DNA phylogeny (identity = 96.7 %), supported by high bootstrap values. Furthermore, C. dirinariae was related to the group consisting of C. everniae and C. rhabdosporum, supported by high bootstrap values (99 %) (Fig. 2). Comparison of other loci sequences also showed that similarity between C. dirinariae and C. lichenicola were 96.7 % in ITS, 98.8 % in LSU, 93.7 % in tef1, 84.9–85.1 % in rpb2. In addition to phylogeny, C. dirinariae is also morphologically distinct. In our morphological observations and the inoculation experiments, C. dirinariae had 20–35 μm tall phialides and produced perithecia on D. applanata, while C. lichenicola had longer phialides (45–60 μm tall), and failed to produce perithecia on D. applanata and P. tinctorum. Hence, we concluded that C. dirinariae represents a new species. In addition, this is the first report of the sexual morph for the genus Cylindromonium. The genus Cylindromonium has been reported from Belgium (Gams 1971, Diederich 1989), Germany (Gams 1971, Brackel 2010), Great Britain (Hitch 1995), France (Roux 2012), Luxembourg (Diederich 1989), the Netherlands (Brand et al. 2013, Crous et al. 2021), Czech Republic (Kocourková 2009), India (Joshi et al. 2016), Ukraine (Khodosovtsev et al. 2018), and Australia (Gams 1971, Crous et al. 2020). This is the first report of the genus Cylindromonium from Japan. Inoculation experiments using single ascospore cultures revealed that this fungus is homothallic. The newly described C. dirinariae was isolated from D. applanata, while the hosts of C. lichenicola are diverse, including the lichens Cladonia, Hypogymnia, Parmelia saxatilis, Tremella cladoniae, lichens overgrowing Stereum species, fungi; Bulgaria inquinans, algal-covered bark, Alnus bark and Betula litter (Gams 1971, Diederich 1989, Hitch 1995, Brackel 2010, Roux 2012, Brand et al. 2013, Tsurykau et al. 2016). Results of our inoculation experiments are suggestive of host specificity; i.e., C. dirinariae was able to form perithecia on its original host lichen, D. applanata, but not on P. tinctorum. Cylindromonium dirinariae could therefore be considered host specific. Glenn et al. (1997) found that Cylindromonium rhabdosporum (as Acremonium rhabdosporum), occurred on healthy-looking thalli in the field, and perithecia of Nectriopsis rubefaciens (as Nectria rubefaciens) appeared on the same thalli in the closed plates. Although they mentioned the relationship between C. rhabdosporum and N. rubefaciens, they did not confirm that they belong to the same holomorph. Further study is therefore needed to determine the homogeneity between C. rhabdosporum and N. rubefaciens. Presently there are only a few reports of inoculation experiments of lichenicolous fungi, and they were conducted in the field (Fatma et al. 2019). Glenn et al. (1997) found that continuously moist conditions probably play a pivotal role for lichenicolous fungi to produce perithecia. Inoculation experiments in the moist condition in Petri dishes using axenic cultures of lichenicolous fungi may therefore be a useful technique for studying morphological, physiological and ecological features of lichenicolous fungi. This work has demonstrated the potential of inoculation experiments to investigate the morphological feature of perithecia and host specificity. Presently the ecology of C. dirinariae remains unclear. Other species of Cylindromonium were reported to be mycophilic or saprophytic (Crous et al. 2019b, 2020). We expect that inoculation experiments will reveal the interaction between lichenicolous fungi and their host lichens which could help us to better understand the ecological role of lichenicolous fungi. The ascomycete family Nectriaceae includes numerous important plant and human pathogens as well as several facultatively fungicolous or insecticolous species (Rossman 1996, Lombard et al. 2015). Members of Nectriaceae are characterised by uniloculate ascomata that are white, yellow, orange-red or purple, unitunicate asci and phialidic asexual morphs (Rossman et al. 1999, Lombard et al. 2015). In many cases ascomata show a change of colour when mounted in KOH (Lombard et al. 2015). In our study, perithecia of the C. dirinariae were orange in colour and did not react in KOH. About 400 genera and 2 300 species of lichenicolous fungi are known from lichens, but the actual number of lichenicolous fungal species could be much higher (Diederich et al. 2018). Diederich et al. (2018) estimated 3 000 – 5 000 lichenicolous fungal species will eventually be described based on Hawksworth’s global estimates of fungal diversity (Hawksworth 1991, 2001) and the total number of lichen species (Lücking et al. 2017a, 2017b). Lichenicolous fungi are assumed to be an important source of new species in many groups of fungi, including Nectriaceae. This research was partially financially supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 21K19286 (IO). Fig. 1. Cylindromonium dirinariae. A. C. dirinariae colonizing on Dirinaria applanata. B, C. Colonies on MEA. B. Surface. C. Reverse. D. Growth habit on Dirinaria applanata. E. Perithecium. F. Conidiogenous cells and conidia. G. Asci. H. Ascospores. I. Conidia. J. Phialide and conidial mass. Scale bars: B, C = 3 cm; D = 0.5 mm; E, F = 0.25 mm; G, I, J = 10 μm; H = 5 μm. Fig. 2. A phylogenetic tree of Cylindromonium dirinariae and other Nectriaceae and hypocrealean fungi constructed from a maximum-likelihood (ML) analysis based on the ITS sequences. The outgroup is Stachybotrys chartarum. Number at the nodes represents the bootstrap value. Fig. 3. A phylogenetic tree for Cylindromonium dirinariae and other Cylindromonium spp. constructed from a maximum-likelihood (ML) analysis based on concatenated sequence dataset of ITS, LSU rDNA and rpb2. The outgroup is Stachybotrys chartarum. Number above a branch represents the bootstrap value. Fig. 4. Lesions on the lichen thalli after inoculation. A. Colony of C. dirinariae on D. applanata. B. Colony of C. dirinariae on P. tinctorum C. Colony of C. lichenicola on D. applanata. D. Colony of C. lichenicola on P. tinctorum. Scale bars = 5 mm. Table 1. Sources of DNA sequence data used in phylogenetic analyses and comparison of sequence data. Species Strain numbers Host/Substrate Collection sites ITS Accession No. LSU Accession No. tef1 Accession No. rpb2 Accession No. References Calostilbe striispora CBS 133491 Erythrina glauca Trinidad and Tobago KM231789 – – – Lombard et al. (2015) Ciliciopodium brevipes CBS 691.83 Fagus sylvatica Netherlands KM231856 – – – Lombard et al. (2015) Ciliciopodium hyalinum CBS 106.13 Soil Switzerland KM231857 – – – Lombard et al. (2015) Cylindrium aeruginosum CBS 693.83 Fagus sylvatica Netherlands KM231854 – – – Lombard et al. (2015) Cylindrium elongatum CBS 685.83A Fagus sp. Netherlands KM231852 – – – Lombard et al. (2015) Cylindromonium alloxyli CPC 38159 Meliola on leaves of Alloxylon pinnatum Austria MW175339 MW175379 – MW173114 Crous et al. (2020) Cylindromonium eugeniicola CPC 37170 Leaf litter of Eugenia capensis South Africa NR166338 – – – Crous et al. (2019b) Cylindromonium everniae CBS 148255 Evernia prunasti Netherlands NR175231 OK664736 – NR175231 Crous et al. (2021) Cylindromonium dirinariae sp. nov. TNS–L–131533 Dirinaria applanata Japan LC731273 LC731274 LC731275 LC744391 This study TNS–L–131534 Dirinaria applanata Japan LC731276 LC744402 LC744396 LC744390 This study TNS–L–131535 Dirinaria applanata Japan LC731277 LC744401 LC744395 LC744392 This study Cylindromonium lichenicola CBS 415.70A Aerial algae Netherlands MH859774 LC744400 LC744397 LC744394 Vu et al. (2019); This study CBS 303.70 Alnus sp. Germany MH859675 – – – Vu et al. (2019) CBS 188.70 Apothecia of lichen Germany MH859549 LC744399 LC744398 LC744393 Vu et al. (2019); This study Cylindromonium rhabdosporum CBS 438.66 Cladonia furcata Austria MH858850 – – – Vu et al. (2019) Falcocladium multivesiculatum CBS 120386 Leaf litter Brazil JF831936 – – – Rungjindamai et al. (unpublished) Falcocladium sphaeropedunculatum CBS 111292 Leaf litter Brazil JF831938 – – – Rungjindamai et al. (unpublished) Falcocladium thailandicum CBS 121717 Eucalyptus camaldulensis Thailand JF831939 – – – Rungjindamai et al. (unpublished) Hyaloseta nolinae CBS 109837 Nolina micrantha USA KM231846 – – – Lombard et al. (2015) Lectera colletotrichoides CBS 109728 Medicago sativa Turkey KM231851 – – – Lombard et al. (2015) Nectria balansae CBS 123351 Coronila sp. France HM484552 – – – Hirooka et al. (2011) Nectria cinnabarina CBS 125165 Aesculus sp. France HM484548 – – – Hirooka et al. (2011) Nectria dacryocarpa CBS 121.87 Tree fern Sulawesi KM231850 – – – Lombard et al. (2015) Nectria mariae CBS 125294 Buxus sempervirens France JF832629 – – – Hirooka et al. (2012) Nectria nigrescens CBS 125148 Wood USA HM484707 – – – Hirooka et al. (2011) Phialoseptomonium eucalypti CBS 145542 Leaves of Eucalyptus Australia MK876402 – – – Crous et al. (2020) Pochonia sp. CBS 634.75 Arcyria sp. Netherlands KM231845 – – – Lombard et al. (2015) CBS 892.7 Myxomycete Netherlands KM231844 – – – Lombard et al. (2015) CBS 401.70 Myxomycete Netherlands KM231843 – – – Lombard et al. (2015) Rodentomyces reticulatus CBS 128675 Rodent dung Italy JF832659 – – – Hirooka et al. (2012) Sarocladium kiliense CBS 400.52 Ficus carica UK KM231849 – – – Lombard et al. (2015) Septofusidium berolinense CBS 731.70 – Germany KM231841 – – – Lombard et al. (2015) Stachybotrys chartarum CBS 129.13 – Unknown KM231858 MH866145 – KM232434 Lombard et al. (2015) Thyronectria lamyi CBS 417.89 Berberis vulgaris Germany KM231837 – – – Lombard et al. (2015) Thyronectria pyrrhochlora CBS 125131 Acer campestre Austria HM484545 – – – Hirooka et al. (2011) Thyronectria quercicola CBS 128976 Quercus ilex Spain JF832624 – – – Hirooka et al. (2012) Thyronectria sinopica CBS 462.83 Hedera helix Netherlands HM484542 – – – Hirooka et al. (2011) Tilachlidium brachiatum CBS 505.67 Hypholoma fasciculare Poland KM231839 – – – Lombard et al. (2015) Trichonectria rectipila CBS 132.87 – USA MH862058 – – – Vu et al. (2019) Trichonectria setadpressa J.E.20–13 Lobariella pallida France MT153969 – – – Flakus et al. (2019) CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; CPC: Culture collection of Pedro Crous, housed at the Westerdijk Fungal Biodiversity Institute; J.E.: The private herbarium of Javier Etayo, Pamplona. Table 2. Comparison of dimensions (μm) between Cylindromonium dirinariae and C. lichenicola. Conidia* Phialide length* Phialide width (Base)* Phialide width (Apex)* Hyphae width C. dirinariae (4.1–)5.9 ± 0.9(–10.3) × (1.5–)2.4 ± 0.4(–3.5) (14.7–)27.4 ± 8.1(–56.7) (1.7–)2.3 ± 0.3(–3.1) (1.0–)1.4 ± 0.2(–2.0) (1.2–)2.0 ± 0.3(–2.6) C. lichenicola (4.1–)6.8 ± 1.1(–10.0) × (1.5–)2.4 ± 0.5(–3.3) (33.9–)51.7 ± 7.6(–69.9) (2.0–)3.2 ± 0.5(–4.0) (1.1–)1.6 ± 0.3(–2.5) (1.5–)2.3 ± 0.5(–3.3) *Significantly different. Table 3. Colonization rate in inoculation experiments. Lichen species inoculated Inoculum Cylindromonium dirinariae Cylindromonium lichenicola Control Dirinaria applanata 16/20* (80 %)** 13/15 (87 %) 3/12 (25 %) Parmotrema tinctorum 4/4 (100 %) 2/2 (100 %) 0/5 (0 %) *Number of colonised lichenicolous fungi/the number of inoculated lichens. **Perithecia produced. Conflict of interest: The authors declare that there is no conflict of interest. ==== Refs REFERENCES Ahmadjian V (1961). Studies on lichenized fungi. The Bryologist 64 : 168–179. Brackel WV (2010). 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==== Front Fungal Syst Evol Fungal Syst Evol FUSE Fungal Systematics and Evolution 2589-3823 2589-3831 Fungal Systematics and Evolution 10.3114/fuse.2023.11.02 Articles New records and barcode sequence data of wood-inhabiting polypores in Benin with notes on their phylogenetic placements and distribution Olou B.A. 1 * Langer E. 2 Ryvarden L. 3 Krah F.-S. 4 Hounwanou G.B. 1 Piepenbring M. 5 Yorou N.S. 1 1 Research Unit Tropical Mycology and Plants-Soil Fungi Interactions (MyTIPS), Faculty of Agronomy, University of Parakou, BP 123 Parakou, Benin 2 Department of Ecology, University of Kassel, Heinrich-Plett-Str. 40, Kassel, Germany 3 Institute of Biology, University of Oslo, P.O. Box 1066, Blindern, N-0316 Oslo, Norway 4 Faculty of Biological Sciences, Institute for Ecology, Evolution and Diversity, Conservation Biology, Goethe University Frankfurt, Frankfurt am Main, Germany 5 Department of Mycology, Goethe University Frankfurt am Main, Biologicum, Max-von-Laue-Str. 13, 60438, Frankfurt am Main, Germany *Corresponding author: borisolou@yahoo.fr Corresponding editor: P.W. Crous 23 1 2023 1 2023 11 1142 16 8 2022 13 1 2023 © 2023 Westerdijk Fungal Biodiversity Institute 2023 https://creativecommons.org/licenses/by-nc-nd/4.0/ Fungal Systematics and Evolution is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License Wood-inhabiting fungi (WIF), such as polypores, are extremely species-rich and play vital roles in the functioning of forest ecosystems as decomposers. Despite the importance of polypores, our knowledge of the diversity and distribution of these fungi is still poor in general and especially for West Africa. To advance our knowledge we here summarise results from field collections between 2017 and 2021 and present (i) a taxonomic overview, (ii) phylogenetic placements and (iii) an illustrated catalogue of wood-inhabiting polypore fungi with colour pictures. During the field sampling campaigns, we collected 647 specimens. Based on morphological characteristics and molecular barcode data, 76 polypore species belonging to six orders, 15 families and 39 genera were identified. Of the 76 species, 30 are new to the West Africa, 69 new to Benin, and two new combinations Fuscoporia beninensis and Megasporia minuta are proposed. With this summary, we provide new data for further research. Citation: Olou BA, Langer E, Ryvarden L, Krah F-S, Hounwanou GB, Piepenbring M, Yorou NS (2023). New records and barcode sequence data of wood-inhabiting polypores in Benin with notes on their phylogenetic placements and distribution. Fungal Systematics and Evolution 11: 11–42. doi: 10.3114/fuse.2023.11.02 catalogue diversity new taxa phylogeny polypore tropical Africa wood-inhabiting fungi ==== Body pmcINTRODUCTION Wood-inhabiting fungi (WIF) such as polypores are ecologically a very diverse group, ranging from saprotrophic, parasitic, to mycorrhizal (Ryvarden & Johansen 1980, Tedersoo et al. 2007). As saprotrophs, they play vital roles in food webs, participate in the recycling of soil carbon and nutrients (Harley 1971), and transform hard-to-digest organic matter (such as lignin and cellulose) into forms usable by other organisms (Stokland et al. 2012). In addition to their ecological importance, polypores have practical importance for human beings as food, medicine and resources for mycoremediation (Gilbertson 1980, Zjawiony 2004, Grienke et al. 2014). Despite the importance of polypores, their diversity and distribution are often overlooked and neglected in the tropics. As result, many species might disappear without been discovered, recorded and identified (Ujang & Jones 2001, Lindenmayer et al. 2013) because of the alarming rate of forest degradation in the tropics and the fact that polypores are partly specialised towards wood from certain plant species (Krah et al. 2018). Likewise, we could not obtain any information on the function of the species in the ecosystem and for human beings (Lindenmayer et al. 2013). Thus, it is important to document the diversity of polypores in less surveyed areas like Benin. Benin is a country located in West Africa and characterised by a great diversity of landscapes and ecosystems. It abounds with a floristic diversity of 2 807 known plant species out of 3 000 estimated (Akoegninou et al. 2006). Despite the floristic diversity coupled with the dependence of polypores on dead or living trees, only 10 species of polypores have been reported up to 2017. These are Favolus tenuiculus, Ganoderma applanatum, Ganoderma lucidum, Ganoderma mbrekobenum, Lentinus squarrosulus, Lentinus tuber-regium, Lentinus velutinus, Nigroporus stipitatus, Pycnoporus sanguineus, and Trametes palisotii (Boa 2004, Ihayere et al. 2010, Eyi-Ndong et al. 2011, Osemwegie et al. 2014, Yorou et al. 2014, Boni & Yorou 2015). These reports were based on field observations and no fungarium material is available for taxonomic revision of some of these species such as G. applanatum and G. lucidum which do not occur in Africa according to recent studies (Cao et al. 2012, Wang et al. 2012). The first mycological investigations in Benin with a focus on basidiomycetes wood-inhabiting polypores started in 2017. From these surveys, 36 species of polypores were fully identified based on morphological examination (Olou et al. 2019a). These species were subsequently listed in the checklist of West African fungi (Piepenbring et al. 2020). Since then, further records and new species of polypores were reported for Benin and West Africa (Olou et al. 2019b, 2020, 2021, Olou & Ryvarden 2021). However, as molecular sequence data are lacking, these specimens have never been included in DNA-based studies of polypores. Knowing that fungal identification based on morphology only sometimes yields unreliable results due to the misleading morphological characteristics (Olson & Stenlid 2002, Giraud et al. 2008, Hughes et al. 2013, Perez et al. 2013, Lücking et al. 2014); there is a need to re-examine previously reported polypore species from morpho-anatomical and molecular perspective. DNA marker sequencing, widely known as barcoding (Hibbett 1992, Bridge et al. 2005, Nilsson et al. 2006, Hibbett et al. 2011, Hibbett & Taylor 2013), has become a popular tool for a variety of studies, including species identification and molecular phylogenetic inference (Hebert et al. 2003, Hebert & Gregory 2005, Savolainen et al. 2005). Different genes or specific DNA regions are used in barcoding application. The ITS region has been widely adopted by the mycological community as the most suitable marker with a high probability of correct identification for species in many groups of fungi (Schoch et al. 2012). Although the ITS region is widely accepted, sequence data for this region are available for less than 1 % (Vu et al. 2014) of the estimated 3.8 million species of fungi (Nilsson et al. 2006, Hawksworth & Lücking 2017, Raja et al. 2017). Moreover nearly 70 % of the described species have not yet been sequenced (Rossman & Palm-hernández 2008). Knowing that African species have been scantily used in DNA based studies, there is no doubt that many of the species not yet sequenced are from tropical Africa. This lack of DNA sequences from African specimens is a problem in phylogenetic analyses in a global context. Considering these issues, this study aims to summarise results from field collections in Benin between 2017 and 2021 and present (i) a taxonomic overview, (ii) phylogenetic placements and (iii) an illustrated catalogue of wood-inhabiting polypores with colour photos. MATERIAL AND METHODS Specimens A total of 647 specimens of WIF were collected in eight different forests of Benin namely the Pahou forest, semi-deciduous dense forest of Lama, the woodlands of Toui-kilibo, Ouémé supérieur, Trois Rivières, Okpara, National Park W, and the gallery forest of Bassila from July to September each year starting from 2017 to 2021. All wood-inhabiting polypores with a focus on basidiomycetes were photographed in their natural environment before recording using a Sony camera, model DSC-HX400V. The geographic coordinates of occurrence of each specimen were recorded. Small pieces of fresh basidiocarps were placed in plastic bags half-filled with silica gel for a later DNA extraction. The rest of basidiocarps were air- or oven-dried at 45–50 °C for 1–2 d depending on the consistency of the basidiomata. The dried basidiomata were then preserved in plastic bags for morphological investigation. Specimens are deposited at the mycological herbaria of the University of Parakou (UNIPAR) in Benin, with duplicates at the University of Kassel (KAS) in Germany, and at the Institute of Biology, University of Oslo (O) in Norway. Wood-inhabiting polypore fungi species identification Macro-morphological descriptions were based on fresh and dried fungarium specimens. Macro-morphological and microstructures descriptions were based on dried fungarium specimens. Macro-morphological characters are described with the aid of a dissecting microscope Leica EZ4 while microstructures are described using a Leica DM500 compound microscope. For the microstructures, fine sections through the basidiomata were prepared for observation using a razor blade under a dissecting microscope Leica EZ4 and mounted in 5 % aqueous solution of potassium hydroxide (KOH) mixed with 1 % aqueous solution of Phloxine. Melzer’s reagent (to test for dextrinoid or amyloid reactions), Cotton Blue (to test for cyanophilic reaction) were used and then examined at a magnification of 1 000× using a Leica DM500 compound microscope. For species identification, we used the identification keys of Ryvarden & Johansen (1980), Gilbertson & Ryvarden (1986, 1987), Bernicchia & Gorjón (2010), Bernicchia & Gorjón (2020). DNA extraction, amplification, and sequencing Genomic DNA of all specimens was extracted using mainly the microwave DNA extraction method (Dörnte & Kües 2013). When microwave DNA extraction did not yield good results, the NucleoSpin Plant II DNA extraction kit (Macherey, Nagel, Germany) and the E.Z.N.A.® Fungal DNA Mini kit according to manufacturer’s instructions were used. An Epoch machine was used to measure the amount of DNA before amplification. The extracted genomic DNA was amplified targeting the nuclear ribosomal DNA region spanning both of the internal transcribed spacers (ITS) for all species with the primer pair ITS-1F/ITS4 (White et al. 1990, Gardes & Bruns 1993). The Polymerase Chain Reaction (PCR) procedure was as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles at 95 °C for 30 s, 52 °C for 30 s, and 68 °C for 1 min, and a final extension of 68 °C for 3 min. For some genera such as Megasporoporia and Microporus, additional regions namely the large subunit 28S nrDNA (LSU) and the translation elongation factor (Tef) with primer pair LR0R/LR5 (Vilgalys & Hester 1990) and EF1-983F/EF1-1567R (Rehner & Buckley 2008) were amplified. The PCR products were further cleaned with a QIAquick PCR Purification Kit according to the manufacturer’s instructions (QIAGEN GmbH, Hilden, Germany) and then sequenced at the company Eurofins Genomics Germany GmbH (https://www.eurofinsgenomics.eu/). Phylogenetic analysis Both sequenced DNA strands (forward and reverse) were used to build the consensus sequences using Geneious v. 5.6.7 (Kearse et al. 2012). Thereafter, the names given to each species on the basis of morphological examination were assigned to each consensus sequence. All newly generated sequences were aligned with similar sequences retrieved from GenBank. The resulting alignment was used to construct a global phylogenetic tree for wood-dwelling fungi. Later, more in-depth phylogenetic trees were constructed for the two most represented orders, namely Hymenochaetales and Polyporales. For Hymenochaetales, 27 ITS sequences retrieved from GenBank were aligned together with 10 ITS sequences from Benin specimens. For the analysis on Polyporales, 93 ITS sequences retrieved from GenBank were aligned together with 49 ITS sequences from Benin specimens. Sequences were aligned with MAFFT v. 7 (Katoh et al. 2017). Then, the alignments were manually adjusted with AliView v. 1.28 (Larsson 2014) and exported as Phylip format. The best-fit evolutionary model was estimated for each alignment using the standard model selection (Kalyaanamoorthy et al. 2017) implemented in IQ-TREE v.1.6.12 (Minh et al. 2020, http://www.iqtree.org/) with the command line –m TESTONLY. Following this substitution model, the phylogenetic tree inference of Maximum likelihood (ML) and Bayesian Inference (BI) were performed to verify the phylogenetic position of all newly generated sequences. On the dataset of all orders, a Maximum likelihood analysis was performed. The Branch support was evaluated using the Ultrafast Bootstrap (UFBoot) (Hoang et al. 2018) with 5 000 replicates and approximate likelihood ratio test (SH-aLRT) (Anisimova et al. 2011) with 5 000 replicates. The analysis was performed in IQ-tree v. 1.6.12 (Minh et al. 2020, http://www.iqtree.org/) with the command line mode. On Hymenochaetales and Polyporales datasets, the branch supports were evaluated using two approaches, the Ultrafast Bootstrap (UFBoot) (Hoang et al. 2018) and posterior probability (PP). The approaches were performed using IQ-TREE v. 1.6.12 (Minh et al. 2020, http://www.iqtree.org/) and MrBayes v. 3.2.7 respectively. The ML was run using IQ-TREE v. 1.6.12 with 5 000 replicates. The BI was executed using MrBayes v. 3.2.7 in command line mode (https://github.com/NBISweden/MrBayes) for five million generations until the standard deviation of split frequencies reached 0.01. Chain convergence was determined using Tracer v. 1.7.1 () and the first 25 % (5 000) trees was discarded as burn-in. The remaining trees were used to build the consensus tree using the Phylogenetic Tree Summarization (SumTrees) program within DendroPy v. 4.3.0. (Sukumaran & Holder 2010, https://github.com/jeetsukumaran/DendroPy). The topology of the ML tree is used, and to add the posterior probabilities (PP) of BI on the ML tree, the Phylogenetic Tree Summarization (SumTrees) program within DendroPy v. 4.3.0. (Sukumaran & Holder 2010, https://github.com/jeetsukumaran/DendroPy) was used. Then, the UFBoot values were added to the ML best tree that already has the posterior probabilities using IQ-TREE v.1.6.12 (Trifinopoulos et al. 2016). The resulting tree with (UFBoot / PP) is presented below and the support values of UFBoot and PP are indicated on each node when they are > = 50 %. For some genera with questionable phylogenetic position of some species, a targeted phylogenetic analysis on each genus was performed using the ITS-LSU combination for Megasporoporia and ITS-LSU-Tef for Microporus. The sequences used in the analyses with the GenBank accession numbers, voucher, and origin of the specimens are presented in the supplementary Table S1. Wood-inhabiting polypore fungi species distribution in Benin The GPS coordinates from the photos of the species taken in the field were extracted using the DNRGPS (https://gisdata.mn.gov/dataset/dnrgps). In addition to the field data, presence records across Africa with their geographic coordinates were downloaded from Global Biodiversity Facilities (GBIF) for each species reported in this study. Moreover, the check-list of fungi of the West-Africa was used to ensure no omission for this region. Duplicate records were deleted and the World Geodesic System 1984 (EPSG 4326) were used to project the geographic coordinates on Africa continent extent under the QGIS v. 3.22.1. We removed erroneous coordinates like coordinates falling in oceans. In order to make the map easier to read, we aggregate occurrences records by genus level. RESULTS Diversity of basidiomycetes wood-inhabiting polypores from Benin In total, 39 genera, 15 families, and six orders representing 76 species of wood-inhabiting polypores are recorded in Benin. The three most diverse genera are Coriolopsis, Perenniporia and Trametes with five species each. Hymenochaetaceae and Polyporaceae are the most dominant families, with respectively 15 and 46 species each. The other 13 families are represented by one, two or three species. Importance of newly generated sequences A total of 152 DNA single direction reads were generated, resulting in 76 consensus sequences namely 59 ITS, 10 LSU, 7 Tef. These sequences are mostly the first ever for wood-inhabiting polypores in West Africa. Before this study, sequences of Microporus concinnus, M. incomptus, and Phellinus beninensis were missing in GenBank and are therefore generated here for the first time. The absence of sequences of African wood-inhabiting polypores has always hampered large-scale studies on this group. With these new sequences generated for the West African specimens, further phylogeographic studies integrating sequences from African specimens will be more easily doable. Phylogenetic placement of Benin wood-inhabiting polypores Phylogenetic analyses revealed distinct clades corresponding to specific taxonomic orders such as Agaricales, Gloeophyllales, Hymenochaetales, Polyporales, and Russulales. Sequences belonging to the orders of Hymenochaetales and Polyporales are the most abundant in our analyses while the other three orders Agaricales, Gloeophyllales and Russulales are less represented (Supplementary Fig. S1). The phylogenetic tree on Hymenochaetales is composed of Hymenochaetaceae and newly generated sequenced are positioned accordingly in the corresponding clades with the exception of sequences named Phellinus beninensis (Fig. 1). Sequences of Ph. beninensis cluster together and fall into the Fuscoporia clade with good branch support (70/1). Further phylogenetic analyses of the genus Fuscoporia confirmed the correct phylogenetic position of Ph. beninensis (Fig. 2). From the phylogenetic tree on Polyporales, newly generated sequences cluster together with other similar sequences retrieved from other studies, except the sequences of Megasporoporia setulosa and sequences of the genus Microporus where species are misplaced (Fig. 3). Sequences of the species identified as Microporus incomptus and M. affinis grouped together with other sequences named M. xanthopus and M. concinnus, while sequences named in this study as Megasporoporia setulosa did not match with other sequences of the same name available in GenBank (Fig. 3). Phylogenetic analyses combining the ITS and LSU regions for Megasporoporia and ITS-LSU-Tef for Microporus resolved the questionable phylogenetic positions of these different species (Fig. 4, 5). Annotated and illustrated checklist of wood-inhabiting polypores An alphabetical list (by genus name) of wood-inhabiting polypores identified in this study is given below. The current name of each species is checked against Index Fungorum and MycoBank. Whenever there is a difference between the two databases, the phylogenetic position of the species in this study or other published studies is used to select the correct current name. Substrate and collection data are provided for each species wherever possible. At this stage, no generic placement can be given to the genus Coriolopsis as the type species (Coriolopsis polyzona) is currently known as Trametes polyzona. As this study did not focus on the genus that may house the Coriolopsis species, we preferred to keep the name Coriolopsis in inverted commas. Species recorded for the first time in Benin, i.e. recorded by us through a series of mycological surveys since 2017 are in bold. Amylosporus campbellii (Berk.) Ryvarden – Fig. 6A. Substrata: On the soil in semi-deciduous dense forest. Material examined: OAB0625, semi-deciduous dense forest of Pahou/ Ouidah (Benin), 8 Jul. 2021; OAB0644, same forest, 8 Jul. 2021. Distribution: Widespread in subtropics and tropics. In Africa, specimens recorded from Nigeria, Kenya, Tanzania, and Ghana (Ryvarden & Johansen 1980, Piepenbring et al. 2020). Climacodon pulcherrimus (Berk. & M.A. Curtis) Nikol. – Fig. 6B. Substrata: On decomposing hardwoods. Material examined: OAB0240, semi-deciduous dense forest of Lama (Benin), 6°57’8’’N, 2°6’12’’E, altitude: 37.8 m a.s.l., 14 Aug. 2018. Distribution: Mainly Tropical, and known from different tropical regions (Moreno et al. 2007). First record in West Africa. “Coriolopsis” byrsina (Mont.) Ryvarden – Fig. 6C. Substrata: Found on dead tree of Diospyros mespiliformis, but maybe found on other angiosperms. Materials examined: OAB0079, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 6°57’59’’N, 2°08’02’’E, altitude: 51. 3 m a.s.l., 3 Aug. 2017; OAB0255, semi-deciduous dense forest of Lama/ Zogbodomey, 6°57’40’’N, 2°8’2’’E, altitude: 154. 3 m a.s.l., 15 Aug. 2018. Distribution: Widespread in tropical Africa, and seen throughout East Africa (Ryvarden & Johansen 1980). First record in West Africa. “Coriolopsis” caperata (Berk.) Murrill – Fig. 6D. Substrata: Found on dead tree of different angiosperm trees. Materials examined: OAB0189, woodland of Trois Rivières in Benin, 10°27′30′′N, 3° 25′11′′E, altitude 386.2 m a.s.l., 27 Aug. 2017; OAB0194, at the same locality, 10°26′53′′N, 3°24′37′′E, altitude 360.9 m a.s.l., 27 Aug. 2017. Distribution: Widespread in tropical Africa and seen throughout East Africa (Ryvarden & Johansen 1980). First record in West Africa. “Coriolopsis” floccosa (Jungh.) Ryvarden Substrata: On angiosperms of all kinds. Material examined: OAB0216, savannah-land of the National Park W (Benin), 11°28’25’’N, 3°3’0’’E, altitude: 281.89 m a.s.l., 31 Aug. 2017. Distribution: Pantropical and common in East Africa in savannah-land and dry forests (Ryvarden & Johansen 1980). First record for the Western African mycobiota. “Coriolopsis” sanguinaria (Klotzsch) Teng – Fig. 6E. Substrata: On dead angiosperms of all kinds. Material examined: OAB0158, woodland of Ouémé supérieur in Benin, 9°46′9′′N, 2°14′40′′E, altitude 382.5 m a.s.l., 25 Aug. 2017; OAB0197, woodland of Trois Rivières in Benin, 10°28′9′′N, longitude 3°24′40′′E, altitude 364.2 m a.s.l., 28 Aug. 2017; OAB0199, woodland of Trois Rivières in Benin, 10°28′8′′N, 3°24′39′′E, altitude 363.4 m a.s.l., 28 Aug. 2017. Distribution: Paleotropical, widespread in East Africa (Ryvarden & Johansen 1980). First record in West Africa. “Coriolopsis” strumosa (Fr.) Ryvarden – Fig. 6F. Substrata: On dead woods. Material examined: OAB0081, semi-deciduous Dense forest of Lama in Benin, 6°57’31’’N, 2°7’59’’E, altitude: 56.1 m a.s.l., 3 Aug. 2017. Distribution: Widespread in the paleotropics from Western Africa to Australia (Ryvarden & Johansen 1980). Cubamyces flavidus (Lév.) Lücking – Fig. 6G. Substrata: On dead wood. Material examined: OAB0047, semi-deciduous dense forest of Lama (Benin), 6°57′58′′N, 2°90′55′′E, altitude 63.5 m a.s.l., 27 Jul. 2017; OAB0090, woodlands of Kilibo (Benin), 8°32’30”N, 2°41’30”E, altitude 334 m a.s.l., 16 Aug. 2017; OAB0196, Trois Rivières (Benin), 10°28′90′′N, 3°24′40′′E, altitude 364.6 m a.s.l., 28 Aug. 2017. Distribution: Tropical species (Zmitrovich et al. 2012). First record in West Africa and species reported from Benin (Olou et al. 2019a, 2020). Cubamyces lactineus (Berk.) Lücking – Fig. 6H. Substrata: On deciduous wood of many kinds. Materials examined: OAB0207, National Park W (Benin), 11°28′13′′N, 3°3′40′′E, altitude 289.7 m a.s.l., 31 Aug. 2017; OAB0232, semi-deciduous dense forest of Lama (Benin), 6°56′40′′N, 2°6′20′′E, altitude 98.3 m a.s.l., 13 Aug. 2018. Distribution: Rare in Africa (Ryvarden & Johansen 1980). First record in West Africa. Diplomitoporus hondurensis (Murrill) Ryvarden – Fig. 6I. Substrata: On dead hardwood. Material examined: Benin, Province Borgou, OAB0183, Woodland of Trois Rivières (Benin), 10°26’46’’N, 3°25’2’’E, altitude: 365.1 m a.s.l., 27 Aug. 2017. Distribution: Mainly distributed in Americas (Kout & Vlasák 2010). First record in tropical Africa. Earliella scabrosa (Pers.) Gilb. & Ryvarden – Fig. 6J. Substrata: On dead deciduous trees. Materials examined: OAB0062, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 6°58’47’’N, 2°5’30’’E, altitude: 58. 4 m a.s.l., 31 Jul. 2017; OAB0067, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 6°57’70’’N, 2°6’30’’E, altitude: 56. 4 m a.s.l., 1 Aug. 2017; OAB0119, Woodland of Toui-Kilibo, 8°32’33’’N, 2°40’47’’E, altitude: 332.8 m a.s.l., 18 Aug. 2017; OAB0169, Woodland of Ouémé Supérieur, 9°46’45’’N, 2°12’49’’E, altitude: 353.2 m a.s.l., 25 Aug. 2017; OAB0186, Woodland of Trois Rivières, 10°26’47’’N, 3°25’30’’E, altitude: 367.3 m a.s.l., 27 Aug. 2017; OAB0212, National Parc W, 11°28’17’’N, 3°30’20’’E, altitude: 285.6 m a.s.l., 31 Aug. 2017. Distribution: Pantropical distribution and quite common in tropical Africa (Ryvarden & Johansen 1980). Echinochaete brachypora (Mont.) Ryvarden – Fig. 6K. Substrata: On dead wood. Materials examined: OAB0071, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 6°56’55’’N, 2°6’4’’E, altitude: 73.3 m a.s.l., 2 Aug. 2017; OAB0147, Woodland of Ouémé supérieur (Benin), 9°45’25’’N, 2°18’38’’E, altitude: 336.2 m a.s.l., 24 Aug. 2017. Distribution: Pantropical, in Africa from Uganda, Tanzania, Malawi, Kenya, Burundi (Ryvarden & Johansen 1980). First record in West Africa. Flavodon flavus (Klotzsch) Ryvarden – Fig. 6L. Substrata: On fallen and standing dead trunks and branches. Materials examined: OAB0005, dry dense forest of Pahou/ Ouidah (Benin), altitude. 17.6 m a.s.l., 18 Jul. 2017; OAB0145, Woodland of Ouémé supérieur (Benin), 9°45’24’’N, 2°18’37’’E, altitude: 336.1 m a.s.l., 24 Aug. 2017; OAB0205, Woodland of Trois Rivières, 10°28’60’’N, 3°24’26’’E, altitude: 350.5 m a.s.l., 28 Aug. 2017; OAB0227, National Parc W, 11°28’14’’N, 3°30’16’’E, altitude: 285.6 m a.s.l., 31 Aug. 2017. Distribution: Throughout tropical Africa (Ryvarden & Johansen 1980). Fomitiporia aff. punctata (P. Karst.) Murrill – Fig. 6M. Substrata: On dead branch of living tree of Isoberlinia doka. Material examined: OAB0208, National parc W, 11°28’14’’N, 3°30’40’’E, altitude: 288.2 m a.s.l., 31 Aug. 2017. Distribution: The distribution of the species is still unknown even though Ryvarden & Johansen (1980) reported the presence of Fomitiporia punctata in East Africa. However, Decock et al. (2007) reported that this species has a distribution restricted to the northern or more temperate areas of the Northern Hemisphere. That means, the specimen identified here as Fomitiporia aff. punctata might be a different or new species and therefore additional specimens and other collections named Fomitiporia punctata in Africa need to be studied in depth morpho-anatomically and molecularly. Fomitopsis ostreiformis (Berk.) T. Hatt. – Fig. 6N. Substrata: Found on dead trunk of Mangifera indica. Material examined: OAB0822, Woodland of Ouémé supérieur (Benin), 9°45’40’’N, 2°25’46’’E, altitude: 342.9 m a.s.l., 19 Jul. 2021. Distribution: Probably a tropical species. Reported mainly in Asia. In Africa, the specimen was reported from Gabon (Liu et al. 2022). This is the first record in West Africa. Fulvifomes fastuosus (Lév.) Bondartseva & S. Herrera – Fig. 6O. Substrata: On dead and living tree of Dialium guineense, and other unidentified angiosperm trees. Materials examined: OAB0015, semi-deciduous dense forest of Pahou/ Ouidah (Benin), 6°23’05’’N, 2°09’22’’E, altitude. 17.6 m a.s.l., 20 Jul. 2017; OAB0043, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 6°57’07’’N, 2°06’03’’E, altitude: 56.8 m a.s.l., 27 Jul. 2017; OAB0235, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 13 Aug. 2018; OAB0237, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 6°56’42’’N, 2°5’49’’E, altitude: 29.6 m a.s.l., 13 Aug. 2018; OAB0252, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 6°57’29’’N, 2°6’4’’E, 15 Aug. 2018; OAB0259, semi-deciduous dense forest of Lama/ Zogbodomey (Benin), 6°58’0’’N, 2°8’3’’E, altitude: 69.3 m a.s.l., 20 Aug. 2018. Distribution: Pantropical and quite common throughout tropical Africa (Ryvarden & Johansen 1980). Fulvifomes indicus (Massee) L.W. Zhou – Fig. 7A. Substrata: On dead and living angiosperm trees. Material examined: OAB0210, National Parc W, 11°28’18’’N, 3°30’20’’E, altitude: 287.5 m a.s.l., 31 Aug. 2017. Distribution: Reported from Asia and Africa (Ryvarden & Johansen 1980, Zhou 2014). New record in West Africa. Fulvifomes rimosus (Berk.) Fiasson & Niemelä – Fig. 7B. Substrata: On dead wood of different kind. Materials examined: OAB0213, National Park W (Benin), 11°28′18′′N, 3°3′30′′E, altitude 285.2 m a.s.l., 31 Aug. 2017; OAB0214, at the same locality, 11°28′18′′N, 3°3′30′′E, altitude 285.2 m a.s.l., 31 Aug. 2017; OAB0215, at the same locality, 11°28′17′′N, 3°3′10′′E, altitude 284.7 m a.s.l., 31 Aug. 2017. Distribution: Cosmopolitan species, reported from Europe, Asia, Africa, and Australia (Ryvarden & Johansen 1980). In West Africa, reported from Togo, Sierra Leone (Piepenbring et al. 2020) and newly reported here from Benin. Fulvifomes yoroui Olou & Langer – Fig. 7C. Substrata: On living tree of Pseudocedrela kotschyi. Material examined: OAB0097, Woodland of Toui-Kilibo in Benin, 8°32′30′′N, 2°40′49′′E, altitude 328.3 m a.s.l., 16 Aug. 2017. Distribution: New to West African mycobiota and presently only reported from Benin (Olou et al. 2019b). Funalia leonina (Klotzsch) Pat. – Fig. 7D. Substrata: On dead wood of different kinds. Material examined: OAB0105, Woodland of Toui-Kilibo in Benin, 8°32′36′′N, 2°41′12′′E, altitude 313.7 m a.s.l., 17 Aug. 2017; OAB0110, woodland of Toui-Kilibo in Benin, 8°32′37′′N, 2°40′13′′E, altitude 329.9 m a.s.l., 18 Aug. 2017; OAB0166, woodland of Ouémé supérieur (Benin), 9°46′50′′N, 2°12′40′′E, altitude 351.1 m a.s.l., 25 Aug. 2017; OAB0181, Trois Rivières (Benin), 10°26′50′′N, 3°25′13′′E, altitude 370.5 m a.s.l., 27 Aug. 2017; OAB0193, Trois Rivières (Benin), 10°26′53′′N, 3°24′37′′E, altitude 348.5 m a.s.l., 27 Aug. 2017. Distribution: Widespread in tropical Africa from Senegal in west to Ethiopia in north and south to South Africa (Ryvarden & Johansen 1980). Fuscoporia beninensis (Olou & Ryvarden) Olou, comb. nov.MycoBank MB 844735. Fig. 7E. Basionym: Phellinus beninensis Olou & Ryvarden, Syn. Fung. (Oslo) 44: 10. 2021. Substrata: On an unidentified dead angiosperm tree. Material examined: OAB0132, Woodland of Toui-Kilibo (Benin), 8°37’06’’N, 2°37’44’’E, altitude: 318.89 m a.s.l., 19 Aug. 2017. Distribution: First record in West Africa and so far, known only from the type locality in Benin (Olou & Ryvarden 2021). Fuscoporia gilva (Schwein.) T. Wagner & M. Fisch. – Fig. 7F. Substrata: On several dead angiosperm tree such as D. guineense, Mimusops andongensis, and other unidentified angiosperm trees. Materials examined: OAB0045, semi-deciduous dense forest of Lama (Benin), 6°57’07’’N, 2°06’03’’E, altitude: 56.8 m a.s.l., 27 Jul. 2017; OAB0070, at the same locality, 6°56’55’’N, 2°06’04’’E, 2 Aug. 2017; OAB0087, at the same locality, 6°56’58’’N, 2°08’18’’E, altitude: 38.5 m a.s.l., 3 Aug. 2017; OAB0108, Woodland of Toui-Kilibo (Benin), 8°32’36’’N, 2°41’13’’E, altitude: 315 m a.s.l., 17 Aug. 2017; OAB0120, at the same locality, 8°32’35’’N, 2°40’48’’E, altitude: 332.8 m a.s.l., 18 Aug. 2017. Distribution: Pantropical and quite common in tropical Africa (Ryvarden & Johansen 1980, Wagner & Fischer 2002). Fuscoporia senex (Nees & Mont.) Ghobad-Nejhad – Fig. 7G. Substrata: On several dead angiosperm tree such as D. guineense, M. andongensis, and other unidentified angiosperm trees. Materials examined: OAB0006, semi-deciduous dense forest of Pahou (Benin), 6°23’30’’N, 2°9’16’’E, altitude: 23.1 m a.s.l., 19 Jul. 2017; OAB0021, dry dense forest of Pahou (Benin), 6°23’30’’N, 2°9’17’’E, altitude: 30.3 m a.s.l., 21 Jul. 2017; OAB0052, semi-deciduous dense forest of Lama (Benin), 6°57’59’’N, 2°9’46’’E, altitude: 56.6 m a.s.l., 28 Jul. 2017; OAB0066, at the same locality, 6°57’70’’N, 2°6’30’’E, altitude: 57.9 m a.s.l., 1 Aug. 2017; OAB0086, at the same locality, 6°56’59’’N, 2°8’16’’E, altitude: 35.3 m a.s.l., 3 Aug. 2017; OAB0106, Woodland of Toui-Kilibo (Benin), 8°32’36’’N, 2°41’12’’E, altitude: 312.89 m a.s.l., 17 Aug. 2017. Distribution: Pantropical and widespread in Africa (Ryvarden & Johansen 1980, Ghobad-nejhad & Dai 2007). First record in West Africa. Ganoderma aridicola J.H. Xing & B.K. Cui – Fig. 7H. Substrata: On dead wood. Materials examined: OAB0233, semi-deciduous dense forest of Lama (Benin), 13 Aug. 2018; OAB0241, at the same locality, 6°57’50’’N, 2°6’80’’E, altitude: 62 m a.s.l., 14 Aug. 2018; OAB0243, at the same locality, 6°57’40’’N, 2°6’30’’E, altitude: 69.2 m a.s.l., 14 Aug. 2018; OAB0254, at the same locality, 6°57’39’’N, 2°6’16’’E, altitude: 58.8 m a.s.l., 15 Aug. 2018. Distribution: So far known from the type locality, South Africa (Xing et al. 2016). First record in West Africa. Ganoderma enigmaticum M.P.A. Coetzee et al. – Fig. 7I. Substrata: On stumps, trunks and dead trees. Materials examined: OAB0063, semi-deciduous dense forest of Lama (Benin), 6°58’47’’N, 2°5’20’’E, altitude: 59.1 m a.s.l., 31 Jul. 2017; OAB0094, Woodland of Toui-Kilibo (Benin), 8°32’30’’N, 2°41’30’’E, altitude: 333.4 m a.s.l., 16 Aug. 2017; OAB0095, at the same locality, 8°32’30’’N, 2°41’30’’E, altitude: 333.2 m a.s.l., 16 Aug. 2017; OAB0099, at the same locality, 8°32’37’’N, 2°41’12’’E, altitude: 314.8 m a.s.l., 17 Aug. 2017; OAB0104, at the same locality, 8°32’36’’N, 2°41’13’’E, altitude: 314.39 m a.s.l., 17 Aug. 2017; OAB0109, at the same locality, 8°32’33’’N, 2°40’53’’E, altitude: 316.39 m a.s.l., 17 Aug. 2017; OAB0113, at the same locality, 8°32’37’’N, 2°40’12’’E, altitude: 328.9 m a.s.l., 18 Aug. 2017; OAB0115, at the same locality, 8°32’36’’N, 2°40’48’’E, altitude: 332.5 m a.s.l., 18 Aug. 2017; OAB0124, at the same locality, 8°37’60’’N, 2°38’31’’E, altitude: 310.6 m a.s.l., 19 Aug. 2017; OAB0136, at the same locality, 8°37’60’’N, 2°37’45’’E, altitude: 318.39 m a.s.l., 19 Aug. 2017; OAB0154, Woodland of Ouémé supérieur (Benin), 9°45’16’’N, 2°8’31’’E, altitude: 324.89 m a.s.l., 24 Aug. 2017; OAB0159, at the same locality, 9°46’90’’N, 2°14’40’’E, altitude: 382.3 m a.s.l., 25 Aug. 2017; OAB0170, Trois Rivières (Benin), 10°26′50′′N, 3°25′17′′E, altitude 373.7 m a.s.l., 27 Aug. 2017; OAB0174, at the same locality, 10°26′49′′N, 3°25′18′′E, altitude 372.5 m a.s.l., 27 Aug. 2017; OAB0175, at the same locality, 10°26′50′′N, 3°25′18′′E, altitude 372.4 m a.s.l., 27 Aug. 2017; OAB0219, National Parc W, 11°28’30’’N, 3°3’40’’E, altitude: 287.5 m a.s.l., 31 Aug. 2017. Distribution: Typified with South African material (Coetzee et al. 2015). First record in West Africa and most abundant Ganoderma species in Benin. Ganoderma aff. lucidum – Fig. 7J. Substrata: On dead wood. Materials examined: OAB0011, dry dense forest of Pahou (Benin), 6°23’57’’N, 2°9’90’’E, altitude: 24.7 m a.s.l., 19 Jul. 2017; OAB0016, at the same locality, 6°23’50’’N, 2°9’22’’E, altitude: 19.7 m a.s.l., 20 Jul. 2017; OAB0017, at the same locality, 6°23’50’’N, 2°9’22’’E, altitude: 15.9 m a.s.l., 20 Jul. 2017; OAB0029, at the same locality, 6°23’50’’N, 2°9’21’’E, altitude: 16 m a.s.l., 20 Jul. 2017; OAB0121, Woodland of Toui-Kilibo (Benin), 8°32’34’’N, 2°41’12’’E, altitude: 321.7 m a.s.l., 18 Aug. 2017; OAB0122, at the same locality, 8°32’34’’N, 2°41’13’’E, altitude: 320.5 m a.s.l., 18 Aug. 2017; OAB0133, at the same locality, 8°37’60’’N, 2°37’44’’E, altitude: 343.3 m a.s.l., 19 Aug. 2017; OAB0139, at the same locality, 8°37’60’’N, 2°37’45’’E, altitude: 317.9 m a.s.l., 19 Aug. 2017; OAB0146, Woodland of Ouémé supérieur (Benin), 9°45’25’’N, 2°18’38’’E, altitude: 336.5 m a.s.l., 24 Aug. 2017. Distribution: Cosmopolitan species but as the interpretation of the name is very variable, the distribution also is quite variable. Thus, the type species is restricted to Europe and part of China (Cao et al. 2012). While the distribution of the species reported here is still unknown. So far, recorded throughout Benin. Ganoderma mbrekobenum E.C. Otto et al. – Fig. 7K. Habitat: On roots and trunks of living or dead trees of several angiosperms. Materials examined: OAB0083, semi-deciduous dense forest of Lama (Benin), 6°57’32’’N, 2°7’42’’E, altitude: 50.2 m a.s.l., 3 Aug. 2017; OAB0123, Woodland of Toui-Kilibo (Benin), 8°37’60’’N, 2°38’32’’E, altitude: 307.89 m a.s.l., 19 Aug. 2017; OAB0143, Woodland of Ouémé supérieur (Benin), 9°45’28’’N, 2°19’58’’E, altitude: 334.6 m a.s.l., 24 Aug. 2017; OAB0187, Trois Rivières (Benin), 10°27′30′′N, 3°25′11′′E, altitude 384.5 m a.s.l., 27 Aug. 2017; OAB0218, National Parc W, 11°28’30’’N, 3°3’40’’E, altitude: 278.7 m a.s.l., 31 Aug. 2017. Distribution: Typified with West African material. The type locality is Ghana (Crous et al. 2016a, b) and this is a new record for Benin. Gloeophyllum striatum (Fr.) Murrill – Fig. 7L. Substrata: On an unidentified dead angiosperm tree. Materials examined: OAB0129, Woodland of Toui-Kilibo (Benin), 8°37’02’’N, 2°38’05’’E, altitude: 308.2 m a.s.l., 19 Aug. 2017; OAB0843, Woodland of Ouémé supérieur (Benin), 9°46’412’’N, 2°14’41’’E, altitude: 364.8 m a.s.l., 20 Jul. 2021. Distribution: Pantropical and widespread through Africa (Ryvarden & Johansen 1980). First record in West Africa. Gloeoporus dichrous (Fr.) Bres. – Fig. 7M. Substrata: On dead wood. Materials examined: OAB0265, semi-deciduous dense forest of Lama (Benin), 20 Aug. 2018. Distribution: Cosmopolitan species (Ryvarden & Johansen 1980). First record in West Africa. Gloeoporus thelephoroides (Hook.) G. Cunn. – Fig. 7N. Substrata: On dead wood. Material examined: OAB0230, semi-deciduous dense forest of Pahou (Benin), 6°22’56’’N, 2°9’12’’E, altitude: 105.9 m a.s.l., 26 Sep. 2017. Distribution: Pantropic, in Africa widespread from Sierra Leone to Rhodesia and Madagascar (Ryvarden & Johansen 1980). Grammothele lineata Berk. & M.A. Curtis – Fig. 7O. Substrata: On deciduous wood of many kinds. Material examined: OAB0515, semi-deciduous dense forest of Lama (Benin), 20 Aug. 2018. Distribution: Widespread in Africa (Ryvarden & Johansen 1980), first record in West Africa. Hexagonia hirta (P. Beauv.) Fr. – Fig. 8A. Substrata: On hard dead wood. Materials examined: OAB0026, semi-deciduous dense forest of Pahou (Benin), 6°23’20’’N, 2°9’14’’E, altitude: 23.2 m a.s.l., 19 Jul. 2017; OAB0140, Woodland of Toui-Kilibo (Benin), 8°37’60’’N, 2°37’45’’E, altitude: 322 m a.s.l., 19 Aug. 2017; OAB0182, Trois Rivières (Benin), 10°26′46′′N, 3°25′30′′E, altitude 365.3 m a.s.l., 27 Aug. 2017. Distribution: Seems to be restricted to Africa (Ryvarden & Johansen 1980). In west Africa reported from Ghana, Sierra Leone and Nigeria (Piepenbring et al. 2020). First record in Benin. Hexagonia hydnoides (Sw.) M. Fidalgo – Fig. 8B. Substrata: On hard dead wood. Materials examined: OAB0201, Trois Rivières (Benin), 10°28′90′′N, 3°24′40′′E, altitude 369.8 m a.s.l., 28 Aug. 2017; OAB0275, semi-deciduous dense forest of Lama (Benin), 6°58′48′′N, 2°40′57′′E, altitude 81.9 m a.s.l., 20 Aug. 2018; OAB0602, Okpara forest (Benin), 9°14′41′′N, 2°43′22′′E, altitude 189.5 m a.s.l., 8 Jun. 2021. Distribution: Quite common in Africa (Ryvarden & Johansen 1980). First record in West Africa. Hexagonia phellinoides Ryvarden – Fig. 8C. Substrata: On dead wood. Materials examined: OAB0203, Trois Rivières (Benin), 10°28′11′′N, 3°24′49′′E, altitude 360.8 m a.s.l., 28 Aug. 2017; OAB0226, National Parc W, 11°28’14’’N, 3°3’16’’E, altitude: 276.8 m a.s.l., 31 Aug. 2017. Distribution: First record in West Africa and known from the type locality Zimbabwe (Ryvarden et al. 2022). Hexagonia tenuis (Hook.) Fr. – Fig. 8D. Substrata: On deciduous wood of all kinds. Materials examined: OAB0012, dry dense forest of Pahou (Benin), 6°23’40’’N, 2°9’21’’E, altitude: 18.6 m a.s.l., 20 Jul. 2017; OAB0054, semi-deciduous dense forest of Lama (Benin), 6°58′50′′N, 2°9′45′′E, altitude 49.6 m a.s.l., 28 Jul. 2018. Distribution: Widespread in Africa (Ryvarden & Johansen 1980). In West Africa reported from Mali, Ghana, Burkina faso, Togo, Senegal, Benin and Gambia (Guissou et al. 2008, Kane & Courtecuisse 2013, Olou et al. 2019a, Piepenbring et al. 2020). Inonotus pachyphloeus (Pat.) T. Wagner & M. Fisch. – Fig. 8E. Substrata: Recorded on dead and living angiosperms trees. Materials examined: OAB0246, semi-deciduous dense forest of Lama (Benin), 6°57′30′′N, 2°6′80′′E, altitude 65.4 m a.s.l., 14 Aug. 2018; OAB0247, at the same locality, 6°57′30′′N, 2°6′80′′E, altitude 41.1 m a.s.l., 14 Aug. 2018; OAB0260, at the same locality, 6°57′40′′N, 2°6′60′′E, altitude 119.5 m a.s.l., 15 Aug. 2018. Distribution: Widespread in Africa (Ryvarden & Johansen 1980). In West Africa, known from Nigeria, Sierra Leone, Burkina Faso and Senegal (Guissou et al. 2008, 2015, Kane & Courtecuisse 2013). First record in Benin. Inonotus rickii (Pat.) D.A. Reid – Fig. 8F. Substrata: On living tree. Material examined: OAB0253, semi-deciduous dense forest of Lama (Benin); 15 Aug. 2018. Distribution: Probably cosmopolitan species. In Africa, records were from Egypt, Guinea, Morocco, and South Africa (Ouabbou et al. 2012, Shehata & Abd El-Wahab 2013, Tchoumi et al. 2020). First record in West Africa. Irpex lacteus (Fr.) Fr. – Fig. 8G. Substrata: On dead wood. Material examined: OAB0176, Trois Rivières (Benin), 10°26′49′′N, 3°25′17′′E, altitude 372.2 m a.s.l., 27 Aug. 2017. Distribution: Cosmopolitan species, in West Africa specimens reported from Benin (Olou et al. 2019a) and Ghana. Laxitextum bicolor (Pers.) Lentz – Fig. 8H. Substrata: On dead wood. Material examined: OAB0126, Woodland of Toui-Kilibo (Benin), 8°37’20’’N, 2°38’50’’E, altitude: 332.2 m a.s.l., 19 Aug. 2017. Distribution: Maybe cosmopolitan species, specimens reported from Africa (Bernicchia & Gorjón 2010). First record in West Africa. Lentinus squarrosulus Mont. – Fig. 8I. Substrata: On dead wood. Materials examined: OAB0263, semi-deciduous dense forest of Lama (Benin), 6°57′16′′N, 2°5′41′′E, altitude 67.59 m a.s.l., 14 Aug. 2018; OAB0761, Woodland of Toui-Kilibo (Benin), 8°32’29’’N, 2°41’30’’E, altitude 323 m a.s.l., 13 Jul. 2021. Distribution: Paleotropical, and widespread in Africa. In West Africa specimens recorded from Benin, Côte d’Ivoire, Ghana, Mali, Nigeria, Burkina Faso, Togo and Sierra Leone (De Kesel et al. 2002, Boa 2004, Guissou et al. 2008, Eyi-Ndong et al. 2011, Kane & Courtecuisse 2013, Boni & Yorou 2015). Lentinus tricholoma (Mont.) Zmitr. – Fig. 8J. Substrata: On dead wood of different kinds. Material examined: OAB0504, Okpara forest (Benin), 9°15′38′′N, 2°43′32′′E, altitude 323.6 m a.s.l., 11 Sep. 2019. Distribution: In Africa, reported from Nigeria (Ryvarden & Johansen 1980) and this is the first record from Benin. Lentinus tuber-regium (Fr.) Fr. – Fig. 8K. Substrata: On dead wood. Material examined: BAA0762, Okpara forest (Benin), 15 Jun. 2021. Distribution: Paleotropical, distribution similar to Lentinus squarrosulus. Lignosus sacer (Afzel. ex Fr.) Ryvarden – Fig. 8L. Substrata: On ground. Material examined: OAB0293, Gallery forest of Bassila (Benin), 11 Aug. 2019. Distribution: Tropical Africa from Sierra Leone to Kenya and south to South Africa (Ryvarden & Johansen 1980, Piepenbring et al. 2020). Megasporia minuta (Y.C. Dai & X.S. Zhou) Olou, comb. nov. MycoBank MB 846986. Basyonym: Megasporoporia minuta Y.C. Dai & X.S. Zhou, Mycol. Prog. 7: 254. 2008. Distribution: So far known from China. Megasporoporia setulosa (Henn.) Rajchenb. – Fig. 8M. Substrata: On fallen and standing dead trunks and branches. Material examined: OAB0060, semi-deciduous dense forest of Lama (Benin), 6°58′47′′N, 2°5′20′′E, altitude 55.9 m a.s.l., 31 Jul. 2017; OAB0065, at the same locality, 6°58′48′′N, 2°5′60′′E, altitude 60 m a.s.l., 31 Jul. 2017; OAB0101, Woodland of Toui-Kilibo (Benin), 8°32’36’’N, 2°41’12’’E, altitude: 315.2 m a.s.l., 17 Aug. 2017; OAB0102, at the same locality, 8°32′37′′N, 2°41′12′′E, altitude: 314.8 m a.s.l., 17 Aug. 2017; OAB0138, at the same locality, 8°37′60′′N, 2°37′45′′E, altitude: 314.2 m a.s.l., 19 Aug. 2017. Distribution: Very common in East Africa (Ryvarden & Johansen 1980). Reported from Ghana in West Africa (Masuka & Ryvarden 1999). Microporus affinis (Blume & T. Nees) Kuntze – Fig. 8N. Substrata: On deciduous dead wood. Material examined: OAB0082, semi-deciduous dense forest of Lama (Benin), 6°57′31′′N, 2°8′00′′E, altitude 56.1 m a.s.l., 3 Aug. 2017. Distribution: Common species throughout the tropics in the old world from western Africa to the Pacific area (Ryvarden & Johansen 1980). First record in Benin. Microporus concinnus P. Beauv. – Fig. 8O. Substrata: On deciduous dead wood. Materials examined: OAB0038, semi-deciduous dense forest of Lama (Benin), 6°57′50′′N, 2°10′50′′E, altitude 55.9 m a.s.l., 27 Jul. 2017; OAB0051, at the same locality, 6°57′59′′N, 2°9′47′′E, altitude 55.6 m a.s.l., 28 Jul. 2017; OAB0269, at the same locality, 6°57′60′′N, 2°60′21′′E, altitude 59.1 m a.s.l., 14 Aug. 2018. Distribution: Tropical species. Specimens reported from Cameroon, Nigeria, Tanzania, Sierra Leone and Zaire (Palisot-Beauvois 1804, Ryvarden & Johansen 1980, Piepenbring et al. 2020). First record in Benin. Microporus incomptus (Afzel. ex Fr.) Kuntze – Fig. 9A. Substrata: On deciduous dead wood. Material examined: OAB0039, semi-deciduous dense forest of Lama (Benin), 6°57′50′′N, 2°10′50′′E, altitude 60.6 m a.s.l., 27 Jul. 2017. Distribution: African species. Specimens reported from Nigeria and Sierra Leone (Piepenbring et al. 2020), and this is the first record from Benin. Microporus xanthopus (Fr.) Kuntze – Fig. 9B. Substrata: On deciduous dead wood. Materials examined: OAB0032, semi-deciduous dense forest of Pahou (Benin), 6°23’30’’N, 2°9’31’’E, altitude: 17.4 m a.s.l., 25 Jul. 2017; OAB0034, at the same locality, 6°23’30’’N, 2°9’30’’E, altitude: 12.6 m a.s.l., 25 Jul. 2017; OAB0056, semi-deciduous dense forest of Lama (Benin), 6°58′28′′N, 2°8′41′′E, altitude 41.8 m a.s.l., 31 Jul. 2017; OAB0075, at the same locality, 6°56′55′′N, 2°60′40′′E, altitude 69.7 m a.s.l., 2 Aug. 2017. Distribution: Tropical species, very common throughout the tropics in the old World, from Western Africa to the Pacific Area (Ryvarden & Johansen 1980, Olou et al. 2019a, Piepenbring et al. 2020). Quite common in southern Benin. Mycobonia miquelii (Mont.) Palacio & Westphalen – Fig. 9C. Substrata: On dead wood of different kinds. Materials examined: OAB0041, semi-deciduous dense forest of Lama (Benin), 6°57′50′′N, 2°10′5′′E, altitude 60.5 m a.s.l., 27 Jul. 2017; OAB0699, semi-deciduous dense forest of Lama (Benin), 6°57′31′′N, 2°80′00′′E, altitude 55.6 m a.s.l., 10 Jul. 2021. Distribution: Pantropic, in Africa reported from Republic of Sierra Leone, Uganda, Tanzania, Kenya Ghana, and Nigeria (Ryvarden & Johansen 1980, Piepenbring et al. 2020). First record in Benin. Neonothopanus hygrophanus (Mont.) De Kesel & Degreef – Fig. 9D. Substrata: On dead wood. Material examined: OAB0676, semi-deciduous dense forest of Lama (Benin), 6°57′51′′N, 2°10′00′′E, altitude 52 m a.s.l., 10 Jul. 2021. Distribution: Tropical species, widespread in tropical Africa (Eyi-Ndong et al. 2011). In West Africa, specimens reported from Benin, Burkina-Faso, Ghana, Guinea, Nigeria, Sierra Leone, Togo, (Boa 2004, Eyi-Ndong et al. 2011, Kamou et al. 2015, Piepenbring et al. 2020). Perenniporia beninensis Olou & Ryvarden – Fig. 9E. Substrata: On deciduous dead wood. Material examined: OAB0050, semi-deciduous dense forest of Lama (Benin), 6°57′59′′N, 2°9′46′′E, altitude 54.9 m a.s.l., 28 Jul. 2017. Distribution: So far only known from the type country, Benin (Olou & Ryvarden 2021). Perenniporia centrali-africana Decock & Mossebo – Fig. 9F. Substrata: On deciduous dead wood. Materials examined: OAB0042, semi-deciduous dense forest of Lama (Benin), 6°57′51′′N, 2°10′00′′E, altitude 62.3 m a.s.l., 27 Jul. 2017; OAB0042, at the same locality, 6°56′55′′N, 2°6′40′′E, altitude 54.2 m a.s.l., 2 Aug. 2017; OAB0190, Trois Rivières (Benin), 10°26′59′′N, 3°25′12′′E, altitude 369.1 m a.s.l., 27 Aug. 2017; OAB0220, National Park W (Benin), 11°28′26′′N, 3°30′10′′E, altitude 275.1 m a.s.l., 31 Aug. 2017; OAB0282, semi-deciduous dense forest of Lama (Benin), 6°58′00′′N, 2°8′80′′E, altitude 57.2 m a.s.l., 20 Aug. 2018. Distribution: Probably a pantropical species. Reported from the type locality, Cameroon (Decock & Mossebo 2001), and also reported from Senegal and Benin in West Africa (Decock & Mossebo 2002, Olou et al. 2019a). Perenniporia miniochroleuca Ryvarden – Fig. 9G. Substrata: On dead wood. Material examined: OAB0072, semi-deciduous dense forest of Lama (Benin), 6°56′55′′N, 2°60′40′′E, altitude 72 m a.s.l., 2 Aug. 2017. Distribution: Specimens have only been seen from Zimbabwe (Ryvarden et al. 2022). First record in West Africa. Perenniporia tephropora (Mont.) Ryvarden – Fig. 9H. Substrata: On deciduous dead wood. Materials examined: OAB0008, semi-deciduous dense forest of Pahou (Benin), 6°23’10’’N, 2°9’27’’E, altitude: 15.3 m a.s.l., 20 Jul. 2017; OAB0036, at the same locality, altitude: 13.5 m a.s.l., 25 Jul. 2017; OAB0202, Trois Rivières (Benin), 10°28′11′′N, 3°24′49′′E, altitude 364.1 m a.s.l., 28 Aug. 2017; OAB0224, National Park W, 11°28′21′′N, 3°3′60′′E, altitude 275.89 m a.s.l., 31 Aug. 2017. Distribution: Pantropical, widespread in East Africa (Ryvarden & Johansen 1980). First record in West Africa. Perenniporia vanhulleae Decock & Ryvarden – Fig. 9I. Substrata: Found on dead tree of Diospyros mespiliformis but maybe found on other kind of angiosperms. Materials examined: OAB0096, Woodland of Toui-Kilibo (Benin), 8°32’30’’N, 2°41’20’’E, altitude: 332.6 m a.s.l., 16 Aug. 2017; OAB0164, Woodland of Ouémé supérieur (Benin), 9°46’48’’N, 2°12’59’’E, altitude: 349.5 m a.s.l., 25 Aug. 2017; OAB0188, Trois Rivières (Benin), 10°27′30′′N, 3°25′11′′E, altitude 353.7 m a.s.l., 27 Aug. 2017. Distribution: Widespread in tropical African countries like Zimbabwe, Namibia, and Senegal (Decock & Ryvarden 2015). First record in Benin. Phanerochaete sordida (P. Karst.) J. Erikss. & Ryvarden Substrata: On dead wood. Materials examined: OAB0018, dry dense forest of Pahou (Benin), 6°23’50’’N, 2°9’21’’E, altitude: 15.1 m a.s.l., 20 Jul. 2017; OAB0048, semi-deciduous dense forest of Lama (Benin), 6°57′58′′N, 2°9′56′′E, altitude 63.4 m a.s.l., 27 Jul. 2017; OAB0160, Woodland of Ouémé supérieur (Benin), 9°46’90’’N, 2°14’40’’E, altitude: 380.9 m a.s.l., 25 Aug. 2017. Distribution: Cosmopolitan species (Eriksson et al. 1978). First record in West Africa. Phellinus carteri (Berk. ex Cooke) Ryvarden – Fig. 9J. Substrata: On dead angiosperms. Material examined: OAB0217, National Park W (Benin), 11°28′30′′N, 3°30′30′′E, altitude 277.5 m a.s.l., 31 Aug. 2017. Distribution: Specimens reported from Ghana in Africa (Ryvarden & Johansen 1980), first record in Benin. Phellinus purpureogilvus (Petch) Ryvarden – Fig. 9K. Substrata: On dead wood. Material examined: OAB0151, Woodland of Ouémé supérieur (Benin), 9°45’16’’N, 2°80’25’’E, altitude: 327.8 m a.s.l., 24 Aug. 2017. Distribution: Reported from the type locality in Sri Lanka and Tanzania (Ryvarden & Johansen 1980). First record in West Africa. Phylloporia beninensis Olou & Langer – Fig. 9L. Habitat: On dead wood or dead parts of living trees of woody angiosperms, including Trichilia emetica. Materials examined: OAB0107, woodlands of Kilibo (Benin), 8°32’36.39”N, 2°41’12.80”E, altitude 312 m a.s.l., 17 Aug. 2017; OAB0142, woodlands of Ouémé Supérieur, 9°45’29.09”N, 2°19’58.78”E, altitude 334 m a.s.l., 24 Aug. 2017; OAB0511, woodlands of Okpara, 9°15’36”N, 2°43’28”E, altitude 330.1 m a.s.l., 11 Sep. 2019. Distribution: Currently known from the type locality in Benin (Olou et al. 2021). Phylloporia littoralis Decock & Yombiyeni – Fig. 9M. Substrata: On living branches, twigs of angiosperm trees. Material examined: OAB0204, Trois Rivières (Benin), 10°28′50′′N, 3°24′33′′E, altitude 345.8 m a.s.l., 28 Aug. 2017. Distribution: Known from the type locality in Gabon (Yombiyeni & Decock 2017) and reported here for the first time outside of the type locality. Phylloporia spathulata (Hook.) Ryvarden Substrata: On dead angiosperm trees. Material examined: OAB0294. Distribution: In West Africa, reported from Ghana (Piepenbring et al. 2020) and Benin. Piptoporellus baudonii (Pat.) Tibuhwa, Ryvarden & S. Tibell – Fig. 9N. Substrata: On the ground either from buried roots or from a pseudosclerotium, more rarely on stumps. It attacks many different forest trees and is locally a serious root pathogen in Africa. In Benin, it attacks plant species of the genus Isoberlinia. Materials examined: OAB0603, woodlands of Okpara; OAB0604, at the same locality; OAB0867, Trois Rivières (Benin), 10°27′90′′N, 3°24′53′′E, altitude 372.3 m a.s.l., 17 Aug. 2021. Distribution: Widespread in Tropical Africa (Ryvarden & Johansen 1980; Tibuhwa et al. 2020). Reported from Burkina-Faso (Guissou et al. 2008) and first record in Benin. Podofomes mollis (Sommerf.) Gorjón – Fig. 9O. Substrata: On dead part of living trees. Materials examined: OAB0238, semi-deciduous dense forest of Lama in Benin, 6°56’41’’N, 2°6’11’’E, altitude: 84.8 m a.s.l., 13 Aug. 2018; OAB0670, same locality, 10 Jul. 2021; OAB0697, same locality, 6°57’50’’N, 2°10’00’’E, altitude: 84.8 m a.s.l., 13 Aug. 2018. Distribution: First record in West Africa. Podoscypha bolleana (Mont.) Boidin – Fig. 10A. Substrata: On dead wood. Materials examined: OAB0683, semi-deciduous dense forest of Lama (Benin), 6°57′51′′N, 2°10′00′′E, altitude 39.2 m a.s.l., 10 Jul. 2021; OAB0702, at the same locality, 6°57′50′′N, 2°10′10′′E, altitude 39.2 m a.s.l., 10 Jul. 2021; OAB0722, at the same locality, 11 Jul. 2021. OAB0725, at the same locality, 11 Jul. 2021; OAB0885, National Park W (Benin), 11°28′26′′N, 3°3′10′′E, altitude 277.1 m a.s.l., 24 Jul. 2021; OAB0885, at the same locality, 11°28′24′′N, 3°30′10′′E, altitude 275.5 m a.s.l., 24 Jul. 2021. Distribution: Widespread in tropical regions. In West Africa, reported from Ghana (Piepenbring et al. 2020) and first record in Benin. Pycnoporus sanguineus (L.) Murrill – Fig. 10B. Substrata: On standing and falling trunks of almost every kind of deciduous wood. Materials examined: OAB0088, woodlands of Kilibo (Benin), 8°32’30”N, 2°41’30”E, altitude 333.3 m a.s.l., 16 Aug. 2017; OAB0184, Trois Rivières (Benin), 10°26′46′′N, 3°25′30′′E, altitude 365.4 m a.s.l., 27 Aug. 2017; OAB0507, woodlands of Okpara (Benin), 9°15’40”N, 2°43’30”E, altitude 336.6 m a.s.l., 11 Sep. 2019; OAB0512, same locality, 9°15’41”N, 2°43’24”E, altitude 360.9 m a.s.l., 11 Sep. 2019. Distribution: Pantropical and common in tropical Africa (Ryvarden & Johansen 1980). Widespread in dry areas, and recorded mainly in dry forests and Savannahs in Benin. Schizophyllum commune Fr. – Fig. 10C. Substrata: On dead wood. Material examined: OAB0112, woodlands of Kilibo (Benin), 8°32’37”N, 2°40’13”E, altitude 329.4 m a.s.l., 18 Aug. 2017. Distribution: Cosmopolitan species and widespread in Africa (Piepenbring et al. 2020). Schizophyllum umbrinum Berk. – Fig. 10D. Substrata: On dead wood. Material examined: OAB0507, woodlands of Okpara (Benin), 9°15’39”N, 2°43’47”E, altitude 329.3 m a.s.l., 11 Sep. 2019. Distribution: Probably a tropical species. First record in West Africa. Serpula similis (Berk. & Broome) Ginns – Fig. 10E, F. Substrata: On a dead stump. Material examined: OAB0266, dry dense forest of Pahou (Benin), 20 Jul. 2018. Distribution: Reported from Gambia, Ivory coast, and Nigeria (Piepenbring et al. 2020) and first record from Benin. Theleporus calcicolor (Sacc. & P. Syd.) Ryvarden – Fig. 10G. Substrata: On dead part of living tree of Drypetes floribunda (Müll.Arg.) Hutch. Material examined: OAB0258, semi-deciduous dense forest of Lama (Benin), 6°57′38′′N, 2°6′60′′E, altitude 67.2 m a.s.l., 15 Aug. 2018. Distribution: Reported from Sri Lanka, Malaya, and Tanzania (Ryvarden 1979, Ryvarden & Johansen 1980). First record in West Africa. Tomophagus colossus (Fr.) Murrill – Fig. 10H. Substrata: On a completely degraded angiosperm stump. Material examined: OAB0774, woodlands of Kilibo (Benin), 8°32’28”N, 2°41’40”E, altitude 338.6 m a.s.l., 13 Jul. 2021. Distribution: Pantropical. Known from several Western and Central African countries (Ryvarden & Johansen 1980). First record in Benin. Trametes cingulata Berk. – Fig. 10I. Substrata: On deciduous wood. Materials examined: OAB0093, woodlands of Kilibo (Benin), 8°32’31”N, 2°41’30”E, altitude 333.1 m a.s.l., 16 Aug. 2017; OAB0114, at the same locality, 8°32’36”N, 2°40’48”E, altitude 330.7 m a.s.l., 18 Aug. 2017; OAB0117, at the same locality, 8°32’35”N, 2°40’48”E, altitude 332.8 m a.s.l., 18 Aug. 2017; OAB0135, at the same locality, 8°37’60”N, 2°37’44”E, altitude 325.1 m a.s.l., 19 Aug. 2017; OAB0155, woodlands of Ouémé Supérieur, 9°46’90”N, 2°14’40”E, altitude 386.1 m a.s.l., 25 Aug. 2017; OAB0161, at the same locality, 9°46’90”N, 2°14’39”E, altitude 380.5 m a.s.l., 25 Aug. 2017; OAB0171, Trois Rivières (Benin), 10°26′50′′N, 3°25′17′′E, altitude 372.6 m a.s.l., 27 Aug. 2017; OAB0173, at the same locality, 10°26′50′′N, 3°25′18′′E, altitude 372.3 m a.s.l., 27 Aug. 2017; OAB0178, at the same locality, 10°26′51′′N, 3°25′13′′E, altitude 373.3 m a.s.l., 27 Aug. 2017; OAB0231, dry dense forest of Pahou (Benin), 6°22’59”N, 2°9’70”E, altitude 100.5 m a.s.l., 26 Sep. 2017. Distribution: Widespread in Africa and Asia (Berkeley 1854, Ryvarden & Johansen 1980). Reported from Ghana, Nigeria (Piepenbring et al. 2020) and Benin. It is the most abundant Trametes species in Benin. Trametes palisotii (Fr.) Imazeki – Fig. 10J, K. Substrata: On deciduous wood of all kinds. Materials examined: OAB0118, woodlands of Kilibo (Benin), 8°32’33”N, 2°40’47”E, altitude 332.7 m a.s.l., 18 Aug. 2017; OAB0153, woodlands of Ouémé Supérieur, 9°45’16”N, 2°8’31”E, altitude 326.1 m a.s.l., 24 Aug. 2017; OAB0198, Trois Rivières (Benin), 10°28′80′′N, 3°24′40′′E, altitude 364.1 m a.s.l., 28 Aug. 2017. Distribution: Pantropical and very common in areas with seasonal drought. Specimens reported from Ethiopia to Malawi (Imazeki 1952, Ryvarden & Johansen 1980). Widespread in tropical Africa. Trametes parvispora Olou, Yorou & Langer – Fig. 10L. Substrata: On dead part of living angiosperm tree D. guineense. Materials examined: OAB0022, dry dense forest of Pahou/ Ouidah (Benin), 6°23’2.97”N, 2°9’15.90”E, altitude: 33.1 m a.s.l., 21 Jul. 2017; OAB0023 at the same locality, 6°23’3.07”N, 2°9’16.32”E, altitude 18.4 m a.s.l., 21 Jul. 2017; OAB0267, same locality, 6°23’2.49”N, 2°9’16.27”E, altitude 33.1 m a.s.l., 20 Jul. 2018; OAB0268, at the same locality, 26 Aug. 2018. Distribution: Apart from the type locality, Benin (Olou et al. 2020), the species has recently been reported from Nigeria. Trametes polyzona (Pers.) Justo – Fig. 10M. Substrata: On deciduous wood of many kinds. Materials examined: OAB0092, woodlands of Kilibo (Benin), 8°32’31”N, 2°41’30”E, altitude 332.5 m a.s.l., 16 Aug. 2017; OAB0128, at the same locality, 8°37’20”N, 2°38’50”E, altitude 307.4 m a.s.l., 19 Aug. 2017; OAB0165, woodlands of Ouémé Supérieur, 9°46’49”N, 2°12’49”E, altitude 348.6 m a.s.l., 25 Aug. 2017; OAB0191, Trois Rivières (Benin), 10°26′53′′N, 3°24′38′′E, altitude 372.6 m a.s.l., 27 Aug. 2017; OAB0195, at the same locality, 10°26′53′′N, 3°24′37′′E, altitude 346.9 m a.s.l., 27 Aug. 2017. Distribution: Pantropical, in Africa reported from almost all countries south of Sahara (Ryvarden & Johansen 1980, Justo & Hibbett 2011). Trametes socotrana Cooke – Fig. 10N. Substrata: On dead wood. Materials examined: OAB0131, woodlands of Kilibo (Benin), 8°37’60”N, 2°37’44”E, altitude 326.2 m a.s.l., 19 Aug. 2017; OAB0165, woodlands of Ouémé Supérieur, 9°46’49”N, 2°12’58”E, altitude 350.3 m a.s.l., 25 Aug. 2017. Distribution: Seen in different parts of Africa from Kenya, Tanzania, Burundi, Malawi and Ghana (Ryvarden & Johansen 1980, Piepenbring et al. 2020). First record in Benin. Tyromyces contractus Olou & Ryvarden – Fig. 10O. Substrata: On dead wood. Material examined: OAB0073, semi-deciduous dense forest of Lama (Benin), 29 Jul. 2017. Distribution: Known so far from the type locality, Benin (Olou & Ryvarden 2021). Distribution of identified species in Africa Figure 11 shows the distribution of the species collected and identified in this study. The widest distributed genus is Schizophyllum, represented in this study mainly by Schizophyllum commune. The genus Microporus clearly shows a tropical distribution and is distributed from Senegal to Madagascar. Benin, which was the subject of our study, and the Congo seem to be the best covered. Several other countries are still unexplored or occurrence data are not publicly available. Future mycological survey series will be extended to these countries where currently we have very little data on wood-inhabiting fungi. When zooming in on Benin where we collected the specimens, several species and genera of polypores have a wide distribution from south to north of Benin. However, some genera and species show a geographical pattern. For example, species of the genus Microporus were only found in southern Benin, while species of the genera Gloeophyllum and Piptoporellus were only found in central and northern Benin (dry area of Benin). Within the same genus it is also quite common to observe that although the genus has a wide distribution from the South to the North, some species have narrow distribution and for the moment never recorded in the South. An example is the genus Trametes where species are distributed throughout the country. However, some species like P. sanguineus, T. polyzona, and T. socotrana occur mainly in the North of Benin. DISCUSSION Phylogenetic position of wood-inhabiting polypores in Benin All investigated specimens were first identified by morpho-anatomical characteristics. Further, we generated DNA sequences for wood-inhabiting polypores collected in tropical Benin (West Africa) and used them for phylogenetic analyses in order to confirm the morphological identification. All newly generated sequences fall correctly into the corresponding clades with the exception of the sequences named Megasporoporia setulosa, Microporus concinnus, M. incomptus, M. affinis and M. xanthopus. For the genus Microporus, we performed further phylogenetic analyses involving the ITS, LSU and Tef. The results of these analyses clearly show that M. affinis, M. cocinnus, M. incomptus and M. xanthopus are four different species (Fig. 5 and Supplementary Fig. S2). Of all the M. xanthopus sequences available on GenBank, only the sequence obtained from a specimen from Nigeria (accession number KT273357) groups with our sequences from Benin (Supplementary Fig. S2). All other M. xanthopus sequences available on GenBank are different from the Benin and Nigeria ones. The type locality of M. xanthopus is Nigeria, West Africa (MycoBank), so it makes sense that Benin and Nigeria sequences group together and are conspecific. Other specimens outside of West Africa bearing this name need to be carefully examined both phylogenetically and morphologically. Therefore, a thorough taxonomic study of the Chinese specimens currently known as M. xanthopus will be crucial to shed light not only on the correct identity of these specimens but also to increase our knowledge of the global distribution of M. xanthopus. Phylogenetic analyses of Microporus based on the ITS and the combination ITS-LSU-Tef gave a poor and unresolved tree while a phylogenetic analysis based on Tef gave a better tree (Fig. 3, Supplementary Fig. S2 vs Fig. 5). This shows that even though the ITS region is recognised as a universal and suitable region for correct identification (Schoch et al. 2012), this region alone is not sufficient for some genera and cryptic species (Badotti et al. 2017, Lücking et al. 2020) and therefore studies combining different DNA regions are needed and important for accurate identification. Microporus affinis has several varieties (MycoBank). From our analysis, several sequences identified as M. affinis do not group with others of the same name. Besides, the M. affinis sequence from Benin forms a distinct lineage in our analysis. Unfortunately, no spores were found from our specimen despite several attempts. We therefore preferred to keep the name M. affinis for the Benin specimen and further studies on this species will be carried out as soon as additional specimens of this species become available. Megasporoporia setulosa was originally described from Tanzania, Africa. Our phylogenetic analyses based on the ITS-LSU loci of Megasporoporia and related genera showed the same results like the one from ITS and the specimens from Benin and the one collected by Ryvarden in 1973 from the type locality (Tanzania) group together and form a distinct group far away from sequences from GenBank annotated as M. setulosa from China, Brazil and the USA (Fig. 3, 4). The sequence named M. setulosa from China falls in Megasporia clade and grouped together with a sequence of Megasporia hexagonoides. This shows that this sequence was misidentified. Nevertheless, the other sequences named M. setulosa from the USA and Brazil are not actually setulosa but rather a recently described new species, M. neosetulosa (Lira et al. 2021). This confirms that the sequences from our Benin and Tanzania specimens newly generated in this study are the true M. setulosa. New combinations in the genera Fuscoporia and Megasporia Our phylogenetic analysis of the genus Fuscoporia places the sequences of the species identified as Ph. beninensis in the Fuscoporia clade. Fuscoporia as well as several other genera such as Fomitiporiella, Fulvifomes, Phylloporia, etc. have been segregated from Phellinus sensu lato (Wagner & Fischer 2001, 2002). These small genera share very similar morpho-anatomical characteristics that complicate their differentiation based on morphology alone. As a result, introducing a new species into one of the small genera based on morphology is very challenging to all polyporologists. Despite above-mentioned consideration, Ph. beninensis was introduced as a new species based on morphological characteristics of a specimen from Benin (Olou & Ryvarden 2021). Here, the sequences of the same species fall in Fuscoporia, underlining the importance of combining molecular and morphological data to accurately assign species to genera. Thus, a new combination Fuscoporia beninensis is proposed (see checklist). Although this study did not focus on the genus Megasporia, our phylogenetic analysis on the genus Megasporoporia and related genera such as Megasporia, and Megasporoporiella showed that the sequences of Megasporoporia minuta cluster together and fall into the clade Megasporia. Megasporia is segregated from Megasporoporia sensu lato and characterised by the acyanophilous, non-dextrinoid hyphae, which are also unbranched to sparingly branched, usually lack hyphal pegs and dendrohyphidia, with a neotropical distribution. However, Megasporoporia is characterised by strongly dextrinoid, cyanophilous hyphae, which are also unbranched to sparingly branched, lack dendrohyphidia, with the presence of hyphal pegs in most of species, with tropical and subtropical distributions (Yuan et al. 2017). Megasporoporia minuta is characterised by an annual to biennial habit, resupinate basidiocarp with a distinct sterile margin, and small, cylindrical to oblong-ellipsoid spores. Unlike most species of Megasporoporia, M. minuta has small pores, oblong-ellipsoid basidiospores, and lacks hyphal pegs and dendrohyphidia (Zhou & Dai 2008). These characteristics fit well the genus Megasporia. Megasporoporia minuta was described based on morphological examination only. Given that the genus Megasporia is segregated from Megasporoporia sensu lato, it can therefore be concluded that in the absence of the molecular data, M. minuta has been misassigned, and that in the presence of the molecular data the correct genus for this species is Megasporia. Therefore, we propose here a new combination Megasporia minuta. Diversity and distribution of wood-inhabiting polypores in Benin In this study we provide for the first time an illustrated catalogue of wood-inhabiting polypores with colour photos and molecular data as well as species distributions in Africa. A total of 76 species of wood-inhabiting polypores belonging to six orders, 15 families and 39 genera are reported for Benin based on own collections from 2017 to 2021. Of the 76 species, 30 are new to West Africa and 69 are recorded for the first time for Benin. Of the 10 species reported from Benin before 2017, only five were recollected and identified in this study. The other five species, namely F. tenuiculus, G. applanatum, G. lucidum, L. velutinus, and N. stipitatus, were not identified during our mycological surveys. Future fieldwork and taxonomic investigation will show whether these species occur in Benin. Several specimens belonging to genera like Coltricia, Favolus, Ganoderma, Lentinus, etc. are still awaiting proper identification. The diversity of wood-inhabiting polypores reported here is very low compared to other regions where the diversity is well explored (Lindblad 2001, Cui & Dai 2007, Robledo & Rajchenberg 2007, Dai et al. 2011, Dai 2012, Niemelä 2013, Ryvarden & Melo 2014, Kunttu et al. 2015, Jang et al. 2016, Ginns 2017, Bernicchia & Gorjón 2020, Gafforov et al. 2020). This low diversity in Benin is partly because this study only focused on basidiomycetes and did not take into account corticoid and ascomycetous wood-inhabiting fungi such Xylariales. In addition, the low sampling effort (only eight partially surveyed forests out of a total of 48 forests existing in Benin), and the relatively short survey period compared to diversity studies in other regions are responsible for the low diversity recorded in Benin. For example, Gafforov et al. (2020) reported 153 species of wood inhabiting fungi based on records from 1950 to 2020. Dai (2012) reported 704 species of polypores in China after 20 years of mycological surveys. Therefore, this checklist on polypores in Benin should be considered as provisional. Considering Benin’s floristic diversity of 2 807 species (Akoegninou et al. 2006), and the fact that a high diversity of plant species leads to a high diversity of associated fungal species (Hawksworth 1991, Hawksworth & Lücking 2017) and the scanty knowledge of the mycodiversity in this area (Piepenbring et al. 2020), we are sure that more species of wood-inhabiting polypores can be discovered in Benin in the future. Most of wood-inhabiting polypores reported in this study are saprotrophs and as such they are key players in wood decomposition, maintenance and functioning of forest ecosystems (Harley 1971, Wei & Dai 2004, Purahong et al. 2018). By decomposing dead plant materials, they increase carbon sequestration below ground in forest ecosystems and thereby reducing greenhouse gas (GHG) emissions. In addition to this, they hold effective mechanisms of lignocellulose-decomposing enzymes useful to attack toxic and non-toxic pollutants (Adaskaveg et al. 1990, Coetzee et al. 2015, Kües 2015). Future studies to evaluate the ability of degradation of plastic waste of new species typified with Benin material will be a new and innovative research application idea to take advantage of the enzymes produced by fungi and decomposers. Apart from saprotrophic species, other species inhabiting living wood such as Ganoderma, Phylloporia, Fulvifomes, Inonotus, and Phellinus are reported. Some species of these genera can cause enormous damage and important economic losses to forest ecosystems (Ganglo & Maître 2003). Some wood-inhabiting polypores are edible and/or medicinal mushrooms. Although in Benin the edibility and medicinal effects of polypores are not well known to the public, some species such as Lentinus squarrosulus and Lentinus tuber-regium are used for food (De Kesel et al. 2002, Boni & Yorou 2015, Fadeyi et al. 2017). Apart from these species, we have other species like Echinochateae brachypora which is not known by the Beninese public but reported as edible in other areas in Africa (Eyi-Ndong et al. 2011). Unlike plants, the use of mushrooms in traditional medicine is still unknown, yet they are very effective in the treatment of certain diseases in many regions of the world. Among the species used are species of the genus Trametes and species of the complex Ganoderma lucidum which are used in the treatment of cancer, diabetes, and AIDS (El-Mekkawy et al. 1998, Akbar & Yam 2011). Species of the G. lucidum complex are regarded as the most valuable medicinal mushrooms and used to treat various diseases (Paterson 2006, Grienke et al. 2014, Zhu et al. 2016). As these species are reported in Benin, further ethnomycological, chemotaxonomic, mycochemical, and pharmacological studies will be important to benefit from these species and achieve the Sustainable Development Goals by directly contributing to human health and well-being (SDG3), decent work and economic growth (SDG8). Indirectly this will also contribute to poverty and hunger eradication (SDG1 and 2) through cash income that will be generated from the cultivation of mushrooms like Ganoderma spp. Hiherto, Hymenochaetales and Polyporales are the two orders with the highest diversity in Benin. These two orders have also been reported as the most dominant in other studies dealing with the diversity of wood-inhabiting fungi (Dai 2012, Gafforov et al. 2020). This could be justified by the fact that the distribution of species of these two orders would be largely determined by their degree of specificity for host species and broad environmental conditions. Of the 76 species reported here, three species, namely F. ostreiformis, G. striatum, P. baudonii are brown rot fungi. Both species are recorded in the central and northern Benin (drier area of Benin). Previous studies have revealed the effect of climate on the type of rot (Gilbertson 1981, Hibbett & Donoghue 2001). The distribution of some species would require host and habitat preference and thus a particular environmental condition (Gilbert et al. 2008). This could explain why species of some genera like Microporus, Gloeophyllum, Piptoporellus are only present in one area or habitat. Thus, to maintain the diversity of basidiomycetes wood-inhabiting polypore fungi, special attention should be given to the protection of their habitats. The distribution map provided here is therefore a baseline for good management and decision making on wood-inhabiting fungi. This research was funded by The German Federal Ministry of Education and Research, BMBF (grant No. 01D20015). Boris Armel Olou also received support from the German Academic Exchange Service, DAAD (grant No. 91651629), and IDEA WILD (grant No. OLOUBENI0220). Supplementary Material: http://fuse-journal.org/ Fig. S1. Maximum likelihood tree with rapid bootstrap values. The species names are followed by voucher or strain number and country of origin. Species names in red are from Benin. Fig. S2. Maximum likelihood (ML) analysis of the genus Microporus with rapid bootstrap values based on the combined ITS-LSU-Tef dataset. Newly generated sequences highlighted in red. The sequence names are followed by voucher or strain number and country of origin. Table S1. Taxon names and GenBank accession numbers of some sequences used in this study. Fig. 1. Maximum likelihood (ML) and Bayesian analysis (BI) analyses of Hymenochaetales based on the ITS dataset. Branch support values given as UFBoot / PP. Newly generated and Benin sequences are highlighted in red colour. The sequence names are followed by voucher or strain number and country of origin. Fig. 2. Maximum likelihood (ML) analysis of the genus Fuscoporia with rapid bootstrap values based on the ITS dataset. Newly generated sequences highlighted in red. The sequence names are followed by voucher or strain number and country of origin. Fig. 3. Maximum likelihood (ML) and Bayesian analysis (BI) analyses of Polyporales based on the ITS dataset. Branch support values given as UFBoot / PP. Newly generated and Benin sequences are highlighted in red. The sequence names are followed by voucher or strain number and country of origin. Fig. 4. Maximum likelihood (ML) and Bayesian analysis (BI) analyses of Megasporoporia sensu lato based on the combined ITS-LSU dataset. Branch support values given as UFBoot / PP. Newly generated sequences highlighted in red and in blue sequences previously named as Megasporoporia minuta. The sequence names are followed by voucher or strain number and country of origin. Fig. 5. Maximum likelihood (ML) analysis of the genus Microporus with rapid bootstrap values based on the Tef dataset. Newly generated sequences highlighted in red. The sequence names are followed by voucher or strain number and country of origin. Fig. 6. Macromorphology of polypores in Benin. A. Amylosporus campbellii. B. Climacodon pulcherrimus. C. Coriolopsis byrsina. D. Coriolopsis caperata. E. Coriolopsis sanguinaria. F. Coriolopsis strumose. G. Cubamyces flavidus. H. Cubamyces lactineus. I. Diplomitoporus hondurensis. J. Earliella scabrosa. K. Echinochaete brachypora. L. Flavodon flavus. M. Fomitiporia punctata. N. Fomitopsis ostreiformis. O. Fulvifomes fastuosus. Fig. 7. Macromorphology of polypores in Benin. A. Fulvifomes indicus. B. Fulvifomes rimosus. C. Fulvifomes yoroui. D. Funalia leonina. E. Fuscoporia beninensis. F. Fuscoporia gilva. G. Fuscoporia senex. H. Ganoderma aridicola. I. Ganoderma enigmaticum. J. Ganoderma lucidum group. K. Ganoderma mbrekobenum. L. Gloeophyllum striatum. M. Gloeoporus dichrous. N. Gloeoporus thelephoroides. O. Grammothele lineata. Fig. 8. Macromorphology of polypores in Benin. A. Hexagonia hirta. B. Hexagonia hydnoides. C. Hexagonia phellinoides. D. Hexagonia tenuis. E. Inonotus pachyphloeus. F. Inonotus rickii. G. Irpex lacteus. H. Laxitextum bicolor. I. Lentinus squarrosulus. J. Lentinus tricholoma. K. Lentinus tuber-regium. L. Lignosus sacer. M. Megasporoporia setulosa. N. Microporus affinis. O. Microporus concinnus. Fig. 9. Macromorphology of polypores in Benin. A. Microporus incomptus. B. Microporus xanthopus. C. Mycobonia miquelii. D. Neonothopanus hygrophanus. E. Perenniporia beninensis. F. Perenniporia centrali-africana. G. Perenniporia miniochroleuca. H. Perenniporia tephropora. I. Perenniporia vanhulleae. J. Phellinus carteri. K. Phellinus purpureogilvus. L. Phylloporia beninensis. M. Phylloporia littoralis. N. Piptoporellus baudonii. O. Podofomes mollis. Fig. 10. Macromorphology of polypores in Benin. A. Podoscypha bolleana. B. Pycnoporus sanguineus. C. Schizophyllum commune. D. Schizophyllum umbrinum. E. Serpula similis. F. Hymenophore of S. similis. G. Theleporus calcicolor. H. Tomophagus colossus. I. Trametes cingulata. J. Trametes palisotii. K. Hymenophore of T. palisotii. L. Trametes parvispora. M. Trametes polyzona. N. Trametes socotrana. O. Tyromyces contractus. Fig. 11. Distribution of polypore genera in Africa. Each coloured circle represents a genus. Table 1. Newly generated sequences with the GenBank accession numbers, vouchers, and origin of the specimens. Species name Voucher Origin Accession numbers (ITS) Amylosporus campbellii OAB0625 Benin ON876004 OAB0644 Benin ON876005 Climacodon pulcherrimus OAB0240 Benin ON876006 Coriolopsis byrsina OAB0079 Benin ON876007 OAB0255 Benin ON876008 Earliella scabrosa OAB0062 Benin ON876009 Echinochaete brachypora OAB0147 Benin ON876010 Flavodon flavus OAB0205 Benin ON876011 OAB0227 Benin ON876012 Fomitopsis ostreiformis OAB0822 Benin ON876013 Fulvifomes fastuosus OAB0043 Benin ON876014 Fuscoporia beninensis OAB0132 Benin ON876015 OAB0782 Benin ON876016 Fuscoporia gilva OAB0087 Benin ON876017 Ganoderma aridicola OAB0233 Benin ON876018 Ganoderma enigmaticum OAB0063 Benin ON876019 Ganoderma mbrekobenum OAB0123 Benin ON876020 Gloeophyllum striatum OAB0129 Benin ON885270 Gloeoporus dichrous OAB0265 Benin ON876021 Gloeoporus thelephoroides OAB0230 Benin ON876022 Hexagonia tenuis OAB0013 Benin ON876023 Inonotus pachyphloeus OAB0246 Benin ON876024 OAB0247 Benin ON876025 Inonotus rickii OAB0253 Benin ON876026 Laxitextum bicolor OAB0126 Benin ON876027 Lentinus squarrosulus OAB0761 Benin ON876028 Lentinus tricholoma OAB0504 Benin ON876029 Lignosus sacer OAB0293 Benin ON876030 Megasporoporia setulosa OAB0060 Benin ON876031 OAB0065 Benin ON876032 Microporus affinis OAB0082 Benin ON876033 Microporus concinnus OAB0038 Benin ON876034 OAB0051 Benin ON876035 Microporus incomptus OAB0039 Benin ON876036 Microporus xanthopus OAB0032 Benin ON876037 OAB0034 Benin ON876038 OAB0056 Benin ON876039 OAB0075 Benin ON876040 Neonothopanus hygrophanus OAB0636 Benin ON876041 OAB0676 Benin ON876042 OAB0855 Benin ON876043 Perenniporia centrali-africana OAB0190 Benin ON876044 OAB0221 Benin ON876045 Perenniporia tephropora OAB0036 Benin ON876046 OAB0202 Benin ON876047 OAB0224 Benin ON876048 Perenniporia vanhullii OAB0096 Benin ON876049 OAB0164 Benin ON876050 OAB0188 Benin ON876051 Phanerochaete sordida OAB0018 Benin ON876052 Podoscypha bolleana OAB0669 Benin ON876053 OAB0725 Benin ON876054 Schizophyllum commune OAB0112 Benin ON876055 Schizophyllum umbrinum OAB0507 Benin ON876056 Theleporus calcicolor OAB0258 Benin ON876057 Trametes sanguinea OAB0506 Benin ON885559 Conflict of interest: The authors declare that there is no conflict of interest. ==== Refs REFERENCES Adaskaveg JE Gilbertson RL Blanchette RA (1990). 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==== Front J Am Coll Emerg Physicians Open J Am Coll Emerg Physicians Open 10.1002/(ISSN)2688-1152 EMP2 Journal of the American College of Emergency Physicians Open 2688-1152 John Wiley and Sons Inc. Hoboken 10.1002/emp2.13006 EMP213006 Original Research Pediatrics Pediatric emergency care coordinator workforce: A survey study FOSTER et al. Foster Ashley A. MD https://orcid.org/0000-0002-3059-6218 1 Ashley.foster@ucsf.edu * Li Joyce MD, MPH 2 Wilkinson Matthew H. MD, MPH 3 Ely Michael MHRM 4 5 6 Gausche‐Hill Marianne MD 7 8 9 Newgard Craig MD, MPH 10 Remick Katherine MD 3 11 12 1 Department of Emergency Medicine University of California San Francisco San Francisco California USA 2 Division of Emergency Medicine Boston Children's Hospital Boston Massachusetts USA 3 Department of Pediatrics, Dell Medical School University of Texas at Austin Austin Texas USA 4 Emergency Medical Services for Children Data Center Salt Lake City Utah USA 5 Department of Pediatrics University of Utah Salt Lake City Utah USA 6 Division of Critical Care University of Utah Salt Lake City Utah USA 7 Los Angeles County EMS Agency Los Angeles California USA 8 Departments of Emergency Medicine and Pediatrics David Geffen School of Medicine at University of California Los Angeles Los Angeles California USA 9 Departments of Emergency Medicine and Pediatrics Harbor‐University of California Los Angeles Medical Center Torrance California USA 10 Center for Policy and Research in Emergency Medicine, Department of Emergency Medicine Oregon Health & Science University Portland Oregon USA 11 Department of Surgery, Dell Medical School University of Texas at Austin Austin Texas USA 12 Emergency Medical Services for Children Innovation and Improvement Center, University of Texas at Austin Dell Medical School Austin Texas USA * Correspondence Ashley Foster, MD, Department of Emergency Medicine, University of California San Francisco, 550 16th Street, Box 0649, San Francisco CA 94143, USA. Email: Ashley.foster@ucsf.edu 18 7 2023 8 2023 4 4 10.1002/emp2.v4.4 e1300614 6 2023 05 3 2023 22 6 2023 © 2023 The Authors. JACEP Open published by Wiley Periodicals LLC on behalf of American College of Emergency Physicians. https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. Abstract Objectives The appointment of pediatric emergency care coordinators (PECC) in emergency departments (EDs) enhances pediatric readiness, yet little is understood regarding this workforce. We describe PECC role characteristics, responsibilities, barriers, and threats to the role among a national cohort. Methods We surveyed a sample of PECCs from all regions of the United States who participated in the Emergency Medical Services for Children PECC Workforce and Trauma Collaboratives (2021–2022). EDs were categorized by annual pediatric patient volume: low (<1800), medium (1800–4999), medium‐high (5000–9999), and high (≥10,000). Trend tests were performed to explore the relationship between pediatric volume and PECC characteristics. Results Among 187 PECCs, 114 (61.0%) responded. The majority (75.2%) identified as a nurse. There was a significant difference in median hours per week spent on PECC activities by pediatric volume ranging from a median of 2 hours (interquartile range [IQR] 0.0–2.3) for low pediatric volume to 16 hours (IQR 4.0–37.0) for high pediatric volume (P < 0.001). Most respondents reported more time was needed for PECC activities (58.4%), and desired additional training to support the role (70.8%). Most (74.6%) felt the PECC position should be paid, yet 30.7% reported the role was voluntary. The most frequently assigned responsibilities were education of staff (77.2%) and oversight of quality improvement (QI) efforts (72.8%). Conclusion Characteristics of PECC workforce vary but PECC activities of education and QI work are common among all. There is a reported need for additional training and support. Further studies will determine the impact of PECC characteristics on pediatric readiness. pediatric emergency care coordinator pediatric emergency medicine pediatric emergency preparedness pediatric readiness Health Resources and Services Administration 10.13039/100000102 #U07MC37471‐01‐00 US Department of Health and Human Services (HHS) source-schema-version-number2.0 cover-dateAugust 2023 details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.3.2 mode:remove_FC converted:18.07.2023 Foster AA , Li J , Wilkinson MH , et al. Pediatric emergency care coordinator workforce: A survey study. JACEP Open. 2023;4 :e13006. 10.1002/emp2.13006 Supervising Editor: Mike Wells, MBBCh, PhD ==== Body pmc1 INTRODUCTION 1.1 Background Most children are cared for in general emergency departments that see fewer than 10–15 pediatric patients per day. 1 , 2 Pediatric‐specific resources within an ED may vary based on ED characteristics, including pediatric annual volume. 2 A joint policy statement co‐authored by the American Academy of Pediatrics (AAP), the American College of Emergency Physicians (ACEP), and the Emergency Nurses Association (ENA) recommends all EDs have both a physician and nurse pediatric emergency care coordinator (PECC) as a critical component of maintaining pediatric readiness. 3 , 4 , 5 This is endorsed by the National Pediatric Readiness Project (NPRP), a quality coalition formed by the Health Resources and Services Administration's Emergency Medical Services for Children (EMSC) Program, AAP, ACEP, and ENA as well as recommended by the National Academy of Medicine (NAM). 2 , 6 The role of the PECC is defined by NAM and the NPRP as a nurse and/or physician who serves as pediatric champion and in an administrative role to coordinate quality improvement and education, ensure policies and procedures to care for children are in place, and liaise between departments and hospitals within a health care system. 6 Joint AAP, ACEP, and ENA policy states, “The physician and nurse PECCs may be concurrently assigned other roles in the ED (eg, frontline staff designated by leadership) or may oversee more than 1 program in the ED (ie, medical or nursing director or as coordinator for trauma, stroke, or cardiac [ST‐segment–elevation myocardial infarction]). PECC roles may be shared through formal agreements with administrative entities, such as within hospital systems, when there is another ED capable of providing definitive pediatric care.” 4 The PECC position might be viewed as the pediatric equivalent of a trauma program manager role in a verified trauma center. The PECC nurse and physician roles are accountable for the development, implementation, and evaluation of pediatric emergency care (coordinating all pediatric‐related services in the ED including overseeing pediatric quality and performance improvement efforts, pediatric patient safety, development of pediatric policies, protocols, and practice guidelines, ensuring pediatric competencies and education for staff, and the availability of pediatric equipment and supplies). 4 The presence of at least 1 PECC is “central to the readiness of any ED that cares for children.” 4 Prior literature details presence of a PECC within an ED is strongly correlated with higher pediatric readiness, independent of other factors. 2 , 7 Importantly, high pediatric readiness EDs are associated with decreased pediatric mortality for children with critical illness and injury compared to facilities with low pediatric readiness. 8 , 9 , 10 , 11 , 12 1.2 Importance Although the PECC role has been recommended within the ED to ensure delivery of high‐quality emergency care of children and contributes to high pediatric readiness, little detail is known about how the PECC position is implemented within the ED setting. Prior literature estimates the number of EDs in the United States with a PECC ranges somewhere between 17.2% and 59.3%, depending on the survey used and the survey participants (ED nurse manager, ED medical director). 2 , 13 , 14 One study describes additional characteristics of the PECC role, including a median of 12 hours per week (interquartile range [IQR], 5.0–40.0) spent on role responsibilities and highlights participation in a wide variety of activities. 13 The COVID‐19 pandemic also potentially affected the PECC role and availability of staffing for this role due to changes in pediatric emergency volume, particularly in year 2020. 15 , 16 Further insight into the existing PECC role, potential responsibilities, funding and support offered for the role, and potential barriers and threats to the sustainability of the role within the ED setting are needed to better inform how to support and increase the PECC workforce. 1.3 Goals of this investigation We survey a national cohort of PECCs, to describe the characteristics of individuals serving in the role, assigned responsibilities and estimated time spent, departmental support, ideal role characteristics, perceived benefits of the PECC role, and potential barriers and threats to PECC role implementation within the ED. 2 METHODS 2.1 Design, setting, selection of subjects We conducted a cross‐sectional survey study of PECCs from September 2021 to March 2022. Individuals approached were participants in the EMSC PECC Workforce Development Collaborative (September 1, 2022) 17 or EMSC PECC Trauma Improvement Sprint (February 23, 2022 and March 2, 2022) 18 and identified as serving in the PECC role within the ED. 16 Both collaboratives focused on providing PECC resources, practices, and processes to ensure compliance with pediatric readiness guidelines, as well as networking opportunities for individuals serving in the PECC role. All individuals within both collaboratives were required to complete an initial intake questionnaire that contained the following questions, (1) “Do you consider yourself a PECC or a pediatric champion?” and (2) “We are currently conducting a survey to characterize the PECC position across the nation. Would you be interested in completing this additional survey?” All respondents who selected yes to both questions received an email with a link to the online survey via REDCap software. 19 The study was deemed exempt by the Institutional Review Board at the University of Texas at Austin. 2.2 Survey instrument Survey questions were designed and informed by prior research, reviewed by ED pediatric readiness experts, and a survey methodologist. 2 , 13 Survey questions were refined via an iterative process involving all co‐authors. A pilot instrument was sent to 5 individuals (former PECCs or pediatric emergency clinicians) who were not included in the study to assess ease of access, clarity, and flow. Survey questions covered topics of participant hospital and ED characteristics, participant ED role, PECC role characteristics and assigned responsibilities, potential optimal roles and responsibilities, barriers to obtaining additional training, potential barriers and threats to the perceived success in the PECC role, and perceived impact of the COVID‐19 pandemic on the PECC role. All survey questions can be found in supplemental material (Supplemental File). Reminder emails were sent weekly for up to 3 weeks. For respondents who selected yes to question 1 (“Do you consider yourself a PECC or a pediatric champion?”) and no to question 2 (“We are currently conducting a survey to characterize the PECC position across the nation. Would you be interested in completing this additional survey?”), 1 additional email was sent to ask if they would reconsider completing the survey. The Bottom Line The deployment of pediatric emergency care coordinators in emergency departments enhances the provision of pediatric emergency care. This paper describes their roles and responsibilities, as well as the barriers and challenges faced by these important members of the ED team. 2.3 Hospital and PECC characteristics Hospital‐level variables selected within the study included hospital location by United States region (Northeast, Midwest, South, West, Islands), annual total ED patient volume, annual pediatric ED patient volume, hospital location (urban, suburban, rural, remote, none), hospital categorization (academic, community, county, critical access, none), ED configuration (general ED, pediatric ED, pediatric ED within children's hospital, standby ED, free‐standing ED, other), and presence of hospital pediatric inpatient services. United States regions were defined by the census division. 20 Annual ED pediatric patient volume was categorized based on prior readiness literature: low (<1800 pediatric visits), medium (1800–4999 pediatric visits), medium‐high (5000–9999 pediatric visits), and high (≥10,000 pediatric visits). 2 PECC‐level variables included current participant position (physician, nurse, physician assistant, nurse practitioner, other), and how participant was assigned to the PECC role (designated by assigned or applied for or full‐time position, volunteer, other). Participants were asked if the PECC role is shared with another individual at the hospital. Respondent‐reported proportions of assigned PECC responsibilities were multiplied by hours spent on PECC activities to determine the median number of hours per week estimated for each activity. A time deficit variable was defined by the difference in actual time spent on PECC activities and ideal time needed for PECC activities to explore potential discrepancies between the 2. The time deficit proportion was determined by dividing the participant's protected hours for the PECC role by the participant's stated ideal hours for the PECC role, per week. 2.4 Data analysis Not all questions were answered by all respondents, so denominators vary in the analyses. Descriptive statistics were used to assess survey responses with Stata V14.2 (Stata Corp, College Station, TX). Analyses using the Kruskal Wallis test, as well as non‐parametric and categorical trend tests were performed to explore the relationship between pediatric annual ED volume and participant characteristics, PECC role characteristics, and PECC responsibilities. Statistical tests were 2 tailed with an alpha at 0.05. The Kruskal–Wallis, Wilcoxon rank‐sum, and non‐parametric trend tests were used to assess the relationship between the time deficit and hospital‐level predictor variables and PECC‐level predictor variables. To ensure significant differences did not exist between individuals who share the PECC role compared to responses for individuals who do not share the PECC role, a sensitivity analysis was performed post‐hoc using the chi‐square test, Fisher's exact test, or non‐parametric trend test, as appropriate. Responses to 2 free text questions (What do you value most about your role as the pediatric champion/PECC; Please describe any challenges of barriers to success in your role as the pediatric champion/PECC) within the survey were systematically analyzed through inductive iterative review for thematic analysis by 2 authors (J.L., M.E.). Responses were put into an Excel spreadsheet to allow for comparative theme coding. Emerging themes were discussed among the 2 authors until consensus was achieved. The primary author (A.A.F.) served as the external reviewer and examined the data and reviewed themes derived by the authors (J.L., M.E.) for exhaustiveness. 3 RESULTS Sixty‐one percent (114/187) of the individuals approached completed the survey. Most (75.2%) identified as an ED nurse and have worked a median of 15 years in the ED (Table 1). The affiliated site demographics were collected for each participant (Table S1). Participants worked within all regions in the United States, with the Midwest most represented (36.0%), and most within a general ED (71.9%). The median time spent in the PECC role was 1 year (Table 2). The majority (54.4%) noted that the PECC role was designated (assigned, applied for, or full‐time position) and 30.7% reported the role was a voluntary position. Twenty‐nine percent of participants shared the PECC position with another individual. There were no statistically significant differences in PECC role characteristics between participants who shared the PECC role compared to those who did not (Table S1). TABLE 1 Participant emergency department role characteristics. n (%) Low pediatric annual volume, n = 22 Medium pediatric annual volume, n = 34 Medium‐high pediatric annual volume, n = 24 High pediatric annual volume, n = 34 P value n (%) n (%) n (%) n (%) Role, n = 113 Nurse 85 (75.2) 16 (72.7) 26 (76.5) 17 (70.8) 26 (76.5) 0.276* Nurse practitioner 2 (1.8) 0 (0.0) 1 (2.9) 1 (4.2) 0 (0.0) Physician 13 (11.5) 3 (13.6) 1 (2.9) 2 (8.3) 6 (17.6) Other 13 (11.5) 3 (13.6) 6 (17.6) 3 (12.5) 1 (2.9) Median (years) providing care in the emergency department n [25th, 75th percentile], n = 113 15 [8,20] 13 [7.75, 20] 15 [8, 21.5] 15 [5, 20] 15 [10, 25.5] 0.439** Required maintenance of certifications/training, n = 113 Total 46 (40.7) 8 (36.4) 8 (23.5) 10 (41.7) 20 (58.8) 0.037*** Nurse 34 (30.1) 4 (18.2) 5 (14.7) 8 (33.3) 17 (50.0) Nurse practitioner 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Physician 8 (7.1) 2 (9.1) 1 (2.9) 2 (8.3) 3 (8.8) Other 4 (3.5) 2 (9.1) 2 (5.9) 0 (0.0) 0 (0.0) * Chi‐square test. ** P trend test. *** Fisher's exact test. John Wiley & Sons, Ltd. TABLE 2 Participant pediatric emergency care coordinator (PECC) role characteristics. n (%) Low pediatric annual volume, n = 22 Medium pediatric annual volume, n = 34 Medium‐high pediatric annual volume, n = 24 High pediatric annual volume, n = 34 P value Median (years) as a PECC n = 113, n [25th, 75th percentile] 1 [0, 4] 1 [0, 2.5] 1 [0, 4] 2 [0, 5] 1 [0, 8.25] 0.163** PECC selection, n = 114, n (%) Designated* 62 (54.4) 9 (40.9) 21 (61.8) 14 (58.3) 18 (52.9) 0.202*** Volunteer 35 (30.7) 10 (45.5) 11 (32.4) 6 (25.0) 8 (23.5) Other 17 (14.9) 2 (9.1) 2 (5.9) 4 (16.7) 9 (26.5) Role adjuncts offered n = 114, n (%) Shift reduction 7 (6.1) 1 (4.5) 1 (2.9) 2 (8.3) 3 (8.8) 0.340** Preferential scheduling 4 (3.5) 1 (4.5) 0 (0.0) 2 (8.3) 1 (2.9) 0.802** Monetary compensation 6 (5.3) 0 (0.0) 0 (0.0) 2 (8.3) 4 (11.8) 0.016** Other 4 (3.5) 0 (0.0) 2 (5.9) 0 (0.0) 2 (5.9) 0.477** None of the above 84 (73.7) 19 (86.4) 27 (79.4) 16 (66.7) 22 (64.7) 0.042** PECC position shared, n = 34, n (%) 6 (27.3) 9 (26.5) 5 (20.8) 14 (41.2) 0.256** * Designated = assigned, applied for, or full‐time position. ** P trend test. *** Chi square test. John Wiley & Sons, Ltd. A median of 4 hours per week were spent on PECC activities, with a statistically significant difference in hours spent among pediatric annual volume categories (2 hours low volume, 4 hours medium volume, 5 hours medium‐high volume, 16 hours high volume, P < 0.001). Approximately one third of participants received protected time for the PECC role (32.4%). Among PECCs with protected time, median hours per week of protected time was also directly proportional to the annual pediatric volume category (2 hours low volume, 9 hours medium volume, 20 hours medium‐high volume, 38 hours high volume, P < 0.001). The most frequently assigned responsibilities for all PECCs were education of staff (77.2%) and oversight of quality improvement (QI) efforts (72.8%) (Table 3). QI also represented the assigned responsibility that required the highest median number of estimated hours per week (1.7 hours, range 0.0–28.8 hours). TABLE 3 Assigned responsibilities as a pediatric emergency care coordinator (PECC), proportion of effort spent on activities, and median number of hours per week estimated for each assigned PECC activity. n (%) Mean proportion of effort spent per PECC % Median number of hours per week estimated for each activity per PECC Education of staff 88 (77.2) 21.0% 1.1 Quality improvement 83 (72.8) 27.5% 1.7 Liaise with other hospital committees/departments 77 (67.5) 13.3% 0.8 Simulation activities or training 64 (56.1) 11.4% 0.5 Evidence‐based guidelines/decision support tools development/maintenance 62 (54.4) 10.9% 0.6 Administration/management of ED policies relevant to pediatric readiness 60 (52.6) 12.4% 0.8 Evaluate/assess pediatric competencies of staff 57 (50.0) 11.0% 0.7 Orientation of staff 55 (48.2) 15.2% 0.8 Pediatric disaster preparedness planning 52 (45.6) 9.0% 0.6 Stocking equipment/medications 47 (41.2) 13.1% 0.6 Interfacility transfer agreement and development and maintenance of pediatric resources not available at your facility 30 (26.3) 4.7% 0.7 Public outreach 26 (22.8) 9.5% 0.9 Research 17 (14.9) 8.8% 1.2 Other 13 (11.4) 45.7% 1.4 None of these apply 7 (6.1) not applicable not applicable John Wiley & Sons, Ltd. Participants were asked to provide information on the ideal PECC position. The majority felt more dedicated time was needed to fulfill PECC activities (58.4%), and also desired additional PECC training (70.8%). One hundred percent of participants felt PECCs should have access to state or regional meetings and conferences specifically for PECCs. Most (74.6%) reported the PECC position should be a paid position. There were substantial differences in participant median time spent per week on PECC activities and ideal time to spend on PECC activities per week (Figure 1). The median time deficit for participants was 6.5 hours per week (IQR 4.0–9.8). The median time deficit proportion was 0.4 (IQR 0.3–0.6). There was no statistically significant association found between the time deficit variable and hospital‐ and PECC‐level characteristics. FIGURE 1 Median hours per week spent on pediatric emergency care coordinator (PECC) activities and median ideal hours per week to spend on PECC activities, by annual pediatric volume. Participant responses to questions, “How many hours per week do you spend on pediatric champion/PECC responsibilities and tasks?” and “How many total hours per week spent on pediatric champion/PECC responsibilities would be ideal?” Participants given option to select 0–50. Participants report median of 4 hours per week spent on PECC activities and median of 12 hours per week ideally on PECC activities. Difference in median hours (H) per week spent on PECC activities and median ideal hours per week to spend on PECC activities represented by Δ. Participants shared what they value most about the PECC role through free text response that was coded into themes (Table 4). Prominent themes included: serving as a role model for others, being an agent of change, providing training, resources, and high‐quality pediatric care, and having a passion for serving as the pediatric champion. TABLE 4 Pediatric emergency care coordinator (PECC) role value and challenges/barriers to success in role as a PECC themes. Values Barriers Role model for others: to improve their own knowledge, leadership skills, and learn from pediatric emergency experts, and to lead within their own EDs Agent of change: to make a difference, inspire others to be better, and improve pediatric care in their EDs Provide training and resources for others: to help others to be better prepared, more confident, and knowledgeable in pediatric care Passion for serving as the pediatric champion: have a true passion for pediatrics and feel it is an essential role to ensure pediatric readiness, participants want to advocate for pediatric care Provide high‐quality pediatric care by ensuring consistent, compassionate, evidence‐based care within their communities and retaining care locally PECC is not a formalized role Impact of the COVID‐19 pandemic Lack of compensation Lack of time: both lack of protected time, insufficient time overall, and having competing priorities Lack of administrative support: a general lack of understanding of the role of PECC and not seeing value in the role (this is also compounded by low pediatric volume within adult oriented facilities), and there is lack of sufficient funds to support efforts ED staff barriers: ED understaffed, high turnover, staff not understanding value of PECC role or importance of pediatric training Lack of PECC training/guidance: lack of expectations, role definition, or general pediatric education Isolation from others: having difficulty accessing resources or knowledge, and difficulty accessing networking with other individuals who are working to accomplish similar goals Resistance/hesitancy to change practice John Wiley & Sons, Ltd. Barriers and threats to the PECC role were explored. The most frequent barriers to obtaining additional pediatric training included allotted time off for professional development (57.5%) and lack of department funding (56.6%) (Figure 2). Participants were asked to describe any challenges or barriers to success in their role as the PECC. Common themes included PECC is not a formalized role, lack of compensation, lack of time, lack of administrative support, lack of PECC training/guidance, isolation from others, resistance/hesitancy to change practice, ED staff barriers (eg, ED understaffed, high ED staff turnover, staff not understanding value of PECC role or importance of pediatric training), and impact of COVID‐19 pandemic (Table 4). Most common ongoing potential threats to keeping the PECC position within the ED included competing programs (32.1%) and change in ED leadership (29.5%) (Figure 3). Respondents reported the COVID‐19 pandemic impacted the PECC position in the ED due to decreased patient volume (38.1%), and reassignment to other duties in the ED or hospital (19.5%) (Table S3). FIGURE 2 Participant‐perceived barriers to obtaining additional pediatric training. Participant responses to question, “What are potential barriers to obtaining additional pediatric training?”. Participants given option to select all that apply. Allotted time off for training represented the most frequently selected answer choice (65/113). FIGURE 3 Threats to keeping the pediatric emergency care coordinator (PECC) position within the emergency department. Participant responses to question, “What are ongoing potential threats to keeping the pediatric emergency care coordinator position within your emergency department?” Participants given option to select all that apply. Competing programs represented the most frequently selected answer choice (36/112). Abbreviation: ED, emergency department. 3.1 LIMITATIONS Our study has several limitations. First, the participants were selected from workforce collaboratives and do not represent all staff serving in the PECC role nationally. However, as the participants elected to participate in the workforce collaboratives, they likely represent the most motivated individuals to improve pediatric care, who we should be serving to support in their activities. Second, there is variability in survey respondent ED roles and individual experience within the ED (ie, nurse, physician) and we did not perform on‐site verification of participant‐reported hospital, ED, or PECC characteristics, which may result in differing perspectives on the questions asked as well as reporting bias. Third, we do not have information on survey non‐respondents and our results may not be generalizable to all EDs. Additional survey response rate could have shifted the survey results. However, our respondents represent all types of EDs from regions throughout the United States and represents a broad sampling of PECCs in the United States. 4 DISCUSSION Historically within the United States, the emergency care of children has been uneven, 6 with consequences of increased mortality for patients that are seen in EDs that are unprepared to care for children (lower ED pediatric readiness). 8 , 9 , 10 Despite the association of PECCs with survival, 12 little has been known about PECC characteristics, the time required for the role, and how this time is spent. Through the first national assessment of PECCs, we demonstrated that PECC role characteristics, support, and activities are highly variable. Furthermore, the PECC role seems to experience high turnover, despite the position being filled by experienced nurses, physicians, and nurse practioners, suggesting instability of the position. Finally, the PECC position is often underbudgeted, requiring volunteer efforts. We found that although participants worked in the ED a median of 15 years, they had spent a median of only 1 year within the PECC position. The difference in time within the role may be explained by high frequency of staff and ED position turnover within a hospital, specifically nursing staff. In recent years, ED nursing turnover has become an escalating problem, with literature identifying several factors that contribute to high turnover rates in the ED: workplace violence, critical incidents in the workplace, and the work environment. 21 Moreover, we found that the majority of respondents did not receive protected time for PECC activities, with many reporting a lack of the PECC as a formalized role, lack of administration support, and lack of compensation. Lack of formalization of the role may create interdependence with other ED positions (eg, clinical nurse educator, trauma program manager). This may also lead to PECC turnover with changes in the ED leadership positions. Furthermore, PECCs identified competing priorities as a threat to the position, which likely worsened during the height of the COVID‐19 Pandemic (survey conducted during this time period), when ED staffing and availability of resources experienced significant strain. 22 , 23 , 24 As the PECC role serves as an important component to ensuring pediatric readiness, EDs can work toward formal establishment of the PECC role with appropriate compensation and protected time for pediatric‐specific work to ensure both sustainability for the individual within the PECC role regardless of stressors to the emergency system and the ED PECC role itself. We also found a significant difference in both the amount of time spent on PECC activities and the amount of protected time for PECC activities. These differences were proportional to annual pediatric volume. Maintenance of resources and personnel can be financially prohibitive for hospitals that care for low volumes of children. Consequently, there is greater reliance on pediatric United States tertiary care centers, evident by closure of pediatric inpatient beds, particularly within rural and low‐volume pediatric hospitals. 25 An increased reliance on pediatric regional centers may impact a hospital's decision to invest in pediatric resources for the hospital, including training, and personnel. However, decrease of pediatric resources within community hospitals may have patient‐centered and financial consequences for the health care system. 26 For example, regionalization of pediatric care may lead to attrition of frontline hospital capacity to care for children with conditions ranging from common pediatric illness to critical illness. 27 This may negatively affect an ED's ability to manage critically ill children until appropriate definitive care can be arranged with interfacility transfer. In 2022, hospitals within the New England region with pediatric inpatient and ICU capabilities were at capacity even outside of expected respiratory surges. 28 Subsequently, surrounding EDs had to manage critically ill children for prolonged periods of time and work to transfer sick children in their ED to hospitals sometimes states away. 29 As demand for limited pediatric resources may increase, ensuring all EDs are prepared for managing critically ill children will continue to be vitally important. Regions should work to develop collaborative networks between academic pediatric medical centers and general EDs (“hubs and spokes”) and lead quality initiatives aimed to improve pediatric readiness among all participating EDs. 30 , 31 Most survey participants reported that more time was needed to fulfill PECC activities and desired additional education and training. Impressively, all (100%) respondents of the survey desired access to conferences specifically for PECCs. This may signify a desire to not only learn specific education and skills for the PECC role but also the need to socialize and organize as a community of health care clinicians. As in‐person conferences can be expensive and require significant time and money to coordinate, state or regional PECC‐specific virtual conferences or forums may be a way to provide PECCs with an opportunity to connect and learn. Additionally, workforce collaboratives aimed to develop individuals who are interested in improving the quality of pediatric emergency care, such as the EMSC PECC Workforce Development Collaborative 17 or EMSC PECC Trauma Improvement Sprint, 18 can supplement educational conferences at no cost to the individual. Respondents also identified barriers to obtaining additional pediatric training including allotted time off for training, lack of department funding, and lack of availability of training. Innovation is needed to bring pediatric‐specific education and training to all EDs and could include previously effective initiatives such as in‐situ simulation, shared quality improvement projects among multiple hospitals, and regional conferences organized by state EMSC program managers. 30 , 31 , 32 Additionally, continued focus on dissemination of existing PECC position resources may be necessary to meet the needs of individuals who may be relatively new to the PECC position. 33 , 34 To our knowledge, this is the first study to provide detail about the ED PECC role, how the role is supported, and perceived “ideal” characteristics, barriers, and challenges an individual PECC experiences in the role. These data suggest substantial variability in PECC role characteristics and support, a need for increased protected time for the PECC role, and additional resources to help the individuals within their role. More formalized PECC training programs, an increase in collaborative shared learning networks at the state or regional level, and innovative means to standardize role characteristics and responsibilities may aid in ED PECC role sustainability. AUTHOR CONTRIBUTIONS Drs. Ashley A. Foster, Joyce Li, and Katherine Remick conceptualized and designed the study. Drs. Ashley A. Foster and Katherine Remick drafted the initial manuscript, coordinated, and supervised data collection, and reviewed and revised the manuscript. Dr. Ashley A. Foster collected the data. Dr. Matthew H. Wilkinson carried out the initial quantitative data analysis, and reviewed and revised the manuscript. Dr. Joyce Li and Mr. Michael Ely performed the qualitative data analysis. Drs. Joyce Li, Marianne Gausche‐Hill, Craig Newgard, and Mr. Michael Ely interpreted the data and critically reviewed and revised the manuscript. All authors have approved the final manuscript as submitted and agree to be accountable for all aspects of the work. PRIOR PRESENTATION Abstract to be presented at Pediatric Academic Societies April 30, 2023. Washington, DC. CONFLICT OF INTEREST STATEMENT The authors have no conflicts of interest relevant to this article to disclose. Supporting information Supporting information Click here for additional data file. Supporting information Click here for additional data file. ACKNOWLEDGMENTS The EMS for Children Innovation and Improvement Center is supported by the Health Resources and Services Administration (HRSA, #U07MC374710100) of the US Department of Health and Human Services (HHS) as part of the Emergency Medical Services for Children Innovation and Improvement Center totaling $3,000,000 with 0% financed with non‐governmental sources. Ashley Foster, MD, is an assistant professor of Emergency Medicine at University of California San Francisco. 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==== Front Transl Anim Sci Transl Anim Sci tranas Translational Animal Science 2573-2102 Oxford University Press US 10.1093/tas/txad059 txad059 Non Ruminant Nutrition AcademicSubjects/SCI00960 Water- and feed-based arginine impacts on gut integrity in weanling pigs Greiner Laura Department of Animal Science, Iowa State University, Ames, IA 50011, USA Humphrey Dalton Department of Animal Science, Iowa State University, Ames, IA 50011, USA Kerr Brian USDA ARS, ARS National Laboratory for Agriculture and Environment, Ames, IA 50011, USA Becker Spenser Department of Animal Science, Iowa State University, Ames, IA 50011, USA Breuer Sophie Department of Animal Science, Iowa State University, Ames, IA 50011, USA Hagen Chloe Department of Animal Science, Iowa State University, Ames, IA 50011, USA Elefson Sarah Department of Animal Science, Iowa State University, Ames, IA 50011, USA Haydon Keith CJ America – Bio, Fort Dodge, IA 50501, USA Corresponding author: greinerl@iastate.edu 1 2023 29 5 2023 29 5 2023 7 1 txad05906 9 2022 25 5 2023 18 7 2023 © The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Abstract Two hundred and forty newly weaned pigs (PIC, Hendersonville, TN) were used to determine if supplementing additional arginine (Arg) either in the water or in the feed, and the combinations thereof, improved intestinal integrity and growth performance in nursery pigs. Each of the 80 pens contained three pigs (21 ± 2 d of age) which were randomly allotted to treatments in 4 × 3 factorial arrangement consisting of four water treatments (0%, 4%, 8%, and 12% Arg stock delivered through a 1:128 medication delivery system) in combination with three dietary Arg treatments (1.35%, 1.55%, and 1.75% standardized ileal digestible Arg; SID). Pigs and feeders were weighed at the d0, d6 (water and diet change), d20 (diet change), and d41 for the calculation of average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (G:F). Eighty pigs, 1 pig/pen, were euthanized at d6 for ileum evaluation of villus height and crypt depth. The remaining pigs were taken off the Arg-water treatment and fed phase-2 diets formulated to contain 1.35%, 1.55%, and 1.75% SID Arg. All pigs received a common diet from d20 to d41. Data were analyzed by pen as repeated measures (SAS 9.4). No interaction between water- and dietary-Arg was detected on nursery pig growth performance. There was a significant quadratic effect of SID Arg in the feed on pig final body weight (BW), ADG, ADFI, and G:F (P ≤ 0.037), where feeding 1.55% dietary Arg tended to improve growth performance compared to the 1.35% level for the 41 d of the trial (P ≤ 0.088). The use of the stock 8% Arg in the water resulted in a reduction in crypt depth (0:132.5, 4:140.7, 8:117.3, 12:132.0; P ≤ 0.01) and an improvement in intestinal permeability. The 4% oral Arg significantly reduced villous height:crypt depth ratio (0:2.50, 4:2.09, 8:2.56, 12:2.43; P ≤ 0.02). In conclusion, the feeding of 1.55% Arg resulted in an improvement in nursery pig ADG, ADFI, G:F, and final BW but did not alter intestinal villi morphology; however, the use of Arg in the water resulted in an improvement in intestinal villi, but no phenotypical change in piglet growth in the nursery. arginine nursery pigs water National Pork Board 10.13039/100008370 ==== Body pmcIntroduction The weaning process for pigs is a stressful event due to changes in diet type and consistency, social distress, environmental challenges, or lack of exposure to dry feed prior to weaning. In addition, various studies have documented that the age of weaning can influence intestinal integrity (Spreeuwenberg et al., 2001; Moeser et al., 2007, 2017; Xun et al., 2018). The disruption of gastrointestinal function can influence piglet growth and livability. Furthermore, the greater the disruption typically is associated with subsequent growth performance. Many dietary strategies have been suggested to prevent or alleviate challenges associated with an impaired intestinal barrier. As the intestinal barrier repairs itself, some amino acids have been shown to have benefits. Low levels of certain amino acids, such as arginine (Arg), from nursery pig diets have been shown to significantly restrict growth (Mertz et al., 1952; Roth et al., 1995). Arginine plays an important role in intestinal immunity and barrier function by stimulating protein synthesis and reducing transepithelial permeability, thus improving intestinal absorptive area (Corl et al., 2008; Zhu et al., 2013). Data are conflicted regarding the appropriate level of amino acids that benefit intestinal repair and growth relative to whole-body tissue deposition. Supplementing Arg in the feed has been shown to negatively impact the growth performance of weaned pigs (Southern and Baker, 1982; Hagemeier et al., 1983). However, little is known about the balance of Arg relative to amino acids besides lysine. More recent work did not indicate an adverse effect on feed intake of up to 2% dietary Arg (Hu et al., 2015). The transition from milk to solid food can be delayed due to the change in the animal’s environment and the stress associated with new social hierarchy development, but newly weaned piglets will seek out water (Brooks et al., 1984). Therefore, many swine production facilities utilize water medication systems to deliver electrolytes and other potentially beneficial nutrients to aid in the transition period. Because Arg can improve intestinal immunity and barrier function, supplementing Arg in water may be a feasible option. However, there are no data published concerning Arg in the water. Therefore, the objective of this study was to determine if supplementing Arg, either in feed, water, or both, improves intestinal integrity and growth performance in nursery pigs. Materials and Methods Animal Care Statement All procedures in this experiment adhered to guidelines for the ethical and humane use of animals for research according to the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010) and were approved by the Institutional Animal Care and Use Committee at Iowa State University (IACUC #20-143). Animals and Experimental Design Two hundred and forty barrows and gilts with an average body weight (BW) of 5.17 kg (PIC 337 × 1050, PIC Genus, Hendersonville, TN) arrived immediately after weaning at approximately 21 d of age. Pigs were randomly allocated (mixed gender and gate cut with three pigs per pen) into 80 test pens at the Iowa State Swine Nutrition Research Facility (Ames, IA) upon arrival. Room configuration allowed for four Arg-water treatments with 20 pens per water line. Within each waterline, pens were randomly assigned into an incomplete randomized design to one of three Arg-dietary treatments across the 20 pens to allow for 20 replications per water treatment, 26–27 replications per feed treatment, and 6–7 replications of water × feed. Once allotted into pens, pigs were placed onto the treatment regimens (4 × 3 factorial). Each pen had one single-hole feeder and two nipple drinkers. The pigs were porcine reproductive and respiratory syndrome and porcine epidemic diarrhea virus negative and were vaccinated against Mycoplasma hyopneumoniae and porcine circovirus prior to weaning. Dietary Treatments The three dietary treatments consisted of 1.35%, 1.55%, and 1.75% standardized ileal digestible (SID) Arg (CJ America – Bio, Fort Dodge, IA) designed to provide 0, 0.33, and 0.65 additional g Arg/d above the NRC (2012) recommendations using feed-grade Arg. For the water Arg treatments, feed-grade Arg was placed into clean, plastic buckets filled with fresh water to constitute the four levels of water treatment (0%, 4%, 8%, and 12% Arg). After the solution was thoroughly mixed, a 1:128 proportioner (D25 Fixed Dosatron Injector, Dosatron International, Clearwater, FL) was utilized to distribute the solution into one of the four water lines. The solution was delivered into each row of trial pens at a final concentration of 0.03%, 0.06%, and 0.09% Arg designed to provide 0, 0.22, 0.44, and 0.66 additional g Arg/d, respectively. Diets were formulated to be isocaloric with a constant level of soybean meal. All nutrients were calculated to meet or exceed NRC (2012) requirements. Upon arrival, pigs were placed on both the water and dietary treatments. Water treatments were removed at the end of phase 1 (days 0–6). Pigs remained on the dietary Arg treatments during phase 2 (days 6–20). Following phase 2, pigs were placed on a common diet for the remaining 21 d of the nursery period (days 20–41). Sample Collection and Analysis Pigs and feeders were weighed at the beginning of the trial, day 6 (water and diet change), day 20 (diet change), and day 41 (end of trial) to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F). On day 6 (prior to diet and water treatment change), one pig per pen was orally administered a solution of lactulose (500 mg/kg BW) and mannitol (50 mg/kg BW) via a gastric tube at a dose of 1 mL/kg of BW. Four hours post-gavage, blood was collected via a sterile jugular venipuncture using a 10-mL serum vacutainer tube (Becton Dickinson, Franklin Lakes, NJ). The blood was separated via centrifugation (2000 × g for 10 min at 4 °C), and the serum was collected and stored at −80 °C for later analysis of lactulose and mannitol concentration. Following bleeding, these same pigs were euthanized by captive bolt stunning, followed by exsanguination. Post-euthanasia, the abdomen was opened, and a 2-cm segment of the terminal ileum was dissected, washed with phosphate-buffered saline (pH 7.4), and stored in 10% neutral buffered formalin solution at room temperature for 24 h before being submitted to Iowa State University Diagnostic Laboratory (Ames, IA) for villus and crypt depth evaluation. In short, the samples were removed from formalin, and the tissue was processed following standard histological procedures. In short, the formalin-fixed ilea were processed and embedded in paraffin wax. A 5-µ transverse section was cut, stained with hematoxylin and eosin, and mounted on glass slides. Images of ileal sections were taken. Ten well-oriented villi and crypt pairs were selected. The height of each villus was measured from the top of the villus to the crypt transition. The crypt depth was defined as the invagination between two villi. The 10 individual measurements were taken from each pig, and the average of the 10 observations was used in the statistical analysis. A villous height:crypt depth ratio was calculated from the measurements of crypt depth and villous height for each sample. The remaining pigs in the pen were then taken off the Arg-water treatment and continued on phase 2 dietary treatments, being the same dietary SID Arg level in phase 1 but a slightly different diet composition, Table 1. On day 20 (prior to diet change), one pig per pen was gavaged with lactulose and mannitol using previously described methods. Four hours post-gavage, blood was collected via sterile jugular venipuncture using a serum vacutainer tube. The blood was separated as described above to determine lactulose and mannitol concentration at day 20. After collecting blood, pigs were taken off phase 2 diets and fed a common diet for the next 21 d, with pigs and feeders weighed on day 41 to determine overall pig performance. Mortalities or removals were weighed with a date recorded to adjust growth and feed intake data. Table 1. Diet composition for the trial to assess arginine levels in nursery pig diets. Ingredients are listed as a percent inclusion in the diet and reported on an “as-fed” basis Ingredient, % Phase 1 (d 0–6) Phase 2 (d 6-20) Phase 3 (d 20-41) 1.35% Arg1 1.55% Arg 1.75% Arg 1.35% Arg 1.55% Arg 1.75% Arg Basal Corn 32.555 32.351 32.150 47.766 47.556 47.356 58.580 Soybean meal 47.5% CP2 20.000 20.000 20.000 28.000 28.000 28.000 34.000 Oat groats 15.000 15.000 15.000 5.000 5.000 5.000 — DairyLac 803 12.906 12.906 12.906 5.406 5.406 5.406 — Fish meal 5.000 5.000 5.000 2.500 2.500 2.500 — Soybean oil 3.000 3.000 3.000 3.000 3.000 3.000 3.000 Plasma protein 3.985 3.985 3.985 1.290 1.296 1.296 — Dried whey 2.500 2.500 2.500 2.500 2.500 2.500 — Monocalcium phosphate 1.270 1.270 1.270 0.957 0.957 0.957 1.410 Calcium carbonate 0.800 0.800 0.800 0.903 0.903 0.903 1.100 Arginine4 0.614 0.817 1.020 0.328 0.531 0.733 — l-Lysine HCL 0.500 0.500 0.500 0.500 0.500 0.500 0.450 Zinc oxide 0.375 0.375 0.375 0.375 0.375 0.375 — Sodium chloride 0.300 0.300 0.300 0.300 0.300 0.300 0.500 dl-Methionine 0.294 0.294 0.294 0.282 0.282 0.282 0.252 Vitamin premix5 0.250 0.250 0.250 0.250 0.250 0.250 0.250 l-Threonine 0.209 0.209 0.209 0.205 0.205 0.205 0.179 Trace mineral premix5 0.150 0.150 0.150 0.150 0.150 0.150 0.150 l-Valine 0.146 0.146 0.146 0.152 0.152 0.152 0.121 l-Tryptophan 0.076 0.076 0.076 0.067 0.067 0.067 0.040 Copper sulfate 0.070 0.070 0.070 0.070 0.070 0.070 — Total 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Calculated composition Metabolizable energy, Mcal/kg 3.32 3.32 3.32 3.39 3.39 3.39 3.45 Crude protein, % 22.48 22.66 22.84 21.67 21.85 22.03 21.00 SID Lys6 1.50 1.50 1.50 1.42 1.42 1.42 1.33 Calcium, % 0.93 0.93 0.93 0.79 0.79 0.79 0.77 Available phosphorus, % 0.60 0.60 0.60 0.40 0.40 0.40 0.37 SID Arg, % 1.35 1.55 1.75 1.35 1.55 1.75 1.30 1Arginine. 2Crude protein. 3DairyLac 80 is a granular, high-lactose ingredient manufactured through the process of dry rolling liquid whey permeate that contains 3% crude protein and 80% lactose. 4Arg was provided as free base. 5Vitamin and trace mineral; provided 7,656 IU vitamin A, 875 IU vitamin D, 63 IU vitamin E, 3.8 mg vitamin K, 70 mg niacin, 33.8 mg pantothenic acid, 13.8 mg riboflavin, 0.06 mg vitamin B12, 165 mg Zn (zinc sulfate), 165 mg Fe (iron sulfate), 39 mg Mn (manganese sulfate), 16.5 mg Cu (copper sulfate), 0.3 mg I (calcium iodate), and 0.3 mg Se (sodium selenite) per kilogram of diet. 6SID, standardized ileal digestible; Lys, lysine. Serum samples were submitted to the University of Illinois (Champaign, IL) for the measurement of lactulose and mannitol concentration via high-performance liquid chromatography (Dionex ICS-3000/ICS-5000; Dionex Corp., Sunnyvale, CA). Measurements below the lower limit of detection (LOD; 0.1 µg/mL) were imputed by randomly sampling from a lognormal distribution starting at zero and truncated at the LOD. The parameters of the lognormal distribution were calculated from the original data, with samples below the LOD set equal to the LOD (Cohen and Ryan, 1989). Feed was manufactured at the Iowa State University Swine Nutrition farm feed mill. Feed samples were collected at the completion of mixing for each phase, and a core feed sample from each treatment was taken from the feed storage containers after each dietary treatment in each phase. Samples were then stored at −20 °C for subsequent analysis. Feed samples were ground to 1 mm particle size (Variable Speed Digital ED-5 Wiley Mill; Thomas Scientific, Swedesboro, NJ). Processed feed samples were homogenized and analyzed, in duplicate, for nitrogen (N; method 990.03; AOAC, 2007; Trumac; LECO Corp., St. Joseph, MI). For N analysis, ethylenediaminetetraacetic acid (9.56% N) was used as the standard for calibration and was determined to contain 9.59 ± 0.02% N. Crude protein was calculated as N × 6.25. Feed samples were analyzed at commercial laboratories for proximate analysis (Eurofins Scientific, Des Moines, IA) and amino acids (University of Missouri, Columbus, MO). Statistical Analysis Growth performance and intestinal integrity data were analyzed as repeated measures according to the following model: yijkl=    μ   +wi+fj+(w*f)ij+dk+(w*d)ik+(f*d)jk+(w*f*d)ijk+eijkl, where yijklyijkl is the observed value for lth experimental unit within ith level of Arg in the water and jth level of Arg in the feed on kth day; μ is the overall mean; wiwi is the fixed effect of the ith level of Arg in the water (i = 1–4); fjfj is the fixed effect of the jth level of Arg in the feed (j = 1–3); w∗fij(w∗f)ij is the interaction between Arg in the water and Arg in the feed; dkdk is the fixed effect of fixed effect of the kth day (k = 1–3); w∗dik(w∗d)ik is the interaction between Arg in the water and day; f∗djk(f∗d)jk is the interaction between Arg in the feed and day; w∗f∗dijk(w∗f∗d)ijk is the three-factor interaction between Arg in water, Arg in the feed, and day; eijkleijkl is the random error associated withyijklyijkl, assuming eijkl~N[0, Il ⊗ARH(1)] for growth performance and eijkl~N(0, Il ⊗CS) for lactulose-to-mannitol ratio. IlIlis the identity matrix, ARH(1) is the first-order autoregressive covariance matrix with heterogeneous variances, and CS is the compound symmetry covariance matrix. Lactulose-to-mannitol ratios were natural log-transformed to achieve normality. Histology data were analyzed according to the following statistical model: yijk=   μ   +wi+fj+(w*f)ij+eijk, where yijkyijk is the observed value for the kth experimental unit within ith level of Arg in the water and jth level of Arg in the feed; μ is the overall mean; wiwi is the fixed effect of the ith level of Arg in the water (i = 1–4); fjfj is the fixed effect of the jth level of Arg in the feed (j = 1–3); w∗fij(w∗f)ij is the interaction between Arg in the water and Arg in the feed; eijkeijk is the random error associated withyijkyijk, assuming eijk~N(0,Iσe2). All models were implemented in SAS 9.4 (SAS Inst., Cary, NC) using the GLIMMIX procedure. Covariance matrices were selected as the best fit for the repeated measures models according to Bayesian Information Criterion for each response variable. Normality of the Studentized residuals was verified using the Shapiro-Wilk test from the UNIVARIATE procedure. Studentized residuals greater than three standard deviations from the mean were considered outliers and excluded from the analysis. Data were reported as least squares means, and the SLICE statement was used to perform F-tests between treatments at each day. Means separation was conducted using the PDIFF option with Tukey adjustment for multiplicity. When protected F-test P ≤ 0.10, orthogonal polynomial contrasts were constructed to test linear and quadratic effects of arginine in the feed or the water. The pen was considered the experimental unit. Results were considered significant if P < 0.05 and a tendency if 0.05 < P ≤ 0.10. Results Pigs arrived from the sow facility with an average weaning weight of 5.17 ± 0.48 kg. At the end of the 41 d trial, pigs weighed approximately 21 ± 3.31 kg. Overall, the pigs had a 0.35 ± 0.08 kg average daily gain and a 0.70 ± 0.04 G:F. Analyzed dietary analysis for the Lys and Arg were lower than that of the calculated diets; however, Lys was consistent across treatments, and the Arg increased across treatments as expected (Table 2). Furthermore, total and SID Arg intake (g/d) was similar using calculated or analyzed feed values across all phases (Table 3). Table 2. Diet calculated total and standardized ileal digestible lysine and arginine compared to analyzed values by phase Item, % Phase 1 Phase 2 Phase 3 1.35% SID1 Arg2 1.55% SID Arg 1.75% SID Arg 1.35% SID Arg 1.55% SID Arg 1.75% SID Arg Basal Calculated  Total   Lysine 1.66 1.66 1.66 1.57 1.57 1.57 1.47   Arginine 1.56 1.76 1.96 1.48 1.68 1.89 1.34   Arg:Lys 0.94 1.06 1.18 0.94 1.07 1.20 0.91  SID   Lysine 1.50 1.50 1.50 1.42 1.42 1.42 1.33   Arginine 1.35 1.55 1.75 1.35 1.55 1.75 1.30   Arg:Lys 0.90 1.03 1.17 0.95 1.09 1.23 0.98 Analyzed  Total   Lysine 1.52 1.52 1.55 1.55 1.53 1.50 1.50   Arginine 1.68 1.93 2.14 1.49 1.69 1.90 1.34   Arg:Lys 1.11 1.27 1.38 0.96 1.10 1.27 0.89  SID3   Lysine 1.37 1.37 1.40 1.40 1.38 1.35 1.34   Arginine 1.45 1.70 1.91 1.36 1.56 1.76 1.30   Arg:Lys 1.06 1.24 1.36 0.97 1.13 1.30 0.97 1Standardized ileal digestible. 2Arginine. 3Estimated from analyzed total values. Table 3. Dietary treatment mean arginine intake by phase based on calculated and analyzed feed values Phase 1 Phase 2 Phase 3 1.35% SID1 Arg2 1.55% SID Arg 1.75% SID Arg 1.35% SID Arg 1.55% SID Arg 1.75% SID Arg 1.35% SID Arg 1.55% SID Arg 1.75% SID Arg Total arginine intake, g/d  Calculated 1.02 1.47 1.40 4.48 5.67 5.95 10.54 11.69 10.75  Analyzed 1.10 1.61 1.53 4.51 5.71 5.98 10.54 11.69 10.75 SID arginine intake, g/d  Calculated 0.89 1.29 1.25 4.09 5.24 5.51 10.22 11.34 10.43  Analyzed3 0.95 1.42 1.37 4.12 5.27 5.53 10.22 11.34 10.43 1Standardized ileal digestible. 2Arginine. 3Analyzed SID Arg in feed estimated from analyzed total values. There were no significant water-by-day or feed-by-day interactions for ADG or ADFI (P ≥ 0.277; Table 4). There was a significant three-way interaction for G:F; however, the lack of significance for ADG and ADFI suggests this interaction was driven by high variability in G:F during the first phase of the study; therefore, only main effect results are presented. Averaged over day, supplementing Arg in the feed tended to improve ADG, ADFI, and G:F (P ≤ 0.087). Means separation indicated that feeding 1.55% SID Arg tended to improve ADG, ADFI, and G:F compared to 1.35% SID Arg (P ≤ 0.088), which was further supported by the significant quadratic effect of feed (P ≤ 0.032). Subsequently, there was a quadratic effect of Arg in the feed on final BW (P = 0.037), where pigs fed 1.55% SID Arg tended to be heavier on day 41 compared to pigs fed 1.35% SID Arg (P = 0.077). Supplementing Arg in the water did not impact nursery pig BW, ADG, ADFI, or G:F (P ≥ 0.444). Additionally, there were no interactions between Arg in the feed and water (P ≥ 0.155). Table 4. Effect of arginine in the water or feed on piglet initial and final bodyweight (BW) and overall (days 0–41) average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed ratio (G:F) Item Arginine in water, %1 SEM SID Arginine in feed, %2 SEM 0 4 8 12 1.35 1.55 1.75 BW  Initial BW, kg 5.21 5.18 5.21 5.10 0.111 5.16 5.19 5.16 0.096  Final BW, kg 20.95 21.20 21.68 20.84 0.706 20.35 22.22 20.93 0.619 ADG, kg 0.28 0.28 0.29 0.26 0.014 0.26 0.30 0.27 0.012 ADFI, kg 0.41 0.40 0.42 0.39 0.416 0.39 0.43 0.40 0.015 G:F 0.56 0.57 0.50 0.49 0.044 0.48 0.60 0.52 0.038 P-values Feed3 Water Feed Lin. Quad. Water × feed Day Water × day Feed × day Water × feed × day BW 0.822 0.139 — — 0.285 <0.001 0.798 0.122 0.718  Initial BW, kg4 0.886 0.970 — — — — — — —  Final BW, kg5 0.838 0.089 0.502 0.037 — — — — — ADG, kg 0.574 0.083 0.583 0.031 0.279 <0.001 0.222 0.473 0.805 ADFI, kg 0.685 0.087 0.624 0.032 0.322 <0.001 0.267 0.277 0.737 G:F 0.444 0.069 0.433 0.031 0.155 <0.001 0.558 0.066 0.013 10%, 4%, 8%, and 12% arginine as the free base stock solution prior to 1:128 dilution into the water system. 2SID, standardized ileal digestible. 3Orthogonal polynomial contrasts tested when protected F-test P ≤ 0.10. 4Main effects and polynomial contrasts sliced at d0 from repeated measures model. 5Main effects and polynomial contrasts sliced from d41 from repeated measures model. The use of 8% Arg in the water resulted in a reduction in crypt depth (P < 0.01; Table 5), and 4% Arg reduced total villous height:crypt depth ratio (P < 0.02). The feeding of Arg did not change the villous height:crypt depth ratio. The delivery of Arg in the water resulted in a significant reduction of lactulose:mannitol (P < 0.01) in serum when Arg was added at the level of 4% (Figure 1), but not when provided in the feed. Table 5. Main effects of arginine in the feed or the water on piglet villus height, crypt depth, and villus height:crypt depth ratio Item Arginine in water, %1 SEM SID Arginine in Feed, %2 SEM 0 4 8 12 1.35 1.55 1.75 Villus height 342.4 294.2 299.7 316.7 15.89 300.2 323.1 316.4 13.94 Crypt depth 132.5a 140.7a 117.3b 132.0a 4.5 127.2 129.0 133.2 3.9 Villus:Crypt 2.5a 2.1b 2.6a 2.4a 0.11 2.4 2.5 2.4 0.10 P-values Water3 Water Lin. Quad. Feed Water × feed Villus height 0.145 — — 0.481 0.291 Crypt depth 0.0051 0.219 0.472 0.545 Villus:Crypt 0.018 0.621 0.206 0.975 0.677 10%, 4%, 8%, and 12% arginine as the free base stock solution prior to 1:128 dilution into the water system. 2SID, standardized ileal digestible. 3Orthogonal polynomial contrasts tested when protected F-test P ≤ 0.10. a,bWithout a common superscript differ (P ≤ 0.05). Figure 1. The impact of oral arginine (A) and dietary arginine (B) on serum lactulose:mannitol. Pigs were orally gavaged with a lactulose-mannitol solution, and blood samples were collected approximately four hours later to determine the lactulose:mannitol in the blood. Discussion While the analyzed values of Lys and Arg differed from the calculated values, the ratios of Arg:Lys increased as expected across the treatments and maintained values higher than NRC requirements. Feeding 1.55% SID Arg improved ADG, ADFI, and G:F, which resulted in a 1.87 kg (9%) increase in final BW compared to the pigs fed the 1.35% Arg diet. Due to the quadratic effect of Arg on ADFI, average SID Arg intake based on analyzed feed values was the highest in pigs fed the 1.55% Arg diet (5.10 vs. 6.01 vs. 5.78 g/d for 1.35%, 1.55%, and 1.75% diets, respectively). Together, this may indicate that the improved growth performance resulting from Arg supplementation was largely driven by improvements in ADFI and, consequently, increased Arg intake. Furthermore, there were no feed-by-water interactions, suggesting the added Arg intake allowed through water was not enough to alter the growth response elicited by altering feed Arg levels. All diets were above NRC (2012) requirement for Arg intake throughout the nursery period (3.72 g SID Arg/d), suggesting current recommendations may be underestimated. In this study, increasing Arg in the feed resulted in improved pig performance, with the optimal response occurring at approximately 2.74 g Arg/d higher than current NRC (2012) recommendations. The literature is conflicting regarding the response of nursery pig growth performance to increased Arg. Some studies have shown reduced performance when feeding excess levels of Arg relative to NRC recommendations, while others have observed no changes or improved performance with increased Arg, indicating the balance of other amino acids relative to lysine may determine the response to Arg supplementation (Southern and Baker, 1982; Hagemeier et al., 1983; Anderson et al., 1984; Zhan et al., 2008; Perez-Palencia et al., 2022). The use of lactulose and mannitol to assess intestinal permeability has been widely documented in human trials (Travis and Menzies, 1992; Mishra and Makharia, 2012; Sequeira et al., 2014). Lactulose is a relatively large sugar molecule that cannot be easily transported across the intestinal lumen, while mannitol is a smaller molecule that can readily move across the intestinal epithelium. However, when the tight cell junctions are impaired, lactulose molecules can paracellularly migrate from the intestinal lumen into the bloodstream. Therefore, the ratio of lactulose to mannitol in circulation provides insight on intestinal permeability. A low lactulose:mannitol ratio indicates a tight intestinal barrier, while a high ratio suggests a leaky gut. Therefore, measuring these sugar molecules within the blood can provide insight into the degree of intestinal permeability. Aside from its role as a constituent of protein synthesis, Arg may play regulatory roles in maintaining intestinal barrier and immune function. As described by Das et al. (2010), Arg is a substrate for inducible nitric oxide synthase, which catalyzes the conversion of l-Arg to nitric oxide and l-citrulline. Nitric oxide is an important component of cytotoxic immune cell killing of pathogens. Therefore, Arg may aid in reducing inflammation in the gut by modulating the immune system through nitric oxide synthesis. Furthermore, nitric oxide stimulates angiogenesis and increased blood flow, which may aid tissue repair (Dai et al., 2013). Consequently, supplemental l-Arg has been shown to improve intestinal development in newly-weaned pigs (Zhan et al., 2008). Additionally, in vivo work has shown that l-Arg and l-Cit improve intestinal tight junction barrier function during hypoxia through nitric oxide production (Chapman et al., 2012). The present study showed no differences in intestinal integrity measures when Arg was added to the feed. In contrast, other work has demonstrated that feeding Arg can maintain barrier function in mice (Viana et al., 2010). Further research has demonstrated that feeding mice a diet supplemented with 2% l-Arg decreased intestinal permeability during hyperthermia (Costa et al., 2014). Liu et al. (2008) demonstrated that feeding 1% supplemental Arg reduced IL-6 production within the intestine during an Escherichia coli challenge in nursery pigs. The use of Arg in the water during the first week after weaning resulted in an improvement in intestinal morphology. These findings support other studies showing that 2% oral Arg can reduce intestinal injury (Sukhotnik et al., 2004; Koppelmann et al., 2012). Similarly, Yang et al. (2016) supplemented l-Arg in milk replacer fed to pre-weaned pigs and observed that 8 g/kg l-Arg increased villus height and relative intestine weight in the subsequent nursery period. The current study included Arg at 0%, 4%, 8%, and 12% in the water stock solution, allowing for approximately 0, 0.22, 0.44, and 0.66 g Arg/d intake through the water. Intestinal permeability was improved at both the 4% and 8% inclusion levels compared to pigs receiving the 0% Arg water. In addition, the crypt:villi ratio was improved at the 4% inclusion level in the water compared to pigs receiving the 0% Arg water, suggesting that pigs may benefit from supplementing Arg in the water at the time of weaning. While the delivery of oral Arg improved intestinal permeability and villus height, this response did not depend on the level of Arg in the feed, further supporting the lack of interactive effects of feed and water Arg. The lack of response of oral Arg on phenotypical changes in growth rate could be because the piglets were not significantly challenged with enteric pathogens. Together, this may indicate Arg supplementation in the water or feed may work through different modes of action. The differences in response to Arg in the feed or the water observed in the present study are not clear; therefore, further research investigating these routes of supplementation is warranted. In conclusion, nursery pig growth performance was improved with no changes in intestinal morphology when Arg was increased in the diet. However, the administration of Arg through the water resulted in improved intestinal integrity and morphology but no phenotypical changes in growth. Acknowledgment The authors would like to thank Trey Faaborg for his input and assistance in the completion of this project. Conflict of Interest Statement The authors disclose that this project was funded by CJ Bio America which is the manufacturer of the arginine tested within the study. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by Iowa State University or the USDA and does not imply approval to the exclusion of other products that may be suitable. The USDA is an equal opportunity provider and employer. Funding Two investigators were funded by the National Pork Board. The animal research was funded by CJ Bio America. ==== Refs Literature Cited Anderson, L. C., A. J. Lewis, E. R. Peo, Jr, and J. D. Crenshaw. 1984. Effects of excess arginine with and without supplemental lysine on performance, plasma amino acid concentrations and nitrogen balance of young swine. J. Anim. Sci. 58 :369–377. doi:10.2527/jas1984.582369x.6423600 AOAC International (AOAC). 2007. Official methods of analysis of AOAC Int. 18th ed. Gaithersburg, MD: AOAC Int. Brooks, P. H., S. J. Russell, and J. L. Carpenter. 1984. Water intake of weaned piglets from three to seven weeks old. Vet. 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==== Front Ophthalmic Res Ophthalmic Res ORE ORE Ophthalmic Research 0030-3747 1423-0259 S. Karger AG Basel, Switzerland 37331334 531143 10.1159/000531143 Research Article Combined Trabeculotomy-Non-Penetrating Deep Sclerectomy for Glaucoma in Sturge-Weber Syndrome CTNS for Sturge-Weber Syndrome 2698043 Huang Lulu a b 2698044 Xu Li a b 2698045 Liu Yixin a b 2698046 Yang Yijie a b 2698047 Wang Ning a b 2698048 Gu Mengyang a b 2698049 Sun Chengyang a b 2698050 Wu Yue a b 2698051 Guo Wenyi a b a Department of Ophthalmology, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China b Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, China Correspondence to: Wenyi Guo, wyguo9h@163.com or Yue Wu, eyedrwuyue@gmail.com 16 6 2023 Jan-Dec 2023 66 1 935944 28 10 2022 5 5 2023 2023 © 2023 The Author(s).Published by S. Karger AG, Basel 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission. Abstract Introduction The aim of the study was to evaluate the efficacy and safety of combined trabeculotomy-non-penetrating deep sclerectomy (CTNS) in the treatment of Sturge-Weber syndrome (SWS) secondary glaucoma. Methods This retrospective study reviewed cases that underwent CTNS as initial surgery for SWS secondary glaucoma at our Ophthalmology Department center from April 2019 to August 2020. Surgical success was defined as an intraocular pressure (IOP) ≤ 21 mm Hg with (qualified success) or without (complete success) the use of anti-glaucoma medications. IOP >21 mm Hg or <5 mm Hg despite 3 or more applications of anti-glaucoma medications on 2 consecutive follow-up visits or at the last follow-up, performance of additional glaucoma (IOP-lowering) surgery, or with vision-threatening complications were classified as failure. Results A total of 22 eyes of 21 patients were included. Twenty-one eyes were of early-onset type and 1 eye was of adulthood onset. For Kaplan-Meier survival analysis, the overall success rates at 1st and 2nd years were 95.2% and 84.9%, while the complete success rates at 1st and 2nd years were 42.9% and 36.7%. At the last follow-up (22.3 ± 4.0 months, range: 11.2∼31.2), overall success was achieved in 19 (85.7%) eyes and complete success in 12 (52.4%) eyes. Postoperative complications included transient hyphema (11/22, 50.0%) and transient Ⅰ degree shallow anterior chamber (1/22, 4.5%), and retinal detachment (1/22, 4.5%). No other severe com plications were detected during the follow-up. Conclusion CTNS significantly reduces IOP in SWS secondary glaucoma patients who have serious episcleral vascular malformation. CTNS in SWS secondary glaucoma patients is safe and effective for short and medium periods. A randomized controlled study comparing the long-term prognosis of SWS early-onset and late-onset glaucoma underwent CTNS is worth conducting. Keywords Sturge-Weber syndrome Glaucoma Combined trabeculotomy-non-penetrating deep sclerectomy This work was supported by the National Natural Science Foundation of China (No. 81970796, No. 82171046, No. 82101114), Clinical Research Program of Shanghai Municipal Health Commission (No. 201940330), Clinical Research Program of 9th People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (No. JYLJ201904), Clinical Research Plan of SHDC (SHDC2020CR6029), Cross-Disciplinary Research Fund of Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine (YG2019QNA18), and the Research Grant of the Shanghai Science and Technology Committee (No. 20DZ2270800). The authors have no financial disclosures to declare in relation to this study. ==== Body pmcIntroduction Sturge-Weber syndrome (SWS) is a rare, congenital, multisystem disorder affecting approximately 1 in 50,000 individuals. It is characterized by facial port-wine stain (PWS), leptomeningeal angiomas, and glaucoma [1]. Clinical manifestations also include cognitive delays, seizures, and choroidal hemangioma. Estimates suggest that 30–71% of patients have glaucoma, with 60% developing it during infancy and 40% having juvenile- or adulthood-onset glaucoma [2–8]. Previously, ophthalmologists believed that abnormal anterior chamber angles were the cause of SWS early-onset glaucoma, while increased episcleral venous pressure (EVP) was responsible for juvenile- or adulthood-onset glaucoma [9–12]. Our previous study found that trabeculotomy had good intermediate-term surgical outcomes for early-onset glaucoma [13]. However, we later discovered that the outcome of trabeculotomy for patients with more severe episcleral vascular malformation was not ideal [14]. This strongly suggests that malformed episcleral vasculatures, which drain aqueous humor, may also contribute to SWS early-onset glaucoma. Researchers have validated the increased scleral venous pressure in the affected eye with the help of specific equipment [11, 12]. Therefore, surgical interventions that address both the elevated EVP and angle deformity effectively may lead to a better prognosis in SWS. Due to the presence of choroidal hemangioma, a sudden drop in intraocular pressure (IOP) during filtering surgery increases the risk of choroidal detachment or choroidal effusion in SWS [15–23]. In 1964, Krasnov reported the first non-penetrating filtering surgery, known as sinusotomy [24]. Since then, the surgical process has been continuously improved, and non-penetrating deep sclerectomy (NPDS) has become one of the most popular non-penetrating filtering surgeries. NPDS involves the removal of the juxtacanalicular trabeculum and Schlemm’s canal endothelium, as well as the creation of an intrascleral space and a trabeculo-Descemet’s membrane (TDM) [25]. The two main hypothetical mechanisms of aqueous resorption are subconjunctival filtering bleb and suprachoroidal filtration, both of which are independent of EVP [26]. Compared to trabeculectomy, NPDS results in similar IOP reduction but with fewer complications in the early postoperative period, as the procedure does not penetrate into the anterior chamber and the TDM allows a slow decrease of IOP [27]. Therefore, we combined trabeculotomy and NPDS to pursue a higher success rate and a lower complication rate in SWS. This study assesses the outcome of combined trabeculotomy-non-penetrating deep sclerectomy (CTNS) in SWS secondary glaucoma patients. Materials and Methods Subjects This retrospective study reviewed 23 eyes of 22 SWS secondary glaucoma patients who underwent CTNS as initial treatment at the Department of Ophthalmology, Shanghai Ninth People’s Hospital, from April 2019 to August 2020. This study was conducted in accordance with the Declaration of Helsinki and was ethically approved by the Institutional Review Board of the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, with the approval number being [SH9H-2021-T253-1]. Written informed consent was waived due to the retrospective nature of the study. The diagnosis, surgery, follow-up, and necessary treatment were all provided by the same ophthalmologist (W.G.). Diagnosis and Eligibility Criteria All patients included in this study had either unilateral or bilateral facial PWS and glaucoma. The diagnosis of port-wine stain was made by the Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital. For infants, only those with multiple episcleral vascular abnormal network underwent CTNS, as per the previously stated criteria for multiple episcleral vascular abnormal network [14]. For children, the diagnosis of glaucoma required the presence of at least two of the following criteria [1, 28]: (1) IOP >21 mm Hg or obvious asymmetry between the 2 eyes (≥6 mm Hg); (2) a cup-to-disc ratio (C/D) > 0.5 or obvious asymmetry C/D between the 2 eyes (≥0.2); (3) cornea findings: diameter enlargement (diameter >11 mm in newborns, >12 mm for children <1 year old, or >13 mm for any age), obvious asymmetry cornea diameter between the 2 eyes, corneal edema, or Haab striae; (4) typical visual field loss and retinal nerve fiber layer defects; (5) progressive myopia or myopia shift with increased ocular dimensions that outpace. For adults, the diagnosis of glaucoma required the presence of at least two of the following criteria: (1) IOP >21 mm Hg; (2) C/D > 0.5 or obvious asymmetry C/D between the 2 eyes (≥0.2); (3) typical visual field loss and retinal nerve fiber layer defects. SWS early-onset glaucoma is defined as meeting the criteria for SWS secondary glaucoma, plus any of the following [1]: (1) age at diagnosis is ≤3 years; (2) corneal diameter enlargement, or the difference in diameter from the unaffected side is ≥0.5 mm. SWS late-onset glaucoma is defined as meeting the diagnosis of SWS secondary glaucoma but not meeting the diagnosis of early-onset glaucoma. Operative Procedure (1) A fornix-based conjunctival flap (Fig. 1a) was created. (2) A 5 × 5 mm limbus-based superficial scleral flap was cut 2 mm anteriorly into the clear cornea, including one-third of the total scleral thickness (Fig. 1b). (3) A sponge soaked in 25 mg/mL solution of 5-fluorouracil was placed under the superficial scleral flap and conjunctival flap for 3 min (Fig. 1c, d). (4) The eye was washed with 20 mL of balanced salt solution. (5) A rectangular deep scleral flap (4 × 4 mm) smaller than the superficial one was created (Fig. 1e). The remaining TDM should be as thin as possible. On reaching the anterior part of the dissection, the outer wall of Schlemm’s canal was left uncut. (6) The incision was deepened at the gray-white junction on both sides of the deep scleral flap until the outer wall of the Schlemm’s canal was cut (Fig. 1f). (7) The trabeculotomy probe was introduced into the temporal canal from the temporal radial incision and rotated into the anterior chamber (Fig. 1g). Similar trabeculotomy was performed from the nasal incision. In total, about 120° of the trabecular meshwork was ruptured. (8) The deep scleral flap was extended anteriorly until the Schlemm’s canal was encountered and unroofed. The deep scleral flap together with the outer wall of Schlemm’s canal were removed at this stage (Fig. 1h). (9) The Schlemm’s canal endothelium and the juxtacanalicular trabeculum were peeled off softly (Fig. 1i). At this procedure stage, there should be an evident percolation of aqueous humor through the remaining trabeculum. (10) The superficial scleral flap was repositioned and sutured. (11) Viscoelasticity was injected under the superficial scleral flap (Fig. 1j). (12) After suturing the conjunctival flap, pilocarpine was applied topically directly to the eye. Fig. 1. Surgical process of combined trabeculotomy-deep sclerectomy. a A fornix-based conjunctival flap is created. b A 5 × 5 mm limbus-based superficial scleral flap. Place the sponge soaked in 5-Fu was in the scleral bed (c) and between the sclera and Tenon’s capsule (d) for 3 min. e A smaller (4 × 4 mm) rectangular deep scleral flap is created, with the outer wall of Schlemm’s canal left uncut. f The incision was deepened at the gray-white junction on both sides of the deep scleral flap until the outer wall of the Schlemm’s canal was cut. g Trabeculotomy is performed. h The deep scleral flap, along with the outer wall of Schlemm’s canal, is removed. i The Schlemm’s canal endothelium and the juxtacanalicular trabeculum are peeled off. j Viscoelasticity is injected under the superficial scleral flap. Data Collection Data were retrieved from clinical records. The parameters recorded included IOP, C/D, anti-glaucoma medications (prior to and after surgery), history of anti-glaucoma surgery, and epileptic seizures. Complications throughout the follow-up period were recorded. Success Criteria Success and failure were defined in accordance to the 2008 World Glaucoma Association consensus [29]. Complete success was defined as IOP ≤21 mm Hg without anti-glaucoma medications. Qualified success was defined as IOP ≤21 mm Hg with anti-glaucoma medication. Overall success included complete success and qualified success. Failure was defined as: (1) IOP >21 mm Hg or <5 mm Hg despite application of 3 or more anti-glaucoma medications on 2 consecutive follow-up visits or at the last follow-up; (2) performance of additional glaucoma (IOP-lowering) surgery; (3) with vision-threatening complications. Exclusion Criteria 1. Follow-up was less than 1 year. 2. Uncompletion of CTNS. Statistical Analysis Statistical analysis was performed using GraphPad Prism software, version 8.0. Normality was assessed with the Shapiro-Wilk test, and quantitative variables exhibiting a normal distribution were reported as the mean and standard deviation. For non-normal variables, the median and interquartile range (IQR) were presented. An unpaired t test was used to compare the preoperative IOP between glaucomatous eyes and fellow eyes, while the Mann-Whitney test was used to compare the corneal diameter and central corneal thickness (C/D) between the two groups. Dunnett’s multiple comparisons test was applied to the IOPs at each follow-up. Results were considered significant with a p < 0.05. Results Overall, a total of 22 eyes from 21 patients underwent CTNS, with a minimum of 1 year of follow-up, where 1 patient (1 eye) was excluded for not completing CTNS. Of those patients, 13 were male and 8 were female; 1 required surgery in both eyes due to bilateral glaucoma and bilateral facial PWS. Altogether, 21 eyes were of early-onset type, 20 (91.0%) eyes had thickened choroid or choroidal hemangioma, 3 (14.3%) patients were detected with clinical seizures, and 7 (31.8%) eyes had corneal edema. The baseline demographic characteristics are summarized in Table 1. The median age of the patients at the time of surgery was 4.6 (IQR: 2.2, 8.0) years, with a range of 0.2–30.3 years. The average preoperative IOP in the glaucomatous eyes was 25.4 ± 5.6 mm Hg and 13.7 ± 5.0 mm Hg in the contralateral eyes. The median C/D in the glaucomatous and fellow eyes were 0.80 (IQR: 0.68, 0.86) and 0.30 (IQR: 0.26, 0.40), respectively. The median corneal diameter of the glaucomatous and fellow eyes were 13.00 (IQR: 12.43, 13.43) mm and 11.75 (IQR: 11.50, 12.00) mm, respectively. Table 1. Baseline demographic and clinical data Preoperative findings Total (N = 22) Age of surgery, years, median (IQR) 4.6 (2.2, 8.0) Sex  Male 13  Female 8 IOP, mm Hg, mean ± SD  Glaucomatous eyes (N = 22) 25.4±5.6*  Fellow eyes (N = 20) 13.7±5.0 C/D, median  Glaucomatous eyes (N = 22) 0.80(0.68, 0.86)a  Fellow eyes (N = 20) 0.30(0.26, 0.40) Corneal diameter, mm, median (IQR)  Glaucomatous eyes (N = 22) 13.00 (12.43, 13.43)a  Fellow eyes (N = 20) 11.75 (11.50, 12.00) Corneal edema (%) 7 (31.8) Unilateral surgery 20 Bilateral surgery 1 Choroidal hemangioma/choroid thickening (%) 20 (90.9) Seizures (%) 3 (14.3) IOP, intraocular pressure; C/D, cup-to-disc ratio; IQR, interquartile range, shown as (25%, 75%); SD, standard deviation. The Shapiro-Wilk test is used for normality tests. *p < 0.0001, compared with the fellow eyes using unpaired t test. a p < 0.0001, compared with the fellow eyes using Mann-Whitney test. For SWS early-onset patients, Kaplan-Meier survival analysis shows the overall success rates at the 1st and 2nd years were 95.2% and 84.9%, respectively, while the complete success rates were 42.9% and 36.7% (Fig. 2). At the final follow-up (22.3 ± 4.0 months, range: 11.2∼31.2), overall success was achieved in 18 (18/21, 85.7%) eyes and complete success in 11 (11/21, 52.4%) eyes. The adult-onset glaucoma eye achieved complete success at the last follow-up (12.4 months). Fig. 2. The cumulative complete success rate and overall success rate for SWS early-onset glaucoma. The cumulative proportions of the overall success rates at the 1st and 2nd years were 95.2% and 84.9%, respectively, while the complete success rates were 42.9% and 36.7%. Low to moderately elevated blebs were seen in 17 (17/22, 77.3%) eyes. The IOP significantly decreased from 25.4 ± 5.6 at baseline to 18.2 ± 5.1 at the last follow-up (p < 0.0001), with significant decreases evident at each follow-up: 1st week (15.6 ± 5.5, p < 0.0001), 1st month (17.1 ± 5.8, p = 0.0001), 6th month (18.2 ± 7.9, p = 0.0004), 1 year (20.1 ± 5.8, p = 0.0179), 1.5 year (19.1 ± 4.8, p = 0.0034), and 2 years (18.9 ± 4.1, p = 0.0031) (Fig. 3 and Table 2). The number of anti-glaucoma medicines in follow-up is shown in Table 3. The number of patients with anti-glaucoma medicines declined from 14 to 10 at the last follow-up. The median (IQR) of the number of medications was 1.5 (0, 3) at baseline and 0 (0, 2.25) at the last follow-up. Fig. 3. IOP at each follow-up visit. IOP per patient at each follow-up visit. Bars represent mean and SD. The green area represents the physiological range where 5≤ IOP ≤21 mm Hg. For all postoperative visits, the p value was significant compared with baseline (p < 0.05). SD, standard deviation. Table 2. IOP at follow-up (N = 22) Time p value Baseline (N = 22)  IOP, mm Hg, mean ± SD 25.4±5.6 N/A Week 1 (N = 20)  IOP, mm Hg, mean ± SD 15.6±5.5 <0.0001 a Month 1 (N = 17)  IOP, mm Hg, mean ± SD 17.1±5.8 0.0001 a Month 6 (N = 21)  IOP, mm Hg, mean ± SD 18.2±7.9 0.0004 a Year 1 (N = 20)  IOP, mm Hg, mean ± SD 20.1±5.8 0.0179 a Year 1.5 (N = 19)  IOP, mm Hg, mean ± SD 19.1±4.8 0.0034 a Year 2 (N = 18)  IOP, mm Hg, mean ± SD 18.9±4.1 0.0031 a Last follow-up (N = 22)  IOP, mm Hg, mean ± SD 18.2±5.1 <0.0001* IOP, intraocular pressure; SD, standard deviation; N/A, not applicable. *Compared with baseline IOP using unpaired t test. aCompared with baseline IOP using Dunnett’s multiple comparisons test. Table 3. The number of medications at follow-up Time Patient with medications, n (%) Number of medications, median (IQR) p value Baseline (N = 22) 14 (63.6) 1.5 (0, 3) N/A Week 1 (N = 20) 3 (15) 0 (0, 0) 0.0027 a Month 1 (N = 18) 4 (22.2) 0 (0, 0.25) 0.0088 a Month 6 (N = 21) 8 (38.1) 0 (0, 1) >0.05a Year 1 (N = 19) 9 (47.4) 0 (0, 2) >0.05a Year 1.5 (N = 19) 10 (52.6) 1(0, 3) >0.05a Year 2 (N = 18) 9 (50.0) 1 (0, 3) >0.05a Last follow-up (N = 22) 10 (45.5) 0 (0, 2.25) >0.05* IQR, interquartile range, shown as (25%, 75%); N/A, not applicable. *Compared with baseline using Mann-Whitney test. aCompared with baseline using Dunn’s multiple comparisons test. No participant received additional interventions, such as bleb needling or second anti-glaucoma surgeries, during the follow-up period. During the surgery, trabecular meshwork tear and iris prolapse occurred in 1 patient (1/23, 4.3%), who was subsequently excluded from the analysis in the article. Postoperative complications included transient hyphema (11/22, 50.0%) and transient I degree shallow anterior chamber (1/22, 4.5%), which resolved without intervention by the first week. One patient with bilateral CTNS had a unilateral retinal detachment (1/22, 4.5%) at the first-week follow-up, which was confirmed by B-scan to have healed during the follow-up. No other serious complications were detected during the follow-up. We conducted a Cox regression analysis on early-onset participants to investigate risk factors for postoperative IOP escalating beyond 21 mm Hg (the time at which the success rate is reduced). PWS (unilateral/bilateral), age, corneal edema (yes/no), corneal diameter, C/D, hyphema (yes/no), choroid thickening (yes/no), gender (male/female), preoperative IOP, preoperative medication (yes/no) were the initial variables included in the model, and stepwise regression based on AIC value was used for variable screening. The results revealed that males were less likely to experience postoperative IOP elevation, while high preoperative IOP and preoperative medication were risk factors for postoperative IOP elevation (p < 0.05), and other variables were not significantly associated with postoperative IOP elevation (Table 4). Table 4. Indicators associated with postoperative IOP elevation greater than 21 mm Hg Estimate Std. error Statistic p value Male −2.410 0.830 −2.902 0.004 Preoperative IOP 0.201 0.076 2.660 0.008 Preoperative use of anti-glaucoma medicine 1.937 0.789 2.456 0.014 Std. Error, standard error; IOP, intraocular pressure. Discussion Glaucoma is a leading cause of irreversible blindness, severely affecting the quality of life of those affected due to progressive visual field defects and the need for lifelong treatment. For patients with SWS, the situation is even worse, with many SWS secondary glaucoma patients developing glaucoma within the first year of life [2, 30]. This can lead to an increased financial burden on families. In addition, topical medications often fail to achieve the desired IOP, particularly in early-onset cases [31], and complications such as choroidal/retinal detachment and suprachoroidal hemorrhage are more common. Ophthalmologists are dedicated to improving the lives of these patients. It was previously thought that angle dysplasia was the primary cause of early-onset glaucoma in Sturge-Weber syndrome (SWS); thus, the treatment often employed was trabeculotomy, similar to what is used for primary congenital glaucoma (PCG). Although there were some studies showing changes in the trabecula of SWS [32], some of our findings suggest that the angle structure of SWS early-onset glaucoma is different from that of PCG: (1) Gonioscopy reveals a lack of mesodermal tissue in SWS; (2) the IOP of SWS early-onset glaucoma is often not very high; Haab’s striae are not as common; (3) after the removal of Schlemm’s canal’s inner wall during CTNS, aqueous humor is able to exudate through the TDM. This implies that the trabeculae of SWS may not be the major site of resistance to the outflow of aqueous humor in SWS. Our prior study demonstrated that SWS early-onset glaucoma patients with pronounced scleral vascular malformations have a relatively poor response to trabeculotomy, indicating that vascular elements may be implicated in the emergence of SWS early-onset glaucoma patients [14]. In addition, we identified a GNAQ R183Q mutation in the scleral vessels of SWS, which is believed to be a major cause of vascular malformations in SWS [33]. Since Schlemm’s canal develops from scleral veins [34], morphological and functional abnormalities may also be present in the Schlemm’s canal in SWS. We suppose that the aqueous outflow resistance of SWS early-onset glaucoma patients is mainly located in Schlemm’s canal. With age, the scleral vascular malformation becomes increasingly severe, and the EVP gradually increases. The distal resistance thus becomes the primary pathogenic factor. Some early-onset patients with a serious episcleral vascular malformation may already have an increasing distal resistance in the early stage. The specific action mechanism of trabeculotomy in SWS may differ from that of PCG. Trabeculotomy mainly resolve the obstruction in trabecular meshwork in PCG, while it may aim at the inner wall of the Schlemm’s canal in SWS. Therefore, trabeculotomy is insufficient for patients with severe episcleral vascular malformations present at birth. In this study, we first described a novel surgical method – CTNS – for SWS adulthood-onset glaucoma and early-onset glaucoma patients with severe episcleral vascular malformation. CTNS combines trabeculotomy and NPDS to address the resistance anterior to the inner wall of Schlemm’s canal and posterior to the outer wall of the canal, respectively. The aqueous humor can either enter the Schlemm’s canal directly from the anterior chamber through the incision of trabeculotomy or enter the scleral concave pool through TDM, then drain into the suprachoroidal space, scleral connective tissue, and subconjunctiva. Finally, the overall success rates at the 1st and 2nd year follow-up points were 95.2% and 84.9%, respectively, while the complete success rates were 42.9% and 36.7%. Of note, the IOP of 3 qualified success patients fell below 21 mm Hg without medicine, after a short-term anti-glaucoma medication therapy. IOP was significantly decreased at each subsequent follow-up. Considering the poor prognosis of adult and child patients with severe episcleral vasculature malformation enrolled in this study, the efficiency of CTNS in SWS is encouraging. Studies in both PCG and POAG have demonstrated that males are at a greater risk for disease progression [35, 36]. In this study, males were found to be less likely to experience an elevation in IOP postoperatively. This may suggest a different pathogenesis of SWS secondary glaucoma. Other gender-related factors, such as hormones and outdoor activities, should not be overlooked, as they may be prime research areas for the future [37, 38]. Due to the presence of choroidal hemangioma, there is an increased risk of exudation during filtration surgery due to the sudden drop in IOP. To the best of our knowledge, choroidal detachment rate (4.5% vs. 10∼57.1%) in our case series is fairly low (Table 5) [15, 17–23]. Though most of the patients (90.9%) had choroidal hemangioma or choroidal thickening, only a 4-year-old male child developed retinal detachment at 1st week after CTNS. We believe this low choroidal detachment rate is likely due to the slight variations of IOP during and after CTNS, as CTNS did not penetrate into the anterior chamber and TDM provides moderate resistance to aqueous humor drainage. Table 5. Choroidal detachment after glaucoma filtering surgery in SWS Author Surgery Choroidal thickening/hemangioma, % Choroidal/retinal detachment, % (n) Success rate, % This study CTNS 90.9 4.5(1/22) 85.7 Hamush et al. [17] 1999 Ahmed NA 27.3(3/11) 79.0 Kaushik et al. [18] 2019 Ahmed 16.67 12.5(3/24) 75.0 Sarker et al. [19] 2021 Ahmed 40 10.0(2/20) 90.0 Trabeculectomy + MMC 30 10.0(2/20) 85.0 Iwach et al. [20] 1990 Trabeculectomy NA 23.5(4/17) NA Budenz et al. [21] 2000 Baerveldt NA 20.0(2/10) 100.0 Ali et al. [22] 1990 Trabeculectomy NA 57.1(4/7) 85.7 Agarwal et al. [15] 1993 CTT NA 16.7(3/18) 61.1 Amini et al. [23] 2007 Molteno 100 44.4(4/9) 97.2 CTNS, combined trabeculotomy-non-penetrating deep sclerectomy; NA, not available; MMC, mitomycin C; CTT, combined trabeculotomy-trabeculectomy. CTNS allows for aqueous humor to enter the subconjunctival space through a scleral incision, thereby creating subconjunctival drainage. Given the rapid wound healing in young patients, we used 25 mg/mL of 5-fluorouracil sponge placed under the conjunctival sac and scleral flap for 3 min after the superficial scleral flap of CTNS was created for anti-scarring treatment. Our findings revealed that in 77.3% (17/22) of eyes, low to moderately elevated blebs were present at the last follow-up. This proportion is similar to that of combined trabeculotomy-trabeculectomy in PCG [39, 40]. It is possible that this proportion is underestimated in the current study, as children unable to cooperate were categorized as not observed. The limitations of CTNS were also present, with NPDS and trabeculotomy having a long learning curve and the combination of the two procedures potentially resulting in trabecular meshwork tear and iris prolapse. To reduce the risk, we performed trabeculotomy before unroofing the Schlemm’s canal, and only 1 patient had iris prolapse. Our current research is a retrospective non-comparative case series, and a prospective randomized controlled study is needed to demonstrate the efficacy and safety of CTNS. Besides, our study is limited by its short follow-up, with a previous study reviewing the outcome of combined trabeculotomy-trabeculectomy over a period of 23 years, finding that more than half (15/26, 57.7%) of SWS secondary glaucoma requires secondary surgery in a mean duration of 23.13 ± 24.41 months [41]. The long-term efficiency of CTNS in young SWS patients with serious episcleral vascular malformation deserves further exploration. We will keep a close and constant observation of these patients and believe that this study still provides useful experience in managing SWS secondary glaucoma. Conclusion CTNS significantly reduces IOP in SWS secondary glaucoma patients who have serious episcleral vascular malformation. CTNS in SWS secondary glaucoma patients is safe and effective for short and medium periods. A randomized controlled study comparing the long-term prognosis of SWS early-onset and late-onset glaucoma underwent CTNS is worth conducting. Statement of Ethics This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, approval number [SH9H-2021-T253-1]. Informed consent is waived due to the retrospective nature of the study. Conflict of Interest Statement The authors declare no competing interests. Funding Sources This work was supported by the National Natural Science Foundation of China (No. 81970796, No. 82171046, No. 82101114), Clinical Research Program of Shanghai Municipal Health Commission (No. 201940330), Clinical Research Program of 9th People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (No. JYLJ201904), Clinical Research Plan of SHDC (SHDC2020CR6029), Cross-Disciplinary Research Fund of Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine (YG2019QNA18), and the Research Grant of the Shanghai Science and Technology Committee (No. 20DZ2270800). The authors have no financial disclosures to declare in relation to this study. Author Contributions L.H., Y.W., and W.G. contributed to study design, data analysis and interpretation, and drafted the manuscript. 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==== Front Intervirology Intervirology INT INT Intervirology 0300-5526 1423-0100 S. Karger AG Basel, Switzerland 37263256 530906 10.1159/000530906 Research Article Impact of Age and Clinico-Biochemical Parameters on Clinical Severity of SARS-CoV-2 Infection Severity of SARS-CoV-2 Infection 2696220 Wani Shariq Ahmad a 2696221 Gulzar Babar a 2696222 Khan Mosin Saleem b 2696223 Majid Sabhiya b 2696224 Bhat Irfan Ahmad a a Department of Medicine, Government Medical College Srinagar and Associated Hospitals, Jammu and Kashmir, India b Department of Biochemistry, Government Medical College Baramulla and Associated Hospitals, Jammu and Kashmir, India Correspondence to: Mosin Saleem Khan, mosinsaleemkhan@gmail.com or Irfan Ahmad Bhat, irfanahmad798@gmail.com Shariq Ahmad Wani, Babar Gulzar, and Mosin Saleem Khan have contributed equally as first authors. 1 6 2023 Jan-Dec 2023 66 1 8896 21 3 2022 13 4 2023 2023 © 2023 The Author(s). Published by S. Karger AG, Basel 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission. Abstract Introduction The surge in novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease-2019 (COVID-19) has overwhelmed the health system. To help health-care workers and policy makers prioritize treatment and to decrease the burden on health systems caused by COVID-19, clinical severity along with various clinico-biochemical parameters was evaluated by designing a cross-sectional study comprising 236 SARS-CoV-2-infected individuals from Kashmir Valley, India. Methods Briefly, real-time polymerase chain reaction (RT-PCR) was used for the confirmation of SARS-CoV-2 infection. The principles of spectrophotometry and chemiluminescent microparticle immunoassay (CMIA) were employed to estimate the levels of glucose, TSH, and 25-hydroxy vitamin D levels in serum of infected patients. Results A total of 236 patients infected with SARS-CoV-2 were taken for this cross-sectional study. Patients with COVID-19 had a male predominance (72.9 vs. 27.1%) and a higher prevalence of 25-hydroxy vitamin D deficiency (72.0 vs. 28.0%) with a mean 25-hydroxy vitamin D levels of 24.0 ± 13.9 in ng/mL. We observed a varied clinical spectrum of SARS-CoV-2 infection with 36.4%, 23.7%, and 29.7% patients having mild, moderate, and severe disease, respectively. We observed that severity of SARS-CoV-2 infection was significantly associated with older age group, hypertension, low TSH levels, and 25-hydroxy vitamin D deficiency. Conclusion We conclude that not only old age but also hypertension and low levels of TSH and 25-hydroxy vitamin D levels could significantly lead to clinical severity of SARS-CoV-2 infection. Keywords Severe acute respiratory syndrome coronavirus 2 Corona virus disease-2019 Clinico-biochemical Angiotensin-converting enzyme 2 Jammu and Kashmir Clinical severity 25-Hydroxy vitamin D This study was funded by the Department of Biochemistry Government Medical College Srinagar and Associated Hospitals. ==== Body pmcIntroduction Novel SARS-CoV-2 is an enveloped positive-sense RNA virus that typically gains entry to the cell via the angiotensin-converting enzyme 2 (ACE2) receptor [1], whereby it predominantly infects the lower respiratory tract, binding to ACE2 on alveolar epithelial cells [1, 2]. According to the latest data of January 2022 from Johns Hopkins University of Medicine Coronavirus Resource Center, a total number of confirmed coronavirus disease-2019 (COVID-19) cases have exceeded 280 million mark toward the beginning of January 2022, which means that one person on the globe has been infected every 4 s since the documented start of COVID-19 and around 797,017 cases have been reported everyday over the same period, with the global fatality rate standing at 2.1% [3]. As the spectrum of signs and symptoms among COVID-19-infected individuals is broad, ranging from mild to severe illness, requiring intensive care, identifying risk factors that may predict the patients’ outcome is imperative for a better resource allocation in mitigating this current COVID-19 outbreak [4, 5]. COVID-19 shows an increased number of cases and a greater risk of severe disease with increasing age and understanding the role of age in transmission and disease severity is critical for determining the likely impact of social-distancing interventions on SARS-CoV-2 transmission [6]. On the one hand, diabetes and its associated comorbidities increase the risk of a more severe course of COVID-19 and increased mortality [7]. Several recent clinical studies have shown that hypertension is one of the most prevalent comorbidities of COVID-19 and a risk factor for severe COVID-19 infection [8]. Abnormal release of thyroid hormone affects the robustness of the immune response that is integral in maintaining COVID-19 outcomes [9]. Moreover, previous studies have linked decreased FT3, TSH, and FT4 with clinical deterioration of COVID-19 patients [10]. Nonetheless, the correlation between thyroid parameters and COVID-19 severity remains elusive as indicated by the variability of reported study results [11]. One of the most overlooked factors that could influence outcome of COVID-19 is the relative vitamin D status of populations [12] as this vitamin exerts well-known immunomodulatory functions spanning from the innate to the adaptive arms of the immune system and including the downregulation of proinflammatory cytokines, such as interleukin-6 [13]. Such a possibility is further supported by recent studies, suggesting a likely relationship between COVID-19 severity and the prevalence of vitamin D deficiency [14, 15], as well as from small reports in patients with or without COVID-19 [16, 17]. Jammu & Kashmir (North India), the place of our study, is also one of the worst-hit states of India with the total number of cases exceeding 3.4 lac among the total population of 12.5 million people with case-fatality rate of 1.4% [17]. Since in Kashmir (North India), India due to its unique geographical location and lifestyle, individuals are more prone to metabolic disorder and vitamin D deficiency; therefore, this cross-sectional study aimed to disentangle the relative contribution of demographic, clinical, and biochemical parameters on the outcome of patients with SARS-CoV-2 infection. Materials and Methods Study Design This was a cross-sectional study conducted by the Department of Medicine in collaboration with the Department of Biochemistry, Government Medical College Srinagar and Associated Shri Maharaja Hari Singh (SMHS) Hospital, J&K, India. The study was approved by the Institutional Ethical Committee, Government Medical College Srinagar as per Helsinki declaration. Informed consent was taken from each participant after following proper procedure. Written informed consent in vernacular (Urdu/Kashmiri) and working language (English) with questionnaire response from SARS-CoV-2-infected patients or their caretakers was taken on record. Study Subjects, Inclusion, and Exclusion Criteria Two hundred thirty-six (n = 236) patients infected with SARS-CoV-2 attending the Medical Emergency were included in the study. The mean age of patients was 49.2 ± 15.4 years with a male:female ratio of 2.7:1. All the study subjects were of Kashmiri ethnicity. Patients with any sort of inflammatory/autoimmune disorders, genetic disorder, and cancer were excluded from the study. Pregnant women with SARS-CoV-2 infection were also excluded from the study. Sample Size Keeping the power of study as 80% and the effect size of 0.4 at type 1 error of 50%, the sample size came out to be 230. The sample size was calculated using the statistical software “G Power v. 16.2.3.” Real-Time Polymerase Chain Reaction for Detection of SARS-CoV-2 A positive real-time polymerase chain reaction (RT-PCR) test was considered as a gold standard for identification of SARS-CoV-2-infected patients who developed COVID-19. Nasopharyngeal swab was taken from individuals attending the Medical Emergency of SMHS Hospital with COVID-19 symptoms and immediately immersed in viral transport medium. The high-quality viral RNA was isolated from viral transport medium using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) following manufacturers’ protocol. The multiplex RT-PCR test intended for the qualitative detection of nucleic acid from SARS-CoV-2 was carried out using TaqPath™ COVID-19 Combo Kit (Applied Biosystems, USA). The RT-PCR was performed using Applied Biosystems™ 7,500 Fast Dx Real-Time PCR instrument in the Department of Microbiology, associated C.D. Hospital, Srinagar, J&K [18]. Sample Collection Blood (03 mL) was collected from each patient with COVID-19 on the next day when he/she came RT-PCR positive for SARS-CoV-2 infection. The blood sample was collected through venipuncture after an overnight fast of at least 8–10 h. Collected blood was immediately transferred into clot activator vials and centrifuged at 4,000 rpm for 2 min and serum was aliquoted into 2 mL microfuge tubes. The serum samples were stored at −20 °C until further analysis. Quantitative Estimation of Glucose in Serum Quantitative estimation of fasting serum glucose (by hexokinase G-6-PDH method) was performed using standard commercially available kits (Abbott, USA). Serum samples were processed and analyzed on an ARCHITECT-C-4000 fully automated biochemistry analyzer (Abbott, USA) in the Biochemistry Diagnostic Laboratory, SMHS Hospital Srinagar, employing the principle of spectrophotometry and following reagent kit instructions. The reference range of fasting serum glucose was taken as 100–126 mg/dL [19]. Quantitative Estimation of Serum TSH and Vitamin D Levels Levels of serum TSH and 25-hydroxy vitamin D levels of COVID-19 patients were estimated using chemiluminescent microparticle immunoassay technology with flexible assay protocols, referred to as Chemiflex. Serum samples were quantitatively analyzed on an ARCHITECT i2000 fully automatic immunoassay analyzer (Abbott, USA) in the Biochemistry Diagnostic Laboratory, SMHS Hospital Srinagar, following the ARCHITECT reagent kit (Abbott, USA) instructions. The reference range of analytes was taken as: TSH: 0.35–5.2 µIU/mL and 25-hydroxy vitamin D: 28–53 ng/mL [20, 21]. Statistical Analysis Statistical analysis was done using SPSS 23.0 statistical package (SPSS Inc., Chicago, IL, USA). Binary logistic regression analysis was used to untangle the association of various demographic and clinico-biochemical parameters with disease severity. Analysis was done by the F-test (ANOVA). The relative risk was estimated by odds ratios (OR) and 95% confidence intervals. p ≤ 0.05 was considered as significant. Results Patient Characteristics In our study, a total of two hundred thirty-six (n = 236) patients infected with SARS-CoV-2 were enrolled. Study included 172 male and 64 female patients. There were equal number of COVID-19 patients in both age groups (<50 years; 50.0%, ≥50 years; 50.0%). 19.5% (46/236) of patients infected with SARS-CoV-2 had T2DM, while 39.0% (92/236) had hypertension. COPD was present in 4.2% (10/236) of patients infected with SARS-CoV-2. TSH levels were high in 12.7% (30/236) of patients infected with SARS-CoV-2. Interestingly, 25-hydroxy vitamin D levels were reduced in 72.0% (170/236) of patients infected with SARS-CoV-2. Table 1 contains the demographic and clinico-biochemical characteristics of patients infected with SARS-CoV-2. Table 1. Demographic and clinico-biochemical characteristics of COVID-19 patients taken for the study Demographic and clinico-biochemical characteristics Cases (n = 236) n (%) Gender  Male 172 (72.9)  Female 64 (27.1) Age group  <50 years 118 (50.0)  ≥50 years 50.0 T2DM  No 190 (80.5)  Yes 46 (19.5) Hypertension  No 144 (61.0)  Yes 92 (39.0) COPD  No 226 (95.8)  Yes 10 (4.2) TSH levels  Normal 206 (87.3)  Low 30 (12.7) 25-Hydroxy vitamin D levels  Normal 66 (28.0)  Low 170 (72.0) COVID-19, coronavirus disease-2019; T2DM, type 2 diabetes mellitus; COPD, chronic obstructive pulmonary disease. Clinical Severity of SARS-CoV-2 Infection As per COVID-19 treatment guidelines, National Institutes of Health, SARS-CoV-2 infection is grouped into following categories as per the “severity of illness”: Asymptomatic or Presymptomatic Infection: individuals who test positive for SARS-CoV-2 using a RT-PCR test but have no symptoms that are consistent with COVID-19. Mild Illness: individuals who have various signs and symptoms of COVID-19 but who do not have shortness of breath, dyspnea, or abnormal chest imaging. Moderate Illness: individuals who show evidence of lower respiratory disease during clinical assessment or imaging and who have an oxygen saturation (SpO2) ≥94% on room air at the sea level. Severe Illness: individuals who have SpO2 <94% on room air at sea level, a respiratory rate >30 breaths/min, or lung infiltrates >50%. Critical Illness: individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction. In our study, 10.2% (24/236) were asymptomatic, 36.4% (86/236) were having mild illness, and 23.7% (56/236) and 29.7% (70/236) were having moderate and severe illness, respectively. Interestingly, none of the enrolled patients had critical illness. Table 2 includes the severity of SARS-CoV-2 infection in enrolled patients. Table 2. Clinical spectrum of SARS-CoV-2 infection Severity of SARS-CoV-2 infection Cases (n = 236) n (%) Asymptomatic 24 (10.2) Mild 86 (36.4) Moderate 56 (23.7) Severe 70 (29.7) Critical 00 (0.0) Association of Demographic and Clinico-Biochemical Parameters with Clinical Spectrum of SARS-CoV-2 Infection Table 3 depicts the association of various demographic and clinico-biochemical parameters with clinical spectrum of SARS-CoV-2 infection. Almost 45.0% (16/36) of asymptomatic individuals belonged to older age group (≥50 years) compared to a higher percentage of older age group (≥50 years) patients having moderate (74.0%; OR = 3.5; p = 0.008) and severe diseases (75.0%; OR = 3.7; p = 0.004). More than 60.0% of patients among the moderate and severe disease groups were found to be hypertensive, which is statistically significant (p < 0.0001). Among asymptomatic SARS-CoV-2-infected patients, only 6.0% (02/36) were having decreased TSH levels as compared to 26.0% (12/46) of moderately ill patients having decreased TSH levels and the difference was statistically significant (OR = 6.0; p = 0.02). Table 3. Association of various demographic and clinico-biochemical parameters with clinical spectrum of SARS-CoV-2 infection Severity of SARS-CoV-2 infection N = 236 (%) Characteristics Or (95% CI) p value Gender, n (%) Male 172 (72.9) Female 64 (27.1) Asymptomatic Mild Moderate Severe 36 (15.3) 98 (41.5) 46 (19.5) 56 (23.7) 26 (72.0) 70 (71.4) 30 (65.0) 46 (82.0) 10 (28.0) 28 (28.6) 16 (35.0) 10 (18.0) Ref. (1.00) 1.04 (0.4–2.4) 1.3 (0.5–3.5) 0.56 (0.2–1.5) 0.9 0.5 0.2 Age group, n (%) <50 years 118 (50.0) ≥50 years 118 (50.0) Asymptomatic Mild Moderate Severe 36 (15.3) 98 (41.5) 46 (19.5) 56 (23.7) 20 (55.0) 72 (73.0) 12 (26.0) 14 (25.0) 16 (45.0) 26 (27.0) 34 (74.0) 42 (75.0) Ref. (1.00) 0.45 (0.2–1.01) 3.5 (1.4–8.9) 3.7 (1.5–9.1) 0.06 0.008 0.004 T2DM, n (%) No 190 (80.5) Yes 46 (19.5) Asymptomatic Mild Moderate Severe 36 (15.3) 98 (41.5) 46 (19.5) 56 (23.7) 30 (83.0) 86 (88.0) 36 (78.0) 38 (68.0) 06 (17.0) 12 (12.0) 10 (22.0) 18 (32.0) Ref. (1.00) 0.7 (0.24–2.0) 1.4 (0.4–4.2) 2.4 (0.8–6.7) 0.5 0.5 0.1 Hypertension, n (%) No 144 (61.0) Yes 92 (39.0) Asymptomatic Mild Moderate Severe 36 (15.3) 98 (41.5) 46 (19.5) 56 (23.7) 32 (89.0) 72 (73.0) 18 (39.0) 22 (39.0) 04 (11.0) 26 (27.0) 28 (61.0) 34 (61.0) Ref. (1.00) 2.9 (0.9–8.9) 12.4 (3.7–41.1) 12.3 (3.8–39.8) 0.06 <0.0001 <0.0001 COPD, n (%) No 226 (95.8) Yes 10 (4.2) Asymptomatic Mild Moderate Severe 36 (15.3) 98 (41.5) 46 (19.5) 56 (23.7) 34 (94.0) 98 (100.0) 44 (96.0) 50 (89.0) 02 (6.0) 00 (0.0) 02 (4.0) 06 (9.0) Ref. (1.00) 0.1 (0.01–1.1) 0.7 (0.15–4.0) 2.0 (0.3–10.7) 0.9 0.8 0.4 TSH levels, n (%) Normal 206 (87.3) Low 30 (12.7) Asymptomatic Mild Moderate Severe 36 (15.3) 98 (41.5) 46 (19.5) 56 (23.7) 34 (94.0) 90 (92.0) 34 (74.0) 48 (86.0) 02 (6.0) 08 (8.0) 12 (26.0) 08 (14.0) Ref. (1.00) 1.5 (0.3–7.4) 6.0 (1.2–28.8) 2.8 (0.5–14.1) 0.6 0.02 0.2 25-Hydroxy vitamin D levels, n (%) Normal 66 (28.0) Low 170 (72.0) Asymptomatic Mild Moderate Severe 36 (15.3) 98 (41.5) 46 (19.5) 56 (23.7) 16 (44.0) 30 (30.6) 10 (21.7) 10 (17.8) 20 (56.0) 68 (69.4) 36 (78.3) 46 (82.2) Ref. (1.00) 1.8 (0.8–3.9) 2.8 (1.1–7.5) 3.6 (1.4–9.5) 0.1 0.03 0.007 COVID-19, coronavirus disease-2019; T2DM, type 2 diabetes mellitus; COPD, chronic obstructive pulmonary disease. We observed a significantly higher percentage of moderately ill patients having decreased 25-hydroxy vitamin D levels compared to relatively lower percentage of asymptomatic individuals with decreased 25-hydroxy vitamin D levels (78.3 vs. 56.0%; OR = 2.8; p = 0.03). Similarly, a significantly higher percentage of severely ill patients were having decreased 25-hydroxy vitamin D levels as compared to asymptomatic individuals having decreased 25-hydroxy vitamin D levels (82.2 vs. 56.0%; OR = 3.6; p = 0.007). We did not observe association of any other parameter with severity of SARS-CoV-2 infection. Discussion Our study involved two hundred thirty-six (n = 236) SARS-CoV-2-infected individuals. A higher percentage of males were observed compared to females signifying that the risk of getting SARS-CoV-2 infection was more in case of males compared to females. Consistent feature of the ongoing COVID-19 pandemic, caused by the SARS-CoV-2, is the male bias toward the transmission [22]. Sex differences in both the innate and adaptive immune systems have been previously reported and may account for the female advantage in COVID-19 as females have higher numbers of CD4+ T cells [23], more robust CD8+ T-cell cytotoxic activity [24], increased B-cell production of immunoglobulin compared to males [25], and decreased expression of ACE2 receptors [26]. Females replete with estradiol have greater number and functionality of ACE2, likely a factor in their greater ability to handle SARS-CoV-2 infections. Additionally, estradiol-mediated RAAS modulatory actions provide further cardiovascular protection to females [27]. Gender-based sociocultural and behavioral differences could contribute to the sex difference seen in COVID-19 transmission. Men are less likely to wash their hands with soap after entering a restroom, and in many cultures, men may be more likely to leave the house and enter crowded areas. In addition, unequal access to healthcare and testing between sexes may skew toward a male bias in infection rates [28]. We observed quite a good number of SARS-CoV-2-infected individuals having hypertension (39%). Growing data show a higher risk of SARS-CoV-2 infections in hypertensive patients. As per previous multicenter study, 30% of COVID-19 patients had previously coexisting hypertension [29]. Another large study reported a prevalence of hypertension as 15% among COVID-19 patients [30]. A weaker immune system is one of the reasons hypertensive individuals are at higher risk for SARS-CoV-2 infections [8]. We observed that more than two-third of the SARS-CoV-2-infected individuals had vitamin D deficiency. Vitamin D deficiency has been shown to potentially increase the risk of severe respiratory infections [31]. According to a previous study, patients with vitamin D deficiency were 4.6 times more likely to be positive for COVID-19 than patients without vitamin D deficiency [32]. Assessing the severity of COVID-19 is crucial to determine the appropriateness of mitigation strategies and to enable planning for health-care needs as epidemics unfold. In our study, most of the SARS-CoV-2-infected patients had mild disease (36.4%), followed by individuals with severe (29.7%) and moderate disease (23.7%), respectively. A small proportion of study population was asymptomatic (10.2%). As of January 2022, more than 270 million cases of COVID-19 have been reported globally, including more than 5.3 million deaths amounting to a global fatality rate of almost 2.1% [33]. According to a previous study from the USA, including 1.3 million COVID-19 cases, 14% of patients required hospitalization, 2% were admitted to the intensive care unit, and 5% died [34]. According to the report from the World Health Organization (WHO) – China Joint Mission on COVID-19, 80% of COVID-19 cases had mild-to-moderate disease, while 13.8% developed severe disease, and 6.1% required intensive care [35]. According to the latest report of Ministry of Health and Family Welfare, India reported total cases of around 40 million with a fatality rate of around 1.3% [17]. When stratified, several factors shape the clinical severity of SARS-CoV-2 infection and outcome of COVID-19 patients. Our findings, together with early evidence, suggest that there is age dependence in susceptibility and clinical severity of COVID-19 [36]. According to the latest report from Centers for Disease Control and Prevention (CDC), the patients above 30 years of age had 2 to 10 times higher rate of hospitalization and a whopping 4 to 370 times higher rate of mortality compared to reference age group of 18–29 years [37]. The hypothesis for age-related difference in the severity of COVID-19 is multifactorial. It has been maintained that elderly people have weak immunity, organ dysfunction apart from the higher frequency of comorbidities, which makes them more susceptible to SARS-CoV-2 severity [38]. Previous study investigating the isolated or direct effect of age on COVID-19 disease severity, after accounting for important age-related risk factors such as diabetes, cardiovascular disease, and chronic pulmonary disease, has observed rather smaller effect of age on disease severity [39]. Moreover, younger individuals have stronger mucosal innate immune response, which helps clear the virus [40]; lower tendency to develop a cytokine storm due to low levels of cytokines [41]; higher lymphocyte counts with a higher proportion of naïve T cells, T regulatory cells, and T follicular helper cells [42]; and less prone to endothelial damage and abnormal clotting [43]. In our study, hypertension was an independent predictor of worst outcome in SARS-CoV-2-infected patients. Hypertension is the single largest global contributor to disability-adjusted life year’s lost and major risk factor for COVID-19 severity [44]. A large case series from China observed an increase in case-fatality rate to 6.0% from 2.3% in COVID-19 patients with hypertension [45]. End organ damage and cardiovascular events are associated with poorer control of high blood pressure and it seems plausible that hypertension leads to more severe SARS-CoV-2 infection [46]. In addition, in hypertensive patients with COVID-19, due to the imbalance in the renin-angiotensin system (RAS), the NADH/NADPH oxidase system may be activated by excessive inflammatory reactions [45], resulting in cell damage and vasoconstriction [47], aggravating lung damage in COVID-19 and leading to a poor prognosis. On the one hand, the use of angiotensin-converting enzyme inhibitors can result in a decrease in ACE and an increase in the expression of ACE2 in the lungs [48]. More ACE2 may mediate the invasion and infection of SARS-CoV-2 into lung cells, resulting in viral spread and aggravation of symptoms [44]. In our study, low TSH levels were significantly associated with moderate disease severity. A previous study on Chinese population showed that TT3 and TSH levels decreased with the severity of COVID-19 [49]. As per a previous report, 56% COVID-19 patients had significantly lower-than-normal TSH levels in the severe and critical group compared with non-COVID-19 pneumonia patients [49]. Additionally, Wei et al. showed that the decreased TSH concentration might be associated with the changes in TSH-secreting cells in the pituitary [50], which might be due to direct viral effect on the pituitary cells or an indirect effect wherein various systemic changes such as the activation of various proinflammatory cytokines caused by the virus infection or its treatment led to hormonal changes in the pituitary-endocrine axis feedback loops [49]. It has been emphasized that thyroid hormone dysregulates RAS, which contributes to increased ACE2 expression [51], thus leading to more severe disease. Several studies along with some meta-analysis have found that thyroid hormones play a role in modulating the cellular level of both innate and adaptive immune responses and abnormal thyroid hormone levels were significantly associated with a higher risk of COVID-19 severity [9, 52]. We observed a significantly strong association of decreased serum 25-hydroxy vitamin D levels with COVID-19 severity. Such a likelihood is more held by recent epidemiology data and small reports, suggesting a likely relationship between the risk of severe respiratory dysfunction/mortality during the course of hospitalization in patients affected by COVID-19 and the prevalence of vitamin D deficiency independently of inflammatory markers, age, or presence of major comorbidities such as obesity, diabetes, and hypertension [16, 53]. Vitamin D supports production of antimicrobial peptides in the respiratory epithelium in response to both viral and bacterial stimuli. Second, vitamin D might help to reduce the inflammatory response to infection, thus reducing the chances of SARS-CoV-2 severity [54, 55]. In a wide variety of animal studies and in in vitro cell models, 25-hydroxy vitamin D has been demonstrated to downregulate the production of inflammatory cytokines, such as TNF-α and IL6, while increasing inhibitory cytokines. These studies promote the chance that sufficient levels of 25-hydroxy vitamin D may reduce the incidence of cytokine storm in COVID-19 [56]. Moreover, vitamin D protected the pulmonary vascular barrier from acute inflammatory injury in mice by locally targeting the RAS [57], whose dysregulation has been implicated in favoring the SARS-CoV-2 entry into alveolar cells with massive cytokine activation and development of ARDS [58]. Added affirmative effects of 25-hydroxy vitamin D include the stimulation of surfactant synthesis by alveolar type-II cells, thus improving alveolar surface tension and exerting a protective role against inflammation and oxidative stress, thereby refining the pulmonary vascular barrier and limiting the cytokine storm [59, 60]. Prevalence of vitamin D deficiency is reported to be high in chronic obstructive pulmonary disease (COPD) patients and increases with the severity of COPD, which, in turn, can influence the prognosis of many COVID-19 patients having COPD as a collateral disease [61]. In fact, as per many previous studies, a high-dose, oral vitamin D supplementation to augment 25-hydroxy vitamin D >50 ng/mL helped to achieve SARS-CoV-2 RNA negativity in greater proportion of asymptomatic vitamin D-deficient individuals with SARS-CoV-2 infection along with a significant decrease in inflammatory marker, while, as per other studies, Vitamin D supplementation showed protective effects against mortality and ICU admission in COVID-19 patients [62, 63]. To our knowledge, this is the first study from this part of the world (Kashmir Valley, North India) to comprehensively summarize the effect of age and clinico-biochemical parameters on clinical severity of SARS-CoV-2 infection. Since the population is ethnic with conserved gene pool, the effect of various factors on severity of COVID-19 disease is more profound. The number of SARS-CoV-2-infected patients enrolled in our study is modest; we need a larger sample size of clinical data to support our conclusions. In addition, we did not analyze complications such as acute renal injury, etc. However, these limitations will not affect the reliability of our overall results. Conclusion We observed an increased clinical severity of SARS-CoV-2 infection in elderly patients: patients having hypertension, decreased TSH, and 25-hydroxy vitamin D deficiency. Identifying the risk factors for clinical severity of SARS-CoV-2 infection will not only attract early intervention and appropriate medical decisions but also assist to design intervention trials aimed at exploring whether vitamin D replacement therapy might prevent the risk of respiratory failure in patients with SARS-CoV-2 infection, in current global pandemic. Acknowledgments We are very much thankful to the study participants attending the tertiary care hospital for their COVID care. Statement of Ethics The research work was in compliance with the guidelines for human studies and was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. The written informed consent and the study protocol were approved by Institutional Review Board of Government Medical College Srinagar vide no. 1010/ETH/GMC dated 12-06-2020. All the samples were collected after taking written informed consent from the patients and proper ethical procedures were followed. Conflict of Interest Statement The authors declare that there is no conflict of interest. Funding Sources This study was funded by the Department of Biochemistry Government Medical College Srinagar and Associated Hospitals. Author Contributions Conceptualization, formal analysis, investigation, methodology, and visualization: S.A.W., B.G., and M.S.K.; data curation and writing – original draft: M.S.K.; funding acquisition, project administration, and supervision: S.M. and I.A.B.; Resources: S.M; Software: M.S.K; validation: M.S.K., S.M., and I.A.B; writing – review and editing: M.S.K. and I.A.B.; and approval of final manuscript: all authors. Data Availability Statement Detailed data are not publicly available due to ethical reasons. The data will be made available upon request. Further inquiries can be directed to the corresponding author. ==== Refs References 1. Majid S , FarooqR, KhanMS, RashidS, BhatSA, WaniHA, . Managing the COVID-19 pandemic: research strategies based on the evolutionary and molecular characteristics of coronaviruses. SN Compr Clin Med. 2020;2 :1767–76. 10.1007/s42399-020-00457-z.32864575 2. Jiang F , DengL, ZhangL, CaiY, CheungCW, XiaZ. 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==== Front Ophthalmic Res Ophthalmic Res ORE ORE Ophthalmic Research 0030-3747 1423-0259 S. Karger AG Basel, Switzerland 37331353 531036 10.1159/000531036 Research Article Comparison of the Intrableb Characteristics of Anterior Segment Optical Coherence Tomography Imaging in Trabeculectomy according to Amniotic Membrane Transplantation Intrableb Characteristics according to Amniotic Membrane Transplantation 2697381 Moon Sangwoo a b 2697380 Kim Jinmi c 2697382 Lee Jiwoong a b a Department of Ophthalmology, Pusan National University College of Medicine, Busan, South Korea b Biomedical Research Institute, Pusan National University Hospital, Busan, South Korea c Department of Biostatistics, Clinical Trial Center, Biomedical Research Institute, Pusan National University Hospital, Busan, South Korea Correspondence to: Jiwoong Lee, glaucoma@pnu.ac.kr 16 6 2023 Jan-Dec 2023 66 1 970982 4 4 2022 8 5 2023 2023 © 2023 The Author(s).Published by S. Karger AG, Basel 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission. Abstract Introduction The purpose of this study was to compare the characteristics of filtering bleb with anterior segment optical coherence tomography (AS-OCT) according to amniotic membrane transplantation (AMT). Methods One hundred and sixteen eyes of 103 glaucoma patients who underwent trabeculectomy with (AMT group; 85 eyes) or without AMT (control group; 31 eyes) were included. Intrableb parameters were evaluated with AS-OCT. Surgical success was defined as an intraocular pressure (IOP) ≤18 mm Hg and IOP reduction ≥20% without medication at the time of AS-OCT examination. Logistic regression analyses were performed to determine factors associated with IOP control. Results In the eyes with successful IOP control, the fluid-filled space area, score, and height were greater for the AMT group than the control group (all ps < 0.001), while stripping layer thickness was greater and bleb wall reflectivity was lower for the control group than the AMT group (all ps < 0.001). Surgical success in the AMT group was associated with greater fluid-filled space score, lower bleb wall reflectivity, and microcyst formation (odds ratio [OR] = 8.016, 0.913, and 16.202, respectively, all ps ≤ 0.041). Lower bleb wall reflectivity alone was associated with surgical success in the control group (OR = 0.815, p = 0.019). Conclusion The extent of the fluid-filled space was associated with successful IOP control after trabeculectomy with AMT. Hyporeflective bleb wall was associated with successful IOP control in AMT and control groups. Keywords Trabeculectomy Amniotic membrane transplantation Bleb Anterior segment optical coherence tomography Primary open-angle glaucoma This research was supported by a grant from Medical big data and AI-based early detection of visual dysfunction funded by Busan and managed by Busan Techno Park and by the Patient-Centered Clinical Research Coordinating Center, funded by the Ministry of Health and Welfare, Republic of Korea (Grant no. HI19C0481, HC19C0276). The funding agencies have played no role in this research. ==== Body pmcIntroduction Trabeculectomy, a procedure introduced in the 1960s, remains the gold standard for glaucoma filtering surgery in patients with medically uncontrolled glaucoma [1–3]. The purpose of trabeculectomy is to allow aqueous humour drainage from the anterior chamber to the subconjunctival or subtenon’s space [3, 4]. The aqueous humour in the filtering bleb passes through the conjunctiva and mixes with the tear film, or is absorbed by vascular or perivascular conjunctival tissue and lymphatic vessels near the surgical area [4]. Failure of trabeculectomy may be due to obstruction of the aqueous outflow at the level of the scleral flap or the ostium, and formation of fibrotic tissue in the subconjunctival and episcleral area [3, 5]. Although the surgical success rate of trabeculectomy increases with the use of antimetabolites, including mitomycin C (MMC) and 5-Fluorouracil, the procedure is associated with an increased risk of complications, such as avascular cystic bleb, bleb leakage, and bleb-related infection [6–9]. Previous studies reported that trabeculectomy with amniotic membrane transplantation (AMT) had a lower complication rate and higher success rate than trabeculectomy without AMT [10–12]. In our previous study, trabeculectomy with AMT was found to be a safe and effective method for intraocular pressure (IOP) reduction in patients with primary open-angle glaucoma (POAG) without the development of avascular cystic blebs or bleb-related infection [12]. Intrableb structures which can be analysed using anterior segment optical coherence tomography (AS-OCT) or ultrasound biomicroscopy (UBM) are associated with the surgical success of trabeculectomy [13–18]. Intrableb morphology, including bleb wall reflectivity, bleb height, bleb wall thickness, and length and height of the fluid-filled cavity, have been associated with bleb function after trabeculectomy [15, 19, 20]. Only one study has evaluated intrableb structures after trabeculectomy with AMT using an anterior segment imaging device [14]. The authors investigated intrableb structures using UBM in patients with glaucoma who underwent trabeculectomy with or without AMT and reported that bleb wall reflectivity in the trabeculectomy alone group and the extent of the subconjunctival fluid-filled space in the AMT-assisted trabeculectomy group were factors associated with long-term IOP control [14]. However, the difference in the type of conjunctival incision between the two groups was a potential limitation of the UBM study as it may influence the IOP-lowering mechanism and intrableb structures associated with IOP control [14]. A fornix-based conjunctival incision was made for trabeculectomy without AMT, while limbal-based conjunctival incision was made for trabeculectomy with AMT [14]. Further studies found that the different types of conjunctival incision in trabeculectomy had distinct features of filtering bleb associated with IOP control [21, 22]. Therefore, the type of conjunctival flaps should be considered in the evaluation of intrableb structures associated with bleb function. Furthermore, bleb wall reflectivity was assessed qualitatively, not quantitatively in the previous study [14]. UBM examination is time-consuming and may be associated with the risk of bleb infection or bleb morphological change as examination requires contact with the eye [22–24]. On the contrary, bleb imaging with AS-OCT is a non-contact and non-invasive examination that allows imaging of filtering bleb with higher resolution than UBM [22]. To our knowledge, AS-OCT imaging of the filtering bleb after fornix-based trabeculectomy with AMT has not been previously evaluated. Therefore, the purpose of this study was to compare the characteristics of the filtering bleb assessed with high-resolution spectral-domain AS-OCT imaging after trabeculectomy with a fornix-based conjunctival flap according to AMT and to evaluate intrableb parameters associated with successful IOP control in patients with POAG. Materials and Methods Study Design and Population This investigation was a retrospective cohort study. The study was conducted according to the guidelines of the World Medical Association Declaration of Helsinki, and approved by the Institutional Review Boards of Pusan National University Hospital (approval no. 2111-004-108). All patients gave written informed consent for the surgical procedures and for their information to be stored in the hospital database and used for research. The patients with POAG who underwent fornix-based trabeculectomy with or without AMT between November 2014 and March 2020 were enrolled in this study. And all the patients were followed-up for at least 1 year postoperatively. Since August 2017, we have performed the trabeculectomy with AMT for all eyes who were eligible for glaucoma surgery and agreed to receive the AMT. Indications for surgery were as follows: (1) IOP not appropriately controlled with maximal tolerated medical therapy or laser trabeculoplasty or both, and (2) intolerance or allergy to glaucoma medication. Exclusion criteria included secondary glaucoma including pseudoexfoliation syndrome, pigment dispersion syndrome, and any other ocular or systemic disorder known to affect the optic nerve head, macula, or visual field. Patients who had undergone previous ocular surgery, except for uncomplicated phacoemulsification, were excluded. Prior to the study, all the participants underwent thorough ophthalmologic examination, including best corrected visual acuity (BCVA), slit-lamp examination, IOP measurement with Goldmann applanation tonometry, gonioscopy, dilated fundus examination, stereoscopic optic disc, and red-free retinal nerve fibre layer photography (AFC-210; Nidek, Aichi, Japan), biometry using the IOL Master (Carl Zeiss Meditec, Dublin, CA, USA), and standard automated perimetry. Central corneal thickness (CCT) was measured using ultrasonic pachymetry (Pachmate; DGH Technology, Exton, PA, USA). Keratometry was performed with an Auto Kerato-Refractometer (ARK-510A; NIDEK, Hiroshi, Japan). POAG was diagnosed by the presence of glaucomatous optic disc changes and corresponding visual field defects as confirmed by two reliable visual field tests and open anterior chamber angle. A glaucomatous optic disc was defined as a disc meeting at least one of the following criteria: (1) a rim notch with a rim width ≤0.1 disc diameter; or (2) a vertical cup-to-disc ratio of >0.7; or (3) a cup-to-disc ratio asymmetry between the 2 eyes ≥0.2; or (3) disc haemorrhage; or (4) a retinal nerve fibre layer defect congruent with visual field defects [25]. Automated perimetry was performed using a Humphrey Visual Field Analyzer 750i instrument (Carl Zeiss Meditec) with the Swedish interactive threshold algorithm 24-2 in all subjects. Glaucomatous visual fields were those that met at least one of Anderson-Patella’s criteria [25]: (1) a cluster of ≥3 points in the pattern deviation plot in a single hemifield (superior/inferior) with p < 0.05, one of which must have been p < 0.01; (2) glaucoma hemifield test result outside normal limits; or (3) abnormal pattern standard deviation with p < 0.05. Reliable visual field tests were defined as false-positive rate <15%, false-negative rate <20%, and fixation loss <20%. Successful IOP control was defined as an IOP ≤18 mm Hg and an IOP reduction ≥20% without glaucoma medication at AS-OCT examination [26]. IOP was measured at 10:00 AM and 5:00 PM at the AS-OCT test date, and if the difference between the two values was greater than 2 mm Hg, a third IOP was additionally measured, the mean of which was used in the analysis [27]. If eyes did not meet the above criteria, the IOP control was considered unsuccessful. Surgical Procedure All procedures were performed by one surgeon (J.W.L). Under local anaesthesia, the limbal conjunctiva was incised 5–6 mm to form the fornix-based conjunctival flap, and the conjunctiva and Tenon’s capsule were separated towards the conjunctival sac. A trapezoidal scleral flap (basal 4 mm, apical 2.5 mm, bilateral 2.75 mm) with 2/3 of the sclera thickness was constructed. Weck-cell sponges soaked with 0.4 mg/mL (0.04%) of MMC were applied between the Tenon’s capsule and sclera for 2–3 min. After the sponges were removed, the area exposed to MMC was irrigated with 20 mL of balanced salt solution. Inner sclerostomy was performed, and then peripheral iridectomy was performed. The scleral flap was closed with two preplaced 9-0 nylon (Ethicon Inc., Johnson & Johnson, Somerville, NJ, USA) releasable sutures. In the AMT group, 15 × 15 mm single layer of cryopreserved amniotic membrane (MS Amnion, MS BIO inc., Seongnam, South Korea) was placed with the stromal side facing up under Tenon’s capsule. The amniotic membrane was secured to the lateral side of scleral flap with two interrupted 10-0 nylon sutures (Ethicon Inc., Johnson & Johnson) (Fig. 1). The anterior chamber was inflated with BSS, and the degree of aqueous outflow through the scleral flap and bleb leakage through the conjunctival sutures were assessed. Postoperatively, administration of topical eye drops, including Levofloxacin (CravitⓇ, Santen Pharm, Co., Osaka, Japan) four times a day and Prednisolone acetate (PredbellⓇ, CKD Pharm, Co., Seoul, South Korea) six times a day for 1 month, was commenced and tapered over 8–12 weeks according to bleb morphology and IOP. Fig. 1. Surgical technique of amniotic membrane transplantation (AMT) over scleral flap during trabeculectomy. a Cryopreserved amniotic membrane was peeled from nitrocellulose filter paper. 15 × 15 mm single layer of amniotic membrane was placed over the scleral flap with the stromal side up. b The limbal side of the amniotic membrane was secured to both sides of the scleral flap margin with two micropoint 10-0 nylon vascular needles. The amniotic membrane was placed under Tenon’s capsule with the stromal side up using a muscle hook. c The conjunctiva and Tenon’s capsule were closed with interrupted micropoint 10-0 nylon vascular needles. For trabeculectomy without AMT, the same procedure was performed except for AMT. AS-OCT Imaging Postoperative blebs were imaged with the Anterior Segment Module (ASM, volume scan vertical-filtering blebs mode, “VolBleb”) of the Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany) which used shorter wavelength light sources (870 nm) and higher speed (40,000 A-scans/second) than time domain OCT. In this study, the high-resolution VolBleb mode with enhanced depth imaging mode and automatic real time of 16 frames was used. OCT scan pattern size was 8.3 × 2.8 mm, and the number of B-scans was 21 with a 139 μm-distance between B-scans. Penetration depth was 1.9 mm with lateral resolution scaling of 10.84 μm/pixel and axial resolution scaling of 3.87 μm/pixel. Images with a quality score >25 dB were included for analysis. When the scleral margin was not apparent in OCT image, manual adjustment of the contrast setting enabled identification of the sclera edge. Intrableb structural parameters were measured with the device’s built-in software (Heidelberg Eye Version: 1.10.2.0), while bleb wall reflectivity was measured with ImageJ software (ImageJ 1.50b, http://imagej.nih.gov/ij/; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Horizontal (tangential to the limbus) and vertical scans (radial perpendicular to the limbus) were taken at the maximum elevation of bleb for each eye. The quantitative parameters included maximum bleb height, maximum bleb wall thickness, maximum striping layer thickness, maximum fluid-filled space height and area, fluid-filled space score (FFSS), and bleb wall reflectivity (Fig. 2). The mean of these horizontal and vertical measurements was used for analysis. The symbols used in the figures were defined as follows: The white and white dotted two-way arrows indicate the bleb wall thickness and fluid-filled space height, respectively. The star indicates the fluid-filled space and the asterisk indicates scleral flap. The black arrow indicates the microcyst and the white arrow indicates visible amniotic membrane beneath the bleb wall. The white arrow head indicates the margin of hyporeflective layers with striping phenomenon. Fig. 2. Representative anterior segment optical coherence tomographic images of fluid-filled space score (FFSS) and measurements of intrableb parameters. a Fluid-filled space was diffuse and extended posteriorly beyond the field of image view (FFSS 2). b The fluid-filled space was limited and demarcated with a clear posterior margin (FFSS 1). c Fluid-filled space was not visible (FFSS 0). The white and white dotted two-way arrows indicate the bleb wall thickness and fluid-filled space height, respectively. The star indicates the fluid-filled space and the asterisk indicates scleral flap. The black arrow indicates the microcyst and the white arrow indicates visible amniotic membrane beneath the bleb wall. Bleb height was measured as the maximal vertical distance between the first reflective signal from the conjunctiva along a straight line perpendicular to a tangent to sclera (Fig. 2). Bleb wall thickness consisting of conjunctiva, Tenon’s capsule, and/or incorporated amniotic membrane was measured as the maximal vertical distance between the first reflective signal of the conjunctiva to the top of the fluid-filled space (Fig. 2). The striping layer was defined as the multiple parallel and fluid-filled channels in Tenon’s capsule, which resembled a honeycombed structure (Fig. 3d) [16, 28]. Fig. 3. Different intrableb structures of eyes in the same patient who underwent trabeculectomy with or without amniotic membrane transplantation (AMT). a Slit-lamp bleb photographic image of the right eye that underwent trabeculectomy with AMT. The bleb had diffuse, moderate height, and mild vascularity based on the Indiana Bleb Appearance Grading Scale (H2 E3 V2 S0) at 1.5 years after trabeculectomy with AMT. b Anterior segment optical coherence tomographic image of the right eye that underwent trabeculectomy with AMT. Diffuse and posteriorly extended fluid-filled space was noted with low bleb wall reflectivity and multiple microcysts. c Slit-lamp bleb photographic image of the left eye that underwent trabeculectomy alone without AMT. The bleb had diffuse, moderate height, and mild vascularity based on the Indiana Bleb Appearance Grading Scale (H2 E3 V2 S0) at 2 years after trabeculectomy alone. d Anterior segment optical coherence tomographic image of the left eye that underwent trabeculectomy alone without AMT. Multiple parallel hyporeflective layers and fluid-filled channels with striping phenomenon in Tenon’s capsule were noted. The star indicates the fluid-filled space, the black arrow indicates the microcyst, and the white arrow indicates visible amniotic membrane beneath the bleb wall. The white arrow head indicates the margin of hyporeflective layers with striping phenomenon. Fluid-filled space height was measured as the maximal vertical distance in the signal void or hyporeflective area between the bottom of the inner bleb wall and the top of the sclera along a straight line perpendicular to a tangent to the sclera (Fig. 2). The fluid-filled space area was measured as the maximal area of signal void or hyporeflective area between the bottom of the inner bleb wall and the top of the sclera. The fluid-filled space score was graded from 0 to 2: (1) score 0, not visible fluid-filled space; (2) score 1, limited and demarcated fluid-filled space with a clear posterior margin; and (3) score 2, diffuse fluid-filled space extending posteriorly beyond the field of image view [14]. Bleb wall reflectivity was analysed using ImageJ software. An ellipse mark of the background near the bleb wall and 3 different evenly spaced ellipse marks in the bleb wall (anterior, middle, and posterior) were placed to measure the bleb wall reflectivity. The value of background was subtracted from the 3 bleb wall reflectivity values, and then the average of the 3 bleb wall reflectivity values was calculated [29]. Microcyst formation was a qualitative parameter in the analysis and defined as a hyporeflective or signal void space directly within or beneath the epithelial layer in the bleb wall [16]. Statistical Analyses Data distribution normality was checked using the Kolmogorov-Smirnov test. Differences between the two groups were analysed with the Mann-Whitney U test or Independent-sample t-test for continuous variables and the χ2 or Fisher’s exact test for categorical variables. Multivariate logistic regression analysis with stepwise forward variable selection method was used to determine intrableb AS-OCT-based structural parameter which was significantly associated with successful IOP control in entire eyes and in each AMT and control group, and the odds ratio for these parameters was calculated. The independent variables included were sex, preoperative lens status, performance of AMT (for analysis in the entire group) as categorical variables. Age, the number of preoperative glaucoma medications, and preoperative IOP were included as continuous, independent variables. The intrableb parameters included as variables were bleb height, bleb wall thickness, striping layer thickness, bleb wall reflectivity, fluid-filled space height, fluid-filled space area, fluid-filled space score, and presence of microcyst formation. All statistical analyses were performed using SPSS software version 22 (IBM Corp., Armonk, NY, USA). p values <0.05 were considered statistically significant. Results Demographics and Clinical Characteristics in All Patients A total of 116 eyes of 103 patients were included in this study. Thirty-one eyes of 27 patients received fornix-based trabeculectomy with MMC alone (control group), and 85 eyes of 76 patients received fornix-based trabeculectomy with MMC and AMT (AMT group). Demographics and clinical characteristics of patients in each group are shown in Table 1. There were no significant differences between the two groups in sex, age at trabeculectomy, MMC soaking time, IOP at AS-OCT test, preoperative IOP, number of preoperative glaucoma medications, preoperative BCVA, preoperative lens status, CCT, axial length, spherical equivalent, and visual field parameters (all ps ≥ 0.053). Overall frequency of successful IOP control (72/85 = 84.7% in the eyes with AMT vs. 23/31 = 74.2% in those without AMT; χ2 test, p = 0.274; Table 1) was similar in the two groups. Table 1. Demographics and clinical characteristics of patients with primary open-angle glaucoma AMT group Control group p value Patients, n 76 27 Sex, female 21 (27.6) 8 (29.6) 1.000 Diabetes mellitus 14 (18.4) 7 (25.9) 0.415 Hypertension 23 (30.3) 5 (18.5) 0.317 Eyes, n 85 31 Age at trabeculectomy, years 59.73±13.87 57.45±14.81 0.369 MMC soaking time, minutes 2.26±0.29 (2–3) 2.33±0.37 (2–3) 0.458 Eye laterality, right 37 (43.5) 12 (38.7) 0.677 IOP at OCT test, mm Hg 13.01±4.80 14.19±5.78 0.103 Successful IOP control, % 72 (84.7) 23 (74.2) 0.274 Preoperative IOP, mm Hg 31.67±9.34 (16–59) 31.32±8.33 (19–55) 0.936 Preoperative medications, n 4.04±0.57 (1–5) 4.00±0.55 (2–5) 0.814 Preoperative visual acuity, logMAR 0.30±0.43 (3-0) 0.42±0.49 (1.7–0) 0.584 Preoperative lens status  Phakia 45 (52.9) 18 (58.1) 0.677  Pseudophakia 40 (47.1) 13 (41.9) Central corneal thickness, μm 533.28±44.97 529.23±38.44 0.653 Axial length, mm 24.80±1.88 25.09±2.41 0.837 Spherical equivalent, dioptre −2.21±3.11 −2.67±2.61 0.238 Visual field parameter  Visual field index, % 51.38±32.43 43.54±28.34 0.312  Mean deviation, dB −17.36±9.39 −19.97±7.44 0.275  Pattern standard deviation, dB 7.38±3.72 8.98±4.08 0.053 Counting fingers at 30 cm was considered equivalent to the Snellen value of 20/2000, which corresponds to a logMAR of 2.0. Hand motion acuity was considered equivalent to the Snellen value 20/20,000, which corresponds to a logMAR of 3.0. Values are presented as mean ± standard deviation (range) or number (%) unless otherwise indicated. AMT, amniotic membrane transplantation; IOP, intraocular pressure; MMC, mitomycin C; logMAR, logarithm of the minimum angle of resolution; OCT, optical coherence tomography. Comparison of AS-OCT Images of the Filtering Bleb after Trabeculectomy AS-OCT-based intrableb structural parameters were compared between eyes with successful IOP control and eyes with unsuccessful IOP control in both the AMT and control groups (Table 2). In the AMT group, eyes with successful IOP control had higher bleb (p < 0.001), thicker bleb wall and striping layer (p = 0.004 and p < 0.001, respectively), lower bleb wall reflectivity (p < 0.001), and more frequent microcyst formation (p = 0.002) than eyes with unsuccessful IOP control. In addition, the area and score of the fluid-filled space were greater in eyes with successful IOP control than in those with unsuccessful IOP control in AMT group (p = 0.043 and p < 0.001, respectively). Table 2. Intrableb parameters assessed with anterior segment optical coherence tomography after trabeculectomy with or without amniotic membrane transplantation Intrableb parameters AMT group Control group Successful, n = 72 Unsuccessful, n = 13 p value* Successful, n = 23 Unsuccessful, n = 8 p value* Bleb height, μm 1,451.60±258.39 1,043.12±379.85 <0.001 1,593.33±192.61 1,104.75±531.53 0.005 Bleb wall thickness, μm 791.56±248.86 559.62±199.34 0.004 1,254.28±388.27 678.69±588.49 0.009 Striping layer thickness, μm 288.09±207.81 32.23±60.85 <0.001 814.59±417.43 204.63±301.07 0.001 Bleb wall reflectivity 99.62±22.35 146.80±12.18 <0.001 77.46±16.84 130.49±38.30 0.003 Fluid-filled space area, mm2 2.66±1.20 1.78±1.96 0.043 1.17±1.31 1.64±2.30 0.982 Fluid-filled space score 1.71±0.43 0.85±0.72 <0.001 0.85±0.76 0.69±0.65 0.674 Fluid-filled space height, μm 660.04±259.36 483.50±483.74 0.140 339.04±320.51 426.06±552.45 0.982 Microcyst formation 66 (91.7) 7 (53.8) 0.002 18 (78.3) 2 (25.0) 0.012 Values are presented as mean ± standard deviation or number (%) unless otherwise indicated. AMT, amniotic membrane transplantation. *Comparison between successful and unsuccessful eyes in each group. In the control group, eyes with successful IOP control had higher bleb (p = 0.005), thicker bleb wall and striping layer thickness (p = 0.009 and p = 0.001, respectively), lower bleb wall reflectivity (p = 0.003), and more frequent microcyst formation (p = 0.012) than eyes with unsuccessful IOP control. However, there was no significant difference in parameters associated with the fluid-filled space between the eyes with successful IOP control and those with unsuccessful IOP control (all ps ≥ 0.674). Logistic Regression Analyses for Determining the Intrableb Parameters Associated with IOP Control Multivariate logistic regression analysis was performed to determine the AS-OCT-based intrableb parameters associated with IOP control in entire eyes and in each AMT and control group (Table 3). In the AMT group, lower bleb wall reflectivity (p = 0.003), greater fluid-filled space score (p = 0.027), and the presence of microcyst formation (p = 0.041) were directly associated with successful IOP control. In the control group, lower bleb wall reflectivity alone was directly associated with successful IOP control (p = 0.019). In the entire group, lower bleb wall reflectivity (p < 0.001), greater fluid-filled space score (p = 0.002), and the presence of microcyst formation (p = 0.034) were directly associated with successful IOP control. Table 3. Intrableb parameters associated with successful IOP control determined with stepwise logistic regression analyses Intrableb parameters Multivariate logistic regression analysis Coefficient Odds ratio (95% CI) p value Entire group (n = 116)  Bleb wall reflectivity −0.066 0.936 (0.909, 0.963) <0.001  Fluid-filled space score 2.320 10.178 (2.357, 43.951) 0.002  Microcyst formation 1.762 5.826 (1.143, 29.690) 0.034 AMT group (n = 85)  Bleb wall reflectivity −0.091 0.913 (0.860, 0.970) 0.003  Fluid-filled space score 2.081 8.016 (1.274, 50.436) 0.027  Microcyst formation 2.785 16.202 (1.114, 235.676) 0.041 Control group (n = 31)  Bleb wall reflectivity −0.205 0.815 (0.687, 0.967) 0.019 CI, confidence interval; AMT, amniotic membrane transplantation. The presence of transplanted amniotic membrane was also evaluated on AS-OCT examination in the AMT group. It was found in 52/72 (72.2%) of the eyes with successful IOP control and 6/13 (46.2%) of the eyes with unsuccessful IOP control. However, there was no significant difference between the two groups (p = 0.065, Fisher’s exact test). On univariate logistic regression analysis, the presence of the transplanted amniotic membrane was not associated with IOP control in the AMT group (p = 0.069). Encapsulated bleb was found only in patients with unsuccessful IOP control in both the control group (3/8 eyes, p = 0.012) and the AMT group (6/13 eyes, p < 0.001). Representative Cases after Trabeculectomy according to AMT Figure 3 showed distinct characteristics of intrableb structures in the same patient who underwent trabeculectomy with or without AMT. A 34-year-old male patient underwent trabeculectomy with AMT in the right eye and trabeculectomy without AMT in the left eye. Slit-lamp photographic images of both eyes showed that the bleb had similar morphologic findings of diffuse, moderate height, and mild vascularity based on the Indiana Bleb Appearance Grading Scale (H2 E3 V2 S0) (Fig. 3a, c) [30]. However, the AS-OCT-based intrableb structures were remarkably different between the two eyes (Fig. 3b, d). Figure 4 showed representative cases of AS-OCT images after trabeculectomy with (Fig. 4a, c, e) or without (Fig. 4b, d, f) AMT. A 75-year-old male patient underwent trabeculectomy with AMT in the left eye 2 years before AS-OCT examination (Fig. 4a). The functioning bleb had a posteriorly extended fluid-filled space (FFSS 2) with successful IOP control of 10 mm Hg without medication. Transplanted amniotic membrane was visible at the inner bleb wall. A 53-year-old female patient underwent trabeculectomy alone in the left eye 2.5 years before AS-OCT examination (Fig. 4b). AS-OCT image showed thick bleb wall with multiple parallel hyporeflective layers and fluid-filled channels within the bleb wall, and IOP was 10 mm Hg without medication. A 68-year-old male patient underwent trabeculectomy with AMT in the left eye 2 years before AS-OCT examination (Fig. 4c). The functioning bleb had a posteriorly extended fluid-filled space (FFSS 2), Tenon’s layer with striping phenomenon, and multiple microcysts with successful IOP control of 13 mm Hg without medication. Transplanted amniotic membrane was visible in the fluid-filled space beneath inner bleb wall. A 69-year-old female patient underwent trabeculectomy alone in the right eye 2 years before AS-OCT examination (Fig. 4d). AS-OCT image showed multiple parallel hyporeflective layers and fluid-filled channels inside Tenon’s layer and successful IOP control of 8 mm Hg without medication. A 70-year-old man underwent trabeculectomy with AMT in the left eye 3 years before AS-OCT examination (Fig. 4e). AS-OCT image showed a thin hyperreflective bleb wall and the fluid-filled space was limited with a clear demarcated posterior margin (FFSS 1). IOP was 21 mm Hg with medications. A 60-year-old male patient underwent trabeculectomy alone in the left eye 1 year before AS-OCT examination (Fig. 4f). AS-OCT image showed low height bleb with a thin and hyperreflective bleb wall and limited fluid-filled space (FFSS 1), which suggested a non-functioning bleb. IOP was 20 mm Hg with glaucoma medications. Fig. 4. Representative cases of anterior segment optical coherence tomography (AS-OCT) images in patients who underwent trabeculectomy with (a, c, e) or without (b, d, f) amniotic membrane transplantation (AMT). a AS-OCT image of the bleb in the AMT group showed diffuse and extended fluid-filled space (FFSS 2) and successful IOP control at 2 years after trabeculectomy. b The filtering bleb in the control group showed thick bleb wall with low reflectivity, multiple parallel hyporeflective layers and fluid-filled channels, microcysts and successful IOP control at 2.5 years after trabeculectomy. c AS-OCT image of the bleb in the AMT group showed bleb wall with low reflectivity and multiple microcysts, posteriorly extended fluid-filled space (FFSS 2), and successful IOP control at 2 years after trabeculectomy. The transplanted amniotic membrane was visible within the suprascleral space. d AS-OCT image of the bleb in the control group showed a thick bleb wall with striping phenomenon and successful IOP control at 2 years after trabeculectomy. e AS-OCT image of the bleb in the AMT group showed a thin and high reflective bleb wall regarded as an encapsulated bleb. IOP control was unsuccessful at 3 years after trabeculectomy. f AS-OCT image of the bleb in the control group showed a high reflective bleb wall with low bleb height and minimal fluid-filled space, and unsuccessful IOP control at 1 year after trabeculectomy. The star indicates the fluid-filled space and the asterisk indicates the scleral flap. The black arrow indicates microcyst and the white arrow indicates visible amniotic membrane beneath the bleb wall. The white arrow head indicates the margin of hyporeflective layers with striping phenomenon. Discussion In the present study, the intrableb structural factors associated with IOP control were investigated using AS-OCT in patients with POAG who underwent fornix-based trabeculectomy with or without AMT. To our knowledge, this is the first study to evaluate intrableb structure after fornix-based trabeculectomy with AMT using AS-OCT. We found that the functioning bleb in both control and AMT groups had common features of intrableb structure which were different from those of the non-functioning bleb. The functioning bleb was higher and had a thicker bleb wall and striping layer, lower bleb wall reflectivity, and more frequent microcyst formation than the non-functioning bleb in both groups. However, there were additional distinct characteristics of intrableb parameters in the functioning bleb in AMT group which were not observed in control group. We found that the fluid-filled space area and score in the functioning bleb were greater than those of the non-functioning bleb in the AMT group. Conversely, there were no significant differences in the fluid-filled space area and score between the functioning bleb and non-functioning bleb in the control group. Multivariate logistic regression analysis confirmed that higher fluid-filled space score, as well as lower bleb wall reflectivity, and microcyst formation were significantly associated with surgical success in the AMT group, whereas lower bleb wall reflectivity alone was associated with surgical success in the control group. These findings are consistent with those of a previous study evaluating the UBM-based intrableb structure associated with IOP control after trabeculectomy with AMT [14]. The authors reported that a wide subconjunctival fluid-filled space was associated with successful IOP control in the AMT group [14]. The results of the present study are also consistent with an earlier study which assessed the filtering bleb after trabeculectomy alone with AS-OCT [20, 24]. Tominaga et al. [24] demonstrated that the IOP levels were significantly lower in eyes with posterior episcleral fluid (PEF) beyond the scleral flap compared to eyes without PEF. Kawana et al. [20] found that IOP was negatively correlated with horizontal and vertical length of the fluid-filled cavity, height of fluid-filled cavity, and volume of the internal fluid-filled cavity. In this study, there were no significant differences in the fluid-filled space area and score between the functioning bleb and non-functioning bleb in the control group. However, the functioning bleb had greater bleb height, bleb wall thickness, striping layer thickness, and lower bleb wall reflectivity than the non-functioning bleb in the control group. These findings are consistent with the results of a previous study using UBM which reported that eyes without AMT had no or minimal fluid-filled space and that a third of the bleb with good IOP had a hyporeflective bleb wall after trabeculectomy alone [14]. There are two possible explanations for our findings. The interval between trabeculectomy and the time of AS-OCT examinations in this study was 2.48 ± 0.73 years in the control group and 2.23 ± 0.74 years in the AMT group. Since post-trabeculectomy changes over time in the AS-OCT parameter have been reported, the possibility that the subconjunctival fluid-filled space may be diminished due to the wound healing process after trabeculectomy alone cannot be excluded [14, 16]. In patients with POAG, reports found that transforming growth factor-β (TGF-β) was elevated in the aqueous humour and was significantly higher in trabecular meshwork cells, inducing fibrotic changes in the trabecular meshwork leading to increased aqueous outflow resistance and IOP elevation [31–33]. In addition, glaucoma filtration surgery such as trabeculectomy was found to cause tissue trauma inducing a localized inflammatory response such as release of cytokines, including TGF-β, recruitment or activation of macrophages, polymorphonuclear cells, and platelets [34]. The transdifferentiation of fibroblasts into myofibroblasts is a major step in wound healing and scar formation, and myofibroblast transdifferentiation is regulated by TGF-β [35]. Therefore, downregulation of TGF-β signalling is a principal strategy for preventing scar formation during wound healing after glaucoma surgery [36]. The amniotic membrane has anti-scarring and anti-inflammatory properties in fibrotic eye disease, including glaucoma surgery [10–12, 37, 38]. The anti-scarring effect of the amniotic membrane is mediated by downregulating TGF-β signalling pathway and myofibroblast differentiation. The amniotic membrane also precludes polymorphonuclear cell infiltration and facilitates macrophage apoptosis [37, 39]. Therefore, we hypothesize that the fluid-filled space in the AMT group persisted after trabeculectomy for a long time due to the anti-scarring and anti-inflammatory effect of the amniotic membrane, although the AMT group revealed a similar interval between surgery and AS-OCT examination to that observed in the control group [14, 40]. Nakamura et al. [14] also suggested that the amniotic membrane may prevent adhesion between the conjunctiva and sclera, which can help maintain the fluid-filled space and subsequent subconjunctival aqueous drainage pathway in the posterior direction. In a rabbit experiment, the preserved human amniotic membrane was decomposed after approximately 3–4 weeks after trabeculectomy [41]. However, in our study, AS-OCT was performed in patients with a minimum follow-up period of 1 year, and the transplanted amniotic membrane was noted in 52/72 (72.2%) in the successful IOP control group and 6/13 (46.2%) in the unsuccessful IOP control group (p = 0.065). Even though the presence of the amniotic membrane was not associated with surgical success (p = 0.069), transplanted amniotic membrane may maintain its anti-fibrosis, anti-inflammatory activity and improve the stability of the bleb wall as a supportive tissue for a considerable period of time. The second explanation for our finding is that the prominent aqueous drainage route may be different between the control group and AMT group [14]. The function of the filtering bleb after trabeculectomy alone may be associated with transconjunctival outflow of aqueous humour [14]. Amar et al. [42] found that microcysts observed at the surface of functioning bleb corresponded to goblet cell containing aqueous humour. The finding of this study is consistent with those of previous AS-OCT imaging studies which evaluated intrableb structural parameters associated with IOP control after trabeculectomy alone [15, 20, 24, 43]. Tominaga et al. [24] demonstrated that thicker bleb wall with lower bleb wall reflectivity was associated with lower IOP. Kawana et al. [20] also reported that volume of the hyporeflective area and microcyst number was negatively related with IOP. Narita et al. [15] found that greater bleb height was significantly associated with surgical success in multivariate analysis. Singh et al. [43] reported that thickening of the bleb wall was found in the majority of successful blebs, whereas bleb wall thickening was typically absent in failed blebs. The main aqueous drainage route may be the subconjunctival pathway in the AMT group [14]. Nakamura et al. [14] suggested that amniotic membrane located between the conjunctiva and sclera may act as a barrier against transconjunctival movement of aqueous humour. Therefore, the authors concluded that the blebs after trabeculectomy with AMT could maintain successful IOP control only when the fluid-filled space was formed posteriorly beyond the field of image view regardless of bleb wall reflectivity [14]. However, we found that a greater fluid-filled score, as well as lower bleb wall reflectivity and more frequent microcyst formation were associated with successful IOP control in the AMT group. Amniotic membrane has a high hydraulic conductivity and is semipermeable to water [44]. Our previous experimental study found that when the amniotic membrane was applied to the upper surface of the Ahmed glaucoma valve body, the fibrous capsule was looser and had a more disorganized collagen architecture in rabbit eyes with AMT compared to eyes without AMT [36]. Although successful IOP control is mainly associated with intact subconjunctival aqueous drainage pathway, transconjunctival drainage of aqueous humour may play a partial role in the AMT group. In contrast to the previous study by Nakamura et al. [14] in the AMT group in our study, both the transconjunctival pathway (lower bleb wall reflectivity and microcyst formation) and subconjunctival pathway (greater fluid-filled space score) were significantly associated with good IOP control in multivariate logistic regression. Even if the eyes had a fluid-filled space score of one and the posterior margin of the fluid-filled space was closed, blebs with lower bleb wall reflectivity and microcyst formation showed successful IOP control. Therefore, in the AMT group, not only subconjunctival absorption but also transconjunctival pathway may play an important role in aqueous outflow. The conflicting findings between this study and the study by Nakamura et al. [14] may be due to differences in demographic and ocular characteristics, surgical technique, and imaging modality. It is recognized that the conjunctival flap technique, such as fornix-based conjunctival flap and limbal-based conjunctival flap, affects bleb morphology [3, 21, 22]. A more diffuse posteriorly draining bleb was formed in the fornix-based trabeculectomy, whereas more cystic blebs and/or ring of steel with more anterior drainage were formed with limbal-based trabeculectomy [3]. The fact that a limbal-based conjunctival flap was made in the study by Nakamura et al. [14] and a fornix-based conjunctival flap was made in this study for trabeculectomy with AMT, suggest that different types of conjunctival incision may result in distinct features of the filtering bleb. In the study by Nakamura et al. [14] among the eyes with AMT, the median number of previous intraocular procedures was two and only 6/28 (21.4%) eyes had POAG. Conversely, in our study, there were no eyes with a history of intraocular surgery, and a diagnosis of POAG was made in 100% of the eyes with AMT. A history of previous ocular surgery and secondary glaucoma may have an impact on bleb morphology [45]. In the study by Nakamura et al. [14], bleb imaging was performed using UBM with a resolution of 50 μm. However, we analysed intrableb structures using AS-OCT with higher resolution than that of UBM, and AS-OCT enabled us to measure intrableb parameters and analyse bleb wall reflectivity quantitatively using the IMAGEJ software. There are some limitations to the present study. As this was retrospective study, only a small sample of patients with unsuccessful IOP control was included (8 eyes in the control group and 13 eyes in the AMT group). Therefore, the findings of this study should be interpreted with caution. However, there was no significant difference in sex, age at trabeculectomy, IOP at AS-OCT test, preoperative IOP, number of preoperative glaucoma medications, preoperative BCVA, preoperative lens status, CCT, axial length, spherical equivalent, and visual field parameters between the control and AMT group. Although the Spectralis AS-OCT used in this study has higher resolution and speed than UBM or time domain OCT such as the Visante OCT (Carl Zeiss AG, Germany) [46, 47], the Spectralis AS-OCT uses shorter wavelength light sources of 870 nm, resulting in a penetration depth of 1.9 mm compared with 6 mm of Visante OCT and 4–7 mm of UBM [46, 47]. Therefore, the possibility that the structure beyond 1.9 mm depth may not be included in the OCT image cannot be excluded. However, the AS-OCT image was taken with EDI and ART modes on to improve image quality, and only OCT images >25 dB were included in the analysis. Manual adjustment of the contrast setting allowed us to identify the scleral edge when the scleral margin was not apparent in the OCT image. An earlier diagnosis of non-functioning bleb may help direct proper management in the postoperative period after trabeculectomy because bleb management procedures are more effective when administered as soon as possible if needed [48]. In the previous study, the tendency towards encapsulation was seen much earlier with AS-OCT, showing thinner bleb wall thickness and higher bleb cavity height (referred as fluid-filled space height in the present study) at 1–2 weeks postoperatively, compared with functioning bleb [17]. However, the interval between trabeculectomy and the time of AS-OCT examinations in this study was 2.48 ± 0.73 years in the control group and 2.23 ± 0.74 years in the AMT group. An early bleb evaluation with AS-OCT at 1–2 weeks was not performed in this study. For future research, we plan to evaluate early intrableb parameters associated with long-term IOP control and bleb management procedures. We used a stringent success criterion for success in this study. Previous studies suggested that IOP <21 mm Hg may not be optimal success criteria for trabeculectomy [49, 50]. Recent clinical trials have adopted cut off points of IOP <18 mm Hg based on long-term outcomes of glaucoma surgery [51, 52]. Nevertheless, several post hoc analyses with more stringent IOP criteria may be needed to apply the results generalizable to patients with glaucoma. Finally, the long-term follow-up periods might affect the intrableb parameters as described in the previous study by Narita et al. [16]. Bleb height, bleb wall thickness, striping layer thickness, and other parameters changed over time. Therefore, the findings of the intrableb parameters in this study should be interpreted with caution. Conclusion Bleb wall reflectivity was significantly associated with bleb function in both the control and AMT group. However, the fluid-filled space score was associated with IOP control only in the AMT group. The transplanted amniotic membrane in trabeculectomy may be involved in IOP control by enhancing aqueous flow through the subconjunctival absorption, as well as by maintaining flow through the transconjunctival pathway. Acknowledgments We would like to thank Editage (www.editage.co.kr) for English language editing. Statement of Ethics The study was conducted according to the guidelines of the World Medical Association Declaration of Helsinki, and approved by the Institutional Review Boards of Pusan National University Hospital (approval No. 2111-004-108). All patients gave written informed consent for the surgical procedures and for their information to be stored in the hospital database and used for research. Conflict of Interest Statement The authors have no conflicts of interest to declare. Funding Sources This research was supported by a grant from Medical big data and AI-based early detection of visual dysfunction funded by Busan and managed by Busan Techno Park and by the Patient-Centered Clinical Research Coordinating Center, funded by the Ministry of Health and Welfare, Republic of Korea (Grant no. HI19C0481, HC19C0276). The funding agencies have played no role in this research. Author Contributions Jiwoong Lee contributed to conceptualization and involved in writing, review, and editing; Jiwoong Lee and Sangwoo Moon contributed to methodology; Sangwoo Moon contributed to investigation and involved in writing original draft preparation; Jiwoong Lee, Sangwoo Moon, and Jinmi Kim contributed to data curation. All authors have read and approved the final manuscript and agreed to publish the manuscript. 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==== Front Intervirology Intervirology INT INT Intervirology 0300-5526 1423-0100 S. Karger AG Basel, Switzerland 37231989 528178 10.1159/000528178 Research Article Hepatitis C Virus Genotype 5 Variability in Treatment-Naïve Patients in South Africa HCV Genotype 5 in South Africa 2651027 Maunye Tshegofatso K. a b 2651028 Gededzha Maemu P. c 2651029 Blackard Jason T. a d 2651030 Rakgole Johnny N. a 2651031 Selabe Selokela G. a b a HIV and Hepatitis Research Unit, Department of Virology, Sefako Makgatho Health Sciences University, Pretoria, South Africa b National Health Laboratory Service, Pretoria, South Africa c Department of Immunology, Faculty of Health Sciences, University of Witwatersrand and National Health Laboratory Service, Johannesburg, South Africa d Division of Digestive Diseases, University of Cincinnati College of Medicine, Cincinnati, OH, USA Correspondence to: Tshegofatso K. Maunye, maunyetk@gmail.com 9 5 2023 Jan-Dec 2023 66 1 7787 2 5 2022 14 11 2022 2023 © 2023 The Author(s). Published by S. Karger AG, Basel 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission. Abstract Introduction Hepatitis C virus (HCV) genotype 5 was originally identified in South Africa, where it represents 35–60% of all HCV infections. There are limited data on resistance-associated variants (RAVs) in South Africa. Thus, we investigated variability within the NS3/NS4A, NS5A, and NS5B genes of treatment-naïve individuals with HCV genotype 5 infection at the Dr. George Mukhari Academic Hospital (DGMAH) in Pretoria, South Africa. Methods Nested PCR was performed to amplify the NS3/4A, NS5A, and NS5B genes. RAVs were evaluated using the Geno2pheno tool. Results In the NS3/4A gene, F56S and T122A were detected in one sample each. The D168E mutation was detected in 7 samples. Within the NS5A gene, the T62M mutation was detected in 2 individuals. In the NS5B gene, 8 of 12 individuals (67%) had the A421V mutation, while all 12 individuals (100%) had the S486A mutation. Discussion RAVs were detected frequently among treatment-naïve individuals with HCV genotype 5 infection in South Africa. Thus, resistance testing may be prudent when initiating treatment of patients with genotype 5 infection. Additional population-based studies are needed to understand the prevalence of these RAVs during HCV genotype 5 infection. Keywords Hepatitis C virus Africa Genotype Drug resistance This work was supported by grants from the Medical Research Council, the National Research Foundation, the National Health Laboratory Service Research Trust, and the Stella and Paul Loewestein Charitable and Educational Trust in South Africa. ==== Body pmcIntroduction There are approximately 71 million people worldwide with chronic hepatitis C virus (HCV) infection [1]. HCV is a leading cause of chronic liver disease, liver transplantation, and hepatocellular carcinoma. The prevalence of HCV varies considerably across African countries with Egypt, Cameroon, and Burundi having the highest prevalence rates (reviewed in [2]). In South Africa, the seroprevalence of HCV infection is 1.8% in healthcare workers, 3.4–13% in HIV-positive individuals, and 28–72% in persons who use drugs [3–6]. The viral genome encodes multiple structural (core, E1, and E2) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [7]. NS3/4A, NS5A, and NS5B are targets for direct-acting antivirals (DAAs). NS3/4A contains protease, RNA helicase, and NTPase activities. The NS5A protein is a key component of replication and viral assembly. The HCV NS5B protein is an RNA-dependent RNA polymerase that is responsible for the synthesis of negative-sense RNA and new positive-sense RNAs that are incorporated into progeny virions [8, 9]. Significant advances have been made in the treatment of HCV infection in recent years; however, DAAs remain costly in some locations and are not available in many resource-limited settings. DAA combinations such as glecaprevir/pibrentasvir and velpatasvir/sofosbuvir achieve viral clearance rates of 98% (reviewed in [10]). Nonetheless, the high genetic variability of HCV can lead to drug-resistant variants. A high genetic barrier to resistance can be achieved by a combination of DAAs with nonoverlapping resistance profiles but requires laboratory monitoring. Multiple studies have reported that HCV genotype is a determinant of treatment response and disease pathogenesis [11–14]. HCV genotype 5 was originally identified in South Africa [15, 16], where it represents 35–60% of all HCV genotypes [16–20]. However, pockets of genotype 5 infections have been reported in France, Spain, Syria, Greece, Botswana, Ethiopia, India, and Belgium [21–32]. There is paucity of data on resistance-associated variants (RAVs) in South Africa. Thus, we investigated variability within the NS3/NS4A, NS5A, and NS5B genes of treatment-naïve individuals with HCV genotype 5 infection at the Dr. George Mukhari Academic Hospital (DGMAH) in Pretoria, South Africa. Materials and Methods Study Population Stored remnant/leftover serum samples from 22 individuals with HCV genotype 5 infection – based on analysis of the 5′ untranslated region [18] – attending DGMAH from January 2007 to October 2010 were included. Sample collection was reviewed, and consent was waived by the Medunsa Research and Ethics Committee (MREC/p/142/2009:PG) and the Sefako Makgatho University Research Ethics Committee (SMUREC/M/03/2017:PG). To ensure patients’ anonymity and maintain confidentiality, patient identifiers were removed prior to any analyses. HCV RNA Extraction and cDNA Synthesis RNA was extracted from serum samples using the QIAamp Viral RNA Mini Kit (Qiagen, Germany). The resulting RNA extracts were converted into cDNA using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer’s instructions. HCV Viral Load Determination HCV viral loads were determined by quantitative real-time PCR on the Eco Real-Time PCR system (Illumina, USA) using HCV reverse primer 5′ – CGC GAC CCA ACA CTA CTC – 3′, HCV forward primer 5′ – CGG GAG AGC CAT AGT GGT – 3′, and HCV probe FAM – TGC GGA ACC GGT GAG TAC ACC – MGB) to target the 5′ UTR [33]. A 10-fold serial dilution of a known HCV RNA-positive control (800,000 copies/mL) was used to generate standard curves for quantification of study samples. In-House HCV PCR Nested PCR was performed using the PicoMaxx High Fidelity system master mix (Agilent Technologies, USA) following the manufacturer’s instructions. Primers to amplify NS3/4A, NS5A, and NS5B complete genes were specific to genotype 5 as published by Ku et al. [34]. An additional primer pair was developed for this study to amplify the NS3 gene (Table 1). The first round PCR products were used as templates for second round PCR using the same conditions as described below. The thermal cycling conditions for both 1st and 2nd rounds were 95°C for 2 min followed by 40 cycles of 95°C for 30 s, annealing temperature depending on primer pair for 30 s and 72°C for 3 min followed by a final extension at 72°C for 10 min. PCR products were visualized following electrophoresis on agarose gels stained with ethidium bromide (Promega, USA). Table 1. Primers for NS3, NS5A, and NS5B for cDNA synthesis and PCR amplification Name Gene PCR round Primer sequence (5′-3′) Annealing temperature (oC) Reference NS3_5A_PCR1F NS3/4A 1st TGC TCC ACC TTG GTA GGC TGA CCG G 52 Ku et al. [34] (2016) NS3_5A_PCR1F2 NS3/4A 1st TGA CCG GAA CGT ACA TTT ATG ACC 52 Ku et al. [34] (2016) 5ANS3_3R NS3/4A 1st ACG CRA TCA GCC TRT TCA TCC 52 This study NS3_5A_PCR2F NS3/4A 2nd CCT ATG GAG ACG AAG GTC ATC ACG 54 Ku et al. [34] (2016) 5ANS3_4R NS3/4A 2nd GCC ACC CAC CCT CCT AGR AT 54 This study NS5A_5A_PCR1F NS5A 1st GAC CTA GTM AAC CTC CTG CC 50 Ku et al. [34] (2016) NS5A_5A_PCR1R NS5A 1st TCA AGC AAG TCC TGC CAC AC 50 Ku et al. [34] (2016) NS5A_5A_PCR2F NS5A 2nd TCT CCG ACR CAC TAC GTG CC 48 Ku et al. (2016) NS5A_5A_PCR2R NS5A 2nd TAC ACA AGA TTG TGG TGG CG 48 Ku et al. [34] (2016) NS5A_5A_PCR2R1 NS5A 2nd GTG ACC TTC TTC TGC CT 48 Ku et al. [34] (2016) NS5B_5A_PCR1F NS5B 1st GCG GCT TCA TAT TCT TCC ATG CC 52 Ku et al. [34] (2016) NS5B_5A_PCR1R NS5B 1st GGA GTG TTT AGC TCC CAG C 52 Ku et al. [34] (2016) NS5B_5A_PCR2F NS5B 2nd GAC CTT TCG TCA GGG TCA TGG T 50 Ku et al. [34] (2016) NS5B_5A_PCR2R NS5B 2nd GGG AGY AAA AAG ATG CCT AC 50 Ku et al. [34] (2016) Phylogenetic Analysis PCR products were purified and sequenced using an ABI Prism Genetic Analyzer 3730XL (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sequence data were edited using ChromasPro v1.5 (Griffith University, Australia). Multiple sequence alignments were performed in Clustal X 2.1 [35] to compare South African sequences to 204 references representing genotypes 1–7 available through the HCV sequence database at https://hcv.lanl.gov/content/sequence/NEWALIGN/align.html. Study sequences were then realigned with a subset of genotype references, as well as additional full-length genotype 5 references. Phylogenetic inference was performed using a Bayesian Markov chain Monte Carlo approach as implemented in the Bayesian Evolutionary Analysis by Sampling Trees (BEAST) version 1.10.1 program [36] with an uncorrelated log-normal relaxed molecular clock, general time-reversible model, and nucleotide site heterogeneity estimated using a gamma distribution. The Markov chain Monte Carlo analysis was run for a chain length of 500,000,000, and results were visualized with Tracer version 1.7.1 to confirm adequate chain convergence. The effective sample size was calculated for each parameter, and all effective sample size values were >1,000, indicating sufficient sampling. The maximum clade credibility tree was selected from the posterior tree distribution after a 10% burn-in using TreeAnnotator version 1.10.1 and visualized in FigTree version 1.4.4 as we have described previously [37, 38]. Determination of RAVs To evaluate the presence or absence of RAVs, all study sequences were submitted to Geno2pheno (hcv) 0.92 [39]. RAVs were also identified by comparing the wild-type amino acids published by Sorbo et al. [40] and evaluated for possible substitutions within the identified locations that are associated with drug resistance. Statistical Analysis Inferential statistical analyses for associations between the viral load and sequence diversity were conducted using SPSS v25. Data were collapsed into binary format and entered into 2 × 2 tables to calculate odds ratios, 95% confidence intervals, and χ2p values. p values ≤0.05 were considered statistically significant. For determining the predictors of sequence diversity, logistic regression was performed. Results Amplification of Genotype 5 Samples The study population consisted of 12 (54.6%) females and 10 (45.4%) males. The mean age was 63 years (range: 21–86). The median HCV viral load was 84,172 copies/mL (range: 152–513,000 copies/mL). Patient demographic and clinical data are provided in Table 2. The NS3/4A, NS5A, and NS5B genes were successfully amplified from 10, 12, and 12 individuals, respectively. Five samples could not be amplified for any of the target regions. The median viral load for these samples was slightly lower than for those samples that did amplify (31,405 copies/mL vs. 91,683 copies/mL). As expected, all samples clustered with genotype 5 references for the NS3/4A, NS5A, and NS5B regions (Fig. 1–3). Table 2. Summary of the characteristics of the study participants Sample ID Antibody titer Age, years Gender Clinical condition Viral load, copies/mL ZADGM0308 33.6 79 Male Jaundice 76,778 ZADGM0518 N/A 55 Male N/A 89,689 ZADGM0525 N/A 75 Female Hepatitis 949,000 ZADGM0651 115.94 73 Male Cirrhosis 4,210,000 ZADGM0869 67.12 66 Female Diabetic 34,746 ZADGM1104 97 50 Female N/A 1,632 ZADGM1707 N/A 65 Female N/A 91,683 ZADGM1908 97.17 86 Male Massive ascites 36,000 ZADGM2088 89.27 53 Female Diabetic 679,000 ZADGM2352 N/A 72 Female N/A 41,205 ZADGM2439 62.05 37 Male Hemophilia A 565,000 ZADGM2582 36.99 58 Female N/A 96,300 ZADGM3013 77.5 63 Male N/A 60,425 ZADGM3073 80.46 60 Female Hepatic encephalopathy 31,405 ZADGM4124 N/A 63 Male Diabetic 246,000 ZADGM4227 N/A 60 Female Congestive heart failure 135,000 ZADGM6485 90 73 Male Thrombosis 152 ZADGM6544 26.97 63 Male Renal failure 12,057 ZADGM7890 114.95 62 Female Massive ascites 5,130,000 ZADGM7938 125.09 75 Female Hepatitis 28,906 ZADGM9150 79.36 62 Female Massive ascites 96,238 ZADGM9684 52.36 21 Male N/A 78,654 N/A, not available. Fig. 1. NS3/4A nucleotide sequences from this study (red) were compared to HCV genotype references (denoted as genotype-country-accession number). Fig. 2. NS5A nucleotide sequences from this study (red) were compared to HCV genotype references (denoted as genotype-country-accession number). Fig. 3. NS5B nucleotide sequences from this study (red) were compared to HCV genotype references (denoted as genotype-country-accession number). Resistance-Associated Variants Eight amino acid positions within the NS3/4A region that are associated with drug resistance were evaluated, including L36, F56, K80, T122, I132, D168, I170, and N174. As shown in Table 3, F56S and T122A were detected in one individual each. The D168E mutation was detected in 7 individuals. Nine amino acid positions within the NS5A gene were evaluated, including Q24, L28, Q30, L31, S38, P58, T62, A92, and Y93. The T62M mutation was detected in 2 individuals. Nineteen amino acid positions within NS5B were evaluated, including E237, Q273, S282, C316, H330, L392, M414, L419, A421, A444, F445, E446, V451, I482, S486, V494, P495, A499, and G556. Of the 12 individuals, 8 (67%) had the A421V mutation, while all 12 (100%) had the S486A mutation. There was no association between HCV viral load and the presence of any RAV detected in this study – F56S (p = 0.73), T62M (p = 0.12), T221A (p = 0.20), Q309R (p = 0.82), Q355I (p = 0.62), Q355T (p = 0.91), A421V (p = 0.53), and S486A (p = 0.32). Table 3. RAVs in the NS3/NS4A, NS5A, and NS5B regions Sample ID NS3/4a NS5A NS5B ZADGM0308 F56S D168E T62M * ZADGM0518 WT WT A421V S486A ZADGM0525 * WT * ZADGM0651 * T62M * ZADGM0869 WT WT A421V S486A ZADGM1104 T122A D168E WT S486A ZADGM1707 * * A421V S486A ZADGM1908 * * A421V S486A ZADGM2088 D168E WT S486A ZADGM2352 * WT S486A ZADGM2439 * WT S486A ZADGM2582 WT WT A421V S486A ZADGM3013 D168E WT A421V S486A ZADGM3073 * * * ZADGM4124 D168E WT * ZADGM4227 D168E * A421V S486A ZADGM6485 * * * ZADGM6544 * * * ZADGM7890 * * * ZADGM7938 * WT * ZADGM9150 D168E * A421V S486A ZADGM9684 * * * With at least one RAV, n (%) 7 of 10 (70) 2 of 13 (15.4) 12 of 12 (100) WT, wild type. *Not amplified and/or not analyzed due to poor sequence quality. Immune Response Epitopes CD8+ T cell responses are critical to the control of HCV infection and include the human leukocyte antigen B57-restricted epitopes NS5B2629–2637 (KSKKTPMGF) and NS5B2936–2944 (GRAAICGKY) but have been based on studies of HCV genotype 1 [41, 42]. As shown in Figure 4a, all genotype 5 study sequences, as well as references, had an A instead of G within the KSKKTPMGF epitope. There were 15 other mutations within this epitope, including two positions with multiple mutations in multiple genotype 5 sequences. The GRAAICGKY epitope had a K to I mutation in all genotype 5 study sequences and references (Fig. 4b). There were 6 other mutations within this epitope, including one position with mutations in multiple genotype 5 sequences. Vaughn et al. [43] identified NS5B positions that contact nascent RNA during RNA synthesis. As shown in Figure 4c, these contacts were completely conserved in all study participants and genotype 5 references with the exception of a single amino acid polymorphism in reference KJ925146 from South Africa. Fig. 4. a Variation in the HLA-B57-restricted epitope NS5B2629–2637 associated with spontaneous viral clearance. The wild-type sequence is KSKKTPMGF. Amino acid variants within consensus sequences are shown as a frequency plot. b Variation in the HLA-B57-restricted epitope NS5B2936-2944 associated with spontaneous viral clearance. The wild-type sequence is GRAAICGKY. Amino acid variants within consensus sequences are shown as a frequency plot. c Variation within RNA channel contact points. The wild-type sequence is RQKKVTFDRLQV. Amino acid variants within consensus sequences are shown as a frequency plot. Discussion A high prevalence of resistance-associated mutations was observed in the present study of treatment-naïve individuals with HCV genotype 5. The NS3/4A gene D168E mutation was observed in multiple individuals. This finding is supported by a previous study in which the D168E mutation was detected in 3 of 6 individuals and the T122A mutation in 2 of 6 individuals [34]. Multiple drug resistance mutations (D168E + T122A + F56S) are critical mutations that confer resistance to a wide range of DAAs in all the HCV genotypes. Mutations at NS3 position D168 confer resistance to multiple DAAs [44–47]. The most common mutation detected in patients failing treatment with NS5A inhibitors is Y93 C/H/N/S [48]. The prevalence of naturally occurring resistance mutations that are associated with NS5A inhibitors is estimated to be 29.6% [49]. In the present study, only one NS5A RAV – T62M – was observed in 2 individuals. However, another study found no RAVs in the NS5A gene [34]. In contrast, another study found two mutations – T62A and S54Y – in one individual [50]. Only two RAVs associated with NS5B resistance were detected in the current study with a prevalence rate of 100% (S486A) and 67% (A421V). In contrast, a previous study by Prabdial-Sing et al. [19] observed no RAVs in the NS5B gene in South African individuals. This may reflect the analysis of a short region (11%) of the NS5B gene in that study compared to the whole NS5B gene evaluated in the present study. Other studies have reported the K72R mutation within the NS5B gene of 2 of 8 (25%) individuals [50]. We observed no significant association between the HCV viral load and the presence of RAVs; however, this likely reflects the small sample size. A larger sample size may be required to rigorously evaluate the relationship between these variables. Previous studies of CD8+ T cell epitopes that are associated with spontaneous clearance of HCV were restricted to genotype 1 infections [41, 42]. However, the NS5B2629–2637 (KSKKTPMGF) and the NS5B2936-2944 (GRAAICGKY) epitopes were not conserved in any individuals. While these findings may suggest different rates of spontaneous clearance for genotype 5 compared to other genotypes, immune responses have not been characterized functionally for genotype 5. Thus, additional studies are required to evaluate immune responses and spontaneous clearance rates in countries in which genotype 5 circulates. Treatment response rates for genotype 5 are poorly studied compared to other HCV genotypes. Sustained virologic response (SVR) rates of 71–77% and 64% after treatment of genotype 5 infections with pegylated interferon + ribavirin have been reported in South Africa and France, respectively [51–53]. DAA treatment of HCV in South Africa has been reported in a pilot study of 21 individuals [20]. The overall SVR was 95%; however, only 8 individuals with genotype 5 were evaluated. A pooled analysis of data from phase 2 and 3 studies evaluating the efficacy of DAAs on genotype 5 or genotype 6 infections showed SVR of 98% [54]. Moreover, HCV genotype 5 subgenomic replicons have been established and will enable additional studies of pan-genotypic DAAs [55]. This study is small in nature and is restricted to a single academic center. This reflects the limited geographic distribution of genotype 5 and its occurrence mainly in resource-limited settings. Other limitations to this study include the inability to amplify all HCV genes from all study samples. The low PCR positivity rate may be due to genetic variability within genotype 5, the study samples having been stored for several years prior to use in this study, and/or freeze-thaw of these samples for use in other studies. Nonetheless, our findings demonstrate the existence of RAVs in all major targets of current HCV therapy among treatment-naïve individuals with HCV genotype 5. These data suggest that resistance testing may be prudent when initiating treatment of patients with genotype 5 infection. Further population-based studies are needed to understand the prevalence of these RAVs during HCV genotype 5 infection. Statement of Ethics Sample collection was reviewed, and consent was waived by the Medunsa Research and Ethics Committee (MREC/p/142/2009:PG) and the Sefako Makgatho University Research Ethics Committee (SMUREC/M/03/2017:PG). To ensure patients’ anonymity and maintain confidentiality, patient identifiers were removed prior to any analyses. Conflict of Interest Statement The authors have no conflicts of interest to report. Funding Sources This work was supported by grants from the Medical Research Council, the National Research Foundation, the National Health Laboratory Service Research Trust, and the Stella and Paul Loewestein Charitable and Educational Trust in South Africa. Author Contributions Tshegofatso K. Maunye contributed to the design of the project, conducted laboratory work, performed data analysis, wrote the initial manuscript draft, and edited/approved the final manuscript. Maemu P. Gededzha contributed to the design of the project, conducted laboratory work, performed data analysis, and edited/approved the final manuscript. Jason T. Blackard performed data analysis and edited/approved the final manuscript. Johnny N. Rakgole contributed to the design of the project, conducted laboratory work, performed data analysis, and edited/approved the final manuscript. Selokela G. Selabe contributed to the design of the project, edited/approved the final manuscript, and obtained funding for the project. Data Availability Statement Study sequences are available in GenBank under accession numbers KC767829–KC767834 and ON228285–ON228302. Further inquiries can be directed to the corresponding author. ==== Refs References 1. World Health Organization . Hepatitis C. WHO; 2019. 2. 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==== Front Ann Med Ann Med Annals of Medicine 0785-3890 1365-2060 Taylor & Francis 37455518 10.1080/07853890.2023.2234936 2234936 Version of Record Research Article Physical Medicine & Rehabilitation Internal consistency and construct validity of the Short Musculoskeletal Function Assessment (SMFA) in older adults S. Teljigovic et al. https://orcid.org/0000-0001-7784-7451 Teljigovic Sanel ab https://orcid.org/0000-0002-9183-083X Lindahl Marianne a https://orcid.org/0000-0002-3964-4642 von Magius Camilla Mølholm c https://orcid.org/0000-0002-2961-7800 Sjøgaard Gisela b https://orcid.org/0000-0003-3968-6364 Søgaard Karen bd https://orcid.org/0000-0001-8436-1046 Sandal Louise Fleng b a Centre for Health and Rehabilitation, University College Absalon, Slagelse, Denmark b Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, Denmark c Center for Sundhed og Ældre, Sundhed & Træning, Slagelse, Denmark d Department of Clinical Research, University of Southern Denmark, Denmark CONTACT Sanel Teljigovic sate@pha.dk 16 7 2023 2023 16 7 2023 55 1 22349365 6 2023 4 7 2023 5 7 2023 KnowledgeWorks Global Ltd.15 7 2023 published online in a building issue15 7 2023 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 2023 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. Abstract Objective The primary objectives of this study were to 1) investigate the internal consistency 2) and construct validity of the Short Musculoskeletal Function Assessment Questionnaire (SMFA) in older adults commencing physical rehabilitation in an outpatient setting. Methods This cross-sectional study recruited older adults who had commenced physical rehabilitation in an outpatient setting. The SMFA consists of two indices: 1) dysfunction capturing the impact of musculoskeletal disorders on physical limitations, and 2) bothering capturing how the individual is emotionally affected by their disorder. SMFA holds four categories: ‘mobility’, ‘daily activities’, ‘emotional status’, and ‘function of the arm and hand’. Participants answered the SMFA alongside other patient-reported questionnaires (such as the 36-Item Short Form Survey, SF-36) and similar) and objectively measured muscle strength for the upper and lower body and functional capacity. Results We included 115 older adults with a median age of 74 years (IQR 9). Adequate internal consistency was seen with Cronbach’s alpha values of 0.90–0.94 for the SMFA indices and 0.77–0.91 for the SMFA categories. The strongest correlations between the SMFA indices were observed with the SF-36 physical component summary (SMFA-Dysfunction r = 0.74, p < 0.05, SMFA-Bother r = 0.72, p < 0.05). Only fair correlations were found between SMFA index scores and clinical outcome measures. Discussion This study demonstrated that the SMFA has adequate internal consistency and construct validity for self-reported health status in older adults, especially when considering components covering physical health status. However, we only observed fair correlations between SMFA and clinical outcome measures, indicating that SMFA does not adequately capture muscle strength and functional capacity. Keywords SMFA older adults construct validity internal consistency The project has been granted 2.589.134 DKK (including 44% overhead) from The Danish Council for Independent Research (ref. nr. 8045-00052B), 290.000 DKK from The Danish Research Committee for Physiotherapists, and 373.026 DKK from University College Absalon. ==== Body pmcIntroduction Older adults in outpatient settings are a heterogeneous group suffering from various health disorders such as musculoskeletal disorders (e.g. back and neck pain, osteoarthritis), chronic obstructive pulmonary disease, and depression. These conditions ultimately disperse the functional abilities. With increasing age, the risk of more than one chronic condition increases steadily, affecting physical functioning and health-related quality of life [1,2]. Furthermore, differences in function and recovery profiles exist among older adults with medical conditions [3]. This indicates different rehabilitation needs within this heterogeneous population. Usually, physical rehabilitation is focused primarily on one condition, and many conditions in one individual challenge the choice of health outcomes to assess progress. Using patient-reported outcome measures (PRO’s) plays an important role in reflecting the context of patients’ everyday lives, as it allows the assessment of an individual’s perception of functional limitations and symptoms and how they think it affects their daily living [4]. The Short Musculoskeletal Function Assessment Questionnaire (SMFA) captures the dysfunction of specific musculoskeletal disorders. The SMFA aims to provide a patient-reported perception of the impact of musculoskeletal disorders on physical limitations (SMFA dysfunction index, SMFA-D) and how the individual is emotionally affected by their disorder (SMFA bother index, SMFA-B) [5]. Using a standardized questionnaire to compare different musculoskeletal diseases and injuries in patients undergoing surgical or conservative inpatient treatment [6]. When choosing PROs that reflect the degree of dysfunction in physical rehabilitation contexts, it is essential to consider the measured construct, its value to the patient, and its psychometric properties. A commonly used holistic framework for classifying and revealing the impact of a condition on health in everyday life is the International Classification of Functioning, Disability, and Health (ICF), developed by the World Health Organization (WHO) [7]. The ICF includes the constructs of body function and structure, activities, and participation while simultaneously considering the effects of the context as environmental and personal factors. Physical and occupational therapists often use the classification during diagnostics to assign information so that specific goals can be targeted for each construct [8]. Using the ICF as a framework provides a common international language for collecting data on disability and functioning in a structured and consistent way. Consequently, investigating the construct validity of the SMFA by examining its association with other health-related questionnaires representing different ICF domains and its association with typically used clinical outcome measures will provide valuable information to physical and occupational therapists regarding the clinical interpretation of SMFA scores. Hence, the primary objectives of this study were to 1) investigate the internal consistency 2) and the construct validity of the SMFA questionnaire in older adults commencing physical rehabilitation in an outpatient setting. Methods The reporting of this paper is based on the ‘COnsensus-based Standards for the selection of health Measurement Instruments’ (COSMIN) reporting guidelines for studies on measurement properties of PRO measures [9]. The body funding this research played no role in this study’s design, conduct, or reporting. Study design and participants This was a multicenter cross-sectional study. Eligible participants were older adults aged ≥ 65 years who were referred for physical rehabilitation in municipality-based health centers from a medium-sized Danish municipality covering both rural and urban populations. Administrative personnel initially screened participants for eligibility in January-September 2021, a period partly characterized by lockdown due to COVID-19. If deemed eligible, older adults were invited to participate in a screening interview conducted at their own homes. The interview consisted of standardized questions, collecting information on the reasons for referral, comorbidities, use of medications, history of falls, and practical information regarding transportation. Risk evaluation for participating in clinical measurements was also performed. Participants were excluded from participation if any of the following criteria were met: inability to speak or read Danish, active cancer, upper or lower limb amputations, hypertension >180/110 mmHg for diastolic/systolic blood pressure, referral to rehabilitation primarily due to gynaecological or neurological conditions (apoplexies), surgeries where movement restrictions prohibit participating in most of the clinical outcome measures, or general practitioner advised against participation. Ethics The study protocol was reviewed and approved by the Ethical Committee in Region Zealand, Denmark (SJ-758), and the General Data Protection Regulation at the University of Southern Denmark, Odense (10.330). Further, the trial has been registered at clinicaltrials.gov (NCT04862481) as a sub-study of our ongoing randomized controlled trial (NCT0491308). Written consent was obtained from all the participants. Furthermore, all participants were informed about their right to withdraw from the study, as explicitly stated by the Ethics Committee in Region Zealand, Denmark. Data collection A standardized data collection protocol was approved by the regional ethical committee and followed across recruitment sites and personnel to ensure reproducibility and avoid selection bias. If eligible and willing to participate in the study, older adults received a paper-based 100-item survey. The survey was answered by participants in familiar surroundings using paper and pencil during home visits [10–13]. If queries arose while answering the survey, participants were instructed to discuss this with the principal investigator. The questionnaire answers were double-entered in Excel (Microsoft Office 365) to blind the outcome assessors from the SMFA scores of each participant before the clinical testing session. Patient-reported outcome measures The 100-item survey consisted of the following questionnaires: the 36-Item Short Form 36 (SF-36), the Tilburg Frailty Indicator (TFI), Program of Research to Integrate Services for the Maintenance of Autonomy (PRISMA7), New Mobility Scale (NMS), and questions on sociodemographic variables). All the questionnaires are described in Table 1. Table 1. Descriptive outcome table. Patient-reported outcome measures Procedure and Scoring Instrument / equipment SMFA The questionnaires consists of 46 items and can be divided into a dysfunction index ‘SMFA-D’ (34 items) and a bother index ‘SMFA-B’ (12 items). Further, items can be clustered into categories: Daily Activities, Mobility, Emotional Status, and Arm/Hand. The total possible score ranges between 0–100, with higher scores indicating higher degree of dysfunction or bother. [5] Paper and pencil SF-36 Generic survey assessing health-related quality of life – The SF36 covers dimensions of Physical Functioning, Role Physical, Bodily Pain, General Health, Vitality, Social Functioning, Role Emotional, Mental Health and two summary scores of Physical health and Mental health. The SF-36 transformed score ranges between 0–100 with higher scores indicating better health-related quality of life. [14] Paper and pencil PRISMA-7 Composed score of frailty – consists of seven items each score either 0 or 1 points. The total possible score ranges between 0 and 7 points. Higher scores indicate increased risk of frailty. [11] Paper and pencil NMS Composed score of physical mobility – consists of three items each score between 0–3 points, depending on the degree of help. The total possible score ranges between 0–9 points. Higher scores indicate better mobility. Paper and pencil TFI Composed bio-psycho-social approach, which measures frailty. It is composed of 15 multidimensional items regarding the physical, psychological, and social aspects of human functioning. The scoring range is between 0–15 points. [12] Higher scores indicate an increased risk of frailty. [15] Paper and pencil Sociodemographic variables Name, sex, personal ID (CPR number), educational level, previous occupation. Paper and pencil Musculoskeletal pain or discomfort The regions of musculoskeletal pain (marked on a body chart), pain intensity and pain duration at the most painful body region in the last three months Paper and pencil Clinical outcome measures      Weight and body composition Measured using a bioimpedance with a segmental analyser. The scale was adjusted for one kg simulating the weight of the clothes. [16] Variables collected were weight (kg.), segmental fat percentage (%) and lean body mass (kg.), body water percentage (%), visceral fat index, and basic metabolic rate (kJ). [16] Tanita 9MC-780U Multi Frequency Segmental Body Composition Analyzer (Tanita, Tokyo, Japan)  Height, meters Height was measured barefooted using a stadiometer, with the back leaned against the wall. [17] Etopoo stadiometer (China)  Handgrip maximal strength, kg. The maximal hand-grip strength test was measured using a hydraulic dynamometer. The participant was placed in a standing position with the elbows fixed and placed in a 90 degrees flexion. The forearm was placed in a neutral position. Three maximal measures were taken with each hand, with a 30 second break between measures. [18] SAEHAN hydraulic dynamometer (Saehan, Masan, South Korea)  Elbow maximal flexion and extension muscle strength, kg. Elbow flexion strength test was conducted in a sitting position with the elbow flexed to 90 degrees. The dynamometer was placed at the anterior aspect of distal forearm, proximal to the wrist and fixed using a belt. The elbow extension strength test was conducted sitting with the elbow flexed to ∼100 degrees. The dynamometer was placed posteriorly of the distal forearm, proximal to the wrist. Three maximal measures were taken with each hand, with a 30 second break between measures. MicroFET2 (Utah, USA)  Shoulder maximal abduction muscle strength, kg. The shoulder abduction strength test was conducted in a sitting position, with the shoulder abducted to ∼90 degrees, and the elbow extended. The dynamometer was fixed using a belt and placed at the distal end of the humerus. Three maximal measures were taken with each hand, with a 30 second break between measures. MicroFET2 (Utah, USA)  MVIC, Nm Participants were seated with knees and hips in a 90-degree angle with the strain gauge applied just above the malleolus. The segmental distance was measured from the fulcrum of the knee to the middle of the strain gauge to calculate the maximal torque. Four maximal measures were taken for the left leg, with a 30 second break between measures. [19] Strain gauge (SDU, Odense, Denmark)  Leg press 5RM strength, kg. Five repetitions maximum strength test in leg press. Participants were instructed to place their feet symmetrically at the footplate, with hip-width apart, and the seat was adjusted forward until ‘os coccyx’ started lifting from the seat. The participants were instructed to apply pressure equally through the entire soles of both feet. All settings were noted down for everyone. [19] Technogym training equipment (Technogym, Cesena, Italy)  Knee extension 5RM strength, kg. Five repetitions maximum strength test in knee extension. The seat and backrest were adjusted, so the tights were supported without restricting knee movement. The ankle pad was adjusted to fit just above the ankles. Participants were instructed to fully extend their knees and return to the starting position in a controlled manner. [19] Technogym training equipment (Technogym, Cesena, Italy)  Calf extension 5RM strength, kg. Five repetitions maximum strength tests in calf extension bilaterally in kilograms. Participants were instructed to place their feet symmetrically, pointing directly forward on the bottom of the footplate, with shoulders width apart and straighten their legs to get in the starting position. The participants plantar flexed art. talocruralis maximally from the starting position while keeping their knees fixed. All settings were noted down for everyone. [19] Technogym training equipment (Technogym, Cesena, Italy)  Tandem test, seconds Static balance in three positions for 10 seconds each (feet together, semi tandem, and full tandem). A point is given for each second stance. The total possible score ranges between 0–30 points. [20] Stopwatch  Two-minute walk test, meters Participants were instructed to walk as fast and long as they could during the test. The participant was informed when one minute was remaining of the test. Otherwise, no encouragement was given. Heart rate (in Beats Per Minute) and perceived exertion (The Borg Rating of Perceived Exertion 6–20 scale) were also collected pre- and post-testing. [21] Course 15.2 meters, Stopwatch, Apple Watch series 5, The Borg Rating of Perceived Exertion 6–20 scale TUG, seconds Time was noted in seconds on how long it took to rise from a chair, walk three meters, turn around and go back to the chair and sit down again. The time was stopped as soon as the participant touched the chair with their gluteal area. The test was conducted three times, with 60 seconds rest between attempts. [22] Course three meters, Chair (46 centimetres in height) with armrests, Stopwatch SMFA: Short Musculoskeletal Function Assessment, SF-36: 36-Item Short Form Survey, PRISMA-7: Program of Research on Integration of Services for the Maintenance of Autonomy, NMS: The New Mobility Score, TFI: Tilburg Frailty Indicator, MVIC: Maximal isometric strength in knee extension, TUG: Timed Up and Go test, CPR: Civil registration number. The SMFA is an abbreviated version of the more comprehensive 101-item Musculoskeletal Function Assessment questionnaire (MFA) and has been developed to render its usage in clinical practice [23]. The SMFA is a 46-item self-reported generic questionnaire and takes approximately 10–20 min to complete. The SMFA consists of two indices:1) dysfunction capturing the impact of musculoskeletal disorders on physical limitations (SMFA-Dysfunction, 34-items) and 2) bother capturing how the individual is emotionally affected by their disorder (SMFA-Bother, 12-items). Items related to the SMFA-B index aim to clarify individuals’ approaches to dysfunction in recreation, housework, and leisure. The SMFA is scored using a 5-point Likert scale (from ‘Unable to do’ to ‘Not at all difficult’ and from ‘Extremely bothered’ to ‘Not at all bothered’), which can be transformed and summarized on a 0–100 score, where higher values indicate higher degrees of dysfunction or bother [23]. The items of the SMFA can be categorized into four categories: ‘mobility’, ‘daily activities’, ‘emotional status’ and ‘function of the arm and hand’. Clinical outcome measures Before initiating the assessment of clinical outcome measures, the participants were asked to hand in signed written consent forms. The clinical test session was booked to 3–7 days after the screening interview. Clinical testing took 90–100 min to complete and was conducted by the principal investigator alone or with a physical therapy student. The equipment was calibrated each morning before initiating the testing procedure. On arrival, all participants were asked not to drink for one hour before the test and were encouraged to empty their bowels. The warm-up program preceded testing. The clinical outcome measures included maximal bilateral handgrip strength, elbow flexion strength, elbow extension strength, and shoulder abduction strength measured using a dynamometer fixed with a belt. Lower extremity strength was assessed as the maximal voluntary isometric contraction (MVIC) of knee extension in the left leg measured using a strain gauge device. Bilateral leg press, knee extension, and calf extension were assessed as five repetition maxima. Balance and functional capacity were assessed using the tandem balance test, two-minute walk test, and timed up and go test (TUG). All outcomes are presented in Table 1. The clinical outcome measures were conducted as described in our study protocol for a randomized controlled trial investigating whether individualized physical exercise programs combined with increased protein intake can improve the measure of all ICF levels when compared with usual care alone or care as usual in combination with increased protein intake [24]. Hypotheses testing for construct validity One method for investigating the construct validity of a measurement instrument is hypothesis testing [25,26]. We hypothesized a strong correlation between the SMFA-Dysfunction index and the SMFA-Bother index, as has previously been shown in other heterogeneous populations [5]. Moreover, we hypothesized that higher scores on the SMFA-Dysfunction and SMFA-Bother would reflect higher scores on the PRISMA7 and TFI and lower scores on the SF-36 and NMS questionnaires, thereby reflecting the ability of the SMFA to measure health status. We expect SMFA-Dysfunction to show stronger correlations with patient-reported outcomes than SMFA-Bother [4]. Additionally, we hypothesized that SMFA-Dysfunction and SMFA-Bother scores are only fairly to moderately correlated with clinical outcome measures. Hence, higher SMFA scores are expected to correlate with lower strength measures, and poor balance and mobility. However, we expected that higher scores on the SMFA would reflect a longer time to complete the TUG test. As the SMFA-Dysfunction and SMFA-Bother are sum scores comprising items covering several body areas, the assessment of a specific function (e.g. strength, balance) is not necessarily correlated. Therefore, we hypothesized that specific clinical outcome measures will not be as strongly correlated to SMFA-Dysfunction and SMFA-Bother as they will be in a specific SMFA category [4,5]. Finally, we expect the SMFA-Dysfunction to correlate more strongly with the clinical outcome measures than the SMFA-Bother. Sample size calculation The sample size calculation was performed using Stata (v. 17) and is based on correlations (Spearman’s rank order correlation coefficient) between SMFA-Dysfunction and subscales from the SF-36 [27]. To detect a correlation coefficient of minimum r = ±0.3 with a significance level of 0.05, and a power of 0.8, between the SMFA-Dysfunction and the generic health-related quality of life questionnaire SF-36, a minimum of 85 participants were needed based on the Fisher’s z test. Some studies have shown a response rate for the SMFA to be approximately 65% for the studies [28]. Consequently, 35% extra participants were added to the calculated sample size, resulting in a target of 115 participants initially included in the study. Statistical analyses Scoring instructions for the SMFA were followed, and missing responses were handled as suggested in the original scoring manual [23]. We anticipated that some participants would not complete all clinical tests due to discomfort, pain, or fear of injury. In these circumstances, participants were excluded from the analysis, as no data were available. All statistical analyses were performed using Stata BE (ver. 17), and the do files were stored in the dataset. Initially, the raw data were inspected for Gaussian distribution and were considered not to be normally distributed. Summary statistics were presented as medians with interquartile ranges (IQR). Categorical data are presented as counts and percentages. Cronbach’s α was calculated to investigate the internal consistency of the specific categories of the SMFA (daily activities, emotional status, arm and hand function, mobility). Internal consistency was considered acceptable, with Cronbach’s α above 0.70, as previously stated in our statistical analyses plan published at clinicaltrials.gov (NCT04862481). Construct validity was investigated using Spearman’s rank-order correlation coefficients. The rho values were interpreted as r = ±0.1 to 0.25 weak, r> ±0.25 to 0.50 a fair, r> ±0.50 to 0.75 moderate, and above r> ±0.75 strong [29]. Results Participants The study included 115 participants with a median age of 74 years (IQR: 9), and 61% were females, Figure 1. In comparison, the excluded older adults had a median age of 76 years (IQR: 10), and 58% were females. Approximately 20% of the participants had a low BMI, 59% had a normal BMI, and 21% had a BMI > 30.9. Regarding physical activity, 29.5% of the participants reported being sedentary, and 50% reported that their physical activity level could be categorized as ‘standing or walking without physical exertion’. The prevalence of musculoskeletal pain or discomfort was highest in the lower extremities (35.7%), followed by the upper extremities (31.3%), spinal region (25.2%), and other body areas (7.8%). Approximately half of the participants (50.9%) reported living alone. More than half (62%) of the participants had a history of falls in the previous year. The median scores (IQR) for the patient-reported outcomes and clinical measurements are shown in Table 2. Figure 1. Flow chart.*Other indicates severe and complex health issues that excluded the older adults from participating in most of the selected clinical outcome measures (<50% of the clinical outcome measures). Table 2. Scores from patient-reported outcome and clinical outcome measures of the participants. Total, n = 115 Median [IQR] SMFA    SMFA-D 24 [25]  SMFA-B 37 [33] SF-36    Physical Functioning 55 [44]  Role Physical 0 [50]  Bodily Pain 41 [40]  General Health 67 [37]  Vitality 55 [35]  Social Functioning 100 [37.5]  Role Emotional 67 [100]  Mental Health 80 [32]  Physical component summary 34 [15]  Mental component summary 54 [16] PRISMA-7 2 [2] NMS 9 [2] TFI    Total score 4 [5]  Physical score 2 [3]  Psychological score 1 [2]  Social score 1 [2] Handgrip strength (kg) 24.5 [17] Elbow extension strength (kg) 9.9 [5.9] Elbow flexion strength (kg) 12.7 [8] Shoulder abduction strength, kg. 9.7 [5.7] MVIC knee extension, N 216 [126] Leg press, kg. 98 [50] Knee extension, kg. 33 [17] Leg extension, kg. 81 [60] Tandem balance test, seconds 30 [6] Two-minute walk test, meters 130 [58] TUG, seconds 8.6 [3.6] SMFA-D: Short Musculoskeletal Function Assessment – Dysfunction index, SMFA-B: Short Musculoskeletal Function Assessment – Bother index, SF-36: 36-Item Short Form Survey, PRISMA-7: Program of Research on Integration of Services for the Maintenance of Autonomy, NMS: The New Mobility Score, TFI: Tilburg Frailty Indicator, MVIC: Maximal Voluntary Isometric Contraction, TUG: Timed Up and Go test. Internal consistency No flooring or ceiling effects were observed in the scores for SMFA-D and SMFA-B [30]. Adequate internal consistency for the SMFA was found with Cronbach’s alpha values of 0.94 for the SMFA-Dysfunction and 0.90 for the SMFA-Bother. Furthermore, Cronbach’s alpha values were calculated for the SMFA categories. The values are 0.91 for the Daily Activities Category, 0.89 for the Mobility Category, 0.77, and 0.90, respectively. This indicates a close relationship between the set of items for specific indices and categories of SMFA in older adults. Construct validity Correlation with PRO measures Our a priori hypothesis that SMFA-Dysfunction and SMFA-Bother are strongly correlated was confirmed (r = 0.87, p ≤ 0.001). As expected, the SMFA indices and SMFA categories were negatively correlated with SF36 and NMS, and positively correlated with PRISMA-7 and TFI (TFI total, physical, and psychological scores) (Table 3). Consequently, if participants reported high levels of dysfunction, they similarly reported lower levels of health and mobility, and higher levels of lost autonomy and frailty. As hypothesized, the correlations between SMFA indices and PRO were generally indicative of moderate construct validity (r > 0.50–0.75, p ≤ 0.001), (Table 3). We hypothesized that SMFA-Dysfunction would show stronger correlations with the listed PROs than SMFA-Bother; however, this was not the case. Both SMFA indices were correlated to a similar extent with the listed PROs (Table 3). The SMFA-Bother was poorly correlated with the SF36 General Health dimension (r = −0.49, p ≤ 0.001), which was contrary to the hypothesis. The TFI social score was not correlated with any SMFA index or category (Table 3). Table 3. Construct validity: Correlation table for Short musculoskeletal function Assessment questionnaire, indices/categories, and other patient-reported outcome measures.   SMFA indices SMFA categories Total, n = 112 Dysfunction Index Bother Index Daily activities Mobility Emotional status Arm and hand function SF-36              Physical Functioning −0.74 −0.69 −0.67 −0.79 −0.62 −0.36  Role Physical −0.58 −0.64 −0.64 −0.34 −0.52 −0.32  Bodily Pain −0.63 −0.70 −0.61 −0.47 −0.54 −0.34  General Health −0.55 −0.49 −0.48 −0.56 −0.46 −0.33  Vitality −0.62 −0.62 −0.56 −0.53 −0.58 −0.35  Social Functioning −0.64 −0.62 −0.61 −0.44 −0.62 −0.35  Role Emotional −0.40 −0.45 −0.37 −0.24 −0.44 −0.27  Mental Health −0.58 −0.59 −0.51 −0.44 −0.56 −0.42  Physical component summary −0.74 −0.72 −0.72 −0.70 −0.59 −0.35  Mental component summary −0.45 −0.50 −0.40 −0.26 −0.50 −0.32 PRISMA-7 0.53 0.59 0.49 0.50 0.53 0.30 NMS −0.63 −0.60 −0.62 −0.56 −0.47 −0.44 TFI              Total score 0.60 0.56 0.55 0.51 0.51 0.43  Physical score 0.60 0.56 0.58 0.56 0.49 0.38  Psychological score 0.56 0.52 0.49 0.37 0.56 0.44  Social score 0.10 0.10 0.07 0.12 0.01 0.15 SMFA: Short Musculoskeletal Function Assessment, SF-36: 36-Item Short Form Survey, PRISMA-7: Program of Research on Integration of Services for the Maintenance of Autonomy, NMS: The New Mobility Score, TFI: Tilburg Frailty Indicator. The presented coefficients are calculated as Spearman’s Rank-order correlation coefficients. All presented coefficients are statistically significant at the level of p ≤ 0.05. Correlation with clinical outcome measures As expected, the SMFA indices and categories correlated negatively with all clinical outcome measures except for the TUG test. Higher SMFA-Dysfunction scores indicated a higher degree of dysfunction and were correlated with lower levels of muscle strength and functional capacity. Overall, the correlations were fair (r > 0.25–0.50, p ≤ 0.001), as hypothesized (Table 4). In addition, stronger correlations were found between SMFA-Dysfunction and clinical outcome measures than between SMFA-Bother and clinical measures. In addition, as hypothesized, clinical outcome measures assessing upper extremity strength showed higher correlations with the SMFA category arm and hand functions than the SMFA indices. Further, the SMFA category Mobility correlated more strongly with the two-minute walk test and TUG compared to the SMFA indices, as hypothesized (Table 4). Table 4. Correlation table for Short musculoskeletal function Assessment questionnaire, indices/categories, and clinical outcome measures.   SMFA indices SMFA categories Total, n = 115 Dysfunction Index Bother Index Daily activities Mobility Emotional status Arm and hand function Handgrip strength, n = 110 −0.45 −0.34 −0.40 −0.26 −0.28 −0.62 Elbow extension strength, n = 101 −0.52 −0.35 −0.44 −0.39 −0.38 −0.61 Elbow flexion strength, n = 102 −0.52 −0.37 −0.48 −0.43 −0.34 −0.63 Shoulder abduction strength, n = 105 −0.50 −0.35 −0.42 −0.50 −0.34 −0.56 MVIC knee extension, n = 110 −0.40 −0.32 −0.39 −0.40 −0.20 −0.38 Leg press strength, n = 75 −0.31 −0.29 −0.39 −0.49 −0.09 −0.07 Knee extension strength, n = 96 −0.35 −0.29 −0.42 −0.43 −0.10 −0.15 Calf extension strength, n = 61 −0.30 −0.28 −0.37 −0.31 −0.11 −0.19 Tandem balance test, n = 115 −0.41 −0.29 −0.36 −0.45 −0.32 −0.27 Two-minute walk test, n = 115 −0.45 −0.38 −0.38 −0.57 −0.37 −0.28 TUG, n = 115 0.44 0.34 0.33 0.59 0.39 0.31 MVIC: Maximal Voluntary Isometric Contraction, TUG: Timed Up and Go test. The presented coefficients are calculated as Spearman’s Rank-order correlation coefficients. All presented coefficients are statistically significant at the level of p ≤ 0.05. Discussion This study investigated the construct validity of the SMFA using a cross-sectional design, including a population of 115 older adults referred for outpatient-based rehabilitation. As hypothesized, SMFA indices showed moderate correlations with PROs assessing health status in older adults. The results demonstrate that the SMFA indices cover many of the same constructs (health status ‘SF-36’, frailty ‘PRISMA-7/TFI’, mobility ‘NMS’) as the presented PROs in older adults commencing physical rehabilitation in an outpatient setting. Consequently, the SMFA indices have adequate construct validity for assessing self-reported health status in older adults, especially when considering components covering physical health status. These results underline the value of using clinical outcome measures in rehabilitation, as SMFA does not capture this and consequently is not closely related to the degree of dysfunction. SMFA indices cover mental and social features to a lesser degree than the components linked to physical ability (Table 3). Our findings support the previous results of SMFA conducted in other populations. This shows that the SMFA can function as a measure of older adults’ health status across different musculoskeletal disorders. In the first validity study of the SMFA questionnaire, Swiontkowski et al. demonstrated sufficient construct validity by comparing scores of the SMFA indices to SF-36 dimensions [5]. Almost equivalent measurement properties were obtained in the present study even though Swiontkowski et al. included younger participants who had sustained musculoskeletal disease or injury. Other studies have demonstrated good reliability, validity, and responsiveness in different populations suffering from musculoskeletal disorders and injuries [4, 6, 31–40]. Concerning the Danish translation, an investigation of the content validity for the SMFA showed that the SMFA covers all the ICF constructs [37]. Similarly, it was recently reported that the SMFA could cover all constructs and contextual factors of the ICF and capture the injuries’ consequences for everyday life in 160 middle-aged fracture patients [41]. Thus, despite the different target groups, the SMFA has the same ability to cover similar constructs to those of the SF-36. However, interestingly, the severity of an injury can perhaps impact the construct validity of the SMFA when compared to SF-36, as some studies have demonstrated significantly lower correlations in participants with severe or acute injuries, although the one-week recall version of SF-36 was used [37]. The lower correlations might partly be explained by the fact that SF-36 Physical functioning mainly includes items related to the function of the lower extremities. In contrast, the SMFA has more detailed upper and lower body items, which is a noteworthy advantage compared to the SF-36 [32]. Nevertheless, this partly explains why no correlation between the TFI and the SMFA indices and categories can be found. However, in a study by Gobbens et al. the authors correlated TFI subscales with adverse outcomes of frailty. They found significant but low correlations between the TFI social frailty subscale and disability measured by the Groningen Activity Restriction Scale (r = 0.257, p < 0.001) in comparison to the TFI physical frailty (r = 0.488, p < 0.001) and TFI psychological frailty (r = 0.361, p < 0.001) [42]. In the present study, the social component of the SF-36 showed moderate correlations with the SMFA indices, which is also supported by other studies, even though the participants in the present study also demonstrated very good scores on the SF-36 social functioning dimension (Table 3) [5, 35, 38]. The explanation might be that the SF-36 is more sensitive for measuring social well-being, making it easier to spot minor differences between individuals than the scale used in TFI. The correlations between the SMFA indices and clinical outcome measures were mostly fair, which was expected because the instruments measured different constructs. We expected stronger correlations between some SMFA categories and clinical outcome measures in the same body area. The SMFA arm and hand function category had the highest correlation with clinical measurements assessing upper extremity strength (handgrip, elbow, and shoulder strength). Swiontkowski et al. compared handgrip strength and range of motion in the upper and lower extremity joints to the SMFA-D, but not the subcategories, and found similar results to those of the present study when comparing SMFA-D and handgrip strength (r = 0.45, p < 0.05). Conversely, correlations between the SMFA mobility category and measures assessing lower extremity function (leg press strength, knee extension strength, calf extension strength, tandem balance test, two-minute walk test, and TUG) were mainly fair. Remarkably, shoulder abduction strength correlated more strongly with the SMFA mobility category than with the MVIC of knee extension. One explanation could be that more than half the participants had a history of falls within the last year, hurting their shoulders, which could have influenced their ability to use them during activities. This could have impaired shoulder function and strength over time. Suppose that impaired function or strength is accompanied by fear of falling. In that case, it can lead to further declines in physical functioning, influencing their ability to perform the activities listed in the mobility category of the SMFA [43]. Kirschner et al. found a stronger correlation between SMFA-D and walking speed than that between the two-minute walk distance and SMFA-D [35]. The difference could be due to different methods of assessing walking. Kirschner et al. used a course of 25 meters to determine gait speed, as in the study by Swiontkowski et al. [35] Strengths and limitations One limitation of this study is the significant number of participants who scored well on the PRISMA-7, NMS, and TFI questionnaires, indicating a lower degree of frailty or immobility. This study was conducted during the COVID-19 pandemic when most older adults did not attend habitual activities. This could have influenced their understanding of the context, looking at the items addressed in the questionnaires, thereby scoring themselves better than they otherwise would have. Another explanation might be that the frailest older adults stayed at home during the pandemic, so we included more functionally fit older adults. Nevertheless, these instruments were considered reasonable for use based on observations of older adults and discussions with health professionals from a specific clinical setting before the COVID-19 pandemic. Another significant limitation of this study is that we only measured reliability as internal consistency, and not as repeated measures. Additionally, in this cross-sectional study, we could not investigate the responsiveness of SMFA, which would have been helpful. We utilized the COSMIN taxonomy and assessed construct validity through hypothesis testing. We chose to present correlations between the SMFA and PROs and clinical outcome measures to cover aspects of construct validity. We acknowledge differences in the research literature regarding validity terminology, as acknowledged by the COSMIN study [44]. However, the strength of this study was the investigation of the association between the SMFA indices, categories, and clinical outcome measures often used in clinical practice. These results provide insight into the relationship between clinical measures, often targeted as primary goals, and patients’ reflection of their level of dysfunction and bother. Overall, the SMFA is a valuable instrument for health professionals to use with older adults in an outpatient rehabilitation setting alongside clinical measurements to measure health status. Still, future research should examine the responsiveness of SMFA in a population of older adults. Acknowledgements The authors thank all funding parties for financially supporting this trial. The authors also thank all the older adults who commenced this study and the Slagelse Municipality for allowing us to conduct the study in a real clinical setting. Author contributors All authors have formed a project description (November 2020). ST wrote the first draft and is the guarantor of the manuscript, and ML, CMM, GS, KS, and LFS elaborated on the final paper. All authors contributed to and approved the final version. None of the sponsors was involved in the trial design, data collection, management, analysis, or interpretation of the data. Disclosure statement No potential conflict of interest was reported by the author(s). Data availability statement Research data supporting this research will be uploaded to the Danish State Archives after the end of the project (https://www.sa.dk/en/research-researchers-research-service-the-danish-national-archives/). ==== Refs References 1 Dillin A, Gottschling DE, Nystrom T. 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==== Front Ann Med Ann Med Annals of Medicine 0785-3890 1365-2060 Taylor & Francis 37455447 10.1080/07853890.2023.2230871 2230871 Version of Record Research Article Surgery Risk factors for fractures following liver transplantation: a population-based cohort study J.-W. Chang et al. Risk Factors for Fractures Following Liver Transplantation https://orcid.org/0000-0002-9462-883X Chang Jei-Wen abc Yang Hui-Hsin cd Lin Niang-Cheng ce Kuo Fang-Cheng cd Lin Tzu-Ching ac https://orcid.org/0000-0002-8147-2629 Tsai Hsin-Lin cde a Department of Pediatrics, Taipei Veterans General Hospital, Taipei, Taiwan b Institute of Emergency and Critical Care Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan c Faculty of Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan d Division of Pediatric Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan e Division of Transplantation Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan CONTACT Hsin-Lin Tsai hltsai@vghtpe.gov.tw Division of Pediatric Surgery, Department of Surgery, Taipei Veterans General Hospital, No. 201, Sec. 2, Shipai Rd., Beitou District, Taipei City, Taiwan; Division of Transplantation Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan; Faculty of Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan 16 7 2023 2023 16 7 2023 55 1 223087116 2 2023 17 6 2023 25 6 2023 KnowledgeWorks Global Ltd.15 7 2023 published online in a building issue15 7 2023 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 2023 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. Abstract Background Liver transplant recipients have an increased risk of osteoporosis and fractures. The aim of this study was to identify risk factors for fractures after liver transplant in a Taiwanese population. Methods We identified newly diagnosed liver transplant recipients from the National Health Insurance Research Database in Taiwan between 2003 and 2015. Risk factors of post-transplant fractures were analyzed using a Cox proportional hazards model. Results A total of 4821 patients underwent liver transplantation, of whom 419 (8.7%) had post-transplant fractures. Independent predictors of post-transplant fractures were age ≥65 years at transplantation (hazard ratio (HR): 1.566; 95% confidence interval (CI) 1.122–2.186), female sex (HR: 1.648; 95% CI 1.319–2.057), fractures within 1 year prior to transplant (HR: 3.664; 95% CI 2.503–5.364), hepatitis C carriers (HR: 1.594; 95% CI 1.289–1.970), alcoholism (HR: 1.557; 95% CI 1.087–2.230) and daily prednisolone dose >1.61–3.78 mg/day (HR: 1.354; 95% CI 1.005–1.824), >3.78–9.18 mg (HR: 4.182; 95% CI 3.155–5.544) and >9.18 mg (HR: 13.334; 95% CI 9.506–18.703). Post-transplant fractures were inversely correlated with tacrolimus (HR: 0.617; 95% CI 0.417–0.913) and sirolimus/everolimus (HR: 0.504; 95% CI 0.391–0.650) treatment. Conclusions The liver transplant recipients, and especially those who were aged ≥65 years, female, hepatitis C carriers, had a history of fractures within 1 year prior to transplant, alcoholism, and higher daily prednisolone dose were associated with an increased risk of post-transplant fractures. Conversely, the use of tacrolimus and sirolimus/everolimus was associated with a decreased risk of fractures. Key Messages This study identified risk factors for fractures after liver transplant in a population-based study in an area with high prevalence of hepatitis B and hepatitis C. Recipients who were aged ≥65 years, female, hepatitis C carriers, had a history of fractures within 1 year prior to transplant, alcoholism, and higher daily prednisolone dose were independent risk factors for post-transplant fractures. Our findings highlight the importance of identifying individuals at high risk of fractures and concomitant tacrolimus and sirolimus/everolimus treatment to avoid the use of high-dose steroids and prevent post-transplant fractures. Keywords Fracture glucocorticoid hepatitis B hepatitis C immunosuppressant liver transplantation This study was supported by a grant from Taipei Veterans General Hospital (V109C-164). ==== Body pmcIntroduction With advances in surgical techniques and immunosuppressant agents, graft and patient survival after liver transplantation have improved dramatically in the past decades. One of the major challenges facing the field of liver transplantation today is the increasing incidence of bone diseases including osteoporosis and fractures which cause significant long-term morbidity [1,2]. Thus, understanding the general, pre- and post-transplant specific risk factors associated with post-transplant fractures may help with an early diagnosis and prompt interventions for optimal patient outcomes. Patients with chronic liver diseases such as non-alcoholic chronic fatty liver disease and those with cholestatic disease are at an increased risk of osteoporosis [3,4]. In addition, decompensated cirrhosis with excess unconjugated bilirubin may interfere with the differentiation of primary osteoblasts [5]. Other known risk factors for osteoporosis include vitamin D and vitamin K deficiency, malnutrition, decreased body weight and hypomagnesemia, which are extremely common among patients evaluated for liver transplantation [6–8]. In general, more severe liver disease and cholestasis are associated with more severe bone loss. Therefore, many patients awaiting liver transplantation already have osteopenia or osteoporosis. Post-liver transplantation factors known to contribute to osteoporosis and fractures include high-dose glucocorticoids, other immunosuppressive agents, particularly cyclosporine and tacrolimus, secondary hyperparathyroidism and hypogonadism [9]. In addition, estrogen deficiency during menopause has also been shown to increase the risk of bone loss. Osteoporosis is common in patients with primary biliary cholangitis, partly because of the high prevalence in perimenopausal women. Post-liver transplant bone loss is most rapid in the first three to six months. The rate of bone loss then slows as the dose of glucocorticoids is reduced and pre-transplant conditions are resolved. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the major causes of cirrhosis and hepatocellular carcinoma leading to the need for liver transplantation. Prior to the introduction of the national HBV vaccination program in Taiwan in 1984, the prevalence of chronic HBV infection in the general population was approximately 15%–20% [10]. The prevalence of HCV infection in Taiwan is estimated to be about 3.3% (1.8–5.5%), which is much higher than the worldwide average [11]. Apart from liver diseases, both HBV and HCV infections have been shown to increase the risk of osteoporosis in non-cirrhotic patients [12,13]. However, few studies have investigated the frequency of fractures among liver transplant recipients in a population-based study in an area with high HBV and HCV carrier rates, such as Taiwan. Furthermore, these studies had relatively small sample sizes, were not population-based, and were conducted in Western countries. There may be differences in the epidemiology and risk factors for osteoporosis and fractures between different ethnicities, races and prevalence of HBV and HCV infection. Therefore, we conducted this large population-based cohort study to identify the frequency of fractures in Taiwanese liver transplant recipients. In addition, we also investigated the general and transplant recipient-specific risk factors for fractures using data from the National Health Insurance Research Database (NHIRD) in Taiwan. Methods Data source Data were obtained from the NHIRD of the Ministry of Health and Welfare of Taiwan. Since November 2015, researchers are asked to perform on-site data analysis of the NHIRD at a Health and Welfare Data Centre. The National Health Insurance (NHI) program in Taiwan is a compulsory single-payer program launched in March 1995 and currently covers approximately 99% of the population in Taiwan. The NHIRD contains complete information on medical service utilization, including demographic information, ambulatory care claims, inpatient claims, procedures, prescription details and diagnoses based on International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes. High accuracy of the diagnosis codes for common conditions and severe diseases in the NHIRD has been demonstrated [14,15]. Under the NHI program, patients with liver transplantation can apply for a catastrophic illness certificate. Catastrophic illness certificates are reviewed by relevant experts after thorough verification of medical records, as well as relevant documents of the liver transplant procedure. As such, the Catastrophic Illness Patient Database of liver transplantation is highly accurate. Furthermore, high accuracy of the NHIRD in recording liver transplantation has been demonstrated [16]. Patient identification The transplant recipients included in this study were patients aged >18 years who underwent a liver transplantation (ICD-9-CM codes V42.7, 996.82) between 1 January 2003 and 31 September 2015, identified through catastrophic illness certification. For domestic transplantation recipients, the index date was defined as the admission date with Taiwan NHI reimbursement order codes 75020B and 75020 A (liver implantation). The NHIRD did not contain official records of the transplantation procedure for overseas transplant recipients. Therefore, the index date for overseas transplant recipients was defined as 1 month before the patient applied for a catastrophic illness certificate, because the length of overseas post-transplantation stay has been estimated to be approximately 1 month. All of the enrolled index cases were followed from the index date until a diagnosis of fractures, death, the end of follow-up in the medical records, or 31 December 2015. The demographic data of the patients including age at transplantation, sex and age at diagnosis of a fracture were extracted. The patients were classified into two age subgroups: 18–64 and ≥65 years. This study was approved by the Institutional Review Board of Taipei Veterans General Hospital (2019-08-004BC). Then, we requested access to the Taiwan’s NHIRD through a formal application and approved by the Health and Welfare Data Science Centre of the Ministry of Health and Welfare, Taiwan. Outcomes and types of fracture Post-liver transplantation fractures were identified using ICD-9-CM codes 805–829 during the follow-up period. The fracture sites were identified from medical service claims based on the ICD-9-CM diagnosis. Fractures were grouped into seven categories: (1) vertebra (ICD-9-CM 805.x-806.x); (2) femur (ICD-9 820.x, 821.x); (3) upper extremity (humerus, radius and ulna) (ICD-9-CM 812.x, 813.x, 818.x); (4) wrist and hand (ICD-9-CM 814.x, 815.x, 816.x, 817.x); (5) rib/clavicle/scapula/sternum (ICD-9-CM 807.x, 810.x, 811.x); (6) lower extremity (patella, tibia, fibula, ankle and foot (ICD-9-CM 822.x, 823.x, 824.x, 825.x, 826.x, 827.x); (7) pelvis (ICD-9-CM 808.x) and others (ICD-9-CM, 809.x, 819.x, 828.x, 829.x). Skull and facial fractures were excluded from the analysis. Chronic liver or biliary disease prior to transplantation Acute and subacute necrosis of the liver (ICD-9-CM 570), alcoholic liver disease (alcoholic cirrhosis [ICD-9-CM 571.0, 571.1, 571.2, and 571.3]), primary biliary cholangitis (ICD-9-CM 571.6), hepatitis B (ICD-9-CM 070.2, 070.3, and V02.61), hepatitis C (ICD-9-CM 070.41, 070.44, 070.51, 070.54, V02.62, and 070.7), Wilson’s disease (ICD-9-CM 275.1) and biliary atresia (ICD-9-CM 751.61) were identified. The diagnosis of chronic non-alcoholic fatty liver disease was based on ICD-9-CM codes 571.8 and 571.9 and no coexisting causes of alcohol-related liver disease, hepatic steatosis and chronic liver diseases mentioned above [17]. We also recorded the claims-based diagnosis of liver malignancy (ICD-9-CM 155.0, 155.1, 155.2). Study variables We assessed risk factors known to increase the risk of fractures in the general population, including female sex, osteoporosis (ICD-9-CM 733.0x) or prior history of fractures, rheumatoid arthritis (ICD-9-CM 714.0) and diabetes mellitus (ICD-9-CM 250). We also analyzed comorbidities that could be associated with fractures including hyperthyroidism (ICD-9-CM 242), hypertension (ICD-9-CM 401-405), systemic lupus erythematosus (ICD-9-CM 710.0), hyperlipidaemia (ICD-9-CM 272.0-272.4), coronary artery disease (ICD-9-CM 410-414), cerebrovascular disease (ICD-9-CM 430–438), chronic obstructive pulmonary disease (ICD-9-CM 491-493, 496), malnutrition (ICD-9-CM 262, 263), testicular hypogonadism in men (ICD-9-CM 257.2 and 257.9), menopausal and postmenopausal disorders in women (ICD-9-CM 627), and alcoholism (ICD-9-CM 303.x, V11.3, 305.0, 291.x, 357.5, 425.5, 535.3, 571.0, 571.2, 571.3). The diagnoses of comorbidities were considered valid based on two outpatient claims or one inpatient claim 1 year before or at the time of transplantation. Glucocorticoids, immunosuppressive drugs and bone protective therapy The dosage of systemic glucocorticoids including that prescribed orally at outpatient visits and that taken orally or intravenously during inpatient treatment were calculated until the date of fracture or the study end date. The glucocorticoids included betamethasone (Anatomical Therapeutic Chemical [ATC] code H02AB01), dexamethasone (ATC code H02AB02), methylprednisolone (ATC code H02AB04), paramethasone (ATC code H02AB05), prednisolone (ATC code H02AB06), triamcinolone (ATC code H02AB08), hydrocortisone (ATC code H02AB09), and cortisone (ATC code H02AB10). All glucocorticoid prescriptions were converted to prednisolone equivalents. The mean daily prednisolone dosage was classified into four groups by quartile: 0–1.61 mg/day, >1.61–3.78 mg/day, >3.78–9.18 mg/day, and >9.18 mg/day. Data on other immunosuppressive drugs used to prevent rejection including tacrolimus (ATC code L04AD02), cyclosporine (ATC code L04AD01), mycophenolate mofetil/mycophenolic acid (ATC code L04AA06) and sirolimus (ATC code L04AA10)/everolimus (ATC code L04AA18) that were prescribed within the follow-up period were extracted for analysis. Data on the drugs most often used in the treatment of osteoporosis including bisphosphonate (ATC codes M05BAxx and M05BBxx), calcium (ATC codes A12AAxx and V03AE07), vitamin D (ATC code A11CCxx) and calcium with vitamin D (ATC code A12AXxx) supplements were also recorded. Statistical analysis Categorical variables are presented as number and percentage, and continuous variables are presented as mean ± standard deviation. Cox proportional hazards regression analysis was performed to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) of risk factors for fractures. First, potential risk factors were screened using a univariable Cox proportional hazards model. Candidate variables with p < 0.05 in the univariable Cox proportional hazards analysis were then included in multivariable Cox proportional hazards analysis to identify independent risk factors for fractures among the liver transplant recipients. All statistical analyses were performed using SPSS version 21.0 (IBM Inc., Armonk, NY). P values of < 0.05 were considered statistically significant. Results Demographics and clinical characteristics of the study population A total of 4228 domestic and 593 overseas liver transplant recipients were identified in the NHIRD during the study period. The cohort included 3568 (73.8%) male and 1253 (26.0%) female patients. The mean age of the recipients was 52.7 ± 9.3 years. A total of 1194 patients died during the study period, and the mean time from transplantation to death was 1.3 ± 2.1 years. During the follow-up period, post-transplant clinical fractures were recorded in 419 (8.7%) patients during a mean follow-up period of 3.7 ± 3.1 years. The mean age at fracture was 56.4 ± 9.2 years, and the average time to fracture after transplantation was 3.0 ± 2.5 years. The highest rate of fractures was in the first 12 months after the transplant. Of the 419 patients with fractures, 101 (24.1%) had a first fracture in the first year, 80 (19.1%) at 1–2 years, 63 (15.0%) at 2–3 years, 49 (11.7%) at 3–4 years, and 45 (10.7%) at 4–5 years post transplant. In addition, 408 patients presented with a single fracture and 11 patients presented with two or three fractures. Among the total 431 fracture sites, fractures most commonly occurred in the upper extremities (humerus/radius/ulna) (n = 107, 24.8%), followed by the lower extremities (n = 91, 21.1%), vertebra (n = 73, 16.9%), clavicle/scapula/sternum/rib (n = 63, 14.6%), and femur (n = 58, 13.5%) (Table 1). Table 1. Classification of all first fracture sites post-transplant. Fracture site n (%) Vertebra 73 (16.9 %) Femur 58 (13.5%) Upper extremity (humerus, radius and ulna) 107 (24.8%) Wrist and hand 32 (7.4%) Clavicle/scapula/sternum/rib 63 (14.6%) Lower extremity (tibia, fibula, patella, ankle, foot) 91 (21.1%) Pelvis and others 7 (1.6%) The demographic characteristics and clinical information of the liver transplant recipients with or without fractures are summarized in Table 2. The recipients who developed post‐transplant fractures tended to be female (38.4% vs. 24.8%), older than 65 years (9.8% vs. 7.2%), and have a fracture 1 year prior to transplantation (7.9% vs. 1.9%). The rates of HBV and HCV infection were 64.6% and 32.9%, respectively. Overall, 2870 (59.5%) and 1671 (34.7%) recipients were diagnosed with preoperative liver malignancy and alcohol-related cirrhosis, respectively. The absolute case numbers of chronic non-alcoholic fatty liver disease among the recipients with fractures were not available, as data on any finding with fewer than three patients cannot be extracted to protect their identification. The rates of alcoholic cirrhosis (37.2% vs. 34.4%), primary biliary cholangitis (11.5% vs. 7.2%), and chronic HCV infection (40.8% vs. 32.2%) before transplantation were higher in the recipients with fractures. However, the recipients with fractures had a lower rate of HBV infection (57.5% vs. 65.3%). There were high prevalence rates of diabetes mellitus (n = 1241, 25.7%), alcoholism (n = 1102, 22.9%) and hypertension (n = 889, 18.4%). Moreover, the recipients with fractures had higher rates of osteoporosis (2.6% vs. 0.5%), hypertension (20.8% vs. 18.2%), diabetes mellitus (28.6% vs. 25.5%), systemic lupus erythematosus (1.0% vs. 0.3%), hyperlipidaemia (3.8% vs. 3.2%), coronary artery disease (4.8% vs. 3.1%), cerebrovascular disease (4.5% vs. 2.0%), chronic obstructive pulmonary disease (5.3% vs. 2.7%), hypogonadism and postmenopausal disorders (0.7% vs. 0.4%), and alcoholism (26.7% vs. 22.5%) than the recipients without fractures. Table 2. Comparison of demographics and comorbidities between the liver recipients with and without a post‐transplant fracture.   Total Fracture Without fracture n = 4821 n = 419 n = 4402 Female recipient 1253 (26.0%) 161 (38.4%) 1092 (24.8%) Age at transplant 52.7 ± 9.3 53.4 ± 9.2 52.6 ± 9.3 Age at transplant ≥ 65 years 356 (7.4%) 41 (9.8%) 315 (7.2%) Overseas transplantation 593 (12.3%) 51 (12.2%) 542 (12.3%) Fracture in 1 year prior to transplant 118 (2.4%) 33 (7.9%) 85 (1.9%) Chronic liver or biliary disease prior to transplantation        Acute and subacute necrosis of liver 555 (11.5%) 38 (9.1%) 517 (11.7%)  Alcohol-related cirrhosis 1671 (34.7%) 156 (37.2%) 1515 (34.4%)  Primary biliary cholangitis 365 (7.6%) 48 (11.5%) 317 (7.2%)  Chronic non-alcoholic fatty liver disease 29 (0.6%) NA NA  Hepatitis B 3115 (64.6%) 241(57.5%) 2874 (65.3%)  Hepatitis C 1588 (32.9%) 171 (40.8%) 1417 (32.2%)  Wilson’s disease 37 (0.8%) 3 (0.7%) 34 (0.8%)  Biliary atresia 25 (0.5%) 0 (0.0%) 25 (0.6%)  Malignancy 2870 (59.5%) 243 (58.0%) 2627 (59.7%) Comorbidity        Osteoporosis 34 (0.7%) 11 (2.6%) 23 (0.5%)  Hyperthyroidism 17 (0.4%) 0 (0.0%) 17 (0.4%)  Hypertension 889 (18.4%) 87 (20.8%) 802 (18.2%)  Diabetes mellitus 1241 (25.7%) 120 (28.6%) 1121 (25.5%)  Systemic lupus erythematous 19 (0.4%) 4 (1.0%) 15 (0.3%)  Hyperlipidemia 158 (3.3%) 16 (3.8%) 142 (3.2%)  Coronary artery disease 156 (3.2%) 20 (4.8%) 136 (3.1%)  Cerebrovascular disease 108 (2.2%) 19 (4.5%) 89 (2.0%)  Chronic obstructive pulmonary disease 139 (2.9%) 22 (5.3%) 117 (2.7%)  Malnutrition 8 (0.2%) 0 (0.0%) 8 (0.2%)  Rheumatoid arthritis 22 (0.5%) NA NA  Hypogonadism and postmenopausal disorder 20 (0.4%) 3 (0.7%) 17 (0.4%)  Alcoholism 1102 (22.9%) 112 (26.7%) 990 (22.5%) Mean dosage of glucocorticoids        0–1.61 mg/day 1205 (25.0%) 90 (21.5%) 1115 (25.3%)  >1.61-3.78 mg/day 1205 (25.0%) 90 (21.5%) 1115 (25.3%)  >3.78-9.18 mg/day 1206 (25.0%) 144 (34.4%) 1062 (24.1%)  >9.18 mg/day 1205 (25.0%) 95 (22.7%) 1110 (25.2%) Bone-protective medications        Bisphosphonate therapy 102 (2.1%) 13 (3.1%) 89 (2.0%)  Calcium 336 (7.0%) 35 (8.4%) 301 (6.8%)  Vitamin D 70 (1.5%) 5 (1.2%) 65 (1.5%)  Calcium and vitamin D 36 (0.7%) 5 (1.2%) 31 (0.7%) Immunosuppressive medications        Tacrolimus 4599 (95.4%) 391 (93.3%) 4208 (95.6%)  Cyclosporine 384 (8.0%) 52 (12.4%) 332 (7.5%)  Mycophenolate mofetil/mycophenolic acid 4064 (84.3%) 372 (88.8%) 3692 (83.9%)  Sirolimus/everolimus 1389 (28.8%) 74 (17.7%) 1315 (29.9%) NA: not available. Glucocorticoids, bone-protective medications and immunosuppressants In the patients with fractures, 21.5%, 21.5%, 34.4% and 22.7% were in the 1st (0-1.61 mg/day), 2nd (>1.61-3.78 mg/day), 3rd (>3.78-9.18 mg/day) and 4th (>9.18 mg/day) quartiles of average daily glucocorticoid dose, respectively, compared to 25.3%, 25.3%, 24.1% and 25.2% in the patients without fractures. Overall, 2.1% of the patients received bisphosphonate treatment after transplantation. Calcium and vitamin D supplements were taken by 7.0% and 1.5% of the patients post-transplant, respectively. The most commonly prescribed immunosuppressive drugs were tacrolimus and mycophenolate mofetil/mycophenolic acid. A higher proportion of the recipients with fractures received cyclosporine (12.4% vs. 7.5%) and mycophenolate mofetil/mycophenolic acid (88.8% vs. 83.9%) compared to those without fractures. Conversely, the prescription rates of tacrolimus (93.3% vs. 95.6%) and sirolimus/everolimus (17.7% vs. 29.9%) were lower in the recipients with factures than in those without fractures. Univariable and multivariable analyses of risk factors for the development of fractures Table 3 shows the results of the univariable and multivariable analyses for associations between post-transplant fractures and demographic and clinical data. The univariable analysis revealed that transplantation at ≥65 years of age, female sex, a history of fractures within 1 year prior to transplantation, alcohol-related cirrhosis, primary biliary cholangitis, HCV infection, osteoporosis, hypertension, diabetes mellitus, systemic lupus erythematous, cerebrovascular disease, alcoholism and mean daily glucocorticoid dose >1.61–3.78 mg, >3.78–9.18 mg and >9.18 mg prednisolone equivalent were associated with a higher risk of developing a fracture. However, the risk of fractures was significantly lower in the patients with HBV infection. Among the prescribed immunosuppressives, the use of tacrolimus and sirolimus/everolimus was negatively correlated with post-transplant fractures. Overseas transplant, acute and subacute necrosis of the liver, chronic non-alcoholic fatty liver disease, Wilson’s disease, malignancy, biliary atresia, hyperthyroidism, hyperlipidaemia, coronary artery disease, chronic obstructive pulmonary disease, malnutrition, rheumatoid arthritis, hypogonadism and postmenopausal disorder and the use of bisphosphonate, calcium, vitamin D supplements, cyclosporine and mycophenolate mofetil/mycophenolic acid were not correlated with post-transplant fractures. Table 3. Analysis of risk factors for post-transplant fractures in the liver transplant recipients.   Univariable Multivariable   HR (95% CI) p value HR (95% CI) p value Age at transplant ≥ 65 years 1.613 (1.168–2.227) *0.004 1.566 (1.122–2.186) *0.008 Female recipient 1.973 (1.620–2.403) *<0.001 1.648 (1.319–2.057) *<0.001 Overseas transplantation 0.773 (0.577–1.037) 0.086     Fracture in 1 year prior to transplant 5.284 (3.7000–7.547) *<0.001 3.664 (2.503–5.364) *<0.001 Chronic liver or biliary disease prior to transplantation          Acute and subacute necrosis of liver 0.750 (0.537–1.047) 0.091      Alcohol-related cirrhosis 1.259 (1.033–1.536) *0.023 0.954 (0.691–1.316) 0.773  Primary biliary cholangitis 1.381 (1.022–1.866) *0.035 1.200 (0.874–1.648) 0.259  Chronic non-alcoholic fatty liver disease 0.989 (0.246–3.968) 0.988      Hepatitis B 0.633 (0.521–0.769) *<0.001 1.076 (0.867–1.335) 0.505  Hepatitis C 1.634 (1.344–1.986) *<0.001 1.594 (1.289–1.970) * <0.001  Wilson’s disease 0.744 (0.239–2.316) 0.609      Biliary atresia 0.049 (0.000–17.340) 0.314      Malignancy 0.969 (0.798–1.177) 0.751     Comorbidity          Osteoporosis 3.049 (1.675–5.551) *<0.001 1.722 (0.909–3.261) 0.095  Hyperthyroidism 0.049 (0.000–62.887) 0.410      Hypertension 1.371 (1.082–1.736) *0.009 1.280 (0.998–1.642) 0.052  Diabetes mellitus 1.349 (1.091–1.667) *0.006 1.140 (0.911–1.426) 0.252  Systemic lupus erythematous 2.900 (1.083–7.765) *0.034 1.569 (0.576–4.276) 0.379  Hyperlipidemia 0.905 (0.549–1.492) 0.696      Coronary artery disease 1.538 (0.982–2.411) 0.060      Cerebrovascular disease 2.227 (1.405–3.529) *0.001 1.595 (0.989–2.571) 0.055  Chronic obstructive pulmonary disease 1.489 (0.969–2.289) 0.069      Malnutrition 0.050 (0.000–13389.353) 0.638      Rheumatoid arthritis 1.113 (0.277–4.466) 0.880      Hypogonadism and postmenopausal disorder 1.180 (0.379–3.676) 0.775      Alcoholism 1.387 (1.116–1.723) *0.003 1.557 (1.087–2.230) **0.016 Mean dosage of glucocorticoids          0–1.61 mg/day 1 1      >1.61–3.78 mg/day 1.476 (1.098–1.982) *0.010 1.354 (1.005–1.824) *0.047  >3.78–9.18 mg/day 4.506 (3.419–5.939) *<0.001 4.182 (3.155–5.544) *<0.001  >9.18 mg/day 13.938 (10.064–19.303) *<0.001 13.334 (9.506–18.703) *<0.001 Bone-protective medications          Bisphosphonate therapy 1.729 (0.995–3.005) 0.052      Calcium 0.852 (0.602–1.206) 0.366      Vitamin D 0.605 (0.250–1.461) 0.264      Calcium and vitamin D 1.094 (0.452–2.646) 0.842     Immunosuppressive medications          Tacrolimus 0.618 (0.420–0.908) *0.014 0.617 (0.417–0.913) *0.016  Cyclosporine 1.322 (0.988–1.770) 0.060      Mycophenolate mofetil/mycophenolic acid 0.987 (0.728–1.337) 0.931      Sirolimus/everolimus 0.592 (0.460–0.762) *<0.001 0.504 (0.391–0.650) *<0.001 CI: confidence interval; HR: hazard ratio. *p < 0.05. Multivariable analysis demonstrated that the independent predictors of post-transplant fractures were an age at transplant ≥65 years (HR: 1.566; 95% CI 1.122-2.186, p = 0.008), female sex (HR: 1.648; 95% CI 1.319–2.057, p < 0.001), fractures in the 1 year prior to transplant (HR: 3.664; 95% CI 2.503–5.364, p < 0.001), HCV carriers (HR: 1.594; 95% CI 1.289–1.970, p < 0.001), alcoholism (HR: 1.557; 95% CI 1.087–2.230, p = 0.016), and daily prednisolone dose >1.61–3.78 mg/day (HR: 1.354; 95% CI 1.005–1.824, p = 0.047), >3.78-9.18 mg (HR: 4.182; 95% CI 3.155–5.544, p < 0.001) and >9.18 mg (HR: 13.334; 95% CI 9.506–18.703, p < 0.001). Post-transplant fractures remained inversely correlated with the use of tacrolimus (HR: 0.617; 95% CI 0.417–0.913, p = 0.016) and sirolimus/everolimus (HR: 0.504; 95% CI 0.391–0.650, p < 0.001). Discussion Various studies have shown that a high percentage of patients with end-stage liver disease referred for liver transplantation have osteoporosis and osteoporotic fractures [18], and that this further increases the risk of fractures after transplantation. Consistent with previous studies, the well-known risk factors for fractures such as older age, female sex and previous fractures were also identified in the current study. Chronic alcohol and nicotine exposure are important risk factors for osteoporosis, and both are common in liver transplant recipients. In addition, malnutrition and magnesium depletion are common in patients with alcoholism and end-stage liver disease. In the present study, compared to the recipients without fractures, those with fracture tended to have more comorbidities. Similar to previous studies, chronic alcoholism predisposed the recipients to a greater risk of fractures. In addition, we found that the patients with osteoporosis, hypertension, diabetes mellitus, systemic lupus erythematous, and cerebrovascular disease had an increased risk of fractures in univariable analysis, but not in multivariable analysis. Hypogonadism due to central suppression of the hypothalamic-pituitary-gonadal axis is common in patients with end-stage liver disease [19]. After liver transplantation, persistent hypogonadism or partial reversibility of the hypothalamic-pituitary-gonadal system dysfunction has been demonstrated. In the present study, we found that hypogonadism and postmenopausal disorders were not associated with an increased risk of fractures. Oestrogen plays an important role in the growth and maturation of bone and also in the regulation of bone turnover via modulation of the production of various cytokines [20]. Exogenous oestrogen supplementation has been shown to prevent oestrogen deficiency-related osteoporosis of various causes [21]. In addition, oestrogen replacement therapy has also shown benefits in the prevention of osteoporosis and fractures following liver transplantation in postmenopausal women [22]. Vitamin D is important for calcium homeostasis and bone mineralization. Chronic obstructive liver disease may interfere with vitamin D metabolism, leading to decreased uptake of vitamin D. In addition, cirrhosis has been shown to impair 25-hydroxylation of vitamin D, consequently resulting in significantly depressed levels of 25-hydroxyvitamin D3 [23]. Previous studies have reported that prophylactic bisphosphonate treatment is effective in preventing bone loss and fractures in liver transplant recipients [24,25]. In the present study, we found that the use of bone protective medications including bisphosphonates, vitamin D supplements and calcium supplements did not have a beneficial effect on decreasing the risk of fractures. However, we only analyzed prescribed calcium and vitamin D supplements, and we did not consider those that could have been bought over the counter or obtained via dietary intake. The pathogenesis of bone loss differs in different chronic liver diseases [26]. Hemochromatosis [27] Wilson’s disease [28] and cholestatic liver diseases are driven mainly by decreased bone formation, and viral hepatitis, transplantation, and the use of glucocorticoids are associated with increased bone resorption. In addition, vitamin K deficiency is frequently observed in patients with cholestasis. Vitamin K can suppress bone resorption by impairing osteoclast maturation and function. The mechanisms involved in osteoporosis in non-alcoholic fatty liver disease include cytokine production in an inflamed liver, vitamin D deficiency, metabolic syndrome and limited physical activity [29]. In patients with non-alcoholic fatty liver disease, significantly higher levels of tumour necrosis factor-alpha, lower levels of osteocalcin and higher levels of osteopontin have been reported. However, controversy exists regarding whether the type of underlying liver disease can predict bone loss and fractures. Some authors have reported higher rates of osteoporosis and fractures in patients with primary biliary cholangitis [30] and non-alcoholic fatty liver disease, especially in older Chinese men [31]. In contrast, Wester et al. reported a similar risk of fractures between patients with non-alcoholic fatty liver disease and the general population [32]. In the present analysis, neither chronic non-alcoholic fatty liver disease nor primary biliary cholangitis were independent factors for the development of fractures. Viral hepatitis causes an inflammatory response with increased production of bone resorption-activating cytokines such as tumour necrosis factor-alpha [33]. Previous studies have confirmed associations between osteopenia and fractures with HBV and HCV infection [13,34–36]. Both HBV and HCV infections were highly prevalent in our study cohort. We found that HBV infection did not increase the risk of fractures, whereas HCV infection was an independent risk factor for fractures. Glucocorticoids are used to prevent or reverse severe rejection following liver transplantation. Low bone mineral density is a well-known risk factor for developing fractures in glucocorticoid-treated patients. The pathophysiology of glucocorticoid-induced osteoporosis includes directly decreasing bone formation by inducing osteoblast apoptosis and increasing resorption by promoting osteoclastogenesis [37,38]. Glucocorticoids also act indirectly by reducing intestinal calcium absorption and renal calcium wasting, thereby leading to secondary hyperparathyroidism. In addition, glucocorticoids decrease the secretion of androgens and oestrogens, primarily by inhibiting gonadotropin secretion. In the present study, we found that the liver transplant recipients with a daily prednisolone dose >1.61–3.78 mg/day, >3.78–9.18 mg and >9.18 mg had 1.354, 4.182- and 13.334-fold increased risks of fractures, respectively. In other words, the risk of fractures increased as the daily dose of steroids increased. Buckley and Humphrey recommended bisphosphonates as the first-line treatment for glucocorticoid-induced osteoporosis [38]. However, other studies have shown that discontinuing denosumab produced a rebound in bone turnover with an increased risk of new vertebral fractures [39]. Among the glucocorticoid-sparing immunosuppressants used to prevent rejection, calcineurin inhibitors including cyclosporine and tacrolimus have been reported to cause osteoporosis [40–42]. Cyclosporine stimulates both osteoclasts and osteoblasts, leading to dose-dependent high-turnover osteoporosis. Tacrolimus has been demonstrated to induce bone loss through an increase in bone resorption only. Monegal et al. reported that tacrolimus offered a significantly better long-term effect on femoral neck bone mineral density in liver transplantation recipients compared with cyclosporine [42]. The effects of the other immunosuppression regimens such as mycophenolate mofetil and sirolimus/everolimus on bone are still controversial [43]. In the present study, the use of tacrolimus and sirolimus/everolimus was beneficial with regards to fractures, which may be due to the lower dose of glucocorticoids. Cyclosporine and mycofenolate mofetil did not significantly influence the occurrence of fractures. Therefore, recipients with risk factors for fractures should pay attention to bone health status and consider the concomitant use of immunosuppressants to minimise the use of glucocorticoids and thereby decrease the risk of fractures. The main strengths of this study include the large sample size, which may have avoided selection and participation bias, and the inclusion of multiple potential clinical covariates in the analysis. However, this study also has several limitations. First, information on the patients’ lifestyle such as smoking, the amount of alcohol consumption and caffeine use, dietary habits, body mass index, physical activity level, and family history of fractures is not available in the NHIRD. We validated the presence of alcoholism by using ICD-9-CM diagnosis codes of alcohol-related diseases and alcohol dependency. Second, bone mineral density and biochemistry data such as calcium, phosphate, 25-hydroxyvitamin D and magnesium were not evaluated. Third, data on the patients’ use of self-paid medications such as calcium and vitamin D supplements were also not recorded in the NHIRD. It is possible that the clinicians may have only offered osteoporosis prevention and/or therapy to the patients who were likely to have a higher fracture risk, which may have resulted in bias related to prescribing decisions. Furthermore, the diagnosis of fractures was based on ICD-9-CM codes rather than being validated by X-ray or other imaging modalities. However, to validity of the diagnosis of a new fracture, new fractures were defined as one inpatient or two outpatient clinical diagnoses of a fracture after the index date. Conclusions In conclusion, we found that female liver transplant recipients aged ≥65 years, patients with prior fractures, chronic HCV infection, alcoholism and a higher daily prednisolone dose were associated with a high risk of fractures. Conversely, tacrolimus and sirolimus/everolimus was associated with a decreased risk of fractures. Clinicians should carefully assess the risk of fractures, monitor bone mineral density and prescribe concomitant immunosuppressant therapy with tacrolimus or sirolimus/everolimus to avoid the use of high-dose glucocorticoids, as these factors remain the most effective way of preventing post-transplant fractures. Further studies are warranted to investigate the role of calcium and vitamin D supplementation and postmenopausal hormone replacement therapy in the prevention of osteoporosis and fractures following liver transplantation. Authors’ contributions JW Chang, HL Tsai, HH Yang, NC Lin, FC Kuo and TC Lin participated in the conception and design of the experiments; JW Chang and HL Tsai were involved in the data analysis and interpretation; JW Chang drafted the manuscript. HH Yang, NC Lin, FC Kuo, TC Lin and HL Tsai critically revised the intellectual content. All authors read and approved the final manuscript and agree to be accountable for all aspects of the work. Disclosure statement No potential conflict of interest was reported by the author(s). Data availability statement The Taiwan’s NHIRD is an anonymized database. Researchers can apply access to Taiwan’s NHIRD database after ethical approval from the institutional review board. The applicant must be Taiwanese or be affiliated with a Taiwanese research institute. After receiving permission of gaining access to Taiwan’s NHIRD database, the Health and Welfare Data Centre asks qualified investigators to conduct on-site data analysis by using provided computers and software including SAS, Stata, R, and SPSS at one branch of Health and Welfare Data Centre through remote connection to Health and Welfare Data Centre servers. No individual-level data may be taken out from centres. 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==== Front Bioengineered Bioengineered Bioengineered 2165-5979 2165-5987 Taylor & Francis 37455672 10.1080/21655979.2023.2234135 2234135 Version of Record Research Article Research Article Optimization of energy recovery efficiency from sweet sorghum stems by ethanol and methane fermentation processes coupling B. HAMADOU ET AL. BIOENGINEERED Hamadou Bakari a b c Djomdi Djomdi c Zieba Falama Ruben a d Djouldé Darnan Roger c Audonnet Fabrice b Fontanille Pierre b Delattre Cedric b Pierre Guillaume b Dubessay Pascal b Michaud Philippe b Christophe Gwendoline b a Energy Research Laboratory, Renewable Energy Section (LRE/SENC), Institute for Geological and Mining Research (IRGM) , Nlongkak Yaounde, Cameroon b Université Clermont Auvergne, CNRS, Clermont Auvergne INP, Institut Pascal , Clermont-Ferrand, France c Department of the Renewable Energies, the National Advanced School of Engineering of Maroua, University of Maroua , Maroua, Cameroon d National Advanced School of Mines and Petroleum Industries, University of Maroua , Maroua, Cameroon CONTACT Gwendoline Christophe gwendoline.christophe@uca.fr Université Clermont Auvergne, CNRS, Clermont Auvergne INP, Institut Pascal , Clermont-Ferrand, France 17 7 2023 2023 17 7 2023 14 1 228244 Integra14 7 2023 Integra14 7 2023 28 3 2023 08 6 2023 04 7 2023 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. 2023 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. ABSTRACT Taken separately, a single sweet sorghum stem bioconversion process for bioethanol and biomethane production only leads to a partial conversion of organic matter. The direct fermentation of crushed whole stem coupled with the methanization of the subsequent solid residues in a two-stage process was experimented to improve energy bioconversion yield, efficiency, and profitability. The raw stalk calorific value was 17,144.17 kJ/kg DM. Fermentation step performed using Saccharomyces cerevisiae resulted in a bioconversion yield of 261.18 g Eth/kg DM, i.e. an energy recovery efficiency of 6921.27 kJ/kg DM. The methanogenic potentials were 279 and 256 LCH4/kg DM, respectively, for raw stem and fermentation residues, i.e. energy yields of 10,013.31 and 9187.84 kJ/kg DM, respectively. Coupling processes have significantly increased yield and made it possible to reach 13,309.57 kJ/kg DM, i.e. 77.63% of raw stem energy recovery yield, compared to 40.37% and 58.40%, respectively, for single fermentation and methanization processes. HIGHLIGHTS Sweet sorghum stem is a viable feedstock source for efficient coproduction of ethanol and methane Sorghum stems calorific value determination revealed an energy potential of 17.15 MJ/kg DM Energy recovery by single methanization yielded 18.03% more than ethanol fermentation Coupling processes has significantly increased energy recovery yield and profitability GRAPHICAL ABSTRACT Keywords Sorghum bioconversion bioenergy ethanol methane energy efficiency biomass Campus France 10.13039/501100006537 Financial support to perform this study was provided by the French government which funded Bakari Hamadou’s research mobility through its Eiffel excellence scholarship program. Campus France (EIFFEL-DOCTORAT 2021 / n°P769728GD21–0000000051). ==== Body pmc1. Introduction World population increase and economic development lead to an exponential growth of the global energy demand. The energy sector, which is essentially based on fossil resources, is the source of about three quarters of GHG emissions and has already raised the mean global temperature by 1.1°C with a lot of negative impacts on the environment [1]. While fossil resources are increasingly depleted, further accentuating concerns about energy and environmental security. Faced with this context, the international community has opted for an energy transition policy targeted to reduce GHG emissions and achieve carbon neutrality in order to cap global warming to 1.5°C by 2050 [2]. The use of renewable energy sources has become an absolute and unavoidable necessity in order to meet this current challenge. Development of energy recovery efficiency from biomass is one of the most important ways of keeping our environment sustainable. Lignocellulosic biomass is one of the most available alternative sources of energy and accounts for approximately 14% of renewable energy consumption worldwide [3]. In the last decade, the use of lignocellulosic biomass agricultural residues as alternative to generate various kinds of energy vector biomolecules, has gained increasing interest because of its several interesting features including diversity, sustainability, and low GHG emissions [4]. Sweet sorghum is one of the most attractive sources of bioenergy because of its versatility, combining the production of starchy grains, high fermentable sugars content stalks and a high green biomass yield [5]. Sorghum multifunctionality gives it the specificity of providing interesting solutions to meet energy and environmental challenges without adversely affecting food chain [6]. Its green biomass productivity varies from 20 to 120 tonnes/ha, depending on the agronomic and environmental conditions as well as the variety’s botanical specificities [7]. In addition, several features make sorghum one of the most interesting cereal crops, including resistance to extreme agronomic conditions, resilience, low input requirements and high production yield. Fifth most produced cereal crops worldwide, sorghum global production in 2020 was estimated at 58.70 million tonnes out of a cropping area of 40.25 million hectares [8]. Sweet sorghum stalks energy recovery like any other lignocellulosic biomass, can be carried out using several techniques, in particular thermochemical conversion (direct combustion, pyrolysis, gasification, liquefaction, etc.) or biological conversion (ethanol fermentation, biomethanization, dark fermentation for hydrogen production, etc.). Thermochemical conversion has low overall energy recovery yields and emits large amounts of pollutants, and therefore many operational and environmental challenges are associated with it [9]. On the other hand, bioconversion presents economic and environmental advantages compared to other technologies [10]. Bioethanol and biomethane are among the most well-known energy vector biomolecules produced from the bioconversion of sweet sorghum stem [11]. Currently, methanization proves to be the more efficient method of lignocellulosic biomass energy bioconversion compared to ethanol fermentation [12]. However, taken separately, a single bioconversion process only leads to a partial conversion of organic matter. More specifically, energy recovery yield from sweet sorghum stems by single ethanol fermentation process is between 6500 and 8900 kJ/kg DM (considering ethanol energy yield at 26,500 kJ/kg) [10,11], while the energy potential or calorific value of sweet sorghum stalk has been estimated at around 18,320 kJ/kg DM [13,14]. This low yield is mainly due to the fact that this lignocellulosic biomass is composed mostly of cellulose, hemicellulose and lignin which integrate strongly forming a complex structure, resistant and difficult to metabolize by microorganisms. A pretreatment step aimed at fractionation is often necessary in order to enhance the accessibility of microorganisms to carbohydrate polymers. Several pre-treatment techniques (mechanical, chemical, thermal, biological, or their eventual combination, etc.) are currently used and have improved performance [15]. However, they have limitations such as high energy or reagent costs, potential pollutants, formation of inhibitor compounds for microbial metabolism, low efficiencies, substrate loss, etc. [4]. In addition, the pretreatment of lignocellulosic biomass, in particular, the production of sugar monomers from the fibrous fraction, is a complex and the costliest part of the biomass energy conversion process [16]. Moreover, the typical process of ethanol production from sweet sorghum stalks proves to be unprofitable because of the very costly juice extraction step. The liquid fraction and the solid residues are usually processed separately. This fractionation step leads to a low energy bioconversion yield, while efficient pathways of biomass energy recovery with lower energy consumption are essential for cost-effective production of bioenergy from lignocellulosic feedstocks [17–19]. Development of more efficient and profitable energy bioconversion process is therefore necessary. This study experiments a combination of direct ethanol fermentation and methanization in order to enhance the overall energy recovery efficiency and profitability of this process. Direct ethanol fermentation (without juice extraction nor pre-treatment step) of crushed whole stem using Saccharomyces cerevisiae coupled with the mesophilic methanization of the subsequent solid residues in a two-stage process has been proposed in this work. The overall objective is to improve the energy bioconversion process yield and performance of sweet sorghum stems. 2. Material and methods 2.1. Sorghum stems collecting and pretreatment The studied sweet sorghum stalks (Sorghum bicolor (L) Moench) were harvested in the locality of ’KODEK’ (10° 39′ 19”N; 14° 24’ 42”E; 880 m), department of DIAMARE, Far North region of Cameroon. Stalk samples were then forthwith stripped, cut out, and kept at −20°C. Ethanol fermentation and methanization have been studied (separately and in combination). Figure 1 shows the overall process diagram. The sorghum variety was first formally identified (species, cultivar, GPS coordinates, etc.), and then a sufficient quantity of stalks was harvested. The stems then underwent a physicochemical characterization. Ethanol production has been experimented on different fractions of the biomass (juice, pith, and whole stem) in order to compare the process efficiency. For this purpose, the stripped stems were cut up, dried (steaming at 60°C until obtaining constant mass) and crushed to a grain size ≤0.5 mm using a mill. A sample of stems was peeled to separate the pith from the bark, and then dried and crushed in the same way. The juice was extracted from wet pith samples by grinding and mechanical squeezing using a mill then characterized before being fermented following the description of route 1 (Figure 1). According to itinerary 2, direct fermentation of crushed pith suspended in water has been experimented. During route 3, single direct ethanol fermentation and direct methanization were experimented on raw shredded whole stalk. Then, the methanization of the subsequent solid residues from ethanol fermentation was also tested in order to compare the efficiencies of the processes separately and the fermentation/methanization coupling process. Figure 1. Overall research implementation flow. 2.2. Sweet sorghum stalk physicochemical characterization The determination of the biomass dry matter content has been carried out according to the Association of Official Analytical Chemists (AOAC) method [20]. Samples were first steamed at 105°C while monitoring the decrease in mass until a stable mass was obtained. The biomass dry matter rate, expressed as a percentage of wet matter, has been estimated after cooling samples in the atmosphere of a desiccator, according to Equation (1):(1) DMr=Mf−TMi−T×100 With, DMr: Dry matter rate (%); Mf: Final crucible mass and its residual dry contents (g); Mi: Initial crucible mass and its wet contents (g); T: Tare of empty crucible (g). The moisture rate of the samples (Mr) has been deduced from the dry matter content according to Equation (2):(2) Mr=100−DMC The ash content determination has been carried out by incineration in an electric muffle furnace (550°C for 6 h) of the subsequent dry residues of the samples. The mineral content has been expressed as a percentage of DM and estimated after cooling in the atmosphere of a desiccator, according to Equation (3):(3) MMr=Mf−TMi−T×100 With, MMr: Mineral matter rate (%); Mf: Final crucible mass and its residual sample contents after incineration (g); Mi: Initial crucible mass and its dry sample contents before incineration (g); T: Tare of empty crucible (g). The organic matter content (OMC) has been deduced from the mineral matter content according to Equation (4):(4) OMC=100−MMr All experiments have been performed in triplicate, and the results are the mean values of the repetitions. The determination of the sorghum stalk biochemical composition has been carried out according to Van Soest [21]. Sorghum stalk total fiber rate, expressed as a percentage of DM, has been evaluated as described by Equation (5):(5) Total Fibers=1−S−W1S∗100 With, S: Initial mass of biomass sample (g) and W1: Residual mass of insoluble in neutral detergent (g). Hemicellulose has been evaluated from the dried residual parts of neutral detergent-insoluble fibers (W1). It was expressed as a percentage of DM and quantified as described by Equation (6):(6) Hemicellulose=W1−W2S∗100 where W2 represents the residual mass of a biomass fraction insoluble in acid detergent (g). Cellulose and lignin have been quantified after sulfuric acid (72%) digestion of residual fractions of acid-detergent-insoluble fibers (W2). They were expressed as a percentage of DM and estimated as described by Equations. (7) and (8), respectively.(7) Cellulose=W2−W3S∗100 (8) Lignin=W3S∗100 where W3 represents the residual mass of a biomass sample after digestion with sulfuric acid (g). The phenolic compounds content was determined by the assay method using Folin Ciocalteu’s reagent according to the protocol described by Gutfinger [22] and expressed in grams of gallic acid equivalent/liter (g GAE/L). Calorific values were determined according to the bomb calorimeter method using an isoperibol calorimeter (calorimètre PARR 6400). To this end, the shredded biomass fractions were previously compressed (3 kPa for 1 min) in the form of pellets using a mechanical press. Then, a complete combustion (in the presence of pressurized oxygen) of a pellet sample was carried out in the bomb calorimeter. The bomb was immersed in the water of the calorimeter, and the heat released by the combustion was transmitted to the water whose temperature rise was measured. The higher calorific value, calculated as gross heat of combustion, was calculated according to Equation (9):(9) HCV=M∗Ce∗ΔT−e1−e2−e3m where HCV is the higher calorific value of the sample (cal/g); M represents the total water mass of the calorimeter (g); Ce is the water specific heat capacity (1 cal/g.°C); ΔT (Tmax − Tmin) is the water temperature increase (°C); e1, e2, and e3 represent, respectively, the heat released by the combustion of nitrogen, sulfur, and fuse wire (cal); m represents the mass of the biomass sample (g). The total mass of water in the calorimeter (M) has been previously determined by the combustion of a mass (m) of benzoic acid of known higher calorific value (HCV = 6319 cal/g), and the value of M was calculated according to Equation (10):(10) M=HCV∗mΔT∗Ce 2.3. Determination of sugar content and carbohydrate profile of biomass The contents of water-soluble sugars and non-water-soluble structural sugars were determined by the enzymatic assay method, using K-SUFRG assay kit (Megazyme, Bray Business Park Bray, Co. Wicklow A98 YV29, Ireland) according to the protocol elaborated by Outlaw and Mitchell [23], Beutler [24], and Kunst et al. [25]. For this purpose, the carbohydrate extracts were previously filtered using Whatman N°1 filter paper and then D-glucose concentration of samples was firstly estimated before and after hydrolysis of sucrose by β-fructosidase (invertase). The samples D-fructose contents were estimated after the D-glucose assay and after isomerization by phosphoglucose isomerase (PGI). Water-soluble sugars were extracted from different shredded biomasses with hot water. To that end, 1 g of ground biomass fraction has been mixed with 50 mL of Milli-Q water in a flask and heated to 90°C for 90 min using a water bath. The solution was filtered under vacuum using a size 3 frit (40–90 μm), and the filtrate was recovered and used for the determination of total water-soluble sugars as well as for the determination of the carbohydrate profile. The subsequent fibrous fractions from the filtration were dried (60°C until mass stabilization) then hydrolyzed with sulfuric acid (0.75 M) in order to extract the structural water-insoluble sugars. To do this, 0.2 g of the dried fibrous fractions have been mixed with 5 mL of sulfuric acid solution (0.75 M) and heated (100°C for 90 min) using a water bath. The hydrolyzates were then subjected to 10 min of centrifugation at 13 000 g. The supernatants were recovered and neutralized (pH = 7.0 ± 0.1) using a 10 M NaOH solution and used for total water-insoluble sugars assay and for the determination of their monosaccharidic composition. The monosaccharidic composition of the different extracts was investigated by the High-Performance Anion-Exchange chromatography (HPAEC-PAD) device, which is an ICS 3000 ion chromatography chain (Dionex Corporation, Sunnyvale (CA), USA). It consists of a pump module that can operate in gradient and a pulsed amperometric detector (HPAE-PAD). The stationary phase consists of polystyrene and divinylbenzene beads 10 μm in diameter on which particles functionalized by NR4+ groups are agglomerated. It reaches stable pressures of 275 bars in a pH range of 0 to 14. The samples were eluted in isocratic mode with a 16 mM decarbonated NaOH solution at 0.5 mL/min of flow rate for 20 min. After each elution, a 30 min chase with a 100 mM NaOH solution was performed to elute any contaminants still interacting with the stationary phase. Before each analysis, the column was equilibrated with the 16 mM NaOH solution for 10 min. All samples have been filtered using 0.22 µm filter before being injected. The injections are performed in triplicate, and the injection loop is 25 µL. Data acquisition and processing have been carried out using chromeleon software (version 6.8). 2.4. Ethanol production from different sorghum stalk fractions Saccharomyces cerevisiae ATCC 7754 was used for ethanol production. The strain was taken up in sterile water and spread on YM-agar agar (yeast Medium, Difco 0712-01-8) and then incubated at 28°C for 24 h. It was then preserved at 4°C and subcultured on a petri dish for 24 h before being used as a preculture to inoculate the reactor [26]. The fermentation was carried out using an aerated and stirred reactor of 6 L total volume (Biostat A, B. BRAUN, Germany), with 4 L of working volume. Culture media consisted of 10% (m/v) of equivalent biomass fraction (particle size of 0.5 mm) or juice diluted at 60 g/L of total sugars content, supplemented with additional nutrients as indicated by Kristiansen [27] with the exception of glucose. A glucose control was carried out in parallel. Culture media have been firstly autoclaved (121°C for 20 min) before being inoculated with 10% (v/v) of a preculture of Saccharomyces cerevisiae in the exponential growth phase. The temperature of the culture medium was set at 30°C, with an aeration of 1.5 L/min, 500 rpm of stirring speed, and the pH was fixed at 5 by the automatic addition of a 2 M NaOH solution. Samples have been taken every 2 h (shredded stalk and marrow) and every 1 h (sorghum juice and glucose control) for fermentation kinetics monitoring (total sugars consumption, microbial growth, and the production of bioethanol). The total sugars consumption (for juice, crushed whole stem and marrow) was monitored by the assay method described by Dubois et al. [28] using sulfuric acid and phenol. Biomass growth was monitored measuring A600 (Biomate 3S, UV–visible spectrophotometer, Thermo Scientific, Lyon, France). Bioethanol production and glucose consumption (for glucose control) were monitored using HPLC (1260 Infinity Quaternary LC system, Agilent Technology, Palo Alto, CA, USA) device. It was provided with two ionic exclusion columns (Rezex ROA 300 × 7.8 mm, Phenomenex, Torrance, CA, USA) interconnected in series in an oven (50°C) and coupled to a refractometer (HP 1100 series, Agilent Technologies, USA) as products detector. The mobile phase involved a solution of 2 mM sulfuric acid at 0.7 mL/min flow rate and 70 bars (7000 kPa). In order to avoid column clogging, all the samples have been firstly deproteinized before injection. To this end, 125 μL of a 0.3 M barium hydroxide solution and 125 μL of zinc sulfate solution (5% w/v) have been mixed to 1 mL of samples and subjected to 10 min of centrifugation (Thermo scientific, Lyon, France) at 10 000 g. The supernatants have been filtered using a 0.2 μm cellulose acetate filter (Chromafil, Steinheim, Germany) before being analyzed. 2.5. Determination of biochemical methane potential The methanogenic potential was determined on the shredded raw stem without ethanol fermentation as well as on the subsequent solid residues from fermentation. Methanization was carried out in a stirred reactor of 2 L total volume with 1.4 L of working volume. The substrate was inoculated by a consortium from the digestate of the Methelec methanization unit. The tests were carried out under mesophilic conditions (37 ± 1°C), with a stirring speed of 120 rpm. The methanogenic potential and the quality of the gas (volume, biogas composition) have been followed according to the French Agency for Ecological Transition (ADEME) method [29]. Data statistical analysis was performed using the Excel spreadsheet of the Microsoft office software version 2013. The experiments have been performed in three repetitions, and results were reported as an average ± standard deviation. 3. Results and Discussion 3.1. Sugar content and monosaccharidic composition of biomass The whole sorghum stem water-soluble sugars composition was sucrose, glucose, and fructose with concentrations estimated, respectively, at 79.29, 199.22, and 196.08 g/kg DM (Table 1). The sugar concentrations of sorghum variety subject to this study are much higher than those of the varieties experimented by Crépeau et al. [30] whose concentrations were 2.4–34.9, 25.2–39.1, and 20.4–32.1 g/kg DM, respectively. However, it is much closer to those of the varieties experimented by Ostovareh et al. [31], with, respectively, 121, 92, and 83 g/kg DM. The same carbohydrate profile was observed in the pith and in the bark. However, the sucrose contents were much higher in the pith and in the bark compared to that of the whole stem. They were 212.83 and 82.05 g/kg DM, respectively, for marrow and bark. This study shows that, sorghum stalk water-soluble sugar content is essentially concentrated in the pith compared to the bark, where the contents were only 82.05, 52.28, and 47.52 g/kg DM, respectively, for sucrose, glucose, and fructose. These results are in accordance with the remarks made by Billa et al. [32], who studied the biochemical composition of the sweet sorghum stem and indicated that the pith was twice as rich in glucose and sucrose (71% of dry weight) compared to bark (34.6%). These results show that this agricultural by-product, due to its high concentration of fermentable water-soluble sugars, constitutes an interesting source of raw material for bioethanol production. This sorghum stem’s high content of free and fermentable sugars makes it suitable for the production of several types of energy vector biomolecules. It is therefore relevant to explore and find the most efficient and profitable energy conversion pathways. Direct ethanol fermentation/methanization coupling process would optimize energy recovery while avoiding the costly steps of fractionation and pretreatment of the biomass. Ethanol fermentation produces not only ethanol but also a large quantity of microbial biomass (Saccharomyces cerevisiae) which is often very little valued. Optimization of energy bioconversion process of fermentable sugars from sorghum stalks would involve the energy recovery of solid fermentation co-products, in particular, the Saccharomyces cerevisiae biomass. The valorization of liquid secondary metabolites from ethanol fermentation, particularly short-chain carboxylic acids (succinate, acetate, propionate, lactate, etc.) is also positioned as a new innovative and promising bioconversion pathway. Medium chain carboxylic acids production from waste biomass through chain elongation reaction has currently attracted the attention of several authors and is considered as a promising way for the future compared to traditional techniques of single ethanol fermentation or methanization pathways [33].Table 1. Carbohydrate profile and sugar content of different biomass fractions.   Deleafed stem Marrow Bark Carbohydrate profile and sugar content of raw biomass (g/kg DM) Glucose 199.22 ± 1.57 165.80 ± 1.41 52.28 ± 0.91 Fructose 196.08 ± 1.26 158.74 ± 1.32 47.52 ± 0.78 Sucrose 79.29 ± 1.05 212.83 ± 1.96 82.05 ± 1.11 Carbohydrate profile and sugar content of bagasse (g/kg DM) Arabinose 17.32 ± 1.08 20.67 ± 1.81 12.85 ± 1.04 Galactose 5.17 ± 0.21 8.21 ± 0.37 3.11 ± 0.14 Glucose 103.61 ± 2.46 303.32 ± 2.98 53.54 ± 1.76 Xylose 142.64 ± 2.69 114.60 ± 2.83 146.41 ± 2.74 Furthermore, analysis of the biomass fibrous fraction (bagasse) hydrolyzate reveals that the water-insoluble structural sugars were composed mainly of xylose, glucose, arabinose, and some traces of galactose. For the crushed whole stem bagasse, the sugar proportions were 53.07%, 38.55%, 6.44%, and 1.92%, respectively, for xylose, glucose, arabinose, and galactose (Table 1). The same carbohydrate profile was observed for the marrow and bark fractions, with a predominance of xylose and glucose contents, evaluated, respectively, at 25.65% and 67.88% for the marrow bagasse and 67.81% and 24.79% for bark bagasse. Sipos et al. [34] also investigated the carbohydrate profile of sweet sorghum stalk bagasse. The sugar concentrations were 44.14, 17.96, and 5.53 g/L, respectively, for xylose, glucose, and arabinose, i.e. 65.26%, 26.55%, and 8.17%, respectively. These results show that sweet sorghum stalk bagasse is a source of additional sugars that could be used to optimize sorghum stem energy bioconversion through a prior saccharification process. However, lignocellulosic biomass saccharification techniques (enzymatic hydrolysis, biocatalyst in particular), although they improve the energy bioconversion yield, prove to be economically unprofitable due to the cost of reagents and operating conditions. Chemical hydrolysis, due to its toxic and corrosive nature, is not environmentally friendly and generates microbial growth inhibitor compounds. It is therefore necessary to reconsider the energy bioconversion strategies and pathways of this biomass. Moreover, due to the presence of a high proportion of xylose in bagasse hydrolyzate, its valorization into bioethanol requires the choice of microorganisms capable of metabolizing pentoses in order to make the bioconversion process more profitable. 3.2. Biochemical composition of sorghum stalk juice The biochemical composition of the biomass is a crucial factor that determines the relevance of the choice of adequate energy recovery technique. The juice sugar content of the S. bicolor studied was 252.51 and 78.46 g/L, respectively, for total water-soluble sugars and reducing sugars. This concentration of total water-soluble sugar is much higher than that indicated by Djomdi et al. [35] on the same variety (98.85 g/L). This sugar content is also higher than those of the varieties experimented by Crépeau et al. [30] and Daniel et al. [36], who reported sugar contents evaluated, respectively, at 60.89–111.10 g/L and 100.3 g/L. Moreover, the sorghum juice carbohydrate profile for this study was mainly composed of glucose, fructose, and sucrose. Sucrose was the dominant sugar, and the proportions were 64.4%, 21.13%, and 14.47%, respectively, for sucrose, glucose, and fructose. Similar compositions, evaluated on other sweet sorghum varieties, were previously indicated by Prasad et al. [37] (60%, 33%, and 7%, respectively, in sucrose, glucose, and fructose) and Serna-Saldívar et al. [38] with proportions estimated at 53–85%, 9–33%, and 6–21%, respectively. This high sucrose content is consistent with other experiments [39,40], and the glucose concentration is very often higher than that of fructose. This carbohydrate profile of sorghum juice is comparable to that of sugar cane juice. However, sugar cane juice has a relatively higher sucrose content (90–98%) with very low proportions of glucose (0.5–4%) and fructose (0.5–6%) [38,41]. The sorghum juice phenolic compounds content was evaluated at 0.32 g/L. A similar observation was indicated by Sereme et al. [42] who reported contents of phenolic compounds <2% DM in the sorghum caudatum stalk. This low content of phenolic compounds in sorghum juice is a major asset for its valorization for ethanol production, since these compounds have detrimental effects on biomass growth and metabolism and can act, at high concentrations, as antimicrobials [43]. Beyond this sorghum stalk juice biochemical composition (high fermentable sugars content with low concentration of phenolic compounds), very suitable for ethanol production, the extraction processes tend to restrict its profitability due to high water and energy consumption. Enzymatic treatment applied to the shredded whole stems without extraction step has been recently demonstrated by Bakari et al. [44]. Admittedly, the authors have demonstrated that the extraction step could be dispensed and therefore significantly improve the process economic profitability, but the energy bioconversion yield remains to be perfected. This explains the need to experiment with the coupling of direct ethanol fermentation of shredded whole stems and the methanization of subsequent solid residues. This would make it possible to valorize both the stem fibrous fraction and the microbial biomass (Saccharomyces cerevisiae) as well as other fermentation secondary metabolites. Avoiding the extraction and hydrolysis steps would make the process more profitable. 3.3. Biochemical composition and calorific value of different biomass fractions Analysis of the chemical composition of raw deleafed sorghum stalk revealed contents estimated at 93.5% and 87.5%, respectively, for dry matter and organic matter. These contents are comparable to those of the varieties experimented by Luna et al. [45], who reported 95.3–95.9% and 94.6–95.6%, respectively, for DM and OM. The solid residues from the ethanol fermentation process have compositions similar to that of the shredded raw stalk. The contents were 96.0% and 86.5%, respectively, for DM and OM. This could be explained by the fact that these solid fermentation residues, although devoid of water-soluble sugars, also contain large quantities of microorganisms (Saccharomyces cerevisiae), and therefore additional organic matter. The hemicellulose, cellulose, and lignin contents of the raw stem were 17.39%, 22.27%, and 5.09% DM, respectively, i.e. 88.61% of these sorghum stem total fibers were made up of hemicellulose (38.85%) and cellulose (49.76%). This represents a potential source of substrates for bio-methane production, since the expensive process of fibrous fraction saccharification is not necessary for energy recovery through methanation pathway. These contents are much higher than those of the variety experimented by Billa et al. [32], who reported 10.2% and 12.4% DM, respectively, for hemicellulose and cellulose. However, they are much closer to those of the varieties experimented by Khalil et al. [40], (11.7–17.2% and 20.1–26.1% DM, respectively, for hemicellulose and cellulose) and those of the variety studied by Billa et al. [32] (4.8% for lignin). Similar experiments performed by several authors indicate that total fibers content as well as its distribution in hemicellulose, cellulose, and lignin vary widely depending on the studied varieties of sorghum. Moreover, the total fiber contents were 81.84% DM and 44.75% DM, respectively, for ethanol fermentation solid residues and raw stems. Ethanol fermentation solid residues cellulose, hemicellulose, and lignin contents were 33.11, 44.87, 3.86% DM, respectively (Table 2), and 95.28% of total fibers consisted of hemicellulose (54.83%) and cellulose (40.45%) compared to 88.61% for raw stem fibers. This confirms that the residual stem fibrous fraction has been enriched with fibers from Saccharomyces cerevisiae growth and is therefore a potential source of additional energy that can be recovered by a subsequent methanation process. In addition to the microbial fiber supply, fermentation process also allowed partial degradation of sorghum stem fibers (Figure 2). However, Saccharomyces cerevisiae not being able to degrade fibers, this degradation observed after fermentation was essentially due to the autoclaving operation. The ethanol fermentation-methanization coupling process effectively improves the energy recovery yield without costly traditional pretreatment methods. The calorific values were 17,144.17 and 18,117.23 kJ/kg DM, respectively, for raw stalk and solid residues from ethanol fermentation process. These results show that ethanol fermentation residues have a slightly higher energy potential than that of the raw biomass. This relatively high energy content of the ethanol fermentation residues could be explained by their high fiber content (81.84% DM) compared to the raw biomass (44.75% DM) (Table 2). Sweet sorghum stem biochemical composition and its calorific value were comparable to those of other lignocellulosic feedstocks such as corn stover, sugarcane, rice straw, switchgrass, etc., estimated at 16.28–18.64 MJ/kg (for calorific value), 35–53% DM (for cellulose), 15–36% DM (for hemicellulose), and 14–32% DM (for lignin) [13,38,46]. Figure 2. Structure of raw sorghum stalk fibers before fermentation (a) and mixture of sorghum stalk fibers and microbial biomass after fermentation (b). Table 2. Calorific value and biochemical composition of sorghum stem biomass before and after ethanol fermentation. References   Calorific value (MJ/Kg) DM (%) OM (%) Total fibers (% DM) Cellulose (% DM) Hemicellulose (% DM) Lignin (% DM) This study Raw sorghum stem 17.15 ± 0.47 93.50 ± 0.93 87.50 ± 0.87 44.75 ± 0.71 22.27 ± 0.18 17.39 ± 0.65 5.09 ± 0.19 Solid residues from ethanol fermentation 18.11 ± 0.32 96.00 ± 0.88 86.50 ± 0.96 81.84 ± 0.73 33.11 ± 0.42 44.87 ± 0.39 3.86 ± 0.11 [14,38] Sweet sorghum 18.32 - - 13.0 25–44.6 22–27.1 11–24.7 [13] Corn stover 17.65 - - - 35–53.0 15–28.0 15–21 [38,46] Sugar cane 17.33 - - 13.5 41.6 19–25.1 20.3–32 [38] Rice straw 16.28 - - - 36.0 28.0 14.0 [13] Switchgrass 18.64 - - - 36.0–43 31.6–36 17–22 3.4. Ethanol production from different stalk fractions Ethanol production was tested on three fractions of sorghum stalk biomass (shredded whole stalk and marrow with particle size ≤0.5 mm and mechanical extracted juice) in order to compare the efficiency of fermentation processes. The fermentation kinetics are presented in Figure 3. The degradation rates (residual mass after fermentation) were 30.47% and 43.12%, respectively, for whole stem and pith. Ethanol fermentation efficiencies were 0.46, 0.43, and 0.44 g Eth/g consumed total sugars, respectively, for whole stem, pith, and juice (Figure 4). These results show that there is no major difference between the fermentation efficiencies of the different stem fractions. The biomass fractionation, in particular, the juice extraction step, is therefore not necessary, thus offering the possibility of fermenting the whole ground stem, improving the process efficiency. In addition, whole stalk fermentation brings additional Saccharomyces cerevisiae fibers to the fibrous sorghum fraction and constitutes a major asset for subsequent methanization step of the ethanol fermentation residues. These results confirm the observations made by Bakari et al. [44] who reported that the very expensive and laborious juice extraction step, consuming energy and water, could be avoided, significantly enhancing the efficiency and sorghum stem fermentation process performance. Similar ethanol production yields, 0.41–0.44 g Eth/g consumed sugar [34]; 0.39–0.48 g Eth/g consumed sugar [47] and 0.42–0.48 g Eth/g consumed sugar [48] have been reported in different studies. The juice fermentation kinetics was similar and comparable to that of the glucose control fermentation (Figure 3b), which implies that the sweet sorghum juice, due to its biochemical composition and in particular its carbohydrate profile, constitutes an adequate substrate for ethanol production using Saccharomyces cerevisiae. Figure 3. Kinetics of different biomass fractions fermentation: (a) shredded whole stalk and marrow, (b) juice and glucose control. Figure 4. Ethanol fermentation and bioconversion yields for different biomass fractions. Furthermore, the bioconversion yields were 261.18, 280.02, and 274.85 g Eth/kg DM, respectively, for whole stalk, marrow, and juice (Figure 4). These yields are much higher than those obtained by Ban et al. [49], who reported 147 g/kg DM. These bioconversion yields, expressed in terms of energy recovery efficiency, were 6921.27, 7420.53, and 7283.52 kJ/kg DM, respectively (considering ethanol calorific value at 26,500 kJ/kg [10]). These results are in accordance with those reported by some authors in the literature [10,11], who indicated that energy recovery efficiency of sorghum stalk bioconversion to ethanol was between 6500 and 8900 kJ/kg DM. These results indicate that sorghum stalk bioconversion by single ethanol fermentation made it possible to recover, respectively, 40.37%, 43.28%, and 42.48% of the raw biomass initial energy content. This justifies the need and the choice to recover the ethanol fermentation solid residues in the form of methane in order to optimize the energy conversion yield of this sorghum by-product. 3.5. Production of bio-methane from different biomass fractions The bio-methanization was tested on the shredded raw stem before and after ethanol fermentation. The methanogenic potentials were (319 ± 11) and (296 ± 23) LCH4/kg of organic matter, respectively, for raw deleafed stalk and ethanol fermentation residues (Table 3). This result is in line with those of several other previously reported studies. Głąb et al. [12] reported that the methanogenic potential of sweet sorghum stalks was 296–342 LCH4/kg total solids. It was 317 LCH4/kg volatile solids according to the study performed by Herrmann et al. [50] on hybrid variety of sweet sorghum (Sorghum bicolor x sudanense). Expressed in terms of raw biomass, the potentials were (279 ± 10) and (256 ± 20) LCH4/kg of raw biomass, respectively, for raw deleafed stalk and fermentation residues. These results are comparable to those reported by Ostovareh et al. [31], who obtained 278.7 LCH4/kg volatile solids after organosolv pretreatment of sweet sorghum stems. This bioconversion by anaerobic digestion process leads to the energy recovery yields of 10,013.31 and 9187.84 kJ/kg of raw biomass, respectively, for raw deleafed stalk and fermentation residues (considering the lower calorific value of methane at 35.89 MJ/m3 [51]). Antonopoulou et al. [10] experimented with methane production from sweet sorghum stem bagasse and reported that the methanogenic potential was 78 LCH4/kg and an energy yield of 9845 kJ/kg DM. This study shows that sorghum stalk bioconversion by anaerobic digestion process made it possible to recover, respectively, 58.40% and 53.59% of raw stalks initial energy content. These results show that the ethanol fermentation residues have similar and comparable methanogenic potential to that of the raw stem. This could be explained by the high fiber content of these residues composed of 44.87% and 33.11%, respectively, in hemicellulose and cellulose, against 17.39% and 22.27%, respectively, for the raw stem fibers (Table 2). The ethanol fermentation allowed an additional microbial fiber supply, significantly improving the methanogenic potential of the solid residues. The partial degradation of stem fibers during the fermentation step also facilitated their decomposition by methanogenic bacteria enhancing energy bioconversion efficiencies. Furthermore, bioconversion by single methanization of raw shredded stem has a better energy recovery yield (10013.31 kJ/kg) compared to single ethanol fermentation (6921.27 kJ/kg). The direct fermentation of shredded stalks coupled with methanization of the subsequent solid residues made it possible to reach 13,309.57 kJ/kg DM, with an energy recovery yield of 77.63% of raw biomass initial energy content (Table 3), i.e. respectively 37.26% and 19.23% more compared to single fermentation and single methanization (Figure 5). Recovering sweet sorghum stems without going through costly extraction or hydrolysis processes significantly improves the overall energy conversion efficiency, making the process more cost-effective. Figure 5. Energy efficiency of different biomass bioconversion processes. Table 3. Sorghum stalks bioconversion yield. References   Total soluble sugars (% DM) Methanogenic potential (LCH4/kg OM) Ethanol yield (g Eth/kg DM) Total energy recovery yield (%) This study Raw sorghum stems 53.82 ± 5.38 319 ± 11 261.18 ± 2.8 77.63 [12,49] Sweet sorghum stalks - 296–342 119.47–147 - [31] Treated sweet sorghum stalks 29.6 278.7 107.3 - [10] Sweet sorghum stems - 107 - 75.02 Figure 6 illustrates the cumulative production kinetics and the evolution of the methanogenic potential of the biomass as a function of the hydraulic retention time. The cumulative production of methane began from the first days of anaerobic digestion without a latency phase. It was relatively low (25%) during the first 11 days for both substrates. The exponential production phase started from the 12th day and the plateau (100%) was reached after 22 days and 25 days of methanization, respectively, for shredded raw stem and ethanol fermentation residues (Figure 6a). These results show that the ethanol fermentation solid residues, because of their high fiber content, constitute a substrate for methanization in the same way and comparable to the raw stem. Thus, highlighting the relevance of recovering them into methane in order to optimize the process energy efficiency. Głąb et al. [12] experimented with the biomethanization of sweet sorghum stalk and noticed that the cumulative methane production increased intensively during the initial phase until the 5th day and the plateau was reached after approximately 30 methanization days. These observations are similar to those reported by Herrmann et al. [50] who studied methanization on certain cereal crops (sorghum, maize, rye, triticale) and noticed a rapid production of methane during the first days of methanization without inhibition or lag phase. This rapid and intense production of methane could be explained by the fact that the organic matter contained in the substrate is easily biodegradable. Figure 6. Kinetics of cumulative methane production (a) and evolution of biomass methanogenic potential (b). Evolution of CH4 and CO2 contents as a function of hydraulic retention time for raw stem (c) and solid ethanol fermentation residues (d). Figure 6 shows the evolution of biogas composition, in particular, the evolution of the CH4 and CO2 contents according to the hydraulic retention time during the bio-digestion process. Biogas composition analysis revealed a high CO2 content at the beginning of the methanization process until the 7th day (62% CO2 and 38% CH4), then this trend was gradually reversed and reached 68% CH4 and 32% CO2 after the 22nd day of methanization of the shredded raw stem (Figure 6c). The same trend was observed for the bio-digestion of ethanol fermentation residues. The composition of the biogas was 53% CO2 and 47% CH4 on the 7th day and 60% CH4 and 40% CO2 after the 25th day of bio-digestion (Figure 6d). These results are comparable to those obtained by Antonopoulou et al. [10] who experimented with the methanogenic potential of sweet sorghum stems and indicated that the methane content was 64%. Takaki et al. [52] experimented with the methane production from the vinasse resulting from ethanol fermentation process from sweet sorghum stalk juice. They obtained biogas with an average methane content of 62.7%. This study shows that solid residues from sorghum stalk ethanol fermentation process constitute a raw material for the production of biogas with a high methane content and comparable to that of raw stalk. 3.6. Economic analysis Several studies have demonstrated the cost-effectiveness of sweet sorghum fermentable sugars bioconversion into ethanol. The cost of ethanol production estimated at 615.4 $/ton (0.49 $/L) was reported by Li et al. [53] using advanced solid state fermentation technology. This study indicated that feedstock cost accounts for 78% of the total cost. A similar ethanol production cost of 0.58 $/L has been reported by Regassa and Wortmann [54] for Florida’s sweet sorghum stems. These cost estimates were made including sweet sorghum production and harvesting, the input for biomass logistics, etc., which were the major expenses. For example, costs were estimated at 54.4% to 57.8% for biomass logistics input factor, and biomass conversion into ethanol accounted for about 38.4% to 42.2% [55]. In fact, although considering all these factors, the cost of producing bioethanol from free sugars of sweet sorghum stems was found to be the lowest and cost-effective compared to other similar lignocellulosic biomass (sugar cane, wheat, cassava, beet, etc.) [53]. The present study focused on sweet sorghum stalks that were agricultural by-products. Generally, in Africa, sorghum was grown mainly for seeds for human consumption, and the stalks were left in the fields in abundance. Therefore, in the economic analysis, the production and harvesting costs have not been considered, but only the costs of transport and other input factors related to the bioconversion of biomass, are considered. Thus, the cost of biomass logistics input factor (representing 54.4% to 57.8% of the total cost), given by the previous authors, would be significantly improved, making the proposed method of the present study more profitable. Moreover, the estimated bioconversion cost made by the authors mentioned above also includes costs linked to the biomass pre-treatment operations (juice extraction, hydrolysis, etc.). Biomass pre-treatment costs (electricity, water, enzymes, reagents, etc.) significantly increase the input factor of bioconversion process. For example, enzymatic hydrolysis leads to an increase in production cost estimated at 0.03–0.2 $/L of ethanol [56,57]. Similarly, an increase of 1.78–1.90 $/L depending on the type of pre-treatment applied was reported by Da Silva et al. [58] for different pre-treatment processes. Devi et al. [59] reported that the cost was 0.57 $/L of ethanol produced from lignocellulosic biomass through enzymatic and chemical hydrolysis. They found that, the major part of the production cost was attributed to the chemical pre-treatment and saccharification of the biomass. The direct fermentation of the shredded whole stems tested in this study made it possible to recover the free sugars while avoiding the juice extraction cost, reducing therefore the total operating cost and improving the economic profitability of the bioconversion process. On the other hand, the methanation of the subsequent solid residues from the direct fermentation has eliminated the costs of biomass hydrolysis. The energy recovery efficiency and the economic profitability could be significantly improved compared to the expensive traditional method of biomass hydrolysis. 4. Conclusion Optimization of energy recovery process from sorghum stalks has been investigated. Physicochemical characterization of raw biomass reveals a high concentration of fermentable free sugars, composed of 199.22, 196.08, and 79.29 g/kg DM, respectively, for glucose, fructose, and sucrose. Its composition (31.74% dry matter) was total fibers, hemicellulose, cellulose, and lignin at, respectively, 63.50%, 16.96%, 18.25%, and 28.60% DM. Its calorific value was 17,144.17 kJ/kg DM. Energy recovery yields by single bioconversion processes of fermentation and methanization were, respectively, 6921.27 and 10,013.31 kJ/kg DM. Coupling processes significantly enhanced yield reaching 13,309.57 kJ/kg DM, with 77.63% energy recovery, i.e. 37.26% and 19.23% more compared to single fermentation and methanation, respectively. Acknowledgment The data that support the findings of this study are available from the corresponding author [G.C.] upon reasonable request. Disclosure statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Credit Authors Contribution Statement Bakari Hamadou, Djomdi Djomdi, and Ruben Zieba Falama: Conceptualization, Methodology, Investigation, Software, Data curation, Writing – Original draft preparation. Fabrice Audonnet and Pierre Fontanille: Visualization, Investigation. Cedric Delattre and Guillaume Pierre: Software, Validation. Roger Djouldé Darnan and Pascal Dubessay: Meyhodology, Writing – Reviewing and Editing. Philippe Michaud and Gwendoline Christophe: Conceptualization, Methodology, and Supervision. ==== Refs References [1] IEA, 2021. World Energy Outlook 2021. IEA, Paris. https://www.iea.org/reports/world-energy-outlook-2021. [2] IEA, 2022. 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Waste Biomass Valor. 2023; doi: 10.1007/s12649-022-02026-y [45] Luna P, Risfaheri H, Charalampopoulos D, et al. Fractionation of carbohydrate polymers from Indonesian sorghum by-products. Food Bioprod Process. 2022;135 :114–122. doi: 10.1016/j.fbp.2022.07.007 [46] Kim M, Day DF. Composition of sugar cane, energy cane, and sweet sorghum suitable for ethanol production at Louisiana sugar mills. J Ind Microbiol Biotechnol. 2011;38 (7 ):803–807. doi: 10.1007/s10295-010-0812-8 20803247 [47] Ratnavathi CV, Suresh K, Vijay KBS, et al. Study on genotypic variation for ethanol production from sweet sorghum juice. Biomass Bioenergy. 2010;34 (7 ):947–952. doi: 10.1016/j.biombioe.2010.02.002 [48] Laopaiboon L, Pornthap T, Jaisil P, et al. Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae. World J Microbiol Biotechnol. 2007;23 (10 ):1497–1501. doi: 10.1007/s11274-007-9383-x [49] Ban J, Yu J, Xu Z, et al. Ethanol production from sweet sorghum residual. Front Chem Sci Eng. 2008;2 (4 ):452–455. doi: 10.1007/s11705-008-0072-6 [50] Herrmann C, Heiermann M, Idler C. Effects of ensiling, silage additives and storage period on methane formation of biogas crops. Bioresour Technol. 2011;102 (8 ):5153–5161. doi: 10.1016/j.biortech.2011.01.012 21334882 [51] Theuretzbacher F, Bauer A, Lizasoain J, et al. Potential of different Sorghum bicolor (L. Moench) varieties for combined ethanol and biogas production in the Pannonian climate of Austria. Energy. 2013;55 :107–113. doi: 10.1016/j.energy.2013.04.020 [52] Takaki M, Tan L, Murakami T, et al. Production of biofuels from sweet sorghum juice via ethanol–methane two-stage fermentation. Ind Crops Prod. 2014;63 :329–336. doi: 10.1016/j.indcrop.2014.10.009 [53] Li S, Li G, Zhang L, et al. A demonstration study of ethanol production from sweet sorghum stems with advanced solid state fermentation technology. 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==== Front Hum Vaccin Immunother Hum Vaccin Immunother Human Vaccines & Immunotherapeutics 2164-5515 2164-554X Taylor & Francis 37455405 10.1080/21645515.2023.2217075 2217075 Version of Record Research Article Rotavirus Cost-effectiveness analysis of introducing rotavirus vaccine into immunization program in Zhejiang province, China: A decision tree-Markov model study H. LV ET AL. HUMAN VACCINES & IMMUNOTHERAPEUTICS Lv Huakun Chen Fuxing Wang Ying Chen Yaping Hu Yu Institute of Immunization and Prevention, Zhejiang Center for Disease Control and Prevention , Hangzhou, China CONTACT Yu Hu husix@163.com Institute of Immunization and Prevention, Zhejiang Center for Disease Control and Prevention , No. 3399 Binsheng Road, Binjiang District, Hangzhou, China. 16 7 2023 2023 16 7 2023 19 2 2217075Integra17 7 2023 Integra17 7 2023 29 1 2023 09 5 2023 19 5 2023 © 2023 The Author(s). Published with license by Taylor & Francis Group, LLC. 2023 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. ABSTRACT Two RV vaccines have been licensed in Zhejiang province, China, including the pentavalent RotaTeq (RV5) and the monovalent RV vaccine (RV1). This study aimed to evaluate the cost-effectiveness of RV1 and RV5 to inform the prioritization of provincial immunization program. A decision tree-Markov model was applied to assess the cost-effectiveness of RV1 or RV5 vaccination, compared to no RV vaccination in a 5-year period. The incremental health cost per disability-adjusted life year (DALY) is averted when each kind of RV vaccine included in the immunization program was estimated. It was also compared to the willingness-to-pay (WTP) threshold of 1 time of the Gross Domestic Product (GDP) per capita of Zhejiang province in 2021 (16865.70US$). The uncertainties of these results were assessed through the one-way sensitivity analysis and probabilistic sensitivity analysis. In the base-case scenario, RV5 was the least costly RV vaccine with a cost-effectiveness ratio of 510 US$ per DALY averted. The other RV1 vaccines provided equivalent benefits but at higher costs, with a cost-effectiveness ratio of 4600 US$ per DALY averted. Compared to no vaccination, the probabilities of being cost-effective were 72.3% for RV1 and 88.4% for RV5, at a WTP threshold of 1 time of the GDP per capita. An RV vaccination program using any of the two China-licensed RV vaccines compared to no vaccination would avert 30–70% of outpatient visits, hospitalizations, and deaths from RVGE. RV5 was currently estimated to be the most cost-effective. KEYWORDS Rotavirus vaccine cost-effectiveness model immunization program a medical and health science and technology project of Zhejiang province 2023KY633 This study was funded by a medical and health science and technology project of Zhejiang province [Grant number: 2023KY633]. ==== Body pmcIntroduction More than 500,000 deaths in children under-five are caused by diarrhea annually worldwide, accounting for 10% of childhood mortality.1 Rotavirus (RV) is one of the major etiology of diarrhea, which comprises 44% hospitalizations and 28% deaths associated with diarrhea worldwide.2 A surveillance study from China indicated that the proportion of RV infection was 30% among all diarrhea cases under 5 years of age.3 Although diarrheal deaths have been decreasing, acute watery diarrhea has remained a leading cause of under-five mortality in China. In 2013 alone, it accounted for 3.8% deaths among under-five children.4 According to the estimates from WHO,5 9072 under-five children died of infectious diarrhea, of which 3191 had RV infections. Despite the efforts to reduce the burden of RV, it was approximately responsible for 12.1 million RV gastroenteritis (RVGE) cases among children under 5 years of age in China in 2008. Besides, China is one of the highest disease burden countries of RVGE worldwide, with a cost of 365 million US$ a year.6 Vaccination is one of the most effective ways to prevent RVGE. WHO recommended the inclusion of RV vaccination into national immunization programs (NIPs) in 2013.7 As of 2022, RV vaccines have been introduced in 114 countries. Several studies have revealed that RV vaccination is a cost-effective intervention for prevention of RVGE, severe diarrhea, relative hospitalizations, and deaths, especially in countries with a high childhood mortality rate.8,9 Many reports have shown that RV vaccination was cost-effective in low- and middle-income countries (LMIC).10 Currently, two RV vaccines have been licensed in mainland of China: the pentavalent RotaTeq (RV5, Merck & Co., USA) and the monovalent RV vaccine from China (RV1, Lanzhou Institute of Biological Products Co., China). The vaccination schedule of RV5 includes three doses at 3, 4, and 5 months of age, while RV1 can be used among children aged 2 months to 3 years and be given one dose in every year. Both two vaccines were not included in the Chinese NIP and were voluntary and self-paid vaccines. To date, there has been no scale-up of the program due to no known published data on cost considerations and local cost-effectiveness of the available RV vaccines. Cost-effectiveness analysis can provide the evidence to policy-makers about the health and cost consequences of the introduction of RV vaccines.11 It can also be used to compare the different product characteristics and help to support national or regional strategic planning and priority setting in the context of a constrained budget for public health. Through a single birth cohort model, we aimed to evaluate the cost-effectiveness and financial implications of the RV vaccination from the societal perspective and to compare the cost-effectiveness of the two RV products that could be used in the future. Methods Study design A decision tree-Markov model was used to estimate the potential cost-effectiveness and health impact of introducing RV vaccines into the Zhejiang provincial immunization program over a 5-year period from 2021 to 2025, compared to no RV vaccination (Figure 1). The length of each Markov state cycled was set as 1 year and the half-cycle correction was used in this model. We compared two RV vaccines to no vaccination, respectively. We also compared the cost-effectiveness of the RV vaccines to the willingness-to-pay (WTP) threshold of one time of the Gross Domestic Product (GDP) per capita of Zhejiang province in 2021 (16865.70US$). The WTP threshold, which was a value to represent “an estimate of what an individual might be prepared to pay for one year lived healthily,”12 allowed cost-effectiveness ratio to be interpreted. Figure 1. Overview of the decision tree-Markov model. International and local literature, government records, and local health database were reviewed. If available, local data, such as the primary data from local health agencies, were prioritized. An expert panel, comprising 10 multidisciplinary specialists from pediatric infectious disease specialists, pediatric gastroenterologists, vaccinologists, maternal and child nurses, health economists, public health practitioners, and immunization program managers were organized. Two workshops were responsible for reviewing the data inputs on disease burden, vaccine coverage, vaccine efficacy, vaccine costs, healthcare costs before our analysis, especially for their accuracy, completeness, and appropriateness. Population size, life-expectancy by single year of age and single calendar year were obtained from Zhejiang provincial bureau of statistics. The potential benefits of RV vaccination among children under 5 years of age were estimated through the number of RVGE case, outpatient visit, hospitalization, death, and disability-adjusted life year (DALY), with and without vaccination. DALY accounted for both life years lost due to early death and life years lived with the disease, which facilitated comparison with other potential public health interventions. RVGE cases referred to children in the community with RVGE. Outpatient visit constituted the case who visited (but not admitted) to outpatient clinic or emergency room. Hospitalization was defined as the case admitted to an inpatient health-care facility. RVGE was further classified as non-severe or severe by Vesikari score13 which was developed to help assess the severity of RVGE on 20 points, combining different symptoms such as diarrhea and vomit episodes, dehydration status, type of treatment and others. The non-severe RVGE was defined as the Vesikari score was <11 points and was presumed to lead to recovery, including cases following homecare and outpatient visits. Severe RVGE was defined as the Vesikari score was ≥11 points and involved outpatient/emergency room visit, hospitalization, and death. The healthcare treatment cost was estimated with and without vaccination intervention. Cost associated with non-severe RVGE homecare and outpatient visit, severe RVGE outpatient visit, and hospitalization was included, using the 2021 US$ for all costs. As recommended by WHO, the time preference for immediate benefits and the opportunity of investing present capital were represented by discounting the future health outcomes and costs at 3%.14 Sensitivity analyses were conducted using the discount rates of 1% and 5%, and ran scenarios using the high and low estimates of the following parameters: RV infection rate, vaccination coverage, vaccine efficacy for RV1 and RV5, vaccine price, health system cost projection, and health-care costs. Probabilistic sensitivity analysis (PSA) was used and ran the simulation 10,000 times to create a cost-effectiveness acceptability curve to calculate the probability of cost-effectiveness of the two RV vaccines at a WTP threshold of one time of the GDP per capita of Zhejiang province in 2021. Model parameters For all parameters which had perceived uncertainty in the data, the low and high ranges were set for the sensitivity analyses. If 95% confidence interval (CI) for parameter was not available, a wide range by subtracting or adding 25% of the base input value was applied (Table 1).Table 1. Input parameters for the cost-effectiveness of the rotavirus vaccination model. Parameters Level Central value Scenarios Source Lower bound Higher bound Demographic            Newborns in 2021   432026 - - Local dataset  Under-five mortality rate (/100000) 0–11 months 593 547 643 [15]   12–59 months 199 170 209    Under-five RVGE rate (/100000) 0–11 months 3805 2909 4894 [16,17]   12–23 months 3564 2725 4584     24–35 months 929 710 1195     36–47 months 482 369 620     48–59 months 152 116 195    Non severe RVGE case (/100000)   7374 5224 10870 [18]  Non severe RVGE outpatient visits (/100000)   685 239 2489 [19]  Severe for outpatient visit (/100000)   2315 1627 2867 [19]  Severe for hospitalization (/100000)   807 605 1009 [20]  Death (/100000)   48 19 90 [21] Cost (US$)            Non-severe RVGE homecare   10.49 7.87 13.12 [24]  Non-severe RVGE outpatient visit   64.34 41.14 95.44 [24]  Severe RVGE outpatient visit   218.27 129.1 331.34 [24]  Severe RVGE hospitalization   476.12 260.74 746.75 [24]  Price of RV1   35.00 30.00 42.00 Expert opinion  Price of RV5   62.00 56.00 69.00 Expert opinion  Vaccination cost   10.10 7.60 12.60 [25] Probability (%)            Vaccine efficacy for full-series of RV1   36.60 5.20 58.60 [23]  Vaccine efficacy for full-series of RV5   78.90 75.50 82.30 [22]  NIP-vaccination coverage   95 91 97 Expert opinion  Discount rate   3 1 5 [12]  Vaccine wastage rate   3 1 5 Expert opinion Health utility            Disability weight (%) Healthy 0.00 0.00 0.00 [26]   Non-severe homecare 18.80 12.50 26.40     Non-severe for outpatient visit 18.80 12.50 26.40     Severe for outpatient visit 24.70 16.40 34.80     Severe for hospitalization 24.70 16.40 34.80     Death 100.00 100.00 100.00    Duration of illness (years) Non-severe homecare 0.01 0.01 0.02 [14]   Non-severe for outpatient visit 0.01 0.01 0.02     Severe for outpatient visit 0.02 0.01 0.03     Severe for hospitalization 0.02 0.01 0.03   Costs originally in CNY, converted to USD using 2021 average exchange rate (1 USD = 6.7 CNY). Demographics A single cohort model was applied in this study. The birth cohort of 2021 of Zhejiang province was obtained from the Zhejiang bureau of statistics. The mortality of children under five years was obtained from the united nation interagency group for child mortality estimation.15 Disease burden To estimate the RV infection rate (per 100,000 per year, under 5 years), the most recent cross-sectional study by Carlos16 was reviewed. The age distribution data of RV infection were adapted from a report from the national diarrhea surveillance in the pre-vaccine period.17 The standard set of age distribution data was adjusted to get a more granular estimation of the proportion of RVGE occurring in every year period under 5 years of age, by using the parametric curve (Burr distribution). The proportion of non-severe RVGE case, including homecare and outpatient visits, was calculated by subtracting the incidence of severe RVGE case from the incidence of any symptomatic RVGE case, obtained from a systematic review and meta-analysis from LMICs.18 The incidences of non-severe outpatient visits and severe for outpatient visit proportion of RVGE outpatient visits were taken from a modeling study by Debellut.19 To estimate the proportion of RVGE hospital admissions, we calculated the number of hospital admissions due to diarrhea in children aged <5 years based on a study by Farthing20 and then multiplied this by the RV-positive proportion mentioned above. The RV mortality rate (for the pre-vaccination era) was obtained from the Global Burden of Disease study (GBD) study.21 All the disease burden input values were adjusted and discussed by the expert panel. The cumulative proportion of the RV infection spectrum was also adjusted to 100%. Vaccine coverage and efficacy Local data on DTP vaccination coverage rates were used as a proxy for the expected RV vaccine coverage. The DTP vaccination rate of 95% was the average annual coverage rate during the 2015–2021 period, which was obtained from the national vaccination coverage surveillance system. The low and high values were the lowest (91%) and highest (97%) rates in the set. Vaccine efficacy of RV5 was obtained from the randomized controlled trial implemented in China.22 The vaccine efficacy of RV1 was from the real-world data, and we assumed that the protective time for the first, second, and third doses could be maintained between 2, 3, and 5 years of age.23 Any herd effect of the RV vaccine was not considered in the analysis. Healthcare cost Briefly, costs were classified as the direct and indirect costs. Direct costs, whether medical or non-medical, included the costs of goods and services used in the entire treatment course. Indirect costs referred to the caregivers’ loss of productivity or income. Costs of homecare and healthcare visits and hospitalization for RVGE were derived from the investigation in two provinces in China in 2015.24 All the cost data is substituted with contemporary (2021) prices. For both health-care visits and hospitalizations, we itemized the costs where applicable. We then constructed interval estimates for each item using the local expert panel. The vaccine cost in this study was assumed to be half of the currently self-paid price per course for any kind of vaccine. This set was considered the previous experience that the prices have fallen by at least 50% if the vaccine was included in Zhejiang provincial immunization program. The wastage rate of RV vaccine was assumed at 3% by the expert panel. Vaccination cost per dose included the cost on vaccine handling, vaccination delivery, cold-chain management, adverse event management, and other service delivery to implement the vaccination strategy. Vaccination cost was calculated based on an investigation from 15 provinces in China in 2015,25 and the cost data was inflated to the level of 2021 year based on the World Bank inflation rate. Health utility To estimate DALYs, we used GBD disability weights for all-cause diarrheal disease as a proxy for RV disability weights,26 which was also used by a previous report from the Philippines.27 In this study, we used the same disability weight for calculating the DALY of non-severe RVGE, including homecare and outpatient visit cases. The duration of RVGE was a composite of the average length of hospital stay based on insurance claims, and the average pre-hospital symptomatic period reported by Carlos.14 Results Impact on healthcare costs and RV related disease events A summary of health-care costs and disease events with and without RV vaccination is presented in Table 2.Table 2. RV-related disease events, healthcare cost, and vaccine program costs over a 5-year period of RV vaccination program. Parameter Scenario No vaccination RV1 RV5 Disease events        Non-severe RVGE homecare 15775 10292 3950  Non-severe RVGE outpatient visit 11419 7452 2862  Severe RVGE outpatient visit 6819 4445 1705  Severe RVGE hospitalization 4248 2769 1067  Death 289 188 71  DALYs 4562.75 2975.93 1136.03 Averted disease events        Non-severe RVGE homecare - 5483 11825  Non-severe RVGE outpatient visit - 3967 8557  Severe RVGE outpatient visit - 2374 5114  Severe RVGE hospitalization - 1479 3181  Death   101 218  DALYs averted - 1586.82 3426.72 Cost (US$)        Healthcare costs 43622214.46 28384609.41 9266518.19  Averted healthcare costs - 15237605.05 34355696.27  Vaccination costs - 22587728.01 36110314.62  Incremental net costs - 7350122.96 1754618.35 All parameters were assumed to have Beta-PERT distribution. DALYs and costs were discounted at a rate of 3%. Estimates of disease events and health cost were based on the 2021 birth cohort (432026 new birth) and presented in absolute counts. No-RV vaccination Over a 5-year period, total health-care costs of RVGE for 2021 birth cohort under the non-universal RV vaccination scenario would be 43.6 million US$. Concurrently, there were estimated 27,194 cases of non-severe RVGE and 11,356 cases of severe RVGE. Furthermore, there would be about 18,238 outpatient visits, 4248 hospitalizations, and 289 deaths due to RVGE. RV1 vaccination With the RV1 vaccination program in place, health-care costs of RVGE would reduce by 35.0% among the 2021 birth cohort during a 5-year period compared to no vaccination, with an estimated 15.2 million US$ averted. Meanwhile, there would be 22.59 million US$ for the procurement and delivery of RV1. It would result in an incremental net cost of approximately 7.35 million US$ for the inclusion of RV1 in the Zhejiang provincial immunization program for a 5-year period. RV vaccination would also result in 9450 cases of non-severe RVGE and 3853 cases of severe RVGE averted over 5 years. Overall, there would be a decrease in the number of the outpatient visit and hospitalization and death by 6341, 1479, and 101, respectively. The corresponding reduction was 34.8%, 34.8%, and 35.0% for outpatient visit, hospitalization, and death. RV5 vaccination With the RV5 vaccination program in place, health-care costs of RVGE would reduce by 78.8% the 2021 birth cohort during a 5-year period compared to no vaccination, with an estimated 34.36 million US$ averted. Meanwhile, there would be 36.1 million US$ for the procurement and delivery of RV5. It would result in an incremental net cost of approximately 1.75 million US$ for the inclusion of RV5 in Zhejiang provincial immunization program for a 5-year period. RV vaccination would also result in 20,382 cases of non-severe RVGE and 8295 cases of severe RVGE averted over 5 years. Overall, there would be a decrease in the number of the outpatient visit, hospitalization, and death by 13,671, 3181, and 218, respectively. The corresponding reduction was 75.0%, 74.9%, and 75.4% for outpatient visit, hospitalization, and death. Cost-effectiveness analyses Base case In the base-case scenario, the cost-effectiveness ratio of RV5 was 512.04 US$ per DALY averted, while the cost-effectiveness ratio of RV1 was 4631.98 US$ per DALY averted. One-way sensitivity analyses Figures 2 and 3 show the sensitivity of the results to changes in key parameters. To illustrate this, each RV vaccine was compared to no RV vaccination, and we estimated sensitivity to the incremental cost-effectiveness ratio (ICER). For RV1 vaccination, the vaccine efficacy had the greatest impact on ICER. The ICER was 4582.65 US$ per DALY averted when the vaccine efficacy was 58.6%, while the ICER was 4965.27 US$ per DALY averted when the vaccine efficacy was 5.2%. For RV5 vaccination, the cost of RV5 had the greatest impact on ICER. The ICER was 469.12 US$ per DALY averted when the cost of RV5 was 56 US$, while the ICER was 561.51 US$ per DALY averted when the cost of vaccine delivery was 69 US$. Both RV1 and RV5 vaccination had ICERs less than 1 time GDP threshold at 1% and 5% discount rates. Figure 2. One-way sensitivity analysis on the cost-effectiveness of RV vaccination compared to the no-RV vaccination. Figure 3. Probability sensitivity analysis on the cost-effectiveness of RV vaccination compared to the no-RV vaccination. Probabilistic sensitivity analyses The lower costly vaccine, RV1, had the 72.3% probability of being cost-effective when compared to no vaccination, at a WTP threshold of 1 time of the GDP per capita of Zhejiang province in 2021 (16865.70US$). RV5 had the 88.4% probability of being cost-effective when compared to no vaccination, at a WTP threshold of 1 time of the GDP per capita. Both two RV vaccines clearly dominated the no-vaccine strategy by providing a DALY at a cost within the range of one time GDP per capita. Discussion We estimated that RV vaccination might have prevented 101 or 218 deaths in a one-year birth cohort. This more than 30% or 70% reduction in deaths was broadly consistent with the impact of RV vaccination observed in the real-world study, which found that the RV vaccines halved the RV-positive proportion among the diarrhea outpatient visits and hospital admissions.28 Compared to no RV vaccine, the use of RV vaccines in Zhejiang provincial immunization program was cost-effective in our study. Even in scenarios with the least or lowest level of the parameters, the cost-effectiveness results were still stable. This timely and province-specific analysis indicated that RV5 would be more cost-effective compared to RV1. Similar studies from China were consistent with our findings. Wang29 reported that including RV5 into nation immunization program was projected to prevent 62.6% of RVGE cases and 72.6% of deaths and RV1 would avert 20.3% of RVGE cases and 22.4% of deaths. The ICERs per quality-adjusted lift year (QALY) gained were 8833 US$ for RV5 and 26,759 US$ for RV1. Cui30 reported that RV5 showed very good cost-effectiveness, with the ICER of RV5 shifting from RV1 of 1715.11 US$ per QALY. RV5 had significantly decreased RVGE incidence compared to RV1. Besides, we should consider that the vaccine price of RV vaccines used in the analysis might not be reflective of the actual future tender prices of the vaccines. Procurement prices for these two RV vaccines were not available as they were self-paid vaccines in current time. The actual tender prices might be lower than those we used in this model, once these vaccines become available in Zhejiang provincial immunization program. It was important to note because the cost-effectiveness ratios from the base-case scenario and the sensitivity and probabilistic analyses showed RV5 had a higher value. In the PSA, RV5 also showed a higher probability of being cost-effective using 1 time GDP per capita as the WTP threshold when compared to no vaccination. Our modeling found that RV5 was the most cost-effective option despite the vaccine-cost was higher than RV1. This difference in health benefits occurred because we assumed that two RV vaccines had a different level of protection at the final dose. In this study, price of RV5 was a crucial determinant of the cost-effectiveness ratio. Due to a higher vaccine price per dose, the overall vaccination program cost for RV5 was among the highest. The importance of vaccine price had been previously illustrated in a previous report,31 who found that a reduction of almost 70% in price for RV5 in 2015 showed to be cost-effective. The vaccine price of RV5 used in this study was 20% lower than those used in that study. In fact, the potential entry of new vaccines into Zhejiang provincial immunization program would encourage more competitive pricing, possibly improving the cost-effectiveness. The cost-effectiveness analysis in health, which was measured by the incremental cost to obtain a unit of health outcome, was used to compare the costs and outcomes between different interventions. The evaluation was made based on a WTP threshold that represented a good value for money. WHO recommended that the cost-effectiveness threshold correspond to up to 3 times a country’s GDP per-capita10. The significance of this threshold was exhibited in an RV vaccination cost-effectiveness analysis in Bhutan in 2020, where the threshold was fixed at 1.5 times the country’s GDP per capita.32 ICERs exceeded the threshold in that analysis, and RV vaccination was not found to be cost-effective. Since there was no explicit threshold in Zhejiang province from the societal perspective, we applied the traditional threshold. As seen on the cost-effectiveness acceptability curve from PSA, both RV1 and RV5 had an optimal probability of being cost-effective. We assumed that the incremental health system costs for all two RV vaccines were the same, using the vaccination cost from an investigation as reference. However, this data would be not suitable as the investigation did not include Zhejiang province. In Zhejiang province, there were island and mountainous areas geographically difficult to access, which meant a vaccine requiring freezer storage conditions from a central area might incur additional substantial costs for transporting vaccines to the peripheral health centers. This would add to the incremental health system costs and could influence the choice of RV vaccination strategy. Based on this analysis, healthcare cost was considerably higher without any RV vaccination strategies. Our findings indicated a large economic burden on the out-of-pocket cost and loss of household income. Such health expenditures for RVGE could result in medical impoverishment of economically marginalized households, and it might also be averted by RV vaccination in the poorest-quintile households in developing countries. Collectively, this body of evidence provided the additional support to the inclusion of RV vaccine in Zhejiang provincial immunization program. Additionally, the selection of RV vaccine should also consider the relevant aspects like feasibility, affordability and other specific factors. Our analysis included some limitations. First, some of the parameters were based on global or local estimates, which would influence the generalizability of our findings. To mitigate this aspect, we were able to share these estimates with an expert team to ensure consensus around the inputs that were selected. Second, we did not account for the herd immunity, effects of breastfeeding, adverse neurodevelopmental outcomes or malnutrition following severe RVGE. Altogether, these aspects constituted current gaps in this study. Third, we made several assumptions to differentiate the current RV vaccines based on the vaccine efficacy and price. These parameters would be updated over time, and the results should be updated with more relevant data when possible. Conclusion Our results supported that a universal RV vaccination strategy was expected to offer good value for cost-effectiveness in Zhejiang province. Using any of the two China-licensed RV vaccines would avert 30–70% of outpatient visits, hospitalizations, and deaths from RVGE, compared to no vaccination. RV5 vaccination had the best value-for-money with the lowest ICER compared to no vaccination strategy. However, given the uncertainty in vaccine price, both RV1 and RV5 were likely to be cost-effective options. The choice of RV vaccine should be continually reevaluated as more evidence on health system costs, wastage rates, vaccine prices, relative health impacts emerge well as the WTP threshold changes. Acknowledgments We would like to thank the following representatives of various organizations who formed our local expert panel and participated in our consultative meetings and interviews and contributed technical support and expert opinions. Author contributions Y.H. and HK. L. conceived and designed the experiments; FX. C and Y.W. performed the experiments; Y.P.C. and Y.W. analyzed the data; FX. C. contributed reagents/materials/analysis tools; Y.H. wrote the paper. Disclosure statement No potential conflict of interest was reported by the author(s). Ethics approval and consent to participate This study was exempted for ethics approval as it did not contain any personal information and include no human research participants. ==== Refs References 1. Collaborators GBDDD. Quantifying risks and interventions that have affected the burden of diarrhoea among children younger than 5 years: an analysis of the global burden of disease study 2017. Lancet Infect Dis. 2020;20 :37–8. doi:10.1016/S1473-3099(19)30401-3.31678029 2. Clark A, Black R, Tate J, Roose A, Kotloff K, Lam D, Blackwelder W, Parashar U, Lanata C, Kang G, et al. Estimating global, regional and national rotavirus deaths in children aged <5 years: current approaches, new analyses and proposed improvements. PloS One. 2017;12 :e0183392. doi:10.1371/journal.pone.0183392.28892480 3. Yu J, Lai S, Geng Q, Ye C, Zhang Z, Zheng Y, Wang L, Duan Z, Zhang J, Wu S, et al. 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==== Front Oncoimmunology Oncoimmunology Oncoimmunology 2162-4011 2162-402X Taylor & Francis 10.1080/2162402X.2023.2233403 2233403 Version of Record Research Article Original Research Longitudinal immune monitoring of patients with resectable esophageal adenocarcinoma treated with Neoadjuvant PD-L1 checkpoint inhibition T. VAN DEN ENDE ET AL. ONCOIMMUNOLOGY https://orcid.org/0000-0003-2830-2310 van den Ende Tom a b Ezdoglian Aiarpi c d e Baas Lisanne M. c e Bakker Joyce c e Lougheed Sinéad M. c e Harrasser Micaela c e f Waasdorp Cynthia e f g van Berge Henegouwen Mark I. h i Hulshof Maarten C.C.M. i j Haj Mohammad Nadia k van Hillegersberg Richard l Mook Stella m van der Laken Conny J. d van Grieken Nicole C.T. e n Derks Sarah c e f Bijlsma Maarten F. e f g van Laarhoven Hanneke W.M. a b de Gruijl Tanja D. c e a Department of Medical Oncology, Amsterdam UMC, University of Amsterdam , Amsterdam, The Netherlands b Cancer Center Amsterdam, Imaging and Biomarkers , Amsterdam, The Netherlands c Department of Medical Oncology, Amsterdam UMC, Vrije Universiteit Amsterdam , Amsterdam, Netherlands d Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Rheumatology and Clinical Immunology, Amsterdam Institute for Infection and Immunity , Amsterdam, The Netherlands e Cancer Center Amsterdam, Cancer Biology and Immunology , Amsterdam, The Netherlands f Oncode Institute , Utrecht, The Netherlands g Laboratory for Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Amsterdam UMC, University of Amsterdam , Amsterdam, The Netherlands h Department of Surgery, Amsterdam Umc, University of Amsterdam , Amsterdam, The Netherlands i Cancer Treatment and Quality of Life, Cancer Center Amsterdam , Amsterdam, The Netherlands j Department of Radiotherapy, Amsterdam Umc, University of Amsterdam , Amsterdam, The Netherlands k Department of Medical Oncology, UMC Utrecht, Utrecht University , Utrecht, The Netherlands l Department of Surgery, UMC Utrecht, Utrecht University , Utrecht, The Netherlands m Department of Radiotherapy, UMC Utrecht, Utrecht University , Utrecht, The Netherlands n Department of Pathology, Amsterdam UMC, Vrije Universiteit Amsterdam , Amsterdam, The Netherlands CONTACT Tom van den Ende t.vandenende@amsterdamumc.nl Department of Medical Oncology, Amsterdam UMC, University of Amsterdam , Meibergdreef 9, Amsterdam 1105 AZ 17 7 2023 2023 17 7 2023 12 1 2233403Integra13 7 2023 Integra13 7 2023 03 3 2023 17 5 2023 02 7 2023 © 2023 The Author(s). Published with license by Taylor & Francis Group, LLC. 2023 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. ABSTRACT The analysis of peripheral blood mononuclear cells (PBMCs) by flow cytometry holds promise as a platform for immune checkpoint inhibition (ICI) biomarker identification. Our aim was to characterize the systemic immune compartment in resectable esophageal adenocarcinoma patients treated with neoadjuvant ICI therapy. In total, 24 patients treated with neoadjuvant chemoradiotherapy (nCRT) and anti-PD-L1 (atezolizumab) from the PERFECT study (NCT03087864) were included and 26 patients from a previously published nCRT cohort. Blood samples were collected at baseline, on-treatment, before and after surgery. Response groups for comparison were defined as pathological complete responders (pCR) or patients with pathological residual disease (non-pCR). Based on multicolor flow cytometry of PBMCs, an immunosuppressive phenotype was observed in the non-pCR group of the PERFECT cohort, characterized by a higher percentage of regulatory T cells (Tregs), intermediate monocytes, and a lower percentage of type-2 conventional dendritic cells. A further increase in activated Tregs was observed in non-pCR patients on-treatment. These findings were not associated with a poor response in the nCRT cohort. At baseline, immunosuppressive cytokines were elevated in the non-pCR group of the PERFECT study. The suppressive subsets correlated at baseline with a Wnt/β-Catenin gene expression signature and on-treatment with epithelial–mesenchymal transition and angiogenesis signatures from tumor biopsies. After surgery monocyte activation (CD40), low CD8+Ki67+ T cell rates, and the enrichment of CD206+ monocytes were related to early recurrence. These findings highlight systemic barriers to effective ICI and the need for optimized treatment regimens. KEYWORDS Chemotherapy esophageal neoplasm immune system immunotherapy Neoadjuvant funded by Hoffmann-La Roche Ltd The PERFECT trial was funded by Hoffmann-La Roche Ltd., Basel, Switzerland. ==== Body pmcIntroduction The main treatment modality for locally advanced resectable esophageal adenocarcinoma (rEAC) is a combination of chemotherapy, radiotherapy, and surgery. In several countries the neoadjuvant CROSS regimen is considered a standard of care for rEAC.1 However, despite the benefit of neoadjuvant chemoradiotherapy (nCRT) over surgery alone, 49% of patients will develop disease recurrence within 5 years.2 Recently, the CheckMate 577 trial established the value of adjuvant nivolumab (anti-PD-1 antibody) in patients with residual disease after nCRT.3 The hazard ratio for disease recurrence or death was 0.69 (95% CI, 0.56 to 0.86) in favor of the nivolumab arm. In a non-randomized phase II study (PERFECT) we investigated whether the addition of an immune checkpoint inhibitor (ICI), atezolizumab (anti-PD-L1), to nCRT enhanced the efficacy of neoadjuvant treatment.4 Treatment was feasible but there was no significant difference in response or survival compared to a propensity matched nCRT cohort.4 However, there is a strong relationship between pathological complete response (pCR) and long-term outcome, not only for chemoradiation in esophageal cancer but also for neoadjuvant ICI.5,6 Identifying factors related to pathological response could lead to better patient selection through biomarkers or identify mechanisms of treatment resistance. In several tumor types flow cytometry of peripheral blood mononuclear cells (PBMCs) has identified a number of checkpoint molecules and cell types which are altered under the influence of systemic therapy.7,8 In patients with lung cancer and melanoma treated with anti-PD-1 or anti-CTLA-4 immunotherapy, complete and partial radiological responders had higher expression of PD-1 on CD8+ on-treatment or more baseline CD69+ natural killer (NK) cells compared to non-responders.9,10 In poor responders higher frequencies of inhibitory myeloid-derived suppressor cells (MDSCs) or regulatory T cells (Tregs) were found in patients treated with an anti-CTLA-4 antibody (melanoma) or a bi-specific T cell engager (B-precursor acute lymphoblastic leukemia).11,12 Flow cytometry of immune cells thus provides important clues for response and resistance mechanisms in the immunotherapy setting. The abundance of certain PBMC subsets in different response groups is most likely driven by inter-compartmental cross-talk between the tumor microenvironment (TME), lymph nodes, and the blood compartment.8 Local immune evasion harnessed by tumor cells can affect immune cell subsets within the TME and, through the lymph nodes, may have a profound influence on the PBMC compartment.8,13 A relevant example of the aforementioned crosstalk in the context of esophageal cancer is the release of TGF-β from cancer cells in response to radiation, which can lead to the expansion of immunosuppressive Tregs in vitro.14 The release of therapy-induced TGF-β has also been linked in rEAC to epithelial to mesenchymal transition (EMT).15 EMT is characterized by the gradual transformation of epithelial cells to a mesenchymal phenotype which endows them with more immunosuppressive and migratory properties.16,17 However, it is not yet known whether the immunosuppression associated with EMT in the TME can be measured in the peripheral blood. Besides offering novel prognostic or predictive parameters, this may have important implications for the design of new therapeutic strategies based on combined immunotherapeutic approaches. We hypothesize that immunosuppressive pathways such as EMT activated within the TME are reflected in PBMC subsets. In this immune monitoring study, we aimed to identify differential immune profiles based on response status through 14-color-flow cytometry phenotyping of PBMCs and serum cytokine measurements of ICI + nCRT treated rEAC patients before and throughout treatment. Flow cytometry results were compared with an nCRT cohort to find signatures specific for the neoadjuvant ICI combination. In addition, an exploratory analysis was performed on the relationship between PBMC subsets and transcriptomic signatures measured in tumor biopsies. Patients and methods This is a translational exploratory immune profiling substudy of the previously published single-arm PERFECT trial (NCT03087864) investigating the safety and efficacy of atezolizumab, a PD-L1 antibody, combined with nCRT, and subsequent esophagectomy for patients with rEAC.4 In this trial we collected blood samples including heparin and serum tubes at baseline (B), on-treatment in week 5 (OT), before surgery (S) and 3 months after surgery (FU). From the heparin tubes, PBMCs were isolated by gradient centrifugation for flow cytometry analysis, and serum was stored at −80°C for cytokine measurements. An exploratory comparison of the flow cytometry results was performed with a previously published rEAC cohort treated with nCRT without ICI.7 Both cohorts included histologically confirmed, stage II and III esophageal- or gastroesophageal junction adenocarcinomas treated with nCRT given according to CROSS.1 All patients provided written, informed consent for study participation. This study was conducted in accordance with the Declaration of Helsinki and the international standards of good clinical practice. Patient groups A selection of 24 patients from the PERFECT trial were included for this immune monitoring study based on response to neoadjuvant treatment. Response was assessed after neoadjuvant therapy by PET-CT to rule out pre-operative progression and for patients who proceeded to surgery by the pathologist in the resection specimen according to the tumor regression grade (TRG). In this study we included the pathological complete responders (pCR; ypT0N0; n = 9) and a representative selection of the non-pCR patients (n = 15) with subtotal (ypT0N+ or TRG2; n = 5), partial responders (TRG4; n = 5) and patients with pre-operative progression (n = 5). From the nCRT-only cohort all 26 patients were included as exploratory comparator to the PERFECT study.7 From the 26 patients, seven had a pCR and 19 had residual disease in the resection specimen (non-pCR). PBMC isolation PBMCs were isolated from heparinized blood (20 ml) and cryopreserved until analysis, all as previously described.18 In brief, a ficoll (Lymphoprep, STEMCELL technologies) gradient centrifugation protocol was used to isolate the PBMC fraction. The PBMC fraction was viable frozen with cell freezing medium (25% dimethyl sulfoxide, 75% fetal calf serum) and stored in liquid nitrogen until defrosted upon analysis. Flow cytometry of PBMCs Flow cytometry was performed on thawed PBMCs with multicolor panels to characterize the frequency and activation status of lymphocytic, dendritic, monocyte, and myeloid subsets as previously published.7 The samples collected before, during, and after treatment were stained according to customized panels (Table S1) and measured on a flow cytometer (LSRFortessa, BD). Due to a remarkably higher activation of monocytes in patients with a poor outcome at FU, the samples were further characterized by a panel based on different macrophage markers (Table S2). Patients were stained in multiple batches in a random order to make sure the response groups were not acquired together. Standard compensations for every channel were made and updated to ensure consistency in the obtained results. Application settings were used to ensure the reproducibility of the experiments. Non-lineage markers were gated against a control tube without the fluorophores of interest. For gating procedures in FCS Express Version 6, see Fig. S1-S3. Serum cytokine measurements Thawed serum samples were only available and used from the PERFECT trial at baseline, on-treatment, and before surgery. Serum samples were tested by enzyme-linked immunoassay (ELISA; R&D Systems, DuoSet, Minneapolis, Minnesota) to measure latent and active TGF-β1 according to the instructions of the manufacturer. A custom cytokine panel (IL-6, IL-8, IL10, CXCL9, CXCL10, CCL2, CCL5, and VEGF) was used to measure serum levels by cytometric bead array (CBA; BD, Flex-set, Franklin Lakes, New Jersey) on the LSRFortessa according to manufacturer’s instructions. Measurement of supernatant through CBA (IL-6, IL-8, IL10, CXCL10, TNF, CCL2, and CCL5) from magnetic-activated cell sorted CD14+ PBMCs from the FU time-point was done before and 24 h after stimulation according to the following conditions: M1 (100 ng/ml LPS and 20 ng/ml IFN-γ) M2a (10 ng/ml IL-4 and 10 ng/ml IL-10) and Poly IC (100 ug/ml). RNA-sequencing data-set The previously reported RNA-sequencing set with baseline and on-treatment biopsies from PERFECT (GSE165252) was used to correlate PBMC subsets to the MSigDB Hallmark gene sets.19 Moreover, exploratory sub-analyses were performed based on a library of EMT signatures (EMTome) and a canonical 16-gene EMT signature validated in a pan-cancer cohort of The Cancer Genome Atlas (TCGA).20,21 This 16-gene EMT signature consists of 13 mesenchymal markers (VIM, CDH2, FOXC2, SNAI1, SNAI2, TWIST1, FN1, ITGB6, MMP2, MMP3, MMP9, SOX10, GCS) and three epithelial marker genes (CDH1, DSP, and OCLN).21 An EMT score per biopsy was calculated by adding up the values of the log2 transformed values for the mesenchymal markers subtracted by the epithelial markers. Changes between baseline and on-treatment were calculated by the following calculation: delta EMT = on-treatment EMT score minus baseline EMT score. A validated three-gene signature (CCR8, MAGEH1, and LAYN) was used to estimate the abundance of intra-tumoral Tregs by calculating a Z-score across biopsies, and this was correlated with the MSigDB Hallmark gene sets.22 Statistical analyses The unpaired T-test (normal distribution) was used to compare the pCR to the non-pCR group for each immune cell subtype and the expression of surface proteins within subsets. For the FU samples from PERFECT we compared patients with a recurrence vs. no recurrence with the unpaired T-test (normal distribution). The Mann – Whitney U test was used to compare changes between time-points and the cytokine levels between the pCR and non-pCR groups as this data was not normally distributed. Longitudinal analyses were tested through a mixed-effects model to assess the overall change over time. Correlations were assessed through the Pearson correlation coefficient. The Cox proportional-hazards model including the logrank test was used to compare progression-free survival (PFS) and disease-free survival (DFS). The application Cutoff Finder or split on the median was used to stratify patients in two groups for Kaplan – Meier analysis.23 Data cutoff for the survival analyses was 04-01-2021. GraphPad Prism version 9.1 and SPSS version 26.0 (IBM) were used for statistical analyses. An α below 0.05 was regarded as statistically significant. All statistical tests were conducted two-sided. Results The baseline characteristics and clinical outcomes of included PERFECT trial patients (n = 24) are described in Table 1. Clinical characteristics from the nCRT-only cohort are also given in Table 1 and were comparable to those of PERFECT patients.7 Baseline and on-treatment samples were available in PERFECT from 24 patients, before surgery from 16 and 3 months after surgery from 15 patients. In the nCRT-only cohort baseline samples were available from 26 patients, on-treatment from six patients and before surgery from 13 patients.Table 1. Baseline characteristics and clinical outcomes from both patient cohorts. Variables PERFECT nCRT-only   (n = 24) (n = 26) Age, years     Median 66 67,5 Range (40-73) (42-79) Sex     Male 21 (87.5) 21 (80.8) Female 3 (12.5) 5 (19.2) Clinical tumor stage     cTx 0 1 (3.8) cT2 6 (25) 2 (7.7) cT3 17 (70.8) 23 (88.5) cT4a 1 (4.2) 0 Clinical nodal stage     cNx 0 1 (3.8) cN0 8 (33.3) 11 (42.3) cN1 11 (45.8) 10 (38.5) cN2 5 (20.8) 2 (7.7) cN3 0 2 (7.7) Tumor location     Mid 2 (8.3) 0 Distal 17 (70.8) 21 (80.8) GEJ 5 (20.8) 5 (19.2) Pathological response     pCR 9 (37.5) 7 (26.9) ypT+ or ypN+ or progression 15 (62.5) 19 (73.1) Recurrence     Yes 15 (62.5) 12 (46.2) No 9 (37.5) 14 (53.8) Abbreviations: GEJ = gastroesophageal junction; nCRT = neoadjuvant chemoradiotherapy; pCR = pathological complete response. Immunosuppressive PBMC profiles observed in non-complete responders Multicolor flow cytometry was performed on PBMCs of rEAC patients to assess systemic immune changes before and throughout treatment, Figure 1a. At baseline we observed distinct differences in the main immune cell subsets between the response groups of the PERFECT trial (pCR vs. non-pCR) as shown in a heatmap, Figure 1b. A higher mean % of immunosuppressive Tregs (3.48% vs. 4.60%, p = 0.02; Figure 1c) was found in the non-pCR group, while the pCR group had a higher % of type-2 conventional dendritic cells (cDC2; 0.45% vs. 0.27%, p = 0.003; Figure 1d). Two suppressive subsets from the myeloid lineage were also more abundant in the non-pCR group: intermediate monocytes (IM; CD14+CD16+, 1.94% vs. 3.68%, p = 0.01; Figure 1e) and early myeloid-derived suppressor cells (eMDSCs; lineage-CD14-HLA-DR-CD33+CD11b+, 0.24% vs. 0.61%, p = 0.04; Figure 1f). Next, we assessed different immune stimulatory and suppressive checkpoints on each subset. Only CTLA-4+ expression on CD8+ T cells (pCR 2.75% vs. non-pCR 5.36%, p = 0.01; Figure 1g) and HLA-DR+ on CD16-CD56+ NK cells (pCR 52.80% vs. non-pCR 36.95%, p = 0.03; Figure 1h) differed significantly between both response groups at baseline. Additionally, we investigated if baseline PBMC subsets were related to survival: a high percentage of Tregs (Log-rank p = 0.04) and low number of cDC2 (Log-rank p = 0.03) were both associated with poor PFS while IM and eMDSCs were not, Fig. S4.Figure 1. Baseline flow cytometry results of PBMC subsets in patients from the PERFECT trial. a) Treatment regimen with blood sampling schedule from patients treated with anti-PD-L1 combined with neoadjuvant chemoradiotherapy. b) Heatmap of baseline PBMC subset frequencies divided by pCR and non-pCR. The response to neoadjuvant therapy is given on the horizontal axis. Values were normalized between 0 and 100 within each subset based on percentages from singlets (CD3, NK, B cells, cDC1, cDC2, pDC, CD14, mMDSC, eMDSC), parent CD3 (CD8, CD4), parent CD4 (Treg, aTreg, and rTreg) and parent CD14+ (NCM, IM, and CM). The asterisk behind subsets denotes significant differences between the pCR and non-pCR groups. c-h) Baseline PBMC subset and marker frequencies with significant differences between the pCR and non-pCR groups. The pCR and non-pCR groups were statistically tested with the unpaired t-test. Abbreviations: cDC = conventional dendritic cells; CM = classical monocytes; eMDSC = early myeloid-derived suppressor cells; ICI = immune checkpoint inhibitor; IM = intermediate monocytes; mMDSC = mature myeloid-derived suppressor cells; NCM = non-classical monocytes; nCR = non-complete response; nCRT = neoadjuvant chemoradiotherapy; NK = natural killer cell; pCR = pathological complete response; pDC = plasmacytoid dendritic cells; Tregs = regulatory T cells. In the PERFECT trial we evaluated the dynamics of the following major PBMC subtypes: T cells (CD3), monocytes (CD14), B cells (CD19), and NK cells (CD56) by a mixed-effects model. A significant drop in T cells was observed irrespective of response on-treatment (Figure 2a). This was accompanied by an increase in the monocyte fraction (Figure 2b). Moreover, on-treatment the % of B cells (Figure 2c) and NK cells (Figure 2d) significantly dropped and recovered in both response groups before surgery. These findings are consistent with myelosuppression due to the co-administration of chemotherapy in the PERFECT trial regimen. Next, we assessed if subsets and checkpoints differed between response groups based on the on-treatment time-point, Figure 2e. A higher percentage of total Tregs (4.13% vs. 6.15%, p = 0.03) and activated Tregs (aTregs; CD45RA-FOXP3++, 2.44% vs. 4.25%, p = 0.003; Figure 2f) was found in the non-pCR group. In matched baseline and on-treatment samples, the aTreg fraction significantly increased in the non-pCR group, p = 0.02; Figure 2g. Interestingly, when we combined a metric for T cell proliferation (CD8+Ki67+) and the aTreg percentage into a ratio, we discovered that the pCR group had a higher ratio compared to the non-pCR group (5.66 vs. 2.79, p = 0.02; Figure 2h), indicating less immune suppression in the pCR group. Findings regarding the three different monocyte subsets were comparable to baseline with a higher percentage of CD14+CD16+/CD16++ (respectively intermediate and non-classical) monocytes in the non-pCR group (Figure 2e-i). Moreover, CD14+CD16- classical monocytes were consequentially higher in pCR patients (p = 0.01; Figure 2j). Other significant findings on-treatment were a higher CD16+CD56+NK cell fraction (p = 0.049; Table S3) and a higher percentage of CD86+ B cells in the pCR group (p = 0.04; Table S3). Correlations between subsets and survival revealed a low CD8Ki67/aTreg ratio on-treatment was associated with poor PFS (Log-rank p = 0.008), while the aTreg delta or non-classical monocytes on-treatment were not associated with long-term outcome, Fig. S4. Figure 2. Longitudinal flow cytometry results of PBMC subset rates and marker expression in patients from the PERFECT trial. a) Longitudinal results of CD3+ T cells in the pCR group (green) and the non-pCR group (red). b) Longitudinal results of CD14+ monocytes in the pCR group (green) and the non-pCR group (red). c) Longitudinal results of CD19+ B cells in the pCR group (green) and the non-pCR group (red). d) Longitudinal results of CD56 natural killer cells in the pCR group (green) and the non-pCR group (red). e) Heatmap of on-treatment PBMC subset rates between the pCR and non-pCR groups. Data were normalized within each subset. For some patients no data were available from specific markers due to insufficient cells for flow cytometry analysis. This is marked by an empty box with a cross. Values were normalized between 0 and 100 within each subset based on percentages from singlets (CD3, NK, B cells, cDC1, cDC2, pDC, CD14, mMDSC, eMDSC), parent CD3 (CD8, CD4), parent CD4 (Treg, aTreg, and rTreg) and parent CD14+ (NCM, IM, and CM). The asterisk behind subsets denotes significant differences between the pCR and non-pCR groups. f) Activated Treg frequencies on-treatment with the individual values for the pCR and non-pCR groups. The pCR and non-pCR groups were statistically tested with the unpaired t-test. g) Changes (delta) between baseline and on-treatment rates of activated Tregs with the individual values for pCR and non-pCR groups. The pCR and non-pCR groups were statistically compared with the Mann–Whitney U test. h) The proliferating (Ki67+) CD8 T-cell to activated Treg ratio between the pCR and non-pCR groups on-treatment. The pCR and non-pCR groups were statistically tested with the unpaired t-test. i-j) The CD14+CD16++ non-classical monocytes and CD14+CD16- classical monocytes on-treatment with the individual values for the pCR and non-pCR groups. The pCR and non-pCR groups were statistically tested with the unpaired t-test. Abbreviations: CM = classical monocytes; ICI = immune checkpoint inhibitor; IM = intermediate monocytes; NCM = non-classical monocytes; non-pCR = non-complete response; nCRT = neoadjuvant chemoradiotherapy; pCR = pathological complete response; Tregs = regulatory T cells. At the before-surgery time-point, the distribution of subsets was again showing higher percentages of CD14+CD16+/CD16++ monocytes but also CD4+CD45RA-CD27+ central memory cells in the non-pCR group. The higher percentage of central memory CD4 T cells after neoadjuvant treatment in non-pCR patients could be related to weak (co-)stimulation of naïve CD4 T cells and their consequent inability to switch to an effector phenotype.26,27 For the main PBMC subsets we also investigated if changes between baseline and on-treatment or before surgery were associated with pathological response. Besides the change in aTregs, between baseline and on-treatment, there were no other statistically significant differences between response groups, Table S4. A full overview of flow cytometry results from PERFECT can be found in Table S3. In summary based on flow cytometry immune profiling of PBMCs, we observed a higher abundance of immunosuppressive subsets in non-pCR patients. We next questioned whether our findings were specific for the anti-PD-L1 regimen combined with nCRT in the PERFECT trial. Therefore, we compared our data with results from 26 rEAC patients treated with nCRT-only without ICI from a previously published immune monitoring study.7 In the nCRT-only cohort we assessed if there was also a difference at baseline in PBMC subsets between response groups, Fig. S5A. Only the percentage of CD8 T-cells was significantly higher in the pCR group (37.54% vs. 21.81%, p = 0.004; Fig. S5B). No significant difference was found between the response groups based on the baseline immunosuppressive subsets (Tregs, CD14+CD16+ IM monocytes, eMDSCs) earlier identified in the PERFECT trial, Fig. S5C-E. Next, we assessed the dynamics in major subsets which roughly resembled the changes seen in the PERFECT trial with a decrease in T cells and an increase in monocytes, Fig. S6A-B. This was accompanied by a decrease in the % of B cells and a relatively stable % of NK cells, Fig. S6C-D. For the on-treatment time-point, the aTregs, CD8+Ki67+/aTreg ratio, aTregs delta, and non-classical CD14+CD16++ monocyte graphs are given in the supplementary for illustrative purposes, Fig. S6E-F. Relatively few samples (n = 6) were available for the on-treatment time-point in the nCRT-only cohort, and therefore no formal statistics were performed. A full overview of flow cytometry results from the nCRT-only cohort can be found in Table S5. These findings suggest that baseline circulating immunosuppressive subsets are associated with therapy resistance in the PERFECT trial but not in patients treated with nCRT-only, Table 2.Table 2. Main baseline flow cytometry findings compared between response groups in both cohorts. Immune subset/marker pCR nCRT+ICI non-pCR nCRT+ICI pCR nCRT-only non-pCR nCRT-only Baseline         CD8+ (of CD3) 25.79 (14.16–37.38) 30.14 (15.19–57.41) 37.54 (15.48–60.3)* 21.81 (8.93–45.6)* Tregs (of CD4) 3.48 (2.46–5.37)* 4.60 (2.92–6.44)* 3.91 (0.96–9.91) 2.67 (0.43–14.06) cDC2 (of singlets) 0.45 (0.31–0.61)* 0.27 (0.10–0.59)* 0.73 (0.11–1.75) 1.04 (0.07–2.96) IM (of CD14) 1.94 (0.41–3.74)* 3.68 (1.50–8.33)* 2.26 (0.1–6.12) 3.79 (0.29–7.26) eMDSCs (of singlets) 0.24 (0–0.64)* 0.61 (0.01–1.34)* 0.07 (0–0.23) 0.13 (0–0.9) Mean and range are given for each group. An asterisk denotes statistical significance between both response groups (T-test). Abbreviations: cDC2 = type-2 conventional dendritic cells; eMDSCs = early myeloid derived suppressor cells; ICI = immune checkpoint inhibitor; IM = intermediate monocytes; nCRT = neoadjuvant chemoradiotherapy; pCR = pathological complete response; Tregs = regulatory T cells. Immunosuppressive cytokines elevated in serum of poor responders We measured several cytokines (TGF-β1, IL-6, IL-8, IL10, CXCL9, CXCL10, CCL2, CCL5, and VEGF) in the serum of patients to assess their relationship to the identified PBMC subsets (observed by cytometry to be related to response) and directly to response to treatment in the PERFECT trial at three time-points: baseline, on-treatment and before surgery. Correlations were assessed between baseline cytokines, Tregs, cDC2, intermediate monocytes, and eMDSCs, Figure 3a. Only activated TGF- β1 positively correlated with the PBMC Treg percentage, r = 0.58, p < 0.01; Figure 3b. No correlation was found between monocytes and cytokines in serum related to monocyte functionality and recruitment, i.e. CCL2, CCL5, VEGF, TGF-β1, and IL10. This indicates that monocytes are not the sole producers or interactors with these molecules. Comparing correlations between the aforementioned baseline subsets, Tregs were negatively correlated with cDC2 abundance,r = −0.47, p = 0.02, Figure 3a. On-treatment there was no significant correlation between PBMC subsets such as (a)Tregs or monocytes and cytokine serum levels, Fig. S7. Figure 3. Cytokine levels in serum samples from patients treated in the PERFECT trial. a) Pearson correlation matrix between baseline subset rates and serum cytokine levels. Statistically significant correlations are highlighted by black outlined squares, p < 0.05. b) Pearson correlation of CD4+ Tregs and activated TGF-β1 at baseline. c-f) Serum concentrations of IL-6, IL-8, IL-10, and VEGF at the baseline of the pCR and non-pCR groups. The pCR and non-pCR groups were statistically compared with the Mann–Whitney U test. Abbreviations: eMDSC = early myeloid-derived suppressor cells; IL = interleukin; IM = intermediate monocytes; non-pCR = non-complete response; PB = peripheral blood; pCR = pathological complete response; TGF-β1 = transforming growth factor β1; Tregs = regulatory T cells. To assess if there was also a difference in serum markers between response groups in the PERFECT trial, we compared serum levels between pCR and non-pCR patients. At baseline several cytokines showed higher levels in the non-pCR group compared to the pCR group (IL-6 p = 0.03, IL-8 p = 0.02, IL-10 p = 0.03 and VEGF p = 0.08; Figure 3c-f). Interestingly, baseline IL-6, IL-10, and VEGF serum levels also correlated with each other, suggestive of a coordinated immune suppressive program in the non-pCR group, Figure 3a, Table S6. The same trend between cytokine levels and non-pCR was observed on-treatment although the difference was not statistically significant (IL-6 p = 0.10, IL8 p = 0.07, VEGF p = 0.08; Fig. S8). At surgery IL-8 (p = 0.03), CCL5 (p = 0.04) and VEGF (p = 0.02) were significantly higher in the non-pCR group, Fig. S8. Immunosuppressive pathways correlate with circulating PBMC subsets From the PERFECT trial patients RNA-sequencing data was available from baseline and on-treatment tumor biopsies. The sequencing data was used to identify pathways correlating with the abundance of the identified PBMC subsets differentially present between pCR and non-pCR patients. Baseline percentage of Tregs, cDC2, intermediate monocytes, and eMDSCs were correlated with the MSigDB Hallmark gene sets of matched baseline biopsies (n = 20). The Wnt/β-Catenin signaling pathway positively correlated with both Tregs and intermediate monocytes, p < 0.05, Figure 4a. Additionally, cDC2 negatively correlated with the pancreas beta cell pathway and intermediate monocytes positively correlated with hedgehog signaling and myogenesis, p < 0.05, Figure 4a. The eMDSC percentage correlated with the estrogen response and P53 pathway, p < 0.05, Figure 4a. These pathways have all been linked to the expansion of immunosuppressive immune cells or intratumoral immunosuppression.28–32 A full overview of the baseline correlations can be found in Table S7. Figure 4. Relationship between transcriptomic signatures in tumor biopsies and PBMC subsets in the PERFECT trial. a) Correlation between the MSigDB Hallmark gene-sets from baseline tumor biopsies and baseline PBMC subsets. Statistically significant correlations are highlighted by black outlined squares, p < 0.05. b) Correlation between the MSigDB Hallmark gene-sets from on-treatment biopsies and on-treatment circulating aTregs which reached a p value < 0.1. c) Correlation between the MSigDB Hallmark gene-sets from on-treatment biopsies and on-treatment intratumoral Tregs which reached a p value < 0.1. d) Correlation between the delta EMT signature between baseline and on-treatment and the delta aTregs measured by flow cytometry of PBMCs. e) Correlation between an EMT signature and the CD8+Ki67+/aTreg ratio measured by flow cytometry of PBMCs on-treatment. The gray lines demarcate the immunosuppressive low (top left corner) and immunosuppressive high groups from each other. f) Kaplan-Meier of PFS between patients with who were immunosuppressive low (CD8Ki67/aTreg ratio >median and EMT low ≤median) compared to immunosuppressive high (CD8Ki67/aTreg ratio ≤median or EMT high >median). In case no biopsy was available to measure EMT (n = 5) the CD8+Ki67+/aTreg ratio was used to classify patients. Abbreviations: aTreg = activated regulatory T cell; EMT = epithelial – mesenchymal transition; OT = on-treatment; PB = peripheral blood; PFS = progression-free survival; Tregs = regulatory T cells. Next, we correlated the circulating aTreg percentage, as the most significant finding on-treatment, to the Hallmark gene sets of matched on-treatment biopsies (n = 19). The top three pathways positively correlating with aTregs were angiogenesis, hedgehog, and TGFβ signaling, Figure 4b. We subsequently investigated if intratumoral Tregs were also associated with the same pathways as circulating aTregs. An intratumoral Treg signature (CCR8, MAGEH1, and LAYN) positively correlated in on-treatment biopsies (n = 31) with the following top three pathways: UV response down, EMT, and angiogenesis, Figure 4c. Thus, the angiogenesis pathway was positively correlated with both circulating aTregs and intratumoral Tregs. Also, TGF-β signaling and EMT, which were previously found to be related to each other, were at the top of positively correlating pathways, Figure 4b-c, Table S8.15 Further characterization of the EMT phenotype was done by correlating a library of EMT signatures across tumor types (EMTome) to the circulating aTregs.20,21 Several EMT signatures positively correlated with circulating aTregs, Table S9. From these signatures we identified a 16-gene pan-cancer EMT signature previously validated in the TCGA dataset including in EAC samples (see methods). This signature was used to estimate the change in EMT between matched baseline and on-treatment biopsies (n = 15). There was a positive correlation between induction of EMT and the change in circulating aTregs, r = 0.54, p = 0.04; Figure 4d. Interestingly, the CD8Ki67/aTreg ratio negatively correlated with the EMT signature, and based on both markers, two subgroups could be demarcated (gray lines: immunosuppressive low top-left corner vs. immunosuppressive high), r = −0.56, p = 0.01; Figure 4e. Having established that EMT and the CD8Ki67/aTreg ratio were related to each other, we investigated if both were related to PFS. After combining the on-treatment CD8+Ki67+/aTreg ratio and EMT signature, a relationship with long-term outcome was observed. Patients who were immunosuppressive low (CD8Ki67/aTreg ratio >median 2.31 and EMT low ≤median 91.1) had better PFS compared to immunosuppressive high (CD8Ki67/aTreg ratio ≤median 2.31 or EMT high >median 91.1), HR = 0.32 95% CI 0.12–0.88, p = 0.04; Figure 4f. The combined analysis performed better than EMT alone (cutoff median 91.1, log-rank p = 0.57) but not better than the CD8Ki67/aTreg ratio alone (cutoff median 2.31, log-rank p = 0.02). These findings suggest that immunosuppressive pathways such as EMT in the TME are related to intratumoral and circulating (a) Tregs and together with the CD8Ki67/aTreg ratio correlates with PFS. PBMC subsets related to recurrence after surgery For the 3-month time-point after surgery, we investigated if PBMC subsets or related markers were associated with recurrence of disease in the PERFECT trial. Out of 15 patients with data available from flow cytometry 11 experienced a recurrence vs. four who remained disease-free after a median of 809 days of follow-up. The % of CD8Ki67 positive T-cells was higher in patients without recurrence (p = 0.04; Figure 5a). In contrast, the % of intermediate monocytes was higher in patients who developed a recurrence (p = 0.04; Figure 5b). Moreover, the % of CD40+ monocytes in all three subsets was related to recurrence (Figure 5c-e). A full overview of flow cytometry results of the follow-up time-point can be found in Table S10. The activation status (CD40 expression) of classical (HR = 0.26 95% CI 0.08–0.88, p = 0.02; Figure 5f) and intermediate monocytes (HR = 0.31 95% CI 0.09–1.01, p = 0.06; Figure 5g) was related to DFS. Patients with low (<median) levels of expression showed superior DFS. To further explore the phenotype of monocytes after surgery based on recurrence status, we developed a flow cytometry panel based on M1 and M2 macrophage markers, Table S2. Only CD206 showed a statistically significant difference between both groups with a higher % of CD14+CD206+ M2-like monocytes in patients with a recurrence, Figure 5h. In the CD14+CD206+ population a higher % of CD163, CD169, PD-L1, and CD80 expression was found compared to the CD14+CD206- group, Figure 5i. These findings suggest that PBMC from patients with a recurrence are enriched for monocytes with an M2-like suppressive phenotype 3 months after nCRT/ICI treatment and surgery. Functional stimulation of CD14+ isolated monocytes from PBMCs with LPS/IFNy (M1-skewing cocktail), IL-4/IL-10 (M2a-skewing cocktail) or poly IC did not reveal any difference in cytokine profile based on recurrence status, Fig. S9. Figure 5. PBMC flow cytometry results three-months after surgery from the PERFECT trial stratified according to eventual recurrence status. a-e) Subset rates and marker expression levels from patients with and without a recurrence (unpaired t-test). Asterisk indicates statistical significance. f-g) Kaplan Maier of DFS between patients who were high or low in their percentage of classical or intermediate monocytes with CD40 expression, based on the median (<median vs. ≥median). h) The percentage of CD14+CD206+ cells in patients with and without a recurrence at FU. i) Macrophage markers across the CD206+ fraction and CD206- fraction from FU PBMC samples. Abbreviations: CM= classical monocytes; FU= three-months after surgery; IM= intermediate monocytes. Discussion In this immune monitoring sub-study of the anti-PD-L1/nCRT PERFECT trial, we investigated circulating immune cells through flow cytometry analysis and cytokine measurements in patients with rEAC. The non-pCR group had higher percentages of immunosuppressive immune cells and elevated cytokine levels compared to patients who had a pCR. Notably, circulating Treg and monocyte subsets were more abundant, while cDC2 cells were lower in the non-pCR group. Interestingly, this was not observed in a cohort of nCRT-only treated patients. Several immunosuppressive pathways in tumor biopsies were positively correlated with circulating Tregs and monocytes. Three months after surgery monocyte activation status, related to an M2-like phenotype, and a lack of CD8+Ki67+ T cells was predictive of recurrence. By flow cytometry and serum analyses, we observed an immunosuppressive subset profile in non-pCR patients. The non-responders from the PERFECT trial had more Tregs at baseline and an expansion of aTregs on-treatment reflected by a greater increase in the non-pCR group versus the pCR group. Tregs and a ratio for effector/regulatory T-cells have previously been linked to poor response and prognosis in several tumor types.18,33–35 Moreover, Tregs have been associated with inhibition of cDC2 cells present in tumor draining lymph nodes.36 The cDC2 cells are in this way not able to support a conventional CD4 T-cell response to anti-PD-1 therapy.36 In our study we also observed a higher percentage of baseline cDC2 in responders with a relatively lower Treg population. This response pathway related to cDC2 and CD4 anti-tumor immunity might also play a crucial role in rEAC patients treated with anti-PD-L1/nCRT and could be measurable in peripheral blood. Other PBMC subsets related to response were from the myeloid lineage the CD16+ monocytes and eMDSCs which showed higher circulating percentages in the non-pCR group. The presence and expansion of CD16-positive monocytes has previously been established in several tumor types and is an instrumental subset in the crosstalk between tumor and immune-system, resulting in tumor progression and immune suppression.37–40 In a recently published article, the intermediate blood monocytes of ovarian cancer patients were related to soluble immunosuppressive mediators and peritoneal tumor burden.41 Another myeloid subset associated with immunosuppressive regulation, the MDSCs were identified in our study as potential mediators of therapy resistance. MDSCs have been linked to immunosuppression and therapy resistance across different cancer types.24,25,42 In line with these flow-cytometry findings, the analyses of serum also provided evidence for systemic immune suppression including higher levels of IL-6, IL-8, IL-10, and VEGF in non-pCR patients. Across tumor types, these have all been related to cancer stage, prognosis, and immune suppression.43 Important to mention is that the flow cytometry findings in our study seem to be specific for the anti-PD-L1 combination regimen of the PERFECT trial. Determinants of response in PERFECT were mostly immunosuppressive subsets, while in the nCRT-only cohort CD8 T cell abundance and previously identified enrichment for effector memory CD8+ T cells were predictive of pathological response.7 It thus seems that in anti-PD-L1 non-responders there are specific immunological barriers to mount an effective immune-response both at baseline and throughout the course of treatment. In the PERFECT patients we established correlations between immunosuppressive pathways from RNA-sequencing data of tumor biopsies and PBMC subsets. At baseline Tregs and circulating monocytes positively correlated with the Wnt/β-Catenin signaling pathway. This pathway can actively regulate immune cell exclusion of the TME by interacting with tumor-associated macrophages and enhance Treg survival through Snail and β-catenin.28 Other pathways identified by us as positively correlating with intermediate monocytes (hedgehog, myogenesis) and eMDSCs (estrogen, p53) or negatively correlating with cDC2 (pancreas beta cells) have also been linked to immune-cell exclusion and expansion of suppressive immune cells in the TME.29–32 Additionally, on-treatment we identified overlap in pathways positively correlating with circulating aTregs and a signature for intratumoral Tregs. One of these was angiogenesis, which indeed can be associated with Treg proliferation; in colorectal cancer VEGF-A induced by the tumor can enhance circulating Treg proliferation by binding to VEGFR2.44 Moreover, anti-VEGF-A (bevacizumab) treatment in colorectal cancer patients reduced Treg proportions in peripheral blood.44 Another interesting observation was the correlation between the induction of EMT and expansion of aTregs. The relationship between EMT and immune suppression is well established across different tumor types.17 In esophageal cancer this relationship may in part be due to the release of TGF-β by cancer cells under the influence of chemoradiotherapy and the subsequent conversion of CD4 T-cells into Tregs.14,15,45 These Tregs are also able to produce TGF-β and thereby even further promote EMT as well as immune suppression.46 Interestingly in our cohort, the induction of EMT due to nCRT was related not just to a Treg signature in the TME, but also to a change in systemic aTreg rates. The measurement of aTregs by flow cytometry of peripheral blood could serve as a marker for EMT induction. New therapy combinations could be explored through on-treatment monitoring and stratification based on a positive delta threshold of aTregs or high CD8Ki67/aTreg ratio on-treatment. Turn-around time from sample to result could be available within 1 or 2 days. A potential clinical study design could be based on a PD-1/PD-L1 chemoradiotherapy backbone and additional escalation in patients with aTreg expansion on-treatment with anti-CTLA4/VEGF-A antibodies to deplete Tregs or selective TGF-β targeting. Important questions do, however, need to be answered in future studies regarding timing, dosing, toxicity, and biomarker validation. Remarkably, 3 months after surgery, the expression of CD40 on monocytes was predictive for eventual disease recurrence in the PERFECT trial. The CD40 costimulatory receptor can be found on a broad variety of antigen-presenting cells including monocytes.47 Activation of CD40 on monocytes can lead to the induction of proinflammatory cytokines (IL-1a, IL-1b, TNF-a, IL-6, and IL-12) and chemokines (IL-8, CCL2, CCL3, CCL4, and CCL5).47 Depending on the context, CD40 on monocytes can be pro-tumorigenic or tumoristatic.47 Evidence is emerging that CD40 may be involved in the activation status of tumor-associated macrophages and systemic immunosuppression.47,48 We also found less proliferating CD8+ T-cells in patients with a recurrence indicating there may be systemic barriers suppressing T-cell activity. The outgrowth of subclinical minimal residual disease into apparent metastases could be related to systemic T-cell suppression induced by monocytes and macrophages.49 In our cohort we found a CD206+ M2-like monocyte subset with potential suppressive capacity (CD163+ and PD-L1+), although we could not establish any functional cytokines which might be involved in such T-cell suppression after M1/M2a stimulation of CD14+ macrophages.49,50 The latter might be related to the low frequency of CD206+ cells or a different mechanism of action through cell–cell interaction or paracrine signaling. Another aspect relevant in this patient group is the relationship between surgery, inflammation, and the release of immunosuppressive factors.51 The recruitment of neutrophils and monocytes to the wound bed can reeducate these cell types to become immunosuppressive and pro-tumorigenic.51 Although it must be noted that surgery-related changes are usually transient, they could nevertheless have created an immune suppressive window.52,53 In conclusion, after additional validation, the expression of CD40 on monocytes could be a new biomarker for recurrence. Conclusions Immuno-monitoring of peripheral blood from rEAC patients by flow cytometry revealed distinct differences in the systemic immune-profile between complete and incomplete responders of the neoadjuvant PERFECT trial. Non-responders were defined by the presence of immunosuppressive subsets such as: Tregs, CD16+ monocytes and eMDSCs as well as elevated levels of immune suppressive cytokines. Pathways activated in the TME and associated with immune-exclusion, including the Wnt/β-Catenin pathway, positively correlated with these subsets. The abundance of aTregs on-treatment was associated with the angiogenesis pathway and induction of EMT. After surgery monocyte activation (CD40), low rates of CD8+Ki67+ T-cells and the enrichment of CD206+ monocytes were related to early recurrence within 2 years. This study identified systemic immunosuppressive barriers to neoadjuvant immuno-chemoradiotherapy and identified potential targets for future clinical studies. Supplementary Material Supplemental Material Click here for additional data file. Acknowledgments The authors would like to acknowledge all the participants of the PERFECT trial and supporting staff. Disclosure statement MIvBH is consultant for Mylan, Johnson & Johnson, Alesi Surgical, BBraun and Medtronic, and received unrestricted research grants from Stryker. All fees paid to institution. NHM has served as a consultant for MSD, BMS, Astra Zeneca, Servier and Lilly. MFB received research funding from Celgene and Lead Pharma and has acted as a consultant for Servier. HWMvL: Consultant or advisory role: BMS, Daiichy, Dragonfly, Eli Lilly, MSD, Nordic Pharma, Servier. Research funding and/or medication supply: Bayer, BMS, Celgene, Janssen, Incyte, Eli Lilly, MSD, Nordic Pharma, Philips, Roche, Servier. Speaker role: Astellas, Daiichy, Novartis. TDdG reports to be in an advisory role for GE Healthcare, Mendus and LAVA Therapeutics, to have received a research grant from Idera Pharmaceuticals, and to be co-founder and owner of stocks of LAVA Therapeutics, outside of the submitted work. The other authors report no conflict of interest. Authors’ contributions All authors contributed to the design of this study. TvdE, MIvBH, MCCMH, NHM, RvH, SM, NCTvG, MFB, HWMvL, and TDdG were involved in the PERFECT trial including patient care and/or translational research. TvdE, JB, SML, and MH were involved in collecting patient samples and/or isolation of PBMCs. JB, SML, and TDdG developed the immune monitoring panel. TvdE, AE, LMB, JB, MH, and CW performed the data acquisition. TvdE, AE, LMB, MH, HWMvL, and TDdG were involved in data analysis. TvdE was responsible for drafting the manuscript. All authors contributed to the manuscript and read and approved the final manuscript. Ethics approval and consent to participate All patients provided written, informed consent for study participation. This study was conducted in accordance with the Declaration of Helsinki and the international standards of good clinical practice. Consent for publication We confirm that the manuscript has been read and approved by all named authors. Availability of data and material The data that support the findings of this study are available from the corresponding author, [TvdE], upon reasonable request. 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PMC010xxxxxx/PMC10353331.txt

==== Front Mitochondrial DNA B Resour Mitochondrial DNA B Resour Mitochondrial DNA. Part B, Resources 2380-2359 Taylor & Francis 10.1080/23802359.2023.2227744 2227744 Version of Record Research Article Mitogenome Announcement Complete mitochondrial genome and phylogenetic analysis of Sineleotris saccharae (Perciformes, Odontobutiae) L. Zhou et al. Zhou Liying ab Wang Minghua a Li Daming a Tang Shengkai a Liu Yanshan a Chen Xiaohui a https://orcid.org/0000-0002-4624-4102 Zhong Liqiang a a Key Laboratory of Fisheries Resources in Inland Water of Jiangsu Province, Freshwater Fisheries Research Institute of Jiangsu Province, Nanjing, China b College of Animal Science and Technology, Yangzhou University, Yangzhou, China Supplemental data for this article can be accessed online at https://doi.org/10.1080/23802359.2023.2227744. CONTACT Liqiang Zhong lqzhongffri@hotmail.com Key Laboratory of Fisheries Resources in Inland Water of Jiangsu Province, Freshwater Fisheries Research Institute of Jiangsu Province, Nanjing, China 16 7 2023 2023 16 7 2023 8 7 715718 9 2 2023 15 6 2023 KnowledgeWorks Global Ltd.15 7 2023 published online in a building issue15 7 2023 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. 2023 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. Abstract The freshwater sleeper, Sineleotris saccharae Herre, 1940 is a member of the Odontobutiae family, widely distributed in southern China. In the present study, we determined the complete mitochondrial genome of S. saccharae for the first time and analyzed its evolutionary relationship. The complete mitochondrial genome of S. saccharae was 16,487 bp long, and had 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNA (rRNAs) and a control region (CR). The mitogenome of S. saccharae shared the same gene organization and orientation as other teleosts. According to phylogenetic research, S. saccharae was sister to S. chalmersi with high support value, providing the monophyly of the genus Sineleotris. These results will be helpful for understanding the systematics of the odontobutids. Keywords Sineleotris saccharae mitochondrial genome odontobutiae phylogenetic analysis the Special Funds for Quality Management and Standardization of Jiangsu Province 2022-140 Freshwater Fishery Ecology and Resource Monitoring Program of Jiangsu Province 2022-SJ-061-02 The present work was supported by the Special Funds for Quality Management and Standardization of Jiangsu Province [2022-140]; and Freshwater Fishery Ecology and Resource Monitoring Program of Jiangsu Province [2022-SJ-061-02]. ==== Body pmcIntroduction Sineleotris saccharae originally described by Herre (1940), was native to Southern China (Figure 1). Based on morphological traits, the species was subsequently assigned to the genera Philypnus (Chen and Zheng 1985), Hypseleotris (Wu 1991), and Sineleotris (Wu and Zhong 2008). Chen et al. (2002) initially classified the genus Sineleotris as belonging to the Odontobutidae family, while Li et al. (2018) verified this classification using molecular data. Information about genetic characteristics of S. saccharae, including mitochondrial genome, genetic diversity, is still not available. In the present study, we determined mitogenome of S. saccharae for the first time and established the phylogenetic relationship of the odontobutids. Figure 1. Specimen of Sineleotris saccharae was collected from the Fengshun County, Guangdong Province, China. The photo was taken by Huiwen Xiao on 11 October 2020. Materials Adult S. saccharae individuals were collected from Fengshun, Guangdong Province, China (23.817546°N, 116.287902E). A specimen and its DNA were deposited at the ichthyoligocal museum of Freshwater Fisheries Research Institute of Jiangsu Province, China (Dr Liqiang Zhong, e-mail: lqzhongffri@hotmail.com) under the voucher number JSFFRI-20008. Methods Total DNA was extracted with Qiagen Blood and Cell Midi Kit. The mitogenome was amplified using 20 pairs of Odontobutis-specific primers (Ma et al. 2015) and 30 sets of fish-universal primers (Miya and Nishida 1999). The gaps were filled with self-designed primers (supplemental Table S1). Using the same PCR primers, the PCR products were sequenced via an Applied Biosystems ABI 3730XL capillary sequencer. After blasting in the GenBank, raw sequencing data were assembled to final mitogenome with manually inspecting. Then MitoFish was used to annotate and visualize it (Iwasaki et al. 2013). To analyze the phylogenetic position of S. saccharae, 13 PCGs from the closest thirteen fishes were downloaded according to blasting results in GenBank (Table 1, 12 species of gobiiformes and an outgroup Eleutheronema rhadinum). Each of 13 PCGs was aligned separately using Clustal W with default settings and then concatenated to a single multiple sequence alignment. The substitution model mtREV + G + I were selected as the best model for analysis. The maximum likelihood (ML) analysis (Felsenstein 1985) was inferred on MEGA 11 (Tamura et al. 2021) with 1000 bootstrap replicates. Table 1. Species and GenBank accession number of mitogenomes used in this study. NO. Species Accession ID References 1 Sineleotris saccharae OP326576 This study 2 Sineleotris chalmersi MH644035 Wang et al. 2019 3 Rhyacichthys aspro AP004454 Miya et al. 2003 4 Eleotris oxycephala KP713717 Xia et al. 2015 5 Bostrychus sinensis JQ665462 Unpublished 6 Oxyeleotris lineolata KP663727 Zang et al. 2016 7 Oxyeleotris marmorata KF711995 Yang et al. 2016 8 Hemieleotris latifasciata MF927495 Alda et al. 2017 9 Ophiocara porocephala MW387001 Amin et al. 2021 10 Perccottus glenii KC292213 Xue et al. 2013 11 Eleotris picta MF927491 Alda et al. 2017 12 Eleotris fusca  KU674798  Unpublished 13 Eleotris acanthopoma AP004455 Miya et al. 2003 14 Eleutheronema rhadinum MW845829 Zhong et al. 2021 Results The entire mitogenome of S. saccharae was 16,487 bp long, and had 13 PCGs, 22 tRNAs, 2 rRNAs and a CR (Figure 2). The mitogenome of S. saccharae shared the same gene organization and orientation as other teleosts (Table 2). The overall base composition was T 25.3%, C 30.0%, A 28.9%, and G 15.8%. Only one of the 13 PCGs, the ND6, was found to be encoded on the light strand (L-strand), with the other 12 being identified on the heavy strand (H-strand). The 850-bp-long CR has the highest A + T concentration (65.0%) in the entire mitogenome. Figure 2. Gene map of the mitochondrial genome of Sineleotris saccharae (GenBank accession number: OP326576), with 13 protein coding genes, 22 tRNAs, 2 rRNAs, and a control region. Genes encoded on light strand and heavy-strand were shown inner and outside of the ring respectively. Table 2. Organization of the mitogenome of Sineleotris saccharae. Gene Position Size (bp) Codon Anti codon Strand Intergenic nucleotide (bp)b From To Start Stopa tRNA- Phe 1 68 68     GAA H   12S rRNA 69 1024 956       H 0 tRNA-Val 1025 1096 72     TAC H 0 16S rRNA 1097 2766 1680       H 0 tRNA-Leu 2767 2841 75     TAA H 0 ND1 2842 3816 975 ATG TAA   H 0 tRNA-Ile 3819 3888 70     GAT H 2 tRNA-Gln 3888 3958 71     TTG L −1 tRNA-Met 3958 4026 69     CAT H −1 ND2 4027 5072 1046 ATG TA-   H 0 tRNA-Trp 5073 5143 71     TCA H 0 tRNA-Ala 5146 5214 69     TGC L 2 tRNA-Asn 5216 5288 73     GTT L 1 tRNA-Cys 5322 5389 68     GCA L 33 tRNA-Tyr 5390 5460 71     GTA L 0 CO I 5462 7015 1554 GTG TAA   H 1 tRNA-Ser 7016 7082 67     TGA L 0 tRNA-Asp 7085 7156 72     ATC H 2 CO II 7160 7850 691 ATG T-   H 4 tRNA-Lys 7851 7923 73     TTT H 0 ATPase8 7925 8092 168 ATG TAA   H 1 ATPase6 8083 8765 683 ATG TA-   H −10 CO III 8766 9550 785 ATG TA-   H 0 tRNA-Gly 9551 9622 72     TCC H 0 ND3 9623 9971 349 ATG T-   H 0 tRNA-Arg 9972 10040 69     TCG H 0 ND4L 10041 10337 297 ATG TAA   H 0 ND4 10331 11711 1381 ATG T-   H −7 tRNA-His 11712 11780 69     GTG H 0 tRNA-Ser 11781 11848 68     GCT H 0 tRNA-Leu 11853 11925 73     TAG H 4 ND5 11926 13764 1839 ATG TAA   H 0 ND6 13761 14282 522 ATG TAG   L −4 tRNA-Glu 14283 14350 68     TTC L 0 Cyt b 14356 15496 1141 ATG T-   H 5 tRNA-Thr 15497 15568 72     TGT H 0 tRNA-Pro 15568 15637 70     TGG L −1 Control region 15638 16487 850       H 0 In the ML phylogenetic tree (Figure 3), S. saccharae was firstly clustered with S. chalmersi with high support value, then together with Perccottus glenii and Rhyacichthys aspro forming the Gobioidei suorder (bootstrap value >80). While the remaining nine sleepers clustered together forming the Eleotroidei suorder. Figure 3. Maximum-likelihood (ML) phylogenetic tree was reconstructed based on the concatenated 13 protein-coding genes of S. saccharae and other 13 fishes. Accession numbers were indicated after the species names. Numbers at the nodes indicated bootstrap support values from 1000 replicates. Discussion and conclusion In this study, the entire mitogenome of S. saccharae was identified for the first time. It was similar to that of other teleosts in terms of gene organization, and composition (Miya et al. 2003). In the ML phylogenetic analysis, S. saccharae was sister to S. chalmersi with high support value, providing the monophyly of the genus Sineleotris. And all sleepers were placed into two well-supported suborder clusters, which was similar to those of previous studies (Zhong et al. 2018a, 2018b). These results will be essential to the species identification and systematics of the odontobutids in the future. Supplementary Material Supplemental Material Click here for additional data file. Acknowledgments We are grateful to Shujie Liu, Binbin Zhan and Huiwen Xiao for help in field assistance and the species reference image. Author contributions ZLQ conceived this study; ZLY, LYS and CXH conducted the experiments, LDM and TSK analyzed the data; ZLY and WMH wrote the drafting of the paper; ZLQ revised it critically, and that all authors agree to be accountable for all aspects of the work. Ethical approval This study was approved by the animal care and Ethical Committee of Freshwater Fisheries Research Institute of Jiangsu Province. Disclosure statement No potential conflict of interest was reported by the author(s). Data availability statement The mitochondrial genome sequence is available on GenBank of NCBI at www.ncbi.nlm.nih.gov with the accession number of OP326576. ==== Refs References Alda F, Adams AJ, McMillan WO, Chakrabarty P. 2017. Complete mitochondrial genomes of three Neotropical sleeper gobies: Eleotris amblyopsis, E. picta and Hemieleotris latifasciata (Gobiiformes: Eleotridae). Mitochondrial DNA B Resour. 2 (2 ):747–750. doi: 10.1080/23802359.2017.1390412.33490475 Amin MH, Lee SR, Irawan B, Andriyono S, Kim HW. 2021. Characterization of the complete mitochondrial genome of the Northern Mud Gudgeon, Ophiocara porocephala (Perciformes: Eleotridae) with phylogenetic implications. Mitochondrial DNA B Resour. 6 (3 ):953–955. doi: 10.1080/23802359.2021.1889415.33796696 Chen IS, Kottelat M, Wu HL. 2002. A new genus of freshwater sleeper (Teleostei: Odontobutididae) from southern China and mainland Southeast Asia. J Fish Soc Taiwan. 29 :229–235. Chen W, Zheng CY. 1985. 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PMC010xxxxxx/PMC10353333.txt

==== Front Ann Med Ann Med Annals of Medicine 0785-3890 1365-2060 Taylor & Francis 37459584 10.1080/07853890.2023.2234392 2234392 Version of Record Research Article Pulmonary Medicine The prevalence of burnout among pulmonologists or respiratory therapists pre- and post-COVID-19: a systematic review and meta-analysis X. Bai et al. Bai Xiaoyin a* Wan Ziqi b* Tang Jieying c* Zhang Dingding d Shen Kaini a Wu Xia e Qiao Lin a Zhou Yangzhong a Wang Yaqi f Cheng Wei g Jiang Wei h Wang Luo f Tian Xinlun f a Department of Internal Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China b Eight-Year Program, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China c Department of Surgery, School of Clinical Medicine, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing, China d Medical Research Center, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China e Department of Medicine, Tufts Medical Center, Boston, MA, USA f Department of Pulmonary and Critical Care Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China g Department of Critical Care Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China h Department of Medical Intensive Care Unit, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China * These authors contributed equally to this work. Supplemental data for this article can be accessed online at https://doi.org/10.1080/07853890.2023.2234392. CONTACT Luo Wang wangluo@pumch.cn Xinlun Tian tianxl@pumch.cn No. 1 Shuaifuyuan, Wangfujing, Dongcheng District, Beijing 100730, China 17 7 2023 2023 17 7 2023 55 1 223439230 5 2023 3 7 2023 4 7 2023 KnowledgeWorks Global Ltd.17 7 2023 published online in a building issue17 7 2023 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 2023 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. Abstract Objectives The coronavirus disease-19 (COVID-19) increased the already heavy workload in the pulmonary and respiratory departments, which therefore possibly increased the prevalence of burnout among pulmonologists or respiratory therapists. We aimed to compare the differences in burnout among pulmonologists or respiratory therapists pre- and post-COVID-19 by doing a systematic review with meta-analysis. Methods We searched pulmonologist, or pulmonary, or respiratory, and burnout up to 29 January 2023 in six databases. We included studies investigating pulmonologists or respiratory therapists and reporting the prevalence of burnout among them. The risk of bias was assessed by a tool for prevalence studies. The overall prevalence of burnout was pooled. Results A total of 2859 records were identified and 16 studies were included in the final analysis. The included studies reported 3610 responding individuals and 2336 burnouts. The pooled prevalence of burnout was 61.7% (95% confidence interval (CI), 48.6–73.2%; I2 = 96.3%). The pooled prevalence of burnout during COVID-19 was significantly higher than it was prior to the outbreak (68.4% vs. 41.6%, p = .01). The result of the meta-regression revealed that COVID-19 coverage was significantly associated with the prevalence of burnout (p = .04). Conclusions Burnout was widely prevalent among pulmonologists or respiratory therapists and increasingly perceived during COVID-19. Therefore, interventions were needed to reduce burnout in this specialty.KEY MESSASGES The coronavirus disease-19 increased the already heavy workload in the pulmonary and respiratory departments. Burnout was widely prevalent among pulmonologists or respiratory therapists and increasingly perceived during COVID-19. Keywords COVID-19 burnout pulmonologists respiratory therapists Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences 2021-I2M-1-048 This work was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (Grant Number 2021-I2M-1-048) and Peking Union Medical College Hospital Teaching Reform in Undergraduate Education (Grant Number 2023zlgl008). ==== Body pmcIntroduction The coronavirus disease-19 (COVID-19) has been declared an end as a global health emergency in May 2023 [1]. However, with 767 million confirmed cases and 6.9 million deaths as of writing, the disease remains a health threat to the public [2]. Life is returning to what it is like before COVID-19 with some vaccines and anti-virus drugs, but a great number of health workers are still working in the frontline and fighting against local surges in cases and deaths. The outbreak once left no room for mental preparedness. COVID-19 increases the workload and stress placed on health workers, which has an impact on their physical and mental health, including professional burnout, depression and anxiety [3,4]. Burnout is a work-related syndrome of emotional exhaustion (EE), depersonalization (DP) and a reduced perception of personal accomplishment (PA) [5]. Multiple studies have indicated that during the COVID‐19 pandemic, nurses [6], physicians [7], surgeons [8], residents [9] and medical students all experience high levels of burnout [10]. Significant differences were noticed across various specialities [11]. The disease causes respiratory-tracts-related symptoms increasing the already heavy workload in the pulmonary and respiratory departments. In 2015, the prevalence was reported as high as 47% among pulmonary medicine [12]. Several studies investigated the burnout of pulmonary physicians [13] and respiratory therapists during COVID-19 [14,15]. They observed that burnout was widely perceived and levelled up during the pandemic. However, there have not been studies to summarize the prevalence of burnout among pulmonologists and respiratory therapists. Therefore, we aimed to report the overall prevalence by conducting a systematic review and performing a meta-analysis. Methods We followed the PRISMA 2020 guideline to report this study [16]. Database search We searched pulmonologist, or pulmonary, or respiratory, and burnout up to 29 January 2023 in the following databases: PubMed, EMBASE, PsycINFO, Cochrane Central Register of Controlled Trials (CENTRAL), Scopus and Web of Science. The search strategy for all databases was provided in the supplementary material. We also screened the references of related publications to identify additional relevant studies. Two researchers independently searched the databases. Inclusion criteria The inclusion criteria of eligible studies were as follows: studies investigating the population of pulmonologists or respiratory therapists; studies reporting the prevalence of burnout among the aforementioned population. Studies were excluded if they (1) failed to state the number of burnouts among pulmonologists or respiratory therapists; (2) were qualitative studies without reporting the prevalence of burnout; (3) only investigated the interventions against burnout without reporting the baseline prevalence of burnout; and (4) were case reports, reviews, comments, editorials, corrections, replies, notes or book chapters. Study selection EndNote 20 (Clarivate PLC, Jersey, UK) was used to manage the records imported from each database. Duplicated publications were initially removed by the same DOI number and then verified manually. Studies were first screened by reviewing the titles and abstracts. The possibly eligible ones subsequently underwent full-text reviews. The selection was independently conducted by the two reviewers (Z.W. and X.B.). A third reviewer (L.W.) was involved in the disagreements of the included studies that could not be resolved through discussion. Data extraction Based on the purpose of the study, the following information was extracted: first author, publication year, study setting, COVID-19 experience, centres, study design, study period and survey response rate. The following information was extracted from the investigated population: specialty, clinical level, burnout measure instrument, the total number of respondents, demographic characteristics of respondents (sex, age, marriage and COVID-19 care), number of burnouts, the prevalence of burnout, risk factors of burnout and their odds ratios. Two reviewers (Z.W. and X.B.) independently collected data using a standard collection form. Risk of bias assessment The risk of bias in included studies was assessed by a tool proposed by Hoy et al. for prevalence studies [17]. This tool consists of 10 items, one point for each, addressing bias of selection, nonresponse, measurement and analysis. The risk of bias was graded as low (score 0–3), moderate [4–6] and high [7–10]. Certainty was graded using a variation version of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) for environmental and occupational health [18]. Statistical analysis R 4.2.1 (R Foundation, Vienna, Austria) was used for all statistical analyses. The prevalence of burnout among pulmonologists and respiratory therapists was pooled by using logit transformation and presented in a forest plot. No study was excluded because all studies included reported their prevalence of burnout. Heterogeneity was evaluated by measure of I2 and considered significant when I2 ≥ 50%. When there was significant heterogeneity between studies, the random-effects model was used for pooling; otherwise, the fixed-effects model was used. The generalized linear mixed model and the logit transformation were used in the meta-analysis of proportions. The subgroup analysis of burnout prevalence was performed based on their COVID-19 coverage, survey respondent rate, respondent size, measurement instrument and department. A meta-regression was conducted to determine if there were any independent risk factors for burnout prevalence. The difference was considered significant when a two-sided p value was less than .05. Influence analysis by leaving out one study at a time was performed to the pooled prevalence to assess its robustness. Results Included studies A total of 2859 records were identified from PubMed (n = 422), EMBASE (n = 820), PsycINFO (n = 77), CENTRAL (n = 27), Scopus (n = 432) and Web of Science (n = 1081). There were 1088 duplicates removed. The remaining 1771 records went through title-and-abstract reviews. A total of 1662 records were excluded owing to the following reasons: inappropriate type of publications (n = 636), uninterested population (n = 521), unspecified clinicians (n = 255), unrelated topics (n = 152), other psychological symptoms (n = 44), interventional studies (n = 32) and pulmonary or respiratory diseases (n = 22). Therefore, 109 records were considered eligible when evaluated by full-text reviews. Ninety-four records were excluded for the following reasons: an unspecified number of pulmonologists or respiratory therapists (n = 52), not reporting burnout (n = 17) or not reporting the prevalence of burnout (n = 25). One additional paper was found by screening the reference lists of the included studies. Therefore, the final analysis comprised 16 studies (Figure 1) [14,19–33]. Risk of bias was assessed as low in 13 studies, and moderate in the other three studies (Supplementary Table 1). Figure 1. PRISMA flow of study selection. A total of 2859 records were identified from PubMed (n = 422), EMBASE (n = 820), PsycINFO (n = 77), CENTRAL (n = 27), Scopus (n = 432) and Web of Science (n = 1081). There were 1088 duplicates removed. The remaining 1771 records went through title-and-abstract reviews. We excluded 1662 records due to reasons presented in the figure. Therefore, 109 records were further evaluated by full-text reviews. Ninety-four were excluded because of reasons in the above column. One additional paper was found by screening the reference lists of the included studies. Thus, 16 studies were included in the final analysis. The 16 studies were conducted in eight countries, including the United States (n = 6), Saudi Arabia (n = 4), Qatar, Italy, China, the United Kingdom, Brazil and Mexico (n = 1 for each). A cross-sectional design was used for 15 investigations, whereas a longitudinal analysis was used in the other study. The cross-sections spanned from 2009 to 2021 with 12 studies covering the outbreak of COVID-19. The median response rate was 67.6% (range: 36.4–96.2%). Six studies had respondent sizes <100, whereas the other 10 studies had sizes >100. While the nine studies were from multiple centres, the other six were from single sites. Twelve studies investigated respiratory therapists (students), while the other four reported fellows, physicians (n = 2) and residents. Five studies focused on the intensive care unit (ICU), six on the pulmonary and respiratory medicine departments, and the remaining five were from unspecified departments. This information is presented in Table 1. Table 1. Information of the included studies (n = 16). Study Period COVID-19 Setting Center Specialty Level Response rate, % Shbeer and Ageel [19] 2021 August–November Amidst Saudi Arabia Multiple ICU RT 69.3 Spirczak et al. [30] 2020 July–August Amidst United States Multiple Respiratory care department RT 84.8 Siraj et al. [31] 2021 March–May Amidst Saudi Arabia Multiple Respiratory therapy programs RT students 69.9 Roberts and coworkers [14] 2020 July–2021 May Amidst United States Single ICU RT NA Kerlin [32] 2020 July, 2020 October, 2021 January Amidst United States Multiple ICU RT 54 Ahmad et al. [33] 2020 November Amidst Saudi Arabia Multiple NA RT 60.8 Omar et al. [20] 2021 January Amidst Qatar Multiple ICU RT 36.4 Castro et al. [21]b 2021 April–May Amidst Italy Single NA RT 84.2 Alhaykan [22] 2020 December–2021 January Amidst United States Multiple NA RT NA Algarni et al. [23] 2021 September–November Amidst Saudi Arabia Single Respiratory care department RT 66.0 Sharp et al. [24] 2019 January–February Pre- United States Multiple Pulmonary, PCCM, CCM Fellow 51.4 Miller et al. [25]a 2021 January–March Amidst United States Multiple NA RT 37.0 Zhou et al. [26] 2020 March–May Amidst China Single Pulmonary Physician 96.2 Piracha et al. [27]a NA NA United Kingdom Multiple NA Physician NA Fumis et al. [28] 2015 August–September Pre- Brail Single ICU RT 72.7 Austria-Corrales et al. [29]b 2009 April–May Pre- Mexico Single Respiratory medicine Resident NA CCM: critical care medicine; ICU: intensive critical care; NA: not available; PCCM: pulmonary critical care medicine; RT: respiratory therapist. a Full texts were not available and information was extracted from the abstract. b Texts were not in English and translated into English by Google translation. Respondent characteristics The included studies reported a total of 3610 responding individuals, with nine studies presenting the demographic characteristics of their respondent population (n = 2100, 58%) [20, 22–24, 26, 29–31,33]. The reported mean or median ages ranged from 20 to 40 years old. The pooled percentage of males was 46.8% (95% confidence interval (CI), 34.4–60.0%; I2 = 95.7%). The pooled percentage of married individuals was 52.7% (95% CI, 26.1–77.9%; I2 = 98.7%). Additionally, the studies reported features of the region, workload, years of experience, education level, night shifts and others. Prevalence of burnout Most studies (n = 10) used the Maslach Burnout Inventory (MBI) to measure burnout. One study used the MBI two-item, an adapted tool from MBI [24]. The instruments of the other five studies are listed in Table 2, including three tools not available owing to restricted access to their full texts. The 10 studies varied in their definitions and grading cutoffs of burnout despite employing the same tool, MBI, to evaluate burnout (Table 2). Collectively, six studies defined burnout as the combination of high EE, high DP and low PA. Another two studies defined less strictly as fulfilling one of the above three manifestations. The other two did not give the exact definition of burnout; however, they presented the respective number of burnouts concerning the three aspects. Table 2. The measure, definition and prevalence of burnout of the included studies (n = 16). Study Instrument Definition of burnout in high level No. of total No. of burnout Prevalence, % High EE High DP Low PA Shbeer and Ageel [19] MBI NA (cut-off, EE ≥ 28, DP ≥ 11, PA ≤ 29) 12 6 50 6 4 4 Spirczak et al. [30] ProQOL Score of burnout questions ≥ 42 218 128 59a NA     Siraj et al. [31] MBI EE ≥ 27, DP ≥ 10 and PA ≤ 33 559 436 78 291 330 307 Roberts and coworkers [14] NA NA 108 82 75 NA     Kerlin [32] WBI, SPFI SPFI ≥ 1.33, or WBI ≥ 4 100 74 74 NA     Ahmad et al. [32] MBI EE ≥ 38, DP ≥ 18 and PA ≤ 33 152 68 45 68 51 71 Omar et al. [20] MBI EE ≥ 27, or DP ≥ 13, or PA ≤ 33 84 45 54 22 8 38 Castro et al. [21] MBI EE ≥ 22, DP ≥ 7 and PA ≤ 34 9 8 89 NA     Alhaykan [22] MBI EE ≥ 27, DP ≥ 10 and PA ≤ 33 295 100 34 197 139 192 Algarni et al. [23] MBI NA (cut-off, EE ≥ 27, DP ≥ 10, PA ≤ 33) 66 65 98 51 65 48 Sharp et al. [24] MBI 2-item Either item ≥ 1 week 502 276 55 NA     Miller et al. [25] NA NA 1114 880 79 NA     Zhou et al. [26] MBI EE ≥ 27 and/or DP ≥ 10 (PA < 33) 125 61 49 56 34 36 Piracha et al. [27] NA NA 110 59 54 NA     Fumis et al. [28] MBI EE ≥ 27, DP ≥ 10 and PA ≤ 33 57 12 21 NA     Austria-Corrales et al. [29] MBI EE ≥ 26, DP ≥ 10 and PA ≤ 34 99 36 36 NA     DP: depersonalization; EE: emotional exhaustion; MBI: Maslach Burnout Inventory; NA: not available; PA: personal accomplishment; ProQOL: professional quality of life scale; SPFI: Stanford Professional Fulfilment Index; WBI: Well-Being Index. a Sum of burnout in medium and high level. The included studies reported 2336 burnouts. The pooled prevalence of burnout was 61.7% (95% CI, 48.6–73.2%; I2 = 96.3%). Influence analysis revealed that the value was not significantly changed by omitting one study at a time. The pooled prevalence was 58.5% (95% CI, 39.5–75.2%; I2 = 95.5%) if only included studies using the MBI instrument and 60.9% (95% CI, 46.0–74.0%; I2 = 94.8%) if only included studies in full text. The subgroup analysis revealed that experiencing COVID-19 was significantly associated with burnout prevalence. The pooled prevalence of burnout during COVID-19 was significantly higher than it was prior to the outbreak (68.4% vs. 41.6%, p = .01, Figure 2). The subgroup analysis showed subgroups separated by the sample size (<100 vs. ≥100), the survey response rate (>50% vs. ≥50%), the measurement instrument (MBI vs. non-MBI) and the department (ICU vs. non-ICU) were not significantly different in the prevalence of burnout (p = .65, .83, .50 and .37, respectively). The outcome of the meta-regression revealed that COVID-19 was significantly associated with the prevalence of burnout (p = .04). The certainty was graded as low by using adapted GRADE tool mainly because all were cross-sectional studies. Figure 2. Forest plot of the overall prevalence of burnout. The included studies reported a total of 3610 responding individuals and 2100 burnouts. The pooled prevalence of burnout was 61.7% (95% CI, 48.6–73.2%; I2 = 96.3%). The pooled prevalence of burnout during COVID-19 was significantly higher than it was prior to the outbreak (68.4% vs. 41.6%, p = .01). Risk factors Eight studies reported the risk factors of burnout [20, 23–26, 30, 31, 33]. Female pulmonologists or respiratory therapists tended to experience more burnout than their male counterparts [20, 23, 26]. In two studies, a heavier weekly workload was reported to exacerbate burnout [24, 26], while another study presented that the weekly workload was not significantly associated with burnout [20]. The coverage system and mental health services were reported to be protective factors from burnout [24]. Age, academic year and nationality were also found to be significant factors associated with burnout [30, 31, 33]. Discussion To the best of our knowledge, this study was the first meta-analysis to provide a summary of the prevalence of burnout among pulmonologists and respiratory therapists. The overall burnout prevalence was determined to be 61.7%, which was higher (68.4%) during the outbreak than it had been before (40.7%). The level of burnout we obtained was consistent with prior reports from a more general medical population. According to a meta-analysis, the prevalence of burnout among European physicians, using a unidimensional definition, was 43.2% [34], which was comparable to what we observed in the pre-COVID-19 subgroup. The prevalence during COVID-19 was approximately 67% [35], which was consistent with the pooled subgroup result we reported. Furthermore, our findings suggested that there was an increase over the past 3 years, which was observed elsewhere. According to a longitudinal cohort study, burnout was more prevalent during COVID-19, with a post-peak rate being approximately 13% higher than before [36]. Other studies among various demographics have reported that COVID-19 has an impact on the increase in burnout [37–39]. Pulmonologists and respiratory therapists work in close relation with healthcare professionals in the ICU or critical care medicine. Both departments have long been reported to present high burnout prevalence owing to extensive workloads, mental stress, emergency burden and the complexity of patients’ conditions [40–43]. The severe acute respiratory syndrome coronavirus 2 enters the respiratory tract, subsequently harming the respiratory system [44]. Therefore, the likelihood of increased stress and burnout levels in several specific departments was high [45]. Our findings were close to the prevalence of burnout among frontline workers in the ICU [43,46]. Therefore, it was possible that both daunting work stress and overwhelming pandemic together put on a heavy burden on pulmonologists and respiratory therapists to level up the perception of occupational burnout. The extent COVID-19 contributed to burnout (if any) could not be determined. The pandemic affected practically every facet of daily life. Thus, it may have been a confounding factor that masked several important factors, such as lack of wellness resources [37], shortage of personal protective equipment [47] and poor leadership [48]. Future individual studies could delve into the greater impact of COVID-19. Immediate interventions are necessary to address burnout. Out of 10 pulmonologists or respiratory therapists, six to seven experience burnout. This number was not only higher than the frontline health workers during COVID-19 [49], but also much higher than nurses (even working in ICU) [6,50]. On the other hand, there is strong evidence linking occupational burnout to clinicians’ less sustainable career development and, consequently, lowering healthcare quality [51]. The high prevalence of burnout and the significant harm to healthcare organizations highlight the need for urgent interventions to improve burnout. Series of studies among Serbian health workers revealed that fostering resilience and mentalizing would be helpful to reduce burnout [52–54]. Clinicians could pay more attention to their own mental status and find their own solutions towards occupational burnout. The healthcare system should also take burnout into account and facilitate clinicians to reduce burnout. It was observed that residents and trainees were more susceptible to burnout [51]. In addition, COVID-19 served as an obstacle to physical skill training [55]. This highlights yet another need for training programs to establish effective individual-focused or organizational strategies to reduce burnout [56]. There were some limitations in our study. A reliable assessment of the prevalence of burnout among physicians was difficult owing to the inconsistency of the definition and cut-off of burnout [57,58]. This circumstance was also observed in our study. Few studies have reported burnout among pulmonologists, which makes further research difficult. Moreover, the participant sizes in about half of the identified studies were <100 and some studies had response rates <50%. These tend to introduce a respondent bias since burnout is self-reported [59]. Conclusions We reported that the overall prevalence of burnout among pulmonologists or respiratory therapists was as high as 61.4%, which was 68.4% during COVID-19 and 40.7% predating the pandemic. The high prevalence and the significant increase in burnout established the need for immediate interventions to reduce burnout in this specialty. Supplementary Material Supplemental Material Click here for additional data file. Supplemental Material Click here for additional data file. Ethical approval Not applicable. Consent form Not applicable. Author contributions Conception and design: XB, ZW, JT and LW. Administrative support: LW and XT. Provision of study materials or patients: XB, ZW, JT and DZ. Collection and assembly of data: XB, ZW and LW. Data analysis and interpretation: DZ, XW, KS, LQ, YZ, YW, WC, WJ and XT. ZW prepared the figures and tables. Manuscript writing and reviewing: all authors. Final approval of manuscript: all authors. Disclosure statement All of us declare that we have no competing interests. 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==== Front Eur J Psychotraumatol Eur J Psychotraumatol European Journal of Psychotraumatology 2000-8066 Taylor & Francis 37458735 2225154 10.1080/20008066.2023.2225154 Version of Record Basic Research Article Research Article Peritraumatic physical symptoms and the clinical trajectory of PTSD after a terrorist attack: a network model approach Síntomas físicos peritraumáticos y la trayectoria clínica del TEPT tras un atentado terrorista: un enfoque de modelo de red恐怖袭击后围创伤性躯体症状和 PTSD 的临床轨迹：网络模型方法EUROPEAN JOURNAL OF PSYCHOTRAUMATOLOGY B. BERTHAIL ET AL. Berthail Benoit ad Trousselard Marion bc Lecouvey Gregory d Fraisse Florence d Peschanski Denis e Eustache Francis d* Gagnepain Pierre d* https://orcid.org/0000-0002-2304-1783 Dayan Jacques df a French Military Health Service Academy, Paris, France b French Armed Forces Biomedical Research Institute, Brétigny-sur-Orge, France c APEMAC, Université de Lorraine, Metz, France d Normandie Université, UNICAEN, PSL Research University, EPHE, INSERM, U1077, CHU de Caen, GIP Cyceron, Neuropsychologie et Imagerie de la Mémoire Humaine, Caen, France e EHESS, CNRS, UMR8209, Université Paris I Panthéon Sorbonne, HESAM Université, Paris, France f Centre Hospitalier Guillaume Régnier, Pôle Hospitalo-Universitaire de Psychiatrie de l’Enfant et de l’Adolescent, Université Rennes 1, Rennes, France CONTACT Jacques Dayan jcdayan@gmail.com Normandie Université, UNICAEN, PSL Research University, EPHE, INSERM, U1077, CHU de Caen, GIP Cyceron, Neuropsychologie et Imagerie de la Mémoire Humaine, Caen 14000, France; Centre Hospitalier Guillaume Régnier, Pôle Hospitalo-Universitaire de Psychiatrie de l’Enfant et de l’Adolescent, Université Rennes 1, Rennes 35700, France * These authors contributed equally. Supplemental data for this article can be accessed online at https://doi.org/10.1080/20008066.2023.2225154. 17 7 2023 2023 17 7 2023 14 2 22251547 1 2023 28 5 2023 28 5 2023 Nova techset21 6 2023 Converted to JATS 1.2 by Nova Techset21 6 2023 © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group 2023 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. ABSTRACT Introduction: Following a mass casualty event, such as the Paris terrorist attacks of 13 November 2015, first responders need to identify individuals at risk of PTSD. Physical peritraumatic symptoms involving the autonomic nervous system may be useful in this task. Objective: We sought to determine the trajectory of physical response intensity in individuals exposed to the Paris terrorist attacks using repeated measures, and to examine its associations with PTSD. Using network modelling, we examined whether peritraumatic physical symptom associations differed by PTSD status. Methods: Physical reactions were assessed using the Subjective Physical Reactions Scale at three time points: peritraumatic by retrospective recall, then current at one year (8–18 months) and three years (30–42 months) after the attacks. Interaction networks between peritraumatic physical reactions were compared according to PTSD status. Results: On the one hand, the reported intensity of physical reactions was significantly higher in the PTSD group at all time points. On the other hand, using the dynamic approach, more robust positive interactions between peritraumatic physical reactions were found in the PTSD group one and three years after the attacks. Negative interactions were found in the no-PTSD group at one year. Peritraumatic physical numbness was found to be the most central network symptom in the PTSD group, whereas it was least central in the no-PTSD group. Discussion: Network analysis of the interaction between peritraumatic physical subjective responses, particularly physical numbness, may provide insight into the clinical course of PTSD. Our knowledge of the brain regions involved in dissociation supports the hypothesis that the periaqueductal grey may contribute to the process leading to physical numbing. Conclusions: Our findings highlight the role of peritraumatic somatic symptoms in the course of PTSD. Peritraumatic physical numbness appears to be a key marker of PTSD and its identification may help to improve early triage. HIGHLIGHTS Physical numbness was found to be a central symptom in people developing PTSD in our study examining peritraumatic physical symptoms related to the 2015 Paris terrorist attacks. Introducción: Después de un evento con víctimas masivas, como los ataques terroristas de París del 13 de noviembre de 2015, los primeros socorristas necesitan identificar a los individuos en riesgo de TEPT. Los síntomas físicos peri traumáticos que implican al sistema nervioso autónomo pueden ser útiles en esta tarea. Objetivo: Buscamos determinar la trayectoria de la intensidad de la respuesta física en individuos expuestos a los ataques terroristas de París utilizando medidas repetidas y examinar sus asociaciones con el TEPT. Mediante el uso de modelos de red, examinamos si las asociaciones de síntomas físicos peri traumáticos diferían según el estado del TEPT. Métodos: Se evaluaron las reacciones físicas mediante la Escala de Reacciones Físicas Subjetivas en tres momentos: peritraumático mediante recuerdo retrospectivo, luego actual al año (8–18 meses) y a los tres años (30–42 meses) después de los ataques. Se compararon las redes de interacción entre las reacciones físicas peritraumáticas según el estado del TEPT. Resultados: Por un lado, la intensidad declarada de las reacciones físicas fue significativamente mayor en el grupo de TEPT en todos los puntos temporales. Por otro lado, utilizando el enfoque dinámico, se encontraron interacciones positivas más sólidas entre las reacciones físicas peritraumáticas en el grupo con TEPT uno y tres años después de los ataques. Se encontraron interacciones negativas en el grupo sin TEPT al año. Se observó que la sensación de anestesia física peri traumática era el síntoma de red más central en el grupo con TEPT, mientras que era el menos central en el grupo sin TEPT. Discusión: El análisis en red de la interacción entre las respuestas subjetivas físicas peritraumáticas, en particular la anestesia física, puede proporcionar entendimiento sobre el curso clínico del TEPT. Nuestro conocimiento de las regiones cerebrales implicadas en la disociación apoya la hipótesis de que la sustancia gris periacueductal puede contribuir al proceso que conduce a la sensación de anestesia física. Conclusiones: Nuestros hallazgos destacan el papel de los síntomas somáticos peri traumáticos en el curso del TEPT. La anestesia física peritraumática parece ser un marcador clave del TEPT y su identificación puede ayudar a mejorar la evaluación precoz. 简介：在发生大规模伤亡事件后，例如 2015 年 11 月 13 日的巴黎恐怖袭击，急救人员需要识别有患 PTSD 风险的个人。涉及自主神经系统的围创伤性躯体症状可能在这项任务有用。 目的：我们试图通过重复测量识别巴黎恐怖袭击暴露个体的躯体反应强度轨迹，并检查其与 PTSD 的关联。使用网络模型，我们考查了围创伤性躯体症状关联是否因 PTSD 状态而异。 方法：在三个时间点使用主观躯体反应量表评估躯体反应：通过回顾性回忆的围创伤性，然后是攻击后一年（8–18个月）和三年（30–42个月）的当前反应。根据PTSD状态比较了围创伤性躯体反应之间的相互作用网络。 结果：一方面，PTSD 组报告的躯体反应强度在所有时间点都显著更高。 另一方面，使用动态方法，在PTSD组中，在袭击发生后一年和三年内发现了围创伤性躯体反应之间更强的正性相互作用。一年后，在无 PTSD 组中发现了负性交互。围创伤性躯体麻木被发现是 PTSD 组中最核心的网络症状，而在非 PTSD 组中它是最不重要的。 讨论：对围创伤性躯体主观反应（尤其是躯体麻木）之间相互作用的网络分析，可能有助于深入了解 PTSD 的临床过程。我们对涉及解离脑区的了解支持这样的假设，即导水管周围灰质可能有助于导致躯体麻木的过程。 结论：我们的研究结果强调了围创伤性躯体症状在 PTSD 过程中的作用。围创伤性躯体麻木似乎是 PTSD 的关键标志，其识别可能有助于改善早期分类。 KEYWORDS Peritraumatic physical reaction physical numbness post-traumatic stress disorder network modelling dissociation PALABRAS CLAVE Reacción física peritraumática anestesia física trastorno de estrés postraumático modelo de red disociación 关键词 围创伤性躯体反应 躯体麻木 创伤后应激障碍 网络模型 解离 ==== Body pmc1. Introduction The analysis of adaptation mechanisms immediately following a life-threatening event is essential to our understanding of the subsequent development of psychopathological states. The same traumatic event may give rise to an adaptive, acute stress reaction in one individual, but prove pathological and maladaptive for another, and many factors (personality, psychological defenses, etc.) can modulate whether the response is effective or not (Crocq, 1999; DiGangi et al., 2013). Very early studies established that a threatening confrontation leads to an adaptive physiological stress response, characterized by an increase in autonomic nervous system (ANS) activity, and an increase in hormones that drive the fight or flight response (Cannon, 1929; Hess, 1957; Selye, 1956). However, prolonged exposure to stress, or the inhibition of the fight or flight response, can result in maladaptive behaviour (Adams et al., 1969; Lacey, 1967). Animal experiments have shown that when confronted with a predator, in some cases, the reaction of the prey follows a predictable sequence. First, the threatened animal stops moving (freezing) and the sympathetic nervous system is stimulated (manifested as trembling, or an increased heart rate); here, the aim is to quickly analyse the danger. In the second stage, it tries to run away or escape. If it cannot escape, it turns to face the predator, to try to regain a dominant position (fight or flight). If neither fight nor flight actions stop the threat, the animal goes into tonic immobility, a state of involuntary paralysis. The purpose of this simulated death is to ‘disinterest’ the predator (Marx et al., 2008); the heart rate slows, along with the animal’s overall activity. The immediate physical response is sideration (Volchan et al., 2011). Physiologically, this suggests a shift from intense activation of the sympathetic nervous system to an equal and opposite activation of the parasympathetic system (Ford, 2009), and research has shown that tonic immobility is associated with decreased autonomic arousal (Gentle et al., 1989; Reese et al., 1982). These early findings are supported by current studies that postulate there are phylogenetic changes to the ANS, notably based on Porges’ polyvagal theory (Porges, 1995, 2001, 2007). Polyvagal theory argues that peritraumatic physical reactions are indicative of neurobiological changes related to ANS activation, when an individual faces a stressor. The ANS is therefore recognized as a key player in the response to a stressor (Godoy et al., 2018), which makes it relevant when studying PTSD severity (Park et al., 2017; Williamson et al., 2015). Other work has shown that the intensity of peritraumatic physical reactions is a powerful predictive factor in the development of PTSD (Greene et al., 2020). However, analyses of the role of peritraumatic physical reactions in the PTSD clinical trajectory have been based on simple linear regression methods. The classic model underestimates or neglects the fact that symptoms may interact with each other and then, once activated, may be self-sustaining (Borsboom & Cramer, 2013). The latter perspective is at the core of the psychopathological network model, which is currently attracting much scientific interest (Nelson et al., 2017). In psychopathology, network modelling is a dynamic approach that is based on network theory. It aims to illustrate the multifactorial complex links between a mental disorder and its symptoms (Borsboom, 2017). More recently, the idea of a dynamic, interacting psychopathological network has emerged. The latter assumes that mental disorders are stable states of closely connected, self-sustaining symptom networks that have been activated by a common cause (Fried et al., 2017). Networks can be estimated using cross-sectional or longitudinal time series data and analysed at the group or the individual level (Rhemtulla et al., 2016). In these networks, nodes represent various psychological variables (e.g. symptoms, behaviours), while connections between nodes represent unknown statistical relationships (e.g. correlations, predictive relationships) that can be estimated from psychopathological data (Borsboom, 2017). There are two types of connections: (i) directed nodes are connected by an arrow, indicating a unidirectional effect, or (ii) undirected nodes are connected with a line that indicates a mutual relationship, but there is no arrow to indicate the direction of the effect (Bringmann et al., 2016). The analysis of these networks can be divided into three fundamental steps. First, the structure of the network is estimated, based on a statistical model that reflects empirical patterns of relationships between variables. Second, this structure is analysed, and third, the accuracy of network parameters and measurements is evaluated. On 13 November 2015, Paris (France) was the target of a multi-site terrorist attacks involving three kamikaze bombs around the ‘Stade de France’, a football stadium in the northern suburbs of Paris, four different shootings and bombings places in the 10th and 11th arrondissements of Paris and the Bataclan theatre, located in central Paris. The total number of victims was 130 dead and 354 injured in hospital, including 94 absolute emergencies and 250 relative emergencies (Hirsch et al., 2015). In a web-based study of 454 people exposed to the Paris attacks conducted 8–11 months after the events, almost 37% of the study sample had probable PTSD according to the PCL-5 (Pirard et al., 2020). In another web-based study of 698 rescue and law enforcement responders who were mobilized on 13 November 2015, the prevalence of PTSD according to the PCL-5 ranged from 3.5% to 9.9% depending on the type of responders (Motreff et al., 2018). In the context of the Paris attacks, the high prevalence of PTSD among exposed individuals underlines the need to better-understand the disorder (Pirard et al., 2020). The need is twofold: at the clinical level, we need to better-determine the trajectory of PTSD, based on the peritraumatic response (O’Donnell et al., 2008), and, at the practical level, we need to help front-line practitioners identify individuals who are most at risk of developing PTSD (Bossini et al., 2016). The first aim of our study is to compare immediate (peritraumatic) and delayed physical reactions according to PTSD status assessed one year and three years after the attacks. We hypothesize that the intensity of the subjective peritraumatic physical reaction is significantly higher for PTSD subjects and that this difference could be observed whatever the period of assessment. The second aim is to use network theory to explore the structure of the associations between peritraumatic physical reactions, taking into account the individual's PTSD status and the period of assessment. 2. Methods 2.1. Participants and procedures This monocentric longitudinal study is part of the REMEMBER (REsilience and Modification of brain control network following novEMBER 13) biomedical research project (Mary et al., 2020; Postel et al., 2021), which received prior approval from the Nord Ouest III Personal Protection Committee (12/2016; ID RCB: 2016-A00661-50). Exposed participants were recruited through the Programme 13-Novembre cross-disciplinary and longitudinal research initiative (http://www.memoire13novembre.fr/), funded by the French General Secretariat for Investment (SGPI) through the National Research Agency (ANR) and the ‘Programme d’investissement pour l’Avenir’ (PIA ANR-10-EQPX-0021-01). REMEMBER study is a component of this Program. All exposed participants met the requirements of DSM-5 Criterion A (they were all directly or indirectly involved in the Paris attacks). Trauma-exposed participants (see Table 1 for demographic and clinical characteristics) were divided into two subgroups: those with PTSD symptoms according to the DSM-5 criterion of PTSD, and those without. Table 1. Socio-demographic statistics for the study population as a function of PTSD according to the two phases of the study (time T1 and time T2).   T1 (one year (8–18 months) after trauma) T2 (three years (30–42 months) after trauma)   No- PTSD (n = 50) PTSD (n = 30) No- PTSD (n = 54) PTSD (n = 15) Gender          Male 30 (60%) 13 (43.3%) 30 (56%) 8 (53%)  Female 20 (40%) 17 (56.7%) 24 (44%) 7 (47%) Age at interview          ≤ 30 12 (24%) 7 (23.3%) 7 (13%) 2 (13%)  31–40 24 (48%) 16 (53.3%) 27 (50%) 9 (60%)  ≥ 41 14 (28%) 7 (23.3%) 20 (37%) 4 (27%) Educational level          < High school (French baccalaureate) 3 (6%) 1 (3.3%) 4 (7.4%) -  High school–High school +5 23 (46%) 20 (66.7%) 24 (44.4%) 11 (73%)  > High school +5 24 (48%) 9 (30%) 26 (48.2%) 4 (27%) Marital status          Single 21 (42%) 12 (40%) 19 (35.2%) 9 (60%)  In a relationship 13 (26%) 7 (23.3%) 17 (31.5%) 2 (13%)  Married or in a civil partnership (known as PACS in France) 14 (28%) 9 (30%) 15 (27.8%) 4 (27%)  Separated or divorced 2 (4%) 1 (3.3%) 3 (5.5%) –  Widower – 1 (3.3%) – – Professional status          Student 2 (4%) 1 (3.3%) 2 (3.7%) –  Working 45 (90%) 24 (80%) 43 (79.6%) 12 (86%)  On sick leave 2 (4%) 1 (3.3%) 5 (9.3%) –  Seeking employment 1 (2%) 4 (13.3%) 4 (7.4%) 2 (14%) Previous studies realized in this research program also define the notion of partial PTSD (or subthreshold or sub-syndromal) to define exposed individuals (criterion A) who have reexperiencing symptoms (criterion B), persisting for more than one month (criterion F), that caused significant distress and functional impairment (criterion G), without yet meeting all the DSM-5 diagnostic criteria (Leone et al., 2022; Mary et al., 2020; Postel et al., 2021). In the specific context of this work, we decided not to consider this population of partial PTSD because the psychopathological characterization of this entity will be the subject of a later specific analysis. All subjects received information on the protocol and gave written consent prior to participation. 2.2. Instruments PTSD status, and peritraumatic physical reactions were measured using validated questionnaires in French and English, chosen for their psychometric and clinical quality. Inclusion, and initial psychological testing took place between 13 June 2016 and 7 June 2017 (Time 1, one year (8–18 months) post-trauma). A second round of testing took place between 6 July 2018 and 29 June 2019 (Time 2, three years (30–42 months) post-trauma). 2.2.1. Measurement of PTSD The Structured Clinical Interview for DSM-5 (American Psychiatric Association, 2013) was used to diagnose possible disorders related to exposure to the attacks. Any participant who met Criterion A was considered to have PTSD if he or she fully met DSM-5 specifications. Exposed participants who did not meet all criteria were considered to have no PTSD. 2.2.2. Measurement of physical reactions Physical reactions were assessed using the Initial Subjective Reaction Physical Scale, which is part of the Potential Stressful Events Interview (Falsetti et al., 1994). The 10-item self-administered questionnaire assesses the subjective, physical reactions of individuals at the time of the event or at its recall. Ten reactions are evaluated: feeling short of breath, choking (1); feeling dizzy, unsteady or faint (2); heart palpitations or rapid heart rate (3); trembling (4); sweating (5); nausea or abdominal discomfort (6); numbness or a tingling sensation (7); hot flushes and chills (8); a feeling of strangulation (9); and chest pain or discomfort (10). Responses are reported on a four-point Likert-type scale (1 = not at all, 2 = a little bit, 3 = moderately, and 4 = extremely). The total score therefore ranges from 10 to 40. The instrument has good internal consistency (Cronbach’s α = 0.86). 2.3. Data analysis The R software package (version 4.0.3) was used in all analyses. The first stage of our analyses compared the intensity of physical reactions over time between exposed individuals with and without PTSD. We retrospectively assessed the self-reported intensity of peri-traumatic symptoms during time of Session 1 (T1), which was held 8–18 months after the trauma (2016–2017); current symptoms at the time of Session 1; and, finally, current symptoms three years (30–42 months) after the trauma during the time of Session 2 (T2; 2018–2019). An ANOVA was followed by the Tukey post hoc test, if the Shapiro–Wilk test determined that the distribution of the data was normal. The second stage compared interaction networks between physical reactions at the time of the trauma for individuals with and without PTSD at time T1 (one year (8–18 months) post-trauma) then at time T2 (three years (30–42 months) post trauma). 2.3.1. Estimation and visualization of networks When estimating interaction networks, it is common to find false connections due to unmeasured, confounding variables. Typically, this problem is overcome by using partial correlations to create relationships between variables. The method estimates the strength of relationships between variables by controlling for the effects of other measured variables in the model. Two nodes are connected if there is a covariance between them that cannot be explained by any other variable in the network (Epskamp et al., 2018). Coefficients range from −1 to +1 and determine the interaction between two nodes. In general, connections are visualized using red lines to indicate negative partial correlations, and green lines to indicate positive partial correlations (Borsboom & Cramer, 2013). The wider the line, the greater the number of connections. The more nodes there are, the more connections need to be estimated: in a 5-node network, 10 connections are estimated; in a 10-node network, 45 connections are estimated; and in a 20-node network, 190 connections are estimated (Epskamp et al., 2018). In order to successfully control for false connections between variables, their significance is examined. The most commonly used approach is the LASSO (Least Absolute Shrinkage and Selection Operator) method, together with the Extended Bayesian Information Criteria (EBIC) (Epskamp et al., 2018). Networks are estimated using polychoric partial correlations for ordinal data, and then modelled using the LASSO method, with the EBIC, using the qgraph package (Epskamp et al., 2012). In this approach, association networks are undirected and weighted with partial correlations, and estimate association parameters between all nodes using a Gaussian graphical model (Epskamp et al., 2018). The model uses the Fruchterman–Reingold algorithm (Fruchterman & Reingold, 1991), which produces easy-to-visualize networks where the edges are of similar length and overlapping edges do not impede the visualization. However, this method may not be appropriate for small sample sizes since it reduces weak connections at zero, which could result in the removal of moderately strong connections. This can lead to an inaccurate estimation of networks in samples with low statistical power, ultimately reducing the reliability of the analysis (Epskamp et al., 2018). To estimate networks, centrality, and connection strengths in small samples, the Information Filtering Networks (IFN) approach can be used (Christensen et al., 2018). IFNs estimate a fixed number of connections based on the formula ‘3 * nodes – 6,’ and the Triangulated Maximally Filtered Graph (TMFG) is a variant of IFN that filters the network by generating a chordal network (Massara et al., 2016). For more details of the methodology, please see the Supplementary Material. 2.3.2. Analysis of the network structure Not all of the nodes in a network are equally important in determining its structure. Centrality indices provide an insight into the relative importance of a node with respect to other nodes (Borgatti, 2005; Freeman, 1978). For example, a central symptom is a symptom with a large number of connections, which can propagate the activation of the symptom network. Different centrality indices are developed to provide insight into different dimensions. These indices are presented as standardized z-scores that provide information about the relative importance and centrality of nodes. They are based on the connection model, in which the node of interest plays a role, and they can be used to model or predict several network processes, such as the amount of flow through a node or the tolerance of the network to the deletion of selected nodes (Borgatti, 2005). In the present study, we used the NetworkComparisonTest (NCT) package in R (Van Borkulo et al., 2016) to directly compare node centralities, with a single invariance measure, expected influence, as the measure of centrality. 2.3.3. Assessment of the model’s accuracy and robustness In the present study, we used the bootnet R package to determine robustness (Epskamp et al., 2018). Specifically, non-parametric bootstrapping was used to calculate the accuracy of edge weights and node centralities. Edge stability was evaluated by calculating the mean correlation with the original sample. Node stability was determined by calculating a correlation stability (CS) coefficient. CS coefficients were calculated for the centrality measure, and for each of the two networks. To compare edge weights and centralities within networks, we used the difference test function with a p-level of .05. Each bootstrap was run 10,000 times for each network. 2.3.4. Network comparison test To compare the interaction network for the two independent groups, a permutation test was developed to be able to make direct comparisons estimated in different subpopulations (Van Borkulo, 2018). A permutation test compares network structures that contain relationships between variables that are estimated from the data (Van Borkulo et al., 2016). In the present study, we used the NetworkComparisonTest (NCT) package in R (Van Borkulo et al., 2016) to estimate between-group network differences, and compare node centralities and edge weights. The significance level was set at p < .05. 3. Results 3.1. Participants Of the 80 participants at time T1, 37 were women (46.3%) and 43 were men (53.7%). Their average age was 36 years. Furthermore, 95% were educated to high school level (the French baccalaureate) and 41.2% had received a further five years (or more) of education. Finally, 53.8% were married or in a common-law relationship, and 86.2% were in employment. 50 of the 80 included subjects (62.5% of the total sample) had no PTSD symptoms, and 30 (37.5% of the total sample) did. At time T2, only 11 individuals were lost to follow up, and of the remainder, 31 were women (44.9%) and 38 were men (55.1%), with an average age of 37 years. Furthermore, 94% were educated to high school level (the French baccalaureate), and 43% had received a further five years (or more) of education. Finally, 55% were married or in a common-law relationship, and 60% were in employment. Among the individuals who suffered from PTSD symptoms one year (8–18 months) post-trauma, 11 were clinically free of PTSD, and 13 still had symptoms three years (30–42 months) later. Among those who had no symptoms of PTSD one year (8–18 months) post-trauma, two had developed symptoms, and 43 were still asymptomatic three years (30–42 months) later. Descriptive statistics regarding the population are detailed in Table 1, for the two sessions. Figure 1 shows the clinical trajectory of subjects who attended both sessions. Figure 1. The clinical PTSD trajectory of exposed individuals between the time T1 (one year (8–18 months) post-trauma) and time T2 (3 years (30–42 months) post-trauma), assessed using DSM-5 diagnostic criteria. 3.2. Univariate longitudinal analysis of the physical reactions' intensity Significant differences were found in the intensity of physical reactions (total score) reported: at the time of trauma, retrospectively reported at time T1 (F(78,1) = 47.10, p < .001); at time T1 (F(76,1) = 41.38, p < .001); and at time T2 (F(67,1) = 66.27, p < .001), between individuals with, and with no PTSD symptoms (Figure 2). Figure 2. Analysis of variance (ANOVA) results of the comparison of the intensity of physical reactions between individuals with, and with no PTSD symptoms for peritraumatic reactions retrospectively reported at time Tl (left); at the time Tl, held 8–18 months later (middle) and at the time T2, held three years (30–42 months) later (right). 3.3. Interaction networks for peritraumatic physical reactions, and the presence (n = 30) or absence (n = 50) of PTSD at time T1 (one year (8–18 months) post-trauma) 3.3.1. Network estimation Based on the reported peritraumatic physical reactions, recorded retrospectively 8–18 months post-trauma and PTSD status at the same time, two networks were obtained. In the PTSD group strong positive correlations were found between: feeling short of breath (1) and a feeling of strangulation (9); and between trembling (4) and numbness (7). Moreover, numbness (7) was positively correlated with most of the other symptoms. In the no-PTSD group, most negative partial correlations related to nausea or abdominal discomfort (6). Strong positive correlations were found between trembling (4) and hot flushes and shivering (8); between trembling (4) and feeling dizzy, unsteady or faint (2); and between chest pain (10) and feeling short of breath (1) and a feeling of strangulation (9). See Figure 3. Figure 3. Interaction networks for peritraumatic physical reactions for PTSD (left) and no-PTSD (right) groups with EBIC lasso method. Green lines represent positive partial correlations, red lines represent negative partial correlations, and the thickness of the line indicates the strength of the correlation. Partial correlations between −1 and +1 are indicated in the middle of the lines. The maximum absolute value of the partial correlation is shown at the bottom right of the network. For the comparison of the two networks, the tuning parameter was set to λ = 0.40. We investigated central indices as the expected influence of the peritraumatic physical reactions in each network. The resulting plot is shown in Figure 4. The analysis of node centralities found that the expected influence of physical reactions was globally superposable, except for numbness (7). The latter factor is the most central in the network of the PTSD group, whereas it is the least central in the no-PTSD group. In individuals with no PTSD, the key impacts were: trembling, chest pain, and feeling short of breath. Figure 4. Expected influence in the network structure of peri-traumatic physical reactions interaction and bootstrap 95% confidence intervals for estimated expected influence (n = 10,000) in PTSD group (left) or no-PTSD group (right) at time Tl (8–18 months post-trauma). 3.3.2. Edge weight accuracy and stability An accuracy analysis (refer to the Supplementary Material for details of the method) revealed that 95% confidence intervals for edge weights mostly overlapped in both networks, which suggests that they are accurate (see Supplementary Material, Figure Aa). However, the large size of bootstrapped CIs implies that interpreting the order of most edges in the network should be done with care. Nevertheless, the bootstrapped significant difference test was used to compare differences in edge weights in each network (see Supplementary Material, Figure Ab). In the PTSD group, no significant differences were found for edge weights after bootstrapping. Concerning edge stability, the mean correlation with the original sample was mostly positive and ranged from 0.75 to 1 which confirmed the robustness of edge weights (see Supplementary Material, Figure Aa). In the no-PTSD group, the significant difference test showed that the negative interaction between nausea (6) and numbness (7) was significantly larger than most of the other interactions, after bootstrapping; this was followed by positive interactions between feeling dizzy, unsteady or faint (2) and trembling (4), and between trembling (4) and hot flushes (8). These three edges are reliably the strongest in the no-PTSD network, since their bootstraped CIs do not overlap with the bootstrapped CIs of most of the other edges. Concerning edge stability, the mean correlation with the original sample was between 0.5 and 1, with a decreasing trend, which suggests that robustness falls as resampling proceeds (see Supplementary Material, Figure Aa). 3.3.3. Centrality stability The case-dropping bootstrap method was used to investigate centrality stability (refer to the Supplementary Material for details of the method). Stability was quantified using the CS coefficient. It should be noted that the CS coefficient should not be below 0.25, and preferably greater than 0.5. The results of the analysis indicated that centrality was stable in both networks, with CS = 0.51 in the no-PTSD group and CS = 0.60 in the PTSD group. These results mean that the order of the expected influence of nodes can be interpreted, although such an interpretation should be done with care. The bootstrapped difference test was used to compare node centralities in each network (see Supplementary Material, Figure B). In the PTSD group, the expected influence of numbness (7) was significantly stronger than that of most of the other nodes. In the no-PTSD group, the expected influence of numbness (7) was significantly lower than feeling short of breath (1), trembling (4), and chest pain (10). 3.3.4. Network comparisons We investigated differences in the network structure, and significant differences in edge weights and node expected influence between the PTSD and the no-PTSD group. The results revealed that the expected influence of numbness (7) was significantly different (p < .01). The results revealed no significant difference concerning the maximum difference in edge weights (p = .39) (see Supplementary Material, Tables A and B). 3.4. Interaction networks for peritraumatic physical reactions, and the presence (n = 13) or absence (n = 43) of PTSD at time T2 (three years (30–42 months) post-trauma) 3.4.1. Network estimation Based on the reported peritraumatic physical reactions, recorded retrospectively 8–18 months post-trauma, and PTSD status three years (30–42 months) post-trauma, two networks were obtained with the triangulated maximally filtered graph (TMFG) method. In the PTSD group strong positive correlations were found between: feeling short of breath (1) and a feeling of strangulation (9) and numbness (7) and between hot flushes and chills (8) and chest pain or discomfort (10) and numbness (7). In the no-PTSD group, strong positive correlations were found between trembling (4) and feeling dizzy, unsteady or faint (2) and hot flushes and chills (8); and between chest pain (10) and feeling short of breath (1) and a feeling of strangulation (9). See Figure 5. Figure 5. Interaction networks for peritraumatic physical sympton1s for PTSD (left) and no-PTSD (right) groups at time 2 (30–42 months post-trauma) with TMFG method. Green lines represent positive partial correlations, red lines represent negative partial correlations, and the thickness of the line indicates the strength of the correlation. Partial correlations between −1 and +1 are indicated in the middle of the lines. The maximum absolute value of the partial correlation is shown at the bottom right of the network. We investigated central indices as the expected influence of the peritraumatic physical reactions in each network. The resulting plot is shown in Figure 6. The analysis of node centralities found that the expected influence of numbness (7) is one of the most central in the network of the PTSD group, as well as feeling short of breath (1), feeling of strangulation (9) and chest pain (10) whereas trembling (4) and nausea (6) were the least central in the network. In the no-PTSD group, trembling (4) and feeling short of breath (1) are the most central whereas numbness (7), sweating (5) and nausea (6) are the least central in the network. Figure 6. Expected influence in the network structure of peri-traumatic physical reactions interaction and bootstrap 95% confidence intervals for estimated expected influence (n = 10,000) in PTSD group (left) or no PTSD group (right) at time T2 (30–42 months post-trawna). 3.4.2. Edge weight accuracy and stability An accuracy analysis revealed that 95% confidence intervals for edge weights mostly overlapped in both networks, which suggests that they are accurate (see Supplementary Material, Figure Ca). However, the large size of bootstrapped CIs implies that interpreting the order of most edges in the network should be done with care. Nevertheless, the bootstrapped significant difference test was used to compare differences in edge weights in each network (see Supplementary Material, Figure Cb). In the PTSD group, the significant difference test showed that the positive interaction between numbness (7) and feeling short of breath (1) and between numbness (7) and feeling of strangulation (9) were significantly larger than most of the other interactions, after bootstrapping. Concerning edge stability, the mean correlation with the original sample was mostly positive and ranged from 0.5 to 1 which confirmed the robustness of edge weights (see Supplementary Material, Figure Ca). In the no-PTSD group, the significant difference test showed that the positive interactions between feeling short of breath (1), chest pain (10); between feeling of strangulation (9) were significantly larger than most of the other interactions, after bootstrapping. Concerning edge stability, the mean correlation with the original sample was under 0.5 which suggests that robustness falls as resampling proceeds (see Supplementary Material, Figure Ca). 3.4.3. Centrality stability The case-dropping bootstrap method was used to investigate centrality stability (refer to the Supplementary Material for details of the method). The results of the analysis indicated that centrality was stable in both networks, with CS = 0.41 in the PTSD group and CS = 0.25 in the no-PTSD group at time T2. These results mean that the order of the expected influence of nodes can be interpreted, although such an interpretation should be done with care. The bootstrapped difference test was used to compare node centralities in each network (see Supplementary Material, Figure D). In the PTSD group, the expected influence of numbness (7) was significantly stronger than that of most of the other nodes. In the no-PTSD group, the expected influence of numbness (7) was significantly lower than feeling short of breath (1), trembling (4), and chest pain (10). 3.4.4. Network comparisons We investigated differences in the network structure and node expected influence between the PTSD and the no-PTSD group. The results revealed that the expected influence of numbness (7) was significantly different (p < .01) as well as trembling (4) which expected influence was significantly different (p = .01) in the network of the no-PTSD group compared to the network of the PTSD group (see Supplementary Material, Table C). 4. Discussion Our results first demonstrate a significant and stable difference in the intensity of peritraumatic physical reactions measured by scores on the Initial Subjective Reaction Physical Scale between individuals with PTSD symptoms and those without PTSD, both retrospectively, at 8–18 months after the event, and up to three years (30–42 months) later. The intensity of physical reactions was significantly lower in the group with no PTSD symptoms, at all measurement times. At follow-up, 11 subjects were lost, 11 no longer met the criteria for PTSD diagnosis at time T2 out of the 50 previously diagnosed at time T1, while 2 who did not meet the criteria at time T1 did at time T2 (Figure 1). Similar patterns of follow-up have been observed for military veterans (Solomon & Mikulincer, 2006) and for a terrorist attack (Gibert et al., 2021). In addition, our study identifies two profiles, as a function of PTSD status at 8–18 months post-trauma as at 30–42 months post-trauma. In the no-PTSD group, connection strengths in the interaction network between peritraumatic physical reactions fell after bootstrapping, suggesting a decrease in the stability of the network as we resampled. This network is characterized by strong negative correlations between certain initial physical reactions (unlike the PTSD group, where positive, stable connections remain following bootstrapping). In addition, the no-PTSD network featured strong centrality and positive connections between certain symptoms that are known to evoke a physiological stress response through activation of the ANS. On the other hand, numbness was the central symptom in the PTSD group, and it was the factor that was most strongly connected with other symptoms in the network whatever the time session. Finally, we found an overall difference in significant connection strengths between PTSD and no-PTSD networks. According to network resilience and vulnerability theory (Borsboom, 2017), a resilient network is a network in which connections between symptoms are too weak to be self-sustaining over time, even in the absence of the stressor (Borsboom, 2017; Cramer et al., 2016; Kalisch et al., 2019). In line with this approach, the observed lack of stability between connections in the no-PTSD network suggests that it is resilient. We also observed negative interactions in the no-PTSD network – to the best of our knowledge, there is no theoretical explanation for this finding. It is possible that the presence of negative connections increases the network’s instability, by modulating some self-activation loops. Although future studies are needed to support this hypothesis, it opens up an innovative perspective in the field of network theory. Finally, if we consider our findings from the perspective of adaptive or maladaptive stress responses, it is possible that the initial stress response of individuals in the no-PTSD group activated the ANS, and, consequently, they were not overwhelmed (reflected in characteristics consistent with a resilient network). Conversely, the initial stress response of individuals in the PTSD group was maladaptive, reflected in a state of sideration and manifested in peritraumatic numbness, along with characteristics consistent with a vulnerable network. Moreover, our results highlight the central role of physical numbness in peritraumatic physical reactions reported by individuals who developed PTSD at time T1 (8–18 months post-trauma) and at time T2 (30–42 months post-trauma). In the context of PTSD, numbness can refer to both emotional and physical numbness. In the literature, numbness is generally considered on its emotional side and often understood as a type of dissociation that can occur in response to trauma (Friedman et al., 2007; Najavits et al., 2015). Emotional numbness refers to a feeling of detachment or disconnection from one's emotions, as if they are not fully experiencing them (Choi & Seng, 2017). A previous network analysis of interactions between posttraumatic stress symptoms and several covariates in individuals exposed to the Oslo terrorist attack showed the centrality of emotional numbness (Birkeland & Heir, 2017). Our results complement these findings, providing evidence for the importance of the role of peritraumatic subjective physical reactions and the centrality of physical numbness in terms of networks analysis. Physical numbness is a loss of sensation in the body (Dworkin et al., 2018). This may be a result of the body's natural response to stress, which involves the diversion of blood flow away from non-essential areas of the body to support the response to a threat (Ginzburg et al., 2002; Lanius et al., 2010) and in which the periaqueducal gray matter may be particularly involved (Brandão & Lovick, 2019; Dampney, 2019). These conditions can also occur in ischemic or traumatic shock, with a redistribution of vascular flow favouring the brain at the expense of the extremities (Marion, 1991). The periaqueductal grey matter, which plays a central role in pain control, is also activated in this response to stroke (Ally et al., 2020; Rossi et al., 1994). The periaqueductal gray matter (PGM) is a complex structure that coordinates the behavioural and autonomic reactions to stress and injury. Previous research on the neurobiological basis of dissociation has highlighted alterations in functional connectivity in key brain regions such as the prefrontal cortex, amygdala, insula, and periaqueductal gray matter (Terpou et al., 2019). While most of these studies have focused on dissociation as a specific alteration of consciousness (Spiegel et al., 2011), our research investigated numbness as a physical symptom, including tingling and physical numbness. Our study suggests that physical numbness may be a central part of the dissociative response which could involve the functional connectivity of key brain regions such as the periaqueductal grey matter. Further studies are needed to integrate these findings into the literature on the role of defensive behaviours in PTSD, and its dissociative subtype (Terpou et al., 2019). Although the study of peritraumatic physical symptoms is challenging, it is a key element not only to improve the follow-up of subjects who have experienced a trauma such as a terrorist attack, but also to expand our pathophysiological understanding of PTSD, and to develop effective treatments. 5. Implications Our study explored subjective peritraumatic physical reactions in the context of a terrorist attack and highlighted some clinical implications. Our findings suggest that the intensity of physical reactions over time is significantly associated with the presence of PTSD. It also suggests that the structure of interactions between peritraumatic physical reactions could be responsible for a form of vulnerability. In practice, our results suggest that screening could be put in place regarding the risk of PTSD development, based on peritraumatic physical reactions in general, and numbness in particular, as the latter appears to be a central element of the development of PTSD. Furthermore, a pathophysiological distinction can be made between no-PTSD and PTSD groups. PTSD has a major impact, not only on the quality of life of those affected, and their personal or professional life, but also a country’s health system. It is therefore a major public health issue. These issues are not negligible, particularly in terms of co-morbidities (anxiety disorders, depression, suicide, substance abuse), and costs (treatment, hospitalization, inability to work). By specifically targeting the type and intensity of peritraumatic reactions that victims experience, frontline practitioners may be able to rapidly detect whether the individual is able to overcome the stress reaction and indicate the nature and urgency of care the psychically injured person needs. This approach could be decisive in limiting, or even avoiding severe PTSD. Further studies are needed to confirm this postulate in the long term, and to verify whether the implementation of screening tools, based on the collection of peritraumatic physical symptoms can really have a predictive impact on the severity of PTSD expression. In addition, future studies are needed to determine whether there is a link between the structure of an individual’s peritraumatic physical symptoms, and the action of central inhibitory control systems (Mary et al., 2020), along with amygdalo hippocampal circuits (Postel et al., 2021; Ressler et al., 2022), which we know are involved in the expression of classic PTSD symptomatology. Furthermore, given the centrality of numbness in networks of peritraumatic physical symptoms in individuals with PTSD, this symptom appears to be a useful therapeutic target, notably as tonic immobility has been associated with the suppression of autonomic activation of trauma-related recollections. In general, cognitive–behavioural treatments aim to decrease any physiological autonomic activation in response to trauma-related recollections, using strategies such as exposure, relaxation and cognitive restructuring (Foa & Rothbaum, 1998), and have been shown to be effective in many cases of PTSD. Conversely, individuals with a diminished physiological autonomic response (as in the case of tonic immobility) may not be the best candidates for these exposure therapies (Hembree et al., 2003). 6. Strengths and limitations Despite some limitations, which we discuss below, our study has a solid methodological basis. It draws upon standardized measurement instruments, which have been scientifically validated as reliable, and interviews were run by professionals trained in the exercise. The sample is relatively homogenous with respect to the time that has passed since the traumatic event, and their characteristics (due to the targeted nature of the attacks with respect to their spatial and temporal dimensions). Although it could be considered a small sample (n = 80), this corpus remains very singular in its dimension given the traumatic event (terrorist attack) and the attrition rate remains low (13.75%). The design of the longitudinal study with repeated measures allowed us to include an early psychopathological analysis of victims, and to assess and confirm the strength of the findings over time. Finally, the network modelling approach that we adopt to analyse interactions between peritraumatic physical symptoms is an original, dynamic advance in the field of trauma semiology. However, the conclusions that can be drawn from our results are limited, for several reasons. First, the participants were relatively young, highly educated, and not necessarily representative of the general population. Caution must therefore be exercised when generalizing our findings. Second, the retrospective measurement of peritraumatic physical reactions may lead to reporting and memorization biases, which must be considered when interpreting the results, particularly in the case of dissociative reactions. Although this bias could be limited by running studies in the context of emergency facilities, this poses both ethical and practical problems, due to the difficulty of integrating any assessment into existing procedures. Third, it is noteworthy that while our findings revealed peri-traumatic numbness to be a central factor in distinguishing the PTSD and no-PTSD groups at both time 1 (8–18 months post-trauma) and time 2 (30–42 months post-trauma), this result should be interpreted with caution. Specifically, it is important to consider that the methodology employed at time T1, the EBIC lasso method, differs from that used at time T2, the TMFG method. Methodologically, the state of the art in modelling psychological interaction networks is based on lasso regularized Markov Random Fields (MRF), which utilize Gaussian graphical models (GGM) for ordinal or continuous data (Epskamp et al., 2018). The EBIC lasso function is commonly used to compute Gaussian graphical model using graphical lasso based on extended BIC criterium. However, this method is not suitable for small sample size since it considers weak connections at zero (Epskamp et al., 2018). Recently, the Information Filtering Networks (IFN) approach has been proposed as a method for estimating networks, centrality, and connection strengths in small samples (Christensen et al., 2018). IFNs estimate a fixed number of connections and the Triangulated Maximally Filtered Graph (TMFG) is a variant of IFN that filters the network by generating a chordal network. The advantage of TMFG is that the number of connections is constant and does not vary with sample size, thus protecting comparability between two samples. However, the limitation of TMFG is that it can add unnecessary connections. Therefore, the consistency of the results across time points may be influenced by the differences in the analytical approaches. Further research is needed to replicate these findings using consistent methodological approaches across both time points. Fourth, our study was based on psychopathological data regarding the development of PTSD and does not explore physiobiological predictive factors. Further research is clearly needed to clarify the peritraumatic mechanisms involved in the clinical trajectory of individuals exposed to a traumatic event, and to confirm their relevance. Supplementary Material Supplemental Material Click here for additional data file. Supplemental Material Click here for additional data file. Acknowledgements We thank all participants for volunteering to contribute to this study and the victims’ associations who supported the project. We are grateful to Carine Klein-Peschanski, General Secretary to the 13-November Program and Equipment Excellence MATRICE and the researchers; psychologists B. Marteau, and L. Besnehard; technicians; and administrative staff at U1077 (Caen), at ‘Programme 13-Novembre’ in Paris, at INSERM ‘Délégation Régionale Nord-Ouest’. We also thank E. Seery for English-language editing of the main text. Disclosure statement No potential conflict of interest was reported by the author(s). Data availability statement Due to the nature of the research, participants in this study did not agree to share their data publicly, consequently, supporting data is not available. ==== Refs References Adams, D. 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==== Front Hum Vaccin Immunother Hum Vaccin Immunother Human Vaccines & Immunotherapeutics 2164-5515 2164-554X Taylor & Francis 37460107 10.1080/21645515.2023.2233398 2233398 Version of Record Research Article Acceptance & Hesitation Parental perceptions, attitudes, and beliefs regarding vaccination of children aged 0–5 years: A qualitative study of hill-tribe communities, Thailand K. MOONPANANE ET AL. HUMAN VACCINES & IMMUNOTHERAPEUTICS https://orcid.org/0000-0002-6753-6382 Moonpanane Katemanee a https://orcid.org/0000-0001-8699-1244 Thepsaw Jintana a https://orcid.org/0000-0001-8992-0011 Pitchalard Khanittha a https://orcid.org/0000-0001-6541-5053 Purkey Eva b a School of Nursing, Mae Fah Luang University , Chiang Rai, Thailand b Department of Family Medicine, Queen’s University , Kingston, ON, Canada CONTACT Katemanee Moonpanane katemanee.moo@mfu.ac.th School of Nursing, Mae Fah Luang University , Chiang Rai 57100, Thailand. 17 7 2023 2023 17 7 2023 19 2 2233398Integra14 7 2023 Integra14 7 2023 19 4 2023 19 6 2023 02 7 2023 © 2023 The Author(s). Published with license by Taylor & Francis Group, LLC. 2023 The Author(s) https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. ABSTRACT The widespread availability and use of vaccines have tremendously reduced morbidity and deaths related to infectious diseases globally. However, in hill-tribe communities in Northern Thailand, vaccination rates remain low, and there is limited literature on parental perceptions, attitudes, and beliefs about vaccination for children under five years of age. We conducted a qualitative study employing semi-structured interviews to understand parents’ perceptions, attitudes, and beliefs about vaccinations. A purposive sample was used to recruit participants. Data were analyzed using thematic analysis. 74 hill-tribe parents (14 Akha, 11 Hmong, 12 Lahu, 13 Lisu, 12 Karen, and 12 Yao) were interviewed. Four themes emerged from the interviews: 1) traditional beliefs, and practices 2) traumatic experiences, 3) lack of information and effective communication, and 4) trust and support from the community. Findings highlight that it is crucial to build trust by providing knowledge, appropriate information, and advice about vaccinations in order to improve vaccine coverage in children under five years of age in the hill-tribe context. KEYWORDS Vaccination hesitancy ethnic group child health qualitative research The Center of Excellence for the Hill Tribe Health Research CEHR No. 37/2561 The work was supported by the The Center of Excellence for the Hill Tribe Health Research [CEHR No. 37/2561]. ==== Body pmcIntroduction Vaccines are among the most effective public health interventions to prevent communicable diseases1–3 preventing an estimated 2–3 million deaths each year globally.4–6 In Thailand, the Ministry of Public Health (MoPH) launched the Expanded Program on Immunization (EPI) in 1977 in public health services nationwide.7 Currently, the MoPH provides 12 basic vaccines free of charge for children from birth to 12 years of age (e.g. tuberculosis vaccine (BCG), Hepatitis B (HB), Diphtheria-Tetanus-Pertussis-Polio (OPV/IPV), Haemophilus influenza type B (Hib), measles-mumps-rubella (MMR), Japanese Encephalitis (JE), Rotavirus, and Human papillomavirus (HPV) vaccine.8,9 Compared to countries within Southeast Asia, Thailand has drastically increased its vaccine coverage rate and has limited diseases such as polio and measles. Since 2005 the EPI has achieved immunization coverage of around 96–99% among Thai children.10 However, vaccine-preventable diseases are still a major cause of morbidity and mortality in sub-populations,11 such as the migrant population, ethnic minorities, and hill-tribe populations, because of limited health infrastructure, decreased accessibility to services, language barriers, low level of awareness, and limited funding.12–14 A recent report in 2018 showed vaccine uptake in hill-tribe children aged 0–5 years of DTP/OPV3 was 70.0%, MMR was 30%, JE3 was 68.22%, and DTP/OPV5 was 4.8%, which is considered much lower than the specified target (and compares poorly with Thai children, for instance, who have a vaccination rate for MMR of over 90%) and reflects a problem in this public health intervention.15,16 According to recent data, it has been estimated that more than 5 million people belonging to various ethnicities lived in Thailand in the year 2020, which accounts for 7.2% of the total population. The Department of Social Development and Welfare, the total officially recognized “hill tribe” population number 925,82517 and 30% live in Chiang Rai province (350,000) which has the largest population of hill tribe people in the country.15 Of this population, approximately 100,000 are children. Most hill tribe people live in remote or mountain top areas along the Thailand – Myanmar–Laos border and under both the national poverty line and national education level.18,19 Hill tribe people face many barriers in accessing healthcare services including vaccination for their children and these barriers can indeed have serious consequences. Without proper vaccinations, hill tribe children are more vulnerable to diseases such as measles, mumps, rubella, and other preventable infections. These diseases can cause severe complications, long-term health issues, and even death.11 According to a report by the Thai Department of Disease Control from 2014–2019, the yearly number of new measles infections was approximately 5,000 nationwide, and more than 10% were found in the ethnic minority population.20 In addition, in 2020, 6.73 per 100,000 hill tribe children were reported to have measles, and 24.8 were under 5 years of age. The unpublished provincial Health Office report from Chiang-Rai, Thailand’s northernmost province, highlights some common health problems among hill tribe children. The reported incidences of measles, tetanus, and outbreaks of diphtheria in remote areas further underscore the vulnerability of hill tribe children to these preventable diseases.21,22 Decreased rates of immunization among hill tribe children may be due to the many barriers they face in accessing healthcare services.13–15 Previous studies, both in the domestic and international literature, found that the lower vaccination coverage rate in children was caused by many factors, such as knowledge, economic status, language barriers, and access to healthcare services.23–28 However, there are no existing studies exploring immunization among the hill tribe population in Thailand, all of whom have different cultures, languages, and beliefs particularly related to healthcare practices. Due to the context-specific nature of vaccine hesitancy in hill tribe parents, healthcare providers as well as policy makers should focus on gaining a better understanding of vaccine hesitancy in order to develop interventions that are appropriate for this context.29 This study aimed to elicit an understanding of hill tribe parental perceptions, attitudes, and beliefs about vaccination for children aged 0–5 years to identify potential areas for intervention and improvement. Methods Study design A qualitative approach with in-depth, semi-structured interviews was used to gain insight into the ethnic hill tribe parents’ perceptions, attitudes, and beliefs toward vaccination for children aged 0–5 years in rural areas in Thailand. The consolidated criteria for reporting qualitative research (COREQ) checklist was used to structurally report the methods and results of this study.30 Setting and participants Participants were recruited from three hill tribe villages, Theng, Mae Suay, and Mae Fah Luang, whose population are made up of Akha, Lisu, Lahu, Hmong, Yao, and Karen people. Prior to recruitment, the research team met with each nurse practitioner or public health officer in the area to explain the study in detail. Information sheets about the project were provided to each sub-district health promotion hospital (SDHPH). Inclusion criteria included 1) hill tribe parents with at least one child (aged 0–5 years); 2) able to speak Thai fluently. A total of 1,576 parents were invited to participate in the study, with 475 of those belonging to the Akha ethnic group, 102 from the Lisu group, 518 from the Lahu, 245 from the Hmong, 91 from the Yao, and 145 from the Karen. Of those, 74 parents who met the inclusion criteria and chose to volunteer for this study were approached by the principal investigator (PI) during an appointment at the SDHPH for health services in non-emergency situations such as developmental screening, dental visit, and vaccination. These discussions were conducted by the PI in accordance with the study protocols. The parents who were approached were provided with detailed information about the study and were asked to provide written informed consent. This study was approved by the Institutional Review Board of the Provincial office, and informed consent was obtained by all participants. (IRB No. CRPPHO NO.3/2562) Data collection In-person interviews were conducted from August 2019 to February 2020 in meeting room at SDHPH or at participants home, according to their preference. The researchers on this project worked at a university and came from various backgrounds with extensive experience with the ethnic minority population in these areas and qualitative method expertise. The primary investigator set up a three-day research workshop for team members to mentor and train junior qualitative researchers in critical and reflexive engagement with the data and other team members, such as trained research assistants, in overcoming language problems during interviews. A semi-structured interview guide was developed during the literature review and through consultation with the hill tribe village leaders. The questions were assessed for appropriateness, including validity, by the research team and three experts working in the field (one hill tribe researcher expert, a public health officer and a nurse practitioner who worked in primary care settings in the hill tribe communities). Questions were open-ended and included questions such as “Could you please tell me about the vaccination experience for your child?,” “Did your child or children experience any adverse effects after vaccination?,” and “Has your child(ren) missed or delayed a vaccine? and could you please tell me the reason.” In order to operationalize empathic neutrality within the interviews, the researchers attempted to understand (not prove preexisting) reasons for deciding not to (or to partially) vaccinate children. There was a non-judgmental stance toward whatever content emerged during the interviews and within our analytical and publication processes. We firmly believe that our empathic neutrality facilitated trust with our participants and led to positive feedback loops within their social networks, facilitating positive responses from subsequent participants. The interviews were carried out by KM and KP, who were qualitative experts with 10 years of experience, and initiated with a broad, open-ended question that provided a flexible framework for the interviews where participants were encouraged to discuss any issue they deemed relevant to the topic of childhood vaccination. As participants narrated their stories, probing questions were designed to provide an opportunity for parents to discuss their perceptions and experiences when their child had vaccinations or did not get vaccinated. The interviews lasted between 45 to 65 minutes and were conducted in Thai. Interviews were audio-recorded and transcribed verbatim. To ensure a comprehensive evaluation of the data, the investigator took into account the perspectives provided by both parents. A total of 60 interviews were conducted with the mothers and an additional 14 with both parents. The investigator also looked for common threads and discrepancies between the responses of both parents, exploring whether one parent had a greater understanding of the issue at hand. After examining the data, the investigator drew conclusions based on both parents’ responses. After conducting a series of interviews, saturation was reached in that no new data, themes, or ideas were emerging from the interviews, and themes were being repeated.31 Following the interview, participants responded to a demographic questionnaire. Data analysis The transcriptions of interviews and field notes were analyzed using thematic analysis and inductive methodology, including the six phases of thematic analysis as described by Braun and Clarke31 assisted by the qualitative software program NVivo version 12. Two of the authors transcribed and familiarized themselves with the data. Following that, the researchers began to identify preliminary codes, which are features of the raw data that appear to be interesting and meaningful. There were 28 codes or key issues obtained from the data. The codes were grouped into major and sub-themes regarding parents’ perceptions of vaccination. Following this phase, data were compared to identify recurring themes and to review relationships between the main theme and sub-themes, which make a logical argument and may contribute to the overall narrative of the data. Consistent and close readings of the transcripts allowed for fidelity to the thematic analysis method and the data. We were careful to preserve the essence of the participants’ voices, using their narratives to develop thematic titles; hence, our results include rich individual quotes (with assigned codes and numbers) as well as combinations of quotes from multiple participants to form a cohesive narrative. Finally, the authors agreed on the final key themes. These themes were repeatedly checked against the transcripts in order to identify patterns. Interpretation of themes was informed by the literature, objectives of the study, and additional discussions with two other authors with ample working experience in hill tribe healthcare practice. Afterward, the results were revised and critiqued by the research team before a conclusion was developed.31 Results Participants’ characteristics Seventy-four semi-structured interviews were conducted with the 74 participants from six ethnic minority groups. Among the participants, 18.92% were Akha, followed by Lisu (17.57%), Lahu (16.22%), Karen (16.22%), Yao (16.22%), and Hmong (14.86%). The majority of participants were women. Their ages varied between 16–62 years. Of these participants, 24.30% had not enrolled in formal education, 31.10% had completed primary school, whereas and 44.6 had graduated from college or higher education and characteristics are provided in Table 1.Table 1. Participants characteristics. Characteristics (n = 74) (%) Gender     Women 60 81.1 Men 14 18.9 Ethnicity     Akha 14 18.9 Hmong 11 14.9 Lahu 12 16.2 Lisu 13 17.6 Karen 12 16.2 Yao 12 16.2 Age (year)     15–24 13 17.6 25–34 24 32.4 35–44 21 28.4 ≥45 (mean age for women ± SD = 33.37 12.57) (mean age for men ± SD = 31.14 ± 10.62) 16 21.6 Education     No Formal Education 18 24.3 Primary School 23 31.1 Junior High School 24 32.4 Senior High School 6 8.1 Collage 3 4.1 M ± SD = mean ± standard deviation. Themes Parents described their negative perceptions, unpleasant feelings, and personal beliefs regarding the vaccination of children aged 0–5 years within the hill tribe community context. Many parents fear that vaccinations could lead to long-term health problems, such as seizures, or other severe side effects. Others are concerned that the vaccines may not be effective or that the benefits of immunization do not outweigh the risks. Some parents have religious or cultural beliefs that prevent them from vaccinating their children. From the interviews conducted, four themes that emerged in the analysis comprised: 1) traditional beliefs, and practices 2) traumatic experiences, 3) lack of information and effective communication, and 4) trust and support from the community. The excerpts from the interviews selected to illustrate each theme are authentic and succinct, remaining faithful to the participants’ overall sentiments and providing explicit examples of each respective theme. Traditional beliefs and practices Traditional beliefs, including ancestor worship was one of the most common forms of traditional belief practiced by hill tribe communities. There is a deep-rooted fear that often arose from a lack of understanding regarding the science behind vaccinations and their benefits, or from superstitious beliefs that vaccinations might have a negative impact on their spiritual wellbeing. Twenty-two participants reported that they refused going for vaccinations because they believed that their family ancestors did not allow young children to leave their homes. Outside the home, the ancestor has no power to protect the child. For example, one mother stated: My grandparents said we would violate the ancestor rules … a child younger than one year old must stay at home and the ancestor will protect my son because he lives close to the ancestor spirits. Apparently, my parents believe my child is being watched over by an ancestor spirit. Introducing something foreign into their bodies could violate this protection, so it’s important to respect parents’ wishes. (Mother, P5.7) Certain hill tribe groups may also refuse a vaccine due to the beliefs that it contains a toxin intended to function as a chemical weapon by the Thai government to eliminate the hill tribe people. Eleven parents interviewed expressed this view, as exemplified below: It is not safe. My nephew had a vaccination one time … After two or three days passed, he had a seizure and was in a coma. The doctor said his brain was damaged … we think because of the vaccine. It contains harmful substances, such as mercury and fetal cells… toxic chemicals… that kill the child’s brain cells … and I try not to bring my child for vaccination. (Father, P1.8) Traumatic experiences Traumatic vaccination experiences, vaccine side effects or adverse events, and negative interactions with healthcare providers were common reasons for denying vaccination. Eighteen parents who refused vaccination for their children expressed feelings that vaccines are unsafe, harmful, and unpleasant for their children. For example, After having a vaccine at two months old, my child had a high fever, swelling, and redness … I brought her to the hospital … The doctor said she was allergic to vaccines … That was the last vaccination … Now she is two years old and healthy. (Father, P4.12) As I waited for my child to receive their vaccination, I saw a hill tribe woman next to me being scolded by a nurse. She had skipped her child’s vaccination schedule. I could feel the great pain and trauma that she was experiencing. (Mother, P1.2) Fourteen participants expressed mistrust in healthcare providers due to language barriers, stigmatization, and socioeconomic status. It may be that they do not listen to me … because they are not familiar with the hill tribe people, who are dirty, impoverished, and different from them … Hence, I believe this is the reason why we do not wish to visit the hospital. (Father, P3.6) Lack of information and effective communication Lack of knowledge about the importance of vaccinating children is a common problem in the hill tribe communities. Some parents are influenced by misinformation about vaccines or may not be aware of how to access accurate information. In this study, a small majority of participants agreed that vaccinations for children are mandatory, but they did not possess any knowledge related to immunizations. Some participants, particularly those without formal education, stated that they did not understand how vaccines worked, what was inside the needle, or why young children need more vaccinations than adolescents or adults. This was exemplified by a participant who explained: No one informed me about the vaccine. I did not ask because I am sure if the nurse explained it to me … I still would not understand … .it is because I have no education. (Father, P5.5) Lack of information and communication were reflected in parents’ decision to vaccinate their children. Lack of information and ineffective communication included language used, inappropriate information delivery, and communication methods between parents and healthcare providers. I could not bring my child to the hospital by myself because I could not read. Even though I can speak Thai, when the nurse advised me to do something … I needed my husband to help me gain understanding. If my husband has to work … we have to skip the vaccine schedule. (Mother, P4.5) More than 10 participants with no formal education stated that they could not use “the pink book” (the maternal and child health book). Instead, they asked their neighbors about the next appointments for their children. For example, As an ethnic person, I am unable to read the Pinkbook and it is useless. (Mother, P1.5) Trust and support from the community Support from the community was another consideration mentioned by the participants. Many participants indicated that they mainly sought information related to vaccine benefits and harms from sources such as healthcare providers and community leaders, as well as village health volunteers (VHVs). VHVs are the link between people, the community, and the hospital, but they don’t know about vaccines. They know nothing about mosquito control, disabled people, or assessing basic needs. I think VHVs could help by contacting parents directly or delivering door-to-door reminders before an appointment. (Mother, P4.2) Most of the parents who had previously missed the child’s vaccination indicated that beyond providing information, the community leader could be responsible for the child’s vaccination by reminding parents about the vaccine list and schedule in their specific language (e.g., Akha, Lahu, Hmong) during community meetings. This approach could be effective for raising parents’ awareness, especially for those who live far from the vaccination facility and have low literacy levels. For example, My children were fully vaccinated, even though I could not understand Thai. This is because the community leader reminded them about the vaccine schedule and children on the list. (Father, P4.3) Participants agreed that community health systems are imperative for providing trustworthy and accurate information, since many parents believe vaccines are necessary for their children. In the past, the community leader reminded us through local broadcasting. We were doing well with vaccination, but now we have a newly elected leader, and there is no broadcasting at all. Therefore, we are falling behind. (Mother, P5.4) Discussion The purpose of this study was to explore parental perceptions, attitudes, and beliefs toward vaccination for children aged 0–5 years in a hill tribe community context.32–34 The findings of this study align with studies on ethnic minority populations globally that have demonstrated that the major reason people do not receive vaccines is limited understanding and lack of trust in the effectiveness of the vaccine. A study by Wijayanti et al. (2021) demonstrated that parents who had doubts or insufficient information about vaccines can lead to the individual having an inadequate understanding of the information presented to them.35 The finding also highlights the importance of ensuring that parents have access to accurate information about the safety and efficacy of vaccines so that they can make informed decisions. Some parents believed that the vaccine was unnecessary for their children and valued natural treatment, such as breast milk, good care, and hygiene, which they felt were more effective in building the child’s immune system. Moreover, the low vaccine uptake by hill tribe children under five years old was also related to parental beliefs that these children are vulnerable and too fragile to handle immunization.36,37 When studying parents’ views on vaccination, Burghourts et al. (2017) similarly observed a fear of adverse effects and the beliefs that vaccines could harm their child, that their child was too young (under five years old), and that their child had a low risk of catching diseases, which led parents to refuse vaccination.38 Some parents needed reassurance from a trustworthy person and knowledgeable healthcare provider that the vaccine was safe. Studies on vaccine coverage in ethnic children have shown that a lack of trust in vaccine effectiveness and fear of side effects leads to a low rate of vaccine uptake and a high rate of vaccine refusal.39,40 In this study, one of the main findings was that some parents declined vaccines due to significant negative experiences when dealing with healthcare providers, largely due to their hill tribe background. These experiences created a mistrust of the healthcare system, leading to a refusal of vaccines. Moreover, in some parents, vaccines are seen as a product containing toxins and vaccinations are considered to be a violation of ancestral worship rules. Deep-rooted personal beliefs can make it difficult for healthcare providers to solve the problems of parents’ vaccine hesitancy.41–43 Providing effective communication and education, as well as raising public awareness through campaigns by trusted healthcare providers and community leaders, would reduce the vaccine hesitancy of hill tribe parents at the community level, helping limit the spread of rumors and distrust.44 In this study, the participants did not vaccinate their children for a wide range of reasons, such as adverse effects following vaccinations, poverty, and geographic difficulties. However, some hill tribe parents are very interested in vaccinations. Providing information to tribal communities in an easy-to-understand format that is tailored to their specific culture is essential to ensuring that the message being communicated is successful and that parents are able to make informed decisions and could improve vaccination rates among hill tribe children.45–47 In this study, to improve access, the study participants also supported initiatives such as direct personal contact or door-to-door campaigns, using VHVs to promote immunization in children, broadcasting vaccine information within specific languages. Improving vaccine accessibility in hard-to-reach areas should increase service provision and thereby improve the initial low vaccine uptake.48,49 Limitations This study has several limitations. The major one is that it used purposive sampling method within six hill-tribe groups who were mostly women with low education levels, which may not be generalizable to other hill tribe populations.31 Another limitation is that we may be limited by our positionality as outsiders ourselves. Nonetheless, the researchers in our group have a long history of health research in hill tribe populations. The researchers tried to be reflexive, open-minded, and build trust with this community. Recommendations As a result of these findings, strategies to increase the vaccination rate of children have been suggested, including the use of VHVs and local radio broadcasts, which are culturally appropriate for hill tribes. In considering possible interventions, we hypothesized that community-based interventions such as vaccination programs in school, provider reminders, and use of a language intervention would support hill tribe parents in childhood vaccination. Ultimately, these findings address collaboration with community leaders or key stakeholders to provide information support and door-to-door interventions led by VHVs to address the vaccination gap, improve trust between parents and healthcare providers, and provide the parents with a sense of safety when vaccinating their children. Further training of healthcare providers to ensure that they can provide culturally appropriate care which is non-stigmatizing to hill tribe people would also be an important intervention. Conclusion The results of this study show the parental perception toward vaccination for children aged under five years in the hill tribe community context. Parental beliefs, lack of knowledge, lack of information, difficult previous experiences and poor communication resulted in vaccine hesitancy and lack of trust among hill tribe parents, which explains at least in part why they choose to forgo vaccination for their children, along with challenges related to hill tribe geography. These findings can help us to imagine multiple strategies and interventions, targeting both hill tribe parents and health professionals, which would help reduce parental hesitancy. Providing clear information and advice about vaccinations to parents in language that parents can understand is crucial for building relationships and trust with parents and improving the vaccine coverage rate in hill tribe children aged under five years. Acknowledgments We want to thank the parents for participating in our study as well as nurse practitioners for their help in this study. Abbreviations EPI Expanded Program on Immunization SDHPH Sub-District Health Promotion Hospital VHVs Village Health Volunteers Disclosure statement No potential conflict of interest was reported by the author(s). Data availability statement The audio recordings and transcripts analyzed in this study are not publicity available as it is possible to sufficient deidentify them to maintain participant privacy and confidentiality. Addition deidentify information can be obtained from the corresponding author upon reasonable request (email: katemanee.moo@mfu.ac.th). ==== Refs References 1. Rémy V, Zöllner Y, Heckmann U. Vaccination: the cornerstone of an efficient healthcare system. J Mark Access Health Policy. 2015 Aug 12;3 (1 ):27041. doi:10.3402/jmahp.v3.27041. 2. Kaufman J, Ryan R, Walsh L, Horey D, Leask J, Robinson P, Hill S. Face-to-face interventions for informing or educating parents about early childhood vaccination. 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==== Front Case Rep Neurol Case Rep Neurol CRN CRN Case Reports in Neurology 1662-680X S. Karger AG Basel, Switzerland 530812 10.1159/000530812 Single Case – General Neurology Cerebral Sinus Vein Thrombosis following Sneezing: Case Report Extensive CVT following Sneezing 2690813 Nashef Helal 2690814 Haratz Salo 2690815 Mendel Rom Department of Neurology, Assuta Ashdod Medical Center, Ashdod, Israel Correspondence to: Helal Nashef, helaln@assuta.co.il 12 6 2023 Jan-Dec 2023 12 6 2023 15 1 108112 24 2 2023 13 4 2023 2023 © 2023 The Author(s). Published by S. Karger AG, Basel 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission. Abstract Cerebral sinus vein thrombosis (CVT) is a relatively rare neurovascular entity, usually associated with acquired or genetic hypercoagulable states, and in many cases it remains idiopathic. Trauma is also associated with CVT among patients with major head or neck trauma, including penetrating injuries. However, CVT associated with acceleration trauma has only been described in few cases so far. We present an unusual case of a 19-year-old woman with no past medical history, admitted with an extensive CVT following sneezing. A thorough investigation did not reveal any other potential etiology or risk factor other than estrogen-containing oral contraceptives. The patient was treated with anticoagulation and improved clinically with complete recanalization on follow-up imaging. This case suggests acceleration trauma may be a potential factor of risk for CVT. Keywords Cerebral sinus vein thrombosis Acceleration trauma Case report This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. ==== Body pmcIntroduction Cerebral sinus venous thrombosis (CVT) accounts for approximately 1% of all forms of stroke [1]. It is an important cause of cerebrovascular disease in young adults, with a mean age of 33 years old and a two-thirds female preponderance [2]. CVT is associated with various etiologies and conditions including coagulopathies, malignancy, concurrent infection, pregnancy and peripartum period, estrogen-containing oral contraceptives, smoking, and connective tissue disorders among others [3, 4]. CVT can also be associated with trauma; however, it mainly refers to major head or neck trauma, including penetrating injury to skull fracture, dural sinuses, jugular veins, or during neurosurgical procedures [5]. CVT associated with acceleration or minor trauma is limited to a few case reports [6–8]. CVT is frequently missed or diagnosed late since it can mimic other acute neurological conditions and can only be recognized with a specific brain image [9]. We report the occurrence of CVT secondary to acceleration trauma which may be an under-recognized precipitating factor. Case Report A 19-year-old patient with no past medical history presented with a severe right temporal headache one day following sneezing. The headache began immediately after sneezing and was accompanied by nausea with no vomiting or focal neurological symptoms. Treatment with analgesics resulted in some improvement. On the day of admission, a second sneeze resulted in an acute worsening headache, however, with no improvement with treatment. The patient denied preceding trauma other than sneezing, upper respiratory symptoms, smoking, or substance abuse. There was no family history of thrombotic events or known hypercoagulable states. She had no risk factors other than oral contraceptives. On neurological examination, she was fully conscious and oriented, without neck stiffness, swollen discs, or focal neurological signs. Non-contract computerized tomography showed an abnormal hyperdense signal in the right transverse dural sinus compatible with an acute sinus thrombosis (Fig. 1). Computed tomography angiography demonstrated an extensive filling defect in the right jugular vein, sigmoid sinus, and transverse sinus, as well as in the superior sagittal. An empty delta sign in the superior sagittal sinus was also demonstrated (Fig. 2a–c). No anatomical abnormality or other vascular lesions were shown. Laboratory results showed no signs of infectious or inflammatory disease, and no thrombophilia was found; ANA, ANCA, antiphospholipid antibodies, lupus anticoagulant, protein C, factor VIII, factor V-Leiden, antiprothrombin, and MTHFR were all negative. Low levels of protein S were probably due to anticoagulation treatment started earlier. JAK2 mutation was also negative. D-dimer level was elevated (1.07 mg/L; reference range 0.05–0.55). Fig. 1. Non-contrast head CT demonstrates an abnormal hyperdense signal in the right transverse sinus compatible with an acute sinus thrombosis. CT, computed tomography. Fig. 2. a CTA confirms a filling defect in the right jugular vein, sigmoid sinus, and transverse sinus. b The superior sagittal sinus shows a classic empty delta sign. c Coronal image demonstrates a filling defect in the right jugular vein with no evidence of venous dissection. CTA, computed tomography angiography. The patient was treated with low molecular weight heparin and discharged 4 days later with complete resolution of headaches and without evidence of swollen discs on follow-up examination. At follow-up, she remained well with no headaches or new neurological symptoms. She continued treatment with warfarin at therapeutic INR levels. Follow-up magnetic resonance venography demonstrated complete recanalization (Fig. 3). The CARE Checklist has been completed by the authors for this case report, attached as online supplementary material (for all online suppl. material, see https://doi.org/10.1159/000530812). Fig. 3. Follow-up brain MRV demonstrates complete recanalization. MRV, magnetic resonance venography. Discussion CVT is an uncommon neurovascular disorder associated with a wide range of conditions and etiologies including trauma. We present a unique case of a young female patient with extensive dural sinus thrombosis following sneezing. There are few published reports which describe the association between CVT and acceleration or minor trauma, such as sneezing, golf swing, or jumping from a low altitude [6–8]. Similar to our case, all of these patients had at least one predisposing factor such as oral contraceptive use, smoking, or a moderate anticardiolipin antibody level. The exact mechanism for the development of thrombosis following acceleration trauma is unclear. Rottger et al. [6] suggested the mechanism to be a combination of an acceleration trauma, where brain structures impact against fixed dural structures, and eventually a sudden rise in ICP causing damage to the endothelial layer of sinuses and cerebral veins. Bone fragments, sinus dissection, and sinus distortion that may create obstructions to flow and thrombus formation were mentioned by D’Alise et al. [8]. The close temporal association between two sneezing and headache episodes and CVT diagnosis in our case strongly suggests a causal relationship. Moreover, abnormal hyperdensity on initial non-contract computerized tomography is compatible with an acute process, and it seems unlikely that an extensive thrombosis would have been asymptomatic prior to sneezing. This case describes a rare cause of an uncommon neurovascular disorder. We suggest incorporating head acceleration trauma as a potential factor of risk for CVT. Moreover, it is reasonable to consider computed tomography and CVT for patients who present with a headache after acceleration trauma. Statement of Ethics Written informed consent was obtained from the patient for publication of the details of their medical case and any accompanying images. Ethical approval is not required for this study in accordance with local guidelines. Conflict of Interest Statement None of the authors have any financial disclosures or conflicts of interests. Funding Sources This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contributions Helal Nashef conceptualized and designed the work including data acquisition and drafting of the manuscript. Rom Mendel took part in the drafting of the manuscript and in its revision. Salo Haratz took part in the manuscript revision. All authors reviewed and edited the manuscript and approved the manuscript. Data Availability Statement I take full responsibility for the data, the analyses, and the interpretation. All data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author. Supplementary Material ==== Refs References 1. Bousser MG , FerroJM. Cerebral venous thrombosis: an update. Lancet Neurol. 2007;6 (2 ):162–70. 10.1016/S1474-4422(07)70029-7.17239803 2. Coutinho JM , ZuurbierSM, StamJ. Declining mortality in cerebral venous thrombosis: a systematic review. Stroke. 2014;45 (5 ):1338–41. 10.1161/STROKEAHA.113.004666.24699058 3. Towbin A . The syndrome of latent cerebral venous thrombosis: its frequency and relation to age and congestive heart failure. Stroke. 1973;4 (3 ):419–30. 10.1161/01.str.4.3.419.4713031 4. Canhao P , FerroJM, LindgrenAG, BousserMG, StamJ, BarinagarrementeriaF, . Causes and predictors of death in cerebral venous thrombosis. Stroke. 2005;36 (8 ):1720–5. 10.1161/01.STR.0000173152.84438.1c.16002765 5. Ferro JM , CanhãoP, StamJ, BousserMG, BarinagarrementeriaF; ISCVT Investigators. Prognosis of cerebral vein and dural sinus thrombosis: results of the international study on cerebral vein and dural sinus thrombosis (ISCVT). Stroke. 2004;35 (3 ):664–70. 10.1161/01.STR.0000117571.76197.26.14976332 6. Rottger C , TrittmacherS, GerrietsT, KapsM, StolzE. Sinus thrombosis after a jump from a small rock and a sneezing attack: minor endothelial trauma as a precipitating factor for cerebral venous thrombosis? Headache. 2004;44 (8 ):812–5. 10.1111/j.1526-4610.2004.04150.x.15330829 7. Saneto RP , SamplesS, KinkelP. Traumatic intracerebral venous thrombosis associated with an abnormal golf swing. Headache. 2000;40 (7 ):595–8. 10.1046/j.1526-4610.2000.00093.x.10940100 8. D’Alise MD , FichtelF, HorowitzM. Sagittal sinus thrombosis following minor head injury treated with continuous urokinase infusion. Surg Neurol. 1998;49 (4 ):430–5. 10.1016/s0090-3019(97)00182-1.9537663 9. Linn J , Ertl-WagnerB, SeelosKC, StruppM, ReiserM, BrückmannH, . Diagnostic value of multidetector-row CT angiography in the evaluation of thrombosis of the cerebral venous sinuses. AJNR Am J Neuroradiol. 2007;28 (5 ):946–52.17494676

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==== Front 7806090 6166 Neurosci Biobehav Rev Neurosci Biobehav Rev Neuroscience and biobehavioral reviews 0149-7634 1873-7528 35961385 10.1016/j.neubiorev.2022.104811 nihpa1916397 Article Epigenetic mechanisms regulate cue memory underlying discriminative behavior Shang Andrea a Bieszczad Kasia M. abc* a Dept. of Psychology – Behavioral and Systems Neuroscience, Rutgers University – New Brunswick, 152 Frelinghuysen Road, Piscataway, NJ 08854, USA b Rutgers Center for Cognitive Science (RuCCS), Rutgers University, Piscataway, NJ 08854, USA c Department of Otolaryngology - Head and Neck Surgery, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08854, USA * Corresponding author at: Dept. of Psychology – Behavioral and Systems Neuroscience, Rutgers University – New Brunswick, 152 Frelinghuysen Road, Piscataway, NJ 08854, USA. kasia.bie@rutgers.edu (K.M. Bieszczad). 11 7 2023 10 2022 09 8 2022 18 7 2023 141 104811104811 https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). The burgeoning field of neuroepigenetics has introduced chromatin modification as an important interface between experience and brain function. For example, epigenetic mechanisms like histone acetylation and DNA methylation operate throughout a lifetime to powerfully regulate gene expression in the brain that is required for experiences to be transformed into long-term memories. This review highlights emerging evidence from sensory models of memory that converge on the premise that epigenetic regulation of activity-dependent transcription in the sensory brain facilitates highly precise memory recall. Chromatin modifications may be key for neurophysiological responses to transient sensory cue features experienced in the “here and now” to be recapitulated over the long term. We conclude that the function of epigenetic control of sensory system neuroplasticity is to regulate the amount and type of sensory information retained in long-term memories by regulating neural representations of behaviorally relevant cues that guide behavior. This is of broad importance in the neuroscience field because there are few circumstances in which behavioral acts are devoid of an initiating sensory experience. Epigenetics Behavior Memory Cue memory Learning Neurophysiology Sensory systems Neuroplasticity Neuroscience Histone acetylation DNA methylation ==== Body pmc1. Introduction Since the late 1990s, findings in behavioral neuroscience have shown that epigenetic mechanisms are essential to learning and memory processes (Miller, 2010; Duke et al., 2017). Post-translational modifications to histones and to the DNA backbone act at the level of the genome without altering the DNA sequence itself (Allis et al., 2007). A generally agreed upon role for histone and DNA marks that can modify and remodel chromatin in the adult brain is to set a permissive state for event-related gene expression, which is the molecular basis for long-term memory (LTM) formation (Agranoff et al., 1967; Alberini, 2009; Goelet et al., 1986; McGaugh, 2000). For example, histone deacetylation by some histone deacetylases (HDACs) and DNA methylation by DNA methyltransferases (DNMTs) and can work together either synergistically or independently (c.f., Zovkic and Sweatt, 2013) to typically restrict and block access, respectively, to gene promoters and enhancers required for activity-dependent transcription in critical circuits underlying memory consolidation. The combinatorial logic of such marks and interacting enzymes on chromatin structure offers the possibility of an extremely precise code for uniquely controlling key genes and gene families within cells and circuits without pan effects on the genome and brain. What appears to be critical is the neural locus of the permissive state set by epigenetic regulators to enable experience-driven and activity-dependent processes (Fig. 1). As such, the study of behavioral epigenetics has impacted multiple fields broadly related to experience-dependent learning processes in the brain, including those for understanding stress and anxiety (e.g., reviewed in (Kwapis and Wood, 2014; Kim et al., 2018), drug addiction (e.g., Rogge and Wood, 2013; López and Wood, 2015; Mews and Calipari, 2017), including habit formation and relapse (Malvaez et al., 2018; Pribut et al., 2021; reviewed in Werner et al., 2021), as well as for understanding fundamental memory processes like its formation (e.g., Levenson and Sweatt, 2005; Oliveira, 2016; Woldemichael et al., 2014), extinction (e.g., Malvaez et al., 2013; reviewed in Marshall and Bredy, 2019), reconsolidation, forgetting or extinction (Hemstedt et al., 2017; Hitchcock et al., 2018; Stafford and Lattal, 2011; Lattal and Wood, 2013), the distinction between recent versus remote memories (Gräff et al., 2014; Parrish et al., 2010; Day and Sweatt, 2011; White and Wood, 2014; Zovkic and Sweatt, 2013; Jarome and Lubin, 2014), and for different types of memory (e.g., pain, Rahn et al., 2013; aversive, Kwapis and Wood, 2014; appetitive, Tuesta and Zhang, 2014) and memory disease (e.g., Fischer, 2019). Recent literature suggests that the impact of epigenetic processes on learning and memory extends to the sensory systems. This review focuses on a process central to all experience-dependent processes that begins with cue-evoked activity in the sensory brain. Sensory brain areas from the cortex to even the earliest stages of sensory processing are well-accepted as critical sites of learning-induced plasticity in the adult brain (McGann, 2015; Weinberger, 2004, 2007). Thus, the sensory systems of the brain are capable of transforming transient sensory cue-evoked neural activity into persistent memory of those same sensory experiences. The integration of molecular epigenetic effects on systems-level plasticity processes in the sensory brain may be essential for weaving sensory features into the fabric of lasting memory to robustly guide cued behaviors over the long term. The purpose of this review is to showcase evidence from different sensory models of learning and memory that together indicate a remarkable epigenetic contribution to experience-dependent sensory system activity and its subsequent impact on highly discriminative cued behavior. A unifying theme that emerged from this literature survey is that epigenetic mechanisms appear to regulate the amount and type of detailed sensory cue information that is retained from experience into long-term memory. This effect is revealed behaviorally by the ease with which subjects acquire and perform discriminative stimulus-cued behaviors, and physiologically by lasting changes to cue-specific sensory evoked neural activity. Discriminative behavior is guided by the formation of cue-specific memory, which itself does not require discriminative learning per se. For instance, when first interrogated with a behavioral discrimination, animals previously trained to associate just a single-stimulus with an outcome like reward have been shown to respond discriminatively to the learned signal vs. novel test stimuli (e.g., Rotondo and Bieszczad, 2020; Shang et al., 2019; Biergans et al., 2012, 2015; Lockett et al., 2014). The implication of these findings in toto is that learning-induced epigenetic processes could change the post-mitotic function of learning-activated cells in the sensory brain long after the cue stimulus has been removed, dependent on which cells were activated by the sensory identity of the cue (Fig. 1). The role that different chromatin modifications or remodeling processes have on the sensory brain, either directly or indirectly, is broadly important because there are few circumstances, if any, in which behavior is devoid of an initiating sensory “cue” stimulus. Particularly exciting is the current availability of modern tools to manipulate epigenetic processes that, when appropriately harnessed, may be used to exploit the brain’s ability to store highly precise sensory information from significant life experiences with more cues, more detail, and for longer periods of time. With this in mind, the research highlighted here focuses on behavioral and neurophysiological studies that directly manipulate currently tractable epigenetic mechanisms from a selection of epigenetic players that have been implicated in learning or memory formation in non-sensory models including DNA methylation (e.g., via DNA methyltransferases, DNMTs) and histone acetylation (e.g., via histone acetyl transferases, HATs; or histone deacetylases, HDACs). While beyond the scope of the present review, other chromatin modifications like phosphorylation, or methylation; chromatin remodeling involving nucleosomes and tertiary structure; and small-molecule non-coding RNA are also likely at play in the sensory brain, which will require future study. Foundational studies have taken advantage of approaches that can block the function of transcriptional repressors to set a permissive state for gene expression. Many approaches have been used to block DNMT or HDAC enzymes that catalyze the addition of repressive epigenetic marks to DNA or chromatin, respectively, which normally restricts activity-dependent gene expression. Research that identifies correlative relationships without direct experimental manipulations of epigenetic molecules or that investigates downstream molecular pathways to explicate cellular changes in function have been reviewed elsewhere (e.g., Campbell and Wood, 2019; Feng et al., 2007) and are beyond the scope of this focused review. Evidence across sensory modalities together support that epigenetic players may be key to transforming highly precise sensory information (i.e., sensory features) from transient experience into long-term memory that mimics the sensory precision at the time of learning to support the later recall of accurate and precise stimulus identity. 2. Sensory models of memory implicate an epigenetic mechanism of experience-dependent effects 2.1. Auditory models Some of the first studies to implicate epigenetic mechanisms in memory for behaviorally-relevant sensory cues are in auditory models of associative learning. Findings from tone-cued fear conditioning and tone-cued reward learning experiments identified that histone acetylation (Guan et al., 2009; Bredy et al., 2007; Bredy and Barad, 2008), DNA methylation (Day et al., 2013; Monsey et al., 2011) and some forms of histone methylation (Gupta-Agarwal et al., 2012, 2014) are involved in long-term memory (LTM) formation. While many of these studies used systemic administration of pharmacological inhibitors or forebrain-wide genetic deletion approaches to target a candidate epigenetic mechanism, some targeted specific brain regions to show epigenetic contributions within select fear and reward systems, like the lateral amygdala (LA; Monsey et al., 2011; Maddox and Schafe, 2011; Maddox et al., 2014; Gupta-Agarwal et al., 2012, 2014) and ventral tegmental area (VTA; Day et al., 2013), respectively. Together, these reports introduce the idea that epigenetic effects are complex and likely act across a network of brain regions and interacting neural mechanisms involved in experience-dependent processes. As such, other studies have focused more deliberately on auditory system function (Table 1). A 2013 study (Gervain et al., 2013) examined absolute pitch learning in humans. Absolute pitch is the ability to identify or vocally recreate a particular pitch in the absence of a reference pitch, which can be learned in an early life critical period (Levitin and Zatorre, 2003), though studies in adulthood have had mixed success training absolute pitch (Levitin and Rogers, 2005; but see also Wong et al., 2020). Gervain and colleagues (2013) found that musically naïve adults that were treated with a class I HDAC inhibitor (valproate) while undergoing two weeks of absolute pitch training developed an exceptional ability to correctly identify absolute pitch during a later identification test (compared to placebo-treated controls undergoing the same training). These findings were interpreted as evidence for HDAC inhibition to facilitate auditory system function by reopening critical periods for experience-dependent plasticity in adulthood, a hypothesis that is supported by a similar demonstration using VPA in music-exposed mice (Yang et al., 2012). This finding is broadly consistent with the idea that epigenetic players in the adult brain can set up a permissive state for experience to modify sensory neural function and – ultimately – behavior. Studies that have focused on auditory system function are congruent with reports from tone-cued fear-conditioning paradigms that demonstrate how blocking class I HDACs broadly (Bredy et al., 2007; Bredy and Barad, 2008) or specific HDACs like HDAC3 (Kwapis et al., 2017) enhances sound-cued freezing behavior. Similar effects of enhanced tone-cued freezing and extinction of freezing have been reported with genetic knockout of HDAC2 (Guan et al., 2009; Morris et al., 2014). Interestingly, conditional knock-out of Dnmt3a, which encodes a DNA methyltransferase that normally adds methyl marks to directly repress gene expression, but not Dnmt1, has been reported to impair the extinction of cued fear memory (Morris et al., 2013), suggesting distinct roles for different DNMTs in cued associative learning and memory. What this means is that at least two of the most studied epigenetic mechanisms in the adult brain, histone acetylation and DNA methylation, are part and parcel to auditory cue learning. Animal studies have been critical to understanding the physiological underpinnings of epigenetic effects on behavior. Some inhibitors available commercially have been designed to selectively target different HDACs or DNMTs, which adds insight to the complexity of epigenetic effects on the brain and behavior. For example, of the class I HDACs that are found in the nucleus with direct access to chromatin, HDAC3 is the most highly expressed in the brain (Broide et al., 2007) and may be an essential player in memory processes, especially since it is not involved in cellular differentiation or identity like other class I HDACs (e.g., HDAC2; see Fischer et al., 2010). As such, HDAC3 has been singled out as a “molecular brake pad” on gene expression that is necessary for long-term memory formation in the adult brain (McQuown and Wood, 2011). Links between memory formation and neurophysiological plasticity in the adult sensory brain have been most extensively studied in the auditory system (McGann, 2015; Weinberger, 2004, 2007). Various forms of learning-induced auditory cortical plasticity, such as sound-specific tonotopic map expansions, receptive field shifts or changes in receptive field shape, like bandwidth for acoustic frequency, are known substrates of auditory associative learning and memory for simple tones (reviewed in Weinberger, 2015). Thus, a series of studies in adult rats learning about simple sound cues examined the physiological effects of pharmacological inhibition of HDAC3 on sound cue-responsivity in the auditory cortex. These were the first to establish a role for epigenetic processes to regulate typical neurophysiological substrates of learning and memory in the sensory brain. In the first of these studies, Bieszczad et al. (2015) trained adult rats to perform a simple appetitive operant task: lever-press to a tone cue for reward. Rats were treated daily with systemic post-training session injections of either a selective HDAC3-inhibitor (RGFP966) or with vehicle. Remarkably, treatment with the HDAC3-inhibitor caused the auditory cortex to remodel in an unusually multidimensional way (relative to vehicle treatment) by expanding the cortical map of sound frequencies and sound levels associated with reward while also “tuning-in” those receptive fields to respond to the tone cue more selectively by narrowing response bandwidths around the cue’s acoustic frequency. Furthermore, these neural changes were mirrored in behavior: the same animals treated with the HDAC3-inhibitor were also more likely (vs vehicle controls) to selectively lever-press to the precise sound cues linked with reward during prior training relative to novel sound cues. The more selective the auditory cortical response to the trained tone cue (i.e., narrower bandwidth), the more specific the behavioral response to the same tone cue. Subsequent auditory studies corroborated this brain-to-behavior relationship of increased sound cue-selectivity mediated by HDAC3, extending its experience-dependent physiological influence also to subcortical auditory processing (Rotondo and Bieszczad, 2020) and to tasks that establish inhibitory associations between sound and reward, like in tone-cued extinction (Rotondo and Bieszczad, 2021a). What this means is that HDAC3 appears to facilitate cue-selective neural responsivity across the auditory system and across different kinds of tone-cued memory that similarly guide discriminative behaviors, regardless of associative meaning (excitatory vs. inhibitory) or the “direction” of behavior (go vs. no-go). Interestingly, in many of the studies described, the behavioral effects of HDAC-inhibition were not evident during task acquisition nor in the absolute levels of correct performance achieved with training: whether or not treated with the HDAC3-inhibitor, animals learned at the same rate and to the same high level of absolute performance. Increased cue-selectivity was revealed only in behavioral memory tests that required discrimination, i.e., in which animals were probed with the training vs. novel sound cues to discover if the treatment had caused behavioral responding to be highly selective to only the trained sound and impervious to lures. Therefore, if an epigenetic player like HDAC3 regulates the encoding specificity for sensory cues in memory, then tasks that require overt discrimination to incrementally achieve better correct performance would be expected to show effects during task acquisition. This hypothesis was supported in a sound cue discrimination task that required animals to learn and perform an acoustic frequency discrimination. Shang et al. (2019) used a similar protocol for systemic inhibition of HDAC3 using RGFP966 in animals learning an easy two-tone discrimination task (this task was easy in the sense that the two training cues were sound frequencies easily distinguishable even to a naïve listener, human or rodent). The animals were required to respond to one sound frequency for reward, while inhibiting responses to the second sound frequency to avoid an error-signaled time-out period. In this case, task performance was facilitated by post-session administration of RGFP966 that became evident only on the third day of discrimination training (i.e., after two sessions of training with post-session treatments), which was corroborated by frequency-specific effects on auditory cortical plasticity and behavior weeks later. This supports that the effects of systemic HDAC3 inhibition are on memory consolidation, and not task acquisition or performance per se, though studies with appropriate manipulations using genetic knock-outs or viral perturbations to determine any potential effects of HDAC3 function on within-session learning have yet to be accomplished. That auditory cued behavior is influenced by epigenetic processes more broadly is supported by findings that pharmacological DNMT inhibition, shown to block activity-dependent intragenic DNA hypermethylation at key learning-related plasticity genes, also blocks behavioral tone-reward learning and task performance (Day et al., 2013). Further, epigenetic effects have also been found for discriminative behaviors guided by sound cues other than spectral frequency, including for temporal cues like amplitude-modulated (AM) sounds (Rotondo and Bieszczad, 2021b). Remarkably, animals learning an appetitive instrumental task that requires discrimination between two different AM rates (e.g., fast vs. slow) developed highly specific memory for the AM rate paired with reward when treated with an HDAC3-inhibitor. The behavioral effects were correlated with neurophysiological enhancements for temporal processing in the auditory system. Together, these auditory-focused studies lead to the hypothesis that epigenetic mechanisms impact the precision of the experience-to-memory transformation for the multidimensional and complex sensory cues of our daily lived experiences by regulating the relevant neurophysiological parameters of cue-selective responsitivity in the sensory brain. From an auditory perspective, this hypothesis can be tested using spectro-temporally rich and complex acoustic communication sounds like vocalizations. Indeed, using a zebra finch birdsong model of experience-dependent memory for conspecific voices, Phan et al. (2017) found that treatment with an HDAC3-inhibitor in adult birds enabled a 10-fold reduction in the exposure threshold required to induce neurophysiological changes to responses evoked by the heard conspecific vocalizations. Interestingly, the effect was only in the left (but not right) caudal medial nidopallium (NCM), a higher order auditory area in birds known to support memory for complex song stimuli (Vates et al., 1996; Eda-Fujiwara et al., 2003) and with a long history of gene expression events necessary for neurophysiological and behavioral experience-dependent changes (Mello et al., 1992; Jarvis et al., 1995; Chirathivat et al., 2015). While long-term effects (lasting >20 h) typically require ~200 repetitions, in birds treated with the HDAC3-inhibitor, only 20 repetitions of a conspecific song was able to induce changes in gene expression and sound-evoked neural activity. Related studies in the developing zebra finch support the ability of epigenetic regulation to promote very long-lasting effects from juvenile experience with spectro-temporally rich auditory cues onto adult behaviors (Kelly et al., 2018). Changes in chromatin were found to occur in juvenile birds listening to tutor songs with delayed, yet profound, effects on adult song-specific singing behaviors. These results are interpreted as a form of “epigenetic priming” in which chromatin landscapes, including marks incurred by histone acetylation, DNA methylation, and secondary chromatin structure, can set the threshold for subsequent sensory or learning experiences to induce activity-dependent gene expression. The major benefit of epigenetic priming over transcription factor activity are the long versus short, respectively, time scales at which these molecular mechanisms prime the system for gene expression induced by sensory stimulation. That they can operate in highly stimulus-specific ways is remarkable and in need of further investigation (Burns and Gräff, 2021). Overall, these findings in auditory models are particularly relevant to human auditory brain function for acoustic communication and comprehension, which demands a balance between highly specific encoding of acoustic cue features to identify a speaker’s voice or their emotional context (e.g., Dietrich et al., 2008; Jiang and Pell, 2015), and more general cue encoding strategies that allow humans to understand the same words from different speakers (e.g. Von Kriegstein et al., 2010). Because auditory neurophysiological effects of HDAC-inhibition are evident using non-invasive recording strategies like the auditory brainstem response (ABR) and frequency-following response (FFR) (Rotondo and Bieszczad, 2020, 2021a, 2021b) that are reliably detectable in humans (e.g., White-Schwoch et al., 2013), future investigation of the neurophysiological correlates of epigenetic manipulations on memory and discriminative behavior such as those originally reported in Gervain et al. (2013) in humans is tractable and feasible even in clinical settings. Auditory models of learning and memory have been critical to introduce an epigenetic role in the physiological function of the adult sensory system. They support that a behavioral consequence of epigenetic action in the sensory brain is to regulate the formation of cue-memory and subsequent discriminative behavior. Moreover, an epigenetic contribution appears across different types of tasks, whether appetitive or aversive, to direct behavior to either promote or inhibit cue responding, and across various behaviorally-relevant features and complex multidimensional features of a heard sound cue. 2.2. Olfactory models Research in olfactory models of learning and memory, particularly in honeybees, have been especially useful in examining the behavioral consequences of epigenetic action on cue memory (Table 2). Studies have demonstrated the involvement of histone acetylation (Merschbaecher et al., 2012, 2016; Lockett et al., 2014) and DNA methylation (Lockett et al., 2010; Biergans et al., 2012, 2015) in regulating long-term associative memory for olfactory cues, which can be elegantly revealed behaviorally in the honeybee’s proboscis extension response (PER). In the 2010 study by Lockett et al., researchers implicated DNMT3, which is one of four DNA methyltransferases in honeybees (others are DNMT1a, DNMT1b, and DNMT2; in concert with the Ten-eleven translocation or Tet enzymes, which contribute to the biochemical cycle of active demethylation; Biergans et al., 2015) that participate in de novo DNA methylation in memory processing. Generally, DNMT enzymes add methyl marks on DNA that silence gene expression required for memory formation (for a broad review of DNA methylation in behavioral epigenetics see Sultan and Day, 2011; Zovkic and Sweatt, 2013; and Campbell and Wood, 2019). Dnmt3 transcription was found to be upregulated selectively in the mushroom bodies of the honeybee brain—an important locus of experience-dependent effects in the olfactory system—after training to link a particular odor with reward. Interestingly, administering the DNMT inhibitor zebularine did not impair learning during odor-reward training, but instead decreased the retention of the learned association over time. For example, if administered immediately after odor-reward training, the treated honeybees were faster to decrease cued proboscis extension responses when presented with odor alone (without rewards) during extinction training, which suggests that the DNMT inhibitor produced a weaker odor-reward associative memory. In contrast, if zebularine was administered 1 h after odor-reward training, or before extinction training, then the opposite effect was evident: extinction learning was either delayed or more weakly retained (relative to vehicle controls). Under these time-dependent conditions, treated animals had a stronger memory for the original odor-reward association than for the odor-“no-reward” association learned during extinction training. The authors interpreted these findings to support the idea that epigenetic marks from an initial learning experience (here, the acquisition of an odor-reward association) have a time-dependent influence on how subsequent epigenetic activity modulates re-learning (so-called memory “updating”) of previously learned associations (e.g., extinction to associate a previously rewarded odor with reward omission). Experience- and time-dependent expression of both Dnmt3 and Dnmt1b (as well as Tet) have since been implicated also in odor-reward memory (Biergans et al., 2015). Indeed, DNMT activity does not appear to be critical for odor-reward acquisition per se. It is involved in the longevity of newly formed olfactory memories over time and interference (Lockett et la, 2010; Biergans et al., 2012; Biergans et al., 2016), which is consistent with studies that demonstrate DNMT inhibition impairs task reversal learning without simultaneously affecting the ability to acquire new and unique odor-outcome associations (Biergans et al., 2016). Olfactory models also provide support for the hypothesis supported by auditory studies that an essential behavioral consequence of epigenetic action in the sensory brain is to establish highly precise cue memory for discriminative behavior. A series of studies also in honeybees showed that DNA methylation impacts discriminative cue memory to drive learned odor-cued behaviors. In these studies, behavioral memory testing occurs after a training phase and consists of presentations of the trained odor or a novel odor to determine whether the behavioral response will be limited to only the previously experienced odor cue. If discriminative memory is formed, behavior should not generalize to the novel odor. Animals treated with DNMT inhibitors (either zebularine or another a potent DNMT inhibitor called RG108) later generalize their responses to both odors, which demonstrates impaired discriminative behavior at a long-term memory time point (i. e., 24-hours after training; Biergans et al., 2012, 2015). Consistent with these studies, Biergans et al. (2016) found that treatment with RG108 impaired discriminative long-term memory for odor cues, but only after intensive training. Interestingly, the same DNMT inhibitor treatment in animals that had undergone a weak training paradigm instead facilitated odor cue memory during a discrimination test 24-hours later. This is not entirely surprising considering reports of increased histone acetylation with increased training strength, and that HDAC inhibition has been consistently shown to promote long-term memory formation also in honeybee odor memory, particularly under weak training conditions (Merschbaecher et al., 2012, 2016) as in rodents (Stefanko et al., 2009; Federman et al., 2009). Bidirectional effects of DNA methylation or HDAC inhibition on stimulus-specific discriminative behavior may be to promote ecologically beneficial foraging in honeybees. Odor cues consistently associated with a reward outcome might engage epigenetic action to activate a transcriptional program that is fundamentally different from the transcriptional activity invoked by odor cues that are less predictive of reward (Biergans et al., 2016). In addition to training strength, different training paradigms can influence the timepoint at which Dnmt3 increases in expression following training (e.g., at 30 min following odor discrimination training: Lockett et al., 2010; vs. after 5 h after classical conditioning of odor cues: Biergans et al., 2015). This is important evidence to show that the same epigenetic player can regulate different waves of experience-dependent gene expression over time and task type. Furthermore, it is likely that the same epigenetic regulators can influence largely independent networks of genes required for different types of sensory cued behaviors, for example those that adaptively require approach (as in reward learning) or retreat (as in avoidance learning), consistent with at least one report of differential effects of HDAC inhibitors in the honeybee for odor-cued memory for aversive compared to appetitive outcomes (Lockett et al., 2014). That said, extensive research using HDAC inhibition in rodent models of aversive (fear) conditioning (reviewed in Kwapis and Wood, 2014) and reward learning (reviewed in Elvir et al., 2019) including odor-reward training in honeybees (Merschbaecher et al., 2012, 2016) have shown enhancements to long-term memory formation. Thus, the actual source of differential effects of HDAC inhibition on aversive vs. reward learning are yet unclear, especially without full consideration of the extensive interplay between histone acetylation and DNA methylation on experience-dependent processes (Miller et al., 2008). It is important to highlight that the predominant effect of epigenetic action in the adult brain is on long-term memory for discriminative cued behavior, and not learning ability nor on perceptual skill (Biergans et al., 2012, 2016, 2017). This differs from studies using an olfactory model that show trans- and inter-generational effects of olfactory learning that are associated with heritable DNA methylation events (for review, see Dias and Ressler, 2014). These elegant trait inheritance studies show that epigenetic mechanisms can alter perceptual acuity by modifications to the number and sensitivity of receptors in the olfactory bulb in the progeny of odor-cue trained animals, without actual transfer of the learned discriminative behavior itself (Dias et al., 2013, 2015; Klengel et al., 2016). These studies are particularly useful in behavioral epigenetic study because of the one-to-one mapping of gene to odorant receptor. In contrast, the physiological consequence of DNMT activity within the “experienced” olfactory system of an odor-cued trained animal appears to involve effectors of synaptic plasticity that influence encoding in excitatory-inhibitory neuronal networks (Biergans et al., 2015, 2017). Interestingly, the network changes influenced by epigenetic action in the antennal lobe (a homolog of the mammalian olfactory bulb) of an odor-trained honeybee are reminiscent of pattern separation (vs. pattern completion) processes in the hippocampus that are essential to complex discriminative behaviors even in humans (Yassa and Stark, 2011). Overall, that epigenetic regulators can influence cue memory to bidirectionally promote or impair odor cue memory depending on time, training intensity, and training paradigm, adds to our appreciation of their complex actions on experience-dependent modifications in the sensory brain for discriminative behavior. 2.3. Visual models Since seminal experiments by Hubel & Wiesel in the 1960s, experience-induced changes in the visual system — particularly in visual cortex (e.g., primary, V1) — have been extensively investigated to demonstrate the profound influence of visual stimulation on cortical circuits and function early in postnatal life (e.g., Wiesel and Hubel, 1963; Faglioniet al., 1994; Frenkel and Bear, 2004). There is a wealth of neurophysiological studies that implicate epigenetic regulation in the developing visual system. Monocular deprivation (MD) is a classic, experimental paradigm first used by Hubel and Wiesel (1963) in kittens to study how visual inputs influence early postnatal neural function. Depriving one eye of visual stimulation leads to an imbalance in the inputs and shifts responding to favor signals from the nondeprived eye, known as ocular dominance (OD) plasticity (Wiesel and Hubel, 1963; Gordon and Stryker, 1996) and likely cortically mediated (Sawtell et al., 2003, in Fischer et al., 2007). OD plasticity decreases with age: that is, longer periods of deprivation are required to induce OD plasticity in adult animals compared to juveniles (Sawtell et al., 2003; Hofer et al., 2006), which is consistent with the idea that there is a higher threshold for experience-dependent plasticity with increasing age. Furthermore, monocular deprivation causes behavioral deficits in visual acuity that are more resistant to recovery in adulthood (Putignano et al., 2007; Silingardi et al., 2010; Sale et al., 2007; Gotou et al., 2021). Thus, researchers have been particularly motivated to identify and block the molecular ‘brakes’ that normally regulate the decline in age-dependent visual system plasticity in adulthood, in particular for their potential to recover visual deficits (reviewed in Baroncelli and Lunghi, 2020). Epigenetic enzymes may be ideal candidates as they are differentially modulated by visual experience in adult compared to juveniles. For example, histone acetylation and DNA methylation levels in the visual cortex are regulated in juveniles by visual stimulation alone, but not in adult rodents (Putignano et al., 2007; Vierci et al., 2016; Tognini et al., 2015). Further, manipulating levels of histone acetylation by treatments that manipulate DNA methylation (Apulei et al., 2018) or with general HDAC inhibitors (e.g., VPA, Silingardi et al., 2010; Lennartsson et al., 2015; or trichostatin A (TSA), Putignano et al., 2007; Maya-Vetencourt et al., 2011; or sodium butyrate, Silingardi et al., 2010) have been reported to enable OD plasticity in adult animals and restore visual acuity after early-life deprivation compared to untreated controls. Thus, HDAC inhibition may be an attractive potential therapeutic target by its apparent action to “reinstate” early-life susceptibility to experience-dependent visual cortical plasticity. While a complete account of the molecular mechanisms underlying epigenetically-regulated neuroplasticity is beyond the scope of this review, it should be noted that OD plasticity can also be reactivated in the adult brain by environmental enrichment (Sale et al., 2007; Tognini et al., 2012), which itself has been shown to induce hyperacetylation (Baroncelli et al., 2016) and increase the expression of bdnf (Maya-Vetencourt et al., 2011). Furthermore, increases in bdnf expression that permit adult OD plasticity mediated by the neuromodulator, serotonin, have been associated with a temporary increase in histone acetylation at the bdnf promoter that can be mimicked by application of the HDAC inhibitor, TSA (Maya-Vetencourt et al., 2011; see also Tiraboschi et al., 2013). Visual learning experiences continue to drive plasticity in the adult visual brain (reviewed in Karmarkar and Dan, 2006), with changes that can affect both perceptual and cognitive processes related to visual discriminative behavior. The past two decades have also introduced studies that implicate epigenetic mechanisms in long-term memory formation for visual stimulus cues in adult animals (Table 3). For example, in the invertebrate crab model, a looming opaque sheet can be used as a visual danger cue to provoke an initial escape response. The behavioral response to escape eventually habituates—that is, it decreases after repeated presentations of the same visual stimulus once animals learn and remember the visual cue is not associated with a negative (aversive) outcome, like an approaching predator. Fifteen trials of the looming sheet spaced over time are typically required to form long-term memory that habituates behavior. However, crabs treated with a general class I HDAC inhibitor (either sodium butyrate or TSA) that increased histone acetylation were able to consolidate the visual cue memory after just five trials (Federman et al., 2009). Remarkably, despite the reduced threshold for its induction (from 15 to just 5 trials), the memory formed out-competed memory of a prior event learned under normal conditions (Federman et al., 2012). This supports that HDACs (and histone acetylation) can regulate the exposure threshold necessary for visual cue memory formation, which is consistent with memory induced by sound exposure in bird models (Phan et al., 2017) and with object recognition studies in mammals (Stefanko et al., 2009). In a seminal study in the mammalian visual system, Yeh et al. (2004) found that treatment with the HDAC inhibitor, TSA, facilitated long-term memory for light-cued startle in which the autonomic startle response is dampened by the brief presentation of a light-cue after training. Interestingly, the effect of HDAC inhibition on behavior was only evident when tested 24 h after training, and not detected 2 h after training. This difference supports the putative role for HDACs in consolidation for long-term memory, which is transcription-dependent, and not at a short-term memory time point that does not require de novo gene expression. Furthermore, the behavioral effect was explained by an HDAC3-mediated mechanism that engaged the histone acetyltransferase, CREB-binding protein (CBP), during a time window critical for memory consolidation following training. Finally, long-term potentiation (LTP)—a widely-accepted physiological correlate of learning and memory processes in the brain—at synapses between the sensory cortex and amygdala thought to support the light-cued startle response, was strengthened with TSA treatment. Thus, it is possible that neuroplastic potentiation of sensory cortical outputs can drive physiological changes underlying visually-cued learned behaviors. Indeed, learning-induced cortical physiological changes in the visual system have been extensively studied. For example, in V1 neurons of the adult brain, the amplitude of visual-evoked potentials (VEPs) are increased to select visual stimulus cues after learning (e.g., Shuler and Bear, 2006; Hager and Dringenberg, 2010, 2012). This form of plasticity is a stimulus-selective response potentiation, which is defined by characteristic increases in VEP response magnitude only to the specific features of the visual cue paired with a behaviorally-relevant outcome like reward (Cooke and Bear, 2012). As such, epigenetic manipulations (like HDAC or DNMT inhibition) may also feasibly enable activity-dependent changes in cue-selectivity in the visual system that permit discriminative visually-cued behavior, and remains to be tested experimentally. 2.4 Other sensory models suggest an epigenetic role for cue-dependent discriminative behavior 2.4.1. Gustatory: conditioned taste aversion (CTA) Studies from another important sensory model of long-term cue memory showcases conditioned taste aversion (CTA) and its neural correlates in the gustatory (insular) cortex. Gustatory circuits appear to involve a diverse set of epigenetic molecules to regulate taste cue memory at various timescales to vary the dynamics and lastingness of associative memories formed between specific taste cues and malaise. Early work in this gustatory model by Swank and Sweatt (2001) was first to demonstrate the involvement of histone acetyltransferase and lysine acetyltransferase activity in signal transduction cascades necessary for novel taste learning. The authors combined in vivo and in vitro experiments to show that CTA is dependent on the MAP kinase cascade in the insular cortex (IC), where taste-evoked neurophysiological activity coincides with histone acetyltransferase (HAT) activity. Subsequent studies have expanded on the role of epigenetic mechanisms in the IC – in particular, histone acetylation – to regulate aversive memory for conditioned taste. A 2014 study by Nuñez-Jaramillo and colleagues (Núñez-Jaramillo et al., 2014) found that infusing the general class I HDAC inhibitor, sodium butyrate (NaB), in the IC during CTA strengthened the associative memory between the taste cue and malaise, which was revealed behaviorally as delayed extinction of the taste aversion. The same effect of HDAC inhibition on CTA memory was also reported by (Rodríguez-Blanco et al., 2019) with post-training IC injections of the HDAC1/3-selective inhibitor MS-275 (“entinostat”). In this study, the authors determined that at least two rounds of transcription – one immediately, and one 7 h after CTA acquisition – were needed to strengthen CTA memory consolidation for a specific flavor. Furthermore, the authors found that infusion of MS-271 immediately or 7 h after acquisition enabled the formation of CTA memory after weak taste aversion training (0.1 M injections of the malaise-inducing agent, lithium chloride, which is below the normal molar threshold needed for taste aversion). This finding is again consistent with reports from other modalities of subthreshold experiences being promoted to long-term memory with HDAC inhibition (e.g., Stefanko et al., 2009; Phan et al., 2017). However, inconsistencies exist that may be related to distinct roles of different epigenetic players. For example, Morris et al. (2013) knocked out HDAC2 in mouse forebrain, which resulted in accelerated CTA extinction (this was not found in HDAC1 knockouts). Regardless, this body of work suggests a role for histone acetylation events in de novo transcription at multiple time windows after CTA acquisition that are sufficient to influence memory consolidation either by strengthening or weakening long-term memory. More studies are warranted to investigate whether any epigenetic mechanism that regulates CTA produces the same kind of cue-specific neurophysiological effects on cortical taste representation as in other sensory cortices. The field appears to be primed to address this question since there is evidence that taste representation in insular cortex is similarly sparsely organized as are odor representations in the olfactory system (Chen et al., 2021). Further, there is potential for cue-selective learning-induced insular plasticity within the gustatory circuit (Haley et al., 2020) that is casually related to behavioral choices for specific flavors (Vincis et al., 2020). 2.4.2. Somatosensory and sensorimotor Studies implicating epigenetic control of the somatosensory organization of the brain are few and far between. This is somewhat surprising considering the overwhelming evidence for experience-dependent plasticity of somatosensory cortical areas, which was first in the field to establish the highly dynamic nature of the sensory brain to altered peripheral inputs and sensory learning experiences in monkeys (Merzenich et al., 1983a, 1984, 1983b). Rodent studies have been important to establish cue-selective effects of experience to particular whisker representations in barrel cortex, where—like in auditory cortex—the cortical area of whisker representation can change with its behavioral relevance (Frostig, 2006) and is under tight molecular and transcriptional control (Siucinska and Kossut, 1996; Gao et al., 2018; Vallès et al., 2011). Whether this lack of evidence is due to the novelty of its study, or due to some fundamental ontogenic difference in somatosensory areas is currently difficult to discern, though critical periods do appear to close earliest there across development (Reh et al., 2020). That said, there is some evidence consistent with auditory, olfactory and visual models that epigenetic mechanisms are in play to regulate touch-selective responses. Feng et al. (2017) used an associative learning paradigm in mice that paired whisker and tail stimulation with a particular odorant. In some animals, this training led to odor-induced motion of the tail and/or whiskers. Remarkably, barrel cortical neurons in the animals that expressed the behavior recruited new synapses from the olfactory (piriform) and somatosensory “tail” cortical areas that was dependent on the activity of several non-coding RNAs (miRNA-324 and −133a), which epigenetically regulate activity-dependent transcription. Outside of the somatosensory cortex and instead in the cerebellum, Yamada et al. (2019) used a novel tactile-startle associative learning paradigm to train mice to associate a light cue with a startling nose-touch that induces a backwards (escape) movement. After training, mice made escape movements to the light cue alone, indicating that they could associate the light cue with the startling touch. The authors show a crucial role for a cerebellar circuit where learning induces changes to neuronal chromatin remodeling of its tertiary structure to alter sensorimotor-stimulus dependent gene expression: When a chromatin remodeler called Cohesin is selectively perturbed using a conditional CRISPR knockout strategy, the light-touch associative memory is disrupted. Together, these studies establish basic proof-of-concept that learned associative relationships between somatosensory cues and behavioral outcomes may be under epigenetic control. It also brings to question whether different sensory systems have similar or distinct libraries of available epigenetic molecules with which to alter chromatin dynamics in the service of stimulus-induced gene expression. Extending this line of inquiry may be a fruitful line of future research to build on sensory models of epigenetic influence (Table 4). 2.5. Multi-modal models Sensory models of learning and memory in which animals learn to associate discrete stimuli (e.g., a tone cue, a cue light, an odorant) with relevant outcomes (i.e., reward or aversion) have been discussed here as highly tractable approaches to examine cue memory and discriminative behavior. They have the benefit of equally tractable neurophysiological correlates of precise stimulus-related evoked activity within unimodal sensory systems. However, almost all associative learning experiences involve multi-sensory information. ‘Real-life’ experiences rarely, if ever, comprise of stimuli from just one sensory domain. In this section, we highlight studies that have investigated the role of epigenetics in memory for multimodal stimuli like objects and contexts (Table 5). Here we define “objects” as three-dimensional physical entities (like a lego block or a glass beaker), and “contexts” devoid of particular spatial location information per se to avoid the need for a higher-level representation than is required for the examples at hand. As such, both objects and contexts have multiple sensory features, including visual appearance, smell, texture, etc., and as such, are considered here as multimodal stimuli. As a particularly potent example paradigm, novel object recognition (NOR) capitalizes on a rodent’s tendency to explore novel objects and can be used to study how epigenetic mechanisms modulate object memory (e.g. Vogel-Ciernia and Wood, 2014). If animals spend more time exploring a new vs. previously experienced object, then the interpretation is that they consider the new object “novel” and remember the “familiar” object. Thus, performance in NOR requires sensory discrimination between the multimodal features of the two presented objects. Initial experience with an object that a subject actively explores by looking, sniffing, whisking, touching, and other interaction, can alter epigenetic marks including DNA methylation (Muñoz et al., 2010), histone acetylation, and phosphorylation (Gräff et al., 2012) and induce the expression of immediate early genes (e.g., Tanimizu et al., 2018) requisite for long-term memory formation. Seminal studies have implicated histone acetylation in the formation of exceptionally long-lasting object memory under training conditions that are normally insufficient for long-term memory consolidation. Stefanko et al. (2009) demonstrated that mice given 3 min of exploration time with an object are normally unable to form object memory assessed by NOR 24 h after training. In contrast, mice treated with sodium butyrate (NaB) were able to form memory at this long-term timepoint, which required effectively discriminating the novel from the familiar multisensory features of the objects presented at test. Moreover, the treated animals retained object memory for up to a week, a time point at which normal memory usually fails. A 2013 study by Federman and colleagues (Federman et al., 2013) further investigated the effects of training strength on memory persistence. They found that strong training (longer time to explore an object, typically 10 min) that increased hippocampal histone H3 acetylation was able to induce object memory that lasted beyond 24 h and up to 7 days, while weaker training (i.e., less exploration time, typically <5 min) was insufficient. Gräff et al. (2012) also found that an intensive training protocol in mice (spaced 15 min vs. massed 5 min inter-trial intervals) known to induce robust memory at 7 days, enhanced post-translational modifications including histone phosphorylation, acetylation and methylation (pH3S10, AcH3K14, 3MeH3K36) in the prefrontal cortex. Concordantly, inhibition of protein phosphatase 1 (PP1), a regulator of histone post-translational modifications, in a different group of mice learning with a weaker training protocol was able to successfully increase histone marks and enable the persistence of memory. Post-translational modifications were critical for the behavioral effect, which could be blocked with pharmacological agents that interfered with the marks. That said, effects on NOR do not appear to be limited to post-translational modifications since microRNAs also appear to play a role (e.g., Hansen et al., 2010). Interestingly, ‘strong’ training experiences are not affected by HDAC inhibitors that facilitate long-term memory formation following partial or weak training experiences (Biergans et al., 2016; Heinrichs et al., 2013; McQuown and Wood, 2011; Merschbaecher et al., 2012, 2016; Monsey et al., 2011; Stefanko et al., 2009; Rodríguez-Blanco et al., 2019), which suggests that when an experience-dependent threshold is reached, the system is no longer sensitive to further epigenetic perturbation. Overall, these findings implicate a variety of epigenetic modifications in the formation of memory that persists over longer periods of time. A plausible explanation for the persistence of memory is that multimodal objects can be more robustly recalled when memory is attributed with more details of the sensory features characteristic of that object. This hypothesis is consistent with Tulving’s theory of cue-dependent forgetting, which posits that memory retrieval depends on the availability of the “right” cues. A successful retrieval cue is consistent with any of those available and abundant during the original experience (Tulving, 1974). Additionally, there is widespread multisensory convergence and integration across brain regions, including sensory-specific areas (reviewed in Driver and Noesselt, 2008). As such, it has been suggested that multisensory stimulation at the time of learning has better memory outcomes than for learning about unimodal sensory stimuli (reviewed in Matusz et al., 2017). As such, epigenetic manipulations that enable unusually persistent object memory may do so by creating a larger memory trace across the multiple sensory modalities that encompass the many various sensory features of that object. Nonetheless, many NOR studies focus on the role of the hippocampus, rather than the sensory systems, as a site of multimodal integration to underlie observed neural and behavioral effects (e.g., Gräff et al., 2012; Hansen et al., 2010; Frick, 2013). Yet, under circumstances where sensory systems are not interrogated directly, i.e., in studies involving hippocampal-dependent tasks and the more traditionally interrogated medial temporal lobe “memory” systems, it is important to remember that these areas are highly interconnected and dependent on initial processing in the sensory regions of the brain. Furthermore, while the hippocampus and surrounding entorhinal cortical areas are long-standing candidates for memory processes, it is important to remember that systemic applications of HDAC or DNMT inhibitors cross the blood-brain-barrier with broad access to multiple brain systems, including primary sensory areas. Altogether this means that behavioral effects observed could be due to epigenetic action in any number of independent or coordinated brain areas together and in parallel. There is a compelling example of the parallel processes that systemic epigenetic manipulations activate in multiple memory systems. Malvaez et al. (2013) cleverly combined two behavioral paradigms (NOR, and conditioned place preference, “CPP”) to determine independent effects of the same injection of the selective HDAC3-inhibitor, RGFP966, on memory for either task. Interestingly, what they found was that the same systemic injection of RGFP966 enhanced the acquisition of NOR memory while simultaneously promoting the behavioral extinction of CPP. This finding is remarkable because the two memories affected had opposite influences on behavioral performance: to increase behavioral responding in NOR while reducing behavioral responding in CPP. It is likely that the inhibition of HDAC3 altered gene expression in multiple brain regions that were important for either or both tasks, and that the genes activated (whether unique or not) within those systems altered neural processing to ultimately induce unique (and in this case opposite) patterns of behavioral expression. An important study by (McNulty et al., 2012) highlights the dependence on brain region for epigenetic effects on transcription. This study focused on the expression of nr4a1 and nr4a2, which are known to increase in the hippocampus after learning and are both regulated by class I HDACs (Vecsey et al., 2007). The class I HDAC3 specifically has been shown to modulate long-term memory that depends on nr4a2 expression (McQuown and Wood, 2011). McNulty et al. (2012) used siRNA to selectively block the expression of nr4a1 and nr4a2 either in the hippocampus or broadly in the cortex (by intracerebroventricular infusion, i.c.v.) in animals performing novel object recognition or a novel object location task. What they found is that reducing either nr4a1 or nr4a2 expression in hippocampus blocked hippocampal-dependent object location memory. However, for object recognition, only reducing nr4a2 expression in cortex was effective to block memory formation—reducing nr4a1 there had no effect on object recognition. What this means is that different brain regions under similar epigenetic control (e.g., by class I HDACs) critically regulate unique and sometimes non-overlapping sets of genes important for the consolidation of memory for different tasks. By extension, epigenetic controllers like HDACs and DNMTs may differentially regulate the same genes or completely different sets of genes within the different sensory systems depending on the sensory features used to perform the required task, and the demands on the neurophysiological response evoked by those sensory features. As such, it will be important to consider the possible contributing role of unique transcriptional profiles within sensory brain regions that work in concert with more traditionally implicated memory systems of the hippocampus and medial temporal lobe to contribute to the overall memory-enabling effect of epigenetic manipulations and their downstream effectors. 3. Discussion 3.1. Epigenetic mechanisms regulate discriminative behavior via the sensory systems Epigenetic mechanisms are critically involved in memory formation. This review has emphasized a putative role for epigenetic players like HDACs and DNMTs to regulate the sensory characteristics of long-term memory. Behavioral epigenetic studies from across different sensory models of learning support the view that the type and amount of detail remembered about experienced sensory cues is regulated epigenetically to promote or impede subsequent discriminative behavior at recall. As demonstrated by the breadth of the studies highlighted in this review, epigenetic mechanisms can facilitate or impair long-term cue memory at multiple stages of memory formation (e.g., at encoding and/or during consolidation), based upon whether the manipulation is expected to promote or repress de novo gene expression, respectively. Strong examples are from studies using blockade of HDAC or DNMT activity, which releases the brakes on gene expression required for memories to consolidate to the long term. In parallel, this review has showcased the currently known effects of such manipulations on robust changes to neurophysiological responses within the sensory systems themselves (Table 6). Accumulating evidence supports that the removal of epigenetic brakes on transcription is likely setting a permissive state in the sensory brain for activity-dependent neural processes evoked by cue presentation to subsequently “tune in” cue-selective neurophysiological activity to more selectively, strongly, or persistently respond to a subsequent bout of experience with the learned sensory cue. These actions appear behaviorally as a bias to respond selectively to the cue (vs. non-cue stimuli) regardless of the direction of the behavior to “go” or “no-go” and regardless of the type of learning experience, whether by passive exposure or by associative learning, and irrespective of tasks being appetitive (approach) or aversive (avoid), excitatory (as in positive reinforcement) or inhibitory (as in extinction). The other features of memory that provide control over these other task-related aspects of behavior and emotional valence are likely provided by distinct brain regions that cooperate in the network model of memory (Fig. 2). Interestingly, the facilitation of long-term memory by epigenetic manipulation appears to require subthreshold training experiences that on their own would not give rise to long-term memory, but could produce short-term effects. Taken together, these studies promote that a key function of epigenetic mechanisms in memory is to regulate the specificity of long-term memory for sensory cues that can effectively drive highly discriminative behavior. As such, this body of research points to a key role of epigenetic mechanisms to regulate neural substrates of cue memory within the sensory systems themselves, which are then integrated with other more traditional associative memory areas (Fig. 2). That epigenetic mechanisms are involved in regulating the specificity of memory for sensory information raises the question of the benefits that memory for a highly specific cue confers behaviorally. Highly discriminative cue memory without regard for context is not necessarily beneficial. For example, spoken language and communication requires a balanced memory that is both highly specific (e.g., for individual voices) and generalized (e.g., to understand language despite variations in speech). Indeed, the neural mechanisms underlying how some experiences are generalized in memory, as opposed to their maintenance with high veridity to actual stimulus events (by their specificity and precision, as well as the related dimension of accuracy) is not well understood. One candidate mechanism appears to be epigenetic. Thus, the function of standing epigenetic “brakes” on gene expression and their consequent impediment to neurophysiological changes in the naturally-learning, unmanipulated, sensory brain may be to initially bias the system against forming highly specific cue memories in the absence of exceptionally salient experiences (i.e., many trials of repetition, or highly arousing and stressful experiences modelled by paradigms such as CTA or one-trial fear conditioning; see White and Wood 2009; Vogel-Ciernia and Wood, 2014), in which highly discriminative behavior might be more adaptive. In any case, establishing an appropriate balance between plasticity and stability is an essential component of lifelong sensory system function (e.g., Elhilali et al., 2007; Takesian and Hensch, 2013) that might now be informed through the lens of epigenetic control. 3.2. Epigenetic memory modulation for sensory cues is timed to activity-dependent sensory processing A key open question in the neuroepigenetics of learning and memory is how broad manipulations of epigenetic players like HDACs and DNMTs can result in highly stimulus-specific effects on cue memory and discriminative behavior. A likely explanation is that epigenetic manipulations around the time of learning and memory consolidation assign a so-called “permissive state” to select genes that thereby could be successfully activated if initiated by a stimulus-evoked neural response. In line with this idea, evidence exists that manipulating epigenetic mechanisms without a learning event does not influence subsequent memory and behavior (e.g. Maddox and Schafe, 2011; McNulty et al., 2012; reviewed in Burns and Gräff, 2021). Importantly, it is also now well-accepted that broad manipulations even by general class HDAC or DNMT inhibitors do not have pan effects on chromatin modification, but that their marks (e.g., acetyl and methyl groups, respectively) remain circumscribed to select genes or gene families, likely by differential protein-protein interactions of the various HDAC and DNMT enzymes within distinct cell types, circuits, and neural systems (e.g., McNulty et al., 2012). Thus, a potential model to explain selective effects on sensory system plasticity, cue memory, and subsequent discriminative behavior is an activity-dependent process that depends on the confluence of chromatin state with stimulus-evoked neural activities. In this view, epigenetic regulators serve a permissive role, while stimulus-evoked neural activity serves an instructive role on sensory neuroplasticity. The result is a cooperative system that is sufficient to support the later recall of a sensory cue with a high-level of fidelity to the neural activity evoked by cue presentation during the learning event itself. This model relies on a systems-level conceptualization of memory that embraces the possibility that learning events. including sensory events, are—at least in some part—stored where they are initially processed (Nadel and Hardt, 2011). 3.3. The dynamic nature of experience-dependent epigenetic modifications Chromatin modifications for permissive states of transcription-dependent sensory neuroplasticity can be transitory, which is important to note to appropriately interpret the effects of HDAC or DNMT inhibitors on sensory system neuroplasticity, and by extension, epigenetic regulation of neural function and behavior more broadly. For example, non-permanent changes in chromatin state imply that the removal or blockade of HDACs or DNMTs in the adult brain does not fully revert the system to an early-life state of permissive plasticity characteristic of a critical period, but rather does so only temporarily (during a brief time) and partially (in terms of genomic loci). In line with this idea, Benoit et al. (2016) found very few differentially expressed genes in adult mice who had been treated with the HDAC inhibitor, TSA, compared to controls, indicating that HDAC inhibition alone does not induce the reactivation of distinct networks of genes typically activated during the early-life critical period. This is consistent with evidence that patterns of activity-dependent post-translational chromatin modifications are likewise distinct between the juvenile vs. adult state (Putignano et al., 2007; Vierci et al., 2016). Thus, the permissive state for epigenetically regulated learning-induced plasticity in the adult brain is molecularly and genetically different to that in developing brains. This is not entirely surprising given that the distinction is evident also neurophysiologically. For example, ocular dominance (OD) plasticity in the developing visual brain is characterized by a weakening of the synaptic connections from the deprived eye followed by a strengthening of the neural response to the nondeprived eye (Fagiolini et al., 1994). In comparison, OD plasticity in the adult brain involves an increase in the response to the nondeprived eye without weaking the response to the deprived eye. Despite these differences, both forms of plasticity have the same effect on visual function revealed in behavior. That different forms of synaptic changes support the same sensory-behavioral function is evidence for redundancies in sensory system capabilities that can lead to a common functional output, in line with the known structural and functional differences in developing vs. adult brains. What is more likely to happen with HDAC or DNMT inhibition in the adult is a change in the threshold for normal experience-dependent activation of genes typically associated with functional plasticity, which are effectively induced sooner, in larger magnitude, or for longer periods of time following a learning event (McQuown and Wood, 2011). Moreover, ongoing work highlights that the histone acetylation and DNA methylation state of cells in the sensory brain, and more broadly, epigenetic mechanisms that coordinate the function of the participating enzymes there (e.g., like the age-dependent expression of micro-RNAs including miR29a; Napoli et al., 2020), likely act within the normal constraints of the adult brain to balance stability with activity-dependent plasticity. Experimentally enabling chromatin modifications may be a way for the system to be artificially “primed for plasticity” with recent sensory experiences (e.g., Hofer et al., 2006) that ultimately promote changes for adaptive neural and behavioral function based on a personal history with salient sensory cues. 4. Conclusion Accumulating evidence supports a behavioral function of epigenetic control within sensory systems. The literature reviewed showcases that epigenetic mechanisms like histone acetylation and DNA methylation can ultimately regulate the amount and type of sensory memory retained in long-term memories by “fine-tuning” neural representations of behaviorally-relevant sensory features that ultimately guide cued behavior. Studies across sensory systems converge on the idea that epigenetic regulation can facilitate highly precise memory recall for transient sensory cue features experienced in the “here and now” by maintaining a sensory cue’s neurophysiological signature to support the later recall of accurate cue identity up to weeks and even months later. Such research has informed studies on the neurophysiological substrates of cue-specific memory and presents opportunities for future investigation. Questions about molecular and neurophysiological processes for memory specificity versus generalization, how the same epigenetic regulator may modulate different genes in different systems, and individual differences in memory despite identical learning experiences are ripe for study. Future research on whether these manipulations mimic ‘natural’ processes of memory formation could have translational importance for preventing and treating learning, memory and sensory disorders. Funding sources NIH (NIDCD) R01-DC018561 and NIH (NIA) R01-DC018561-S1 to K. M.B. Fig. 1. Experience elicits cue-dependent neurophysiological responses that can drive activity-dependent gene-expression gated by epigenetic mechanisms. A simplified schematic in an auditory model shows the experiential effect of learning about an acoustic frequency cue on sound-elicited neurophysiological activity (highly activated vs. less highly activated vs. non-activated sensory cells; sp/s = spike rate). In cells highly responsive to the cue frequency (“BF cells”), activity-dependent gene expression is possible unless blocked by the presence of chromatin modifying enzymes (e.g., HDACs and DNMTs) that often work together to constrict chromatin conformation and repress transcription (A). If epigenetic repressors are removed naturally either by prior history (B) or by experimental manipulation (C), then cue-elicited activity in active BF cells (B) and even moderate activity in similarly tuned “nearby non-BF cells” (C) could sufficiently activate gene expression within those sensory cells to promote lasting changes that support the formation of cue-specific memory. The overarching message in this schematic is that the cue specificity of memory depends on whether cue responsive cells reach the threshold for de novo gene expression events. The hypothetical premise of this framework is two-fold: first, that gene expression within sensory cells activated by cues can initiate neuroplasticity events within sensory system circuits themselves that ultimately support the formation of cue-specific memory and behavior (e.g., top vs. middle row) including the degree of cue specificity in memory and behavior (e.g, middle vs. bottom row); and second, that all sensory systems participate broadly in memory system networks by contributing what cues and how much sensory detail is encoded in newly formed memories to underlie their sensory richness (c.f., Phan and Bieszczad, 2016). Fig. 2. Sensory systems are an initial part of the distributed network of memory. Experience elicits lasting physiological changes in the sensory brain (c.f., Table 6) that are inherited to downstream brain regions. It is important to note that traditional associative regions (e.g., HPC, AMY) are dependent on initial processing of sensory cues within the sensory regions of the brain that have undergone an experience-dependent physiological change. While there is massive feedback to sensory systems from extra-sensory regions, these influences on memory function are likely secondary to sensory system mediated processes at the time of experience. Thus, a network of physiological changes across multiple sensory and non-sensory associative brain regions together constitutes the characteristics of memory that ultimately control behavior. Abbreviations: HPC – hippocampus, AMY – amygdala, NAc – nucleus accumbens, VTA – ventral tegmental area, PFC – prefrontal cortex, Cer – cerebellum, ERC – entorhinal cortex, IC – insular cortex. Table 1 Epigenetic manipulations and their effects on cue memory in auditory models. HDAC and HAT manipulations Paper Model How Where When Behavioral Training Memory Assessment Effect on Cue Memory Bredy et al. (2007) Mouse HDAC inhibition (VPA) Systemic Pre-training Partial TCF extinction Cued fear @ 7d Increased extinction HDAC inhibition (NaB) Increased extinction Bredy and Barad (2008) Mouse Inhibition (VPA) Systemic Pre-training Partial TCF Cued fear @ 7d Increased cued fear in both same and novel context Partial TCF extinction Cued fear @ 7d Increased cued fear in acquisition context, but decreased cued fear in extinction context Bieszczad et al. (2015) Rat HDAC3 inhibition (RGFP966) Systemic Immediately post-training Tone reward SGT @ 24 h Increased cue specificity Bowers et al. (2015) Mouse HDAC inhibition (RGFP963) Systemic 5 min post-training TCF extinction Extinction retention @ 24 h Decreased freezing HDAC3 Inhibition (RGFP966) No effect Gervain et al. (2013) Human Inhibition (VPA) Systemic Continuous Auditory discrimination SGTa Increased cue specificity Guan et al. (2009) Mouse HDAC1 overexpression Global Continuous TCF Cued fear @ 24 h @ 3 h No effect No effect HDAC2 overexpression Cued fear @ 24 h @ 3 h Decreased freezing No effect HDAC2 KO Cued fear @ 24 h Increased freezing Heinrichs et al. (2013) Mouse Inhibition (NaB) Systemic Immediately post-training Strong extinction of TCF Extinction acquisition @ >24 h to 11d No effect Extinction retention @ 24 h No effect Inhibition (VPA) Immediately post-training Strong extinction of TCF Extinction acquisition @ >24 h to 12d No effect Extinction retention @ >24 h to 2 weeks No effect Inhibition (VPA) Immediately post-training Weak extinction of TCF Extinction acquisition @ >24 h to 18d Increased extinction Extinction retention @ 24 h Increased retention Kwapis et al. (2017) Mouse HDAC2 block (AAV) HPC Basal AMY Continuous TCF Cued fear @ 28 h @ 28 h No effect No effect Lateral AMY @ 28 h Increased Freezing Maddox and Schafe (2011) Mouse Inhibition (TSA) LA 1 h post-training TCF reactivation TCF @ 24 h Increased cued fear 1 h post-training TCF @ 3 h No effect 1 h post-training No reactivation TCF @ 24 h No effect 6 h post-training TCF reactivation TCF @ 24 h No effect Monsey et al. (2011) Rat Inhibition (TSA) LA 1 h post-training Weak TCF TCF @ 24 h Increased cue memory @ 2 h No effect Morris et al. (2013) Mouse HDAC1 CKO Forebrain Continuous TCF Cued fear @ 24 h No effect HDAC2 CKO Cued fear @ 24 h No effect @ 48–96 h Decreased freezing Rotondo and Bieszczad (2020) Rat HDAC3 inhibition (RGFP966) Systemic Immediately post-training Tone reward SGT @ 48 h Increased cue specificity Rotondo and Bieszczad (2020) Rat HDAC3 inhibition (RGFP966) Systemic Immediately post-training Tone extinction SGT @ 48 h Increased cue specificity Rotondo and Bieszczad (2021a) (2021b) Rat HDAC3 inhibition (RGFP966) Systemic Immediately post-training AM rate discrimination SGT @ 48 h Increased cue specificity Shang et al. (2019) Rat HDAC3 inhibition (RGFP966) Systemic Immediately post-training Tone discrimination SGT @ 24 h Increased cue specificity Vecsey et al. (2007) Mouse HDAC inhibition (TSA) HPC Immediately post-training TCF Cued Fear @ 24 h No effect DNA Methylation Manipulations Paper Model How Where When Behavioral Training Memory Assessment Effect on Cue Memory Day et al. (2013) Rat DNMT inhibition (RG108) VTA 15 min pre-training Tone reward association Cued response @ >24 h to 5d Impaired association @ 7d Impaired association 24 & 1 h pre-memory test @ 7d No effect NaC 15 min pre-training @ 7d No effect Maddox and Schafe (2011) Rat DNMT inhibition (5-AZA) LA 1 h post-reactivation Reactivation of TCF Cued fear @ 3 h No effect DNMT inhibition (RG108) 1 h post-reactivation @ 24 h Decreased freezing DNMT inhibition (RG108) 6 h post-reactivation @ 24 h No effect Maddox et al. (2014) Rat DNMT inhibition (RG108) LA 1 h post-training TCF Cued fear @ 3 h @ 24 h No effect Decreased freezing DNMT inhibition (5-AZA) Cued fear @ 3 h @ 24 h No effect Decreased freezing DNMT inhibition (RG108) 1 h post-re-activation Reactivation of TCF Cued Fear @ 3 h @ 24 h No effect Decreased freezing DNMT inhibition (5-AZA) Cued Fear @ 3 h @ 24 h No effect Decreased freezing Monsey et al. (2011) Rat DNMT inhibition (5-AZA) LA 1 h post-training TCF Cued Fear @ 2 h @ 24 h No effect Increased Freezing Morris et al. (2014) Mouse DNMT1 CKO Forebrain Continuous TCF Cued Fear @ 24 h No effect DNMT3a CKO Cued Fear @ 24 h No effect TCF Extinction @ >24 h to 5d Increased Freezing Other Manipulations Paper Model How Where When Behavioral Training Memory Assessment Effect on Cue Memory Gupta-Agarwal et al. (2012) Rat G9a/GLP block (BIX01294; [prevents H3K9me2) ERC 1 h pre-training TCF Cued Fear @ 24 h Increased freezing Gupta-Agarwal et al. (2014) Rat H/KMT-G9a inhibition (UNC0224; [decreases H3K9me2)) LA 1 h pre-training TCF Cued Fear @ 24 h Decreased freezing H/KDM-LSD1 inhibition (t-PCP; [enhances H3K9me2)] Increased freezing Konopka et al. (2010) Mouse Dicer1 disruption Forebrain Continuous TCF Cued Fear @ 48 h Increased Freezing Abbreviations: HDAC – histone deacetylase, HAT – histone acetyltransferase, DNMT – DNA methyltransferase, TCF – tone cued fear, VPA – valproate, NaB – sodium butyrate, SGT – stimulus generalization test, KO – knockout, TSA – trichostatin A, LA – lateral amygdala, AMY – amygdala, NaC – nucleus accumbens, VTA – ventral tegmental area, HPC – hippocampus, ERC – entorhinal cortex, H3K9me2 – histone H3 lysine 9 dimethylation, H/KMT-G9a – histone lysine methyltransferase G9a, H/KDM-LSD1 – histone lysine demethylase LSD1, t-PCP – trans-2-phenylcyclopropylamine. a After 2 weeks of training and VPA treatment. Table 2 Epigenetic manipulations and their effects on cue memory in honeybee models of olfactory learning and memory. DNA methylation manipulations Paper Howa When Behavioral Training Memory Assessment Effect on Cue Memory Biergans et al. (2012) Dnmt inhibition (zebularine) 1 h pre-ACQ & immediately post-ACQ Odor-reward pairing Cue discrimination @ 30 min @ 24 h @ 7 h No effect Impaired discrimination Impaired discrimination Biergans et al. (2015) Dnmt inhibition (zebularine) 2 h post-ACQ Odor-reward pairing Cue discrimination @ 48 h Impaired discrimination Dnmt inhibition (RG108) Impaired discrimination Biergans et al. (2016) Dnmt inhibition (RG108) 2 h post-ACQ 1 tr odor-reward pairing Cue discrimination @ 24 h Enhanced discrimination 6 tr odor-reward pairing Impaired discrimination Odor discrimination Contingency reversal @ >24 h to 3d Impaired associative relearning Lockett et al. (2010) Dnmt inhibition (Zebularine) 24 h pre-ACQ Odor reward ACQ Within session EXT @ 24 h post-odor reward ACQ Enhanced EXT learning 1 h pre- & immediately post-ACQ No effect 1 min post-ACQ Enhanced EXT learning 1 h post-ACQ No effect 2 h pre-EXT Impaired EXT learning 24 h pre-ACQ Odor reward EXT EXT retention @ 5 h post-EXT training No effect 1 h pre-& immediately post-ACQ Impaired EXT memory 1 min post-ACQ No effect 1 h post-ACQ Impaired EXT memory 2 h pre-EXT No effect HDAC and HAT manipulations Paper How a When Behavioral Training Memory Assessment Effect on Cue Memory Lockett et al. (2014) HDAC inhibition (APHA compound 8) 1 h pre-ACQ Odor cue discrimination Cue discrimination @ 24 h Impaired inhibitory memory and discrimination 1 h post-ACQ Impaired inhibitory memory and discrimination 1 h pre-memory assessment Impaired inhibitory memory and discrimination HDAC inhibition (phenylbutyrate) 1 h pre-ACQ Impaired inhibitory memory and discrimination 1 h post-ACQ No effect 1 h pre-memory assessment No effect HDAC inhibition (NaB)b 1 h pre-memory assessment Impaired inhibitory associative memory Merschbaecher et al. (2012) HAT inhibition (Garcinol) 30 min post-ACQ 1 tr odor-reward pairing Cue retention @ 2 h @ 24 h @ 48 h No effect Enhanced cue memory No effect 3 tr odor-reward pairing Cue retention @ 2 h @ 24 h @ 48 h No effect No effect Enhanced cue memory HDAC inhibition (TSA) 1 tr odor-reward pairing Cue retention @ 2 h @ 24 h @ 48 h No effect Impaired cue memory No effect 3 tr odor-reward pairing Cue retention @ 2 h @ 24 h @ 48 h No effect Impaired cue memory Impaired cue memory Merschbaecher et al. (2016) HAT inhibition (Garcinol)b 30 min post-ACQ 1 tr odor-reward pairing Cue retention @ 2 h @ 24 h @ 48 h No effect Impaired cue memory No effect 3 tr odor-reward pairing Cue retention @ 2 h @ 24 h @ 48 h No effect Impaired cue memory Impaired cue memory HAT inhibition (C646)b 1 tr odor-reward pairing Cue retention @ 2 h @ 24 h @ 48 h Impaired cue memory Impaired cue memory Impaired cue memory 3 tr odor-reward pairing Cue retention @ 2 h @ 24 h @ 48 h Impaired cue memory Impaired cue memory Impaired cue memory Abbreviations: ACQ – acquisition, EXT – extinction, HAT – histone acetyltransferase, HDAC – histone deacetylase, DNMT – DNA methyltransferase. a Unless specified, manipulations were applied topically to the thorax b Injected into the thorax Table 3 Epigenetic manipulations and their effects on cue memory in visual models. Paper Model How Where When Behavioral Training Memory Assessment Effect on Cue Memory Federman et al. (2009) Crab HDAC inhibition (NaB) Systemic Immediately pre-ACQ Weak cued fear Cued escape @ 24 h Partial enhancement of cued escape Immediately post-ACQ Enhanced cued escape 3 h post-ACQ No effect 6 h post-ACQ Enhanced cued escape 12 h post-ACQ No effect HDAC inhibition (TSA) Immediately post-ACQ Partial enhancement of cued escape Yeh et al. (2004) Rat HDAC inhibition (TSA) AMY 30 min pre-ACQ Cued fear Fear potentiated startle @ 24 h Enhanced fear-potentiated startle HDAC inhibition (kB decoy DNA) Impaired fear potentiated startle HDAC inhibition (TSA) No effect HDAC inhibition (kB decoy DNA) No effect Abbreviations: AMY – amygdala, ACQ – acquisition, HDAC – histone deacetylase, TSA – trichostatin A. Table 4 Epigenetic manipulations and their effects on cue memory in other sensory models. Paper Model How Where When Behavioral Training Memory Assessment Effect on Cue Memory Feng et al. (2017) Mouse anti-miRNA-324 and anti-miRNA-133a Barrel cortex Continuous Cross modal sensory association Cross modal sensory association @ 10d Impaired associative memory Kwon and Houpt (2010) Rat HDAC inhibition (NaB) Systemic 10 min post-ACQ CTA Taste preference @ 1–6d Enhanced taste aversion, delayed extinction Morris et al. (2013) Mouse HDAC2 CKO Forebrain Continuous CTA Taste preference @ 72 h Impaired taste aversion Morris et al. (2014) Mouse Dnmt3a CKO Forebrain Continuous CTA Taste preference @ 48 h Impaired taste aversion Dnmta CKO No effect Núñez-Jaramillo et al. (2014) Rat HDAC inhibiti on (NaB) Bilateral IC 60 min pre-ACQ CTA Taste preference @ 24 h No effect Rodríguez-Blanco et al. (2019) Rat HDAC inhibition (MS275) Bilateral IC Immediately post-ACQ Strong CTA Taste preference @ 96 h No effect Immediately post-ACQ Weak CTA Enhanced taste aversion 7 h post-ACQ Weak CTA Enhanced taste aversion Yamada et al. (2019) Mouse Conditional Crispr KO of Rad21 Anterior dorsal cerebellar vermis Continuous Delay tactile startle response Cued response acquisition @ >24 h to 5d Impaired cued response Abbreviations: ACQ – acquisition, CKO – conditional knockout, CTA – conditioned taste aversion, IC – insular cortex, DNMT – DNA methyltransferase, HDAC – histone deacetylase Table 5 Epigenetic manipulations and their effects on cue memory in multi-modal models. Paper Model How Where When Behavioral Training Memory Assessment Effect on Cue Memory Bahari-Javan et al. (2012) Mouse HDAC2 inhibition HPC Continuous OR NOR @ 24 h No effect Federman et al. (2013) Mouse HAT inhibition (c646 *) HPC Post-training Strong OR training NOR @ 24 h @ 7d No effect Impaired object memory HDAC inhibition (NaB or TSA) Weak OR training NOR @ 24 h Enhanced object memory Gräff et al. (2012) Mouse Histone PTM inhibition Systemic Immediately post-ACQ OR NOR @ 7d Impaired object memory Hansen et al. (2010) Mouse miR132 overexpression Forebrain Continuous OR NOR @ 30 min Impaired object memory Kong et al. (2020) Mouse Dnmt3b KO Dorsal CA1 Continuous OR NOR @ 24 h No effect Malvaez et al. (2013) Mouse HDAC3 inhibition (RGFP966) Systemic Post-training Weak OR training NOR @ 24 h Enhanced object memory Pre-training & Post-training Weak OL training NOL @ 24 h Enhanced object location memory Immediately post-extinction Cocaine CPP CPP extinction @ 24 h & 48 h 8 h post-extinction Cocaine CPP CPP extinction @ 24 h & 48 h No effect Morris et al. (2014) Mouse Dnmt3a CKO Dnmt2 CKO Forebrain Continuous OR NOR @ 24 h @ 24 h No effect No effect Oliveira et al. (2011) Mouse p300 CKO Superficial cortex & CA1 layers Continuous OR NOR @ 30 m No effect Ploense et al. (2013) Rat HDAC inhibition (NaB or VPA) Systemic 2 h pre-random conditioning Cue-reward pairing Cue-induced reinstatement @ >7d Enhanced cue-induced reinstatement Stefanko et al. (2009) Mouse HDAC inhibition (NaB) Systemic Immediately post-ACQ Weak OR training NOR @ 90 m Enhanced object memory @ 24 h No effect CBPKIX/KIX Mouse @ 7d Enhanced object memory 1 h before 7 day retention Strong OR training NOR @ 7d @ 8d No effect Enhanced object memory Post-OR Strong OR training NOR @ 7d Rescued object memory Abbreviations: HPC – hippocampus, HAT – histone acetyltransferase, HDAC – histone deacetylase, PTM – post-translational modifications, ACQ – acquisition, OR – object recognition, NOR – novel object recognition, OL – object location, NOL – novel object location, CPP – conditioned place preference, OLM – object location memory, NOL – novel object location, OL – object location, NaB – sodium butyrate, VPA – valproate, CKO, conditional knockout, Dnmt – DNA methyltransferase, TSA – trichostatin A. Table 6 Epigenetic manipulations and their effects on sensory systems neurophysiology. Auditory System Paper Model Experience Manipulation Where When Neurophysiological Sensory Systems Effect Bieszczad et al. (2015) Rat Tone reward training HDAC3 inhibition (RGFP966) Systemic Immediately post-training Signal-specific decreases in ACtx acoustic frequency tuning BW and signal-specific increase in tonotopic A1 representational area [24–48 h post-memory test] Monsey et al. (2011) In vitro LTP Dnmt inhibition (5-AZA) Bath application Impaired LTP at thalamic and cortical inputs to LA Weaker LTP induction HDAC inhibition (TSA) Enhanced LTP at thalamic and cortical inputs to LA Phan et al. (2017) Zebra finch Subthreshold (20X) conspecific song exposure HDAC3 inhibition (RGFP966) Systemic Immediately post-training Enabled SSA in left NCM Rotondo and Bieszczad (2020) Rat Tone reward training HDAC3 inhibition (RGFP966) Systemic Immediately post-training Enhanced signal-specific changes in subcortical (ABR) response [24 h post-training] Signal-specific decreases in ACtx acoustic frequency tuning BW [24–48 h post memory test] Rotondo and Bieszczad (2021a) Rat Tone extinction training HDAC3 inhibition (RGFP966) Systemic Immediately post-training Enhanced signal-specific changes in subcortical (ABR) response [24 h post training] Signal-specific decreases in ACtx acoustic frequency tuning BW [24–48 h post memory test] Rotondo and Bieszczad (2021b) Rat AM rate discrimination training HDAC3 inhibition (RGFP966) Systemic Immediately post-training Enabled signal-specific enhancements in cortical [24–28 h post memory test] and subcortical [24 h post training] phase locking, and increased trial-to-trial cortical response consistency. Shang et al. (2019) Rat Tone discrimination training HDAC3 inhibition (RGFP966) Systemic Immediately post-training Signal-specific decreases in ACtx acoustic frequency tuning BW [24–48 h post memory test] Maddox et al. (2014) Rat TCF DNMT inhibition (RG108/5-AZA) LA 1 h post-training @ 3 h: No effect on AEFPs during STM test [3 h]. @ 24 h: Decreased AEFP amplitudes in LA during LTM test. Reactivation of TCF DNMT inhibition (RG108/5-AZA) LA 1 h post-reactivation @ 3 h: No effect on AEFPs in LA @ 24 h: Decreased AEFP amplitudes in LA Olfactory System Paper Model Experience Manipulation Where When Neurophysiological Sensory Systems Effect Biergans et al. (2017) Honeybee Odor-reward extinction DNMT inhibition (RG108) Systemic 2 h post-ACQ Decreased recruitment of glomeruli responding to the novel odor over extinction training Feng et al. (2017) Mouse Cross modal sensory association anti-miRNA-324 & anti-miRNA-133 Barrel cortex Continuous Blocked recruitment of BC neurons that respond to cross-modal stimuli Visual System Paper Model Experience Manipulation Where When Neurophysiological Sensory Systems Effect Apulei et al. (2018) Mouse MD (4d) Gadd45b overexpression Visual cortex Continuous Promoted OD plasticity Maya-Vetencourt et al. (2011) Rat MD (1 week) HDAC inhibition (TSA) Systemic 6d (concurrent with start of MD) Promoted OD plasticity Putignano et al. (2007) Mouse MD (3d) HDAC inhibition (TSA) Systemic 4d (beginning 1 day before MD) Promoted OD plasticity Silingardi et al. (2010) Rat RS HDAC inhibition (VPA/NaB) Systemic 25d (beginning 5d after RS) Promoted OD plasticity and recovered VEP acuity in deprived eye Yeh et al. 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==== Front Med Sci Monit Med Sci Monit Medical Science Monitor Medical Science Monitor : International Medical Journal of Experimental and Clinical Research 1234-1010 1643-3750 International Scientific Literature, Inc. 37438946 10.12659/MSM.940296 940296 Clinical Research Deciphering Pediatric Root Canal Practices in Turkey: A Comparative Study Bridging the Gap between Practice and Literature Şahin Tuğçe Nur https://orcid.org/0000-0002-7617-9719 ABCDEF Çakir Asu https://orcid.org/0000-0003-1276-9709 BDEF Department of Pediatric Dentistry, Karamanoglu Mehmetbey University Ahmet Kelesoglu Dentistry Faculty, Karaman, Turkey Corresponding Author: Tuğçe Nur Şahin, e-mail: tugcenpekdemir@gmail.com A Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2023 13 7 2023 20 6 2023 29 e940296-1e940296-10 09 3 2023 05 6 2023 © Med Sci Monit, 2023 2023 https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Background Pediatric endodontics is a critical area of dental practice, involving the treatment of root canals in primary teeth. Treatment approaches can significantly vary due to a range of factors, potentially impacting the success of the procedure and patient comfort. This variability, often influenced by regional practices, individual dentist preferences, and the pace of dental technology advancements, warrants detailed examination to improve standardization and care outcomes. Accordingly, our study aims to scrutinize the endodontic practices of pedodontists in Turkey and compare them with contemporary literature. Material/Methods We conducted an online survey with 15 questions that 217 Turkish pedodontists answered. The questionnaire sought information about their preferences in canal length measurement, canal sealer, irrigation solution, and other procedural decisions. Chi-square test was used to analyze the responses, with a significance level of p<0.05. Results The majority of respondents were associated with universities (123 out of 217). 103 preferred rotary file sets, and 114 used apex locators for canal length measurement. Iodoform pastes (160) and sodium hypochlorite (180) were the most popular choices for canal sealers and irrigation solution, respectively. 152 participants did not prefer using rubber dam, and 186 did not favor sonic-ultrasonic activators. Many reported using hand files as students (184), with 39 continuing this practice. Post-graduation, 64 participants took root canal treatment courses, while 72 were reluctant to perform the procedure under general anesthesia. Conclusions No single method stood out as superior for primary root canal treatments in deciduous teeth. Incorporating advanced technology into dental practice may potentially improve treatment success and patient comfort. Pediatric Dentistry Root Canal Therapy Surveys and Questionnaires Tooth, Deciduous ==== Body pmcBackground Healthy primary dentition plays a key role in the development of the jaws in terms of preserving occlusion and arch length. The most important factor threatening primary dentition health is dental caries. Although preventive measures have reduced caries, early loss of primary teeth with pulp-involving caries remains a common problem. Fortunately, with successful root canal treatments, professionals can keep most of these deciduous teeth in the mouth until they normally fall out [1,2]. Root canal treatment of deciduous teeth should be performed within a short period and with minimal complications. Effective shaping and debridement without weakening the tooth structure and damaging the permanent tooth germ is crucial [3–5]. The first step of root canal treatment is a good isolation, which is ideally only possible with a rubber dam [6]. In addition to preventing the patient from talking and covering his or her mouth, it provides the clinician with a dry, safe surgical field, improves visibility, and thus increases the success of the procedure. Moreover, a rubber dam is also the most effective means of preventing cross-infection during dental treatments [7]. Root canal treatment progresses in stages, and the success of the treatment depends on the careful implementation of all these steps. As in permanent teeth, correct determination of the working length in primary teeth is also very important for the success of the next steps of root canal treatment [8,9]. However, as soon as the deciduous tooth roots have completed their formation, they begin to resorb [10]. With the progression of root resorption, the apical foramen begins to be positioned more coronally than the anatomical apex of the root. Radiographically, this makes determining the root canal length difficult [11]. There may be resorption along the root and into the root canal, and different connections can form with periapical tissues other than the apical foramina, lateral canals, and accessory canals. Therefore, the use of an electronic apex locator is not recommended for determining the root canal length in deciduous teeth [10]. There are, however, some studies that recommend using this method to determine the length of canals in deciduous teeth root canal treatments without exposing the patient to radiation [8]. Another factor in chemomechanical preparation is the canal file [12]. For this purpose, conventional hand files are still widely used in the endodontic treatment of devital primary teeth. However, with the developing technology, the use of rotary file systems developed for permanent teeth has become routine in endodontic treatment for adults over the past decade and it is well known that modern rotary file systems minimize procedural errors [13]. However, there is a lack of laboratory data showing how modern rotary filing systems alter deciduous tooth root morphology [14]. The use of rotary instruments in pediatric endodontics was first introduced by Barr et al [15]. They stated that the natural flexibility of these files preserves the original anatomy of the curved canals in deciduous molars [16]. On the other hand, Kuo et al [17] suggested that a rotary file with modified length, taper, and tip size would be more effective for pulpectomy in deciduous teeth. Different pediatric file sets have been developed under different brand names by many researchers who share this idea [18]. Since mouth opening is limited in pediatric patients, rotary files with longer lengths are difficult to use. Pediatric rotary files are designed with a shorter length. This feature provides ease of use when working with pediatric patients [16]. One of the most important steps of the endodontic treatment procedure is to remove the debris, pulp tissue, and microorganisms in the root canal system with chemomechanical preparation and to give the root canal a uniform canal shape [12]. Currently, sodium hypochlorite is the most widely used irrigation solution because it possesses many of the desired properties of an ideal irrigation solution in root canal treatments. Sodium hypochlorite has a broad spectrum and nonspecific ability to kill all microorganisms and dissolve necrotic tissues [19]. On the other hand, chlorhexidine has a bacteriostatic effect at low concentrations and a bactericidal effect at high concentrations by causing cell damage, cytoplasm coagulation, and precipitation of proteins and nucleic acids [20]. Chelating agents such as EDTA, which have a limited antibacterial effect, are frequently recommended, especially in narrow and calcified canals, for facilitating canal preparation with its lubricating properties and removing smear deposits [21–23]. Recent studies have indicated that the smear layer and debris can be removed more effectively by activating irrigation solutions in root canals and suggest manual or mechanical activation techniques [24]. The aim of primary tooth root canal treatment includes instrumentation with appropriate file and removal of pulp, bacteria, and bacterial toxins with appropriate irrigation solution and filling the canal with a resorbable material [18]. The ideal deciduous tooth root canal filling material should be antiseptic and nontoxic, easily filled into the canal or easily removed, resorb at the same rate as the deciduous tooth root, be harmless to periapical tissues and permanent tooth germ, and resorb when it overflows from the apex [25]. Zinc oxide eugenol (ZOE), iodoform, and calcium hydroxide paste are the most commonly used filling materials for root canal treatment. It has been found that ZOE is more effective than others in the hyperemic deciduous pulp. However, when it protrudes from the apex, it may alter the eruption path of the permanent tooth as it forms a hard mass and resorbs very slowly compared with physiological root resorption [26]. Although the effect of endodontic instruments and materials on permanent tooth germs is not fully known and compliance problems often seen in pediatric patients cause root canal treatment not to be preferred by some dentists, most dentists apply pulpectomy treatment as an alternative to extraction and space maintainer application [1]. In the literature review, there was no study evaluating the approach of pedodontists in Turkey to the root canal treatment of deciduous teeth and the procedures they follow. Therefore, the present questionnaire-based study aimed to examine the guidelines and approaches of pedodontists in our country when performing primary root canal treatment and to compare them with the current literature on the subject. Material and Methods Ethics Approval Approval for the study was obtained from the Clinical Research Ethics Committee of Karamanoglu Mehmetbey University, Faculty of Medicine, with decision no. 06-2022/18. Study Design and Data Setting The sample size for the descriptive sampling technique was calculated using an expected response rate of between 60% and 80% and a 90% power calculation with a 95% confidence interval. For this purpose, a self-administered and online 15-question 2-part questionnaire (Table 1) prepared using Google Forms (Google, Inc., 2017, California, USA) was randomly sent to 300 pediatric dentists via e-mail and message with the help of the Turkish Pediatric Dentistry Association. To preserve the accuracy and confidentiality of the data, the questionnaire was made anonymous. After the literature search, 15 questions about the primary tooth root canal treatment procedure and the demographic variables of the participants were prepared and emailed to 3 experts to validate the content using a 5-point Likert scale. As a result of the evaluation, each question was found appropriate and included in the study. The reliability of the questionnaire was checked with the help of the Cronbach α value using the test-retest method. Numbers between 0.697 and 1 and the median 1 were considered reliable. The first 4 questions in the questionnaire consisted of questions about demographic variables such as age, gender, place of work and working year of pediatric dentists. The next questions question the attitudes and preferences of physicians regarding the choice of materials and techniques used during primary tooth root canal treatment, participation in any workshop of primary tooth root canal treatment after the completion of specialty/doctoral training, or application preferences under general anesthesia. The questionnaire consisted of open-ended and multiple-choice questions. Participants who answered “I want to participate” in the consent form in the first part moved on to the second part, answered the questions, and participated in the study. Forty-two people who answered “I do not want to participate” to the consent form in the first section of questionnaire and 41 people continuing their specialization or doctorate education were excluded from the study. Responses of 217 pediatric dentists who were answered were statistically analyzed. Statistical Analyses For statistical analysis, participants were divided into 4 groups according to the institutions (University, Oral and Dental Health Center/Oral and Dental Health Hospital (ODHC/ODHH), Private outpatient clinic, Own Clinic) they worked in. The chi-square test was used to determine the association between categorical variables, and p<0.05 was considered significant. The data were analyzed using the IBM SPSS Statistics Version 27 (IBM SPSS Inc., Chicago, IL, ABD) package program. Results The answers of 217 participants were evaluated for this study, which aimed to evaluate the materials and methods used by pedodontists during the primary tooth root canal treatment process and their approaches, as shown in Table 1; 73.7% of the 217 participants were between the ages of 25–35 and 78.8% were female pedodontists (p<0.05). When evaluated according to institutions, as shown in Figure 1, the most participation was from universities (p<0.05). When the participants were asked whether they preferred to use the rubber dam during root canal treatment of deciduous teeth, it was found that most of the participants did not prefer to use the rubber dam (146), except for pedodontists working in their private clinic, whereas only 35% of the pedodontists working in their private clinic did not (p<0.05). Table 2 shows that the majority of institutions found only apex locators sufficient for canal length determination (p<0.05). When examining the file preferences according to the institutions, it was found that pedodontists working in universities and ODHC/Hs mostly preferred rotary file sets, while pedodontists working in their private clinics and private outpatient clinics mostly preferred pediatric canal files (p<0.05). It is shown in Table 3 that approximately 85% of the participants used only hand files during primary tooth root canal treatment when they were interns and only 39 people continued with the same system, while none of the participants who used a rotary or pediatric file set during their education preferred to use only hand files for primary tooth root canal treatment after graduation, and all 6 people who had experienced the pediatric file set during their student years continued their treatment with the same method (p<0.05). There was no significant difference in the preference for irrigation solutions between the institutions, with sodium hypochlorite being the most preferred in each institution, while EDTA was not preferred by any of the pedodontists (p>0.05). As a final irrigation solution, serum was preferred by the majority regardless of the canal irrigation solution (p<0.05). Table 4 also shows that the number of participants who preferred to use a single solution during treatment was 103 (p<0.05). While there was no significant difference between the institutions regarding the use of sonic or ultrasonic activators during treatment, 86% of the participants did not use them (p>0.05). Although there was no significant difference in preference for root canal paste between the institutions for deciduous tooth root canal treatment, 73.7% of the participants preferred iodoform paste (p>0.05). The mean duration of root canal treatment for deciduous teeth, excluding the restoration phase, was calculated to be 22 minutes, with pedodontists working in ODHC/Hs having the shortest mean duration (p>0.05). Based on the file system used, the mean duration of root canal treatment was 22.33 minutes with rotary files, 22.24 minutes with hand files alone, and 22.16 minutes with pediatric root canal files (p>0.05). It was found that 33% of the participants preferred extraction to root canal treatment under general anesthesia, preferring a more radical approach than most participants (p<0.05). The majority of the participants who worked in the state institution and 70% of the participants regardless of their institutional affiliations did not attend any canal treatment courses after they completed their specialization training (p<0.05). Discussion Survey studies enable us to assess whether our views or approaches are in accordance with current trends [27]. The aim of this study was to compare the procedures followed by pedodontists in Turkey when applying root canal treatment to deciduous teeth considering the current literature. There are several factors that complicate the use of rubber dams in the studies published in the literature, including increased costs, difficulty in accessing rubber dams, inadequate education, and patients’ refusal [28,29]. Patients’ low acceptance of rubber dams has been generally reported in the literature as the most important reason for not using rubber dams [30]. However, many studies that question patients’ approaches to rubber dams found that patient acceptance was high [31,32]. Likewise, in our study, most of the pedodontists did not prefer rubber dam isolation for similar reasons. In some studies, the rubber dam is reported to cause less stress to patients than cotton rolls [33] and reduces stress for physicians during treatment as well [34]. According to the 2020 AAPD guidelines [35], rubber dam isolation is the gold standard for pulpal treatments. Nonetheless, many studies have reported, in accordance with our findings, that rubber dams are highly preferable in pediatric patients [27,36,37]. It has been reported in a survey study of 100 dentists in India that half preferred rotary files [38], whereas in a study of 50 dentists, 62% preferred only using hand files [39]. According to a retrospective study examining pediatric dentists’ file preferences [40], 86% of pedodontists preferred rotary files when treating their patients. Similarly, in our study, in which the participants reported that rotary files were preferred the most (47%), 85% of them used only hand files during deciduous root canal treatment when they were trainee students, but today they predominantly use rotary or pediatric file sets, suggesting that physicians prefer more technological techniques thanks to the reduced risk of aspiration of the instruments and the shorter treatment period. It is noteworthy that although we did not observe a significant difference in the duration of treatment when we compared the file preference with the duration of treatment in our study, many recent studies have reported that a pediatric canal file or rotary file facilitates a faster preparation process [5,41–45]. Moreover, in studies comparing the preparation and filling quality of hand files, rotary files, and pediatric files, it has been found that pediatric files were generally superior to the others, and the use of these files is recommended during root canal treatment [16,46,47]. Meanwhile, the AAPD guideline [35] states that hand files or rotary files are preferred. Although there are studies in the literature [39,48] reporting that the most preferred deciduous tooth root canal paste by participating dentists is eugenol paste, unlike this result, similar to our study, in a survey of 360 pedodontists [27], iodoform paste was the most preferred, and in a retrospective study of 2496 case papers [49], Metapex was the most preferred (96.6%). A recent meta-analysis [50] reported that there was no difference between the short- and long-term outcomes of iodoform and pastes with iodoform, but added that the certainty of the evidence in the studies reviewed ranged from low to very low. In another meta-analysis [51], there was no significant difference between iodoform and eugenol paste, but the long-term success of eugenol paste was higher, and the researchers suggested that iodoform paste should be preferred if the tooth to be treated is close to the physiologic fall time, and eugenol paste should be preferred if it is not. In light of the lack of absolute evidence that one paste is superior to another, many publications and books leave it up to the clinician to choose the paste [10,35,52,53]. However, the AAPD [35], in its guideline, added that eugenol paste is more successful in the long run than other pastes. Because ZOE paste is resorbed later than deciduous tooth roots, there have been reports of cases where the material left after pulpectomy caused delayed eruption, resulting in moderate foreign body reaction in the patient and the eruption of the permanent tooth from more palatal or anterior crossbite [54,55]. Based on our study, we believe that participants preferred iodoform paste over ZOE paste due to the mentioned disadvantages. In a study of 2400 patients in South India [56], it was reported that serum was preferred over sodium hypochlorite during root canal treatment of deciduous teeth of patients aged 0–5 years. Likewise, in a study [48] of 221 dentists, serum was again the most preferred irrigation solution. Meanwhile, in our study, it was found that the participants mostly used sodium hypochlorite as the irrigation solution and serum as the final irrigation solution. This may have been caused by the fact that sodium hypochlorite is also mostly preferred during root canal treatment in permanent teeth [57]. The most recent study [58] and many other studies have determined that sodium hypochlorite is the best agent for disinfection, and the AAPD guideline [35] reports that there is no significant difference between solutions and that sodium hypochlorite should be used carefully. The use of apex locators to determine the canal length of deciduous teeth is still a matter of debate. Although there are resources in the literature that do not recommend its use [59], resources that suggest its use but argue that there is not enough research on the subject [60], and resources that recommend its use [61] and references report that apex locators are much more acceptable by children than traditional radiography [62], no conclusive evidence has been provided as to which of the methods is the best. In a survey of 237 physicians [63], the use of apex locators was reported to be less common than the radiographic technique, but due to the limitations of radiographic interpretation and the high probability of overinstrumentation and consequent flood filling of unevenly resorbed roots, the use of apex locators has been recommended regardless of the stage of root resorption. Similarly, the participants (52.5%) preferred apex locators alone most for this purpose because of their advantages, such as saving time for the physician and protecting patients from radiation. Although there are few studies on the use of ultrasonic/sonic activators in primary teeth, it has been found to be quite successful in the studies in which it has been evaluated [64,65]; however, 85% of the participants in our study did not prefer to use it. It is thought that this might have been caused by the divergent and narrow structure of the deciduous tooth roots. The results of a study [66] conducted in Saudi Arabia aimed at assessing the level of knowledge of participants regarding root canal treatment of deciduous teeth were similar to our results, in that most participants did not attend courses or workshops after graduation. The reason for this can be attributed to the fact that the pedodontists participating in the study found their specialist training sufficient or that there are few courses or workshops in our country about primary tooth root canal treatment. The reason why the courses on new developments in the field of pedodontics are quite limited in our country may be that features such as low-noise instruments [67] or computerized anesthesia devices [68] are new, and more studies are needed. An alternative treatment method for primary tooth root canal treatment is extraction [69]. In the early extraction of primary teeth, the need for orthodontic treatment may occur in the future [70]. To prevent this, space maintainers are used. Similar to the results of our study (67%), it has been reported in many studies that instead of extracting the primary tooth, it is preferred to keep the tooth in the mouth with root canal treatment, and the survival rates after primary tooth root canal treatment under general anesthesia are high and the techniques are successful and reliable [68,69]. The limitations of the study are that although the study group comprised an estimated 10.5% [71] of the pedodontists in the country, it was small study group, the participants were mostly from the central, southeast, and western regions of the country, and age distribution is not homogeneous, which may affect experience and preferences. Conclusions There is no compelling evidence to prove the absolute superiority of any irrigation solution, root canal paste, root canal length determination method, or file system in terms of deciduous tooth root canal treatment. We are of the opinion that keeping up to date with current information, technological advancements, and changes will increase the dentist’s chances of success in treatment as well as the patient’s comfort during treatment. Acknowledgments We would like to thank Türkay Kölüş, the Turkish Pediatric Dentistry Association, Mustafa Ülker, Nurhan Uslu Özalp, Tuğba Bezgin, Akif Demirel, Burak Buldur, and İzzet Yavuz. Figure 1 Age distribution of participants by institution. Table 1 Responses of the participants to the questions. Variables Subcategory Number of participants (%) 1. Gender Female 171 (78.8%) Male 46 (21.2%) 2. Age 25–35 160 (73.7%) 36–45 40 (18.4%) 46 and above 17 (7.9%) 3. Years of practice 1–5 years 167 (77%) 6–10 years 34 (15.7%) More than 10 years 16 (7.3%) 4. Which institution do you work for? University 123 (56.7%) ODHC/ODHH 26 (12%) Private outpatient clinic 51 (23.5%) Own Clinic 17 (7.8%) 5. What was the file system you used when you were a student (before specialization) while doing canal preparation for deciduous teeth? Hand file only 184 (84.8%) Rotary file set 27 (12.5%) Pediatric canal file set 6 (2.7%) 6. Have you attended a root canal treatment course/training in the residency or post-residency? Yes 64 (29.5%) No 153 (70.5%) 7. What is the file system you use for root canal preparation of deciduous teeth? Hand file only 39 (18.0%) Rotary file set 103 (47.5%) Pediatric canal file set 75 (34.5%) 8. Do you use ultrasonic/sonic activation for root canal treatment of deciduous teeth? Yes 7 (3.3%) Sometimes 186 (85.7%) No 24 (11.0%) 9. Which canal irrigation solution do you use for root canal treatment of deciduous teeth? Sodium hypochlorite 180 (83.0%) Chlorhexidine 6 (2.7%) Serum 30 (13.5%) Distilled water 1 (0.8%) EDTA 0 (0.0%) 10. Which one do you use as the final irrigation solution when performing root canal treatment on deciduous teeth? Sodium hypochlorite 73 (33.6%) Chlorhexidine 8 (3.7%) Serum 117 (53.9%) Distilled water 17 (7.8%) EDTA 2 (1.0%) 11. How do you determine the length of the canal when performing root canal treatment on deciduous teeth? Dental digital radiographs (RadioVisioGraphy) 65 (30.0%) Dental digital radiographs (RadioVisioGraphy) and apex locator 38 (17.5%) Only apex locator 114 (52.5%) 12. Which root canal paste do you use for filling when performing root canal treatment on deciduous teeth? Eugenol paste 13 (6.0%) Calcium hydroxide paste 44 (20.3%) Iodoform paste 160 (73.7%) 13. Do you use rubber dams for root canal treatment of deciduous teeth? Yes 14 (6.0%) No 152 (70.5%) Sometimes 51 (23.5%) 14. How many minutes does it take you to complete the root canal treatment (excluding the restoration)? 10–15 min 31 (14.3%) 15–20 min 73 (33.6%) 20–30 min 79 (36.4%) More than 30 minutes 34 (15.7%) 15. Do you perform root canal treatment under general anesthesia? Yes 94 (43.3%) Sometimes 51 (23.5%) No 72 (33.2%) Table 2 Distribution of canal length determination technique preferences by institutions. Canal length determination technique Total Dental digital radiographs Only apex locator Dental digital radiographs and apex locator Institution University Count 44 56 23 123 Expected count 36.8 64.6 21.5 123 ODHC/ODHH Count 10 11 5 26 Expected count 7.8 13.7 4.6 26 Private outpatient clinic Count 8 33 10 51 Expected count 15.3 26.8 8.9 51 Own clinic Count 3 14 0 17 Expected count 5.1 8.9 3 17 Total Count 65 114 38 217 Expected count 65 114 38 217 Table 3 Comparison of the file systems that the participants used when they were students and the file systems they currently use. The file system currently using Total Hand file only Rotary file set Pediatric canal file set File system used as a student Hand file only Count 39 84 61 184 Expected count 33.1 87.3 63.6 184 Rotary file set Count 0 19 8 27 Expected count 4.9 12.8 9.3 27 Pediatric canal file set Count 0 0 6 6 Expected count 1.1 2.8 2.1 6 Total Count 39 103 75 217 Expected count 39 103 75 217 Table 4 Comparison of the canal irrigation solution preferences of the participants with the final irrigation solution preferences. Final irrigation solution Total Sodium hypochlorite Chlorhexidine Serum Distilled water EDTA Canal irrigation solution Sodium hypochlorite Count 71 6 86 15 2 180 Expected count 60.6 6.6 97.1 14.1 1.7 180 Chlorhexidine Count 0 2 3 1 0 6 Expected count 2 2.2 3.2 0.5 0.1 6 Serum Count 2 0 28 0 0 30 Expected count 10.1 1.1 16.2 2.4 0.3 30 Distilled water Count 0 0 0 1 0 1 Expected count 0.3 0,1 0.5 0.1 0 1 EDTA Count 0 0 0 0 0 0 Expected count 0 0 0 0 0 0 Total Count 73 8 117 17 2 217 Expected count 73 8 117 17 2 217 This study was presented as an oral presentation at the 2nd International Necmettin Erbakan Dentistry Congress Conflict of interest: None declared Publisher’s note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher Declaration of Figures’ Authenticity All figures submitted have been created by the authors, who confirm that the images are original with no duplication and have not been previously published in whole or in part. 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==== Front Med Sci Monit Med Sci Monit Medical Science Monitor Medical Science Monitor : International Medical Journal of Experimental and Clinical Research 1234-1010 1643-3750 International Scientific Literature, Inc. 37448107 10.12659/MSM.939920 939920 Review Articles Ultrasound-Guided Brachial Plexus Block by Costoclavicular Space Approach: A Narrative Review Xing Taotao 1BEF Ge Lan 12AD 1 Department of Anesthesia, The Second Hospital Affiliated to Zhejiang University School of Medicine, Hangzhou, Zhejiang, PR China 2 Department of Ophthalmology, The Second Hospital Affiliated to Zhejiang University School of Medicine, Hangzhou, Zhejiang, PR China Corresponding Author: Taotao Xing, e-mail: 2520116@zju.edu.cn A Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection 2023 07 6 2023 14 7 2023 29 e939920-1e939920-9 19 2 2023 22 5 2023 © Med Sci Monit, 2023 2023 https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under Creative Common Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) In recent years, ultrasound-guided costoclavicular brachial plexus block (CCB) has gained attention as a novel approach for brachial plexus nerve block. Human anatomy studies have identified the costoclavicular space as the area between the midpoint of the clavicle and the first rib. This space accommodates the brachial plexus, axillary arteries, and veins. Its superficial and fixed position makes it a promising option for infraclavicular brachial plexus blockage, providing a safe and reliable analgesic effect. CCB combines the benefits of real-time ultrasound visualization of the nerve block needle, avoidance of peripheral blood vessels, and targeted delivery of local anesthetics to the nerve. Consequently, it significantly reduces the associated complications of other classical approaches such as interscalene brachial plexus block (ISB), supraclavicular brachial plexus block (SCB), lateral sagittal infraclavicular brachial plexus block (LS-ICB), and axillary brachial plexus block. These complications include phrenic paralysis, incomplete brachial plexus block, and pneumothorax. This narrative review examines the literature on brachial plexus block in the costoclavicular space, discussing the anatomical position, the procedure, clinical indications, choice of local anesthetic concentration and volume, and continuous nerve block of CCB. The aim is to provide a basis for future clinical practice and enhanced safety. Brachial Plexus Block Nerve Block Ultrasonography ==== Body pmcBackground Brachial plexus block is a well-established anesthetic and analgesic technique for upper-limb surgery and is widely used by clinicians [1]. However, the classical brachial plexus approach has advantages and disadvantages depending on individual patients and the surgical area involved. Therefore, selecting the most appropriate brachial plexus block method is crucial to avoid complications [2]. The costoclavicular space has gained recognition in human anatomy studies, and with advancements in ultrasound technology, more brachial plexus blocks are being performed under ultrasound guidance [3]. This approach has reduced puncture-related complications and improved the speed and safety of the brachial plexus block [4,5]. Costoclavicular brachial plexus block (CCB) is a new technique for infraclavicular brachial plexus block, providing anesthesia and postoperative analgesia. Ultrasound-guided CCB has gained maturity and has become a research focus in recent years. Compared to other approaches, CCB offers advantages such as clear visualization of the brachial plexus, simplicity of the procedure, rapid onset, and reliable analgesic effects. Additionally, it reduces complications such as phrenic paralysis, pneumothorax, and nerve block catheter-related issues, thus improving safety and patient comfort [6]. With the increasing number of anatomy and clinical studies conducted in recent years, our understanding of CCB has become more comprehensive. Anatomy of the Brachial Plexus Nerve in the Costoclavicular Space The brachial plexus consists of the anterior branch of the C4 nerve, the C5 to C8 nerves, and the anterior branch of the T1 nerve [7,8]. It primarily innervates the upper limbs and controls the motor and sensory functions of the hands and arms. In the infraclavicular approach [9], the brachial plexus nerve is divided into lateral, medial, and posterior bundles, which extend downward into the axilla through its upper region. The lateral cord of the brachial plexus originates from nerve fibers spanning from C5 to C7 and innervates the following muscles: the biceps brachii muscle, supplying motor innervation to the muscles responsible for elbow flexion; the brachialis muscle, involved in elbow flexion; the coracobrachialis muscle, assisting in shoulder flexion; the Pronator teres muscle, contributing to forearm pronation; the flexor carpi radialis muscle, responsible for wrist flexion; the palmaris longus muscle, involved in wrist flexion; and the radial wrist extensor muscles, supporting elbow, wrist, finger, and thumb extension. The medial cord of the brachial plexus receives nerve fibers from C8 to T1 and provides innervation to the following muscles: the flexor digitorum profundus muscle, controlling flexion of the thumb, index finger, and middle finger; The ulnar nerve supplies the deep flexor muscles of the little finger and ring finger, as well as the intrinsic muscles of the hand, and the pectoralis major muscle, specifically its clavicular portion. The posterior cord of the brachial plexus includes the following nerves: The axillary nerve, originating from C5 to C6 nerve fibers, innervates the deltoid and teres minor muscles; The radial nerve, receiving contributions from C5 to T1 nerve fibers, predominantly C7, controls muscles involved in elbow and wrist extension, finger extension, and thumb extension; The thoracodorsal nerve, derived from C6 to C8 nerve fibers, innervates the latissimus dorsi muscle. Upon the application of electrical stimulation to the brachial plexus bundles, the muscles innervated by these nerve bundles elicit a contraction response [7]. The costoclavicular space is situated behind the midpoint of the clavicle and is a narrow area between the lateral and medial aspects of the clavicle and the first rib. The distribution of blood vessels and the brachial plexus within the costoclavicular space is relatively fixed, with minimal variation [10]. Karmakar et al [11] previously showed that the lateral, medial, and posterior bundles of the brachial plexus in this region are compact and superficial. Under ultrasound guidance, a suitable puncture angle can avoid the initial and axillary veins, leading to an optimal brachial plexus block. By examining fresh autopsy specimens, Sala-Blanch et al [12] observed the brachial plexus, axillary arteries, and veins crossing within the costoclavicular space. The 3 bundles of the brachial plexus are positioned outside the axillary artery: the lateral bundle is the most superficial, the medial bundle is deeper, and the posterior bundle is the most external. Connective tissue envelopes the medial and posterior bundles, separating them from the lateral bundle, in accordance with the microscopic dissection results reported by Monzó et al [13]. Carles et al [14] further indicated that the costoclavicular space lies beneath the pectoralis major and subclavian muscle, and performing continuous nerve block in this area can enhance catheter stability and reduce the risk of catheter displacement. Moreover, the lack of fixed devices in the patient’s neck retains their mobility unaffected. Hence, the clinical application of CCB is supported by solid anatomical foundations. Clinical Applications of CCB Ultrasound-Guided CCB Ultrasound is a non-invasive, real-time, and dynamic visualization technique that allows anesthesiologists to directly visualize the relationship between nerves, blood vessels, and surrounding tissues during brachial plexus block [15]. This improves the success rate and reduces complications [4], serving as a valuable tool in the costoclavicular brachial plexus block procedure. Ultrasound provides clear visualization of the anatomical location, depth, and relationships of the brachial plexus bundle with surrounding muscles and blood vessels. The ultrasound-guided CCB procedure can be performed as follows: Posture Based on existing studies, most patients had their affected upper limbs abducted at a 90° angle while tilting their head to the opposite side [16]. This position brings the costoclavicular space closer to the body surface, allowing for better stretching of the brachial plexus and axillary arteries and veins. This positioning ensures accurate relative positioning of nerves and vessels, producing a clear ultrasound image. Method Similar to the technique described by Li et al [17], a high-frequency linear array probe is placed at the midpoint of the clavicle. By sliding the probe downward, various structures such as the pectoralis major muscle, subclavius muscle, brachial plexus nerve, axillary artery, and vein in the costoclavicular space, as well as other tissue structures like the cephalic vein, thoracoacromial artery, and serratus anterior, can be identified. The probe should be adjusted slightly from a superficial to a deep angle, revealing the pectoralis major, brachial plexus, and pleura. Under the optimal ultrasound image [17,18] (Figure 1), the needle is inserted using an in-plane technique from the outside to the inside of the probe. Once the needle tip is visualized, it is inserted into the middle of the 3 bundles or between the lateral and medial bundles, and the medication is administered while ensuring there is no blood return. To prevent nerve injury, the injection pressure should be adjusted [19], keeping it below 15 psi, and the injection time should be longer than 2–3 min. By evaluating the sensory and motor blockade responses of the ulnar nerve, radial nerve, musculocutaneous nerve, and median nerve, the efficacy of the nerve blockade can be determined [18]. In cases where the tip of the coracoid process joint appears unclear, or the development of nerves and vessels is not well-defined due to the depth of the lateral subclavian fossa in certain patients, some researchers [20] have proposed an alternative puncture path using the opposite direction in-plane needle approach. The procedure is similar to the previously-described method. Once the best image is obtained, the needle is inserted inside the high-frequency linear array probe to avoid the surrounding blood vessels, yielding successful outcomes [21]. Medication regimen In a prospective study involving 40 patients, Kewlani et al. [22] used 0.5% ropivacaine for the block and determined that the 95% effective volume was 18.9 mL. Other studies have also used a 20 mL volume with satisfactory results within 30 min. However, there is limited research on brachial plexus block through the costoclavicular space using an out-of-plane needle or solely relying on a nerve stimulator [23–25], warranting further investigation. Duration of anesthesia The duration of anesthesia in the brachial plexus block varies depending on the type of local anesthetic used and the technique employed. Typically, anesthesia lasts 4–16 hours. Short-acting local anesthetics like lidocaine [28] provide anesthesia for 2–4 h, whereas longer-acting agents such as ropivacaine [22] can provide anesthesia for 8–16 h or even longer. Adding epinephrine to the local anesthetic solution can prolong the duration of the block by slowing absorption of the anesthetic agent. Clinical Indications of CCB In clinical practice, the CCB approach, specifically the lateral sagittal infraclavicular plexus block (LS-ICB) [26], is commonly used for distal upper-arm surgery. This is because the C5 to C7 nerve branches [9], including the suprascapular, subscapular, axillary, and lateral thoracic nerves, have already separated before reaching the subclavicle. Thus, the ICB approach typically covers anesthesia limited to the elbow, forearm, wrist, and hand. With its anatomical structure, the costoclavicular space is a suitable alternative for LS-ICB as a new infraclavicular brachial plexus block approach. Leurcharusmee et al analyzed 90 patients undergoing distal upper-limb surgery and found that CCB offers advantages over the traditional lateral sagittal process approach, including shorter operation time and faster fulfillment of surgical needs [27]. Other studies on CCB [11,17,27,28] have primarily focused on forearm surgery. For shoulder joint and proximal upper-limb operations, where the suprascapular nerve innervates 70% of sensory function [29] and the remaining by the axillary nerve, the brachial plexus nerve block via the subclavian approach often results in incomplete anesthesia. Interscalene brachial plexus block (ISB) or supraclavicular brachial plexus block (SCB) are usually employed to achieve satisfactory blockade [30–32]. In 2017, Carles García-Vitoria proposed that the costoclavicular space can serve as a channel connecting the supraclavicular and subclavian areas. Placing a catheter in the infraclavicular space allows for continuous nerve block in the supraclavicular area, providing effective analgesia and benefits from the protection offered by the pectoralis major and subclavian muscles. This significantly reduces the likelihood of catheter displacement and prolapse after brachial plexus catheterization via the subclavian approach [33]. In 2020, Koyyalamudi et al [34] injected a cadaver with 20 mL of 0.1% methylene blue solution 5 times and dissected the brachial plexus and its branches from the C4 level to the lower armpit, documenting the spread of dye and staining of nerve trunks, bundles, and branches, including the suprascapular nerve. Aliste [23] also confirmed that the costoclavicular space could be used for anesthesia in patients undergoing shoulder surgery by comparing ISB and CCB in a study involving 44 patients. Luo et al [16] evaluated the non-inferiority of brachial plexus block via CCB and ISB in 212 patients in 2023, demonstrating that CCB could provide a nerve block method with fewer complications for high-risk patients undergoing shoulder surgery. Furthermore, several case reports [35–37] have shown that brachial plexus block via the costoclavicular space approach allows for drug spread to the supraclavicular area, meeting the needs of shoulder and upper-arm humerus surgery. Therefore, CCB surpasses the block range of the traditional subclavian approach and fulfills surgical requirements for the forearm and proximal upper arm, including the humerus and shoulder. Concentration and Volume of CCB Local Anesthetic Determining the optimal concentration and volume of local anesthetics for brachial plexus block has been a significant research focus in recent years. Lidocaine, a classic choice for nerve block, has been favored by researchers. Studies by Tran et al [38,39] and González et al [40] investigated brachial plexus block via SCB, ICB, and axillary approaches using sequential lidocaine administration. The effective volume of 1.5% lidocaine ranged from 23.5 mL to 35 mL. In a study conducted by Sotthisopha et a. [28] in 2017, sequential CCB was performed on 57 patients using 1.5% lidocaine, with a 90% effective volume (ED90) of 34 mL. Ropivacaine, known for its unique sensorimotor separation effect at low concentrations [41], has gained popularity among surgeons, especially orthopedic surgeons, and patients [42,43]. The concentration of ropivacaine is often determined based on its pharmacological properties, with intraoperative concentrations ranging from 0.5% to 0.75% [41]. Recent studies [23–25] have utilized a 20 mL dose of 0.35–0.5% ropivacaine, achieving excellent analgesic effects. In a study by Kew et al [22] involving 40 patients undergoing distal upper-limb surgery, the half-effective dose (ED50) of 0.5% ropivacaine was found to be 13.5 mL, while the 95% effective dose (ED95) was 18.9 mL. Wong et al [44] also reported similar findings in a study of 48 patients undergoing hand surgery, where the minimum effective volume of 0.5% ropivacaine in 90% of cases was comparable to results of Kew et al. For postoperative analgesia, a lower concentration of 0.2% ropivacaine can selectively block sensory nerves while preserving limb motor function [45,46]. Multipoint Injection of CCB Numerous studies [40,47,48] have examined brachial plexus block through interscalene, supraclavicular, axillary, and lateral sagittal infraclavicular approaches. Most of these studies indicate that multipoint injection offers advantages such as lower total drug volume and faster onset of effectiveness than single injections. However, it also presents disadvantages, such as increased needle punctures for nerve blocks and a higher risk of vascular puncture complications. In the case of CCB, both anatomical and clinical studies have identified a tissue septum [13] between the medial, lateral, and posterior bundles of the brachial plexus within the costoclavicular space. This septum can impact the diffusion of local anesthetics and the onset time between nerve bundles. Li et al [17] observed that while the success rate of CCB is nearly 97%, the effectiveness of each bundle block differs. The medial and posterior bundles exhibit faster motion block speeds than the lateral bundle. This discrepancy may be attributed to the fact that the posterior and medial bundles are uniformly wrapped within the same diaphragm, while connective tissue separates the lateral bundle as a whole. Building upon previous research, Monzó et al [49] found that 92.5% of patients had a rebound diaphragm within the costoclavicular space, leading to an illusion of drug diffusion upon injection. It was further proposed that the best ultrasound-guided image could be obtained for multipoint injection. Local anesthetics (5 and 10 mL) were administered to the lateral and posterior bundles to counteract the diaphragm effect and achieve a more effective nerve block. Layera et al [50] also demonstrated that the success rate of 2-point injection is similar to that of Monzó. Using 2 injections enables faster anesthesia onset compared to a single injection while achieving similar success rates in terms of surgical anesthesia and analgesia. However, 2 injections increase the number of punctures and the incidence of complications such as Horner’s syndrome. Whether a 3-point injection for the brachial plexus 3-bundle block can yield a faster and more effective anesthetic effect and whether it leads to other complications remains unclear. Further research is required to address these questions. Continuous Brachial Plexus Block of CCB Continuous nerve block [51] involves placement of a nerve block catheter near the target nerve, providing long-term and effective analgesia. It is commonly used for treatment of chronic pain conditions such as complex regional pain syndrome [52], refractory phantom limb pain [53], advanced cancer [54], trigeminal neuralgia [55], and patients requiring prolonged postoperative analgesia for rehabilitation [56]. Continuous brachial plexus block is frequently employed for postoperative pain management and upper-limb rehabilitation exercises [37,58,59]. Due to the long block time, it can significantly reduce postoperative rebound pain [57] and promote recovery of postoperative function, thus improving patient satisfaction [43]. Traditional approaches include interscalene, supraclavicular, and infraclavicular techniques [60]. However, due to the superficial positioning of most cervical catheters, weak muscle support, and frequent neck movement, there is a high risk of catheter displacement. Studies have shown that the catheter displacement rate for the interscalene approach is 35% [61]. Therefore, clinical anesthesiologists often prefer the lateral sagittal infraclavicular approach for continuous nerve block [62]. This approach provides greater stability and lower catheter detachment rates due to the presence of the muscle tunnel formed by the pectoralis major and subclavian muscles [63]. Nevertheless, the catheter transposition rate for the infraclavicular approach was found to be 22% [64]. Carles’s anatomical study of the costoclavicular space in 2017 [14] laid the groundwork for subsequent continuous nerve blocks in this region, located between the pectoralis major, subclavian, and anterior serratus muscles. During ultrasound-guided continuous brachial plexus block in the costoclavicular space, the nerve block needle passes through the pectoralis major and subclavian muscles, and the catheter is secured within the muscle tunnels, resulting in low catheter transposition rates postoperatively. There have been no reported cases of catheter displacement, dislodgment, or catheter-induced nerve injuries associated with continuous nerve blocks in the costoclavicular space [65,66]. Moreover, the variability of the position of the brachial plexus within the costoclavicular space is minimal, with the nerve bundles and blood vessels being relatively concentrated and fixed. Anesthesiologists with knowledge of the relevant anatomy can perform brachial plexus blocks more efficiently compared to other nerve blocks [66]. In addition, compared to the interscalene or supraclavicular approaches, continuous nerve block in the costoclavicular space requires a lower puncture position, has less impact on neck movement, and offers a better overall experience. This approach has also been proven safe and effective for single-dose nerve blocks in pediatric patients [65]. However, the precise efficacy of continuous nerve blocks [66–68] in the costoclavicular space is yet to be determined. In the past, continuous nerve blocks via the interscalene approach were prone to catheter prolapse due to the extensive range of neck motion. The effectiveness of continuous nerve blocks via the costoclavicular approach has been demonstrated [37,69], but further randomized controlled trials are needed to establish its safety. Incidence of Hemi-Diaphragmatic Paralysis at CCB The diaphragm is the primary inspiratory muscle in the human body, accounting for two-thirds of total ventilation [70]. Hemi-diaphragmatic paralysis (HDP) is a common complication of brachial plexus block [29], mainly when local anesthetic drugs spread to block the phrenic nerve, resulting in a 30% reduction in vital capacity and a 20% decrease in ventilation [71]. While most patients are asymptomatic due to compensation from the contralateral diaphragm, individuals with respiratory diseases, severe heart conditions, or obesity may experience chest tightness and even respiratory failure [72]. The phrenic nerve originates from C3 to C5, runs adjacent to the sternocleidomastoid muscle, descends laterally to the anterior scalene muscle, and is located 0.18 cm from the brachial plexus. Studies have shown that the interscalene approach for brachial plexus block can lead to 100% HDP [73]. Experts suggest that reducing the volume of local anesthesia and minimizing drug spread to the septal nerve can help decrease the incidence of HDP. However, even when the total amount of local anesthetic in the interscalene approach is reduced to 5 mL, a 27% incidence of HDP remains [74]. Furthermore, the phrenic nerve separates from the brachial plexus nerve at a distance of 3 mm/cm as it descends from the anterior scalene muscle. Some researchers propose that blocking the brachial plexus away from the phrenic nerve can achieve adequate analgesia while reducing complications such as phrenic paralysis. Although effective in reducing postoperative pain, the supraclavicular approach still results in a 45% incidence of HDP [75,76]. Therefore, careful consideration is necessary when performing brachial plexus blocks in patients with lung disease. In recent years, the costoclavicular space has gained recognition due to its anatomical distance from the diaphragm and the low rate of phrenic nerve block. A randomized controlled trial by Boohwi Hong involving 80 patients who received an injection of 25 mL of 0.375% ropivacaine demonstrated that costoclavicular space block reduced the incidence of diaphragm paralysis to 3% [25]. Similarly, Aliste et al reported a 0% incidence of phrenic nerve block using 20 mL of 0.5% ropivacaine for CCB injection in 2019 [23]. Luo et al revealed that in shoulder arthroscopic surgery, the incidence of HDP in CCB was 7.55%, significantly lower than 92.45% for the interscalene approach. The incidences of shortness of breath and Horner syndrome were also lower in CCB, at 0% and 10.38%, respectively, compared to 18.87% and 10.38%, respectively, in the interscalene approach4[16]. The clinical applications of CCB have recently expanded from the distal to the proximal upper limb and shoulder region [23,77], providing effective anesthesia and analgesia. Its low incidence of diaphragmatic paralysis offers a reliable and safe nerve block method for patients with lung diseases undergoing upper-limb surgeries such as shoulder and humerus procedures. However, for continuous nerve block in the costoclavicular space, there is no available information on the potential accumulation of local anesthetic drugs during long-term infusion and its relation to HDP. Nonetheless, some studies have suggested that diluting the concentration of local anesthetic drugs with normal saline during infusion can alleviate symptoms of limb numbness [78,79]. Therefore, this approach could potentially be used as a solution if the accumulation of local anesthetics leads to HDP. Limitations of CCB Limitations in Visualizing CCB While the costoclavicular space can be easily identified, performing a nerve block in thin or obese patients presents challenges [80]. Thin patients often have a deep paraclavicular fossa behind the coracoid process, resulting in a large ultrasound shadow area and unclear tissue development. Moreover, some patients may have a protruding coracoid process, making it difficult to visualize the needle tip clearly [20]. In obese patients, the costoclavicular space is situated within a layer of fat, the pectoralis major, and the subclavian muscle, which can impede proper needle visualization. These patients are at a higher risk of multiple needle attempts, puncturing the cephalic, axillary veins, and even the acromial artery [81]. To overcome these challenges, some researchers suggest approaching the subclavicular region from medial to lateral to enhance needle visualization by avoiding interference from the coracoid process. However, this technique may result in slower blockade of the medial bundle due to the distribution of nerves within the costoclavicular space, where the medial bundle is located more superficially than the lateral bundle. Thoracic Outlet Syndrome Thoracic outlet syndrome [82] is a challenging clinical condition to diagnose, characterized by the compression of neurovascular bundles in the thoracic outlet, leading to pain and numbness in the shoulder and upper limbs. Possible causes of thoracic outlet syndrome include clavicle trauma, malunion of clavicle fractures, and hemorrhage in the costoclavicular and subclavicular space. In the case of CCB, the neurovascular structures within the costoclavicular space are closely situated and immobile. However, during the nerve block procedure, there is a risk of inadvertently puncturing blood vessels due to the unclear visualization of tissue vessels or needle tips [81]. This can result in bleeding and subsequent compression of the brachial plexus. Therefore, patients who experience prolonged numbness in the upper limbs following CCB should consider the potential for nerve injury during the procedure and the possibility of developing thoracic outlet syndrome. Hemodialysis Patients Using a Double-Lumen Catheter The number of patients with end-stage renal disease (ESRD) requiring kidney replacement therapy has increased recently, and hemodialysis has become a widely used method for managing acute and chronic kidney failure [83]. In clinical practice, hemodialysis access can be achieved through an arteriovenous fistula or the placement of a double-lumen catheter. Among these options, the placement of a double-lumen catheter is preferred by patients due to its minimal impact on blood circulation, effectiveness in preventing infections, and convenience of use [84]. The common sites for inserting a double-lumen catheter in hemodialysis patients include the internal jugular, subclavian, and femoral veins [85]. However, when a double-lumen catheter is placed in the subclavian vein, it can occupy the costoclavicular space, increasing the risk of nerve injury or displacement by interfering with the insertion and angle of the nerve block needle. Furthermore, the insertion site of the double-lumen catheter is deeper and may involve deeper tissues [86], raising the risk of nerve injury. Hemodialysis patients who receive double-lumen catheters also face complications such as catheter-related infections [87], subcutaneous tunnel infections [88], and catheter dysfunction, with catheter-related bacteremia being the main reason for catheter removal [89]. Therefore, caution should be exercised when performing CCB procedures to minimize the risk of skin puncture, catheter-related infections, and systemic complications. Antithrombotic Therapy With the increasing incidence of cardiovascular diseases, more patients are being prescribed antithrombotic medications to prevent thrombosis. However, when these patients undergo regional anesthesia, their abnormal coagulation function poses an increased risk. While studies have shown that bleeding complications following peripheral nerve block (PNB) in such patients are rare, with an estimated incidence rate of 0.67%, the occurrence of a hematoma can lead to serious adverse consequences [90–92]. ESAIC/ESRA and ASRA guidelines classify peripheral nerve blocks based on their potential risk of severe bleeding complications in patients on antithrombotic therapy. Superficial nerve blocks, such as routine interscalene, supraclavicular, axillary, sciatic, and femoral nerve blocks, are considered low-risk, allowing for broader use of perioperative anticoagulant drugs, including high-dose low-molecular-weight heparin. Deep nerve blocks like the lateral sagittal infraclavicular brachial plexus block (ICB) are classified as high-risk, and antithrombotic drugs should be managed according to recommendations for neuraxial procedures [93–95]. While there have been reports of bleeding-related complications associated with superficial nerve blocks, such as nerve damage and significant bleeding, studies have also shown that bleeding complications following peripheral nerve block are rare in patients receiving antithrombotic therapy [91,96–103]. In contrast, complications associated with deep nerve blocks can be more severe, including bleeding and even death [92,104]. CCB has been considered a shallower and safer approach than ICB, with vascular injuries mainly involving the puncture of the axillary vein and artery. Studies have demonstrated that these vessels can be promptly compressed in case of a puncture, suggesting that CCB may be a low-risk superficial nerve block with compressible vessels and a lower risk of bleeding than ICB [20,105,106]. However, there is limited research on the use of antithrombotic drugs in patients undergoing CCB, as most studies exclude patients using these medications. Some studies have reported successful cases of CCB in patients receiving anticoagulant therapy, highlighting its potential as a viable option with good outcomes and no perioperative bleeding complications [35]. Further research is needed to explore the clinical application of CCB in the presence of anticoagulant therapy. Selecting the best imaging method under ultrasound guidance is essential to avoid blood vessels, ensure clear visualization of the needle tip position, and achieve a safe and effective block. Additionally, in the costoclavicular space thoracic outlet syndrome can be caused by bleeding, hematoma, or nerve compression. Therefore, when performing CCB on patients on anticoagulant therapy, it is crucial to use ultrasound guidance to select the optimal image, ensuring avoidance of blood vessels, clear visualization of the needle tip position, and successful and safe nerve blocking. Conclusions The increasing number of cadaver specimens and clinical studies on CCB has enhanced our understanding of various aspects, including ultrasound manipulation, clinical indications, drug concentration and dose, continuous nerve block, and hemidiaphragm paralysis. CCB has expanded its clinical indications to include shoulder joints and humerus, offering a valuable anesthetic option for patients with pulmonary conditions and contraindications for brachial plexus block. However, further research is required to establish its safety profile. Additionally, there is a lack of clinical data regarding the safety and effectiveness of CCB for continuous nerve block, highlighting the need for future investigation in this area. Figure 1 This image was created using the Sonosite SII ultrasound system provided by FUJIFILM Sonosite. AA – axillary artery; LC – lateral cord; PC – posterior cord; MC – medial cord. Conflict of interest: None declared Publisher’s note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher Declaration of Figures’ Authenticity All figures submitted have been created by the authors, who confirm that the images are original with no duplication and have not been previously published in whole or in part. Financial support: This work was supported by a grant from the Zhejiang Medical and Health Science and Technology Plan Project (N0. 2023KY763) ==== Refs References 1 Hsu AC Tai YT Lin KH Infraclavicular brachial plexus block in adults: A comprehensive review based on a unified nomenclature system J Anesth 2019 33 3 463 77 31076946 2 Neumeister EL Beason AM Thayer JA El Bitar Y Perioperative pain management in hand and upper extremity surgery Clin Plast Surg 2020 47 2 323 34 32115058 3 Gamble SG Costo-clavicular syndrome Arch Phys Med Rehabil 1951 32 8 516 22 14857925 4 Gu L An H Zhang X Jiang W Clinical application of ultrasound microscopy-guided pediatric brachial plexus nerve block anesthesia Contrast Media Mol Imaging 2022 2022 3383898 5 Zadrazil M Opfermann P Marhofer P Brachial plexus block with ultrasound guidance for upper-limb trauma surgery in children: A retrospective cohort study of 565 cases Br J Anaesth 2020 125 1 104 9 32340734 6 Kumari P Kumar A Sinha C Kumar A 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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4664 10.12998/wjcc.v11.i19.4664 Case Report Hypothetical hypoxia-driven rapid disease progression in hepatocellular carcinoma post transarterial chemoembolization: A case report Yeo et al. Rapid disease progression in hepatocellular carcinoma Yeo Kai-Fuan Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung 402306, Taiwan Ker Amy School of Medicine, Chung Shan Medical University, Taichung 402306, Taiwan Kao Pei-En School of Medicine, Chung Shan Medical University, Taichung 402306, Taiwan Wang Chi-Chih Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung 402306, Taiwan School of Medicine, Chung Shan Medical University, Taichung 402306, Taiwan. bananaudwang@gmail.com Author contributions: Yeo KF, Ker A, and Kao PE reviewed the literature and contributed to manuscript drafting; Wang CC were responsible for the revision and final approval of the manuscript; This article supported by Yeo KF, Yeo KF, and Ker A contributed equally to the manuscript as the first authors. Corresponding author: Chi-Chih Wang, PhD, Associate Professor, Director, Division of Endoscopy, Department of Internal Medicine, Chung Shan Medical University Hospital, No. 110 Sec. 1, Jianguo N. Rd., Taichung 402306, Taiwan. bananaudwang@gmail.com 6 7 2023 6 7 2023 11 19 46644669 18 2 2023 25 5 2023 6 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Transarterial chemoembolization (TACE) is widely performed for intermediate-stage or unresectable hepatocellular carcinoma (HCC), but approximately half of patients do not respond to TACE treatment. We describe a case of rapidly progressing of HCC after TACE and provide a possible hypothesis for this condition. The finding may contribute to identifying patients who obtain less benefit from TACE, thus avoiding the unnecessary waste of medical resources and treatment during the golden hour window. CASE SUMMARY A 61-year-old woman had been diagnosed with chronic hepatitis B infection and HCC at Barcelona Clinic Liver Cancer stage B, which had been treated by segmental hepatectomy 14 mo ago. The tumor recurred in the two months after surgery. She received an initial TACE and then underwent systemic therapy with lenvatinib 8 mg daily due to an increased level of alpha-fetoprotein (AFP) after the first TACE. However, the tumor continued to progress with an increased level of AFP, and she underwent a second TACE, after which the tumor volume did not obviously decrease on the contrast-enhanced computed tomography image. One month later, she had a third TACE to control the residual HCC tumors. Two weeks after that, the HCC had increased dramatically with tea-colored urine and yellowish skin turgor. Eventually, the patient refused further treatment and went into hospice care. CONCLUSION Intense hypoxia induced by TACE can trigger rapid disease progression in infiltrative HCC patients with a large tumor burden Carcinoma hepatocellular Transarterial chemoembolization Tumor hypoxia Disease progression Tumor burden Case report ==== Body pmc Core Tip: We report an hepatocellular carcinoma (HCC) case with a large tumor burden and infiltrative tumor pattern who exhibited rapidly increased tumor volume within two weeks after undergoing a third trans-arterial chemoembolization (TACE). Although the Barcelona Clinic Liver Cancer staging system classifies multinodular HCC without portal invasion or extrahepatic spread in stage B, it appears that TACE is not suitable for those with a large tumor burden or infiltrative tumor pattern. In addition, hypoxia is an important factor for tumor development, metastasis, and drug resistance. Our case suggests that intense hypoxia induced by TACE may lead to the rapid progression of HCC. INTRODUCTION Primary liver cancer was the third-leading cause of cancer death globally in 2020, with an age-standardized incidence rate of 19.3 cases per 100000 people and an age-standardized mortality rate of 17.7 per 100000 people[1]. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for approximately 75%–85% of cases. Clinically, trans-arterial chemoembolization (TACE) is used as the first-line treatment for patients with Barcelona Clinic Liver Cancer (BCLC) stage B and some unresectable HCC[2,3]. TACE injects cytotoxic agents into the arteries, followed by the embolization of tumor blood vessels, which induces strong cytotoxic and ischemic effects to destroy tumor cells. However, the response rate of intermediate-stage HCC patients receiving TACE is low for unclear reasons, with a pooled objective response rate of about 52%[4]. To our best knowledge, only a few studies have investigated the baseline characteristics of those not benefitting from TACE, such as large tumor volume, high tumor number, and poor performance status[5,6]. In our case, an intermediate-stage HCC patient underwent a third TACE and experienced rapid disease progression within two weeks. This case may provide information on the failure of and resistance to TACE in HCC treatment. CASE PRESENTATION Chief complaints Progressive yellowish skin turgor and firm sensation over the epigastric area. History of present illness A 61-year-old woman had been diagnosed with HCC at BCLC stage B as well as chronic hepatitis B infection 14 mo earlier. Because the typical image of contrast-enhanced abdominal computed tomography (CT) showed three heterogeneous arterial enhancing lesions with delayed phase wash out in segments S4 and S8, with the largest measuring 7.3 cm, she received segmental hepatectomy and cholecystectomy on October 5, 2021 based on the extensive criteria of the University of California San Francisco[7]. Tumor recurrence emerged two months later, and we performed a TACE followed by systemic therapy of lenvatinib 8 mg daily due to an elevated alpha-fetoprotein (AFP) level after the TACE. The subsequent CT confirmed the progression of HCC volume, so we arranged a second course of TACE after the lenvatinib treatment. History of past illness The patient had a history of major depression with good medication control. Personal and family history The patient denied smoking, alcohol use, and betel nut use. She also denied any family history of malignancy. There was no family history of hepatitis B or hepatitis C. Physical examination The patient’s height and weight were 162 cm and 60 kg. Her vital signs were stable, with a body temperature of 37.5°C, pulse rate of 90 bpm, respiration rate of 17 breaths per minute, and blood pressure of 136/76 mmHg. A physical examination revealed icteric sclera on the day of admission. A palpable firm mass lesion was observed at the epigastric area. Laboratory examinations The laboratory data revealed mild anemia, including a low red blood cell count of 392 × 106 cells/μL and a high mean corpuscular hemoglobin of 32.7 pg. The hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin concentrations were within normal values. Coagulation tests showed a low platelet count of 126000 platelets/μL and a prothrombin time/international normalized ratio within the normal value. Alanine aminotransferase and aspartate aminotransferase were elevated at 273 U/L and 272 U/L, respectively, while total bilirubin was 4.7 mg/dL. The renal function tests and electrolyte tests were within normal ranges. Imaging examinations An abdominal contrast CT showed increased residual HCC volume (Figure 1A) after the second TACE course. The coronal view showed a patent portal vein and normal bile ducts (Figure 1B). Contrast-enhanced magnetic resonance imaging (MRI) was arranged taken two weeks after a third TACE course because of an episode of jaundice. It revealed rapidly progressing of HCC volume in a short time, with left intra-hepatic duct (IHD) and portal vein tumor invasions (Figure 2). Figure 1 Abdominal computed tomography scans following the second transarterial chemoembolization. A: Transverse plane showing multiple hepatocellular carcinoma nodules in the right lobe of the liver (red arrows); B: Coronal plane showing an unobstructed portal vein and typical bile ducts (red arrow). Figure 2 Contrast-enhanced magnetic resonance imaging showed rapid progression of hepatocellular carcinoma volume with involvement of the left intra-hepatic duct and portal vein tumor invasions (red arrow) two weeks after the third transarterial chemoembolization. FINAL DIAGNOSIS HCC with rapid, extensive progression and left IHD invasion after TACE. TREATMENT The abdominal enhanced CT still showed increased residual HCC volume and a high AFP level after the second TACE. Therefore, a third TACE was done one month later, after which a firm abdomen over the epigastric area, tea-colored urine, and yellowish skin turgor appeared within two weeks. Abdominal sonography showed dilated left IHDs and a greatly increased liver tumor burden, which were confirmed by a contrast-enhanced MRI. Although we hypothesized that mutations in hypoxia-related genes may contribute to the disease progression after TACE, we did not perform liver biopsies to confirm these mutations due to laboratory limitation in our hospital. We described an endoscopic retrograde cholangiopancreatography intervention for bile duct drainage and immunotherapy to the patient and her husband, which had previously been described in the outpatient clinic before the series of TACE, but she refused and opted for hospice care. OUTCOME AND FOLLOW-UP For personal and religious reasons, the patient decided to pursue hospice care and was referred for home hospice care. DISCUSSION TACE is considered a first-line treatment for unresectable, multinodular, or intermediate-stage HCC, according to the European Association for the Study of the Liver and the American Association for the Study of Liver Diseases[2,3]. A systematic review of TACE therapy’s efficacy for HCC found an objective response rate of 52.5% (95% confidence interval: 43.6-61.5), and overall survival was 70.3% at one year and 32.4% at five years[4]. However, approximately half of intermediate-stage HCC patients had a poor response to TACE, and the best window for other anticancer therapies was missed due to TACE. In this report, our patient with extensive multinodular HCC had a poor response to the second TACE and rapidly progressed to BCLC stage C HCC within two weeks after the third. Despite the probability of the natural course of the disease, it appeared that TACE stimulated the tumors and rapidly led to this upward stage of transition. In the past decade, several studies have revealed that hypoxia can cause tumor development, tumor angiogenesis, and drug resistance and even promote metastasis, which are mediated by hypoxia-markers including hypoxia-inducible factors (HIF), COX-2, AMP-activated protein kinase and glucose transporter[8-14]. It was reasonably hypothesized that the strong tumor hypoxia induced by TACE may cause drug resistance, rapid growth, invasion, angiogenesis, and metastasis of tumors instead of killing tumor cells. Therefore, these hypoxia-markers might serve as indicators of an unfavourable prognosis in patients who undergoing TACE. While we speculated that mutations in hypoxia-related gene could potentially contribute to an increased tumor burden and disease progression, the clinical use of tests to assess these hypoxia markers is still limited in most hospitals. This may tend to occur in multinodular, large HCC tumors because of multiple regions of intra-tumoral hypoxia and increased expression of HIF[12]. In the prospective study of Tsai et al[5], 746 newly diagnosed HCC patients were enrolled, including 624 who received TACE as the primary therapy and 122 who received the best supportive care. Of 624 patients, 102 had a poor response at three months[5]. Among them, 44 died within three months, and 58 patients with rapid disease progressions had contraindications a subsequent TACE for residual tumors, including a high serum bilirubin level, distant metastases, cachexia, main portal vein thrombosis, and regional extrahepatic invasion[5]. The patients with poor responses were of a higher proportion of performance status ≥ 1, albumin ≤ 3.8 g/dL, Child-Turcotte-Pugh class B, AFP > 40 ng/mL, total tumor volume > 65 cm3, and vascular invasion, than those without poor responses[5]. Furthermore, a retrospective study found that, among intermediate-stage HCC patients who received repeated TACE as the primary therapy, a multiple tumor number of ≥ 4 and a large tumor size of ≥ 5 cm were independent risks for a shorter time of progression from BCLC stage B to stage C than that of their counterparts[6]. Both studies showed that TACE may be ineffective for intermediate-stage HCC patients with a large tumor burden and may even lead to rapid disease progression, such as portal invasion, extrahepatic spread, and worse liver function. However, the mechanisms are still poorly understood and should be explored in future studies to improve the treatment outcomes of HCC. Due to the nature of the case report, causality cannot be established and external validity is limited. However, it was unusual for tumors to grow so rapidly in two weeks, indicating that they were likely caused by the third TACE. Large cohort studies and clinical trials are required to explore this relationship. A strength of this study is that our case identified a potential causal relationship in which TACE stimulates tumors to grow rapidly with portal invasion, bile duct invasion, or extrahepatic spread—that is, the upward stage of transition—among specific intermediate-stage patients with a large tumor burden. This case may raise global awareness of the current limitations of the BCLC staging system and contribute to reducing the incidence of ineffective and even harmful TACE treatment in specific patients. CONCLUSION Several guidelines recommend TACE as the first-line treatment for intermediate-stage HCC patients. However, even without portal invasion or extrahepatic spread, cases with a large tumor burden and multi-foci tumor infiltration tends not to respond to TACE, and HCC may even increase dramatically. ACKNOWLEDGEMENTS We extend our gratitude to the reviewers and editor for their valuable feedback. Informed consent statement: Informed written consent was obtained from the patient for publication of this report and any accompanying images. Conflict-of-interest statement: The authors declare that they have no conflict of interest to disclose. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised in accordance with it. Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: February 18, 2023 First decision: May 16, 2023 Article in press: June 6, 2023 Specialty type: Gastroenterology and hepatology Country/Territory of origin: Taiwan Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): D Grade E (Poor): 0 P-Reviewer: koganti SB, United States; Radhakrishnan K, South Korea S-Editor: Liu JH L-Editor: A P-Editor: Liu JH ==== Refs 1 Sung H Ferlay J Siegel RL Laversanne M Soerjomataram I Jemal A Bray F Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries CA Cancer J Clin 2021 71 209 249 33538338 2 Heimbach JK Kulik LM Finn RS Sirlin CB Abecassis MM Roberts LR Zhu AX Murad MH Marrero JA AASLD guidelines for the treatment of hepatocellular carcinoma Hepatology 2018 67 358 380 28130846 3 European Association for the Study of the Liver EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma J Hepatol 2018 69 182 236 29628281 4 Lencioni R de Baere T Soulen MC Rilling WS Geschwind JF Lipiodol transarterial chemoembolization for hepatocellular carcinoma: A systematic review of efficacy and safety data Hepatology 2016 64 106 116 26765068 5 Tsai YJ Hsu CY Huang YH Su CW Lin HC Lee RC Chiang JH Huo TI Lee SD Early identification of poor responders to transarterial chemoembolization for hepatocellular carcinoma Hepatol Int 2011 5 975 984 21533669 6 Kim HY Park JW Joo J Jung SJ An S Woo SM Kim HB Koh YH Lee WJ Kim CM Severity and timing of progression predict refractoriness to transarterial chemoembolization in hepatocellular carcinoma J Gastroenterol Hepatol 2012 27 1051 1056 22098152 7 Decaens T Roudot-Thoraval F Hadni-Bresson S Meyer C Gugenheim J Durand F Bernard PH Boillot O Sulpice L Calmus Y Hardwigsen J Ducerf C Pageaux GP Dharancy S Chazouilleres O Cherqui D Duvoux C Impact of UCSF criteria according to pre- and post-OLT tumor features: analysis of 479 patients listed for HCC with a short waiting time Liver Transpl 2006 12 1761 1769 16964590 8 Godet I Shin YJ Ju JA Ye IC Wang G Gilkes DM Fate-mapping post-hypoxic tumor cells reveals a ROS-resistant phenotype that promotes metastasis Nat Commun 2019 10 4862 31649238 9 Gilkes DM Semenza GL Wirtz D Hypoxia and the extracellular matrix: drivers of tumour metastasis Nat Rev Cancer 2014 14 430 439 24827502 10 Erin N Grahovac J Brozovic A Efferth T Tumor microenvironment and epithelial mesenchymal transition as targets to overcome tumor multidrug resistance Drug Resist Updat 2020 53 100715 32679188 11 Lai JP Conley A Knudsen BS Guindi M Hypoxia after transarterial chemoembolization may trigger a progenitor cell phenotype in hepatocellular carcinoma Histopathology 2015 67 442 450 25425262 12 Semenza GL Intratumoral Hypoxia and Mechanisms of Immune Evasion Mediated by Hypoxia-Inducible Factors Physiology (Bethesda) 2021 36 73 83 33595388 13 Huang M Wang L Chen J Bai M Zhou C Liu S Lin Q Regulation of COX-2 expression and epithelial-to-mesenchymal transition by hypoxia-inducible factor-1α is associated with poor prognosis in hepatocellular carcinoma patients post TACE surgery Int J Oncol 2016 48 2144 2154 26984380 14 Qu K Yan Z Wu Y Chen Y Qu P Xu X Yuan P Huang X Xing J Zhang H Liu C Zhang J Transarterial chemoembolization aggravated peritumoral fibrosis via hypoxia-inducible factor-1α dependent pathway in hepatocellular carcinoma J Gastroenterol Hepatol 2015 30 925 932 25641377

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4579 10.12998/wjcc.v11.i19.4579 Evidence-Based Medicine Network pharmacology and molecular docking to explore Polygoni Cuspidati Rhizoma et Radix treatment for acute lung injury Zheng JL et al. Polygoni Cuspidati Rhizoma et Radix for lung injury Zheng Jia-Lin Department of Respiratory, The First College of Clinical Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China Wang Xiao Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China Song Zhe Department of Respiratory, The First College of Clinical Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China Zhou Peng Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China Zhang Gui-Ju Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China Diao Juan-Juan Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China Han Cheng-En Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China Jia Guang-Yuan Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China Zhou Xu Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China Zhang Bao-Qing Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, Shandong Province, China. mihuai8556@126.com Author contributions: Zhang BQ and Zhou X proposed the concept of this study; Zheng JL and Wang X have contributed to data collection; Song Z, Zheng JL, and Zhou P contributed to formal analysis; Zheng JL and Zhang GJ contributed to the investigation; Zheng JL, Diao JJ, and Han CE have contributed to these methods; Zheng JL, Jia GY, Zhou X, and Zhang BQ supervised the study; Zhang BQ validated this study; Zheng JL and Wang X contributed to the visualization of this study; Zheng JL and Zhou P wrote the first draft of the manuscript; Zheng JL, Wang X, Song Z, Zhou P, Zhang GJ, Diao JJ, Han CE, Jia GY, Zhou X, and Zhang BQ reviewed and edited the manuscript. Supported by Shandong Province Integrated Traditional Chinese and Western Medicine Professional Disease Prevention and Control Project, No. YXH2019ZXY010 . Corresponding author: Bao-Qing Zhang, MD, Attending Doctor, Department of Respiratory, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, No. 42 Wenhua West Road, Jinan 250014, Shandong Province, China. mihuai8556@126.com 6 7 2023 6 7 2023 11 19 45794600 4 5 2023 15 5 2023 25 5 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Polygoni Cuspidati Rhizoma et Radix (PCRR), a well-known traditional Chinese medicine (TCM), inhibits inflammation associated with various human diseases. However, the anti-inflammatory effects of PCRR in acute lung injury (ALI) and the underlying mechanisms of action remain unclear. AIM To determine the ingredients related to PCRR for treatment of ALI using multiple databases to obtain potential targets for fishing. METHODS Recognized and candidate active compounds for PCRR were obtained from Traditional Chinese Medicine Systems Pharmacology, STITCH, and PubMed databases. Target ALI databases were built using the Therapeutic Target, DrugBank, DisGeNET, Online Mendelian Inheritance in Man, and Genetic Association databases. Network pharmacology includes network construction, target prediction, topological feature analysis, and enrichment analysis. Bioinformatics resources from the Database for Annotation, Visualization and Integrated Discovery were utilized for gene ontology biological process and Kyoto Encyclopedia of Genes and Genomes network pathway enrichment analysis, and molecular docking techniques were adopted to verify the combination of major active ingredients and core targets. RESULTS Thirteen bioactive compounds corresponding to the 433 PCRR targets were identified. In addition, 128 genes were closely associated with ALI, 60 of which overlapped with PCRR targets and were considered therapeutically relevant. Functional enrichment analysis suggested that PCRR exerted its pharmacological effects in ALI by modulating multiple pathways, including the cell cycle, cell apoptosis, drug metabolism, inflammation, and immune modulation. Molecular docking results revealed a strong associative relationship between the active ingredient and core target. CONCLUSION PCRR alleviates ALI symptoms via molecular mechanisms predicted by network pharmacology. This study proposes a strategy to elucidate the mechanisms of TCM at the network pharmacology level. Traditional Chinese medicine Acute lung injury Infections Database Network pharmacology Molecular docking ==== Body pmc Core Tip: This study used network pharmacology and molecular docking to explore the treatment of acute lung injury (ALI) using Polygoni Cuspidati Rhizoma et Radix (PCRR), a traditional Chinese medicine (TCM). Functional enrichment analysis suggested that PCRR alleviated ALI symptoms by modulating multiple pathways, including inflammation, immune modulation, and drug metabolism. We identified 13 bioactive compounds and 60 therapeutically relevant genes. Molecular docking further confirmed the strong association between the active ingredients and core targets. This study provides insights into the mechanisms of TCM at the network pharmacology level for the treatment of ALI. INTRODUCTION Acute lung injury (ALI) is a severe and potentially fatal condition that poses a significant threat to health, with high rates of morbidity and mortality[1,2], which is characterized by an excessive lung inflammatory response. Research indicates that the prolonged quality of life is negatively impacted, even in individuals who recover from ALI[3,4]. ALI can be caused by various factors including severe lung infections caused by viruses, Mycoplasma spp., and bacteria. ALI is characterized by pulmonary edema, tissue infiltration by inflammatory cells, and arterial hypoxemia. These clinical features result in damage to the alveolar epithelium and vascular endothelium, ultimately leading to a decrease in lung function[5-7]. Polygoni Cuspidati Rhizoma et Radix (PCRR) has been used in traditional Chinese medicine (TCM) to treat infections and inflammation[8]; its extracts and bioactive constituents exhibit these pharmacological properties[9,10]. However, the effect of PCRR on the pathogenesis of ALI remains unclear, and currently, no effective treatment has been identified. Network pharmacology is a novel and powerful method in which bioinformatics integrates chemoinformatics, network biology, network analysis, and traditional pharmacology[11]. It is an interactive network that operates on the basis of the 'disease-gene-target-drug' concept. It offers the ability to predict the mechanism of action of drugs to treat diseases from a macro perspective[12]. The advancement of systems biology, bioinformatics, and pharmacology has led to the emergence of network pharmacology as a viable, efficient, and cost-effective approach to drug development. Our study aimed to identify the ingredients related to PCRR using multiple databases to determine potential targets for target fishing. In order to study the interactions between potential PCRR and ALI T targets, we integrated a multi-source database and screened the targets related to ALI. Using topology, we built a protein-protein interaction (PPI) network to analyze the interactions between these targets and the hub targets that were screened. Then the study utilized the Database for Annotation, visualization and integrated discovery (DAVID) bioinformatics resource for gene ontology (GO), biological process (BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Molecular docking techniques were also employed to verify the combination of major active ingredients and core targets. MATERIALS AND METHODS Figure 1 presents a schematic representation of the PCRR network pharmacology and molecular docking study for the treatment of ALI, which was established through four main steps: Data preparation, construction of pharmacological networks, cluster analysis, and enrichment analysis. Figure 1 Schematic representation of network pharmacology study of Polygoni Cuspidati Rhizoma et Radix in the treatment of acute lung injury. PCRR: Polygoni Cuspidati Rhizoma et Radix; ALI: Acute lung injury; PPI: Protein–protein interaction; KEGG: Kyoto Encyclopedia of Genes and Genomes. Databases Traditional Chinese medicine database and analysis platform version 2.3 (TCMSP, https://tcmsp-e.com/), PubChem (https://pubchem.ncbi.nlm.nih.gov), STITCH version 5.0 (http://stitch.embl.de/), Therapeutic Target Database (TDD, http://db.idrblab.net/ttd/ last updated September 29, 2021), DrugBank (https://go.drugbank.com/); DisGeNET version 7.0 (https://www.disgenet.org/), Online Mendelian Inheritance in Man (OMIM, https://omim.org/, updated November 19, 2021), Genetic Association Database (https://geneticassociationdb.nih.gov/); STRING version 11.5 (https://www.string-db.org/), UniProt (http://ctdbase.org/, updated December 18, 2019), DAVID version 6.8 (https://david.ncifcrf.gov/home.jsp), Protein Data Bank (https://www.rcsb.org/) Cytoscape 3.7.2, AutoDock Tools 1.5.6, and Pymol2.1 software were used in this study. Data preparation Construction of a database of PCRR components: Data related to the chemical compounds were derived from TCMSP, a unique systems pharmacology platform for Chinese herbal medicines that captures the relationships between drugs, targets, and diseases[13]. In total, 62 PCRR compounds were identified after removing duplicate data. Screening for active ingredients of PCRR: The active ingredients of PCRR were collected from TCMSP based on the following absorption, distribution, metabolism, and excretion (ADME) parameters: Oral bioavailability (OB) > 30% and drug likeness (DL) > 0.18. The OB value was calculated on a silicon system using the OBioavail 1.1 model to determine the relationship between the compound, cytochrome P450 3A4 enzyme, and transporter (p-glycoprotein). The DL value is used to determine the ability of a prospective compound to become a drug by calculating the similarity between a potential compound and a known drug. Although drug-like compounds are not drugs, they have the potential to become drugs. For example, a bioactive molecule with high OB displays good DL, which is a qualitative concept utilized in drug design to optimize the pharmaceutical and pharmacokinetic properties of molecules, such as chemical stability and solubility. We selected 22 compounds with OB values of > 30% as screening targets. Next, we used TCMs with DL > 0.18 as the selection criteria and obtained 43 potential compounds. Predicting PCRR targets: We searched for active chemical targets using the TCMSP2.3 (https://tcmsp-e.com/), PubChem (https://pubchem.ncbi.nlm.nih.gov), and STITCH5.0 (http://stitch.embl.de/) databases. Targets associated with ALI: We collected therapeutic target data for ALI treatment from five resources: (1) Therapeutic Target Database (TTD, http://db.idrblab.net/ttd/); (2) DrugBank (https://go.drugbank.com/); (3) DisGeNET (https://www.disgenet.org/); (4) Online Mendelian Inheritance in Man (OMIM, https://omim.org/); and (5) Genetic Association Database (GAD, https://geneticassociationdb.nih.gov/). In these databases, we used the keyword “acute lung injury” to search for known therapeutic targets of diseases, and all target names were entered into the UniPort website (http://www.uniprot.org/) to search for human target gene names. Then, the ALI-related targets were imported into the DAVID database (http://david.abcc.ncifcrf.gov, version 6.7) for GO enrichment and KEGG network pathway analysis. PPI network construction PPIs between human proteins were acquired from the STRING11.5 database (https://www.string-db.rg/). STRING consists of physical and functional interactions integrated from experimental repositories, public text collections, and computational prediction methods[14]. The action target was imported into the STRING database, defined as a human protein, and an interaction relationship was obtained. The result was saved in TSV format, which retained the node (nodes 1 and 2) and combined rate score information. Network construction was performed in three steps. First, a PPI network of compound targets was developed by linking the compound targets. Second, a PPI network of ALI targets was constructed by linking known PCRR-related targets. Third, a PPI network of the ALI and PCRR targets was developed by intersecting these two networks. Graphical and diagrammatic networks were constructed using Cytoscape (version 3.7.2), a software package used to visualize network analysis. Cluster analysis The network analyzer function in Cytoscape 3.7.2 was used to further analyze mutual relations in the network model. Three topological properties, “degree,” “betweenness centrality,” and “closeness centrality” were adopted to estimate target topological importance. In addition, we selected significant cluster modules from the constructed PPI network using CytoHubba in Cytoscape 3.7.2. GO pathway enrichment analysis We used DAVID 6.8[15,16] (https://david.ncifcrf.gov/) for GO and KEGG (http://www.kegg.jp/) pathway enrichment analyses. Smaller P values indicated greater enrichment, and only functional annotations with enrichment P values corrected by both Bonferroni and Benjamini (P < 0.05) were selected for further analysis. Molecular docking verification Six potential action targets were selected from the top 10 median values in the Cytoscape 3.7.2 software analysis results. We performed molecular docking of six potential targets and key active ingredients. Crystal structures of the target proteins were obtained from the Protein Data Bank (https://www.rcsb.org/). After removing extraneous small molecules from the protein molecules using Pymol 2.1 software, the protein molecules were imported into the AutoDock Tools 1.5.6 software to remove water molecules, add hydrogen atoms, set the atom types, and finally saved as pdbqt files. Compound structures were imported from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and energy minimization of the downloaded compounds was performed using Chem3D, followed by conversion to the mol2 format. The small-molecule compounds were imported into the AutoDock Tools 1.5.6. Atomic charges were added, atomic types were assigned, all flexible bonds were made rotatable by default, and finally saved as pdbqt files. Finally, bulk molecular docking was performed using AutoDock Vina. The molecular docking results were analyzed, and the binding interactions of the compounds with the proteins were visualized using the Pymol 2.1 software. RESULTS Screening of active PCRR ingredients A search of the TCMSP database yielded 62 active ingredients for PCRR. Of these, 10 were initially screened based on their ADME parameters, specifically their oral bioavailability (OB) and drug-likeness (DL) (Figure 2). In addition, through comparison with literature data, polydatin[17,18], resveratrol[19,20], and emodin were identified as active ingredients[21,22] with anti-inflammatory effects. After identifying the relevant structure and target data from PubChem, 13 active ingredients and their corresponding targets were identified after the final screening (Figure 3). Figure 2 The active ingredients of Polygoni Cuspidati Rhizoma et Radix, orange round rectangles represent the 13 active ingredients selected finally. Figure 3 Thirteen active compounds from Polygoni Cuspidati Rhizoma et Radix. OB: Oral bioavailability; DL: Drug likeness. Putative target of active PCRR ingredients PCRR protein targets were searched using the TCMSP, STITCH, and PubChem databases for each of the 13 active chemical components. A total of 433 targets of the active ingredients of PCRR were identified after high-possibility screening and deduplication. In our study, we found that 184 out of the 433 putative targets were shared by two or more of these components (Figure 4). This indicates that these components are likely to be involved in similar biological processes (Figure 5A). This reflects the synergistic effects of multiple components on inflammation and the importance of these targets in the pathogenesis of ALI. These target genes include vascular endothelial growth factor A (VEGFA)[23], transforming growth factor beta-1 (TGFβ1)[24], and acetylcholinesterase[25], which are involved in the response to hypoxia, inflammation, and cell proliferation. Additionally, 184 targets were imported into the DAVID database to conduct KEGG network pathway analysis. The top 10 signal transduction pathways were selected by excluding a wide range of pathways from KEGG enrichment results: Tumor necrosis factor (TNF) signaling pathway, hypoxia-inducible factor-1 (HIF-1) signaling pathway, hosphoinositide 3-Kinase (PI3K)–Akt signaling pathway, estrogen signaling pathway, NF-kappa B (NF-kB) signaling pathway, prolactin signaling pathway, neurotrophin signaling pathway, sphingolipid signaling pathway, ErbB signaling pathway, and VEGF signaling pathway (Figure 5B). Figure 4 Construction of the protein-protein interaction network of durg active compounds and associated targets. Orange represents active compounds of durg. Blue represents gene symbols of targets Figure 5 The putative target of active Polygoni Cuspidati Rhizoma et Radix ingredients. A: Biological processes analysis for targets of active ingredients of Polygoni Cuspidati Rhizoma et Radix; B: Kyoto Encyclopedia of Genes and Genomes pathways through enrichment method to indicate the top 10 signaling pathways of active ingredients of Polygoni Cuspidati Rhizoma et Radix. HIF-1: Hypoxia-inducible factor-1; TNF: Tumor necrosis factor; FDR: False discovery rate. Identification of ALI-related targets ALI-related targets were identified using various databases, including TTD, DrugBank, DisGeNET, OMIM, and GAD. After compensating for redundant data, 128 ALI-related targets were identified (Figure 6A). Because the database names were irregular, official symbols were converted into UniProt IDs for subsequent analyses. The targets related to ALI were imported into the DAVID database to perform KEGG network pathway analysis. To narrow down the KEGG enrichment results, a selection was made to exclude certain pathways. As a result, the top 10 signal transduction pathways were identified: TNF signaling pathway, NOD-like receptor signaling pathway, NF-kB signaling pathway, HIF-1 signaling pathway, toll-like receptor (TLR) signaling pathway, PI3K–Akt signaling pathway, cytosolic DNA-sensing pathway, T cell receptor signaling pathway, JAK–signal transducer and activator of transcription (STAT) signaling pathway, and p53 signaling pathway (Figure 6B). Figure 6 Identification of acute lung injury-Related targets. A: The related targets of acute lung injury; B: Kyoto Encyclopedia of Genes and Genomes pathways through enrichment method to indicate the top 10 signaling pathways of the related targets of acute lung injury. HIF-1: Hypoxia-inducible factor-1; TNF: Tumor necrosis factor; ALI: Acute lung injury. PCRR target screening of PCRR in ALI treatment We identified 60 common targets between PCRR and ALI based on the intersection of protein targets (Figure 7). To construct and analyze the PPI network, we imported fifty eight target genes associated with the active ingredients and ALI into the STRING database. Figure 7 Venn diagram summarizing differentially expressed targets of Polygoni Cuspidati Rhizoma et Radix and acute lung injury. A: Indicates gene targets of Polygoni Cuspidati Rhizoma et Radix; B: Indicates gene targets related to acute lung injury. The common part of A and B indicates the 58 overlapping genes between the disease and drug. ALI: Acute lung injury. Construction of PPI network of compound targets In order to determine the interactive effects of compounds modulated by PCRR, PPI networks of compound targets were developed. These networks consisted of 422 nodes (364 compound targets and 58 compound/ALI targets) and 16702 edges (Figure 8A) and were used to identify the interactions of PCRR-related proteins. To identify significant PCRR-related targets, we considered the median values for degree (39), betweenness centrality (0.003), and closeness centrality (0.45). As a result, we found 25 highly connected nodes with degree greater than 117, betweenness centrality higher than 0.003, and closeness centrality greater than 0.45. (Figure 8B). These targets included STAT3, cellular tumor antigen p53, receptor tyrosine-protein kinase erbB-2, epidermal growth factor receptor, interleukin (IL)-1β, caspase-3 (CASP3), peroxisome proliferator-activated receptor gamma (PPARG), heat shock protein HSP 90-alpha (HSP90AA1), NAD-dependent protein deacetylase sirtuin-1, catenin beta-1, serine/threonine-protein kinase mTOR (MTOR), prostaglandin G/H synthase 2 (PTGS2), transcription factor AP-1, proto-oncogene tyrosine–protein kinase Src, IL6, TNF, nuclear receptor subfamily 3 group A member 1 (ESR1), proto-oncogene c-Fos, RAC-alpha serine/threonine-protein kinase (AKT1), Myc proto-oncogene protein, VEGFA, C-X-C motif chemokine 8, G1/S-specific cyclin-D1, matrix metalloproteinase-9 (MMP9), and mitogen-activated protein kinase 3. Figure 8 Construction of protein-protein interaction network of compound targets. A: The protein-protein interaction networks of compound targets. The purple nodes represent compound targets, and the pink nodes stand for common targets of disease and drugs; B The significant Polygoni Cuspidati Rhizoma et Radix-related targets after topology analysis. Identification of candidate protein targets associated with ALI PPI networks of ALI-related targets were developed to identify the interactions of ALI-related targets with 125 nodes (ASCL1, TFF1, and TFF2 were not involved in protein interactions) and 3498 edges (Figure 9A). Significant ALI-related targets were identified as highly connected nodes with degree > 27, betweenness centrality > 0.49, and closeness centrality > 0.5, based on the median values for these measures (Figure 9B). These targets included PTGS2, cytochrome P450 2E1, PPARG, CASP3, IL1β, IL-2, heme oxygenase 1, platelet endothelial cell (EC) adhesion molecule, kinase insert domain receptor, MMP9, intercellular adhesion molecule 1 (ICAM1), TGFβ1, caveolin-1, integrin beta-2, nitric oxide synthase, IL-10, prothrombin, TLR4, TLR2, interferon beta, VEGFA, C-C motif chemokine 2 (CCL2), serum albumin, catalase, TNF, and IL-6. Figure 9 Identification of candidate protein targets associated with acute lung injury. A: The protein-protein interaction networks of acute lung injury-related targets. The node color changes from light to dark reflect the degree value changes from low to high in the network; B: The significant Polygoni Cuspidati Rhizoma et Radix-related targets after topology analysis. Construction of active ingredient-disease-target network A network of interacting targets was identified using Cytoscape, comprising of 58 targets. PIM1 and DRD1 were not involved in any protein interactions. The network consisted of a total of 1144 edges, which represented the interactions between the proteins (Figure 10). Figure 10 Protein–protein interaction Network graph of 58 Hub Nodes based on their direct interactions for Polygoni Cuspidati Rhizoma et Radix in acute lung injury. Cluster analysis The analysis of the active ingredient-disease-target PPI network was conducted using three key parameters: degree, betweenness centrality, and closeness centrality, to identify topological features.In order to identify important targets, we considered those with median values above certain thresholds (“degree” ≥ 19.72, “betweenness centrality” ≥ 0.014, and “closeness centrality” ≥ 0.585). The results yielded 27 hub nodes and 296 edges (Figure 11A). The 27 key targets were analyzed using CytoHubba and ranked by MCC for the top 10 nodes (Figure 11C). The top ten targets were MMP9, CCL2, IL6, IL1B, ICAM1, TNF, VEGFA, PPARG, CASP3, and PTGS2 (Figure 11B). Figure 11 Cluster analysis. A: The 27 active ingredient-disease- targets of after topology analysis; B: The top 10 active ingredient-disease- targets after cytoHubba and MCC tool; C: Identifcation of candidate Polygoni Cuspidati Rhizoma et Radix -related targets for acute lung injury treatment through protein–protein interaction network. 27 candidate targets are finally predicted. BC: Betweenness centrality; CC: Closeness centrality. GO BP and KEGG pathway enrichment analysis Enrichment analysis was conducted using DAVID 6.8 on 58 targets, with a screening threshold of FDR < 0.01. Our analysis identified 83 GO items and eight KEGG pathways that were deemed relevant for further investigation. GO BP enrichment analysis: We ranked the 83 projects based on their FDR values and identified the top 25 biological processes (Figure 12A). They are primarily involved in apoptosis, proliferation, inflammatory responses, angiogenesis, and drug response. We identified the extrinsic apoptotic signaling pathway in the absence of a ligand (GO:0097192), negative regulation of the apoptotic process (GO:0043066), and the intrinsic apoptotic signaling pathway in response to DNA damage (GO:0008630). Regarding the regulation of proliferation, we identified positive regulation of cell proliferation (GO:0008284), regulation of cell proliferation (GO:0042127), positive regulation of cell migration (GO:0030335), positive regulation of gene expression (GO:0010628), and positive regulation of transcription from RNA polymerase II promoter (GO:0045944). To regulate the inflammatory response, we identified the cellular responses to lipopolysaccharide (GO:0071222), inflammatory response (GO:0006954), response to lipopolysaccharide (GO:0032496), and positive regulation of the nitric oxide biosynthetic process (GO:0045429). Finally, in drug response regulation, we identified drug metabolic process (GO:0017144), exogenous drug catabolic process (GO:0042738), and response to drugs (GO:0042493). Based on these five aspects, the therapeutic effect of PCRR on ALI may result from a complex multi-pathway synergetic effect. Figure 12 Gene ontology biological process and Kyoto encyclopedia of genes and genomes pathway enrichment analysis. A: The gene ontology Biological process enrichment analysis of 58 targets. The top 25 significantly enriched terms in Biological process; B: The Kyoto encyclopedia of genes and genomes pathway enrichment analysis of 60 targets. The top 8 significant pathways for 60 targets; C: Target-Pathway Network. The orange rounds represent Polygoni Cuspidati Rhizoma Et Radix-related targets. The node size is proportional to the target degree in the network. The green round rectangles represent the related pathways. TNF: Tumor necrosis factor; NOD: NOD-like receptor; NF-kappa B: Nuclear factor kappa-B; HIF: Hypoxia-inducible factor-1. KEGG pathway enrichment analysis: To investigate the possible mechanism behind the therapeutic effect of PCRR on ALI, we performed a KEGG pathway enrichment analysis on 60 targets. We screened eight pathways based on a threshold of FDR < 0.01 after excluding a wide range of pathways, including the TNF signaling pathway (hsa04668), HIF-1 signaling pathway (hsa04066), NF-kB signaling pathway (hsa04064), NOD-like receptor signaling pathway (hsa04621), pathways in cancer (hsa05200), T cell receptor signaling pathway (hsa04660), PI3K–Akt signaling pathway (hsa04151), and TLR signaling pathway (hsa04620) (Figure 12B). We also established a target pathway network based on the PCRR targets in each pathway (Figure 12C). The study's inclusion of mainstream pathways for ALI clinical research and drug treatment lends credibility to its findings. PCRR has multiple anti-inflammatory targets that are abundant and have a highly reliable mechanism of action. Molecular docking results and analysis In this study, four compounds (emodin, polydatin, quercetin, and resveratrol) were molecularly docked to IL-6 (PDB ID:4O9H), AKT1 (PDB ID:2UZW), VEGFA (PDB ID:5DN2), PI3K (PDB ID:6PYS), MMP9 (PDB ID: AF-P41245-F1), and HIF-1α (PDB ID:5L9B). According to our findings, the docking score between components and proteins is considered meaningful when it is below -5 kcal/moL. The molecular docking results revealed that most compounds bonded well to the target proteins, with a good match (binding energy of < -5 kcal/moL). These six targets exhibited different binding abilities for the active components of PCRR. Among them, the lowest binding energy was that of polydatin to AKT, with a binding energy of -9.97 kJ/moL, followed by emodin to PI3K with a binding energy of -9.18 kJ/moL, polydatin to HIF-1α at -8.74 kJ/moL, polydatin to MMP9 at -8.61 kJ/moL, polydatin to IL-6 at -7.49 kJ/moL, and polydatin to VEGFA at -7.17kJ/moL. The molecular docking of the ingredients with key targets is demonstrated in Figures 13. Figure 13 Molecular docking results. A: The binding mode of RAC-alpha serine/threonine-protein kinase with Polydatin; B: The binding mode of Phosphoinositide 3-Kinase with Emodin; C: The binding mode of hypoxia-inducible factor-1a with Polydatin; D: The binding mode of matrix metalloproteinase-9 with Polydatin; E: The binding mode of IL6 with Polydatin; F: The binding mode of VEGFA with Polydatin. “a” is the 3D structure of the complex, “b” shows the hydrogen bond donor receptor network of the complex, “c” is the 2D binding mode of the complex. AKT: RAC-alpha serine/threonine-protein kinase; PI3K: Phosphoinositide 3-Kinase; HIF1a: hypoxia-inducible factor-1a; MMP9: Matrix metalloproteinase-9; IL6: Interleukin-6; VEGFA: Vascular endothelial growth factor A. DISCUSSION Network pharmacology has attracted increasing attention as a tool for analyzing the potential mechanisms of TCM in the treatment of diseases. TCM is a systematic theoretical approach. Herbal medicines contain various components that have coordinated and integrated roles. Thus, fully elucidating the mechanisms of action of TCMs by analyzing individual ingredients or targets is difficult. To establish the molecular mechanism of TCM in treating diseases, it is crucial to utilize network pharmacology based on big data bioinformatics. The chemical components isolated from PCRR include polydatin, resveratrol, and emodin, which are used to treat inflammatory diseases[26,27]. The main mechanism of ALI treatment using PCRR should be characterized by multiple components, targets, and pathways. Therefore, we explored this potential mechanism using a network pharmacology approach. Through data screening, 13 active ingredients were identified: Quercetin, resveratrol, luteolin, emodin, beta-sitosterol, rhein, physovenine, (+)-catechin, polydatin, 6, 8-dihydroxy-7-methoxyxanthone, picralinal, physcion diglucoside, and torachrysone-8-O-beta-D-(6´-oxayl)-glucoside. Among them, quercetin has been proven to reduce inflammation and enhance immune function by inhibiting the levels of TNF-α and IL-1α via the TNF, PI3K-Akt, or TLR signaling pathways[28,29]. Resveratrol is well known for its anti-inflammatory and anti-apoptotic properties and has been demonstrated to significantly improve sepsis-induced acute respiratory distress syndrome by decreasing the release of proinflammatory factors and the ratio of alveolar macrophage apoptosis[30]. The attenuation of lung injury by luteolin may be explained by the attenuation of the inflammatory response via the ICAM-1, NF-kB, and partial iNOS signaling pathways[31]; emodin can inhibit the mTOR/HIF-1/VEGF pathway to alleviate ALI[32]. In conclusion, these active ingredients form the basis for the potential effects of PCRR on ALI. Based on the analysis of the target-pathway network and GO-BP analysis results, the potential mechanism of treating ALI using PCRR is probably related to its participation in cell apoptosis, proliferation, inflammatory response, and angiogenesis. Cell apoptosis Excessive or inadequate apoptosis contributes to the pathogenesis of various diseases[33]. ECs express abundant VEGF, which promotes EC survival and maintains normal pH. When ECs are affected by adverse external factors, various factors (TNF-α and angiotensin II) are produced, resulting in cell apoptosis. Transcription factors such as HIF[34] regulate VEGFA through direct promoter binding. Experimental studies have indicated that the components that act on VEGF-A and include luteolin, quercetin, resveratrol, and emodin. Cell proliferation Lung epithelial injury results in the release of various soluble factors from fibroblasts, ECs, macrophages, injured and adjacent epithelial cells, underlying stroma, and plasma exudates. The PI3K-Akt signaling pathway plays a crucial role in regulating cell proliferation and is also involved in the development of inflammatory diseases[35]. The MMP family of enzymes is not normally expressed in healthy tissues[36]. After epithelial cell damage, MPP9 expression is elevated, and its inhibition enhances wound repair. Components such as luteolin, quercetin, and resveratrol in PCRR may affect cell proliferation by inhibiting MMP9 expression via the PI3K-Akt signaling pathway. Inflammatory response Inflammatory responses play important roles in its initiation and maintenance[37]. Proinflammatory cytokines, such as TNF-α and IL-1β, increase lung epithelium permeability, further inducing lung tissue injury and neutrophil accumulation, leading to pulmonary edema[38]. Increased IL-6 levels in ALI are associated with increased mortality and have been identified as biomarkers for monitoring ALI[39]. Pulmonary infections caused by polybiliary lipid compounds are among the most common causes of ALI. Lipid-polybiliary compounds bind to cell-surface receptors, which may increase inflammation and activate oxidative stress and an inflammatory cascade via the TLR4/NF-kB signaling pathway[40]. Quercetin suppresses TNF-induced apoptosis and inflammation by blocking the NF-kB signaling pathway[41]. Therefore, PCRR can inhibit inflammation by reducing TNF-α, IL-1β, and IL-6 levels by regulating the NF-kB signaling pathway. Angiogenesis Hypoxia, which is a low oxygen tension, results in the activation of genes that are responsible for various functions such as angiogenesis, iron metabolism, glucose metabolism, and cell proliferation/survival. The primary factor mediating this response is HIF-1[42]. VEGF plays a crucial role in promoting angiogenesis by exerting mitogenic and anti-apoptotic effects on endothelial cells (ECs), enhancing vascular permeability, and facilitating cell migration. The possible mechanism of PCRR in ALI treatment is related to upregulated antioxidant activity, angiogenesis promotion, and endothelial barrier repair through the HIF-1α/VEGF signaling pathway. CONCLUSION Using network pharmacology and molecular docking, we predicted the targets of the PCRR ingredients and explored the underlying mechanism of their potential therapeutic effects on ALI, focusing on four aspects: Inhibition of abnormal cell apoptosis, promotion of cell proliferation, reduction of systemic inflammatory response, and promotion of angiogenesis. After conducting a thorough analysis of signaling pathways, it was determined that the PI3K-Akt, HIF-1, and NF-kB pathways were the primary pathways responsible for the therapeutic effects of PCRR in ALI. PCRR may act on relevant targets, such as IL-6, AKT1, VEGFA, PI3K, MMP9, and HIF-1α, to inhibit abnormal cell apoptosis, promote cell proliferation, reduce systemic inflammatory response, and inhibition angiogenesis. TCMs consist of a combination of intricate ingredients that target various pathways and receptors, posing a challenge when attempting to analyze the mechanisms of action of their active components. The biological processes, molecular functions, and signaling pathways enriched by these targets were used to clarify the integral regulation of PCRR in ALI treatment, especially in the induction of infectious diseases, providing an important theoretical basis for subsequent experimental studies. This study had some limitations. In this study, we employed network pharmacology and molecular docking techniques to forecast the mechanism of action of PCRR in the treatment of ALI. However, we did not conduct experimental verification to support our findings. Therefore, future studies should include cellular or animal experiments to confirm the conclusions of this study. ARTICLE HIGHLIGHTS Research background The background of this study is focused on Polygoni Cuspidati Rhizoma et Radix (PCRR), a traditional Chinese medicine (TCM) known for its anti-inflammatory effects in various human diseases. However, the anti-inflammatory effects and mechanisms of action of PCRR on acute lung injury (ALI) remain unclear. This study aimed to identify the active ingredients of PCRR for the treatment of ALI using multiple databases to obtain potential targets. In this study, a combination of network pharmacology, bioinformatics, and molecular docking techniques were used to investigate the mechanism of action of PCRR in ALI. The identification of these active ingredients and potential targets for treatment could lead to further research to evaluate the efficacy of PCRR as a therapeutic option for ALI. Research motivation The research motivation for this study is to explore the anti-inflammatory effects and underlying mechanisms of PCRR on ALI. Despite the known benefits of PCRR in the treatment of various inflammatory diseases, its effectiveness and mechanism of action in ALI are not fully understood. The objective of this study was to use network pharmacology and molecular docking techniques to identify active compounds and potential targets of PCRR for the treatment of ALI. The ultimate goal of this study was to propose a strategy to elucidate the mechanisms of TCM at the network pharmacology level and to provide insights into the development of novel therapeutic options for ALI. Research objectives The active compounds in PCRR are involved in the treatment of ALI. Multiple databases were used to identify potential targets for fishing and target genes closely associated with ALI. Network pharmacology and bioinformatics resources were utilized to predict the molecular mechanisms of PCRR in ALI. Verification of the combination of major active ingredients and core targets using molecular docking techniques. In this study, we present a proposed strategy to better understand the mechanisms of TCM through network pharmacology. Additionally, we emphasize the potential therapeutic benefits of PCRR in treating ALI. Research methods The active compounds of PCRR were identified through data collected from various databases including Traditional Chinese Medicine Systems Pharmacology, STITCH, and PubMed. Target ALI databases were built using the Therapeutic Target, DrugBank, DisGeNET, Online Mendelian Inheritance in Man, and Genetic Association databases. Network pharmacology analysis Network construction, target prediction, topological feature analysis, and enrichment analysis were conducted to determine the potential targets and molecular mechanisms of PCRR in ALI. Bioinformatics analysis: Gene Ontology biological processes and Kyoto Encyclopedia of Genes and Genomes network pathway enrichment analyses were performed using bioinformatics resources from the Database for Annotation, Visualization, and Integrated Discovery. The combination of major active ingredients and core targets was verified using molecular docking techniques. Research results Thirteen bioactive compounds corresponding to the 433 PCRR targets were identified. A total of 128 genes closely associated with ALI were identified, out of which 60 genes overlapped with potential therapeutic targets identified by PCRR. These 60 genes were considered to be relevant for developing therapeutic interventions for ALI. According to functional enrichment analysis, PCRR was found to have pharmacological effects on ALI by regulating various pathways such as the cell cycle, apoptosis, drug metabolism, inflammation, and immune modulation. Molecular docking results revealed a strong associative relationship between the active ingredient and core target. This study suggests a method for understanding how TCM works at the network pharmacology level. Additionally, it emphasizes the potential of PCRR as a treatment for ALI. Overall, these results provide important insights into the potential therapeutic effects and mechanisms of action of PCRR in ALI, which could lead to further research evaluating the efficacy of PCRR as a therapeutic option for ALI. Research conclusions This study identified 13 bioactive compounds in PCRR that are related to the treatment of ALI. Using network pharmacology and molecular docking techniques, we identified potential targets of PCRR in ALI and predicted the molecular mechanisms underlying its therapeutic effects. The findings of this study indicate that PCRR may have a beneficial impact on reducing inflammation in patients with ALI by regulating various pathways such as the cell cycle, cell apoptosis, drug metabolism, inflammation, and immune modulation. Overall, This study presents a network pharmacology-based approach to understand the mechanisms of TCM and emphasizes the therapeutic benefits of PCRR in treating ALI. Research perspectives However, further experimental validation is required to confirm the predicted molecular mechanisms and therapeutic effects of PCRR in ALI. Future studies should explore the optimal dosage and administration methods for PCRR in treating ALI, as well as the potential adverse effects. Novel active ingredients and targets related to PCRR for the treatment of ALI can be identified through database mining and experimental verification. The network pharmacology approach used in this study can be applied to investigate the mechanisms of action of other TCMs and their potential therapeutic effects in various diseases. The integration of multiple omics data and machine learning techniques could enhance the accuracy and efficiency of predicting drug-target interactions and uncover the molecular mechanisms of TCM. ACKNOWLEDGEMENT In the process of researching and writing this manuscript, I would like to express my gratitude to all those who have helped me. Data sharing statement No additional data are available. Institutional review board statement: This study did not involve human experiments. Informed consent statement: Not applicable. Conflict-of-interest statement: The authors declare no conflicts of interest. STROBE statement: The authors have read the STROBE Statement—checklist of items, and the manuscript was prepared and revised according to the STROBE Statement—checklist of items. Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: May 4, 2023 First decision: May 12, 2023 Article in press: May 25, 2023 Specialty type: Medicine, research and experimental Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Mitsakakis K, Germany; Rombach M, Germany S-Editor: Ma YJ L-Editor: A P-Editor: Zhang XD ==== Refs 1 Ng CS Wan S Arifi AA Yim AP Inflammatory response to pulmonary ischemia-reperfusion injury Surg Today 2006 36 205 214 16493527 2 de Perrot M Liu M Waddell TK Keshavjee S Ischemia-reperfusion-induced lung injury Am J Respir Crit Care Med 2003 167 490 511 12588712 3 Rubenfeld GD Caldwell E Peabody E Weaver J Martin DP Neff M Stern EJ Hudson LD Incidence and outcomes of acute lung injury N Engl J Med 2005 353 1685 1693 16236739 4 Dowdy DW Eid MP Dennison CR Mendez-Tellez PA Herridge MS Guallar E Pronovost PJ Needham 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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4477 10.12998/wjcc.v11.i19.4477 Review Diffusion tensor imaging in the courtroom: Distinction between scientific specificity and legally admissible evidence van Velkinburgh JC et al. Science publication, medical practice and litigation van Velkinburgh Jennifer Christine Science Communication, Filipodia Publishing LLC, Santa Fe, NM 87505, United States. jcv@filipodia.com Herbst Mark D Diagnostic Radiology, Independent Diagnostic Radiology Inc, St Petersburg, FL 33711, United States Casper Stewart M Personal Injury Law, Casper & DeToledo LLC, Stamford, CT 06905, United States Author contributions: van Velkinburgh JC and Casper SM conceptualized the review; van Velkinburgh JC performed the data collection, formal analysis and interpretation in science/medicine communication and publication topical matters; Herbst MD and Casper SM performed the data collection, formal analysis and interpretation in neuroimaging topical matters; Herbst MD generated the figure; Casper SM performed the data collection, formal analysis and interpretation in legal topical matters, drafted the first version of the manuscript, and generated the Table; All authors reviewed and edited each version of the manuscript for important intellectual content and provided approval of the final version. Corresponding author: Jennifer Christine van Velkinburgh, PhD, Academic Editor, President, Science Editor, Science Communication, Filipodia Publishing LLC, 1000 Cordova Pl 22, Santa Fe, NM 87505, United States. jcv@filipodia.com 6 7 2023 6 7 2023 11 19 44774497 10 4 2023 26 5 2023 13 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. Interest and uptake of science and medicine peer-reviewed literature by readers outside of a paper’s topical subject, field or even discipline is ever-expanding. While the application of knowledge from one field or discipline to others can stimulate innovative solutions to problems facing modern society, it is also fraught with danger for misuse. In the practice of law in the United States, academic papers are submitted to the courts as evidence in personal injury litigation from both the plaintiff (complainant) and defendant. Such transcendence of an academic publication over disciplinary boundaries is immediately met with the challenge of application by a group that inherently lacks in-depth knowledge on the scientific method, the practice of evidence-based medicine, or the publication process as a structured and internationally synthesized process involving peer review and guided by ethical standards and norms. A modern-day example of this is the ongoing conflict between the sensitivity of diffusion tensor imaging (DTI) and the legal standards for admissibility of evidence in litigation cases of mild traumatic brain injury (mTBI). In this review, we amalgamate the peer-reviewed research on DTI in mTBI with the court’s rationale underlying decisions to admit or exclude evidence of DTI abnormalities to support claims of brain injury. We found that the papers which are critical of the use of DTI in the courtroom reflect a primary misunderstanding about how diagnostic biomarkers differ legally from relevant and admissible evidence. The clinical use of DTI to identify white matter abnormalities in the brain at the chronic stage is a valid methodology both clinically as well as forensically, contributes data that may or may not corroborate the existence of white matter damage, and should be admitted into evidence in personal injury trials if supported by a clinician. We also delve into an aspect of science publication and peer review that can be manipulated by scientists and clinicians to publish an opinion piece and misrepresent it as an unbiased, evidence-based, systematic research article in court cases, the decisions of which establish precedence for future cases and have implications on future legislation that will impact the lives of every citizen and erode the integrity of science and medicine practitioners. Diffuse axonal injury Mild brain injury Magnetic resonance imaging Neuroimaging Medicolegal Litigation Medical jurisprudence Ethics Peer review Publishing ==== Body pmc Core Tip: Transcendence of an academic publication over disciplinary boundaries faces the challenge of application by a group that inherently lacks in-depth knowledge on the scientific method, the practice of evidence-based medicine, or the peer-reviewed publication process. A modern-day example of this is the ongoing conflict between diffusion tensor imaging (DTI) publications and legal standards for admissibility of evidence in personal injury litigation cases in the United States. We have amalgamated the peer-reviewed research on DTI in mild traumatic brain injury with the court’s rationale underlying decisions to admit or exclude evidence of DTI abnormalities to support claims of brain injury. INTRODUCTION In 2013 and 2014, the number of emergency department visits, hospitalizations, and deaths related to traumatic brain injury (TBI) approached 2.9 million per year[1,2]. Unsurprisingly, a significant portion of those injuries lead to lawsuits. In addition, such accidents also result in an incalculable number of TBIs, which are characterized by non-specific early symptoms (e.g., amnesia, confusion, dizziness, headache, nausea), no loss of consciousness, and initial imaging studies interpreted as normal. While such patients can later suffer a spectrum of lifelong disabilities, application of the poorly defined label of “mild TBI (mTBI)” complicates both diagnosis and prognosis. The determination of whether there was a concussive blow revolves around the mechanism of injury and the body’s response to the injury. In turn, those issues can be illuminated by input from witnesses. Unfortunately, there are many unwitnessed injuries. Aside from any issues related to the mechanism of injury, in litigated matters involving mTBI, there are two primary areas of controversy: (1) Was there a concussion/mTBI? and (2) Is there a natural course and recovery for the injury? The latter topic implicates the issue of structural brain damage during the chronic stage. The aggregation and evaluation of all data points that go into the complex mosaic of information that is the clinical diagnosis of an mTBI[3] is beyond the scope of this paper. However, those determinations can be made by treating healthcare providers along the course of injury and recovery. Alternatively, a diagnosis and course of recovery might be assessed retrospectively by clinical consultants, researchers, and/or expert witnesses in the context of litigation. The contested field in these cases that has generated the greatest controversy over the last decade is advanced neuroimaging, and diffusion tensor imaging (DTI) in particular. DTI is widely recognized as the most sensitive method to identify white matter disruption in the brain[4] (Figure 1), and the structural neuropathology identified by DTI is positively associated with cognitive dysfunction in attention, memory, processing speed, and executive function[5]. In concussion, DTI plays a complementary role in understanding the physical, cognitive, and emotional disruption in the post-injury chronic stage[6]. Yet, conflict has emerged in the litigation setting by those fearful that the weight of sensitivity will be confused for scientific specificity. Figure 1 Imaging evidence of a traumatic brain injury in a representative patient. A: Bright spot on T2-weighted images near the gray-white junction, consistent with traumatic axonal injury (orange arrow); B: Close-up of lesion shown in Panel A (orange arrow); C: Microhemorrhage near the gray-white junction shown on a microhemorrhage-sensitive magnetic resonance image (orange arrow); D: Gaps in the normally continuous distribution of tracts that pass through the corpus callosum; E: Diffusion tensor imaging (DTI) tractography of the relatively normal right fronto-occipital fasciculus; F: DTI tractography of the left fronto-occipital fasciculus showing decreased tracts compared to the right side shown in Panel E. Recently, Shenton et al[7] argued that because the data generated by DTI might be misconstrued or misinterpreted, it should not be admitted into evidence. However, this perspective obfuscates the difference between scientific standards and legal standards, possibly based on a simple misunderstanding of the manner in which evidence is actually offered and admitted into evidence in civil litigation. Regardless, it is important to initially recognize that one cannot equate degrees of proof required by law with the statistical probabilities about the number of patients who have sustained a TBI or the number of patients who suffer from persistent post-concussion symptoms (PPCS). As has been demonstrated from contemporary longitudinal outcome studies, the probability of PPCS in a head injury patient hover around 50%[8]. Undoubtedly, clinical assessments are influenced by the expertise and/or biases of clinicians. The opinions of treating providers often acquire the imprimatur of reliability. That stamp lasts only so long as it remains unchallenged or the appearance of objective evidence influences those opinions. All clinical data collected following a head injury should be considered in an evolving diagnostic process, where the diagnostic impression can change. While head injury is classified at the time of injury by a system that largely depends upon loss of consciousness and/or the length of post-traumatic amnesia[9], the outcome of the condition does not necessarily correspond with the initial classification[10]. The fact that some symptoms and findings are non-specific[11] does not alter their utility, particularly if they can be contextualized (i.e., used not as a pathognomonic sign but within the context of a larger clinical profile). Medicine does not disregard non-specific symptoms but instead considers and attempts to reconcile them. Because many TBIs are the result of actionable wrongful conduct and because TBI is heterogeneous, the types of evidence gathered are very important for both care of the injured party and the objective benefits of litigation. Individual outcomes following TBI can range from recovery of clinical symptoms to long-term disability, lasting months to years, related to underpinning physical, cognitive, emotional/behavioral and sleep disturbances[11]. DTI abnormalities correlate positively with post-concussion symptoms and severity[12], including self-reported quality of sleep and depression[13]. DTI abnormalities may also account for mTBI-related headache[14,15]. In cases where there are persistent symptoms, a challenge, like Shenton’s, to an arguably objective basis for assessing injury poses a serious threat to the welfare of an injured party, and can deprive that person of meaningful and legally authorized compensation. QUANTITATIVE DTI Neuroscience has recognized that structural white matter damage, known as traumatic axonal injury (formerly or alternatively called shear injury or diffuse axonal injury), often follows TBI at all levels of severity including mTBI. This has been documented in human histological[16] and postmortem[17] and in animal[18] histological studies. Reliance on histology is necessary because of, among other reasons, the low rate of confirmation using standard clinical neuroimaging such as computed tomography (CT) scan and standard magnetic resonance imaging (MRI)[19,20]. DTI is the most sensitive technology available for drawing compelling inferences about the structural integrity of damaged axons in vivo, based upon quantitative assessment of water molecule diffusivity within white matter fibers[21]. When fully intact inside the axonal membrane and the surrounding myelin sheath, water molecules are restricted to intra-axonal movement. When those structures are disrupted through injury, the water molecules become free of spatial restriction and diffuse randomly. Thus, DTI reveals disturbances from the normal patterns of water dispersion consequent to shearing or other damage[22]. Importantly, DTI reveals microstructural pathology that may or may not be visible on routine MRI scans[23,24]. Relying on the metric of fractional anisotropy (FA)[19], neuroscientists have been able to measure whole-brain and regions of interest (ROIs), voxel-wise, and tract-based spatial statistics white matter architecture in mTBI[23,25]. It has been suggested that comparing individual mTBI patients to group norms may lead to “overlook[ing] individual profiles of injury, which are inherently more subtle and heterogeneous across individuals”[26]. However, pre-morbid DTI imaging is rare. To address these issues, a “subject-specific approach” has been proposed[19]. However, given the general absence of pre-morbid DTI data, other clinical diagnostic factors serve as subject-specific markers. As recently noted in a paper published in Neurology[27] on serum biomarker findings related to the neurofilament light chain polypeptide, a marker of axonal damage, DTI is not part of the common imaging protocols following TBI because it has “limited availability, high cost, and cumbersome image processing.” It is not at all surprising that the clinical use of this potential diagnostic technique revolves, at least in part, around cost and reimbursement. Validating microstructural brain damage is not likely to change patient care in most circumstances, so there is little incentive for major medical insurers to pay the expense. While the defense in TBI litigation has popularized a mantra that quantitative DTI “is not in clinical use,” that statement is not only flatly incorrect but overlooks the fact that there is also no legal requirement for clinical use as a prerequisite for the admission of an expert opinion under either the Daubert or Frye standards that will be addressed below. ADMISSIBILITY OF EVIDENCE In the law, several types and forms of evidence can be admitted into the record over objection when the evidence is considered “relevant.” Conceptualizing the conflict that arises in the face of competing expert opinions on scientific matters by merely reading the case law without practical experience can be challenging for neuroscientists, even those with a law degree. More than two centuries of common law have taught us that to highlight a potential weakness in otherwise relevant evidence, it should be framed as a challenge to the weight to be given to that evidence by the jury, and not to the admissibility of the evidence[28]. The evidence should be relevant to such questions as: Did the plaintiff suffer a brain injury? Is there any structural evidence of that brain injury? Rule 401 of the Federal Rules of Evidence provides that evidence is relevant if: it has any tendency to make a fact more or less probable than it would be without the evidence; and the fact is of consequence in determining the action. The same language is mirrored in the rules of evidence in many states. State courts are not bound by the Federal Rules of Evidence (FRE) but many states have adopted the FRE. For example, the North Carolina Code of Evidence mirrors the Federal Rules of Evidence and its Rule 401 provides: “Relevant evidence” means evidence having any tendency to make the existence of any fact that is of consequence to the determination of the action more probable or less probable than it would be without the evidence”[28]. Abnormalities in brain white matter that are consistent with the injury would be considered relevant evidence. A jury’s belief that there is white matter damage consistent with the injury, even if inferred, makes the fact-brain injury-more probable than it would be without the evidence. Whether the same evidence (white matter damage) can be attributed to other causes depends on the specific facts and circumstances of the case. Thus, there are a constellation of factors that may be considered, including but not limited to: The patient’s age; the patient’s prior medical history, including previous concussive events; co-morbidities including co-occurring injuries; pre-existing abuse of drugs, alcohol, and other substances; and the imaging and post-processing techniques employed. In the courtroom, these factors relate to the weight of the evidence. THE DIAGNOSTIC KEY Some health care providers claim, without evidence, that the consequences of an mTBI/concussion resolve within 3 mo of injury and that an injury, if there was one, has no lasting consequences[29,30]. Some neuropsychologists still testify about and perpetuate the 3-mo recovery myth[31]. By contrast, other healthcare providers and neuroscientists believe that some constellation of post-concussion symptoms can and do last beyond the unproven 3-mo period, extending to 6 mo and 12 mo[8], and beyond. Often, a patient undergoes advanced neuroimaging, including DTI, well into the chronic stage of recovery at 2 or more years post-injury, and the observed white matter lesions may be viewed as consistent with the original injury. Litigated cases can be reduced to a battle among expert witnesses who rely upon some objective findings, self-reports, and collateral opinions about whether there is or was a TBI. Given the above paradigm, there is little wonder that reliance on any data generated through diagnostic testing, including MRI and DTI, can take on great importance in aggregating the evidence of injury. However, to support a claim of neural structure damage, even if standing alone, the test and the results are not considered diagnostic like a pathognomonic biomarker. Rather, DTI evidence of white matter damage (reduced FA) in certain patterns and/or in certain structures tends to make the existence of brain injury more or less likely. Thus, while DTI evidence is not a stand-alone diagnostic test (i.e., a biomarker) for brain damage, the DTI results do provide data that can be further analyzed (and be the subject of cross-examination), to determine if the damage is more likely than not consistent with trauma. Indeed, the spatial pattern of white matter abnormalities may vary among patients with similar presenting symptoms (e.g., nausea, vomiting, headache, disrupted sleep, or neurocognitive disorder) based upon the nature of the mechanical perturbation that caused the injury, the manner in which the patient’s brain responded to the perturbation, and genetic vulnerabilities. Also, there are recognized patterns of low FA that are consistent with traumatic axonal injury[26,32]. By addressing and analyzing the actual or potential non-specific signs and/or symptoms, including patterns consistent with traumatic injury, clinicians engage in substantially the same differential diagnostic process that is routinely used in medicine. There has been ongoing legal conflict between proponents of the use of quantitative DTI to aid not only in the diagnosis of mTBI but also as admissible evidence for the fact-finder (usually a jury), and the opponents who claim that although DTI is the most sensitive technique currently available to assess the structural integrity of white matter in the brain, DTI should not be admitted into evidence in TBI trials. It is the latter position that was most recently advocated by Shenton et al[7] in a paper published in the International Journal of Law and Psychiatry, titled “Mild traumatic brain injury: Is DTI ready for the courtroom?” As Shenton’s paper is a more contemporary reiteration of an opinion previously stated by others including Wortzel et al[26] but without new science to support their position, it has not been well embraced by the vast majority of courts (> 90%) that have considered efforts to exclude DTI evidence. Unfortunately, such papers always raise new controversy, with defense experts citing Shenton’s paper in their efforts to exclude DTI evidence for reasons that benefit the defense (i.e. lowering or eliminating damages of the responsible party)[7]. At minimum, Dr. Shenton should have disclosed, but did not, the parameters of her “conflict of interest” as she has, in the past, served as an expert witness for the defense in opposition to admission of DTI evidence. The earliest contribution to this debate cited in Shenton’s paper was from Wortzel et al[26], who advanced the argument (without the benefit of a legal expert as co-author) that DTI should not be used in TBI litigation because, in their opinion, it might fail the standards of admissibility in federal court (i.e., the Daubert admissibility test), the putative findings are non-specific, and group norms should not be used to diagnose an individual patient. Unaware of efforts to reduce the costs of DTI analysis[33], at the North American Brain Injury Society’s conference in New Orleans on September 20, 2013, Dr. Wortzel, a forensic neuropsychiatrist, who failed to disclose his conflicts of interest in his mTBI litigation paper, admitted that one reason the defense in DTI cases does not obtain their own competing DTI assessments is because it is “expensive”[34]. Of course, obtaining a second study commissioned by the defense using a different imaging venue with laboratory-specific protocols and controls would afford an opportunity to determine whether the contest is legitimate or whether the effort to preclude the plaintiff’s evidence is simply a ruse. At least, a second study might either confirm or purport to refute the claimed damage to white matter. In any case, Wortzel’s paper, like Shenton’s paper, failed to distinguish between evidence that contributes to the diagnostic formulation and evidence that is a diagnostic biomarker, failing to recognize that no litigant has claimed that DTI results standing alone are diagnostic for TBI. PUBLICATION ALONE DOES NOT CONVEY RELIABILITY Great care should always be taken to determine the reliability of opinions expressed in any publication. Shenton’s paper, for example, does not reflect new or novel bench science, as would a systematic review that adheres to Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) standards[35] and reports the basis of inclusion/exclusion criteria, nor does it purport to reflect the analyses from any of the 70-plus court decisions denying efforts to exclude DTI evidence. Beyond the documented DTI decisions, it is difficult to speculate on the number of similar decisions that have been made but for which transcript evidence was not obtained and/or reported. Because the supporting documents for DTI decision have been filed in multiple cases around the country as part of a public record, those records are available to anyone desiring to inspect them. From a scientific perspective, the paper is not a systematic review or meta-analysis, an inception cohort study, a cross-sectional study with consistently applied reference standards and blinding, a randomized trial, or even a case series. From the standpoint of evidence-based medicine, it ranks at the bottom of the weighted system (Level 5) established by the Oxford Centre for Evidence-Based Medicine (OCEBM) (2011) because it is a position paper involving “mechanism-based reasoning”[36]. Like the subject article, other papers cited by Shenton et al[26,30,33,37-40] are also the lowest level under ratings of evidence-based medicine. Some authors, such as Drs. Shenton and Wortzel, work as defense forensic witnesses on this or similar issues, embodying potential bias for the non-evidence-based opinions they are publishing[26]. Other citations have included articles by neuropsychologists with similar conflicts of interest (based upon personal experience[37]), but that topic is beyond the scope of this paper. Those papers have not explored or considered the potential bias that favors limiting the use of DTI for research with attendant funding of grants and/or remuneration with expert witness fees. Moreover, while the list of co-authors for Shenton et al[7] includes Judith G. Edersheim, MD, JD, who is both a psychiatrist and a law school graduate, analyses of the applicable rules of evidence do not reflect how the issues have actually been addressed by at least 70 trial courts that have rendered decisions favorable for admitting DTI evidence in mTBI cases. The court rules that govern admissibility of the portion of an expert’s opinion involving new, recent, novel, or innovative science fall primarily under either the Frye[41] or Daubert[42] standard, depending upon the jurisdiction in which the case is pending and whether the matter is in state or federal court. Frye largely governed the admissibility of expert opinions dealing with new or allegedly novel science in the United States prior to adoption of the Daubert rules for application to the Federal Rules of Evidence[31] and which has been largely incorporated, to some extent, in roughly 40 states. For purposes of this paper, the discussion will focus upon the Daubert standard. The decision in Daubert was originally heralded as a sea-change in the rules of evidence but it was really an unmuted and flexible way for a trial judge, acting as a gatekeeper, to address the factors that render a scientific methodology valid. In cases that started with Daubert, the courts have made clear that the gatekeeper’s assessment is not to substitute the judge’s views for that of the expert, choose the more persuasive expert, or penalize the proponent because the science is new or still evolving. In fact, the Daubert court emphasized that issues challenging the application of the science relate to the weight of the evidence that can be explored on cross-examination. THE CASE FOR EXCLUDING DTI EVIDENCE IN THE COURTROOM Shenton et al[7] argued that factors that should justify excluding admission of DTI evidence under the Daubert rule in mTBI cases include standardization, individual comparison to group norms, specificity of findings, and qualifications of the expert. The authors asserted that the concerning issues about DTI have generated various professional responses, including a 2012 conference held at Emory University (Atlanta, GA, United States) and purportedly summarized in the “Meltzer paper”[40]. Unfortunately, this paper was riddled with inaccurate characterizations of the Emory meeting, if one credits the sworn deposition testimony of one of its co-authors, Gordon Sze, MD[43]; one of the attendees, neurologist Randall Benson, MD[44]; as well as a published paper written by an attorney in attendance, William G Jungbauer, Esq[45]. Contrary to the implication of the paper’s title, no consensus was reached at the meeting, no actual votes were taken, and the only opinions considered for the paper were those of the neuroradiologists in attendance. Moreover, no minutes were taken, and opposing views expressed during the conference were not described. Unfortunately, invitation and attendance at the meeting were based upon a somewhat randomized and preferential approach that excluded recognized luminaries, such as Michael Lipton, MD, PhD (whose attendance was precluded because the conference conflicting with the Jewish Sabbath), Erin D Bigler, PhD[43,44] who then resided in Utah, and other neuroradiologists who incorporate DTI into their clinical practices. Of note, and also disregarded, is the fact that DTI has also been used clinically by the United States military[46]; neither the Department of Defense nor the Department of Veterans Affairs were represented at the Emory conference. Meltzer, the lead author, was deposed in 2017 concerning, among other issues, the meeting at Emory, and she verified that DTI is useful for identifying white matter irregularities in the brain; although, she stated that it is not diagnostic but “shows any kind of disruption of the alignment of white matter tracts…[that may be] reported to be abnormal in many diseased states.” Meltzer testified that DTI is not a “disease marker” for any condition[47] and that the “Guidelines” article is not a “scientific paper”[47]. To resolve any possible ambiguity, the proponents of using DTI in mTBI cases do not contend that DTI standing alone is “diagnostic”. Meltzer later clarified her view that “DTI is not appropriate on an individual basis for diagnosing mTBI”[47]. She also admitted that none of the co-authors of the paper, including herself, had any experience using DTI in the TBI population and she was unable to provide a justification for excluding Randall Benson, MD from the authorship group although he was in attendance, perhaps because he was a behavioral neurologist who routinely employs quantitative DTI in his clinical practice and not a neuroradiologist[47]. With regard to the Consensus Conference outcome, Meltzer conceded that the attendees refused to even consider voting on whether DTI should be admitted into evidence in individual cases of head trauma, almost certainly because there was no consensus. Of the six co-authors of the paper, four were affiliated with Emory University’s Center for Ethics and were not neuroradiologists, neuropsychiatrists, neurologists (like Benson), or neuropsychologists[47], and two were neuroradiologists-Meltzer and Gordon Sze, MD of Yale University (New Haven, CT, United States). Dr. Sze testified at his deposition that he did not write any part of the Meltzer paper; this is itself a problematic issue, in violation of the International Committee of Medical Journal Editors standards and norms of science publication[48]. That leaves Meltzer as the sole composing author, despite having no experience working with DTI, although she may have supervised some researchers who did. The combination of the process that gave rise to the Meltzer paper and the deposition testimony of Drs. Meltzer and Sze raises concern about important issues related to the publication, not the least of which is whether the paper should have been designated a consensus report in the first place. THE REAL ACCOUNT OF DTI IN THE COURTROOM Shenton et al[7] minimize the history of the use of DTI in the courtroom because they report only five cases involving DTI and mTBI. The early scientific history of using DTI in mTBI was documented in 2013 by Hulkower et al[49] in “A decade of DTI in traumatic brain injury: 10 years and 100 articles later.” The courtroom history of the use of advanced neuroimaging is less formal but verifiable, in that there are several “unofficial” reports of decisions on the admissibility of DTI (or other advanced neuroimaging) evidence. Members of the Traumatic Brain Injury Litigation Group (TBILG) of the American Association for Justice have created and maintained a databank for court decisions on the subject, which have been documented in a written order, written decision, or transcript of the proceedings. That documentation remains accessible to members of the TBILG and the contents have been filed in a number of cases throughout the United States. Beyond the documented DTI decisions, it is difficult to speculate on the number of similar decisions that have been made but for which transcript evidence was not obtained and reported. A recent decision denying a motion to preclude the use of DTI in a mTBI case was in Amidon v. The Goodyear Tire & Rubber Company, No: 1:18-CV – 02138 (September 3, 2021)[50]. Those files are publicly available. To date, there are approximately 70 documented trial court decisions either admitting DTI evidence or denying a motion to exclude DTI evidence in mTBI cases; by contrast, there have been only four decisions excluding DTI evidence on the basis of methodology (Table 1). Table 1 Diffusion tensor imaging orders by state courts State Cases Date Court Ruling Documents Frye/Daubert CA Gutcher vs Toyota Motor Sales, UNITED STATES, Inc. 8/26/2013 CA Superior Court - County of Kern Supportive Reporter's transcript of testimony and proceedings denying MIL to preclude Dr Wu Daubert CO Whilden vs Kimberly Cline, Elmer Dudden & Colorado Cab Company, LLC 5/10/2010 USDC - Jefferson County Supportive Order - Denying MIL to preclude DTI evidence – Dr Orrison Daubert Woods vs Ruth 2/14/2014 USDC - County of Arapahoe Supportive Order - Motion to Strike Opinions of Plaintiff's Expert Dr Benson - denied (Brimmer, DJ) Daubert White vs Deere & Company, John Deere Limited & John Does 1-5 2/08/2016 USDC - CO Supportive Order - Denied Motion to Exclude Dr Benson's opinions Daubert Siewald vs Flores 5/17/2019 DC - Larimer County Supportive Denial Notice to Strike/Preclude Snyder, Omnibus Order - Motions to strike expert testimony Daubert CT Vizzo vs Fairfield Bedford, LLC 11/2/2016 Superior Court - Judicial District of Stamford/Norwalk - CT Supportive Transcript - Motion to Preclude Daubert/Porter denied Daubert FL Hammar vs Sentinel Insurance Company, LTD 9/27/2010 FL Circuit Court - 13th Judicial Circuit - Hillsborough County Civil Division Supportive Order - Denying Defendant's Frye challenge Frye Sworin vs Harris 4/04/2014 FL Circuit Court - 20th Judicial District - Collier County Supportive Order - Defendant's Motion to Exclude Opinions and Expert's Report of Plaintiff's expert Dr Benson Daubert Marsh vs Celebrity Cruises, Inc. 12/15/2017 USDC- Southern District of FL Supportive Order - Daubert Motion denied (Ungar, DJ) Daubert Sutter vs Youngman 2/25/2019 Florida Circuit Court - 7th Judicial Circuit - Volusia County Supportive Order - Defendant's Frye Motion, Hearing Transcript, Plaintiff's Opposition to Defendant's Frye Motion, Deposition of Dr Wintermark Frye Ward vs Carnival Corp. 3/14/2019 USDC – SD - FL Supportive Written opinion denying defendant’s motion to strike DTI expert (Andrew Walker, M.D.) Daubert Caslow vs Orange Christian School 3/26/2018 Circuit Court, 8th Judicial District Unsupportive DTI precluded Daubert IL Weyer vs SAIA Motor Freight Line, LLC 6/02/2019 USDC- NDIL Supportive Motion in limine to preclude Benson DTI evidence denied Daubert Snyder vs Vital 11/2021 Third Judicial Circuit, Madison County Supportive Order - DTI & 3D Tractography (Dr Ammar Chaudhry) Frye IN Ruppel vs Kucanin USDC – Norther District - IN Supportive Order - Motion to Exclude Dr Benson's opinions denied (Moody, DJ) Daubert IA Lamphier vs Schlee Masonry 4/16/2012 IA - Worker's Compensation Supportive Order for 3T DTI Dr Benson (approved) DTI approved; test not specified Henrickson vs Department of Human Services, State of Iowa 8/14/2012 IA - Worker's Compensation Supportive Order for 3T DTI Dr Benson (approved) DTI approved; test not specified KY Roach vs Hughes 3/09/2016 USDC – W.D.KY; 2016 U.S. Dist. LEXIS 192835 Supportive Order denying to preclude “DTI” Analysis and causation opinion Daubert LA Leboeuf vs B&K Contractors, Inc 5/27/2009 Court of Appeal of LA, 4th Circuit - 10 So. 3D 897 Supportive Judgment of trial court affirmed DTI not contested Andrew, et al vs Patterson Motor Freight, Inc., et al 10/23/2014 USDC – Western District of LA - Lafayette Division - 2014 WL 5449732 Supportive Motion to preclude DTI evidence denied Daubert Barnett vs National Continental Insurance Co. 1/08/2019 USDC – Middle District of LA, 2019 WL 126732 Supportive Motion to preclude DTI evidence denied Daubert Hall vs Landstar Ranger Inc, et al 12/26/2019 USDC – Western District of LA - Lafayette Division - No. 6i18-CV-0410 Supportive Magistrate Judge, Minutes of Court & Order 12/26/19 Daubert Meadors vs D'Agostino 10/29/2020 USDC – Middle District LA Supportive Daubert challenge to non-MD testimony and all DTI issues denied Daubert Hernandez vs Scales 12/15/2021 Court of Appeal of La, 1st Circuit cert denied by SCT Supportive Reversing decision of trial court to preclude DTI & QEEG LSA-C.E. Art. 702 MA Zawaski vs Gigs, LLC & Wendell Lee Zorman 11/04/2010 Superior Court - Commonwealth of Ma Supportive Notice to Preclude denied Daubert Wu vs Lauriat 9/05/2012 Superior Court - Middlesex County Supportive Plaintiff's Opposition to Defendant's Motion, partial transcript Daubert Chiulli vs Newbury Fine Dining d/b/a Sonsie's Restaurant 10/17/2012 USDC - Massachusetts Supportive Denied MIL to preclude DTI (Benson) (Tauro, DJ) Daubert Irwin vs Eclectic Dining Inc., d/b/a Atlantica's Olde Salt House 6/23/2015 USDC - MA - Daubert Supportive Defense Motion to preclude Dr Benson, Plaintiff's Opposition to Motion to Preclude, Defense Reply Brief Daubert Malagic vs Suffolk Construction 8/05/2022 Superior Court - Middlesex County Supportive Oder (Benson) - not diagnostic but consistent Daubert Craffey vs Embree Construction Group, Inc. 10/26/2015 Superior Court - Commonwealth of MA Supportive Order - MIL to preclude DTI denied (Benson) Daubert MI Rye vs Kia Motors America, Inc. 2/16/2010 Circuit Court - Wayne County Supportive Order - Defendant's MIL to preclude testimony of Dr Benson Daubert Ikola vs Matthew Wright, R&L Trainer, Inc & R&L Carriers Shared Services, LLC 6/23/2015 Circuit Court - County of Berrien Supportive Order - Denying Defendant's MIL to Exclude evidence regarding Dr Benson's quantitative DTI Daubert MN Wills vs Benjamin Vincent Sullivan & Pan-O-Gold Baking CO. 9/13/2010 3rd Judicial District - County of Wabasha Supportive Motion to Exclude or limit testimony of [Joseph] Wu denied Frye Hansen vs Crain 4/04/2011 2nd Judicial District - County of Ramsey Supportive Transcript, Motion to preclude Dr Wu on PET and DTI denied Frye Nelson vs BNSF Railway Company 10/01/2013 4th Judicial District - County of Hennepin Supportive MIL barring Dr Wu denied Frye Nordstrom vs Fleet & Farm of Menomie, Inc. 1/7/2014 10th Judicial District - County of Washington Supportive Motion to preclude DTI denied Frye Hyman vs David T. Chang, M.D. 7/18/2014 2nd Judicial District - County of Ramsey Supportive Order - Defendant's MIL (context) Frye Peddycoart vs Lombardo 2/13/2018 1st Judicial District - County of Dakota Supportive Order denying Motion to Exclude Dr Benson Frye MT Kreiman vs C & CB LLC 10/17/2021 MT Seventh Judicial District Court Supportive Cross MIL re DTI; Dr Wortzel barred from testifying DTI not generally accepted Daubert NJ Ferrante vs City of Atlantic City 5/29/2014 Superior Court of NJ - Mercer County - Law Division Supportive MIL to bar Dr Benson's testimony on DTI denied Daubert NM Booth vs Kit, Inc. 3/23/2009 USDC- NM Supportive Order - Denying Joint Motion to Strike/Limit – Orrison (Parker DJ) Daubert Ebel vs Joe F. Apache, Turner Enterprises, Inc. 12/11/2013 State of NN County of Santa Fe, 1st Judicial District Supportive Order - Denying Defendant's Motion to Exclude DTI Daubert NY LaMasa vs Bachman 11/20/2008 Supreme Court, Appellate Division First Department, NY Supportive Judgment affirmed, DTI evidence - 56 A.D. 3d 340, 869NYS (2008) Frye Girgs vs Snapple Distribution Corp. et al 5/22/2014 Civil Court of The City of New York - County of Queens: Part 30 Supportive Decision and Order - MIL to preclude Lipton, DTI and discovery dispute denied Frye Sullivan vs Daniel I. Walters 7/09/2014 Supreme Court of The State of NY; County of Nassau Supportive MIL to preclude DTI (Dr Lipton) denied, Notice of Motion, Affirmation in Opposition, Affirmation in Reply Frye Velez vs Merejo 11/06/2014 Supreme Court of The State of NY - County of NY Supportive Request for Frye DTI hearing denied Frye Klipper vs Liberty Helicopters, Inc. 1/12/2015 Supreme Court of The State of NY - County of NY Supportive Cross motions under Frye, DTI evidence permitted Frye Melendez vs Patrick McCrowell, et al 11/30/2015 Supreme Court of The State of NY - County of Rockland Supportive Hearing and denial of Defendant's Motion to preclude Dr Lipton Frye Jean-Francois vs The Port; Authority of New York and New Jersey 3/16/2016 New York Supportive Supreme Queens; Normative data not in Dr Lipton's control; No showing that normative data needed Frye Betty Casas as Guardian of Luis Casas vs Consolidated Edison 3/24/2017 Supreme Court of The State of NY - County of New York Supportive Decision and Order - MIL DTI denied (Dr Lipton) Frye Brouard vs Convey 2/09/2018 Supreme Court of The State of New York - Suffolk County Unsupportive DTI precluded Frye Aravindakshan vs Martello 3/15/2018 Supreme Court of The State of NY - County of Nassau Supportive DTI admitted (Dr Lipton) and Frye Frye Caramanica vs Rhim 3/23/2018 NY State Supreme Court -NY County Supportive Defendant's experts testimony limited Frye Barney-Yeboh vs Metro-North Commuter Railroad 4/08/2018 NY State Supreme Court - NY County Supportive Cross motions re: DTI preclusion denied Frye Konstantinov vs MTLR Corp., Mehadrin Dairy Corp., & Joel Daskal 1/02/2019 Civil Court of The City of NY - County of Kings: Part 30 Supportive Cross motions re: DTI preclusion denied Frye Davis vs U-Haul of Arizona 7/30/2019 NY State Supreme Court - Bronx Supportive Motion to preclude DTI (Dr Lipton) denied Frye Reichmann vs Whirlpool Corporation 5/20/2020 USDC – Eastern District of New York - 2:16-CV-05151 - AYS Supportive Magistrate judge, Order - Denying Motion for Disclosure Daubert Blake vs New York Central Mutual Fire Ins. Co. 12/3/2021 NY State Supreme Court – Westchester County Supportive Cross Motions (Dr Lipton) - Plantiff's motion for general acceptance granted; Defense motion denied Frye Lee vs Troge 2/22/2022 NY State Supreme Court - Duchess Supportive Defense Motion to Preclude Dr Lipton Frye hearing; Denied; Wintermark white paper found not to be peer reviewed Frye Tardif vs City of New York 6/17/2022 Southern District NY 13-CV-4056 (KMW) Supportive MIL to Preclude DTI denied Daubert Fall vs Greyhound Lines, Inc. 3/9/2023 NY State Supreme Court -NY County Supportive Summary judgment on no-fault threshold denied; Lee vs Troge cited over the Brouard decision Frye PA Barbara Rotunda vs Susan Petruska, DMD 4/2013 Court of Common Pleas - Allegheny County Supportive DTI, Frye hearing (Dr Wu) Frye Amidon vs Goodyear Tire & Rubber Company 9/2021 USDC – Middle District of PA Civil No. 1:18-CV-02138 Supportive Daubert - Motion to Preclude DTI testimony and to preclude Dr Benson denied Daubert Malone vs Taylor 5/7/2019 Tennessee Circuit Court; 2019 WL 6456250 Unsupportive MIL to preclude DTI; DTI excluded Daubert Williams vs Crawford 3/02/2018 TX Court of Appeals, 2018 WL 1124306 Supportive Opinion affirming the trial court judgment subject to a remittitur unrelated to DTI Daubert Gregg vs Covert 11/2021 USDC – ED TX; 2021 WL 5140799 Supportive MIL denied except for causation not addressed by expert Daubert UT Andrus vs Fulgham & Standard Plumbing Supply, Inc. 7/17/2006 District Court of the 3rd Judicial District - Salt Lake County Supportive MIL DTI denied Frye WA Shannon vs Columbia River Basin Railroad 01/23/2013 USDC – Western District of Washington at Tacoma Supportive Order – MIL – reserving on preclusion of DTI witness Daubert Peach vs RLI Insurance Company 5/2019 Superior Court of WA for King County Supportive Order - Denying Defendant's Motion to Exclude testimony of Dr Raji Frye WI Lewis vs Wisconsin Bell, Inc., d/b/a AT&T Wisconsin 12/2014 WI Circuit Court – Milwaukee County Supportive Order - Hearing MIL to ban Dr Benson (DTI) denied Daubert Hoerth vs Allmerica Financial Benefit Insurance Co 3/2023 WI Circuit Court - Winnebago County Supportive Order - Hearing MIL to ban Dr Benson (DTI) denied Daubert WY Wagoner vs Schlumberger Technology Corp 6/20/2008 WY; USDC - WY Supportive Order limiting defense neuroradiology witness testimony because he lacked experience with DTI Daubert Dates are presented as month/day/year. CA: California; CO: Colorado; CT: Connecticut; DTI: Diffusion tensor imaging; FL: Florida; IL: Illinois; IN: Indiana; IA: Iowa; KY: Kentucky; LA: Louisiana; MA: Massachusetts; MD: Medical doctor; MI: Michigan; MIL: Motion in limine; MN: Minnesota; MT: Montana; N/A: Not applicable; NJ: New Jersey; NM: New Mexico; NY: New York; PA: Pennsylvania; PET: Positron emission tomography; QEEG: Quantitative electroencephalogram; TN: Tennessee; TX: Texas; USDC: United States District Court; UT: Utah; VA: Virginia; WA: Washington; WI: Wisconsin; WY: Wyoming. As will be noted hereafter, the most probable points of confusion relate to the role of DTI evidence and misplaced reliance on published papers that do not accurately describe the science. For the former, it must be emphasized that quantitative DTI, standing alone, is not sufficient to diagnose traumatic brain damage. Rather, it is but one item to be considered by a clinician in the diagnostic process. For the latter, the published papers that criticize the use of DTI[7,51,39] are themselves not scientific, as will be the subject of analysis in this paper hereafter. Moreover, those papers conflict with the great weight of scientific authority that validates DTI’s sensitivity to white matter damage, a crucial and relevant factor in these cases. OTHER STANDARDS One of Shenton et al’s critiques of DTI in the courtroom is that the process lacks adequate standardization[7]. Since the global population of researchers and clinicians who use DTI employ a systematic approach to their assessments, Shenton is referring to a formalized consensus standard. This opinion, however, confuses recommendations for uniform standards by implying that systems that have been and are currently in use are faulty or unreliable. While the existence of multiple platforms and protocols confound the direct comparison of results obtained by different DTI scanners and software, as does the use of different subject and control groups and pre- and post-processing techniques in research studies, these can be controlled for (or minimized) in a properly designed comparative analysis. Indeed, such a study is ongoing, involving multiple centers and TBI subjects[52] assessed with different scanners at each site. There are at least 16 different manufacturers of 3T MRI machines, but GE Healthcare (Chicago, IL, United States), Siemens Healthineers (Erlangen, Germany), and Philips Healthcare (Amsterdam, The Netherlands) account for a lion’s share of the market. In all, the hardware settings include those required for each sequence of software. Post-processing software allows for the quantification of data and generation of images. While in a perfect world, universal or consensus standards would be based upon a large population studied in a strictly controlled analysis (i.e., identical scanners, software sequences, computational settings, and post-processing software techniques), such a goal seems unrealistic. It is more realistic to expect that there might be publication of standardized MRI sequences (e.g., “Common Data Elements in Radiologic Imaging of Traumatic Brain Injury”)[53], research with protocols standardized among multiple institutions [e.g., TRACK-TBI studies[52] and Enhancing Neuroimaging Genetics through Meta Analysis (ENIGMA)[54]] or accessible databases [e.g., CARE Consortium maintained at the National Institute of Health (NIH) Federal Interagency Traumatic Brain Injury Research Information System[55] the Laboratory of Neuro Imaging[56], or the data community of the Mind Research Network, Collaborative Informatics and Neuroimaging Suite[57], the last of which charges a royalty to use its control group]. Importantly, even before the Emory meeting in late 2012, DTI guidelines for clinical application had been published (March 8, 2012) by the American Society of Functional Neuroradiology, titled “ASFNR Guidelines for Clinical Application of Diffusion Tensor Imaging”[58]. The recognition of such among TBI researchers and clinicians is highlighted by the Meltzer paper and the White Paper (to be discussed below). It is important to note that while both papers are primarily used in litigation (and heavily relied on by the defense), the latter is a White Paper and not a peer-reviewed systematic review or research article. Both papers discuss the goal of improving DTI standards (particularly of the specificity or diagnostic capabilities). That goal, however, is universal among imaging modalities, both solidly established and newly emerging, and did not negate the findings of white matter abnormalities using the ASFNR guidelines. The Wintermark White Paper[39] was followed by publication of another opinion piece, titled “Traumatic Brain Injury Imaging Research Roadmap”[24]. This Research perspective fomented during a workshop that followed the American Society of Neuroradiology[58] convention in Montreal, Canada (May 2014) and dealt with several advanced imaging processes, including DTI. It provided an exposition of available databases, recommendations for imaging protocols, and suggestions for further stratification of both control group participants and subjects. The Roadmap article summarized the acute stage imaging methods commonly used for the assessment of patients following TBI at that time, including CT and MRI with “conventional” imaging sequences. The authors distinguished the conventional sequences from the “advanced” methods including DTI, stating they “do not yet play a central standardized role in diagnosis and management of mild TBI because they require further validation.” The language stops short of contending that quantitative DTI provides no data that can be useful for clinicians. The Roadmap authors advocated the development of “well-characterized methods for quantitative analysis of advanced imaging data,” “well-accepted, uniform, cross-platform, and user-friendly analysis tools,” and “a large data base of normal individuals to which patients with mild TBI could be compared” that includes “standard variation of normal values in an age-stratified fashion and sufficient representation of abnormal.” The authors also recommended stratification by age, sex, handedness, race, ethnicity, socioeconomic status, and academic achievement, as well as core and preferred imaging protocols. They did not, however, determine that existing advanced neuroimaging data acquisition failed to provide any relevant data, but rather that whether for research purposes or for clinical purposes, by standardizing the data acquisition, neuroscientists could facilitate data sharing across platforms and enhance the collective scientific efforts. CONFLICTING EVIDENCE For the past several years, the defense in mTBI cases has complained that quantitative DTI is an unreliable methodology that should not be used in personal injury lawsuits because the findings (generally reduced FA) are non-specific, and the comparison of a single individual to group normative data is unreliable. Notwithstanding these complaints, and despite suggestions to the contrary in the White Paper and the Roadmap paper, quantitative DTI is widely used clinically for mTBI in multiple locations in the United States and for various other neurologic conditions including multiple sclerosis, Alzheimer’s disease, dementia, Parkinson’s disease[59,60], and planning for brain tumor surgery in both adults and children[61]. Certainly, the quantitative DTI methodologies currently in use can be improved through widespread collaborative efforts to refine the analytic process, or by relying upon larger or better stratified control groups. However, there is no reason to believe that the multisequence brain MRI assessments that include DTI generate false or unreliable data to be interpreted by clinicians. In fact, it is impossible to reconcile the clinical use of DTI in the aforementioned neurological conditions and the exclusion of medicolegal use of DTI in TBI cases. Because there does not appear to be a dispute that DTI is the most sensitive technology available to identify white matter abnormalities in the brain in vivo, whether there is a legitimate conflict about the existence of a brain injury in any specific case, is anyone’s guess. The Shenton paper[7] references the American College of Radiology (ACR) Appropriateness Criteria (AC). Shenton treats the AC ratings as an argument against using DTI in the context of a litigated matter involving head trauma. There are major questions about whether clinicians actually rely upon the AC, and there is little literature on the topic. In 2008, the American Journal of Roentgenology suggested that clinician utilization of the appropriateness criteria is “low”[62]. No follow-up peer-reviewed studies have been published that refute this point. While the ACR may have originally developed the AC as a service to the medical profession that remains underutilized in clinical practice, the AC may not have been a compelling need until the issue of overutilization came to the forefront[63-66]. THE INTERSECTION BETWEEN LAW AND SCIENCE Central to understanding the debate about the use of quantitative DTI in personal injury litigation is the distinction between concepts of proof[67] in science and the law and the requisite levels of proof comparing the 95% confidence level widely used in science to the “greater than 50%” standard applicable in civil lawsuits. Confidence level is a function of statistics and represents the frequency, expressed as a percentage, that the target population would produce a result within the specified confidence level and that the finding was not attributable to chance. Both science and law are focused on evidence that often involves uncovering and exploring facts. Generally, progress in science follows use of the scientific method in research. Research begins with an observation that evolves into a question and further evolves into a hypothesis or “working hypothesis” for an observed condition or event, either naturally or through experimentation. A hypothesis can be proved or disproved and should be accompanied by a “null hypothesis” – the opposite of the hypothesis, that can also be proved or disproved. The research may produce a theory. Using the scientific method, priority is given to research record keeping so that experiments and outcomes can be reproduced and verified. The process must be neutral and free of bias[68,69]. To this end, serious scientific contributions require contributors to fully participate in conflict of interest disclosure mandated by the International Committee of Medical Journal Editors[48]. While meaningful scientific contributions generally find their way into peer-reviewed publications, the rigor of peer review varies journal-to-journal and publisher-to-publisher. Thus, what remains unknown are the studies that did not produce a paper for various reasons including but not limited to the failure to prove the hypothesis or conversely, proving the null hypothesis. Such selective reporting, sometimes referred to as the file drawer effect, can bias the entire trend of reporting on a particular subject[69]. By contrast, in legal matters, opposing parties martial the evidence using relevant documents, photographs, video and by presenting testimony of fact and expert witnesses. The issues to be resolved in an injury case generally involve liability, causation, and damages. The factuality of something is resolved (i.e., deemed more likely than not) by the fact finder (usually a jury) as a result of evidentiary hearings. The evidence and fact finding that occurs in an individual case generally governs only in that case. RESOLVING THE ADMISSIBILITY OF DTI EVIDENCE One of the dangers of a paper like Shenton’s, whose title is “Mild Traumatic Brain Injury: Is DTI ready for the courtroom?”[7] is that it takes a somewhat academic approach to the issue while ignoring the practical application of the law and the way trials actually work. Shenton et al[7] theorize that DTI evidence does not pass the admissibility tests promulgated by the federal courts with the landmark decision by the U.S. Supreme Court in Daubert v. Merrell Dow Pharmaceuticals Inc[42]. They argue that there are questions to be resolved before DTI evidence should be submitted in a personal injury case and considered by a jury. But the law does not defer to science to anticipate and resolve in the laboratory every issue or controversy in a young field of science[42] before the issue is litigated in the courtroom. The distinctions or open issues can be contested through the use of expert preparation, practical experience, and “vigorous cross-examination”[42]. As the Supreme Court stated in Daubert: Respondent expresses apprehension that abandonment of “general acceptance” as the exclusive requirement for admission will result in a “free-for-all” in which befuddled juries are confounded by absurd and irrational pseudoscientific assertions…In this regard respondent seems to us to be overly pessimistic about the capabilities of the jury and of the adversary system generally. Vigorous cross-examination, presentation of contrary evidence, and careful instruction on the burden of proof are the traditional and appropriate means of attacking shaky but admissible evidence[42]. Daubert and the federal cases that followed, revised the gatekeeper status of the trial judge, requiring the judge to determine whether the proposed methodology was sufficiently reliable to be a component of the proffered expert’s opinion. Because most personal injury cases are heard in state courts, it remained the prerogative of each state jurisdiction to either adopt a Daubert-like analysis or to retain its then current standard – usually an iteration of the Frye “general acceptance” rule. In Connecticut, for example, the Supreme Court abandoned the Frye standard and adopted the essence of the Daubert standard in State v. Porter[70]. The essential gatekeeper function under Daubert is to assess the methodology at issue to determine its reliability but not to interject his or her own opinion about the expert’s conclusions. To do so, the Daubert court advocated considering a set of non-exclusive factors that include: Whether the theory or technique on which the expert relies has been or could be tested; whether the theory or technique has been subjected to peer review and publication; the known or potential rate of error of the technique or theory when applied; the existence and maintenance of standards controlling the technique's operation; and whether the theory or technique has been generally accepted in the scientific community[31,42]. This test of reliability is a “flexible” one depending on the “nature of the issue, the expert’s particular expertise, and the subject of his [or her] testimony,” and therefore, no one factor will necessarily be determinative of the reliability of an expert’s testimony[70,71]. The rejection of expert testimony is the exception rather than the rule[71-73].The Second Circuit Court of Appeals has noted that “Daubert reinforces the idea that there should be a presumption of admissibility of evidence,” and it has interpreted Daubert as having “advanced a bias in favor of admitting evidence short of that solidly and indisputably proven to be reliable”[74]. Other Circuit Courts have found that challenges to the methodology used by an expert are usually adequately addressed by cross-examination rather than exclusion. As scientists, Shenton et al[7] overlooked the presumption for the admissibility of such evidence, perhaps throwing out the good for the sake of the perfect. The law does not embrace that philosophy. Contrary to the suggestion of Shenton et al[7], judges need not be trained as scientists. They have historically performed quite well in civil and criminal cases dealing with countless scientific and/or technical evidence issues. The law neither requires nor permits the question of admissibility to turn on whether there is more for science to learn or discover. The arc of science, and neuroscience in particular, will be very long. Indeed, scientists may be frustrated at the “hubris” of the law[75] that has rejected the notion that judges are incapable of becoming sufficiently familiar with complex areas of science to determine which side has the better argument about the reliability of the methodology at issue. Scientists generally do not realize that trial judges are not required to decide the validity of specific scientific proposition but rather whether there are “sufficient indicia of legitimacy”[70] to permit the opinions derived from the methodology to be considered by the trier of fact. Nor should there be limitations on an attorney’s ability to handle cases involving medicine including neuroscience or other complicated matters. Indeed, like in any profession, the roster of trial lawyers includes a broad spectrum of talent, levels of experience, and sophistication. The fact that TBI cases are more complex than a case involving a fractured femur does not demand greater regulation of the practice of law, or a higher level of attorney scientific competency as a pre-condition for a trial lawyer accepting a case. Ethical constraints should suffice to prevent a lawyer from handling a TBI case beyond his or her competency. WHITE PAPERS ARE NOT PEER-REVIEWED SCIENTIFIC ARTICLES There is a misconception on the part of Shenton et al[7] as to both the position advocated in the White Paper[39] as well as the way the White Paper should be applied in the courtroom, given that to date, courts have overwhelmingly admitted DTI evidence in mTBI cases. The two compelling reasons that DTI has a nearly unbroken record of admission over defense objections is beyond question: DTI is recognized as the “most sensitive neuroimaging tool available today to detect microstructural integrity and diffuse axonal injury, the most common injury observed in mTBI”[7]. In addition, without much more evidence, the existence of a white matter abnormality causally connected on a more probable than not basis by an expert in the field, should suffice for evidence to reach the jury. Using the White Paper to anchor the argument that DTI is not ready for the courtroom reflects a lack of insight about publishing in science. First, a White Paper is generally known to be a non-scholarly/unscientific advocacy publication, frequently issued on behalf of a group. Scholarly/scientific papers, in contrast, present new research or review research conducted by others and contribute new, evidence-based information to the body of scientific knowledge. As such, a major difference between the two types of publications is that a White Paper is biased to a pre-determined conclusion and accomplishes that goal by cherry-picking references to lead to a pre-specified conclusion. A scholarly scientific paper states its objective including the working hypothesis; presents a comprehensive, balanced, and impartial assessment of the published literature including the literature that is inconsistent with the authors position; reports on the methodology employed; discusses the findings; and reports on a reasoned and unbiased conclusion. A scholarly scientific paper is also submitted for rigorous peer review that conforms to the Committee on Publication Ethics (COPE) Ethical Guideline for Peer Reviewers[76]. While some journals and organizations publish standards to govern White Papers, neither the American Journal of Neuroradiology nor the American Society of Neuroradiology had such standards when the Wintermark White Paper[39] was published. Whether the White Paper was submitted for anything more than editorial (and not peer-) review became clearer when a subpoena was issued to the journal for any sort of record of peer review, which the journal was unable to supply. In fact, Dr. Wintermark testified in a deposition that the White Paper “is not a scientific paper…It reports evidence and the recommendations, and we are not held to any standard except the peer review process”[77]. Any paper that presents a one-sided view, omitting reference and citation to any peer-reviewed journal articles that validate the opposing perspective such as the sensitivity of DTI, should not be included among the compendium of supporting evidence for a bona fide body of scientific evidence in the field of scientific research, clinical practice, or legal trials. For these reasons, science shies away from White Papers, and instead uses the systematic review, which minimizes or removes bias from the study design, including in the obtainment and analyses of data/evidence and the interpretation of findings. When properly performed, the systematic review is recognized in science as the gold standard for reporting the current state of the science on a particular issue[78]. While neither the paper by Shenton et al[7] nor the White Paper by Wintermark[39] are systematic reviews, both are structurally organized to look like such. However, both papers fail to adhere to PRISMA protocols (see above), including the PRISMA checklist[35]. Furthermore, the best that can be said about the peer review process for the White Paper is that it is superficial. For the Wintermark White Paper[39], in particular, Dr. Wintermark himself testified about how the final version of the paper went to press: “So people who are listed basically got a copy of the article, got an opportunity to suggest edits, addition, deletions. We did our best to integrate as many of those comments as we could knowing that sometimes it is not possible. And the modified version was circulated again and then at the end the author, all the people listed as the authors, mentioned that they were satisfied with the text as it stands, and we submitted it for publication. And then when it was submitted for publication, it underwent another round of peer review. I cannot remember exactly what were the comments we got back, and we did our best to incorporate those, redistributed the paper and the editor of the journal decided to accept it”[77]. The type of review described within this professional group does not qualify as independent peer review[76]. By contrast, a relatively contemporary systematic review of the changes that follow the three most recognized classifications of TBI (mild, moderate, and severe) was published in 2018 (referred to here as the Wallace Paper)[79]. The thrust of that DTI study was to examine the dose-dependent relationship of white matter changes among the three TBI classifications by examining the specific regions of interest, timing of the scans, and effects of the injury. Unlike Shenton et al[7] and the Wintermark White Paper[39], the Wallace Paper strictly adhered to PRISMA reporting requirements. The final sample of studies in the meta-analysis included 29 mTBI studies involving DTI. The authors abandoned the effort to combine the mTBI data with the moderate/severe data after it became evident that the majority of the latter classifications were scanned on 1.5 T scanners whereas most of the mTBI scans were performed on 3.0 T scanners. The Wallace paper reported that 26 ROIs were examined in multiple studies, while 9 ROIs were examined in single studies. For the most part, the authors documented only reduced FA in the ROIs examined, with the exception of slightly increased FA in two ROIs, but with small effect size and results that did not reach the threshold for statistical significance. The Wallace paper reported that the mTBI group had significantly lower FA (vs controls) in the cerebrum (centrum semiovale, corpus callosum, and forceps major). The authors of the Wintermark White Paper[39] declared that the inability of neuroscientists to resolve the matter of crossing white matter fibers posed a problem using DTI for clinical purposes. They noted that there were qualitative differences in the approaches used to address crossing fibers and that “[f]urther studies…were required to determine whether one of these has a clear advantage compared with the others”[39]. Here again, the law requires reasonable probability and not certainty, and any equivocation resulting from areas involving crossing fibers can be minimized by focusing an assessment on alternate regions. The authors also advocated for “[i]mprovements in data quality with high-order eddy current correction, distortion correction, and high-order shimming beyond second order should further improve data fidelity and increase the reliability of subsequent data processing”[39]. Again, there is no doubt there can always be improvements in data control, but such criticism would not render forensic assessments of individual patients compared to group norms invalid based upon this general recommendation. The authors of the Wintermark White Paper[39] also complained that based upon group data acquired through research, there are conflicting findings about whether FA, can increase and/or decrease in the acute and chronic stages of TBI and for different structures within the brain. Those issues did not appear to be significant in the later Wallace study[79]. For the most part, the issues of increased FA seem to be less prominent in DTI studies during the chronic stage. The authors of the White Paper argued that “DTI metrics including FA are not specific to TBI …” but may be found in other neurological disorders that affect the white matter[39]. They further asserted that there is “insufficient evidence” to use quantitative DTI for “routine clinical evaluation of TBI at the individual patient level for diagnosis and/or prognostication”[39]. Possible alternative causes can be analyzed as part of the differential diagnostic process. Furthermore, no one has advocated for the use of quantitative DTI on a routine basis in the context of concussion or mTBI. The process should be reserved for the cohort of subjects with persistent post-concussion symptoms. To the best of our knowledge, no papers have adequately articulated the concerns raised by either Shenton et al[7] or Wintermark et al[39] about DTI being used to provide imaging evidence to be relied upon for the differential analysis necessary to make a diagnosis of mTBI and/or persistent post-concussion symptoms. Similarly, to the best of our knowledge, no papers have closely examined proper scientific publication standards that can be easily evaded by authors and publishers. Again, it is important to not obfuscate the difference between scientific certainty and legal proof – the fair preponderance of the evidence or > 50%; the former being essentially a diagnostic biomarker, as opposed to the latter describing something more likely true than untrue. The law generally recognizes a hierarchy of burdens of proof ranging from the highest burden in criminal cases; an intermediate burden of proof – “clear and convincing evidence” in a claim of civil fraud; and the lowest level – “fair preponderance of evidence” in civil cases[80]. Second, it is critical to not confound the difference between a test that is diagnostic and one that provides medical evidence for ruling out or confirming a diagnosis – even a diagnosis based upon inferences as permitted by the rules of evidence. Third, by placing a premium on the idea of routine clinical use, the authors paint a bona fide clinical care technology, such as MRI/DTI, as a litmus test for the admissibility of an opinion about such at trial. That which is used routinely in the clinic may vary by clinician, care site management, geographic region, and/or socioeconomic factors. Much of medicine is limited by reimbursement rates. Traditionally, corroboration of white matter damage has aided in fashioning rehabilitation of a patient[81] and provided validation of the diagnosis for the patient and clinician. It has also been recently proposed as informative of the persistent physiological effects of concussion after clinical symptoms have abated. This assertion, if accurate, would aid in decisions about return to play/work as well as inform the period of vulnerability of a second concussion[82]. Fourth, it has been shown that DTI abnormalities persist after recognizable symptoms have abated and may be the precursor for degenerative changes[83]. Sixth, the addition of the DTI sequence to a brain trauma MRI protocol should add nominal cost for the facility. While the cost of post-processing of DTI may require a specially trained radiologist, technologist, or statistician, the patient (or attorney) can elect to incur that cost. While Shenton et al[7] cited Wortzel because of his authorship about advanced neuroimaging, they neglected to cite the post-Emory Conference article by DTI luminaries Lipton and Bigler, titled “Clarifying the Robust Foundation for and Appropriate Use of DTI in mTBI patients,”[84] which was a direct response to Wortzel (i.e., being inexorably linked digitally to the paper in the literature databases). Lipton and Bigler pointed out the same weak underpinnings of Wortzel’s arguments in terms of persistence of symptoms of mTBI in some patients; Wortzel’s “straw man” argument denigrating quantitative DTI because it requires quantification; the fact that despite methodological variance among studies the same conclusion is reached – low FA is consistent with TBI; and that the “diagnosis of mTBI, or any other disorder, is based on integration of clinical information, not the result of one diagnostic test.” The Shenton authors instead offer another “straw man” argument that insinuates DTI should not be used as a standalone definitive diagnostic test, a use for which it has not been proposed[7]. A more systematic response to the problematic issues in the Wortzel and Meltzer papers was presented in Hulkower’s “A Decade of DTI in Traumatic Brain Injury: 10 Years and 100 Articles Later”[49]. CONTEMPORARY DTI PAPERS It appears that little has altered the overall landscape for DTI in mTBI litigation since Shenton et al[7] was accepted for publication. That paper, like the White Paper, was devoid of contributions to the practical field of DTI science that validates the reliability of DTI as a measure of structural abnormalities of white matter in the brain. It is concerning, however, that Shenton failed to acknowledge the problems inherent in demanding the standardization of DTI protocols. While large consortiums of clinicians and researchers, like TRACK-TBI[52] (elaborated below) and ENIGMA[54], can collaborate on brain research projects, independent clinicians and those not affiliated with a consortium are left to their own protocols. That said, the imaging protocols for 3T MRI for head trauma used by those consortiums are available online from both the TRACK-TBI and ENIGMA websites. Regarding DTI, the peer-reviewed literature has consistently documented its utility in brain injury/concussion and other neurologic conditions in papers such as two in which Dr. Wintermark was a co-author[51,85]. In “White Matter Asymmetry: A Reflection of Pathology” in Traumatic Brain Injury”[30] the authors reported that the symmetry levels among various studied white matter tracts were lower in the mTBI cohort, a finding that can be interpreted as consistent with trauma. Dr. Shenton herself was a co-author of a paper that reported a study, funded by the Veterans Administration and the NIH, which relied upon DTI to assess the microstructure of limbic and paralimbic structures in the context of PTSD severity[83]; again, not as a pathognomonic biomarker but as part of the pathological profile. The assessment of the white matter microstructure can correlate with chronic post-concussive symptom severity[51] and reveals evidence consistent with persistent neurological disruption[82,85,86]. Another example of the bias in the paper by Shenton et al[7] is the authors’ failure to include in their review a paper entitled “White matter alterations in youth with acute mild traumatic brain injury”[87]. This was a prospective observational case-control study of previously healthy children ages 11–16, who presented to the emergency department within 6 h of an mTBI between December 2010 and August 2012. The study concluded that “white matter alterations” were identified in the subjects based upon MRI with DTI performed an average of 2 d following injury. The study also concluded that there was a poor correlation between symptoms and diffusion changes. This was a TBI group reported to be healthy and without co-morbidities that could cause white matter alterations prior to injury. The DTI confirmation of damaged axons in a pediatric population lacking in co-morbidities and white matter changes seen in an aging population supports the notion that similar traumatic damage can be caused in an adult population. The momentum supporting the continued use of DTI as advocated in this paper has not slowed. Last year, the TRACK-TBI study group published recent findings[88]. This appropriately controlled large multicenter cohort study conducted at 11 trauma centers and with a total of 391 mTBI patients (17-60-years-old) at 2-wk to 6-mo post-injury confirmed DTI to be a reliable imaging tool detecting dynamic white matter microinfrastructure following mTBI. In addition, in 2022, Medeiros et al[89] published a systematic review related to the construction of a neuroimaging-based profile including DTI detection of white matter organization of the neural correlates of neuropsychiatric complications following TBI (focusing on depression in their study). Their findings again support the practical value of a pathological profile and the absence of a single pathognomonic imaging biomarker for brain injury outcomes. Finally, just this year (2023), Graham et al[90] published their findings of a distinct neuroimaging (including DTI) pattern of post-TBI neurodegeneration involving white matter atrophy, with higher shear forces at time of injury correlating to more progressive atrophy many years later. Of note, while DTI analysis of the brain is superfluous in TBI cases that show more classic evidence of traumatic axonal injury including microhemorrhages or bright T2 foci at or near the gray-white junction[91,92] where aging changes rarely occur[93], DTI abnormalities showing truncated tracts that end at abnormal foci on routine MRI images indicate a strong correlation between these MRI methods. However, DTI abnormalities such as truncated tracts, asymmetrical numbers or volumes of tracts provide evidence of white matter damage even in the absence of abnormalities on standard MRI images. It is true that DTI abnormalities, bright T2 spots, and microhemorrhages may sometimes occur in brains of patients in the absence of trauma. As with all imaging findings, however, a radiologist must consider artifacts, congenital anomalies, infectious diseases, and various other alternative diagnoses before arriving at a diagnosis of TBI. Clinical signs of TBI and positive imaging findings, whether from routine imaging or non-routine methods that indicate brain damage consistent with trauma, should not be disconnected, ignored, or devalued merely because they are presented in a courtroom. Summary The undisputed sensitivity of DTI to provide evidence of white matter abnormalities in the brain during the chronic stage of post-concussion symptoms has been the focus of legal conflict for more than 10 years. This article has summarized the technical properties that make DTI one tool available to clinicians seeking to corroborate a brain injury diagnosis and direct rehabilitation. We have discussed how, although papers have criticized the forgoing use of DTI technology, those papers are rife with shortcomings in their publication processes, weakening their integrity among the peer-reviewed literature. As the three fields of science, medicine and law come together to find accurate and valid resolutions to issues that arise in legal proceedings and to guide patient treatment and management, it is critical to amalgamate the knowledge among the three on how scientific proof differs from proof in a courtroom; indeed, as presented herein for DTI in particular an overwhelming majority of judicial decisions have validated admitting scientific and medical evidence for the purposes advocated. CONCLUSION If the concern of Shenton et al[7] is merely that the adversarial process that includes trial lawyers, judges, and jurors may get something wrong, then the argument should fail. Their paper accomplished little other than to stir up trouble, obfuscating the topical issue for non-scientists, particularly judges and juries. While science may evolve to provide the means for corroborating trauma-induced white matter damage documented on quantitative DTI, in the meantime, it is likely that judges will continue to execute their gatekeeping responsibilities to distinguish between scientific methods that have been embraced as reliable and conflicts that revolve around the application of those reliable methods. In the interval, hopefully neuroscientists can accept that our federal and state constitutions ensure that resolving claims involving the weight of evidence remains in the hands of the judge and jury. ACKNOWLEDGEMENTS The authors would like to thank the following individuals for their intellectual contributions to this review: Andrew M Abraham, JD (repository for the databank of DTI decisions); Kenneth B Goldblatt, JD; Kelie M Reece, PhD; Dorothy C Sims, JD; Bruce H Stern, JD. Conflict-of-interest statement: Dr. van Velkinburgh has been engaged as an expert witness in litigated matters concerning authorship and publication of papers in scientific and science related journals including testimony concerning her extended research on Wintermark M, Sanelli PC, Anzai Y, Tsiouris AJ, Whitlow CT; American College of Radiology Head Injury Institute. Imaging evidence and recommendations for traumatic brain injury: advanced neuro- and neurovascular imaging techniques. AJNR Am J Neuroradiol. 2015 Feb;36(2):E1-E11. Dr. Herbst has served as an expert witness in litigated matters interpreting diffusion tensor imaging of the brain in both clinical and forensic settings. Mr. Casper is a trial attorney who represents personal injury plaintiffs alleging various classifications of traumatic brain injury. Some of his clients have undergone advanced neuroimaging ordered by clinicians. Provenance and peer review: Invited article; Externally peer reviewed. Peer-review model: Single blind Corresponding Author's Membership in Professional Societies: American Association for the Advancement of Science (AAAS); American Society for Biochemistry and Molecular Biology (ASBMB); Society for Scholarly Publishing (SSP); Council of Science Editors (CSE); Committee on Publication Ethics (COPE). Peer-review started: April 10, 2023 First decision: May 15, 2023 Article in press: June 13, 2023 Specialty type: Medicine, legal Country/Territory of origin: United States Peer-review report’s scientific quality classification Grade A (Excellent): A Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Stoyanov D, Bulgaria; Zhou Y, United States S-Editor: Ma YJ L-Editor: A P-Editor: Ma YJ ==== Refs 1 Taylor CA Bell JM Breiding MJ Xu L Traumatic Brain Injury-Related Emergency Department Visits, Hospitalizations, and Deaths - United States, 2007 and 2013 MMWR Surveill Summ 2017 66 1 16 2 Center for Disease Control and Prevention Surveillance Report of Traumatic Brain Injury-related Hospitalizations and Deaths by Age Group, Sex, and Mechanism of Injury—United States, 2016 and 2017 2021. 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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4670 10.12998/wjcc.v11.i19.4670 Case Report Metastatic colon cancer treated using traditional Chinese medicine combined with chemotherapy: A case report Deng CG et al. Metastatic colon cancer and TCM Deng Chen-Geng School of Life Sciences, Beijing University of Chinese Medicine, Beijing 102401, China Tang Meng-Yuan Department of Breast and Thyroid Surgery, China-Japan Friendship Hospital, Beijing 100029, China Pan Xue Post-Doctoral Mobile Station,Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing 100053, China Liu Zhao-Heng School of Life Sciences, Beijing University of Chinese Medicine, Beijing 102401, China. zliu@bucm.edu.cn Author contributions: Deng CG, Tang MY, and Pan X contributed equally to this work; Deng CG drafted and edited the original manuscript; Tang MY edited the manuscript and analyzed the data; Pan X collected the data; Liu ZH treated the patient and supervised the study; all authors have read and approved the final manuscript. Supported by The National Natural Science Foundation of China, No. 81904049 ; Beijing Natural Science Foundation, No. 7202118 ; The National Traditional Chinese Medicine Inheritance Innovation Project, No. ZYYCXTD-C-202006-9 ; and The Basic Scientific Research Foundation of Beijing University of Chinese Medicine, No. 2021-JYB-XJSJJ-033 . Corresponding author: Zhao-Heng Liu, MD, Assistant Professor, School of Life Sciences, Beijing University of Chinese Medicine, Northeast Corner of the Intersection of Yangguang South Street and Baiyang East Road, Fangshan District, Beijing 102401, China. zliu@bucm.edu.cn 6 7 2023 6 7 2023 11 19 46704676 20 2 2023 12 4 2023 26 5 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Colon cancer (CC) is one of the leading causes of cancer-related morbidity and mortality worldwide. Traditional Chinese medicine (TCM) is widely used in the treatment of various chronic diseases. CC easily metastasizes and results in high morbidity and mortality rates. CASE SUMMARY A 71-year-old man with a 12-year history of old myocardial infarction and a 7-year history of type 2 diabetes mellitus was diagnosed with CC and underwent right hemicolectomy 1 year ago. Tumor biopsy revealed moderately poorly differentiated adenocarcinoma. Subsequently, chemotherapy with oxaliplatin and paclitaxel was administered. Anastomosis recurrence and pelvic metastasis were noted 37 d later. The patient received eight 21-d cycles of adjuvant chemotherapy with oxaliplatin and capecitabine after recurrence. However, the tumor persisted, and chemotherapy-related liver damage developed gradually. Thus, he was advised to take TCM for the recurrence and pelvic metastasis. The patient’s metastatic CC was cured after receiving TCM combined with long-term chemotherapy. CONCLUSION TCM may be an effective adjunct therapy in the treatment of patients with metastatic CC. Metastatic colon cancer Traditional Chinese medicine Tumor Pelvic metastasis Chemotherapy Case report ==== Body pmc Core Tip: Herein, we report a case of a patient with metastatic colon cancer (CC) who underwent right hemicolectomy and received 8 cycles of chemotherapy coupled with traditional Chinese medicine (TCM). After treatment, the patient was cured and there was no recurrence during the subsequent follow-up. We believe that TCM can provide a new postoperative treatment modality for CC, as the patient's enteroscopy revealed that the anastomotic stoma of the transverse colon had become smooth 7 mo after treatment. INTRODUCTION Colon cancer (CC), the 3rd most common cancer worldwide, is one of the leading causes of cancer-related morbidity and mortality. It generally occurs among people aged > 70 years. Common therapies for CC include adjuvant chemotherapy, palliative chemotherapy, and palliative targeted therapy[1]. However, palliative chemotherapy only provides limited benefits in terms of prolonging progression-free survival, alleviating symptoms, and improving the quality of life of patients, without offering a cure for the disease. Surgery for CC that invades other organs or structures can be challenging[2]. Traditional Chinese medicine (TCM) offers specific treatment approaches for delaying disease progression, and therefore, may emerge as a significant alternative treatment for CC. Right-sided cancers are typically reported in CC. Herein, we report a case of a 71-year-old man with a 12-year history of remote myocardial infarction and a 7-year history of type 2 diabetes mellitus whose metastatic CC was successfully treated with a combination of TCM and long-term chemotherapy. The details of the case are presented in accordance with the CARE reporting checklist. CASE PRESENTATION Chief complaints A 71-year-old male patient with a 12-year history of prior myocardial infarction and a 7-year history of type 2 diabetes mellitus was admitted to our hospital for further evaluation and treatment of postoperative metastatic CC. History of present illness Upon admission to our hospital, the patient was diagnosed with CC and had undergone right hemicolectomy 6 mo earlier. He also complained of numbness and tingling in the limbs for the past 3 years and had lost 10 kg in the past 6 mo. He denied fever, cough, chest pain, chest tightness, abdominal pain, or abdominal distention. Tumor biopsy revealed moderately to poorly differentiated adenocarcinoma (Figure 1) with invasion of the deep muscular layer extending to the serosal layer (Figure 2). Figure 1 Initial X-ray and endoscopy findings. A: X-ray; B: Endoscopy. The patient’s tumor was located in the hepatic flexure of the right colon. Figure 2 Histopathology of the patient’s tumor. Hematoxylin and eosin staining of a biopsy of the patient’s tumor suggested moderately to poorly differentiated adenocarcinoma. Anastomosis recurrence and pelvic metastasis were noted 37 d later (Figure 3). Meanwhile, chemotherapy-related liver damage developed slowly. During a hospital visit, an irregular-density shadow of soft tissue was observed near the anastomotic stoma of the transverse colon, with a size of approximately 1.4 cm × 0.9 cm (Figure 4). Figure 3 Computed tomography findings at postoperative day 37. A: Anastomosis recurrence; B: Pelvic metastasis. Figure 4 Computed tomography before and after traditional Chinese medicine. The patient presented to our hospital after receiving eight cycles of adjuvant chemotherapy with oxaliplatin and capecitabine. A: Initial computed tomography results revealing pelvic metastasis near the anastomotic stoma of the transverse colon, with a size of approximately 1.4 cm × 0.9 cm; B and C: Traditional Chinese medicine led to a gradual reduction in the tumor size according to the re-examinations performed 2 mo (B) and 4 mo (C) after presentation. History of past illness The patient received eight cycles of adjuvant chemotherapy with oxaliplatin and capecitabine after right hemicolectomy. Personal and family history The patient had no personal or family history. Physical examination His vital signs were as follows: temperature, 36.1 °C; blood pressure 117/62 mmHg; heart rate 86 beats/min; and respiratory rate 20 breaths/min. Laboratory examinations The levels of tumor markers, including alpha-fetoprotein, carcinoembryonic antigen, and cancer antigen 199, showed an increasing trend (Table 1). Other laboratory tests were within normal limits. Table 1 Tumor markers Marker Reference range September 2020 November 2020 January 2021 March 2021 May 2021 AFP in ng/mL 0.00–7.00 2.31 10.361 1.95 1.64 1.35 CEA in ng/mL 0.00–4.30 4.521 6.891 4.931 4.801 4.21 CA199 in U/mL 0.00–27 18.43 26.4 33.321 22.85 22.97 1 High level, changes in this patient’s tumor marker levels. AFP: Alpha-fetoprotein; CEA: Carcinoembryonic antigen; CA199: Cancer antigen 199. Imaging examinations The patient’s previous medical records suggested moderately to poorly differentiated adenocarcinoma (Figure 1). Postoperative immunohistochemical results revealed tumor invasion of the deep muscular layer, extending to the serosal layer (Figure 2). Anastomosis recurrence and pelvic metastasis were noted 37 d later (Figure 3). Imaging revealed an irregular-density shadow of soft tissue was observed near the anastomotic stoma of the transverse colon, with a size of approximately 1.4 cm × 0.9 cm (Figure 4A). FINAL DIAGNOSIS The final pathological diagnosis after laboratory and imaging examinations was metastatic CC (stage T2N2bM1a). TREATMENT We advised the patient to initiate TCM and prescribed a regimen consisting of Huangqi (45 g), Chenpi (15 g), Daxueteng (30 g), Baijiangcao (10 g), and Shancigu (15 g). OUTCOME AND FOLLOW-UP Computed tomography revealed that the recurrent neoplasm disappeared after 37 d of sustained medication, and a reexamination performed 4 mo later revealed no recurrence (Figure 4B and C). Additionally, enteroscopy performed 7 mo later showed that the anastomotic stoma of the transverse colon had become smooth (Figure 5). These findings were consistent with the changes in the patient’s tumor marker levels (Table 1). Figure 5 Follow-up colonoscopy finding. Colonoscopy was performed 7 mo after traditional Chinese medicine was given. The anastomotic stoma of the transverse colon has become smooth. DISCUSSION Here, we reported the case of a patient with recurrent metastatic CC after right hemicolectomy who received 8 cycles of chemotherapy combined with TCM and was finally cured. No recurrence was noted during subsequent follow-up. TCM may provide a new treatment modality for CC postoperatively, as the patient’s enteroscopy revealed that the anastomotic stoma of the transverse colon had become smooth 7 mo after treatment. Currently, the recommended treatment for CC includes surgical resection of cancer and counseling of patients to receive adjuvant chemotherapy, palliative chemotherapy, or palliative targeted therapy[3,4]. The current management of disseminated metastatic CC involves various active drugs (either in combination or as single agents), including 5-fluorouracil/leucovorin, capecitabine, irinotecan, oxaliplatin, bevacizumab, cetuximab, panitumumab, ziv-aflibercept, ramucirumab, regorafenib, trifluridine–tipiracil, pembrolizumab, and nivolumab[5]. Treatment personalization allows patients to maximize benefits while minimizing harm, thereby enabling optimal survival and quality of life[6]. Most patients show good curative effects and high long-term survival rates. The number of patients undergoing surgical resection has increased the associated mortality and morbidity, although approximately 10.8% of patients who undergo colectomy for CC have metastatic diseases[7]. However, combination chemotherapy is associated with additional toxicity, which is harmful to patients[8]. Our treatment approach aimed to achieve several goals: Elimination of recurrence and metastatic CC in the pelvis; reduction of chemotherapy-related liver toxicity; and improvement of patient overall quality of life. To achieve these goals, we utilized TCM to dispel cold, remove dampness, reduce phlegm, and resolve masses, as well as to provide detoxifying effects. Huangqi is widely used as an immune stimulant, tonic, antioxidant, hepatoprotectant, diuretic, expectorant, and antidiabetic and anticancer agent[9]. It can enhance the body’s natural defense mechanisms. Astragaloside might contribute to the immunostimulating and anticancer effects of Huangqi, which have been demonstrated in clinical trials and animal experiments[10]. Shancigu extract can significantly inhibit the proliferation of human CC SW480 cells and induce apoptosis[11]. Another study revealed the role of active components of Baijiangcao in CC treatment[12]. Other herbs, such as Daxueteng, are also known to exert antitumor effects. Daxueteng and Baijiangcao can play an important role in intestinal diseases and enhance human immunity. The addition of TCM in the treatment regimen enhances the abovementioned effects. This report has several limitations that preclude us from drawing definitive conclusions. Firstly, it is a single case report, and the evidence level of this treatment approach is insufficient. While we can adjust the prescription slightly based on different conditions, more research is needed to establish the efficacy of TCM combined with chemotherapy. Secondly, the follow-up period is limited, and long-term follow-up is required to confirm the clinical validity of this treatment modality. CONCLUSION TCM may be an effective adjunct therapy to current standard-of-care chemotherapy in the treatment of metastatic CC, as suggested by the resolution of our patient’s metastatic CC after laparoscopic radical resection followed by TCM. However, further investigations are warranted to confirm the efficacy and mechanisms of TCM treatments for CC. Informed consent statement: Written informed consent for the publication of medical information was obtained from the patient. Conflict-of-interest statement: The authors declare that they have no competing interests. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016). Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: February 20, 2023 First decision: March 24, 2023 Article in press: May 26, 2023 Specialty type: Oncology Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C, C Grade D (Fair): D Grade E (Poor): 0 P-Reviewer: Al-Emam AMA, Saudi Arabia; Bacharaki D, Greece; Cheng TH, Taiwan S-Editor: Ma YJ L-Editor: Filipodia P-Editor: Zhao S ==== Refs 1 Moth EB Vardy J Blinman P Decision-making in geriatric oncology: systemic treatment considerations for older adults with colon cancer Expert Rev Gastroenterol Hepatol 2016 10 1321 1340 27718755 2 Klaver CEL Kappen TM Borstlap WAA Bemelman WA Tanis PJ Laparoscopic surgery for T4 colon cancer: a systematic review and meta-analysis Surg Endosc 2017 31 4902 4912 28432461 3 Ku G Tan IB Yau T Boku N Laohavinij S Cheng AL Kang YK de Lima Lopes G Jr Management of colon cancer: resource-stratified guidelines from the Asian Oncology Summit 2012 Lancet Oncol 2012 13 e470 e481 23117002 4 Banerjee A Pathak S Subramanium VD G D Murugesan R Verma RS Strategies for targeted drug delivery in treatment of colon cancer: current trends and future perspectives Drug Discov Today 2017 22 1224 1232 28545838 5 Benson AB 3rd Venook AP Cederquist L Chan E Chen YJ Cooper HS Deming D Engstrom PF Enzinger PC Fichera A Grem JL Grothey A Hochster HS Hoffe S Hunt S Kamel A Kirilcuk N Krishnamurthi S Messersmith WA Mulcahy MF Murphy JD Nurkin S Saltz L Sharma S Shibata D Skibber JM Sofocleous CT Stoffel EM Stotsky-Himelfarb E Willett CG Wu CS Gregory KM Freedman-Cass D Colon Cancer, Version 1.2017, NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw 2017 15 370 398 28275037 6 Benson AB Venook AP Al-Hawary MM Cederquist L Chen YJ Ciombor KK Cohen S Cooper HS Deming D Engstrom PF Garrido-Laguna I Grem JL Grothey A Hochster HS Hoffe S Hunt S Kamel A Kirilcuk N Krishnamurthi S Messersmith WA Meyerhardt J Miller ED Mulcahy MF Murphy JD Nurkin S Saltz L Sharma S Shibata D Skibber JM Sofocleous CT Stoffel EM Stotsky-Himelfarb E Willett CG Wuthrick E Gregory KM Freedman-Cass DA NCCN Guidelines Insights: Colon Cancer, Version 2.2018 J Natl Compr Canc Netw 2018 16 359 369 29632055 7 Moghadamyeghaneh Z Hanna MH Hwang G Mills S Pigazzi A Stamos MJ Carmichael JC Outcomes of colon resection in patients with metastatic colon cancer Am J Surg 2016 212 264 271 27094117 8 Meyers BM Cosby R Quereshy F Jonker D Adjuvant Chemotherapy for Stage II and III Colon Cancer Following Complete Resection: A Cancer Care Ontario Systematic Review Clin Oncol (R Coll Radiol) 2017 29 459 465 28341242 9 Fu J Wang Z Huang L Zheng S Wang D Chen S Zhang H Yang S Review of the botanical characteristics, phytochemistry, and pharmacology of Astragalus membranaceus (Huangqi) Phytother Res 2014 28 1275 1283 25087616 10 Liu P Zhao H Luo Y Anti-Aging Implications of Astragalus Membranaceus (Huangqi): A Well-Known Chinese Tonic Aging Dis 2017 8 868 886 29344421 11 Wang Y Tang J Sun P Lin SH Zhang X [Effect of Pleurotus eryngii extract on proliferation and apoptosis of human colon cancer SW480 cells] Zhongguo Zhongyi Jichu Yixue Zazhi 2021 27 1754 1758 12 Yang H Cheung MK Yue GG Leung PC Wong CK Lau CB Integrated Network Pharmacology Analysis and In Vitro Validation Revealed the Potential Active Components and Underlying Mechanistic Pathways of Herba Patriniae in Colorectal Cancer Molecules 2021 26

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4713 10.12998/wjcc.v11.i19.4713 Case Report Tuberculosis-induced aplastic crisis and atypical lymphocyte expansion in advanced myelodysplastic syndrome: A case report and review of literature Sun XY et al. TB-induced aplastic crisis in MDS Sun Xiao-Yun Department of Hematology, The Central Hospital of Qingdao West Coast New Area, Qingdao 266555, Shandong Province, China Yang Xiao-Dong Department of Hematology, The Central Hospital of Qingdao West Coast New Area, Qingdao 266555, Shandong Province, China Xu Jia Department of Hematology, The Central Hospital of Qingdao West Coast New Area, Qingdao 266555, Shandong Province, China Xiu Nuan-Nuan Department of Hematology, The Central Hospital of Qingdao West Coast New Area, Qingdao 266555, Shandong Province, China Ju Bo Department of Hematology, The Central Hospital of Qingdao West Coast New Area, Qingdao 266555, Shandong Province, China Zhao Xi-Chen Department of Hematology, The Central Hospital of Qingdao West Coast New Area, Qingdao 266555, Shandong Province, China. zhaoxichen2003@163.com Author contributions: Zhao XC and Sun XY developed the idea; Sun XY, Yang XD and Xu J analyzed the data and drafted the manuscript; Sun XY, Yang XD, Xu J, Xiu NN and Ju B participated in the treatment; Zhao XC revised the manuscript; all authors have read and approved the final manuscript. Supported by The Specialized Scientific Research Fund Projects of The Medical Group of Qingdao University, No. YLJT20201002 . Corresponding author: Xi-Chen Zhao, MD, Chief Physician, Department of Hematology, The Central Hospital of Qingdao West Coast New Area, No. 9 Huangpujiang Road, Qingdao 266555, Shandong Province, China. zhaoxichen2003@163.com 6 7 2023 6 7 2023 11 19 47134722 10 4 2023 22 5 2023 31 5 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Myelodysplastic syndrome (MDS) is caused by malignant proliferation and ineffective hematopoiesis. Oncogenic somatic mutations and increased apoptosis, necroptosis and pyroptosis lead to the accumulation of earlier hematopoietic progenitors and impaired productivity of mature blood cells. An increased percentage of myeloblasts and the presence of unfavorable somatic mutations are signs of leukemic hematopoiesis and indicators of entrance into an advanced stage. Bone marrow cellularity and myeloblasts usually increase with disease progression. However, aplastic crisis occasionally occurs in advanced MDS. CASE SUMMARY A 72-year-old male patient was definitively diagnosed with MDS with excess blasts-1 (MDS-EB-1) based on an increase in the percentages of myeloblasts and cluster of differentiation (CD)34+ hematopoietic progenitors and the identification of myeloid neoplasm-associated somatic mutations in bone marrow samples. The patient was treated with hypomethylation therapy and was able to maintain a steady disease state for 2 years. In the treatment process, the advanced MDS patient experienced an episode of progressive pancytopenia and bone marrow aplasia. During the aplastic crisis, the bone marrow was infiltrated with sparsely distributed atypical lymphocytes. Surprisingly, the leukemic cells disappeared. Immunological analysis revealed that the atypical lymphocytes expressed a high frequency of CD3, CD5, CD8, CD16, CD56 and CD57, suggesting the activation of autoimmune cytotoxic T-lymphocytes and natural killer (NK)/NKT cells that suppressed both normal and leukemic hematopoiesis. Elevated serum levels of inflammatory cytokines, including interleukin (IL)-6, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), confirmed the deranged type I immune responses. This morphological and immunological signature led to the diagnosis of severe aplastic anemia secondary to large granule lymphocyte leukemia. Disseminated tuberculosis was suspected upon radiological examinations in the search for an inflammatory niche. Antituberculosis treatment led to reversion of the aplastic crisis, disappearance of the atypical lymphocytes, increased marrow cellularity and 2 mo of hematological remission, providing strong evidence that disseminated tuberculosis was responsible for the development of the aplastic crisis, the regression of leukemic cells and the activation of CD56+ atypical lymphocytes. Reinstitution of hypomethylation therapy in the following 19 mo allowed the patient to maintain a steady disease state. However, the patient transformed the disease phenotype into acute myeloid leukemia and eventually died of disease progression and an overwhelming infectious episode. CONCLUSION Disseminated tuberculosis can induce CD56+ lymphocyte infiltration in the bone marrow and in turn suppress both normal and leukemic hematopoiesis, resulting in the development of aplastic crisis and leukemic cell regression. Myelodysplastic syndrome Aplastic crisis Atypical lymphocyte Leukemic cell regression CD56+ lymphocyte expansion Disseminated tuberculosis Case report ==== Body pmc Core Tip: In patients with myelodysplastic syndrome, bone marrow cellularity and myeloblasts usually increase with disease progression. An advanced myelodysplastic syndrome patient experienced an episode of aplastic crisis. During the aplastic crisis, leukemic cells regressed. The bone marrow was infiltrated with atypical lymphocytes that expressed high frequencies of cluster of differentiation (CD)3, CD5, CD8, CD16, CD56 and CD57. Antituberculosis treatment led to reversion of the aplastic crisis, disappearance of the atypical lymphocytes, increased marrow cellularity and 2 mo of hematological remission, suggesting that disseminated tuberculosis was responsible for the development of aplastic crisis, regression of leukemic cells and activation of CD56+ cells. INTRODUCTION Myelodysplastic syndrome (MDS) is a heterogeneous disease of hematopoietic progenitor cells (HPCs) resulting from malignant proliferation and ineffective hematopoiesis. Oncogenic somatic mutations and increased apoptosis, necroptosis and pyroptosis lead to the accumulation of earlier HPCs and impaired productivity of mature blood cells[1,2]. Morphologically, MDS is characterized by one or more lineages of dysplasia, and an increased percentage of myeloblasts is the most significant dysplastic feature[3,4]. The bone marrow in MDS patients is usually hyperplastic, consistent with dysregulated proliferation and differentiation. However, a fraction of MDS patients can be hypoplastic[5,6]. The identification of cytogenetic abnormalities and oncogenic somatic mutations, especially unfavorable ones, is helpful in the determination of leukemic hematopoiesis[1,2,5-7]. With regard to immunological signatures, patients with low-risk MDS, especially hypoplastic MDS, exhibit autoimmune-predominated responses resembling those in severe aplastic anemia (SAA), whereas patients with advanced MDS demonstrate negative regulator-predominated immune exhaustion resembling that in acute myeloid leukemia (AML)[1,5,6]. An increased percentage of myeloblasts and the presence of unfavorable somatic mutations are signs of leukemic hematopoiesis and indicators of entrance into an advanced stage[2,3,4,6,7]. In MDS patients, bone marrow cellularity and the percentage of myeloblasts usually increase with disease progression[1,3,4], which is generally attributed to clonal evolution or immune escape via the acquisition and accumulation of novel oncogenic mutations under chronic immune pressure[2,7,8]. However, some patients, especially in the end stage, experience an episode of progressive cytopenia, usually concomitant with severe inflammatory conditions. In this situation, the bone marrow can be hypoplastic, leukemic clones are concealed, and the immunological signature changes from an exhausted phenotype to deranged autoimmune responses resembling those in autoimmune hematopoietic failure. Severe infections during aplastic crisis are generally ascribed to functional defects in the myeloid lineage and severe neutropenia[9-11]. A patient with definitively diagnosed MDS-EB-1 experienced an episode of aplastic crisis with concomitant infiltration of atypical lymphocytes and regression of leukemic cells in the bone marrow. The atypical lymphocytes expressed high frequencies of cluster of differentiation (CD)3, CD5, CD8, CD16, CD56 and CD57. Antituberculosis treatment led to not only reversion of the aplastic crisis but also disappearance of the atypical lymphocytes, increased marrow cellularity and 2 mo of hematological remission. This treatment response indicated that disseminated tuberculosis infection, especially gut involvement, was responsible for the development of the aplastic crisis, the regression of leukemic cells and the activation of CD56+ atypical lymphocytes. The reversible aplastic crisis suggested that autoimmune responses due to infectious diseases represent the major pathophysiology in patients with end-stage MDS. Leukemic cell regression during aplastic crisis and a short duration of hematopoietic remission following the resolution of infectious episodes suggest that autoimmune hematopoietic failure likely functions as an inflammatory stress-fueled antileukemic mechanism; the relapse similar to that in transient “spontaneous remission” suggests that the inflammatory stress-fueled antileukemic activity is unable to eradicate the leukemic clones; and the genetically damaged HPCs, together with the active inflammatory condition, contribute to the development of autoimmune hematopoietic failure. CASE PRESENTATION Chief complaints Aggressive pancytopenia for 4 weeks. History of present illness Two years earlier, a 72-year-old Chinese man sought medical help for gradually worsening fatigue and cytopenia. At that time, the complete blood cell count (CBC) showed the following results: White blood cells (WBCs), 1.44 × 109/L; absolute neutrophil count (ANC), 0.62 × 109/L; red blood cells (RBCs), 2.74×1012/L; hemoglobin level (Hb), 97 g/L; platelets (Plts), 175 × 109/L; and absolute reticulocyte count (Ret), 44.62 × 109/L. He was diagnosed with MDS-EB-1 based on hypercellular bone marrow and an increased percentage of myeloblasts (accounting for 8.5% of all nucleated cells) on morphological evaluation, an increased percentage of CD34+CD33+ cells (6.82%) on immunotyping analysis, a normal 46,XY karyotype on cytogenetic analysis, and the identification of somatic mutations in ASXL1, RUNX1, STAG1 and N-RAS genes on molecular analysis. In the following 2 years, the patient was treated with hypomethylation therapy [a total of 14 courses of decitabine (25 mg/d × 5 d)] as a solitary antileukemic agent and was able to maintain a steady disease state. Four weeks prior to this hospitalization, the patient’s fatigue was aggravated, performance status deteriorated, and cytopenia significantly worsened. The degree of fatigue was far more severe than the degree of anemia. History of past illness The patient had a 6-year history of leukocytopenia prior to the diagnosis of MDS-EB-1. He denied having diseases affecting the cardiovascular, endocrine, respiratory, gastrointestinal, urogenital or musculoskeletal systems. Personal and family history No family history of inherited, hematological or neoplastic diseases was recorded. Physical examination The patient was 172 cm tall and weighed 63.5 kg. Upon admission, his vitals were as follows: Temperature, 36.4 °C; respiratory rate, 18 breaths per minute; heart rate, 88 beats per minute; and blood pressure, 114/76 mmHg. Physical examination revealed the presence of mild tenderness of the right lower abdomen. No significant signs involving the nervous, respiratory, cardiovascular, or musculoskeletal systems were present. Laboratory examinations Routine laboratory examinations: At hospitalization, the CBC showed the following results: WBCs, 0.93 × 109/L; ANC, 0.34 × 109/L; RBCs, 1.57 × 1012/L; Hb, 48 g/L; Plts, 16 × 109/L; Ret, 18.50 × 109/L; and C-reactive protein, 263.62 mg/L. The coagulation profile showed hyperfibrinogenemia (4.100 g/L), with a D-dimer level of 0.38 mg/L. Urine analysis revealed occult blood (2+) and protein (1+). Biochemical tests showed moderate hypoalbuminemia (26.1 g/L) and low serum levels of potassium (2.97 mmol/L), sodium (128 mmol/L) and chlorine (94 mmol/L), with the absence of abnormalities in markers of liver and renal function. Pathogenic cultures of blood samples were sterile. Immunological tests for hepatitis A, B, and C virus and human immunodeficiency virus were negative. Tests for Epstein-Barr virus and parvovirus B19 DNA were negative. The IFN-γ release assay yielded a positive result. The pleural effusion in aspirates was bloody, with an elevated level of adenosine deaminase. The serum levels of IL-6, IFN-γ and TNF-α were elevated, suggesting the presence of deranged type I immune responses. Specific laboratory examinations for blood diseases: A morphological evaluation was performed. The bone marrow became aplastic, with the appearance of sparsely distributed atypical lymphocytes that accounted for 77% of all nucleated cells (Figure 1). Of the atypical lymphocytes, the chromatin was highly concentrated, the cytoplasm was excessively abundant, with eosinophilic shade on the basophilic background and most prominently adjacent to the nucleoli without visible granules, and the membrane was canthous. Basophilic substances were present in the cytoplasm of almost all erythrocytes, suggestive of dysplasia in erythropoiesis. Notably, the leukemic cells disappeared. Immunotyping analysis of bone marrow samples revealed decreases in the frequencies of CD34 (0.24%) and CD19 (14.28%) expression and increases in the frequencies of CD3 (54.15%), CD5 (47.23%), CD8 (28.49%), CD16 (28.33%), CD56 (22.16%) and CD57 (41.91%) expression. The immunological signature indicated the activation and expansion of autoimmune cytotoxic T-lymphocytes (CTLs) and NK/NKT cells, resembling that in patients with “SAA” secondary to large granular lymphocyte leukemia (LGLL)[12,13]. The massively reduced frequencies of CD34 and CD19 expression indicated the exhaustion of HPCs and B lymphocytes. Next-generation sequencing analysis revealed a novel somatic mutation in the KMT2D gene in addition to the previously reported mutations. These laboratory data indicated the development of an “aplastic crisis”, in which the activated CTLs and NK/NKT cells were likely responsible for the autoimmune suppression of normal and leukemic hematopoiesis. Figure 1 Morphological evaluation of bone marrow and blood smears during aplastic crisis. A: The bone marrow was aplastic, with sparsely distributed atypical lymphocytes, which accounted for 77% of all nucleated cells. The chromatin of the atypical lymphocytes was highly concentrated, the cytoplasm was excessively abundant, with eosinophilic shade on the basophilic background and most prominently adjacent to the nucleoli without visible granules, and the membrane was canthous, indicating the activation of lymphocytes. Basophilic substances were present in the cytoplasm of almost all mature erythrocytes, suggestive of dysplasia in erythropoiesis. The leukemic cells disappeared. Immunotyping analysis revealed that these atypical lymphocytes expressed a high frequency of cluster of differentiation (CD)3, CD4, CD8, CD5, CD56 and CD57, indicating the activation and expansion of autoimmune cytotoxic T-lymphocytes and natural killer (NK)/NKT cells that suppressed both normal and leukemic hematopoiesis; B: There were predominately atypical lymphocytes in the blood smears, and granulocytes were seldom visualized. Dyserythropoiesis can also be visualized in the blood smears. Imaging examinations Computed tomography (CT) scans were performed in the search for an inflammatory niche, and the chest (Figure 2) and abdominal (Figure 3) CT features, together with the IFN-γ release assay positivity and bloody pleural effusion, suggested a diagnosis of disseminated tuberculosis involving the lungs, pleura, hilum, mediastinum, gastrointestinal tract, peritoneum and urinary tract[14,15]. Figure 2 Chest computed tomography during aplastic crisis. A-H: Multiple calcified lesions were present in the lungs, hilum and mediastinum (yellow arrows), indicating the existence of old tuberculosis. The adjacent exudative lesions indicated reactivation of the old tuberculosis condition. A massive exudative lesion was present in the right lung (green arrows), and there was a similar exudative lesion on the pleuron adjacent to the massive exudative lesion; I: Hypertrophic lesions with pleural effusion were present in the bilateral pleura (red arrows), indicating pleural involvement of the tuberculosis infection. The pleural effusion was bloody, with an elevated level of adenosine deaminase, which was detected in aspirates and reinforced the diagnosis of tuberculosis. Figure 3 Abdominal computed tomography during aplastic crisis. A-E: The wall of the distal ileum was hypertrophically thickened and adhered, and the lumen of the terminal ileum was collapsed (yellow arrow). The lumen of the proximal small intestine was filled with liquid, with several gas—liquid levels. Hypertrophic lesions were also present in several segments of the colon (red arrows), and the lumen of the colon was dilated. In other segments of the colon, the wall showed fibrotic thickening. Notably, panabdominal silt-like fat proliferation led to widening of the bowel loop, forming the so-called “creeping fat sign” and indicating the presence of active chronic gut inflammatory conditions in the small intestine; F-I: In the right iliac fossa, there was an effusive lesion (purple arrow), indicative of peritoneal involvement. A gas—liquid level was present in the distal ileum (green arrows), proximal to which the ileal wall showed hypertrophic thickening (white arrows). The wall of the bladder also showed hypertrophic thickening, with an exudative lesion outside of the hypertrophic bladder wall (blue arrows). An adhered bowel loop was present in the proximal ileum (orange arrows), with the fibrotic thickening of the peritoneum forming the so-called “abdominal cocoon”. These imaging features, together with the imaging features on chest computed tomography, suggested a diagnosis of tuberculosis infection involving the gastrointestinal tract, peritoneum and urinary tract. FINAL DIAGNOSIS The patient was diagnosed with SAA secondary to LGLL. TREATMENT The patient was tentatively treated with a combination of intravenous antibiotics [kanamycin (0.5 g/d) and levofloxacin (0.5 g/d) for 2 wk] for the inflammatory episode, antituberculotics [a combination of rifampicin (0.45 g/d), isoniazid (0.3 g/d), ethambutol (1.0 g/d), and pyrazinamide (1.0 g/d) for 2 mo and subsequently a combination of rifampicin and isoniazid for 6 mo] for the suspected tuberculosis infection, cyclosporine (100 mg bid) to repress the deranged cellular immune responses[16,17], and recombinant human granulocyte colony-stimulating factor (rhG-CSF) for the severe neutropenia. Supportive care was mandatory, without hemolysis or platelet transfusion refractoriness. OUTCOME AND FOLLOW-UP Three weeks of treatment led to significant improvement in the hematological parameters, disappearance of the expanded atypical lymphocytes, and an increased marrow cellularity. The patient’s inflammatory symptoms were gradually ameliorated, and his performance status was significantly improved. The patient was released, and antituberculosis treatment was continued. One month after release from our hospital, he achieved hematological remission. CBC monitoring at the peak showed the following: WBCs, 4.72 × 109/L; ANC, 3.35 × 109/L; RBCs, 3.64 × 1012/L; Hb, 137 g/L; and Plts, 281 × 109/L. This treatment response indicated that disseminated tuberculosis was responsible for the aplastic crisis, leukemic cell regression and atypical lymphocyte expansion. However, the hematological response lasted for only 2 mo, and cytopenia reemerged, with an increase in bone marrow cellularity and the percentage of myeloblasts. Hypomethylation therapy was reinstituted. The patient had received 19 mo of hypomethylation therapy [a total of 3 courses of decitabine (25 mg/d × 5 d) and 10 courses of azacitidine (100 mg/d × 7 d)]. The disease phenotype transformed into AML during hypomethylation therapy. After the transformation, He denied further antileukemic treatment and eventually died of disease progression and an overwhelming infectious episode. DISCUSSION In this manuscript, we describe an episode of reversible aplastic crisis in a patient with advanced MDS. The patient was definitively diagnosed with MDS-EB-1 primarily based on an increase in the percentage of myeloblasts on morphological examination of bone marrow smears and slices and the identification of unfavorable somatic mutations in myeloid neoplasm-associated genes, the two most significant parameters in the diagnosis and risk stratification of MDS[1-4]. Initially, the bone marrow was hypercellular. With the development of an inflammatory episode during hypomethylation therapy, the bone marrow became aplastic, with the infiltration of morphologically atypical lymphocytes. Meanwhile, the leukemic cells regressed. Immunotyping analysis of the atypical lymphocytes revealed high CD3, CD8, CD5, CD16, CD56 and CD57 expression. Disseminated tuberculosis was suspected in the search for an inflammatory niche. Tentative treatment with antituberculotics resulted in the reversion of bone marrow cellularity, disappearance of atypical lymphocytes and reappearance of leukemic clones, providing strong evidence for disseminated tuberculosis as the contributor to the phenotypic transformations. This case study revealed the following attractive points: The first attractive point of this case study is that active tuberculosis infection can trigger bone marrow-predominated autoimmunity in patients with advanced MDS, inducing an episode of aplastic crisis. In this patient, aplastic crisis developed during an inflammatory condition, and autoimmune pathogenesis predominated in the bone marrow. Organ-specific autoimmunity suggests that the targeted antigens are located in the bone marrow. Antituberculosis treatment reversed the marrow-predominated autoimmunity, providing strong evidence that active tuberculosis was responsible for the development of the aplastic crisis. The relief of hematopoietic suppression after successful treatment of the underlying infection suggested that the autoimmune hematopoietic failure was fueled by inflammatory stresses. Tuberculosis-associated aplastic cytopenia has been reported in a few studies[18-22]. Tuberculosis-associated aplastic cytopenia has even been reported in Bacillus Calmette-Guerin vaccination[23]. Tuberculosis infection can trigger cellular immune responses and lead to the excessive production of proinflammatory cytokines[24,25], thereby inducing immune-mediated hematopoietic injury[26,27]. Gut involvement of tuberculosis infection likely plays a more important role because tuberculosis-induced aplastic cytopenia has been reported merely in disseminated tuberculosis instead of isolated pulmonary tuberculosis. Gastrointestinal tuberculosis induces not only inflammatory lesions of its own but also gut dysbiosis, resulting in increased epithelial permeability and compromised barrier function in infected and downstream intestinal segments[28,29]. The gastrointestinal tract is an organ that hosts the most abundant lymphatic tissues and microbial community and therefore can provide sufficient activated immune cells and continuously supply exogenous antigens from both pathogenic and commensal microbes to initiate and perpetuate deranged autoimmunity in the context of increased epithelial permeability and gut inflammatory conditions[30-33], leading to autoimmune hematopoietic failure[34-37]. In our retrospective study to explore the inflammatory conditions of SAA patients during inflammatory flare-ups, 5 of 17 recruited patients exhibited imaging abnormalities suggestive of tuberculosis infection, all involving the gastrointestinal tract and suggesting a high frequency of tuberculosis infection in autoimmune hematopoietic failure[38]. Gut inflammatory conditions in the sustenance of autoimmune responses have been demonstrated by the fact that the onset of an autoimmune disease cannot occur in a germ-free state[39-41]. The second attractive point is that active tuberculosis infection can induce CD56+ lymphocyte proliferation. The concentrated chromatin, abundant cytoplasm, canthous membrane and eosinophilic shade indicated that the lymphocytes were activated. Immunological analysis confirmed that the atypical lymphocytes were activated autoimmune CTLs and NK/NKT cells. The disappearance of atypical lymphocytes with reversion of the aplastic crisis following antituberculosis treatment provided convincing evidence that tuberculosis infection activated CTLs and CD56+ cells. It is reasonably concluded that active tuberculosis stimulated CD56+ cell expansion and in turn suppressed normal and leukemic hematopoiesis. NK and NKT cells are important effector cells in the defense against tuberculosis infection, and tuberculosis antigens can directly stimulate NK/NKT cell proliferation[24,25]. Activated NK/NKT cells can suppress host hematopoiesis via secretion of excessive hematopoietic inhibitors, resulting in autoimmune hematopoietic failure[12,13]. However, in patients with SAA secondary to LGLL, tuberculosis is seldom considered the original trigger. More interestingly, visible granules were absent from the activated CD56+ cells, which is distinctive from LGLL with respect to morphology. The third attractive point is that the leukemic clones were concealed during aplastic crisis, and following antituberculosis treatment, the leukemic clones reemerged after a short duration of hematological remission. This finding suggests that tuberculosis-induced autoimmunity suppressed both normal and leukemic hematopoiesis, with preferential suppression of leukemic cells[42-44]. Neoplasm-associated antigens or damage-associated molecular patterns on genetically defective HPCs likely initiate the primary inflammatory niche in the bone marrow environment and determine organ specificity[45-47]. However, engagement of an exogenous stimulation is critically required in the induction of autoimmune hematopoietic failure[48-50]. Inflammatory stresses due to disseminated tuberculosis fuel organ-specific autoimmunity, resulting in the development of an aplastic crisis. In this sense, autoimmune hematopoietic failure may function as an antileukemic mechanism[51], and disseminated tuberculosis may strengthen immune surveillance against malignant proliferation. However, the leukemic clones cannot be eradicated, resulting in relapse after a short duration of hematological remission following antituberculosis treatment, similar to the process of transient “spontaneous remission”[52,53]. Chronic autoimmune hematopoietic failure is likely caused by inflammatory stress-fueled antileukemic activities targeting genetically damaged HPCs in the context of an active chronic inflammatory condition. We previously reported a patient with refractory SAA who progressed to a myelodysplastic/myeloproliferative neoplasm during the consecutive administration of gut-cleansing preparations (GCPs) and microbiota-modulating treatments[54], but prior intermittent GCPs achieved several cycles of hematopoietic recovery and disease relapse without evident leukemic cell expansion[55]. These data indicated that inflammatory stresses rather than a specific infection fuel organ-specific autoimmunity[34,45,48,50,56]. Limitations of the case study include the lack of an etiopathological diagnosis for disseminated tuberculosis and lack of identification of proinflammatory inhibitors. In addition, the clonality of the atypical lymphocytes was not determined. CONCLUSION Taken together, the results of this case study show that disseminated tuberculosis infection can stimulate the activation and expansion of CD56+ cells and that activated CD56+ cells can suppress both normal and leukemic hematopoiesis, resulting in aplastic crisis and leukemic cell regression in advanced MDS. Antituberculosis treatment can reverse autoimmunity. This inflammatory stress-fueled autoimmunity may represent an antileukemic mechanism, which could be helpful in elucidating the pathogenesis of autoimmune hematopoietic failure and immunological treatments for myeloid neoplasms. Disseminated tuberculosis infection, especially gut involvement, should be given particular attention in autoimmune hematopoietic failure. ACKNOWLEDGEMENTS The authors thank Bin-Han Gao (Department of Radiology, The Central Hospital of Qingdao West Coast New Area) for assistance in the reassessment of the computed tomography images. Informed consent statement: Informed written consent was obtained from the patient to publish this case report and any accompanying laboratory data. Conflict-of-interest statement: The authors have no conflicts of interest to declare that are relevant to the content of this article. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016). Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: April 10, 2023 First decision: May 12, 2023 Article in press: May 31, 2023 Specialty type: Hematology Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Kupeli S, Turkey; Oley MH, Indonesia S-Editor: Ma YJ L-Editor: A P-Editor: Zhao S ==== Refs 1 Li H Hu F Gale RP Sekeres MA Liang Y Myelodysplastic syndromes Nat Rev Dis Primers 2022 8 74 36396662 2 Cook MR Karp JE Lai C The spectrum of genetic mutations in myelodysplastic syndrome: Should we update prognostication? 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A report of 2 cases S Afr Med J 1986 69 451 452 3961639 21 Demiroğlu H Ozcebe OI Ozdemir L Sungur A Dündar S Pancytopenia with hypocellular bone marrow due to miliary tuberculosis: an unusual presentation Acta Haematol 1994 91 49 51 8171938 22 Mangion PD Schiller KF Disseminated tuberculosis complicated by pancytopenia Proc R Soc Med 1971 64 1000 23 Long HJ Aplastic anemia, a rare complication of disseminated BCG infection: case report Mil Med 1982 147 1067 1070 6817203 24 Kathamuthu GR Sridhar R Baskaran D Babu S Dominant expansion of CD4+, CD8+ T and NK cells expressing Th1/Tc1/Type 1 cytokines in culture-positive lymph node tuberculosis PLoS One 2022 17 e0269109 35617254 25 Cao X Xin H Zhang H Liu J Pan S Du Y Feng B Quan Z Guan L Shen F Liu Z Wang D Zhang B Guan X Yan J Jin Q Gao L The Association Between Mycobacteria-Specific Antigen-Induced Cytokines and Host Response to Latent Tuberculosis Infection Treatment in a Chinese Population Front Microbiol 2021 12 716900 34484159 26 Li F Liu X Niu H Lv W Han X Zhang Y Zhu B Persistent stimulation with Mycobacterium tuberculosis antigen impairs the proliferation and transcriptional program of hematopoietic cells in bone marrow Mol Immunol 2019 112 115 122 31082645 27 Li F Ma Y Li X Zhang D Han J Tan D Mi Y Yang X Wang J Zhu B Severe persistent mycobacteria antigen stimulation causes lymphopenia through impairing hematopoiesis Front Cell Infect Microbiol 2023 13 1079774 36743311 28 Jensen SK Pærregaard SI Brandum EP Jørgensen AS Hjortø GM Jensen BAH Rewiring host-microbe interactions and barrier function during gastrointestinal inflammation Gastroenterol Rep (Oxf) 2022 10 goac008 35291443 29 Lu Y Li Z Peng X Regulatory effects of oral microbe on intestinal microbiota and the illness Front Cell Infect Microbiol 2023 13 1093967 36816583 30 Guven-Maiorov E Tsai CJ Nussinov R Structural host-microbiota interaction networks PLoS Comput Biol 2017 13 e1005579 29023448 31 Kayama H Okumura R Takeda K Interaction Between the Microbiota, Epithelia, and Immune Cells in the Intestine Annu Rev Immunol 2020 38 23 48 32340570 32 Mu Q Kirby J Reilly CM Luo XM Leaky Gut As a Danger Signal for Autoimmune Diseases Front Immunol 2017 8 598 28588585 33 An J Liu Y Wang Y Fan R Hu X Zhang F Yang J Chen J The Role of Intestinal Mucosal Barrier in Autoimmune Disease: A Potential Target Front Immunol 2022 13 871713 35844539 34 Zhao XC Sun XY Zhao L Meng FJ Gut inflammation in the pathogenesis of acquired aplastic anemia Chin Med J (Engl) 2020 133 1878 1881 32568881 35 Naithani R Mahapatra M Kumar R Rai S Aplastic anemia and Crohn's disease - coincidence or association? 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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4729 10.12998/wjcc.v11.i19.4729 Academic Writing Revitalizing case reports: Standardized guidelines and mentorship Jeyaraman M et al. Revitalizing case reports in medical literature Jeyaraman Madhan Department of Orthopaedics, ACS Medical College and Hospital, Chennai 600056, Tamil Nadu, India. madhanjeyaraman@gmail.com Ramasubramanian Swaminathan Department of General Medicine, Government Medical College, Omandurar Government Estate, Chennai 600018, Tamil Nadu, India Jeyaraman Naveen Department of Orthopaedics, Shri Sathya Sai Medical College and Research Institute, Sri Balaji Vidyapeeth, Chengalpet 603108, Tamil Nadu, India Nallakumarasamy Arulkumar Department of Orthopaedics, All Indian Institute of Medical Sciences, Bhubaneswar 751019, Odisha, India Sharma Shilpa Department of Paediatric Surgery, All India Institute of Medical Sciences, New Delhi 110029, India Author contributions: All authors contributed equally to writing the manuscript. Corresponding author: Madhan Jeyaraman, MS, PhD, Assistant Professor, Research Associate, Department of Orthopaedics, ACS Medical College and Hospital, Dr. MGR Educational and Research Institute, Chennai 600056, Tamil Nadu, India. madhanjeyaraman@gmail.com 6 7 2023 6 7 2023 11 19 47294733 3 5 2023 30 5 2023 9 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. Despite a decrease in publication, case reports remain essential in medical literature as they offer detailed descriptions of individual patient cases and valuable insights for future management. These reports adhere to a standardized structure comprising sections such as abstract, introduction, case report, discussion, and conclusion. Obtaining informed consent and adhering to guidelines is essential. Case reports contribute to evidence-based medicine by detecting new therapies and adverse events. They also facilitate clear reporting, guideline adherence, and mentorship programs. These reports are vital for documenting rare occurrences, assisting clinicians in timely management, and communicating novel information to busy medical professionals. Following case report guidelines ensures comprehensive and standardized reporting, enhancing the acceptance and quality of case reports, and advancing medical knowledge. Case report Case report guidelines Literature Research ==== Body pmc Core Tip: Despite occupying a lower tier within the evidence hierarchy, case reports serve as one of the foundational element for generating hypotheses that can be explored through more rigorous research methodologies. Adherence to the case report guidelines is crucial, alongside obtaining informed consent from patients, in order to maintain ethical standards and ensure the legitimacy of the case report. To guarantee the quality and rigor of the case report, it is essential to meticulously document the details in accordance with the specific requirements set forth by the target academic journal. INTRODUCTION Case reports have long been regarded as an essential component of medical literature. They provide detailed descriptions of individual patient symptoms, diagnoses, treatments, and follow-ups, offering valuable lessons and experiences that can guide future management of similar situations or cases. Although case reports may be at the bottom of the literature hierarchy, they can serve as the starting point for other evidence which builds on top of them, helping to identify new clinical conditions, observations, treatments, or complications[1]. In recent years, however, the publication of case reports has drastically reduced due to a variety of factors, including a reduction in the impact factor of journals as case reports are less cited than original articles, plagiarism, and authorship conflicts. This paper aims to discuss the format of a case report, common reasons for rejection, and ways to improve acceptance. It focuses on improving writing skills for novice authors and emphasizes the importance of reporting rare or unusual clinical conditions, previously unreported diseases, unusual side effects of therapy, and the unique use of diagnostic tools. STRUCTURE OF A CASE REPORT A standardized case report generally adheres to a specific structure, encompassing five primary sections: Abstract, Introduction, Case Report, Discussion, and Conclusion. Each section plays a crucial role in the overall presentation and coherence of the case report. Abstract: The abstract concisely summarizes the case, capturing its key elements within a limited word count. It includes the patient's demographics, clinical presentation, diagnostic investigations, treatment approach, and notable findings or outcomes, aiming to captivate readers and encourage further exploration of the paper's contents. Introduction: The Introduction contextualizes the case by providing background information and its significance within the existing literature. It incorporates a comprehensive review of relevant published information, summarizing it coherently to set the stage for the presentation of the case. The Introduction establishes the case's context, existing knowledge, and highlights the unique contributions and novel information it offers to the medical community. Case Report: The Case Report section provides essential information about the case, divided into several subsections: Demographics: Covers patient's age, gender, and ethnicity while ensuring confidentiality. History: Details the patient's medical history, including chief complaint, symptom duration, and relevant medical, surgical, and family history. Clinical Examination: Describes the patient's vital signs, general appearance, and system-specific findings, emphasizing unique aspects and using concealed patient images with consent. Investigations: Outlines diagnostic tests conducted, including laboratory tests and imaging studies, along with rationale and results. Treatment: Discusses therapeutic interventions, including modality, dosage, duration, rationale, efficacy, side effects, and modifications. Follow-up: Documents the patient's progress and outcomes during subsequent visits, especially for chronic conditions, including histopathologic examination results for surgical cases and relevant management. Discussion: The Discussion section analyzes and interprets the case findings in relation to existing knowledge and similar reported cases. It incorporates a literature review and focuses on the unique aspects of the case, comparing and contrasting it with previous cases, and providing potential explanations for the observed findings. It addresses the strengths and limitations of the case, mentions encountered challenges or pitfalls, and discusses the implications for clinical practice and research. Conclusion: The Conclusion section summarizes the key points and findings of the case report, highlighting its significance and unique features. It may include recommendations or suggestions for future research, clinical practice, or patient management. The specific content of this section can vary based on the target journal for submission. In addition to the abovementioned sections, obtaining documented informed consent from the patient is an indispensable prerequisite before publishing a case report. Reporting a case without informed consent is deemed unethical. Authors should safeguard the patient's anonymity and secure consent for any images used in the report. By adhering to the structure and guidelines outlined above, authors can ensure a coherent and well-structured case report that may have a higher likelihood of acceptance in medical journals. Each section should be written in an academic tone, using clear and concise language that effectively conveys pertinent information while maintaining the reader's interest. STRATEGIES FOR PUBLISHING A CASE REPORT Figure 1 outlines the strategies involved in publishing a case report. The initial step in writing a case report involves finding a suitable mentor, as research has demonstrated that mentorship positively impacts the quality of case reports[1]. Ensuring the novelty of the case is crucial, necessitating a comprehensive literature review. Subsequently, the report can be drafted in accordance with the provided guidelines and the case report guidelines (CARE guidelines). Once the report is written, it is important to select an appropriate journal that aligns with the specific case. During this process, potential pitfalls should be considered, and measures should be taken to maximize the chances of publication, which are explained below. Maintaining patient privacy is of paramount importance, and it should never be compromised. Professional ethics must be upheld throughout the publication process by keeping all relevant parties well-informed and providing appropriate acknowledgment in the final publication. Additionally, proofreading the manuscript and ensuring its adherence to the journal's guidelines are crucial steps before submission and publication. Figure 1 Strategies for publishing a case report. Several pitfalls may be encountered during the publication process of case reports, such as poor manuscript quality, failure to conform to journal requirements, and rejection due to the low level of evidence provided by case reports in the literature hierarchy. To avoid these issues, authors can follow the CARE guidelines, which provide a 13-point checklist for writing case reports[1-3]. Adherence to the submitting journal's requirements also improves the likelihood of acceptance. Furthermore, finding an experienced mentor is crucial for success in the academic realm, as they can guide authors through the process of selecting a case, writing the report, and finding an appropriate journal for publication. Despite their limitations, case reports play a crucial role in medical research. Case reports, due to their narrow scope and limited readership, typically have limited generalizability and make minimal contributions to the scientific knowledge base, resulting in infrequent citations and potential negative impact on a journal's Impact Factor, while their specialized nature attracts a small number of readers with specific expertise in the field[4]. Nonetheless, case reports serve as essential tools for medical progress by describing unusual or novel occurrences, and providing rapid communication between busy clinicians who may lack the time or resources for large-scale research[1]. A well-written case report can demonstrate the coexistence of evidence-based medicine and the rare findings of an individual patient[5]. Similarly, case reports and case series in dermatology and medicine can serve as the first line of evidence for new therapies and help detect rare adverse events[6]. To improve the reporting of case reports and series, authors should provide a clear and concise title, fully describe the patient's history and intervention, report adverse events, discuss relevance to current medical knowledge, obtain informed consent, and follow the CARE guidelines. Workshops and mentorship programs can help trainees and academic faculty improve their skills in writing case reports[7]. A retrospective pre-post survey has shown that participant confidence in identifying and writing case reports improves with mentorship[7]. Writing and publishing case reports in the medical field remain vital, despite the challenges and limitations associated with them. Case reports are easy to write, relevant to daily clinical practice, and provide a foundation for further research. Reasons for case report rejection may include inadequate formatting, poor manuscript writing, plagiarism, data manipulation, or a low level of evidence. To overcome these issues, authors should follow the submitting journal's guidelines, edit their submissions, avoid plagiarism, ensure data integrity, and focus on novel techniques, unexpected outcomes, or therapeutic challenges[8]. CONCLUSION Ultimately, the importance of case reports in medical research cannot be understated. Despite their limitations and the challenges associated with their publication, case reports offer valuable insights into rare clinical conditions, previously unreported diseases, unusual side effects of therapy, and the unique use of diagnostic tools. By adhering to the CARE guidelines, obtaining proper mentorship, and focusing on the quality and novelty of the case report, authors can increase the likelihood of acceptance and contribute to the advancement of medical knowledge. Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article. Provenance and peer review: Invited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: May 3, 2023 First decision: May 25, 2023 Article in press: June 9, 2023 Specialty type: Medicine, research and experimental Country/Territory of origin: India Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Mahmoud MZ, Saudi Arabia; Watanabe T, Japan S-Editor: Li L L-Editor: A P-Editor: Ju JL ==== Refs 1 Sun Z Tips for writing a case report for the novice author J Med Radiat Sci 2013 60 108 113 26229618 2 Riley DS Barber MS Kienle GS Aronson JK von Schoen-Angerer T Tugwell P Kiene H Helfand M Altman DG Sox H Werthmann PG Moher D Rison RA Shamseer L Koch CA Sun GH Hanaway P Sudak NL Kaszkin-Bettag M Carpenter JE Gagnier JJ CARE guidelines for case reports: explanation and elaboration document J Clin Epidemiol 2017 89 218 235 28529185 3 CARE Case Report Guidelines CARE Case Report Guidelines. [cited 3 June 2023]. Available from: https://www.care-statement.org 4 Khalil S Mishra D Sharing Clinical Experience with the Scientific Community: How to Write a Case Report? Indian Pediatr 2016 53 513 516 27376606 5 Guidelines To Writing A Clinical Case Report Heart Views 2017 18 104 105 29184619 6 Albrecht J Werth VP Bigby M The role of case reports in evidence-based practice, with suggestions for improving their reporting J Am Acad Dermatol 2009 60 412 418 19231639 7 Gupta S Kimble A Henry T Burger A Mhaskar R Block L A Case for Case Reports: How to Write One and Promote Mentorship, Scholarship and Faculty Development Cureus 2023 15 e33299 36741651 8 Aneja K Shyam A Case Reports: The Art of Writing, the Importance of Publishing it and Avoiding Facing Rejection J Orthop Case Rep 2022 12 106 107

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4504 10.12998/wjcc.v11.i19.4504 Minireviews Long-term effectiveness, outcomes and complications of bariatric surgery Gulinac M et al. Bariatric surgery state-of-the-art Gulinac Milena Department of General and Clinical Pathology, Medical University of Plovdiv, Plovdiv 4000, Bulgaria. mgulinac@hotmail.com Miteva Dimitrina Georgieva Department of Genetics, Sofia University "St. Kliment Ohridski", Faculty of Biology, Sofia 1164, Bulgaria Peshevska-Sekulovska Monika Department of Gastroenterology, University Hospital Lozenetz, Sofia 1407, Bulgaria Novakov Ivan P Department of Thoraco-abdominal Surgery, Medical University Plovdiv, Plovdiv 6000, Bulgaria Antovic Svetozar University Clinic for Digestive Surgery, Medical Faculty, Skopje, Skopje 1000, North Macedonia Peruhova Milena Department of Gastroenterology, Heart and Brain Hospital, Burgas, Burgas 1000, Bulgaria Snegarova Violeta Clinic of Internal Diseases, Naval Hospital - Varna, Military Medical Academy, Medical Faculty, Medical University, Varna 9000, Bulgaria Kabakchieva Plamena Clinic of Internal Diseases, Naval Hospital - Varna, Military Medical Academy, Varna 9010, Bulgaria Assyov Yavor Clinic of Endocrinology, University Hospital "Alexandrovska, Sofia 1431, Bulgaria Department of Internal Diseases, Medical University – Sofia, Sofia 1431, Bulgaria Vasilev Georgi Clinic of Endocrinology and Metabolic Disorders, UMHAT "Sv. Georgi", Plovdiv 4000, Bulgaria Sekulovski Metodija Department of Anesthesiology and Intensive Care, University Hospital Lozenetz, Sofia 1407, Bulgaria Medical Faculty, Sofia University, St. Kliment Ohridski, Sofia 1407, Bulgaria Lazova Snezhina Department of Pediatric, University Hospital "N. I. Pirogov", Sofia 1606, Bulgaria Department of Healthcare, Faculty of Public Health "Prof. Tsekomir Vodenicharov, MD, DSc", Sofia 1527, Bulgaria Tomov Latchezar Department of Informatics, New Bulgarian University, Sofia 1618, Bulgaria Velikova Tsvetelina Medical Faculty, Sofia University St. Kliment Ohridski, Sofia 1407, Bulgaria Author contributions: Milena G, Peshevska-Sekulovska, Novakov I, Antovic S, and Velikova T contributed to conceptualization; Peruhova M, Peshevska-Sekulovska M, Snegarova V, Sekulovski M and Velikova T contributed to resources; Lazova S, Miteva D, Gulinac M, Tomov L, Assyov Y, Kabakchieva P, Georgiev T, Visualization, Miteva D, Peshevska-Sekulovska M contributed to data curation; Gulinac M, and Velikova T contributed to writing – original draft preparation; Miteva D, Peshevska-Sekulovska M, Snegarova V, Peruhova M, Sekulovski M and Velikova T contributed to writing – review & editing; Velikova T contributed to supervision; All authors revised and approved the final version of the manuscript; All of the authors approved the final version of the paper prior to submission. Supported by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, No. BG-RRP-2.004-0008-C01. Corresponding author: Milena Gulinac, MD, PhD, Academic Research, Assistant Professor, Department of General and Clinical Pathology, Medical University of Plovdiv, Bul. Vasil Aprilov 15A, Plovdiv 4000, Bulgaria. mgulinac@hotmail.com 6 7 2023 6 7 2023 11 19 45044512 1 4 2023 17 5 2023 6 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. Dietary imbalance and overeating can lead to an increasingly widespread disease - obesity. Aesthetic considerations aside, obesity is defined as an excess of adipose tissue that can lead to serious health problems and can predispose to a number of pathological changes and clinical diseases, including diabetes; hypertension; atherosclerosis; coronary artery disease and stroke; obstructive sleep apnea; depression; weight-related arthropathies and endometrial and breast cancer. A body weight 20% above ideal for age, gender and height is a severe health risk. Bariatric surgery is a set of surgical methods to treat morbid obesity when other treatments such as diet, increased physical activity, behavioral changes and drugs have failed. The two most common procedures currently used are sleeve gastrectomy and gastric bypass. This procedure has gained popularity recently and is generally considered safe and effective. Although current data show that perioperative mortality is low and better control of comorbidities and short-term complications is achieved, more randomized trials are needed to evaluate the long-term outcomes of bariatric procedures. This review aims to synthesize and summarize the growing evidence on the long-term effectiveness, outcomes and complications of bariatric surgery. Bariatric surgery Long-term outcomes Obesity Roux-en-Y gastric bypass sleeve gastrectomy Effectiveness Safety Quality of life ==== Body pmc Core Tip: The method of bariatric surgery is associated with dramatic weight loss, overall health improvement and, in many cases curing obesity-related comorbidities (i.e., diabetes, high blood pressure, sleep apnea, asthma and other breathing disorders, arthritis, cholesterol problems, gastroesophageal reflux disease, fatty liver, urinary stress incontinence, brain pseudotumor, and more). Furthermore, in cases with a high degree of obesity, undergoing this type of surgical intervention also decreases overall mortality. Simultaneously, physical quality of life improved faster than mental quality of life. INTRODUCTION An imbalance in dietary patterns and excessive nutritional intake may give rise to ailments such as obesity. Despite any purely aesthetic considerations, obesity can be described as an excess of adipose tissue that endangers a person's health; specifically, a body weight exceeding 20% of the ideal weight determined by age, gender, and height is deemed to pose a significant health risk[1]. Bariatric surgery is a set of surgical methods for treating morbid obesity. This procedure has gained popularity recently and is generally considered safe and effective. In addition, some of the authors in recent scientific studies claim that it is effective in the short term, although long-term complications have not been described or are rare[1]. The method is associated with dramatic weight loss, improvement and, in many cases curing obesity-related comorbidities. These diseases include diabetes, hypertension, sleep apnea, bronchial asthma and other respiratory disorders, arthritis, cholesterol-related disorders, gastroesophageal reflux disease, fatty liver disease, urinary stress incontinence, brain pseudotumor, and more. Furthermore, in cases with a high degree of obesity, undergoing this type of surgical intervention also decreases overall mortality[2]. Because of this, the term "metabolic surgery" is becoming more and more critical. There are many different causes of obesity, but it is a fact that the number of people suffering from obesity is constantly increasing. According to the World Health Organization, obesity has tripled in many countries of the European region since 1980, with overweight and obesity affecting 50% of the population in most European countries. Furthermore, it is projected that a significant portion of the global population, precisely 60%, or 3.3 billion individuals, may suffer from the effects of excessive weight gain, with 2.2 billion individuals deemed overweight and an additional 1.1 billion individuals classified as obese, by the year 2030[3]. Bariatric surgery is applied to people who cannot lose weight with diets and sports; have a body mass index (BMI) of 35 or higher, and have accompanying diseases such as metabolic syndrome, diabetes, hypertension, and others; as well as people who have an index above 40 but do not have accompanying diseases. Generally speaking, bariatric surgery aims at one single thing - to reduce the volume of food taken in and its absorption[3]. The global guidelines for bariatric surgery were established after a National Institutes of Health consensus conference in 1991. Even then, early bariatric procedures such as jejunoileal bypass were recognized to carry substantial long-term risks. However, the idea of surgical obesity treatment was primarily rejected. Over the next several years, the limited evidence regarding the safety and effectiveness of bariatric surgery was denied. Subsequently, a consensus panel decided that procedures such as Roux-en-Y gastric bypass (RYGB) and vertical banded gastroplasty (VGB) are safe and effective for patients with body mass index (BMI) ≥ 40 kg/m2 or with BMI ≥ 35 kg/m2 and concomitant medical complications of obesity[4]. In this way, a practice standard was established that had previously been lacking, and at the same time, this type of surgery was legitimized as a surgical discipline. Since then, the clinical evidence base for bariatric surgery has grown tremendously. Bariatric surgery has significant health benefits, such as lowering hyperglycemia or normalizing blood glucose levels, lowering blood pressure and cholesterol, and improving obstructive sleep apnea and diabetes-related micro- and macrovascular complications. The surgical techniques involved in bariatric interventions are varied and involve different interventions[5]. When they include "bypassing" the duodenum, there is also a decrease in the hormone ghrelin and an increase in GLP-1 (Glucagon-like peptide 1) and PYY (Gut hormone peptide, tyrosine-tyrosine peptide), which improves insulin sensitivity, as well as other metabolic parameters. Two recently published meta-analyses of studies following patients for up to 5 years also showed reasonable glycemic control in patients with type 2 diabetes after bariatric surgery[5-8]. During the coronavirus disease 2019 (COVID-19), most surgical procedures, especially elected, were postponed because of the health crisis and public health measures[9], including bariatric surgery. Studies have shown that bariatric surgery leads to more significant weight loss and is more effective in treating type 2 diabetes in obese patients than non-surgical treatments. Long-term observations have also shown that these benefits persist for over five years after the surgery. However, the predicting factors of long-term complications, long-term survival, and the impact on patient mental health and costs must be constantly updated[10]. Nevertheless, like any other surgical procedure, this one hides its own risks, some of which are life-threatening, long-term nutritional complications. Often after this procedure, patients develop anemia and have calcium and vitamin losses, which must be compensated by taking substitutes throughout their lives. Some of the potentially severe complications of bariatric surgery are micro- and macronutrient deficiencies. In addition, stenosis and ulceration of the anastomosis, reflux esophagitis, cholelithiasis, steatohepatitis, and altered pharmacokinetics and dynamics may occur[11-13]. Two additional risks are often overlooked: recurrent calcium oxalate urolithiasis and osteoporosis. Both complications are described more often after RYGB, which bypasses the duodenum, where the calcium is mainly absorbed. Unfortunately, an elevated oxalate excretion after RYGB was described, which is usually challenging to treat. Consistent with calcium malabsorption is the low level of urine calcium. Even so, RYGB-related osteoporosis is not associated with changes in weight or vitamin D metabolism[14]. In this review, we focus on the long-term outcomes, complications – rates and management, and quality of life (QoL) and psychological aspects in patients after bariatric surgery. LONG-TERM OUTCOMES AFTER BARIATRIC SURGERY A systematic review by O'Brien et al[15] reviewed 33 datasets reporting data beyond 10 years after bariatric surgery. There were no surgical fatalities. At 20 years, the weight reduction was 30.1 kg, excess weight loss (EWL) was 48.9%, and total weight loss (%TWL) was 22.2%. Although the reoperation rate was first high, currently, it is significantly lowered with a better band and aftercare follow-up. They included 18 gastric bypass reports, 16 of which were RYGB and two for the laparoscopic One-Anastomosis Gastric Bypass (OAGB) version. At 10 years or longer, the mean %EWL for all gastric bypass combinations was 56.7%, with a mean of 55.4% EWL (for RYGB), 80.9% EWL (for OAGB) and 45.9% (for laparoscopic adjustable gastric banding, LAGB). In addition, the mean weight reduction in the two randomized control trials (RCTs) with LAGB was 55.9% EWL [15]. While long-term efficacy was encouraging, most studies exerted inadequate quality, with typically absent controls and several data gaps (incl. missing % follow-up, reoperation rates, perioperative mortality and morbidity, etc.) in most publications. Just two RCTs were included[16,17]. Angrisani et al[17] found 69% EWL and 46% EWL in RYGB and LAGB, respectively. At 10 years in Australian research comparing LAGB to optimum medical care, the LAGB group had 63% EWL. Three noteworthy RCTs of 5-year results with RYGB vs SG have been reported[18-20]. In 5 years, the SLEEVEPASS research[18] revealed 57% and 49% EWL for RYGB and SG, respectively. The SM BOSS research[19] found that RYGB had a 68% excess BMI loss (EBMIL), and the SG had a 61% EBMIL. Finally, the STAMPEDE research[20] found that RYGB had a TWL of 21.7% and SG had a TWL of 18.5%. We anticipate a lengthier follow-up from this and other similar research. Likely, widespread adoption of bariatric surgery will not occur until more high-quality evidence is released. Van Veldhuisen et al[21] also performed a systematic review and meta-analysis of cardiovascular diseases and bariatric surgery. They include 39 prospective or retrospective cohort studies, but no RCTs. Bariatric surgery lowered the risk of all-cause death [pooled hazard ratio (HR) of 0.55, P < 0.001 vs controls] and cardiovascular mortality [HR 0.59, P < 0.001]. Moreover, bariatric surgery lowered the incidence of heart failure [HR 0.50, P < 0.001], myocardial infarction [HR 0.58, P < 0.001], and stroke [HR 0.64, P < 0.001], but not atrial fibrillation (HR 0.82, P = 0.12) [21]. The extent to which the favorable impact of bariatric surgery is related to total weight loss or if additional ancillary benefits also play a role is an essential consideration. According to a new small mechanistic investigation, the advantages of bariatric surgery are entirely connected to weight loss, with no additional objective benefits[22]. However, numerous other studies have shown that ancillary aspects of the surgery, such as a changed gut hormone production, increased insulin sensitivity, and altered gut microbiome[23], impact the outcomes. Therefore, bariatric surgery is referred to as metabolic surgery[20]. Nonetheless, there is no doubt that the degree of weight loss is critical. For example, one study demonstrated that in non-surgical obese patients, a 20% weight loss was required though rarely achieved, to decrease the long-term major cardiovascular events rate. In contrast, at least 10% weight loss was needed in surgical patients, which is usually easily accomplished[23] and supports the hypothesis that additional metabolic processes improve the benefits of surgery[21]. Despite the potential benefits of bariatric surgery for preventing (and maybe treating) cardiovascular illness, no randomized controlled trials have primarily studied the influence of surgery on cardiovascular events or outcomes. According to the European Society of Cardiology's newly issued guideline for cardiovascular disease prevention, "bariatric surgery for obese high-risk people should be considered when lifestyle adjustment does not result in maintained weight loss," i.e., it is a 2A recommendation[24]. This is a significant departure from the former guideline of 2016[25], in which diet and lifestyle changes were encouraged as mainstay therapeutic choices, and bariatric surgery was not formally recommended. Therefore, preventing or treating cardiovascular illness has had little effect on surgical recommendations thus far[26]. The most significant advice for metabolic surgery is for individuals with obesity and Type 2 diabetes. It is currently regarded as a suitable adjunct to existing conventional care in this patient population[27]. In summary, the findings of this systematic review and meta-analysis demonstrate that bariatric surgery, compared to non-surgical therapy, lowers mortality and the incidence of cardiovascular diseases in individuals with obesity. Therefore, in some instances, bariatric surgery should be explored[21]. In their systematic review and meta-analysis, Buchwald et al[28] focused on the 30-d surgical mortality of 0.1%, 0.5% and 1.1% for simply restrictive surgeries, gastric bypass, and biliopancreatic diversion or duodenal switch, respectively. Diabetes, sleep apnea and hypertension were cured in 76.8%, 85.7% and 61.7%, and improved in 86.0%, 83.6% and 78.5% of patients, respectively. Seventy percent or more of individuals observed an improvement in their hyperlipidemia. The authors concluded that after bariatric surgery, morbidly obese people lost weight effectively. Furthermore, most individuals with diabetes, hyperlipidemia, hypertension, and obstructive sleep apnea had their symptoms entirely resolved or improved[28]. Kang et al's systematic review and meta-analysis examined the efficacy and safety of the three most popular bariatric surgery procedures: RYGB, SG, and LAGB[29]. The findings revealed that BMI reduction and %EWL differed substantially between RYGB and LAGB but not between RYGB and SG. No significant difference in weight loss between RYGB and SG was demonstrated, even though they showed superiority to LAGB. While less efficient in weight reduction, LAGB caused fewer problems than the other two surgical techniques[29]. Still, the outcomes of bariatric surgery depend primarily on the multidisciplinary approach[30]. The desirable outcomes following bariatric surgery are presented in Figure 1. Figure 1 Outcomes and effects of bariatric surgery. COMPLICATIONS AFTER BARIATRIC SURGERY Bariatric surgery is generally safe and, in most cases, effective, but this type of surgical procedure can be associated with severe complications, some of which can be fatal[31]. In general, the complications of bariatric surgery are divided into two major groups: early and late. Early postoperative complications include leaks, stenoses, bleeding, and venous thromboembolic events, while late complications include band erosion, acute obstruction, gallstone disease, Dumping syndrome, ischemia, and megaesophagus or pseudoachalasia and death. Regarding the complications of this type of operative manipulation, many scientific groups work in the field, and publications are increasing yearly. One of the most detailed publications on this topic is the following. Chang et al[32] evaluated 30-d significant complications related to bariatric surgeries. This study comprised 71 American studies from 2003 to 2014 and 107874 patients with a mean age of 44 years and a pre-surgery BMI of 46.5 kg m-2. They received gastric bypass, adjustable gastric banding, or SG[32]. The rate of less than 30-day anastomotic leak was 1.15%, of myocardial infarction - 0.37%; and of pulmonary embolism - 1.17%, where the death rates were 0.12%, 0.37%, and 0.18%, respectively, among all patients[33]. Although, among surgical procedures, SG had a greater anastomotic leak rate (1.21%) than gastric bypass (1.14%), gastric bypass exerted a higher risk for myocardial infarction and pulmonary embolism than other techniques. However, we discovered that complication reporting is poorer than other outcomes. The 30-d rates of the three primary problems following each operation vary from 0% to 1.55%, and the mortality rate after these complications ranges from 0% to 0.64%. Future research documenting issues following bariatric surgery should increase the quality of their reporting[32]. On the other hand, in the scientific work of Stone et al[33], a fascinating relationship between the skin color of the patients, respectively the nationality, with the complications that have occurred. They demonstrated that most studies revealed that Black patients had higher 30-d mortality, morbidity, and duration of stay than White patients. The differences between White and Hispanic patients were mainly non-significant in these outcomes. In conclusion, during 30-d of bariatric surgery, black individuals may encounter a greater risk of adverse events than white patients. Explanations for this discrepancy were limited due to the limits of large multicenter datasets. Longer-term studies that cover additional races and ethnicities and take socioeconomic considerations might help future studies[33]. Chierici et al[34] included 39 papers in their systematic review and meta-analysis to demonstrate that biliopancreatic diversion with duodenal switch provides the best weight loss results (total weight loss on the 1st and 3rd year, %TWL 12.38 and 28.42), followed by single-anastomosis duodenoileal bypass (%TWL 9.24 and 19.13), one-anastomosis gastric bypass (%TWL 7.16 and 13.1), and RYGB (%TWL 4.68 and 7.3) were all superior to re-SG. Furthermore, compared to re-SG, duodenal switch and RYGB are linked with an increased risk of late morbidity (OR: 3.07 and 2.11, respectively). However, no significant difference was seen for the other surgeries. In addition, weight recidivism is most common after re-SG; individuals receiving single-anastomosis duodenal bypass had the lowest rate[34]. Poole et al[35] conducted a survey completed by 138/715 eligible surgeons (19.3%) to evaluate the management of bariatric surgery complications. Approximately 54.3% of respondents had not received training in bariatric surgery during their residency or fellowship. Among the 108 respondents, 66.7% believed that general surgeons should handle post-bariatric surgery complications, while 25.9% lacked confidence in addressing such issues. Most respondents (65.7%) expressed interest in pursuing additional continuing professional development options for managing these complications. The most preferred educational modalities for this purpose included hands-on workshops, online resources, and live webinars, with 67.1% of the participants willing to invest 1-3 h and 42.9% ready to pay over $100 for such resources[35]. Some of the complications following bariatric surgery are presented in Figure 2. Figure 2 Early and late complications of bariatric procedures. QUALITY OF LIFE AFTER BARIATRIC SURGERY A systematic review by Hachem and Brennan revealed that bariatric surgery improved QoL more than other obesity therapies. There were significant disparities in QoL improvements across various forms of bariatric surgery. Improvements in QoL were more likely to happen within the first 2 years after surgery, with physical QoL improving faster than mental QoL[36]. According to the study of 18 identified papers by Sierżantowicz et al[37], bariatric therapy appears to produce a long-term advantage in health-related (HRQOL), particularly its physical component score. However, due to psychological predispositions, certain individuals are less likely to benefit from bariatric therapy, whether in terms of HRQOL or weight loss. In addition, studies with inconsistent and imprecise designs may limit the usefulness of the conclusions. Therefore, the author concluded that early identification of such individuals and providing physical and psychological counseling would improve bariatric treatment results[37]. The first systematic evaluation to present data from all quantitative designs on studies that assessed personality traits was conducted by Summerville et al[38]. Overall, neuroticism appeared to be related to worse HRQOL, whereas extraversion was shown to be associated with greater HRQOL. However, null relationships for these two qualities were also detected. Many authors claim that patients frequently rated bariatric surgery as a life-changing intervention that reduces weight, improves obesity-related comorbidities, and improves the QoL. Yet, many poll respondents considered bariatric surgery hazardous or harmful. Patients from racial minority groups expressed more anxiety about mortality risk, lower weight reduction goals, and varied reasons for pursuing bariatric surgery. Female patients were likelier to have good impressions of bariatric surgery and higher weight loss aspirations[39]. The research revealed a disparity between patient views and bariatric surgery's clinical safety and effectiveness profile. Overestimation of the hazards, false expectations, and unfamiliarity with the outcomes of bariatric surgery were all prevalent findings. These negative attitudes toward bariatric surgery may contribute to its underutilization among eligible patients. In addition, views and motives frequently differed by race, location, gender, and age, highlighting the importance of patient-centered education during the prereferral stage. The literature also revealed widespread misunderstandings about bariatric surgery. More studies should be conducted to investigate the influence of education on patient and public attitudes[39]. Despite the good results of bariatric procedures, the current data show that in a small part of all cases, the most severe complication is a fatal outcome. Perioperative mortality rates improved significantly since the early 2000s, ranging from 0.03% to 0.2%[40]. However, as Chang et al[32] maintained, the mortality rate after late complications ranges from 0% to 0.64%. Based on the meta-analysis by Robertson and colleagues and their studies involving over 3.6 million patients, the deaths were 4707. The pooled analysis showed an overall mortality of 0.08 percent. They found no statistically significant difference between overall, 30-d, 90-d, or in-hospital mortality. However, they found a relationship between the types of surgical interventions and mortality, such as 0.03% for gastric band, 0.05% for SG, 0.09% for one-anastomosis gastric bypass, 0.09% for RYGB, and 0.41% for duodenal switch[41]. CONCLUSION Studies demonstrate that bariatric surgery, compared to non-surgical therapy, lowers mortality and the incidence of cardiovascular diseases in individuals with obesity. Most individuals with diabetes, hyperlipidemia, hypertension, and obstructive sleep apnea had their symptoms entirely resolved or improved. At the same time, the mortality rate after these complications ranges from 0% to 0.64%. QoL improvements, especially mental health. In conclusion, we could say that, according to the literature, bariatric surgery is relatively safe. However, there is still a lack of data describing the frequency of obesity recurrences and persistent conditions. We believe there should be an individual selection of a surgical approach for different patients, according to the degree of obesity, because, as we have seen, some procedures have a much higher risk of complications. But following the analyzes described in detail regarding the ratio of long-term complications and mortality, it becomes clear that these complications have decreased to the extent that the benefit of the manipulation itself is greater than the risks involved. That is the main reason why bariatric surgery is increasingly used every year as a means of choice for patients with morbid obesity. Conflict-of-interest statement: The authors declare no conflict of interest. Provenance and peer review: Invited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: April 1, 2023 First decision: April 13, 2023 Article in press: June 6, 2023 Specialty type: Surgery Country/Territory of origin: Bulgaria Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C Grade D (Fair): D Grade E (Poor): 0 P-Reviewer: Emran TB, Bangladesh; Gupta R, India S-Editor: Liu JH L-Editor: A P-Editor: Liu JH ==== Refs 1 Alfonso Enrique Martínez-Núñez Oscar Ernesto Gamboa-López Montserrat Bacardí-Gascón Arturo Jiménez-Cruz Long-term complications and side effects of bariatric surgery: a systematic review JONNPR 2017; 2: 410-415 2 Frigolet ME Dong-Hoon K Canizales-Quinteros S Gutiérrez-Aguilar R Obesity, adipose tissue, and bariatric surgery Bol Med Hosp Infant Mex 2020 77 3 14 32115585 3 Courcoulas AP Yanovski SZ Bonds D Eggerman TL Horlick M Staten MA Arterburn DE Long-term outcomes of bariatric surgery: a National Institutes of Health symposium JAMA Surg 2014 149 1323 1329 25271405 4 Finks JF Dimick JB An updated National Institutes of Health consensus panel on bariatric surgery JAMA Surg 2014 149 1329 1330 25272144 5 Sheng B Truong K Spitler H Zhang L Tong X Chen L The Long-Term Effects of Bariatric Surgery on Type 2 Diabetes Remission, Microvascular and Macrovascular Complications, and Mortality: a Systematic Review and Meta-Analysis Obes Surg 2017 27 2724 2732 28801703 6 Wu GZ Cai B Yu F Fang Z Fu XL Zhou HS Zhang W Tian ZQ Meta-analysis of bariatric surgery versus non-surgical treatment for type 2 diabetes mellitus Oncotarget 2016 7 87511 87522 27626180 7 Heneghan HM Nissen S Schauer PR Gastrointestinal surgery for obesity and diabetes: weight loss and control of hyperglycemia Curr Atheroscler Rep 2012 14 579 587 23054661 8 El Khoury L Chouillard E Chahine E Saikaly E Debs T Kassir R Metabolic Surgery and Diabesity: a Systematic Review Obes Surg 2018 28 2069 2077 29679334 9 Gulinac M Novakov IP Antovic S Velikova T Surgical complications in COVID-19 patients in the setting of moderate to severe disease World J Gastrointest Surg 2021 13 788 795 34512902 10 Crozet J Pasquer A Pelascini E Robert M Factors influencing bariatric surgery outcomes J Visc Surg 2023 160 S7 S11 36922261 11 Nuzzo A Czernichow S Hertig A Ledoux S Poghosyan T Quilliot D Le Gall M Bado A Joly F Prevention and treatment of nutritional complications after bariatric surgery Lancet Gastroenterol Hepatol 2021 6 238 251 33581762 12 Chevallier JM [From bariatric to metabolic surgery: 15 years experience in a French university hospital] Bull Acad Natl Med 2010 194 25 36; 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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4498 10.12998/wjcc.v11.i19.4498 Minireviews Inequity in the global distribution of monkeypox vaccines Tovani-Palone MR et al. Distribution of mpox vaccines Tovani-Palone Marcos Roberto Department of Research Analytics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Chennai 600077, India. marcos_palone@hotmail.com Doshi Neel Department of Medicine, Pravara Institute of Medical Sciences, Ahmednagar 413736, India Pedersini Paolo IRCCS Fondazione Don Carlo Gnocchi, Milan 20148, Italy Author contributions: Tovani-Palone MR contributed to study conception and design, writing of the original draft, and critical review and editing of the final paper; Doshi N contributed to the writing of the manuscript; Pedersini P contributed to the writing of the manuscript and critical review. Corresponding author: Marcos Roberto Tovani-Palone, PhD, Academic Research, Department of Research Analytics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, 162 Poonamallee High Road, Velappanchavadi, Chennai 600077, India. marcos_palone@hotmail.com 6 7 2023 6 7 2023 11 19 44984503 28 12 2022 26 4 2023 15 5 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. Monkeypox (mpox) has been a public health emergency of international concern that emerged in mid-2022 and has spread to 110 countries. The clinical findings of the disease vary according to the seriousness of the cases. Although its case fatality risk has not been high, a significant percentage of patients require hospitalization. In this context, local initiatives were taken to extend the limited supply of vaccines against the disease; however, such measures have not been sufficient to contain the spread of cases and ensure an equitable distribution of health resources. As a result, endemic regions of low-income countries continue to have insufficient access to mpox vaccination. Despite this and considering the global scope of the disease, there is still little discussion in the literature about the difficulties in achieving adequate vaccination coverage rates for the target population of interest. In this article, we briefly discussed general aspects of the disease, including its surveillance, the current global context of challenges for mpox vaccination, and issues on global allocation of health resources as well as proposed related recommendations. Monkeypox Smallpox vaccine Health inequities Vaccination coverage Vaccination hesitancy Healthcare financing ==== Body pmc Core Tip: The development and implementation of new global public health policies, to ensure greater equity in global health financing in the short term, may lead to positive impacts in the global distribution of monkeypox vaccines. This would provide safer and more appropriate management of both this outbreak and future pandemics. INTRODUCTION Along with the ongoing coronavirus disease 2019 (COVID-19) pandemic, a recent public health threat has put the world on high alert: the global monkeypox (mpox) outbreak. According to World Health Organization (WHO) data, confirmed cases of the disease exceeded 86000 in April 2023, with a spread across 110 countries and a total of over 110 deaths. Based on WHO regions, the Americas and Europe have been the two most affected regions, accounting for over 90% of all cases. However, as an endemic disease in West and Central Africa, greater attention from local governments and health authorities is needed during the current period[1]. In an attempt to gain greater support, sufficient production of vaccines, more equitable access by the target population, and the implementation of medical countermeasures for coordinated global action aimed at combating the spread of cases of the disease, the WHO declared mpox a public health emergency of international concern in July 2022[2,3]. In light of this, local initiatives were taken to extend the limited supply of vaccines during the early outbreak period. Although dose-saving approaches have been implemented in some countries including the United States, such measures have not been sufficient to contain the spread of the disease[4] and ensure equity in the global distribution of mpox vaccines. As a result, African countries in particular have had insufficient access to these vaccines[5,6]. Although the number of cases reported globally peaked in August 2022[7], an unequal distribution of health resources, negating any equitable prospects, could have the capacity to potentially threaten different regions of the world with significant limitations in this regard. Despite this and considering the global scope of the disease, there is still little discussion in the literature about the difficulties in achieving adequate vaccination coverage rates for the target population of interest. Here, we briefly discussed general aspects of the disease, including its surveillance, the current global context of challenges for mpox vaccination, and issues on global allocation of health resources as well as proposed related recommendations. ETIOLOGY AND SPREAD OF MPOX Mpox is a zoonotic viral infection caused by the mpox virus (MPXV). Patients present different symptoms and clinical involvement, which can lead to death in severe cases. Although a smallpox-like rash can be characteristically observed in affected patients[5], the mpox transmission and its case fatality rates are substantially lower compared to smallpox infection[1]. MPXV, in turn, corresponds to a double-stranded DNA orthopoxvirus, belonging to the same genus as smallpox, vaccinia, and cowpox viruses[5]. During the outbreak in 2022, a new lineage and sub-lineages of MPXV were identified. The B.1 lineage, classified as clade IIb for its close relationship to clade II, has been predominant in the worldwide spread of mpox[8,9]. Although this suggests that the current outbreak has a major source of infection[9], knowing how such variants might affect transmissibility, virulence and human adaptation remains a challenge. TRANSMISSION, DIAGNOSIS, AND CLINICAL COURSE OF MPOX Among the means of transmission of MPXV, the most common include skin-to-skin and sexual contact[2]. Similar to severe acute respiratory syndrome coronavirus 2, the diagnosis of MPXV infection is primarily made by polymerase chain reaction, using lesion swabs or body fluids[5,10]. Different studies report incubation periods for MPXV ranging from 7-10 d, with most affected patients having systemic disease and approximately 40% progress to complications. Although mpox-related mortality rates are less than 1%, about 1%-13% of cases may require hospitalization, which is expected to significantly burden health systems[5,11]. Figure 1[2,5,11] illustrates a proposed general mechanism for the transmission cycle and clinical manifestations of mpox in humans. Figure 1 Basic mechanism related to monkeypox in humans. LS: Lymphatic system; mpox: Monkeypox. SURVEILLANCE, CASE INVESTIGATION, AND CONTACT TRACING According to the latest WHO recommendations published in 2022, the following actions of response to the multi-country outbreak of mpox should be prioritized and implemented: (1) Provide accurate information to people who may be at increased risk of simianpox virus infection; (2) Ensure access to pre- and post-exposure mpox vaccination for at-risk populations; (3) Prevent the spread of MPXV; and (4) Protect vulnerable individuals and frontline healthcare workers. From this perspective, it is imminent that doctors working in private or public settings, including hospital surveillance services, immediately report suspected cases to local and national public health authorities and to the WHO. In suspected cases of the disease, the investigation should consist of a clinical examination of the patient in a well-ventilated single room, using appropriate personal protective equipment. Patients should be questioned about possible sources of exposure, and a safe specimen collection should be obtained and forwarded to the respective laboratories[7]. As soon as a suspected case is identified, identification and tracing of all possible contacts should be initiated. Contacts of probable and confirmed cases must be monitored or self-monitored daily for any signs or symptoms for a period of 21 d from the last contact with confirmed cases or contaminated materials during the infectious period. However, in the absence of symptoms, quarantine or exclusion from work is not required during the monitoring period, which in turn differs from initial recommendations for COVID-19 surveillance. In this stage, asymptomatic contacts are encouraged to practice hand hygiene and respiratory etiquette and to avoid contact with children, immunocompromised individuals, and pregnant women. Moreover, known contacts are advised to avoid sexual contact with others during this period. This is due to the fact that the evidence on the possible transmission of MPXV before the onset of symptoms continues to be reviewed. Therefore, regardless of whether or not symptoms are present, it is strongly encouraged that this advice is followed. Finally, non-essential travel is also discouraged[7]. CURRENT CONTEXT OF MPOX VACCINATION In the current context of the global mpox outbreak, the non-replicating modified vaccinia Ankara (MVA) vaccine has been one of the most used for both pre- and post-exposure prophylaxis for people at high risk for mpox. This vaccine is commonly administered subcutaneously in two doses, with a 4-wk interval between them. However, given the shortage of vaccine supplies, different countries have authorized single-dose intradermal administration of the MVA vaccine for adults. Thus, a possible increase in vaccine coverage in target groups could be achieved. In this case, only one-fifth of the volume of the subcutaneous route is needed[5]. Recent research conducted by Bertran et al[12] demonstrated that a single dose of the attenuated MVA-Bavaria Nordic smallpox vaccine is highly protective against symptomatic mpox, making it a useful tool for controlling disease outbreaks when rapid protection is required[12]. Moreover, the use of the intradermal route may increase immunogenicity and induce antibody responses similar to the subcutaneous route[5]. The main target groups for mpox vaccination in newly affected countries are homosexuals, bisexuals, and others at a higher risk of contracting mpox. Health professionals and people who have had contact with infected individuals have also been included due to the possibility of medium and high risk of exposure to MPXV infection. However, despite the worldwide concern of mpox vaccination for vulnerable populations, adequate access to both antiviral treatment and vaccines remains an obstacle in different endemic countries[5,6]. Even with strong recommendations for the need to increase the coverage of mpox vaccines on a global scale from the Global Alliance for Vaccines and Immunization, the Vaccine Alliance, and the Global Fund to Fight AIDS, Tuberculosis and Malaria, their administration has been made available for the most part to populations in North America and Europe, with a severe limitation of supplies to low-income countries[5]. Although the outbreak has subsided in countries such as the United Kingdom and the United States due to the roll-out of vaccines and therapies as well as changes in awareness and social behavior, the same has not occurred in countries in West and Central Africa. According to the only announcement made in 2022 by the African Centers for Disease Control and Prevention regarding the shipment of mpox vaccines, the African Union should receive a donation of only 50000 doses from South Korea by then. These vaccines may be the first in the continent not intended for research, but the number of vaccines is too small for the target population of mpox vaccination campaigns across the continent[6]. In addition to vaccine shortages, delays in the access of tecovirimat, an antiviral authorized for emergency use to treat severe mpox[13], have also been reported in African countries[6]. It is therefore plausible to affirm that Africa still lacks adequate access to vaccination and antiviral treatment[5,6], which is essential for patients with severe manifestations and people at risk of serious disease[5]. However, this was similar to the events that happened during the COVID-19 pandemic when countries in Africa received low numbers of vaccines at a delayed timeframe[14]. It is also possible that cases were underreported[15]. The existence of inequity in the global distribution of mpox vaccines and possible underreporting[6] together with the increase in vaccine hesitancy since the beginning of the COVID-19 pandemic[16] and the resurgence of other previously eradicated infectious diseases[17,18] should call for swift and urgent action. Although mass vaccination of the population is neither required nor recommended for mpox, efforts to contain this global outbreak must focus primarily on ensuring equitable vaccine distribution, with concern focused on low-income and middle-income countries. In addition, other relevant measures must be taken, such as massive global support to enable affected countries to respond equitably and effectively to the outbreak, including increased funding for mpox screening centers. This last action is expected to strengthen the disease surveillance system and contribute to access to both adequate treatment and preventive health services. In short, to effectively control the spread of mpox, broad and efficient measures involving a variety of specific measures (Table 1)[2,4,9] should be implemented. Table 1 Key points to contain the spread of monkeypox[2,4,9] No. Key points 1 Massive fight against misinformation 2 Strong information campaign strategy 3 Support from global health agencies 4 Effective collaboration with local laboratories, hospitals, and universities 5 Commitment of Ministries of Health of affected countries and partners 6 Implementing genome surveillance 7 Standardized and robust pharmacovigilance 8 Collaborative efficacy studies of vaccines with standardized protocols and data collection tools IMPORTANCE OF GLOBAL ALLOCATION OF HEALTH RESOURCES AMID THE MPOX OUTBREAK The lack of equity in the global allocation of health resources may impede the implementation of many relevant actions, especially in low-income and middle-income countries. The conclusions of a recent evidence-based review demonstrate that as global health expenditures continue to grow, they remain unequally distributed around the world[19]. As a result of this issue, a worse situation for the mpox outbreak may be expected in several countries beyond what has been reported, particularly in low-income countries. This is due to the potential for underreporting of cases, which may be related to the occurrence of the disease in rural areas[20] in addition to the scarcity of health resources. This context, added to the persistent weaknesses of many health systems, should generate additional limitations that strongly impact the capacity of surveillance systems and the implementation of effective vaccination programs. In order to improve the situation, comprehensive actions, including a planned allocation of financial resources aimed at implementing new interventional measures and surveillance programs using cheap and quick strategies, such as wastewater monitoring of MPXV DNA[9], are also essential. In addition to countries in Africa not receiving enough mpox vaccines in a timely manner, the recent knowledge gained about MPVX did not change the course of the outbreak in Africa. Therefore, even though there was an increase in world attention focused on MPXV, the resulting benefits were observed, at least initially, in developed countries only. Most research on this topic has been published by researchers based in high-income countries[6], which implies the need for more investments as well as better-targeted funding towards robust and globalized scientific advances aimed at the proper management of the disease and the control of its spread. Furthermore, even though many uncertainties remain about mpox therapies and comparative data on safety and efficacy related to the treatment of affected individuals, this should be yet another viable opportunity to fund the development of predesigned adaptive trial protocols that may enable faster discovery of effective drugs against viruses with pandemic potential[21]. CONCLUSION In light of the current situation, the WHO together with other international alliances could play a key role in the development and implementation of global public health policies to ensure greater equity in global health funding in the short term, which should positively impact the global distribution of mpox vaccines as well as provide better management of this outbreak and future health emergencies. Only after building better health systems will be possible to satisfactorily control this and other imminent global emergencies and move closer to meeting the related sustainable development goals[22]. ACKNOWLEDGEMENTS We thank the Italian Ministry of Health-Ricerca Corrente 2023 and Saveetha Institute of Medical and Technical Sciences for supporting this study. Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article. Provenance and peer review: Invited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: December 28, 2022 First decision: March 28, 2023 Article in press: May 15, 2023 Specialty type: Medicine, research and experimental Country/Territory of origin: India Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Gajdács M, Hungary; Giacomelli L, Italy; Haque N, Bangladesh S-Editor: Fan JR L-Editor: Filipodia P-Editor: Fan JR ==== Refs 1 World Health Organization 2022 Mpox (Monkeypox) outbreak: global trends [Internet]. [cited 11 April 2023]. Available from: https://worldhealthorg.shinyapps.io/mpx_global/ 2 Ren SY Li J Gao RD 2022 Monkeypox outbreak: Why is it a public health emergency of international concern? World J Clin Cases 2022 10 10873 10881 36338228 3 Wenham C Eccleston-Turner M Monkeypox as a PHEIC: implications for global health governance Lancet 2022 400 2169 2171 35926551 4 Gruber MF Current status of monkeypox vaccines NPJ Vaccines 2022 7 94 35977979 5 Mitjà O Ogoina D Titanji BK Galvan C Muyembe JJ Marks M Orkin CM Monkeypox Lancet 2023 401 60 74 36403582 6 Kozlov M WHO may soon end mpox emergency - but outbreaks rage in Africa Nature 2023 614 600 601 36765257 7 World Health Organization Surveillance, case investigation and contact tracing for mpox (monkeypox): interim guidance, 22 December 2022 [Internet]. [cited 11 April 2023]. Available from: https://www.who.int/ 8 Patiño LH Guerra S Muñoz M Luna N Farrugia K van de Guchte A Khalil Z Gonzalez-Reiche AS Hernandez MM Banu R Shrestha P Liggayu B Firpo Betancourt A Reich D Cordon-Cardo C Albrecht R Pearl R Simon V Rooker A Sordillo EM van Bakel H García-Sastre A Bogunovic D Palacios G Paniz Mondolfi A Ramírez JD Phylogenetic landscape of Monkeypox Virus (MPV) during the early outbreak in New York City, 2022 Emerg Microbes Infect 2023 12 e2192830 36927408 9 Gao L Shi Q Dong X Wang M Liu Z Li Z Mpox, caused by the MPXV of the clade IIb lineage, goes global Trop Med Infect Dis 2023 8 10 Tan NK Madona CP Taylor JF Fourali LH Sehmi JK Stone MJ Pond MJ Cliff PR Pope CF Performance evaluation of the Viasure PCR assay for the diagnosis of monkeypox: a multicentre study J Clin Virol 2023 158 105350 36473345 11 Thornhill JP Barkati S Walmsley S Rockstroh J Antinori A Harrison LB Palich R Nori A Reeves I Habibi MS Apea V Boesecke C Vandekerckhove L Yakubovsky M Sendagorta E Blanco JL Florence E Moschese D Maltez FM Goorhuis A Pourcher V Migaud P Noe S Pintado C Maggi F Hansen AE Hoffmann C Lezama JI Mussini C Cattelan A Makofane K Tan D Nozza S Nemeth J Klein MB Orkin CM SHARE-net Clinical Group Monkeypox virus infection in humans across 16 Countries - April-June 2022 N Engl J Med 2022 387 679 691 35866746 12 Bertran M Andrews N Davison C Dugbazah B Boateng J Lunt R Hardstaff J Green M Blomquist P Turner C Mohammed H Cordery R Mandal S Campbell C Ladhani SN Ramsay M Amirthalingam G Bernal JL Effectiveness of one dose of MVA-BN smallpox vaccine against mpox in England using the case-coverage method: an observational study Lancet Infect Dis 2023 13 Tempestilli M Mondi A Matusali G Mariotti D Pinnetti C Beccacece A Cimini E Mazzotta V Carletti F Faccendini P Maggi F Girardi E Nicastri E Vaia F Antinori A Tecovirimat concentrations and viral suppression in seminal fluid from patients with mpox Lancet Infect Dis 2023 23 531 532 37004668 14 Uwishema O Onyeaka H Alshareif BAA Omer MEA Sablay ALR Tariq R Mohamed RIH Zahabioun A Yousif MYE Chalhoub E Tovani-Palone MR Current context of pneumonia amidst the COVID-19 pandemic in Africa J Contemp Stud Epidemiol Public Health 2021 2 ep21007 15 Tovani-Palone MR Garoli F Shah PA Kamal MA Nawaz FA Underreporting of cases during the COVID-19 pandemic: a worrying warning for Africa J Contemp Stud Epidemiol Public Health 2022 3 ep22001 16 Khawaja UA Franchi T Pedersini P Tovani-Palone MR Declining rates of global routine vaccination coverage amidst the COVID-19 syndemic: a serious public health concern Einstein (Sao Paulo) 2022 19 eED6552 35019041 17 Hill M Pollard AJ Detection of poliovirus in London highlights the value of sewage surveillance Lancet 2022 400 1491 1492 36243023 18 The Lancet Polio eradication: falling at the final hurdle? Lancet 2022 400 1079 36183713 19 Global Burden of Disease 2020 Health Financing Collaborator Network Tracking development assistance for health and for COVID-19: a review of development assistance, government, out-of-pocket, and other private spending on health for 204 countries and territories, 1990-2050 Lancet 2021 398 1317 1343 34562388 20 Moore MJ Rathish B Zahra F Mpox (Monkeypox). 2022 Nov 30. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan- 21 Lindholm DA Kalil AC Déjà vu all over again? Monkeypox and the urgent need for randomised controlled trials Lancet Infect Dis 2023 23 e56 e58 36400066 22 United Nations Sustainable Development Goals. The 17 goals [Internet]. [cited 23 April 2023]. Available from: https: https://sdgs.un.org/goals

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4635 10.12998/wjcc.v11.i19.4635 Case Report Unique Roberts syndrome with bilateral congenital glaucoma: A case report Almulhim A et al. Unique Roberts syndrome with congenital glaucoma Almulhim Amar Department of Ophthalmology, King Saud University, Riyadh 11411, Saudi Arabia. dr.ammar1412@gmail.com Almoallem Basamat Department of Ophthalmology, King Saud University, Riyadh 11411, Saudi Arabia King Saud University Medical City (KSUMC), Riyadh, Saudi Alsirrhy Ehab Department of Ophthalmology, King Saud University, Riyadh 11411, Saudi Arabia Osman Essam A Department of Ophthalmology, King Saud University, Riyadh 11411, Saudi Arabia Author contributions: Almulhim A and Almoallem B contributed to manuscript writing and editing; Osman E and Alsirrhy E contributed to conceptualization and supervision; and all authors have read and approved the final manuscript. Corresponding author: Amar Almulhim, MD, Academic Fellow, Department of Ophthalmology, King Saud University, King Abdullah Road, Riyadh 11411, Saudi Arabia. dr.ammar1412@gmail.com 6 7 2023 6 7 2023 11 19 46354639 29 12 2022 19 3 2023 20 4 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Congenital glaucoma associated with Roberts syndrome (RS) is an unusual and unique condition. No previous report describes this association. A multidisciplinary approach including molecular studies were conducted to reach the final diagnosis. CASE SUMMARY We present a rare case of a 1-wk-old male with RS associated with bilateral congenital glaucoma, left ectopic kidney, and left-hand rudimentary digits. A comprehensive approach was applied by which bilateral non-penetrating glaucoma surgery was performed with good control of intraocular pressure for more than 6 mo. Cytogenetic and molecular testing were conducted and revealed normal measurements. CONCLUSION This report described a case of a male baby with clinical features of RS but with a negative molecular analysis, presenting with left-hand rudimentary digits, bilateral congenital glaucoma, and left ectopic kidney. To the best of our knowledge, this is the first case reported with phocomelia, bilateral congenital glaucoma, and unilateral ectopic kidney. Roberts syndrome Roberts-SC phocomelia syndrome Phocomelia Congenital glaucoma Karyotype Whole exome sequencing Case report ==== Body pmc Core Tip: Roberts syndrome (RS) is an extremely rare disease characterized by a combination of deformities in the lower and/or upper extremities in association with other organ abnormalities. We provide here the first reported case of RS associated with bilateral congenital glaucoma. Bilateral non-penetrating glaucoma surgery was performed to control intraocular pressure and the outcome was excellent. INTRODUCTION Phocomelia with ocular and internal organ abnormalities are reported as part of Roberts syndrome (RS). RS (OMIM #268300; also known as Roberts-SC phocomelia syndrome) was reported by John Roberts in 1919 and is an autosomal recessive condition characterized by prenatal and postnatal growth retardation, skeletal malformation including tetraphocomelia or mesomelia, mental retardation, and craniofacial dysmorphic features[1]. The clinical presentation is widely variable, from mild cases that can be difficult to confirm without a molecular test to the severe spectrum of diseases that present with a typical phenotype. Babies born with severe RS usually die in utero or shortly postpartum, while mildly affected people can survive to middle age[2]. Herein, we report an unusual case of a male baby with clinical features of RS but with a negative molecular analysis, presenting with left-hand rudimentary digits, bilateral congenital glaucoma, and left ectopic kidney. CASE PRESENTATION Chief complaints A 1-wk-old male baby was referred to our hospital in June 2021 with a picture of bilateral congenital glaucoma. History of present illness According to the parents, the deformed left hand with bilateral cloudy cornea were noticed immediately after birth. Personal and family history The baby was the second child of a healthy non-consanguineous couple and a product of an uneventful pregnancy with spontaneous vaginal delivery at 40 wk. Birth weight was 3 kg. There was neither a family history of similar conditions nor a prenatal history of exposure to any known teratogenic medication. Physical examination On physical examination, left-hand rudimentary digits were noticed (Figure 1). The child was active and sucking well. Examinations of the central nervous system, cardiovascular system, abdomen, and genitalia showed all to be normal. Figure 1 Left-hand rudimentary digits. An ocular examination was performed under general anesthesia and showed bilateral buphthalmos (Figure 2), significant corneal edema, and corneal diameter of 11 mm right/11.5 mm left. The patient was treated with travaprost and combined dorzolamide and timolol, and the intraocular pressure (IOP) was 25 mmHg right/39 mmHg left. Fundus examination showed a cup disc ratio of about 0.2 in both eyes. Central corneal thickness ranged between 900 microns and 930 microns in both eyes. Figure 2 Bilateral buphthalmos and corneal haze. Imaging examinations Echocardiogram findings were normal. Abdominal ultrasound showed a left ectopic kidney located in the ipsilateral pelvic region and not in the lumbar area (Figure 3). Both kidneys appeared normal in size and echogenicity. Figure 3 Abdominal ultrasound. The left ectopic kidney was located in the ipsilateral pelvic region (instead of the normal lumbar region). FURTHER DIAGNOSTIC WORK-UP Based on the given detailed ophthalmological and medical assessment, we applied comprehensive genetic testing by performing karyotyping and chromosomal microarray analysis. Karyotype showed normal male (46, XY) with no evidence of clinically significant numerical or structural chromosome abnormalities. Moreover, we performed whole-exome sequencing (WES) that allowed us to screen the whole exome and to focus in particular on the ESCO2 gene that is known to be linked with RS and other genes related to primary congenital glaucoma such as the CYP1B1 and LTBP2 genes. Despite our detailed review of the WES data, negative results were found, with no observed genetic variants or incidental findings. Differential diagnoses Thalidomide-induced phocomelia; Holt-Oram syndrome; Thrombocytopenia with absent radius syndrome; sporadic phocomelia. FINAL DIAGNOSIS Based on the clinical and diagnostic findings, the final diagnosis was RS with bilateral congenital glaucoma and left ectopic kidney. TREATMENT Bilateral non-penetrating deep sclerectomy plus mitomycin C was applied. The IOP was stable for more than 6 mo postoperatively. Written informed consent was obtained from the parents of the baby. The Institutional Review Board reviewed and approved this study. Permission to publish photographs was also obtained. OUTCOME AND FOLLOW-UP At 6 mo, 9 mo, and 1 year postoperatively, the IOP was stable without anti-glaucoma medication. DISCUSSION RS is an extremely rare autosomal recessive condition that presents with widely variable prenatal and postnatal growth retardation, skeletal malformation, and mental retardation. It was first described by Roberts in 1919 in two children of consanguineous parents. The children presented with upper and lower limb anomalies, skull deformity, exophthalmos, cleft lip, and palate bilaterally[3]. In 1969, Afifi et al[1] reported a syndrome with milder features and labeled it as SC phocomelia syndrome. A systematic review and severity scoring system was published by Van Den Berg and Francke[3] in 1993 that included 100 cases. Goh et al[2] and Zhou et al[4] conducted a review of the literature on RS in adult patients. None of the previously reported cases were associated with congenital glaucoma. Ocular manifestations reported in the literature to date have been hypertelorism in 86.7% of patients, exophthalmia in 69.4% of patients, cloudy cornea in 68.1% of patients, optic nerve cavernous hemangioma in 1 patient, and blue sclera with bilateral macular dysfunction in 1 patient. Renal abnormalities were reported in 50% of patients[1-4]. Worldwide about 157 cases of RS have been reported, and the most recent case was published in October 2020 regarding a 30-wk gestation stillborn male who had the typical phenotype of RS with a normal molecular analysis[5]. In our case, we applied comprehensive genetic testing by performing karyotyping and chromosomal microarray analysis. Chromosomal analysis revealed normal male (46, XY) without any detected abnormalities. WES revealed no abnormalities in the ESCO2 gene (linked to RS) or the CYP1B1 and LTBP2 genes (linked to primary congenital glaucoma). Although this patient had normal molecular study findings, the diagnosis of RS can be established based on the phenotype because RS cases without any molecular abnormality have been reported in the literature[5]. CONCLUSION This report describes a case of a male baby with clinical features of RS but with a negative molecular analysis. He presented with left-hand rudimentary digits, bilateral congenital glaucoma, and left ectopic kidney. To the best of our knowledge, this is the first reported case of RS with a combination of phocomelia, congenital glaucoma, and ectopic kidney without any detected molecular abnormality. Informed consent statement: Informed written consent was obtained from the parents for publication of this report and any accompanying images. Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016). Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: December 29, 2022 First decision: February 17, 2023 Article in press: April 20, 2023 Specialty type: Medicine, research and experimental Country/Territory of origin: Saudi Arabia Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Malik S, Pakistan; Morya AK, India S-Editor: Liu XF L-Editor: A P-Editor: Chen YX ==== Refs 1 Afifi HH Abdel-Salam GM Eid MM Tosson AM Shousha WG Abdel Azeem AA Farag MK Mehrez MI Gaber KR Expanding the mutation and clinical spectrum of Roberts syndrome Congenit Anom (Kyoto) 2016 56 154 162 26710928 2 Goh ES Li C Horsburgh S Kasai Y Kolomietz E Morel CF The Roberts syndrome/SC phocomelia spectrum--a case report of an adult with review of the literature Am J Med Genet A 2010 152A 472 478 20101700 3 Van Den Berg DJ Francke U Roberts syndrome: a review of 100 cases and a new rating system for severity Am J Med Genet 1993 47 1104 1123 8291532 4 Zhou J Yang X Jin X Jia Z Lu H Qi Z Long-term survival after corrective surgeries in two patients with severe deformities due to Roberts syndrome: A Case report and review of the literature Exp Ther Med 2018 15 1702 1711 29434756 5 Salari B Dehner LP Pseudo-Roberts Syndrome: An Entity or Not? Fetal Pediatr Pathol 2022 41 396 402 33026893

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4601 10.12998/wjcc.v11.i19.4601 Randomized Controlled Trial Ulinastatin in the treatment of severe acute pancreatitis: A single-center randomized controlled trial Wang SQ et al. UTI against severe AP Wang Su-Qin Department of General Surgery, The 904th Hospital of Joint Logistic Support Force, Wuxi 214044, Jiangsu Province, China Jiao Wei Department of Nursing, The 904th Hospital of Joint Logistic Support Force, Wuxi 214044, Jiangsu Province, China Zhang Jing Department of Nursing, The 904th Hospital of Joint Logistic Support Force, Wuxi 214044, Jiangsu Province, China Zhang Ju-Fen Department of Neurosurgery, The 904th Hospital of Joint Logistic Support Force, Wuxi 214044, Jiangsu Province, China Tao Yun-Na Department of Neurosurgery, The 904th Hospital of Joint Logistic Support Force, Wuxi 214044, Jiangsu Province, China Jiang Qing Department of General Surgery, The 904th Hospital of Joint Logistic Support Force, Wuxi 214044, Jiangsu Province, China Yu Feng Department of General Surgery, The 904th Hospital of Joint Logistic Support Force, Wuxi 214044, Jiangsu Province, China. yufeng904@21cn.com Author contributions: Wang SQ and Jiao W contributed equally to this work; Wang SQ, Jiao W, Zhang J, Tao YN and Yu F designed the research study; Wang SQ, Zhang JF, Tao YN and Jiang Q performed the research; Wang SQ and Zhang JF contributed new reagents and analytic tools; Wang SQ, Jiao W and Yu F analyzed the data and wrote the manuscript; All authors have read and approve the final manuscript. Supported by Wuxi Science and Technology Development Fund, No. WX03-02B0205-072001-10 . Corresponding author: Feng Yu, MM, Doctor, Department of General Surgery, The 904th Hospital of Joint Logistic Support Force, No. 101 Xing Yuan North Road, Wuxi 214044, Jiangsu Province, China. yufeng904@21cn.com 6 7 2023 6 7 2023 11 19 46014611 22 3 2023 16 5 2023 2 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Severe acute pancreatitis (AP) is one of the most common diseases of the gastrointestinal tract and carries a significant financial burden with high disability and mortality. There are no effective drugs in the clinical management of severe AP, and there is an absence of evidence-based medicine concerning the treatment of severe AP. AIM To explore whether ulinastatin (UTI) can improve the outcome of severe AP. METHODS The present research included patients who were hospitalized in intensive critical care units (ICUs) after being diagnosed with severe AP. Patients received UTI (400000 IU) or placebos utilizing computer-based random sequencing (in a 1:1 ratio). The primary outcome measures were 7-d mortality, clinical efficacy, inflammatory response, coagulation function, infection, liver function, renal function, and drug-related adverse effects were evaluated. RESULTS A total of 181 individuals were classified into two groups, namely, the placebo group (n = 90) and the UTI group (n = 91). There were no statistically significant differences in baseline clinical data between the two groups. The 7-d mortality and clinical efficacy in the UTI group were remarkably improved compared with those in the placebo group. UTI can protect against hyperinflammation and improve coagulation dysfunction, infection, liver function, and renal function. UTI patients had markedly decreased hospital stays and hospitalization expenditures compared with the placebo group. CONCLUSION The findings from the present research indicated that UTI can improve the clinical outcomes of patients with severe AP and has fewer adverse reactions. Ulinastatin 7-day mortality Severe acute pancreatitis Randomized controlled trial Outcome ==== Body pmc Core Tip: One of the most prevalent gastrointestinal tract gastrointestinal disorders, severe acute pancreatitis (AP), is the leading cause of hospitalization due to gastrointestinal diseases. The present study suggests that ulinastatin (UTI) can reduce the risk of overall 7-d mortality in severe AP patients and is associated with a higher total effective rate vs the placebo control group. We also found that UTI can protect against hyperinflammation. It also did not significantly increase the incidence rate of severe adverse effects. INTRODUCTION One of the most prevalent gastrointestinal tract gastrointestinal disorders, severe acute pancreatitis (AP), is the leading cause of hospitalization due to gastrointestinal diseases, and carries a significant financial burden[1-3]. The incidence of AP worldwide is approximately 34 per 100000 people per year and continues to rise, and the health care system costs huge billion annually[4]. Although it is generally considered a mild disease with a good prognosis in most AP patients, 15% to 20% of AP patients develop a severe condition with high morbidity and mortality with systemic and local complications[5,6]. Pancreatitis is aggravated by inappropriate treatment, which is responsible for over 25% of mortality associated with severe AP[7,8]. Although surgical treatment is currently the mainstay in clinical practice, a recent meta-analysis confirmed the presence of multiple complications, postoperative body pain, and prolonged postoperative recovery duration after surgical intervention[9]. More researchers are calling for conservative treatment with drugs, especially for severe AP patients who prefer not to undergo surgery, which might be a good try[1,5,7,10,11]. The pathological mechanisms of AP include immune system mediation and complex cascade reactions of inflammatory activation[12]. Severe stress may trigger systemic inflammatory reactions, and hyperinflammation might disrupt coagulation functioning, leaving the patient at a greater risk of diseases. Long-term patient prognosis is affected by disease progression and multiple organ dysfunction syndromes (MODS) caused by an imbalance and high intensity of the systemic inflammatory response syndrome and the compensatory anti-inflammatory response syndrome 13. The ability to prevent and alleviate MODS and successfully manage hyperinflammation after severe AP are crucial early-stage therapy goals[13]. Ulinastatin (UTI) is an inhibitor of serine proteases that has a molecular weight of 67000 and is purified from human urine. This compound's most important pharmacological properties include anti-inflammation, immunomodulation, and protection of organs[14-16]. In the management of acute inflammatory diseases like sepsis and ischemia-reperfusion damage, pharmaceuticals may be used as antiapoptotic and anti-inflammatory agents[17]. By conducting a meta-analysis that included 15 randomized controlled trials (RCTs), He et al[18] found that patients who underwent cardiac surgery could gain benefit from UTI by suppressing postoperative inflammation and offering lung protection[18]. In China, UTIs are extensively utilized in the management of inflammatory diseases, organ preservation during surgery, and the management of shock[19]. A retrospective study enrolled 130 SAP patients and showed that UTI can improve the clinical outcomes of SAP patients, but efficacy varies with the dosage[1]. Another retrospective study enrolled 78 SAP patients and showed that UTI combined with glutamine is effective in treating severe pancreatitis, which efficiently facilitates the recovery of immune, metabolic, and liver functions[5]. Yang and Zhao[7] also reported a retrospective study that enrolled 100 severe AP patients and showed that somatostatin plus UTI is a viable alternative in the treatment of severe AP patients. A meta-analysis of RCTs suggested that UTI demonstrated a favorable benefit in treating acute respiratory distress syndrome patients, but this conclusion has not been verified without a larger sample size of RCTs[20]. However, the effectiveness of UTI following severe AP is still uncertain since these studies had limited sample numbers and there are no evidence-based clinical trials with large sample sizes. Consequently, the focus of this research was to determine whether or not treating severe AP with UTIs would improve patient outcomes and attenuate hyperinflammation. MATERIALS AND METHODS Study design A placebo-controlled, parallel-arm, randomized experiment was carried out in Jiangsu from October 2018 to December 2021. A total of 217 patients were screened over this period, and 181 of these were initially enrolled in the study to form the ITT population. To determine if the intervention is superior, the present research was conducted. It was registered with the registration number CWXH-IPR-2018015 (date: 9/Sep/2018) with protocol approval from the Clinical Research Ethics Committees of the 904th Hospital of PLA endorsed the methodology used in the present research (2018-YXLL-097) and was following the Declaration of Helsinki. The protocol for the research was subjected to approval granted by the Ethics Committees of all the collaborating centers. Those patients whose competence could be demonstrated by their comprehensive awareness of time, place, and person, along with their comprehension of the investigator's explanation of the trial, were asked to obtain written informed consent for the study. In addition, patients were allocated at random (1:1) and were administered an intravenous infusion of either 400000 IU UTI (Guangdong Tianpu Biochemical Pharmaceutical Co., Ltd., H20040506, 2 mL:100000 IU) or placebos dissolved in 250 mL of 0.9% saline given intravenously over 1 h every 12 h for 7 d, the total daily dose is 800000 units (400000 units administered twice)[16]. Infusion could be interrupted for 1 d if there was a greater than the threefold-fold increase in liver enzymes over baseline levels following the procedure (Figure 1). All other treatments were the same. The last check-up was performed 30 d following the procedure. Figure 1 Study design. AP: Acute pancreatitis; UTI: Ulinastatin. Patients enrolled in the study and sample selection procedures Patients were included in the present research in the ICU. SAP was diagnosed following the criteria of the Revised Atlanta Classification[21]. The following were the criteria for inclusion: (1) Patients aged 25 to 70 years; (2) patients who met the diagnostic criteria of severe AP[2]; (3) patients with complete data; and (4) patients who were randomly assigned to receive either UTI or a placebo. The exclusion criteria were as follows: (1) Patients who were unlikely to be salvaged upon admission; (2) allergic to UTI; (3) received anticoagulant medication within 48 h before hospitalization; (4) pregnant women and patients with malignant tumors; (5) treatment with immunosuppressive drugs; (6) multiple organ dysfunction before illness; and (7) other explanations were discovered by researchers. Randomization and concealment With the aid of SPSS software (version: 14.0) (SPSS Institute, Hefei, Anhui Medical University), permuted-block randomization was carried out based on a computer system that used an allotment list to produce random numbers (in a one-to-one ratio). This was carried out by a statistician who was not a member of the research team to maintain the integrity and blinding of the research. The outcomes of the random sampling process were enclosed in prenumbered envelopes and kept at the location of the research until the study's conclusion was reached. The study medicines were delivered by a research nurse following the random assignment sequence. Both the research participants and the patients were unaware of which medicine was being applied in the trial. In the event of an emergency, such as acute hepatic failure, two experts might recommend that the treatment allotment be unmasked and that the study medicine be adjusted or discontinued if needed, according to the protocol. All of the occurrences were recorded in detail. Then, we acquired information on the patient's demographics, medical histories, and pertinent investigation findings. Outcome assessment All clinical and imaging data and treatment were subjected to assessment by a masked independent diagnostic and assessment committee. This committee included two researchers who were trained before the start of the present research and did not engage in the clinical care of patients. The primary endpoint of this study was 7-d mortality. Additionally, we also evaluated the clinical efficacy, and the specific evaluation methods referred to in the previous literature[7] included cured, effective, and ineffective. Cured as follows: Clinical symptoms (including fever, abdominal distension, abdominal pain, etc.) disappeared basically, blood amylase level returned to positive Ranson scores < 3 and Acute Physiology and Chronic Health Evaluation (APACHE)-II scores < 8, vital signs are stable; Effective as follows: The clinical symptoms improved obviously, and the blood amylase level decreased, Ranson score and APACHE-II score decreased significantly. Ineffective as clinical symptoms, laboratory indicators and related scores not improvement. Total effectiveness included cure and effectiveness. The secondary endpoints included: (1) Inflammatory cytokine levels (pretherapy and posttreatment), such as serum tumor necrosis factor-α and interleukin-6; (2) coagulation function, such as the plasma prothrombin time, activated partial thromboplastin time and fibrinogen levels, was measured using an automatic coagulation analyzer; (3) serum infection index levels, such as white blood cell count, C-reactive protein, and procalcitonin (PCT); (4) liver function index levels, such as alanine aminotransferase and aspartate aminotransferase; and (5) renal function index levels, such as creatinine and blood urea nitrogen. Safety evaluation We kept track of the length of time spent in the intensive care unit, and the most prevalent adverse effects of UTI included granulocytopenia and abnormal liver enzymes. Other rare complications include diarrhea, vomiting, and allergies. All complications were confirmed and recorded by physical examination after two doctors and nurses; granulocytopenia was diagnosed by routine blood level detection. Abnormal liver enzymes were diagnosed by liver function tests. Finally, we check the related index every two days over the first 14 d. Inpatient expenditures and length of stay after surgery Extended hospital stays and greater healthcare costs have previously been recorded for individuals who need ICU[22,23]. This study thus compared the two groups in terms of how long they stayed in the hospital and how much their treatment ultimately cost. Estimation of sample size The primary endpoint of a previous study demonstrated that the 7-d mortality rate among those who received the UTI treatment was 5.36%, but it was 26.92% among those who received the placebo. Our decision to recruit 92 patients (n = 46 per group) was based on the results of a calculation that determined the sample size using an alpha value of 0.05 and a statistical power of 80%. We decided to enroll 181 participants in the study (n = 90 per group). Eventually, 91 patients were recruited in the UTI category, whereas 90 were recruited in the placebo category. Every participant's baseline information was recorded in the study database, and the outcomes were recorded by a research nurse. Statistical analysis Baseline data along with outcome measurements were imported into the database with the aid of a research nurse. The data were collected using paper forms and submitted to a digital database that was secured by passwords. The mean ± SD are the reporting formats for any continuous variables. Analyses of statistical data were executed with the SPSS 19.0 statistical tool (SPSS, Inc., Chicago, United States). M (Q1, Q3) was used to denote measurement data that follow a nonnormal distribution. Quantitative data were evaluated utilizing independent-sample t-tests. The χ2 test or Fisher's exact t-test was conducted to evaluate the qualitative data. The criterion for significance was established at P < 0.05. RESULTS From October to December 2021, we assessed 217 elderly patients. In total, 181 participants were randomized at a 1:1 ratio and given either UTI treatment (n = 91) and receiving a placebo (n = 90). No cases of opening blindness were noted during the research period. Also, no remarkable variations in the preliminary data were found between the two cohorts (Table 1). Within the scope of this study, no patients were ever lost to follow-up. Overall, all the patients were included in the final intention-to-treat analysis (Figure 2). We had a concluding appointment with the last patient chosen at random on February 1, 2022. Figure 2 Trial profile. UTI: Ulinastatin; ITT: Intention-to-treat. Table 1 Comparison of baseline data UTI group (n = 91) Placebo group (n = 90) P value Age (year, mean ± SD) 52.7 ± 3.8 53.5 ± 4.1 0.175 Gender, n (%) 0.705     Male 67 (73.63) 64 (71.11)     Female 24 (26.37) 26 (28.88) BMI (kg/cm2, mean ± SD) 22.6 ± 2.1 23.1 ± 2.5 0.147 Cause of disease, n (%) 0.582     Biliary 61 (67.03) 57 (63.33)     Hyperlipidemic 18 (19.78) 19 (21.11)     Alcoholic 12 (13.09) 14 (15.56) Smoking history, n (%) 0.483     Yes 53 (58.24) 57 (63.33)     No 38 (41.76) 33 (36.67) Drinking history, n (%) 0.371     Yes 58 (58.24) 63 (70.00)     No 33 (41.76) 27 (30.00) Living environment, n (%) 0.604     Town 59 (64.84) 55 (61.11)     Countryside 32 (35.16) 35 (38.89) Past medical history, n (%)     Hypertension 31 (34.07) 29 (32.22) 0.792     Hyperlipidemia 35 (38.46) 37 (41.11) 0.716     Diabetes 28 (30.77) 29 (32.22) 0.833     Cholelithiasis 17 (18.68) 16 (17.78) 0.875 Previous pancreatitis, n (%) 9 (9.89) 10 (11.11) 0.789 APACHE-II score on admission (mean ± SEM) 12.6 ± 0.8 12.2 ± 0.8 0.724 Ranson score on admission (mean ± SEM) 3.3 ± 0.3 3.2 ± 0.4 0.842 Necrotizing pancreatitis 23 (25.27) 20 (22.22) 0.727 Time from the onset to diagnosis (hours) 15.2 ± 6.5 14.6 ± 7.1 0.950 Endoscopic bile duct drainage 37 (40.66) 42 (46.67) 0.455 UTI: Ulinastatin: BMI: Body mass index; APACHE: Acute Physiology and Chronic Health Evaluation. The primary endpoint and clinical efficacy The data showed that the total 7-d mortality rate was 14.36% (26/181). Within the UTI category, the 7-d mortality rate was 7.69% (7/91) in contrast with 21.11% (19/90) within the placebo category. A significantly higher 7-d mortality was seen in the placebo category in contrast with the UTI control (P = 0.010, Table 2). When comparing the UTI category to the placebo category, the total effective rate was greater (P = 0.008, Table 2). Table 2 Comparison of 7-day mortality and clinical efficacy, n (%) UTI group (n = 91) Placebo group (n = 90) P value 7-d mortality 7 (7.69) 19 (21.11) 0.010 Efficacy 0.001     Cured 55 (60.44) 34 (37.78)     Effective 28 (30.77) 35 (38.89)     Ineffective 8 (8.79) 21 (23.33) Total effectiveness 83 (91.21) 69 (76.67) 0.008 UTI: Ulinastatin. The secondary endpoints Before UTI or placebo treatment, there was no variation between the UTI and placebo categories in kidney function index levels, hepatic function index levels, serum infection index levels, coagulation functions, or inflammatory cytokine levels (P > 0.05), but these indices were substantially increased in the UTI category relative to the placebo category (P < 0.05, Table 3). Table 3 Comparison of the secondary end-points Before treatment After treatment UTI group (n = 91) Placebo group (n = 90) P value UTI group (n = 91) Placebo group (n = 90) P value Inflammatory cytokine, mean ± SD     TNF-α (pg/mL) 77.28 ± 19.43 76.91 ± 18.27 0.895 29.67 ± 11.19 43.17 ± 12.08 < 0.001     IL-6 (pg/mL) 89.37 ± 16.28 87.91 ± 16.74 0.553 50.18 ± 12.24 66.35 ± 14.26 < 0.001 Coagulation function, mean ± SD     PT (s) 21.05 ± 3.44 20.62 ± 3.19 0.385 12.16 ± 2.84 16.71 ± 3.15 < 0.001     APTT (s) 39.22 ± 4.26 40.15 ± 4.39 0.150 32.26 ± 3.73 37.25 ± 4.02 < 0.001     D-D (mg/L) 4.26 ± 1.12 4.33 ± 1.27 0.694 1.75 ± 0.59 2.94 ± 0.79 < 0.001     FIB (g/L) 1.07 ± 0.23 1.12 ± 0.26 0.172 3.46 ± 0.60 3.28 ± 0.55 0.037 Infection index levels, mean ± SD     CRP (mg/L) 31.29 ± 4.16 30.98 ± 4.07 0.613 5.19 ± 0.97 8.34 ± 1.33 < 0.001     PCT (ng/L) 2.05 ± 0.21 2.10± 0.19 0.095 0.75 ± 0.23 0.82 ± 0.38 0.135     WBC (× 109/L) 18.05 ± 9.11 17.56 ± 8.97 0.716 12.74 ± 6.41 15.19 ± 7.29 0.017 Liver function index levels, mean ± SD     ALT (U/L) 32.20 ± 3.74 33.15 ± 3.68 0.087 48.19 ± 4.27 53.11 ± 4.53 < 0.001     AST (U/L) 34.17 ± 2.83 34.42 ± 2.99 0.564 49.67 ± 3.88 50.27 ± 4.19 0.230 Renal function index levels, mean ± SD     Scr 81.26 ± 9.91 79.64 ± 9.12 0.254 84.22 ± 10.16 105.42 ± 11.20 < 0.001     BUN 5.37 ± 1.94 5.19 ± 1.73 0.511 8.06 ± 2.99 10.19 ± 3.28 < 0.001 TNF-α: Tumor necrosis factor-α; IL-6: Interleukin-6; APTT: Activated partial thromboplastin time; PT: Prothrombin time; FIB: Fibrinogen; CRP: C-reactive protein; PCT: Procalcitonin; WBC: White blood cell; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BUN: Blood urea nitrogen; UTI: Ulinastatin. Safety evaluation Granulocytopenia and abnormal liver enzymes are the two most common negative outcomes that are caused by UTI. Results showed that granulocytopenia affected 8 patients (8.79%) in the UTI category and 4 patients (4.44%) in the placebo category. Hepatic enzyme abnormalities were more common in the UTI category (34.07% vs 27.77%) than in the control group. No substantial variation in the incidence of granulocytopenia (P = 0.240) or abnormal liver enzymes (P = 0.360) existed between the UTI category and the placebo category (Table 4). Additionally, as illustrated in Table 4, the potential UTI-induced adverse events such as diarrhea (20.00% vs 29.67%, P = 0.132) and vomiting (12.22% vs 20.88%, P = 0.085) were not shown to vary significantly between the two groups. The UTI category had a greater rate of allergic reactions than the placebo category (24.18% vs 10.00%, P = 0.029, Table 4). Throughout the intervention, no blind cases were revealed. Table 4 Comparison of safety evaluation, and postoperative hospital stays and costs, n (%) UTI (n = 91) Placebo (n = 90) P value Granulocytopenia 8 (8.79) 4 (4.44) 0.240 Abnormal liver enzymes 31 (34.07) 25 (27.77) 0.360 Diarrhea 27 (29.67) 18 (20.00) 0.132 Vomiting 19 (20.88) 11 (12.22) 0.117 Allergies 22 (24.18) 9 (10.00) 0.029 Hospitalization stays, day, mean ± SD 17.8 ± 4.2 19.6 ± 5.3 0.012 Hospitalization costs, CNY, mean ± SD 3.94 ± 0.82 3.82 ± 0.99 0.376 UTI: Ulinastatin. Inpatient expenditures and length of stay after surgery There was a significant variation in the length of hospital stay between the UTI category (17.8 d) and the placebo category (19.6 d) (P = 0.012). Costs for inpatient treatment were much lower in the UTI category (38200 RMB) in contrast with the placebo category (39400 RMB), although the variation was insignificant (P = 0.376, Table 4). DISCUSSION The present research demonstrate that UTI is linked to a higher total effective rate compared to the placebo treatment, which could lead to a considerable reduction in the incidence of total 7-d mortality in patients with severe AP. We also discovered that UTI has the potential to enhance kidney function, hepatic function, and coagulation function while also protecting against hyperinflammation and infection. Although the synergistic impact after the simultaneous administration of other medications in certain patients could raise the percentage of patients who develop allergies, UTI did not substantially elevate the incidence rate of serious complications other than allergies, which is worth noting. In addition, it reduced the time patients spent in the hospital. Our results indicated a total 7-d mortality rate of 14.36%, which is in line with previous data[1,24-26]. Severe AP can cause substantial complications with high mortality in up to 25% of those affected with hospitalization periods in an ICU within two weeks[27]. Wang et al[24] reported that the mortality in the basic treatment was 20%, while the mortality was 4.8% in the somatostatin + UTI group. Many causes of severe AP have been recorded in the literature, but the pathogenetic theories and the specific cause of severe AP are still controversial. Whitcomb[28] reported that the development and recurrence of AP were linked to composite factors, including genetics, environmental factors, and metabolic processes. Choledocholithiasis and excess alcohol have become the most common causes of severe AP, especially in developed countries and countries with high alcohol intake[29]. With the in-depth study of AP and the development of modern molecular biology, recent studies have shown that gene variants and specific receptor defects may increase the risk of AP in patients, thereby increasing the risk of developing severe AP[27,30]. Intensive care management, infection prevention and identification, nutritional support, and therapeutic drugs are the cornerstones of treating severe AP; surgery is no longer an early intervention and may not be needed[25]. Further progress will also require the development and clinical testing of medications that target disease mechanisms. In our data, no medication has been shown to consistently modulate the outcomes of severe AP. The main negative result may be due to the small number of enrolled cases, such as antioxidants and vitamin C trials[31] and pentoxifylline trials[32]. Hence, large-sample clinical RCTs are urgently needed. UTI is a 67 kDa glycoprotein isolated from healthy human urine, which is an inhibitor of trypsin and other proteases in the urine applied for the treatment of acute inflammatory diseases, hemorrhagic shock, toxic shock, and sepsis[16,33]. Enhanced splenic proliferative responses and cytokine production after acute AP are two ways in which UTIs aid in the recovery of immune function[34]. Animal research has shown that UTIs can regulate inflammation, oxidative stress, apoptosis, immune regulation, and organ protection[14,35]. UTI treatment was employed to decrease the overall 7-d fatality rate in the current investigation. There are still many controversies in clinical studies in severe AP after UTI treatment. A prospective, randomized, placebo-controlled trial showed that patients at high risk of pancreatitis and hyperamylasemia after endoscopic retrograde cholangiopancreatography (ERCP) did not benefit from low-dose prophylactic treatment with UTI after surgery[36]. Ueki et al[37] also reported that preventive administration of UTI and gabexate mesylate had no significant clinical value on the incidence of post-ERCP pancreatitis. Conclusions from these research reports should be treated with caution, however, because of their limited sample sizes or retrospective nature. Conversely, Wei et al[38] reported that after diabetic ketoacidosis complicated with AP, the recovery period of clinical symptoms and the levels of inflammatory mediators may be reduced by using an insulin pump in conjunction with UTI. Thus, the current investigation approved a large single-center RCT to verify the therapeutic benefits of UTI in severe AP. Additionally, attention should be given to both the prevention and management of allergic responses. Some drawbacks are present in this research. Since this was an RCT conducted at a single site, the findings may not be generalized. The current study only included a single dose of UTI administration. CONCLUSION The results of this research illustrate that UTI treatment could enhance kidney function, hepatic function, and coagulation function and decrease the risk of infection and death associated with hyperinflammation after severe AP. The length of time patients spent in the hospital and their associated costs also dropped significantly. Additional investigation is warranted among individuals receiving varying doses of AP to fully understand the prospective applicability of UTI in these patients. ARTICLE HIGHLIGHTS Research background Severe acute pancreatitis (AP) is one of the most common diseases of the gastrointestinal tract, is the leading cause of hospitalization due to gastrointestinal diseases. Surgical treatment is currently the mainstay in clinical practice, is associated with multiple complications, postoperative body pain, and delayed postoperative recovery. Ulinastatin (UTI) is a serine protease inhibitor, which seemed to show a beneficial effect for acute respiratory distress syndrome patient treatment but lacked a larger sample size of randomized controlled trials. Research motivation To evaluate the clinical value and safety of UTI in severe AP. Research objectives This research was conducted to determine whether UTI might be used to improve the outcomes of patients with severe AP. Research methods Patients with severe AP who were transferred to intensive critical care units were enrolled in the current study. Patients were assigned at random (ratio of 1:1) using a computer to receive either a UTI (400000 IU) or a placebo. The seven-day mortality rate, clinical efficacy, and drug-associated side events were evaluated. Research results No statistically significant differences in baseline clinical data between the two groups. When compared with the results obtained from the placebo category, both the clinical efficacy and the seven-day mortality rate for the UTI category showed significant improvements. UTI therapy was shown to protect against hyperinflammation, attenuate coagulation dysfunction and infection, and even improve liver and kidney functioning. Hospitalization durations for UTI-treated patients were much lower than those in the placebo category. Research conclusions Treatment with UTI may enhance therapeutic efficacy for individuals with severe AP and is associated with fewer side effects. Research perspectives A large number study with varied dosages, and long-term outcome follow-up are needed. ACKNOWLEDGEMENTS We hereby express our gratitude to Jiangsu Brilliant Biological Technology Co., Ltd. for providing technical help. Data sharing statement The datasets used and/or analyzed during the current study, including the redacted study protocol, redacted statistical analysis plan, and individual participant data supporting the results reported, are available from the corresponding authors upon reasonable request. Institutional review board statement: The protocol approval from the Clinical Research Ethics Committees of the 904th Hospital of PLA endorsed the methodology used in the present research (2018-YXLL-097) and was following the Declaration of Helsinki. Clinical trial registration statement: This study was registered at Hospital clinical trial registry (date: 9/Sep/2018), No. CWXH-IPR-2018015. Informed consent statement: Written informed consent was obtained from patients whose competence was established by their accurate orientation for time, place, and person, as well as an understanding of the recruiter’s description of the trial or from their next of kin or legal representative. Conflict-of-interest statement: The authors declare that there are no conflicting interests. CONSORT 2010 statement: The authors have read the CONSORT 2010 Statement, and the manuscript was prepared and revised according to the CONSORT 2010 Statement. Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: March 22, 2023 First decision: May 12, 2023 Article in press: June 2, 2023 Specialty type: Medicine, research and experimental Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B, B Grade C (Good): 0 Grade D (Fair): D, D, D Grade E (Poor): 0 P-Reviewer: Chilimuri S, United States; Gaspar R, Portugal; Iwasaki E, Japan; Kitamura K, Japan S-Editor: Fan JR L-Editor: A P-Editor: Zhao S ==== Refs 1 He HW Zhang H The efficacy of different doses of ulinastatin in the treatment of severe acute pancreatitis Ann Palliat Med 2020 9 730 737 32312068 2 Leppäniemi A Tolonen M Tarasconi A Segovia-Lohse H Gamberini E Kirkpatrick AW Ball CG Parry N Sartelli M Wolbrink D van Goor H Baiocchi G Ansaloni L Biffl W Coccolini F Di Saverio S Kluger Y Moore E Catena F 2019 WSES guidelines for the management of severe acute pancreatitis World J Emerg Surg 2019 14 27 31210778 3 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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4458 10.12998/wjcc.v11.i19.4458 Review Molecular signalling during cross talk between gut brain axis regulation and progression of irritable bowel syndrome: A comprehensive review Singh SV et al. Interaction of gut-brain-axis and IBS progression Singh Shiv Vardan Department of Biochemistry, University of Allahabad, Allahabad (Prayagraj) 211002, Uttar Pradesh, India Ganguly Risha Department of Biochemistry, University of Allahabad, Allahabad (Prayagraj) 211002, Uttar Pradesh, India Jaiswal Kritika Department of Biochemistry, University of Allahabad, Allahabad (Prayagraj) 211002, Uttar Pradesh, India Yadav Aditya Kumar Department of Biochemistry, University of Allahabad, Allahabad (Prayagraj) 211002, Uttar Pradesh, India Kumar Ramesh Department of Biochemistry, University of Allahabad, Allahabad (Prayagraj) 211002, Uttar Pradesh, India Pandey Abhay K Department of Biochemistry, University of Allahabad, Allahabad (Prayagraj) 211002, Uttar Pradesh, India. akpandey23@rediffmail.com Author contributions: Pandey AK and Singh SV conceptualized the idea; Singh SV, Ganguly R, Jaiswal K, Yadav AK, and Kumar R performed the literature search; Singh SV and Ganguly R wrote the first draft of the manuscript and validated the references; Pandey AK critically reviewed and revised the manuscript; and all authors have read and approved the final manuscript. Corresponding author: Abhay K Pandey, PhD, Professor, Department of Biochemistry, University of Allahabad, University Road, Allahabad (Prayagraj) 211002, Uttar Pradesh, India. akpandey23@rediffmail.com 6 7 2023 6 7 2023 11 19 44584476 24 12 2022 9 5 2023 6 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. Irritable bowel syndrome (IBS) is a chronic functional disorder which alters gastrointestinal (GI) functions, thus leading to compromised health status. Pathophysiology of IBS is not fully understood, whereas abnormal gut brain axis (GBA) has been identified as a major etiological factor. Recent studies are suggestive for visceral hyper-sensitivity, altered gut motility and dysfunctional autonomous nervous system as the main clinical abnormalities in IBS patients. Bidirectional signalling interactions among these abnormalities are derived through various exogenous and endogenous factors, such as microbiota population and diversity, microbial metabolites, dietary uptake, and psychological abnormalities. Strategic efforts focused to study these interactions including probiotics, antibiotics and fecal transplantations in normal and germ-free animals are clearly suggestive for the pivotal role of gut microbiota in IBS etiology. Additionally, neurotransmitters act as communication tools between enteric microbiota and brain functions, where serotonin (5-hydroxytryptamine) plays a key role in pathophysiology of IBS. It regulates GI motility, pain sense and inflammatory responses particular to mucosal and brain activity. In the absence of a better understanding of various interconnected crosstalks in GBA, more scientific efforts are required in the search of novel and targeted therapies for the management of IBS. In this review, we have summarized the gut microbial composition, interconnected signalling pathways and their regulators, available therapeutics, and the gaps needed to fill for a better management of IBS. Irritable bowel syndrome Microbiota Gut brain axis Stress Serotonin ==== Body pmc Core Tip: Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder with a dysregulated gut brain communication. Gut microbiota functional characterization is still underappreciated but their roles have been found to be pivotal. Various microbial species and their metabolites with altered composition and diversity have been found to be specific to IBS. Clinical manipulation of these microbial species improved the symptom profile in IBS patients while the associated mechanisms have been identified for a bidirectional communication between gut microbiota and brain. This in turn seems promising for future treatments specific to microbiota manipulation and targeting various cross-talks for the management of IBS and associated symptoms. INTRODUCTION Human body consist of trillions of microbial cells, majority of which inhabit the gastrointestinal (GI) tract thus forming a microbial colonization with a dynamic ecological environment, commonly known as “microbiota”[1,2]. This microbiota is comprised of approximately 500 transient and indigenous species of bacteria, viruses, fungi and protozoa[3,4]. Among all, bacteria are the most abundant microbial community dominated with the members of Firmicutes and Bacteroidetes phyla[5,6]. In recent years, research focusing intestinal microbiota functional characterization led to the identification of a bidirectional crosstalk between brain and gut microbiota, thus forming a gut-brain axis (GBA)[7,8]. Moreover, GBA includes central nervous system (CNS), enteric nervous system (ENS), hypothalamic-pituitary-adrenal (HPA) axis, gut and its microbiota (Figure 1). Interestingly, these components have been identified to be interconnected through various coordinated signalling pathways[8,9]. Abnormalities in these pathways and their regulators have been identified for the etiology of irritable bowel syndrome (IBS). IBS is a common GI bowel disorder featured with altered GI motility, visceral hypersensitivity, post-infection reactivity, small intestinal bacterial overload, carbohydrate mal-absorption and intestinal inflammation[10,11]. These abnormalities result in dysbiosis, recurrent abdominal pain and distressed bowel habits. Figure 1 Regulatory cross talk between gut and brain. The central nervous system and enteric nervous system mainly communicate through vagal and autonomic pathways to control various gastrointestinal functions in a bidirectional approach. These signalling cascades are also regulated through mood and cognitive processes and significantly affected by environmental factors and early adverse events. These signalling crosstalks are significantly altered in irritative bowel syndrome (IBS) thus resulting in abnormal gastrointestinal motility, visceral hypersensitivity and bowel movements. Perturbations in the gut microbiome composition and diversity through various sources alter this cross talk, thus resulting in interceptive feedback to brain and causing functional and neuroplastic changes. Microbial metabolites released from various microbial populations have critical role in these crosstalks, thus having therapeutic values for the management of IBS. ANS: Autonomic nervous system; HPA: Hypothalamic-pituitary-adrenal axis; ECC: Enterochromaffin cells; ICC: Interstitial cells of Cajal; SMC: Smooth muscle cells; SCFAs: Short chain fatty acids; ENS: Enteric nervous system. IBS is affecting 10%-25% of global world population, especially in developed countries with a poorly deciphered pathophysiology[12]. Considering the predominant symptoms and bowel habits, IBS is further classified into four subtypes: diarrhea-predominant IBS (IBS-D), constipation-predominant IBS (IBS-C), mixed IBS (IBS-M), and unclassified IBS[13,14]. Interestingly, women are more susceptible (1.67 times) than men for IBS. In recent years, IBS symptoms have been predominantly found to be associated with environmental factors such as, diet, enteric microbial communities, host genetics and psychology[15]. However, the ways in which these factors contribute to the etiology of IBS are not fully deciphered. The treatments available for IBS are also not very specific. Generally, dietary fiber supplementation for IBS-C, opioids for IBS-D and abdominal pain with bloating, and low doses of antidepressants are recommended for the management of various symptoms associated with IBS[12,13,15]. However, modulating the gut-brain axis seems to be a promising target for the development of novel therapeutic for IBS[16]. In addition to this, various metabolic disorders have also been identified to be massively regulated by gut microbiota and their metabolites[6,17,18]. Involvement of gut microbiota metabolites in maintaining homeostasis including host immunity, physiological functions (digestion and nutrition) and biosynthesis of vitamins have also been recently validated by various research groups[18,19]. Experimental sets of data intoning functional cruciality of microbiota in IBS advocate their widespread interactions not localized only with intestinal cells and ENS, but also directly with CNS through neuroendocrine and metabolic pathways[19,20]. The ENS composed of semiautonomous effector system is also connected to central autonomic network and modulated via afferent and efferent communications through parasympathetic and sympathetic nerves[21,22]. Ongoing bidirectional brain-gut interactions are significantly influenced by serotonergic [5-hydroxytryptamine (5-HT)] pathway, where serotonin also known as 5-HT is an important neurotransmitter and paracrine signalling molecule[23,24]. Serotonin is synthesized by enterochromaffin cells (EC) of the gut and by serotonergic neurons in the CNS[25]. Aberrant 5-HT signalling has been found to be accountable for various GI disorders, including IBS, diarrhea and chronic constipation, and functional dyspepsia[26,27]. Various components of serotonin signalling, including EC cells count, serotonin level, tryptophan hydroxylase activity, and expression of serotonin-selective reuptake transporters have also been found to be altered in IBS[28]. 5-HT signalling in between 5-HT receptors (on postsynaptic and presynaptic neurons at CNS and intestinal serotonergic neurons) and serotonin transporters (SERT) of various cell types of GI tract is crucial for proper functioning of gut-brain communication[29,30]. Additionally, 5-HT is produced by the chemical conversion of tryptophan to 5-hydroxytryptamine, a reaction catalyzed by enzyme tryptophan hydroxylase (TPH1 in EC cells and TPH2 in neurons)[31]. Stored into vesicles formed through the vesicular monoamine transporter (VMAT; VMAT1 in EC cells and VMAT2 in neurons), 5-HT is further released into the extracellular space where it binds to different serotonin receptors (5-HTR)[32]. Further, it is known to regulate peristaltic, secretory, vasodilatory, vagal and nociceptive reflexes (Figure 2)[26,27,33]. Figure 2 Serotonin mediated bidirectional cross talk between gut microbiota and brain. Serotonin (5-HT) plays a key role in microbiota-gut-brain communications to modulate gastrointestinal (GI) and central nervous system (CNS) functions. Serotonin action is mediated through various signalling mechanisms between 5-HT receptors located in postsynaptic and presynaptic neurons at CNS and intestinal serotonergic neurons, and in different cell types of GI tract. Serotonin is synthesized by enterochromaffin cells (EC) in the gut and serotonergic neurons in the CNS. Microbial associated molecular patterns from microbiota directly affect the serotonergic system, mainly through modulating the activity and expression of serotonin transporter (SERT) and serotonin receptors (5-HTRs), as well as the synthesis of 5-HT in GI tract. Stored into vesicles forms through the vesicular monoamine transporter (VMAT; VMAT1 in EC cells and VMAT2 in neurons), 5-HT is further released into the extracellular space where its binds to different serotonin receptors (5-HTR). At the same place taken up by the neurons, enterocytes or platelets through the SERT limit the 5-HT mediated signalling crosstalk. 5-HT: 5-Hydroxytryptamine; TLR: Toll-like receptors; NLR: NOD-like receptors; SFCA: Short-chain fatty acids; MAMP: Molecular patterns from microbiota; SERT: Serotonin transporter; VMAT: Vesicular monoamine transporter. The present review summarizes recent updates consisting key components of microbiota derived GBA, molecular signalling during cross talk between GBA regulation and in the progression of IBS. Herein, we have also discussed the diagnostic and therapeutic implications of microbiota in the management of IBS and related symptoms. IBS IBS is a GI disorder characterized by chronic abdominal pain and altered bowel habits in the absence of any organic cause which significantly affects the quality of life[34]. It is the most diagnosed functional GI disorder accounting for approximately 30 percent of all referrals to gastroenterologists[35]. The pathophysiology of IBS is the result of aberrated and interconnected signalling networks, especially between gut microbiome and brain[34,35]. However, the associated mechanisms and pathways are still unclear. Pathophysiology of IBS Traditionally, IBS was known to be associated with altered GI motility, visceral hypersensitivity and distorted pain perception[34,35]. Studies on IBS indicated the pivotal role of inflammation, alterations in fecal flora, and bacterial overgrowth[36]. IBS is also known as a psychosomatic illness since it is associated with mood disorders with abnormal psychiatric conditions[36,37]. Furthermore, mucosal immune activation, inflammatory cells and elevated inflammatory markers have been recorded in IBS patients[34,35]. Various studies associated with the effect of antibiotics in changing the gut microbiota diversity and complexities are co-related with gut microbiota profiles and IBS symptoms[34,38]. Perturbations in GBA have been also proposed as the main mechanisms in the pathophysiology of IBS. In a recent study, corticotrophin-releasing hormone (CRH) was also found to have an important role in IBS and in augmentation of intestinal mucosal inflammation[39-41]. Role of stress in IBS Studies are suggestive for co-morbidity of stress-related psychiatric illness and IBS, since 50%-60% of IBS patients have been diagnosed with various psychosocial health issues[42,43]. Psychological stress plays an important role in the pathophysiology of IBS, since it critically influences gut-brain axis and its associated metabolism[44]. It is also known to regulate intestinal motility and permeability, visceral hypersensitivity, immune responses, and gut microbiota composition[45,46]. Among the possible mechanisms for this regulation, immune response dependent secretion of various proinflammatory cytokines seems to be the pivotal one[47]. These cytokines activate the HPA and hypothalamic-autonomic nervous system (ANS) axes along with the release of corticotropin releasing factor, adrenocorticotropic hormone and cortisol, and all these subsequently control the gut homeostasis (Figure 3)[48]. It is established that these also alter neurotransmitter release within the enteric nervous system which thereafter affect gut motility, secretion and epithelial permeability via tight junction dysregulation[49]. An abnormal progression of monoamine neurotransmitter systems, including 5-HT and noradrenaline have been noticed in many stress related disorders. Furthermore, serotonin is known for its pivotal role in emotional response and in pain management by modulating brain-gut-microbiota axis, which is also significantly affected by early life stress[50]. Figure 3 Impact of stress on irritable bowel syndrome. A bidirectional signalling has been identified between brain and gut, under stress condition in irritable bowel syndrome (IBS). Stress induces the activation of hypothalamic-pituitary-adrenal axis which results in the release of various stress factors such as corticotropin-releasing factor from hypothalamus, thus causing the release of glucocorticoids from adrenal glands. This collectively alters bowel function and stimulates the upregulation of immune system, which directly or indirectly regulates gut function. In addition to this, stress also modulates the tryptophan metabolism by up-regulating kynurenine pathway which generates neurotoxic and neuroprotective metabolites. Enzymes responsible for the degradation of tryptophan are significantly affected by immune pathways (indoleamine-2,3-dioxygenase) and stress levels (tryptophan-2,3-dioxygenase). Under stressed conditions and due to excess availability of kynurenine and its metabolites, the activation of these pathways lead to potential serotonergic deficiency along with altered enteric nervous system and central nervous system functionalities, thus leading to microbial dysbiosis, as a classical symptom of IBS. CRF: Corticotropin-releasing factor; ANS: Autonomic nervous system; CNS: Central nervous system; ACTH: Adrenocorticotropic hormone; IDO: Indoleamine-2,3-dioxygenase; TDO: Tryptophan-2,3-dioxygenase. MICROBIOTA AND GBA Human GI tract hosts various genera of bacteria that are present on its mucosal surface. The term “microbiota” refers to the ecological system composed of various commensal bacteria in the GI tract, particularly localized on the lining of mucosal surface[51]. The term “microbiome” refers to metagenome of the microbiota and their genetic architecture[52]. Mucosal surface is the only layer separating host tissues from various germ lines by which host immune system remains protective against various pathogens in GI tract[52,53]. Microbial colonization Although, majority of microbiota is still uncharacterized, it consists of a very delicate community of commensal organisms that have evolved over millions of years. Among all, four bacterial phyla including gram-positive Firmicutes (Lactobacillus spp.) and Actinobacteria (Bifidobacterium spp.) along with gram-negative Bacteroides and Proteobacteria are the most prevalent[6,54,55]. In addition to bacterial strains, various viruses, protozoa, archae, and fungus also constitute GI microbiota. Multidisciplinary studies were also performed to characterize the gut microbiota, thus revealing the system as a state of symbiosis[56-59]. Homeostasis of host immune system and mucosal barrier are generally altered by dysbiosis, which in turn promotes the invasion and growth of pathogenic species. Microbiota also regulates gut inflammation with the presence of immune cells in gut wall[60]. Although, the complete sketch of a balanced microbiome is still unknown and each person is assumed to have a distinct microbiota (called as microbiome fingerprint), healthy individuals are generally known to have similar profile and distribution of bacterial phylotypes[61]. Microbial colonization is a vital early life phenomenon as a part of development of a healthy microbiota which seems to be crucial for the development of GBA[62,63]. Altered microbial colonization in human GI tract has been clearly identified to exhibit adverse health effects in the later years of the host[63]. The colonization process is also regulated with various factors such as birth mode and feeding of babies, since the breastfed and vaginally born babies were reported to be initially colonised by the member of Bifidobacteria, Lactobacilli, and Bacteroides species[64]. As compared to pre-term babies, full-term babies are reported for rapid maturation of gut microbiome in their first year of life. There is several evidence supporting the existence of a microbiota and foetal meconium, where the colonisation is reported to start even before the delivery, which suggest that these organisms could also colonise the foetal gut[65]. Various bacteria like Staphylococcus, Streptococcus, Bifidobacterium, and Lactobacillus are also reported in human milk[66]. Thus, colonization is significantly affected in neonatal period by human breast milk and the technique of delivery[67]. Moreover, mononuclear cells carrying bacterial population from the mother’s gut to the mammary gland through an entero-mammary pathway are also playing pivotal role in gut immunological maturation of the infant[68]. This mammary microbiome also possesses anti-infective, anti-inflammatory, immunomodulatory and metabolic activities which also constitute for microbiota colonization in neonatal gut[69,70]. Role of microbiome in GBA The human microbiota play fundamental role in host physiology and pathology, and alteration in microbial population (dysbiosis) has been found to be significantly associated with various GI disorders[71]. A balanced interaction between microbiota and its host seems to be beneficial and essential for intestinal health and host metabolism. Under normal conditions, mucosal microbiota have been found to have significant role in digestion of food, vitamins synthesis, angiogenesis, epithelial cell and in development and maturation of the host defense system[72]. Recently, it has been evident that the intestinal bacterial population significantly affects the CNS physiology and gut inflammation, mainly by a bidirectional communication consisting of various signalling pathways. This communication commonly known as GBA[73], consists of multiple inter-connections including vagus nerve, immune components, and microbial metabolites[74,75]. An enhanced size of microbiota and neuronal development takes place generally in starting five years of an infant, where the neuronal development is mainly configured by maternal microbiota[76]. The effect of gut microbiota on neurodevelopment of an infant was also examined and proved using various experiments on germ-free (GF) or specific pathogen-free mice. These mice were treated with various antibiotics to alter the microbial diversity and thereby to study their importance in gut microbiome[77,78]. These experiments clearly suggested that various neurological problems appeared in the treated animals due to improper maturation of gut microbiome. Results of these studies are suggestive of exaggerated HPA axis in germ free animals with impaired social behaviors, reduced anxiety and increased motor activity[79,80]. In the same experiment, altered neuronal developments and behaviors were found to be improved when the newborn infants were supplemented with various microbial flora[81]. These altered behavioral phenotypes were also found to deregulate various genes and metabolites involved in HPA axis regulation. Among these, some common regulators are adrenaline, 5-hydroxytryptophan, postsynaptic density protein 95, dopamine and synaptophysin[82]. Scientific reports suggest that production of various neuroactive molecules by gut microbes directly or indirectly play critical role in GBA[83]. Among these neurotransmitters, acetylcholine, γ-aminobutyric acid and serotonin are released by various bacterial populations belonging to Bifidobacteria, Lactobacillus, Enterococcus, and Streptococcus species[84]. Interestingly, 90% of total serotonin is produced by gut microbiota, and used for mood disorders and functional regulation of CNS and GI tract[84,85]. Serotonin is known to bind to 5-HT receptors on microglial surface, thus inducing the release of inflammatory cytokines, which in turn play important role in the maintenance of gut inflammation[86]. In addition to 5-HT, tryptophan is also a serotonin precursor and known for influencing microglia activity in gut lumen. Moreover, tryptophan and its derivatives have been found to regulate CNS inflammation, particularly following an aryl hydrocarbon receptor (Ahr) mediated mechanism, which in turn is responsible for microglial activation and transcriptional regulation of astrocytes[87]. The vitality of tryptophan and its metabolism in maintaining CNS homeostasis has been reported recently, where GF animals were found to have augmented 5-HT levels and 5-hydroxyindoleacetic acid in the hippocampus and serum samples, compared to conventionally colonized control mice[87,88]. These findings are suggestive towards the ability of microbiota in maintaining CNS serotonergic neurotransmission, mainly through systemic circulation. These initial findings were also validated by colonizing these animals post-weaning, where they were found to have sufficient levels of tryptophan in peripheral samples and reduced anxiety. Interestingly, this colonization was found ineffective in reversing the CNS neurochemical consequences exhibited by GF animals[89]. In a recent study, one major finding was listed where an intact and diversified microbiota (post birth) was found to produce quinolinic acid and N-methyl-D-aspartate agonist, as a part of tryptophan metabolism regulated by microglial cells[90]. Both have a critical role in the pathogenesis of several neurological conditions such as Huntington’s disease and behavioral disorders[91]. In experimental setup where GF mice were recolonized with bacterial population belonging to Clostridium tyrobutyricum, they exhibited colonized mucus layer, and regulated immune and gut barrier homeostasis[92]. Furthermore, Lactobacillus rhamnosus (JB-1) containing probiotics supplementations in previously colonized mice were found to be able to reduce anxiety and depressive behavior[93]. Recently, researchers also found that microbiota dependent alterations in neural synapses along with fear extinction behavior are not mainly due to altered HPA axis, they are also due to a reduction in various neuroactive metabolites’ levels such as phenyl sulfate, pyrocatechol sulfate, and indoxyl sulfate[94,95]. This was further confirmed with the abraded levels of these metabolites in various samples collected from experimental animals, such as fecal sample, serum, and CSF. Collectively, gut microbiome and its associated bacterial populations were found to be pivotal in maintaining the level of various neuroactive metabolites through which they regulate the behavioral patterns in healthy subjects. Regulatory crosstalk between intestinal microbiome and brain In humans, ENS is generally referred as the second brain which is located inside the digestive tract wall and shielded by a mucous membrane from gut lumen, having five times higher neurons and ganglia than spinal cord. An autonomous system regulating microbiome and GBA is known to be bidirectional whereas somatic sensory system received signals in pathological conditions causes abnormal feelings like discomfort, nausea, and pain which in turn exhibit bowel associated problems[96]. Herein, ANS transports afferent signals received from gut lumen to CNS through various routes such as enteric, spinal and vagal[96]. In these communications, vagus nerve plays a critical role as it has millions of nerve terminals (80% are afferent). Moreover, the same pathway is also used to send efferent signals from CNS to the gut wall[97]. In case of stress and anxiety, neuroendocrine system in response to stress regulates various vital body functions, including digestion and immunity which are mainly dependent on the HPA axis[98]. For example, generation of corticosteroids in response to environmental stress is also found to be dependent on HPA axis[97]. Collectively, neural and hormonal signals regulate brain and microbiome for further regulation of gut cell activity in controlled conditions. In addition, signal transducing cells such as enterochromaffine cells (EC) and dendritic cells (DC) produce various neurotransmitters (Figure 2) viz., 5-HT, somatostatin, cholecystokinin and CRH[99-101]. All these are important for signal-based regulation of microbiota and CNS as these directly affect the microbial behavior. EC serve as luminal sensors for observing a great range of bacteria and microbial compounds in the gut[102]. These microbial populations have neurotransmitter receptors, and their activation is a key factor to understand the mode of microbiota functions and composition. A specialized process called as “inter-kingdom signalling” is a well-known regulatory pathway through which bacteria communicate with gut epithelial cells, mainly by using oligopeptides and monoamines which are also known for their neurotransmitter behavior[103,104]. Numerous neurotransmitters are produced by ENS exhibiting critical roles in GBA regulation, where each has a specific signalling pathway. Some neurotransmitters such as 5-HT, somatostatin, dopamine, neuropeptide Y, peptide YY, cholecystokinin, and corticotropin-releasing factor are also necessary for GBA regulation[96,101,104]. EC in GI tract produces majority of body’s 5-HT and dopamine. An increased synthesis of 5-HT has been found as a key regulator through which microbiota regulates HPA[97]. Additionally, gut microbiota senses the EC cells mainly via short-chain fatty acids (SCFA) (butyrate and acetate) to promote 5-HT synthesis that regulates GI motility, secretion and immunological responses[96]. Hence, a change in microbiota composition significantly affects the levels of 5-HT and its altered level contributes towards the pathophysiology of IBS. Interestingly, microbiota is also connected with CNS through TLR signalling pathway[105,106]. TLR is principally expressed on immune cells of gut wall and neurons of the ENS. Among microbial populations, gram-negative bacterial membrane component lipopolysaccharide (LPS) is selectively detected by TLR which in turn is responsible for the production of proinflammatory cytokines via NF-κB pathway[106]. In colonic biopsies of IBS-D patients, higher TLR expression has been recorded suggesting microbiota and host immune system interaction[102,104]. Furthermore, smooth muscle dysfunction and paralysis in septic ileus is also known to be significantly influenced by the number of cytokines produced by immune cells, in response to LPS through TLR mediated signalling[106]. INTESTINAL MICROBIOME AND SEROTONERGIC SIGNALLING Serotonergic system 5-HT is an important neurotransmitter and paracrine signalling molecule in the gut where its major role has been identified in the modulation of gut-brain communication and in functional GI disorders[21]. In previous studies, altered levels of 5-HT have been reported in various CNS related disorders including anxiety, depression, obsessive compulsive disorder, phobias and in other psychiatric disorders[67,69]. 5-HT modulators including SSRIs and specific 5-HT receptor agonists and antagonists have also been used to treat various metabolic disorders such as, migraine, nausea, obesity, chronic pain, hypertension, vascular disorders, and sexual dysfunction[107]. 5-HT signalling in GI tract motility Among all the neurotransmitter-based regulations of GBA, 5-HT mediated signalling is primary where 5-HT mainly targets receptor subtypes of seven 5-HT family receptors, having 15 subtypes of recognition sites[85]. Additionally, seven types of 5-HT receptors have a wide range of biological tasks including enhancing mucosal permeability and visceral hypersensitivity, inflammation and immune cell activation, and gut motility[107]. Currently, 7 families of 5-HT receptors have been identified, where 5-HT3 and 5-HT4 receptors are in intestine, presynaptic positions and, in sensory and mesenteric neurons[107]. In clinical practices, medications that are known to target these receptors have been frequently used for the management of IBS complications. Enteric microbiota has also been found to control the expression of 5-HT receptors[107]. 5-HT mainly activates peristaltic reflexes in GI tract (Figure 4) thus causing ascending contractile and descending relaxant limbs. 5-HT has been also found to control segmental motor patterns in small intestine. A study performed in tryptophan hydroxylase deficient (Tph2-/- mice) mice demonstrated the functional aspects of 5-HT in GI motility mainly by attributing with significant reductions in contractile complexes and synaptic transmission due to lower level of 5-HT[108,109]. This is further indicative for the significance of serotonergic neurons, comparing to EC cells for constitutive GI motility. Additionally, ENS dopaminergic neurons were found to be immature in Tph2-/- mice, which are responsible for homeostatic GI movement[110]. Moreover, in vivo and in vitro studies have shown decreased intestinal motility in mice with SERT Ala56 mutation which was restored by 5-HT4 receptor antagonists[111]. A SERT antagonist named ‘fluoxetine’ was found to improve GI motility in SERT-/- mice, along with a higher level of 5-HT, thus indicating the importance of 5-HT in GI motility, by targeting SERT[112]. Figure 4 Tryptophan derived serotonergic metabolism in gut brain axis cross talk. Upon absorption in the gut, L-tryptophan is metabolized through three pathways, particular to bacterial cells (indole pathway) of gut microbiota and mammalian cells (kynurenine and serotonin pathways). Tryptophan is converted to 5-hydoxytryptophan (5-HTP) by tryptophan hydroxylase and thereafter converted to -5-HT by the enzyme aromatic L-amino acid decarboxylase. In addition to this, tryptophan is also metabolized to indole derivatives and kynurenine, which also perform various biological functions. Tryptophan derived 5-HT regulates various functions in central nervous system (including emotion, cognition, stress, and visceral perception) and in enteric nervous system (gastrointestinal motility and secretion). CRF: Corticotropin-releasing factor; ANS: Autonomic nervous system; CNS: Central nervous system; GI: Gastrointestinal; 5-HT: 5-Hydroxytryptamine; HPA: Hypothalamic-pituitary-adrenal. Various antibiotic treatments have been found to impede GI motility along with a reduced production of peripheral 5-HT level, which was also confirmed by GF animal studies where a slower GI transit was recorded in these animals compared to control mice having normal gut microbiota[66,112]. In GF mice, treatment with pharmacological blockers of 5-HT4 receptors significantly improved GI transit along with a higher level of luminal 5-HT level[113]. Additionally, 5-hydroxyindole has been produced as a key metabolite of serotonin metabolic pathway (Figure 4), mainly by increasing the colonic motility, especially by activating L-type calcium channels[114]. Recently, in a BTBR (BTBR T+ Itpr3tf/J) mice model of autism spectrum disorder, an altered serotonergic pathway has been found along with downregulation of Tph1 gene, while SERT expression was upregulated along with a reduced level of 5-HT and its producing host (Blautia spp)[115]. A list of various enteric microbiota and their mode of regulating serotonergic pathway are listed in Table 1. Additionally, 5-HT level in combination with gut microbial stimulus can also lift the proportion of M20000 macrophages, which are known to be stimulatory for GI motility particularly in the colonic muscle layer located nearby ENS[117]. Various microbial byproducts such as SCFAs were also found to enhance colonic transit with the release of 5-HT intra-luminally, mainly by targeting GPR43 receptor located on mucosal mast cells[117]. Therefore, these findings clearly suggest that through various mechanisms enteric microbial population controls 5-HT level and associated signalling for the control of GI motility, and dysregulation in this system resulted in abnormal GI movement, which is linked with IBS symptoms[116,117]. It has been widely accepted that IBS patients have altered motor and stool patterns, where enhanced GI motility produce abnormal gut contractions resulting in abdominal pain and discomfort. Table 1 Types of regulation of serotonergic (5-Hydroxytryptamine) pathway by specific enteric microbiota in gastrointestinal tract Microbiota spp. 5-HT pathway Mechanisms of action and observations Ref. Akkermansia muciniphila (Amuc_1100) Upregulation Promote intestinal 5-HT biosynthesis and extracellular availability through TLR2 signalling [129] Akkermansia muciniphila (extracellular vesicles) Upregulation Increase expression of the Htr4 gene, and decreases expression of the Htr2B, Htr3B, and Htr7 genes [129] Bacteriodes thetaiotaomicron Upregulation Restore 5-HT+ EC cells and shape EC networks in the GI tract of GF mice by producing SCFAs [130] Bifidobacterium dentium Upregulation Increase intestinal 5-HT level; expressions of 5-HTra receptors 2a and 4, and SERT by producing acetate [131] Bifidobacterium longum and Downregulation Upregulate SERT expression [132] Lactobacillus acidophilus Bifidobacterium pseudolongum Downregulation Diminish EC cells [133] Clostridium ramosum Upregulation Promote 5-HT synthesis in colonic EC cells and program differentiation of intestinal stem progenitors toward a secretory 5-HT-producing lineage [134] Corynebacterium spp., Enterococcus spp., Streptococcus spp. Upregulation Enable the direct production of 5-HT [135] Escherichia coli Nissle 1917 Upregulation Enhance 5-HT bioavailability in ileal tissue through interaction with compounds secreted from host tissue [136] Indigenous spore-forming bacteria Upregulation Enhance colonic 5-HT pathway by upregulation of Htr4 [137] Lactobacillus acidophilus Down regulation Upregulate SERT expression [138] Lactobacillus plantarum IS-10506 Upregulation Increase gut 5-HT production along with brain 5-HTT, neurotrophin, and brain-derived neurotrophic factor [139] Lactobacillus plantarum PS128 Upregulation Increase 5-HT+ cells in the gut and alter expression levels of Tph1, Chga, Slc6a4, and Htr4 [140] Lactobacillus rhamnosus Down regulation Upregulate gene and protein level of SERT [141] SadA-expressing Staphylococci, Trichinella spiralis and Campylobacter jejuni (pathogens) Upregulation Promote converting 5-HTP into 5-HT; increase EC cell number and reduce SERT expression [141] 5-HT: 5-Hydroxytryptamine; 5-HTP: 5-Hydroxytryptophan; EC: Enterochromaffin cell; SERT: Serotonin transporters. Role of gut microbe-mediated 5-HT signalling in visceral pain sensation Chronic abdominal pain is a crucial aspect of IBS which is also influenced by intestinal gut microbiota[118]. Animal studies are further suggestive for the ability of fecal microbiota to transmit hypersensitivity to colonic distension in rats[119]. These results suggest that abnormal pain perception associated with IBS is also critically derived from gut microbial components[118,119]. Additionally, neurotransmitters derived from gut microbiota have been also found to be decisive for the perception of visceral discomfort. Among the other neurotransmitters, a preferential role of 5-HT has been found to modulate the intestinal pain by activating mesenteric sensory nerve fibers along with the activation of vagal and spinal afferent fibers[120]. Microbial colonization and their commensal activities have been reported to be essential for promoting excitability of the gut sensory neurons, which are responsible for the development of homeostatic pain sensitivity[121]. In GF mice, where a little mucosal inflammation has been found to be associated with visceral hypersensitivity caused by abnormal pain processing in the brain, it can be corrected by fecal microbiota transplantation (FMT) therapies derived from conventional mice[122]. FMTs are also known to stimulate primary nociceptive neurons in the dorsal root ganglia (DRG) and various gut microbial components such as TLR ligands, formyl peptide receptor 1 agonists and SCFAs[122]. These can modulate direct or indirect enhancement of visceral pain sensitivity. Additionally, microbiota derived kynurenic acid, serine proteases and bile acids can reduce pain sensitivity by inactivating DRG neurons or indirectly by releasing opioid-like factors, mainly from the mucosal immune cells[123,124]. Interestingly, the mode of action of 5-HT mediated signalling depends on the type of 5-HT receptor activation and the release of enteric 5-HT, which has been significantly linked to the intensity of abdominal discomfort in IBS patients[125]. The etiology of IBS also includes visceral hypersensitivity along with the nociceptive process and these are mainly regulated with 5-HT3 receptor activation, where it is mainly expressed on peripheral terminals of spinal afferent nerves and vagal afferent nerve endings in stomach[125]. 5-HT3 receptor has been also identified to influence other neurotransmitters in the brain, where a 5-HT3 receptor antagonist (Ramosetron) has been found to lower the visceral hypersensitivity and modifies GI transit in IBS-D patients[126]. Additionally, 5-HT3 receptor antagonists have demonstrated anti-inflammatory effects particularly to the enhanced permeability of gut and mucosal inflammation reported to be caused by various microbial populations[125]. Moreover, an impaired intestinal barrier linked to dysfunctional 5-HT metabolism in IBS patients has been observed recently[126]. Collectively, these findings are indicative of the vitality of gut microbiota in 5-HT-mediated pain perception, where visceral hypersensitivity resulted from the dysfunction of serotonergic pathway has been linked to enteric dysbiosis as reported in IBS cases[127,128]. Role of gut-microbe-mediated 5-HT signalling in mucosal inflammation and immune response Among the various pathways underpinning the IBS pathogenesis, a persistent and low-grade mucosal inflammation has been reported in majority of clinical cases, with aberrant immune cell activation[90-129]. In IBS patients, abdominal pain has been characterized with a significant infiltration of mast cells in the colonic mucosa, which is also accompanied with an augmented level of mucosal 5-HT[28,29]. Although, exact underlying mechanisms and causes of mucosal inflammation are not completely known, dysbiosis of the gut microbiota along with altered serotonergic signalling have been identified as the important contributors for the onset of IBS. Indeed, augmented numbers of mucosal and EC cells have been recorded in various inflammatory diseases, such as inflammatory bowel disease (IBD), where a range of 5-HT receptors are expressed to stimulate intestinal inflammation through serotonergic signalling[118]. In colitis, 5-HT directs proinflammatory cytokines generations by stimulating T cells, peritoneal macrophages and splenic DCs in NFκB-dependent manner[130]. Moreover, enhanced 5-HT availability has been recorded in SERT-deficient animals sensitive to gut mucosal inflammation whereas Tph1-/- mice with a lower level of 5-HT have been found to be resistant to experimental colitis[131]. These results suggest that intestinal inflammation is significantly regulated by enteric 5-HT, mainly by acting as pro-inflammatory immune regulator. Various results derived from a post-inflammatory IBS rat model also reveal the onset of visceral hypersensitivity along with fecal microbial dysbiosis, along with an elevated serum level of 5-HT[132]. Additionally, in recent studies lamina propria mast cells (MC) were found to be significantly co-related with an enhanced and spontaneous release of 5-HT in IBS patients[133]. Interestingly, histamine has also been identified as an important biogenic amine found to have pathophysiological role in IBS, where the exact mechanism is still not fully deciphered. Usually, histamine played critical roles in regulating GI motility, gastric acid and mucosal ion secretion[49,133]. Histamine H1 receptors (H1R) are known to be involved in mediating sensorineural signalling and vascular dilatation, where their activation is known to regulate food and water intake and diurnal feeding rhythm. In addition to this, stimulation of histamine H2 receptors (H2R) have been known for the degranulation of MCs and production of various antibodies, T helper (Th) 1 cytokines, and for T-cell proliferation[133,134]. However, evidences are indicative for the overproduction of histamine by MCs that has been known to cause diarrhea, with an increased neuronal secretomotor function. In constipation, histamine also induces altered enteric neuron function resulting in an excessive segmental contractile colonic motor activity[50,135]. Although, evidences suggested that use of various agents targeting the histamine receptors (HRs) have been found to be potential therapeutic option for IBS patients[134]. In addition to this, upregulated expression of various colonic receptors of 5-HT (5-HT3A/5-HT2B) along with impaired junction proteins has been found in test animals[136]. Additionally, 5-HT3A receptor antagonist administration or FMT derived from the faeces of normal healthy rats, were found to be able to alleviate IBS-like symptoms[136]. Moreover, SCFAs from the gut microbiota have been appeared to have a dual nature directing GI mucosal immunity and inflammation, which are known to be crucial for maintaining gut homeostasis[58]. These cause the over expression of G-protein coupled receptors and induction of regulatory T cells in the gut which in turn increases the integrity of epithelial cell barrier, and thus exhibiting anti-inflammatory effects[136]. In parallel to this, SCFAs are known to cause mucosal inflammation by promoting mucosal 5-HT synthesis (serotonergic pathway) and upregulation of TPH1 transcription[137]. Microbiota mediated serotonergic signalling outside GI tract Various preclinical and clinical data have demonstrated the critical role of 5-HT derived from gut in glucose and lipid metabolism, and in various metabolic disorders[138,139]. Various antagonist specific to 5-HT receptors have been beneficial in reversing the clinical manifestations received by altered 5-HT metabolism. For example, fluoxetine was used to prevent T. sanguinis population by lowering serum triglyceride levels and changing the expression of lipid metabolism genes[139]. Additionally, the gut was found to be significantly regulated by its microbiota member ”Clostridium ramosum”which is found to be associated with lipid transport and storage functions in animals. Furthermore, glucose homeostasis is also controlled by various members of enteric microbiota, mainly with the modulation of EC cell’s 5-HT production where this was mainly targeted with the inhibition or genetic depletion of TPH1[140]. Purine metabolism was also found to be regulated with host-microbial metabolic route in IBS patients[141]. These results are clearly indicative for the 5-HT-dependent regulation of various metabolic pathways in the pathogenesis of IBS. ROLE OF GUT MICROBIOME DERIVED METABOLITES IN IBS The population of gut microbiota has been reported to be massive along with a modular genome which in turn reported to be benefitting the host and adapted to gut environment. These microbes carry out a variety of tasks, such as xenobiotic metabolism, vitamin production, pathogen defense, and dietary fiber fermentation[115,136,142]. Microbiota derived metabolites are small substances synthesized as intermediate or end products of microbial metabolisms, which are also the principal regulators through which gut microbiota plays an important role in host specific metabolism[136]. These metabolites may be produced directly by bacteria, by alteration of host molecules like bile acids, or through bacterial metabolism of food components. Immunological maturation, immune homeostasis, host energy metabolism, and mucosal integrity maintenance are all identified to be influenced by microbial metabolites mediated endocrine signalling[11,12,14]. IBD’s pathophysiology has been principally linked to certain groups of metabolites, such as bile acids, SCFAs, and tryptophan which have been found in a variety of biological tissues, including faeces, urine, serum, liver, and cerebrospinal fluid, thus having significant impact on the physiology of the host[17,136]. Additionally, some metabolites have been also found to be specific to Crohn’s disease patients and non-IBD controls, where individuals with IBD were found to have higher amounts of bile acids, amino acids, and sphingolipids[143]. Interestingly, these individuals have lower levels of cholesterols, phenylbenzodioxanes, indoles, tetrapyrroles and long-chain fatty acids[144]. SCFAs are derived from carbohydrates and are localized to microbiome, regulating the function of intestinal macrophages, appetite, fat accumulation, intestinal motility and energy metabolism[145]. Other SCFAs produced by microbial fermentation include acetate, propionate, and butyrate, as well as the gases methane, hydrogen sulphide and some other intermediates[145,146]. Butyrate, along with other SCFAs, inhibits epithelial stem cells and promotes epithelial homeostasis by producing IL-18 through inflammasome activation, which is known to be the main source of energy in colonic epithelial cells[147]. Like SCFAs, medium-chain fatty acid levels have been found to be depleted in IBD. Thus, it is hypothesized that certain fatty acids, such as conjugated linoleic acid might exert some anti-inflammatory effects by activating the peroxisome proliferator-activated receptor-γ (PPAR-γ)[148]. Additionally, bile acids also influence host metabolism and immune system via interacting with receptors, such as the transmembrane G protein-coupled receptor 5 (TGR5) and farnesoid X receptor (FXR)[149]. By activating type 2 iodothyronine deiodinase, TGR5 enhances insulin sensitivity (via GLP1), thus regulating energy expenditure (in muscle and brown adipose tissue), and gall bladder relaxation[149]. Activation of bile acid receptor (FXR) also affects host metabolism through various ways, including decreased lipogenesis, hepatic gluconeogenesis, and liver regeneration, as well as by generating antimicrobial peptides for liver regeneration[150,151]. Constitutional makeup of gut microbiota is also significantly influenced by bile acid composition. Furthermore, activation of small intestine FXR also reduces bacterial translocation and growth[152]. In addition to other metabolic intermediates, tryptophan is a well-known precursor for the synthesis of several metabolites such as serotonin, melatonin, nicotinamide and vitamin B3[153]. Gut is the main site of tryptophan synthesis and metabolism, where it is converted by commensal microbiota into indoles agonists of pregnane X receptor and AhR, which have pivotal roles in mucosal immunity and homeostasis[154,155]. Moreover, dysbiosis in IBD also causes a significant decrease in microbial tryptophan activation, which in turn increases metabolism (Figure 4)[148,149]. Indole also stimulates GLP1 release while other indole derivatives [such as indoleacetic acid, indole-3-acetaldehyde, indole-3-aldehyde, indoleacrylic acid (IA) and IPA] are also known to act as agonists for AhR[156]. Additionally, AhR also act as a transcription factor which having developmental and tissue-dependent effects on T cell immunity[157]. Reduced AhR expression has been noted in inflamed mucosal samples from Crohn’s disease patients whereas serum samples from these patients were also found to have altered xenobiotic metabolism[158]. Moreover, enhanced catabolism of fatty acids, reduced level of amino acid metabolites including serotonergic and indole derivatives of tryptophan, as well as decreased levels of phenylalanine and histidine metabolite ergothioneine has been also found in these patients[153,155,158]. MICROBIOME TARGETED THERAPEUTICS FOR IBS Restoring the gut microbiome seems to be promising and therapeutic strategy for the management of IBS with diversified clinical scenarios. Moreover, maintaining the GI microbiome to a level of optimal health with the ideal fecal product is known to be challenging and beneficial to human being. In recent years, FMT has been introduced to yield promising results in various gut disorders such as in Clostridioides difficile infection, where FMT has shown high cure rates, compared to other standard therapies[55,159,160]. This initial lead has been also implanted with microbiome manipulation for the treatment of IBD, ulcerative colitis and Crohn’s disease, with a higher rate of cure effectively[161,162]. However, designing FMT formulations as the treatment for IBS is very challenging, since the optimal microbiome composition in healthy people is poorly defined with a higher degree of variability[163]. In addition to this, disrupted microbiota which would be ideally restored via FMT is known to be highly variable. Interestingly, dysbiosis of several inflammatory markers is known to induce the expansion of facultative anaerobes, where the mechanisms by which microbiota are disrupted remain unclear. At the same time, GI tract is known to be contributory for key determinants of FMT success whereas, some patients that received FMT also developed systemic infections owing to the adverse effects of FMT[61,63,148]. To avoid the side effects received from FMT application, one more way is to administer autologous FMT (auto-FMT also known as microbiome restoration), where patient sample is screened for various pathogens, banked and then re-administered to the same patient after disrupting microbiome. In this case, restoring the microbiome samples for a long period of time has storage and safety issues, leading to future health problems. Meanwhile, several IBS therapeutics (Table 2) have been developed which are known to target the gut microbiota and target secondary consequences of alterations in the gut microbiota[164]. Table 2 Various therapies prescribed for the treatment of irritable bowel syndrome along with their possible mode of action Therapy Description Proposed mechanism(s) of action Prebiotics Ingested compounds targeted to stimulate gut microbiota Mechanism of action undefined, but may include: Anti-inflammatory effects; inhibition of pathogen adherence; and growth of intestinal mucosal layer Probiotics Ingested microorganisms (e.g., bacteria) Mechanism of action undefined, but may include: Inhibition of pathogenic microorganism colonization; support intestinal barrier integrity and function; production of beneficial micronutrients; and activation and augmentation of the enteric nervous system Rifaximin Nonabsorbable, bile-soluble antibiotic indicated for the treatment of adults with IBS-D Antibacterial against Gram-positive and Gram-negative bacteria: Modulation of gut-immune signalling; inhibition of bacterial translocation; SIBO eradication (in some patients); causing decreases in GI methane concentrations in combination; and with the antibiotic neomycin (in patients with IBS-C) SBI Prescription medical food for patients with IBS-D Modulation of gut microbiota: Causing decreases in GI permeability SYN-010 Derivative of the HMG-CoA reductase inhibitor lovastatin lactone; currently in development for the treatment of patients with IBS-C Inhibition of methane production by Methanobrevibacter smithii Dietary modification Variable; one example is the low FODMAP diet Causing decreases in GI gas production Causing decreases in intra-luminal fluid production FODMAP: Fermentable oligo-, di-, monosaccharides and polyols; GI: Gastrointestinal; HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A; IBS-C: Constipation-predominant irritable bowel syndrome; IBS-D: Diarrhea-predominant irritable bowel syndrome; SBI: Serum-derived bovine immunoglobulin; SIBO: Small intestinal bacterial overgrowth. CONCLUSION IBS is a prevalent GI disorder where mechanisms regulating the interconnected cross talk between brain and gut microbiota have been found to be altered. Various metabolic pathways and diseases have also been found to be associated with gut microbiota architecture. The pathophysiology of IBS is still not fully deciphered, whereas a complex network of interaction between various genes, metabolic pathways, behavioral events, host immune response and psychosocial factors has been found to be contributory for IBS and its associated symptoms. Interestingly, serotonin (5-HT) as a neurotransmitter secreted by EC cells regulates GI motility, secretion, and sensation, whereas altered 5-HT signalling has been found contributory in the pathophysiology of IBS. Furthermore, environmental stress has a significant impact on IBS etiology where it significantly regulates the neuroendocrine system and gut functions, mainly through immune system mediated mechanisms. The dietary composition and its intake have a pivotal role in the regulation of IBS, hence usage of quality foods which are gluten free, low fat and FODMAP content, tryptophan and fiber rich may be prominent approach for the management of IBS. ACKNOWLEDGEMENTS Shiv Vardan Singh acknowledges University Grants Commission (UGC), New Delhi, India for Dr DS Kothari Fellowship. Risha Ganguly, Kritika Jaiswal and Ramesh Kumar acknowledge financial support from UGC/Council of Scientific and Industrial Research, New Delhi, India in the form of Junior and Senior Research Fellowships. Aditya Kumar Yadav acknowledges financial support from UGC in the form of CRET fellowship. All the authors also acknowledge DST-FIST and UGC-SAP facilities of the Department of Biochemistry, University of Allahabad, Prayagraj, India. Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article. Provenance and peer review: Invited article; Externally peer reviewed. 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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4692 10.12998/wjcc.v11.i19.4692 Case Report Morbihan disease misdiagnosed as senile blepharoptosis and successfully treated with short-term minocycline and ketotifen: A case report Na et al. Report of a rare Morbihan disease Na Jun Department of Dermatology, Peking University First Hospital, Beijing 100034, China Wu Yan Department of Dermatology, Peking University First Hospital, Beijing 100034, China. 3437477565@qq.com Author contributions: Jun Na contributed to manuscript writing and editing, and data collection; Yan Wu contributed to conceptualization and supervision; all authors have read and approved the final manuscript. Corresponding author: Yan Wu, MD, Chief Physician, Department of Dermatology, Peking University First Hospital, No. 8 Xishiku Street, Xicheng District, Beijing, China. 3437477565@qq.com 6 7 2023 6 7 2023 11 19 46924697 13 3 2023 31 5 2023 14 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Morbihan disease is a rare skin condition with diagnostic and therapeutic challenges. Facial nonpitting erythematous edema is usually considered to be a characteristic manifestation and diagnostic clue for the Morbihan disease. Treatment of Morbihan disease remains a dilemma due to its long course, poor response, and high recurrence rate. CASE SUMMARY We report the case of a 69-year-old man with Morbihan disease. The patient presented ptosis as the first and main symptom. There was no obvious edema or other skin lesions. The patient was misdiagnosed with senile blepharoptosis based on eyelid performance and no treatment was administered to him. After continuous progressive asthenia of eye-opening and ptosis for more than one year, a skin biopsy was recommended. Histopathological analysis showed edema in the dermis, lymphatic hyperplasia and dilatation, and perivascular lymphocytic infiltration. An obvious increase in toluidine blue-stained mast cells was observed. The patient was finally diagnosed with Morbihan disease. Minocycline and ketotifen were prescribed based on the increase of mast cells in skin tissue slices. The patient experienced rapid relief seven days later and complete remission after 40 d from the commencement of the treatment. CONCLUSION Ptosis without obvious swelling could be the only or main clinical manifestation of Morbihan disease in rare conditions. An increase of mast cells was an important therapeutic clue to the rapid and remarkable efficiency of the combination therapy of minocycline and antihistamine. Morbihan disease Ptosis Minocycline Mast cells Treatment Case report ==== Body pmc Core Tip: Morbihan disease is a rare skin disease that is mainly characterized by nonpitting erythematous edema on the upper two-thirds of the face. We report an atypical case of progressive blepharoptosis without obvious edema, as a reminder of the existence of this uncommon condition to dermatologists, neurologists, and ophthalmologists to reduce chances of misdiagnosis. Usually long-term minocycline is used to treat Morbihan disease but has uncertain efficacy. We successfully treated our patient with a combination of minocycline and ketotifen. We also summarized previous reports on the use of minocycline in Morbihan disease and noted that mast cells might be key histological predictors of the treatment response to minocycline. INTRODUCTION So far, less than 100 cases of Morbihan disease have been reported. The clinical manifestation of this rare disease has not been fully described. Based on the limited available reports, Morbihan disease classically occurs in middle-aged to elderly males, with chronic persistent nonpitting erythematous edema on the upper two-thirds of the face being the main diagnostic clue in most cases[1]. Ptosis has rarely been considered as a hint of Morbihan disease unless accompanied by extreme periocular swelling[2]. However, diagnosis is more difficult for the atypical cases without obvious edema. The treatment of Morbihan disease also remains controversial. Therapeutic strategies that include drugs, laser, and surgical techniques have been used for Morbihan disease but these often end in long courses and unsatisfactory efficacy[3]. More evidence is urgently needed for the diagnosis and treatment of this disease. Here, we report a special case of Morbihan disease with misleading ptosis and without obvious erythematous edema in a 69-year-old man who was successfully treated with short-term minocycline combined with ketotifen. CASE PRESENTATION Chief complaints A 69-year-old man presented to our Department of Dermatology with progressive asthenia of eye-opening and ptosis for more than one year. History of present illness Ptosis for over one year was the only complaint that the patient presented. There were no obvious lesions on periocular skin or other areas of the face. The patient denied the existence of discomfort such as itching, swelling, or pain. He attended the Neurological Department and Ophthalmology Department frequently with no significant positive changes in his condition. Initial considerations of neurogenic and myogenic ptosis, for example, oculomotor paralysis, myasthenia gravis, and hyperthyroid ophthalmomyopathy, were excluded after a series of examinations including neuroelectromyography, neostigmine test, myasthenia gravis-related antibodies, thyroid function, and craniocerebral imaging. The patient was diagnosed with senile blepharoptosis, and he refused treatment. However, his ptosis aggravated so a skin biopsy was finally recommended. History of past illness The patient reported well-controlled diabetes and hypertension. He denied other systemic diseases or any history of dermatoses such as rosacea, acne, erysipelas, contact dermatitis, angioedema, and skin lupus erythematosus. The patient also denied preceding periocular rash, trauma, surgery, or self-conscious swelling. Personal and family history The patient denied bad personal habits and genetic diseases in his family. However, he reported that his father had similar progressive ptosis but did not attend a hospital for diagnosis and treatment before his death. Physical examination Upon physical examination, moderate bilateral blepharoptosis was observed without obvious edema, erythema, papules, or masses on his face (Figure 1A). Further careful examination of the periocular and upper facial skin led to the observation of shallow glabellar wrinkles and obvious infiltration by touch. It was important to note that erythematous lesions were inspired for the first time after the skin biopsy of the upper eyelid and forehead (Figure 1B). Figure 1 Clinical manifestations of the patient before and after treatment. A: Initial clinical presentation. Moderate ptosis (yellow lines), suspected edema inside bilateral upper eyelids and shallow glabellar wrinkles were observed; B: Seven days after treatment of minocycline and ketotifen. Erythema (yellow arrow) triggered by biopsy and obvious improvement of ptosis and deepened glabellar wrinkles; C: 40 days after treatment of minocycline and ketotifen. The bilateral eyelids basically recovered to the pre-illness state. Laboratory examinations No abnormalities were observed after routine examination of the blood, liver and kidney function, and electrolyte levels. Antinuclear antibody, anti-DNA antibody, anti-ENA antibody, muscle enzymes, and immunoglobulin E (IgE) tests were all negative. There were no obvious abnormalities from neuroelectromyography, neostigmine test, myasthenia gravis-related antibodies, and thyroid function. Upper eyelid and forehead histopathological results showed manifestations such as edema in dermis, lymphatic hyperplasia and dilatation, as well as perivascular lymphocytic infiltration (Figure 2A). Toluidine blue-stained mast cells increased with a mean number of over five in each high-power field (Figure 2B). Figure 2 Biopsy specimen from patient's upper eyelid. A: Hematoxylin–eosin staining (×40) showing edema and dilated lymphatics in dermis (orange arrows). B: Increased infiltration of mast cell (orange arrows) in toluidine blue (×400). Imaging examinations There were no significant abnormalities in craniocerebral imaging. FINAL DIAGNOSIS By combining the typical pathological features with the less conspicuous skin manifestations, which included infiltration by touch, together with subsequent erythema and slight edema induced by biopsy, the patient was diagnosed with Morbihan disease. Given the less typical clinical appearance, differential diseases that could cause periorbital edema were considered, and these included blepharochalasis syndrome, angioedema, contact dermatitis, dermatomyositis, lupus erythematosus and Melkersson-Rosenthal Syndrome. However, the patient’s inconsistent history (e.g. onset age, persistent course, no allergen exposure, no wheals, normal muscle strength, no tongue fissures or facial nerve defects) and associated examinations (e.g. normal eosinophils, IgE, muscle enzymes, autoantibodies, unsupported pathological findings) eliminated the possibility of these differential diseases. TREATMENT The patient was treated with a combination therapy of oral minocycline at 100 mg/d and oral ketotifen at 1 mg/d. His ptosis was rapidly relieved and forehead wrinkles also deepened seven days later (Figure 1B), accompanied by progressive gradual improvement in the condition, overall (Figure 1C). OUTCOME AND FOLLOW-UP Bilateral ptosis improved and the eyelids reassumed the pre-disease state. The administration of the drugs was stopped 40 days after the commencement of the treatment. No recurrence of the signs and symptoms was observed during a six-month follow-up period. DISCUSSION Morbihan disease, also known as rosacea lymphedema or solid persistent facial edema, is a rare entity and few cases have previously been reported. The precise diagnosis of Morbihan disease remains a challenge in clinical practice. Eyelids are most frequently involved, followed by the forehead, nose and cheeks[4]. Swelling can be found in almost all previous periocular Morbihan disease cases as the typical feature. There have been no reports of Morbihan disease with ptosis as the first or only manifestation. When ptosis exists without obvious edema, it is more likely to be overlooked and mislead ophthalmologists, neurologists and dermatologists to confused directions. Skin inflammation can also cause ptosis, which is attributed to transient dysfunction of aponeurosis or levator palpebrae. This dysfunction may be due to inflammatory cell infiltration or edema-induced mechanical weight increase[5]. The inflammatory ptosis should be considered as a diagnostic prompt in our atypical case. The histopathological features of Morbihan disease include dermal edema, lymphatic hyperplasia and dilatation, perivascular infiltration of lymphocytes, perifollicular fibrosis and occasionally perilymphatic or intralymphatic epithelioid cell granulomas[6]. The infiltration of increased mast cells was a histological feature in our patient. Activation of mast cells in Morbihan disease[7-9] can promote vasodilation, edema, and lymphocytic infiltration in the dermis. In addition, mast cells are a key mediator of cutaneous pathogenic fibrosis. These changes are associated with injury and obstruction of lymphatics, ultimately triggering dermal lymphedema and inflammatory ptosis when occurring in eyelids[10]. The treatment of Morbihan disease remains a dilemma with no recognized guidelines[11,12]. Corticosteroids, antihistamines, antibiotics, isotretinoin, and combination regimens have been used but these are long-term and are associated with unsatisfactory outcomes. Carbon dioxide laser blepharoplasty and surgical eyelid debulking[1,13] are other treatment options that are employed when drugs are ineffective. However, recurrence is common when these interventions are used, especially in patients with obvious inflammatory cell infiltration. Minocycline has been used in twelve patients with Morbihan disease in previous English language reports (Table 1), mainly as an oral monotherapy, but only six of twelve patients achieved significant improvement to variable extents. The histopathological features might guide and predict the therapeutic effects of various interventions. The positive effects of minocycline were associated with the presence of mast cells (4/6). The patients without mast cell infiltration failed in treatment with minocycline even at high doses or with long-term administration, except one patient who was infected with increased Demodex mites and was administered minocycline combined with topical metronidazole[14]. This suggested that mast cells might be a histological therapeutic clue for Morbihan disease. The anti-mast cell activities of minocycline indicate their significant role in the treatment of Morbihan disease[15]. Tetracycline can inhibit cytokine production of mast cells in vitro[8], and minocycline has been shown to inhibit IgE responses and mast cell mediated cutaneous edema[16]. However, treatment with minocycline often requires a long period of four to six months, which inevitably exposes the liver to toxicity issues. In our patient, a seven-day rapid response was observed, and a 40-d short course was achieved when minocycline was combined with ketotifen. Ketotifen is a strong mast cell stabilizer that is used in many allergic diseases. It can inhibit mast cell degranulation during the early stages of inflammation. It suppresses pathological edema and fibrosis by reducing the release of inflammatory factors IgE, tumor necrosis factor-α, interleukin (IL)-4, and IL-13[17,18]. The success of the combination therapy of minocycline and ketotifen in our patient may be a good option that reduces the duration of antibiotic administration, especially in the early stages of the disease. Table 1 Use of minocycline and its effects in Morbihan disease Sex/age (yr) Clinical features Mast cell Dose Course Effect M/51[10] Erythema and edema on left upper eyelid with complete left ptosis - N/A N/A Ineffective M/70[6] Erythematous edema on forehead, nose and periorbital areas - 200 mg/d, 300 mg/d Over 2 mo Ineffective M/54[12] Erythema and edema on cheek and periorbital area - N/A 6 to 7 mo Ineffective M/30[13] Edema on bilateral eyelids - 100 mg/d, 50 mg/d 7 mo Discontinued due to insignificant improvement and abnormal liver enzyme levels M/74[7] Swelling on right cheek and left upper eyelid + 50 mg/d, 100 mg/d 4 mo Partially effective F/64[15] Swelling on entire face + 100 mg/d 6 mo Significantly effective M/38[18] Periorbital indurated edema + N/A 3 mo Ineffective M/56[8] Swelling on both eyelids and cheeks + 100 mg/d 7 mo Effective but discontinued due to abnormal liver enzyme levels F/41[4] Erythematous edema on upper eyelids, forehead, nose and cheeks + N/A N/A Effective F/40[11] Bilateral periorbital edema and intermittent blurry vision - N/A A short period Discontinued due to urticaria N/A/88[1] Swelling on left eyelid N/A N/A N/A Partially improved M/28[14] Edema and erythema on periorbital region, nose and cheeks; telangiectasias and pustules - 100 mg/d Over 2 mo Partially improved F: Female; M: Male; N/A: Not applicable. CONCLUSION Morbihan disease with blepharoptosis as the only or main manifestation rather than facial edema has not been previously reported. This rare condition provided supplementary clinical manifestation and diagnostic clues of Morbihan disease for physicians in dermatology, neurology, and ophthalmology. There is no exact effective treatment strategy for Morbihan disease. However, histopathology is an important reference for the selection of treatment strategies. Based on literature review and the successful treatment of our patient, minocycline combined with antihistamines should be the prior treatment choice in Morbihan disease with increased mast cells. ACKNOWLEDGEMENTS We are very grateful to the patient for agreeing to publish of case details. Informed consent statement: Informed written consent was obtained from the patient for publication of this report and any accompanying images. Conflict-of-interest statement: The authors declare that they have no conflict of interest to disclose. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016). Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: March 13, 2023 First decision: May 19, 2023 Article in press: June 14, 2023 Specialty type: Dermatology Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Alkhatib AJ, Jordan; Emran TB, Bangladesh S-Editor: Ma YJ L-Editor: A P-Editor: Ma YJ ==== Refs 1 Yvon C Mudhar HS Fayers T Siah WF Malhotra R Currie Z Tan J Rajak S Morbihan Syndrome, a UK Case Series Ophthalmic Plast Reconstr Surg 2020 36 438 443 31990896 2 Kim JE Sim CY Park AY Hong SA Park YL Jang SY Lee SY Case Series of Morbihan Disease (Extreme Eyelid Oedema Associated with Rosacea): Diagnostic and Therapeutic Approaches Ann Dermatol 2019 31 196 200 33911568 3 Kim JH Treatment of Morbihan disease Arch Craniofac Surg 2021 22 131 134 34225403 4 Ramirez-Bellver JL Pérez-González YC Chen KR Díaz-Recuero JL Requena L Carlson JA Llamas-Velasco M Clinicopathological and Immunohistochemical Study of 14 Cases of Morbihan Disease: An Insight Into Its Pathogenesis Am J Dermatopathol 2019 41 701 710 31567295 5 Koursh DM Modjtahedi SP Selva D Leibovitch I The blepharochalasis syndrome Surv Ophthalmol 2009 54 235 244 19298902 6 Nagasaka T Koyama T Matsumura K Chen KR Persistent lymphoedema in Morbihan disease: formation of perilymphatic epithelioid cell granulomas as a possible pathogenesis Clin Exp Dermatol 2008 33 764 767 18627384 7 Fujimoto N Mitsuru M Tanaka T Successful treatment of Morbihan disease with long-term minocycline and its association with mast cell infiltration Acta Derm Venereol 2015 95 368 369 25510871 8 Okubo A Takahashi K Akasaka T Amano H Four cases of Morbihan disease successfully treated with doxycycline J Dermatol 2017 44 713 716 28150340 9 Ludgate M Baker G Unlocking the immunological mechanisms of orbital inflammation in thyroid eye disease Clin Exp Immunol 2002 127 193 198 11876739 10 Lai TF Leibovitch I James C Huilgol SC Selva D Rosacea lymphoedema of the eyelid Acta Ophthalmol Scand 2004 82 765 767 15606479 11 Boparai RS Levin AM Lelli GJ Jr Morbihan Disease Treatment: Two Case Reports and a Systematic Literature Review Ophthalmic Plast Reconstr Surg 2019 35 126 132 30252748 12 Hu SW Robinson M Meehan SA Cohen DE Morbihan disease Dermatol Online J 2012 18 27 23286817 13 Kou K Chin K Matsukura S Sasaki T Nozawa A Aihara M Kambara T Morbihan disease and extrafacial lupus miliaris disseminatus faceie: a case report Ann Saudi Med 2014 34 351 353 25811210 14 Rizzo A Fiorani D Lazzeri L Taddeucci P Rubegni P Flori ML Russo F Usefulness of in vivo reflectance confocal microscopy in Morbihan syndrome Skin Res Technol 2021 27 974 976 33455034 15 Kabuto M Fujimoto N Honda S Tanaka T Successful treatment with long-term use of minocycline for Morbihan disease showing mast cell infiltration: A second case report J Dermatol 2015 42 827 828 25917342 16 Joks R Durkin HG Non-antibiotic properties of tetracyclines as anti-allergy and asthma drugs Pharmacol Res 2011 64 602 609 21501686 17 Yuan W Hou S Jia H Qiu Z Liu T Chen X Li H Sun Y Liang L Sui X Zhao X Zhao Z Ketotifen fumarate attenuates feline gingivitis related with gingival microenvironment modulation Int Immunopharmacol 2018 65 159 173 30316074 18 Rebellato PR Rezende CM Battaglin ER Lima BZ Fillus Neto J Syndrome in question An Bras Dermatol 2015 90 909 911 26734879

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4684 10.12998/wjcc.v11.i19.4684 Case Report Integrated Chinese and Western medicine in the treatment of a patient with podocyte infolding glomerulopathy: A case report Chang MY et al. Podocyte infolding glomerulopathy Chang Mei-Ying Department of Nephrology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, China Zhang Yu Department of Nephrology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, China Li Ming-Xu Department of Nephrology, First Medical Center of Chinese PLA General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China Xuan Fang Department of Nephrology, First Medical Center of Chinese PLA General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, Beijing 100853, China. xuanfangbasa@qq.com Author contributions: Chang MY performed information collection and drafted the manuscript; Chang MY, Zhang Y, Li MX, and Xuan F contributed to the literature review and manuscript preparation; all authors have read and approved the final manuscript. Corresponding author: Fang Xuan, MM, Attending Doctor, Department of Nephrology, First Medical Center of Chinese PLA General Hospital, Nephrology Institute of the Chinese People’s Liberation Army, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease Research, No. 28 Fuxing Road, Wanshou Road Subdistrict, Southern Haidian District, Beijing 100853, China. xuanfangbasa@qq.com 6 7 2023 6 7 2023 11 19 46844691 28 2 2023 16 5 2023 31 5 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Podocyte infolding glomerulopathy (PIG) is a newly described and rare glomerular disease. To date, only approximately 40 cases have been reported globally. CASE SUMMARY A 26-year-old female patient presented to our hospital with a complaint of intermittent edema of both lower limbs over the past 2 years. The patient was diagnosed with PIG. She was prescribed corticosteroid therapy in other hospitals during the initial stage, to which she had responded poorly and had developed femoral head necrosis. Therefore, we administered immunosuppressants, renin-angiotensin system inhibitors, combined with traditional Chinese medicine. The patient was followed for 1 year, during which her clinical condition improved. CONCLUSION Integrated Chinese and Western medicine may be effective for PIG treatment, which requires active intervention to improve prognosis. Corticosteroid therapy Immunosuppressive agents Podocyte-infolding glomerulopathy Renin-angiotensin system Traditional Chinese medicine Case report ==== Body pmc Core Tip: Due to the limited number of reported cases, insufficient information on the characteristics, diagnosis, and treatment of podocyte infolding glomerulopathy is available. Based on our case and those reported in PubMed, we believe that treatment with corticosteroids, immunosuppressants, and renin-angiotensin system inhibitors is effective. Some patients cannot tolerate corticosteroids and immunosuppressants. When adverse effects occur, clinicians should avoid making negative treatment. Doctors should actively intervene and offer patients treatment suggestions, among which traditional Chinese medicine may be an effective treatment method. INTRODUCTION The concept of podocyte infolding glomerulopathy (PIG) was put forward by the Japanese Society of Nephrology in 2008[1]. PIG can be diagnosed based on histopathological findings. Its pathological feature is the presence of microspheres, microtubules, or both in the glomerular basement membrane (GBM) under electron microscopy, and podocyte infolding into the GBM[1]. Knowledge of the pathogenesis and progression of PIG is rare, due to the limited number of reported cases[1]. Thus, integrated and definitive immunological therapies are not yet available. Some case reports have suggested that corticosteroid therapy can be used to treat this disease. However, it is difficult to assess its efficacy because of the limited number of cases and the lack of long-term follow-up of cases[2-15]. Overall, 15 consecutive cases of PIG, most of which were from Asia, have been reported since the initial study conducted by Joh et al[1] in 2008. Due to the unclear etiology and varied clinical presentations, PIG is more likely a pattern of injury than an etiological diagnosis[7,11]. Therefore, further clinical data need to be accumulated to gain a better understanding of this type of glomerulopathy. Herein, we report a case of PIG in a 26-year-old woman with nephrotic syndrome without renal functional impairment. Along with a literature review of previous cases, we summarize the clinical features and treatment methods for PIG. CASE PRESENTATION Chief complaints A 26-year-old Chinese woman presented to our hospital with intermittent edema of both lower limbs of 2 years’ duration. History of present illness The patient developed pitting edema of the lower extremities in January 2020. She went to the Sixth Medical Center of Chinese PLA General Hospital. Laboratory data are presented in Table 1. A renal biopsy was performed. She was prescribed 80 mg valsartan and 60 mg prednisone daily. After 8 wk of treatment, the dose of prednisone was reduced by 10 mg every 2 wk. However, she had poor compliance and stopped the medication when the dose had been reduced to 20 mg daily in May 2020. Laboratory tests were not performed thereafter. The patient later developed intermittent edema but ignored it. Table 1 Laboratory data before the renal biopsy Test Results 24-h proteinuria, mg 8730 Hemoglobin, g/L 101 (normal: 115-150) White blood cells, × 109/L 3.06 (normal: 3.5-9.5) Platelets, × 109/L 386 (normal: 125-350) Cholesterol, mmol/L 6.96 (normal: 3-5.7) Triglyceride, mmol/L 2.36 (normal: < 1.7) Creatinine, μmol/L 67.4 (normal: 53-97) Albumin, g/L 13 (normal: 40-55) ANA Negative ANCA Negative Anti-double stranded DNA antibody Negative Rheumatoid factor Negative C3, mg/dL 98.4 (normal: 90-180) C4, mg/dL 33.6 (normal: 10-40) IgA, mg/dL 292 (normal: 70-400) IgM, mg/dL 119 (normal: 40-230) Anti-PLA2R antibody Negative ANA: Antinuclear antibodies; ANCA: Antineutrophil cytoplasmic antibodies; IgA: Immunoglobulin A; IgM: Immunoglobulin M; PLA2R: Phospholipase A2 receptor. In December 2021, she was re-hospitalized due to bilateral lower extremity edema, and her 24-h proteinuria level was 4840 mg and serum albumin level was 16.8 g/L. This time, she was treated with 40 mg of intravenous methylprednisolone and 240 mg of oral allisartan isoproxil daily. Seven days later, she developed hip pain and weakness. Hip magnetic resonance imaging showed bilateral ischemic necrosis of the femoral head with joint cavity effusion; therefore, the intravenous methylprednisolone was discontinued, and oral methylprednisolone 36 mg/day was initiated and rapidly tapered (1 tablet per week, or 4 mg). Simultaneously, she was administered 100 mg cyclosporine twice a day while continuing allisartan isoproxil. Her 24-h proteinuria was 5803-7650 mg. In February 2022, her 24-h proteinuria was 6370.65 mg and she came to our hospital for treatment. At admission, the patient had mild edema of both lower limbs, hip pain, and foamy urine. The tongue was pale, with a thin white coating, and her pulse was weak. History of past illness One year earlier, the patient had developed an increase in blood pressure, reaching 170/100 mmHg. She was treated with 240 mg of oral allisartan isoproxil daily. Personal and family history The patient denied any family history of kidney disease. Physical examination On physical examination, her vital signs were as follows: Body temperature, 36.8 ℃; blood pressure, 120/86 mmHg; heart rate, 90 beats per min; respiratory rate, 18 breaths per min. Furthermore, mild edema was observed in both lower limbs. Laboratory examinations The results of laboratory examination were as follows: Urinary protein = 3+, red blood cells = 50/μL, 24-h proteinuria = 6165 mg, serum creatinine = 70.7 μmol/L, serum albumin = 24.2 g/L, cholesterol = 8.03 mmol/L, and triglyceride = 3.24 mmol/L. Imaging examinations Under a light microscope, 28 glomeruli with no spherical sclerosis and three small cell crescents were visible. The mesangial area showed mild focal segmental proliferation and matrix thickening. Capillary loops were well open, and focal epithelial cell vacuolar degeneration was present. The basement membrane was mildly thickened in focal segments. No significant atrophy or inflammatory cell infiltration was seen in the renal interstitium. No significant thickening of the renal vasculature was observed (Figure 1A). Under an electron microscope, extensive podocyte foot-process effacement with infolding into the GBM was seen. The GBM was diffusely thickened with numerous microspherical and microtubular structures (Figure 1B-D). Immunofluorescent staining of the biopsy specimen showed granular deposition of immunoglobulin (Ig)G along the mesangial areas, but with no staining for C3, IgA, IgM, C1q, or fibrinogen (Supplementary Figure 1). Figure 1 Kidney biopsy findings. A: Light microscopic findings on periodic acid-Schiff staining. The mesangial area showed mild focal segmental proliferation and matrix thickening (arrows). The basement membrane is mildly thickened (original magnification, × 400); B: Electron microscopy of kidney biopsy specimen. Thickened glomerular basement membrane (GBM) with numerous diffusely scattered microspherical or microtubular structures (arrows), characteristic of podocyte infolding glomerulopathy (original magnification, × 12000); C: Extensive podocyte foot-process effacement with infolding into the GBM (arrows) (original magnification, × 12000); D: There are no electron-dense deposits (original magnification, × 8000). FINAL DIAGNOSIS Combined with the patient’s medical history, a final diagnosis of PIG was made. TREATMENT We continued allisartan isoproxil treatment and increased the cyclosporine dose to 150 mg in the morning and 100 mg at night. Oral mycophenolate mofetil (500 mg twice a day) was also administered. In addition, we treated the patient with traditional Chinese medicine. First, we diagnosed the patient as having blood stasis and water stagnation, and treated her with Danggui-Shaoyao-San, a compound formula in traditional Chinese medicine, comprised of six herbs: Paeonia lactiflora Pall. 20 g, Alisma orientalis (Sam.) Juzep. 10 g, Angelica sinensis (Oliv.) Diels. 10 g, Poria cocos (Schw.) Wolf. 20 g, Atractylodes macrocephala Koidz. 20 g and Ligusticum chuanxiong Hort 10 g. Over the course of treatment, the edema of the patient was gradually reduced, and the patient’s condition was subsequently mainly characterized by deficiency of the spleen and kidney, manifested as fatigue. We differentiated the syndrome as deficiency of the spleen and kidney, combined with wind evil, and treated her with Modified Huangqi Chifeng decoction to strengthen qi further. The whole prescription was composed of seven herbs: Astragalus membranaceus Bge 30 g, Euryale ferox Salish 20 g, Rosae Laevigatae Fructus 10 g, Radix Paeoniae Rubra 10 g, Saposhnikoviae Radix 10 g, Rhizoma Dioscoreae Nipponicae 20 g, and Hedyotis Diffusae Herba 20 g. OUTCOME AND FOLLOW-UP The patient was followed-up for 1 year, during which we adjusted the drugs slightly according to her symptoms. As shown in Figure 2, her serum albumin levels gradually increased and her urinary protein excretion gradually decreased, with an increase in October 2022, which was considered to be related to an upper respiratory infection. Overall, her clinical condition improved. Figure 2 Changes in 24-h proteinuria (mg), serum creatinine (μmol/L), and serum albumin (g/L) over time. DISCUSSION The concept of PIG, as a newly recognized form of glomerular disease, was first proposed by Joh et al[1] in 2008. Although PIG cases are increasing, it remains unclear whether PIG is a new disease entity or simply a transient morphological phenomenon[2,15]. Hence, it is important to discuss the salient features of this case to gain a better understanding of the nature of the disease. Microstructures typical of PIG were first described in the 1970s[13], referred to as rounded extracellular particles (REPs)[16]. According to Dales and Wallace, REPs in a patient with membranous nephropathy (MN) are nuclear pore organelles based on their morphologic similarity and the presence of autoantibodies in cytoplasmic and nuclear organelles of the patient’s serum[17]. Zhang et al[8] considered that PIG was actually first identified in 1992 when Sato et al[18] described patients with collagen diseases whose renal biopsies demonstrated fine electron-dense deposits in the GBM. It is difficult to determine when PIG first appeared; however, in 2008, a new terminology for PIG was proposed by Joh et al[1], who defined it as microstructures (microspheres and/or microtubules) associated with podocytic invagination (large cytoplasmic projections from podocytes) into the GBM. In contrast to granular debris commonly seen in diseases such as MN, these microstructures are thought to originate from the primary podocyte cell membranes. To date, 40 cases of PIG have been reported globally: 36 from Asia (28 from Japan, 6 from China, and 1 each from Korea and India), 2 from North America (1 from the United States, 1 from Canada), 1 from Argentina, and 1 from Europe[1-15]. We summarized the clinical profiles of the 40 cases, 25 of which were included in the study by Joh et al[1], while the remaining 15 cases are shown in Table 2. Table 2 Clinical profile of podocyte infolding glomerulopathy (15 cases, Case No. 26-40) Case No. Country of the study Year of the study Age (years) Sex Initial proteinuria (mg/day) Hematuria Renal function (Creatinine, μmol/L) Blood pressure (mmHg) Treatment Concomitant diseases Case 26[2] Japan 2013 14 F 3060 1-4 48.6 PSL (40 mg/day) First biopsy: np; Second biopsy: FSGS Case 27[3] Japan 2014 79 M 1426 113.2 140/67 PSL (20 mg/day) HTN, MM Case 28[4] South Korea 2016 44 F 39.8 100/70 PSL (10 mg/day); ARB (15 mg/day) SLE Case 29[5] India 2018 45 F 5800 20 145.9 130/80 High-dose PSL, MMF, 1 g of rituximab UCTD Case 30[6] China 2018 52 F pSS, HT Case 31[7] Germany 2019 56 F 35 U/L 386.3 High dose Pred, rituximab (1 g × 2 ) RA Case 32[8] China 2019 27 F 629 168.0 PSL (48 mg/day), HCQ (0.2 g twice a day) pSS, HT Case 33[8] China 2019 23 F 16796.8 + 46.9 PSL (40 mg/day), HCQ (0.2 g twice a day) tacrolimus (1 mg twice a day) SLE Case 34[9] Argentina 2020 38 F SLE Case 35[10] China 2020 4 M 3670 Given the resistance to steroids and tacrolimus, only treated him with diuretics and (ACEIs) SIOD, hypothyroidism Case 36[11] China 2020 33 F 2124 - 36.2 ARB, Radix Astragali and Huangkui UCTD Case 37[12] Japan 2020 35 F pSS, scleroderma Case 38[13] United States 2021 60 F 2286 61.9 Rituximab (1 g × 3) PLA2R antibody(+) Case 39[14] Canada 2021 52 F 11-20 111.4 158/97 ARB (300 mg/day); spironolactone (25 mg/day); Pred (1 mg/kg) HBcAb(+); HBsAb(+) Case 40[15] China 2021 61 F 2060 19 75.1 ARB (100 mg/day), HCQ (100 mg twice a day), PSL (40 mg/day), CTX (600 mg/month) SLE, HTN ACEIs: Angiotensin-converting enzyme inhibitors; ARB: Angiotensin II receptor blocker; CTX: Cyclophosphamide; F: Female; FSGS: Focal segmental glomerulosclerosis; HCQ: Hydroxychloroquine; HT: Hashimoto’s thyroiditis; HTN: Hypertension; M: Male; MM: Multiple myeloma; MMF: Mycophenolate mofetil; pSS: Primary Sjögren’s syndrome; PLA2R: Phospholipase A2 receptor; Pred: Prednisone; PSL: Prednisolone; RA: Rheumatoid arthritis; SIOD: Schimke immuno-osseous dysplasia; SLE: Systemic lupus erythematosus; UCTD: Undifferentiated connective tissue disease. As shown in Table 2, the age range of the remaining 15 cases was 4-79 years, which was greater than that reported by Joh et al[1], indicating a higher prevalence of PIG and an incidence in a broader population. Significantly more female than male patients were noted (female:male 13:2). Proteinuria reached 16798.8 mg/day, which was markedly higher than the maximum value of 7500 mg/day reported by Joh et al[1]. Three patients, with a mean age of 42.7 years, had increased serum creatinine levels exceeding 1.5 mg/dL (133 μmol/L). On the one hand, this suggests the need for early follow-up of patients and early intervention to slow down the progression of the disease. On the other hand, as PIG is not yet widespread, health education and regular medical check-ups of healthy individuals are important. In Joh et al's report, 17 of the 25 cases were treated with corticosteroids alone or in combination with immunosuppressants[1]. In the 15 newly reported cases of PIG summarized in Table 2, prednisolone (or prednisone) was administered to nine patients (two were treated with prednisolone or prednisone alone[2,3], five were treated with prednisolone/prednisone combined with immunosuppressants, such as mycophenolate mofetil, rituximab, hydroxychloroquine, tacrolimus, and cyclophosphamide[5,7,8,15], one was treated with prednisolone combined with the angiotensin II receptor blocker (ARB) fimasartan[4], one was treated with prednisolone combined with the ARB irbesartan and spironolactone[14]). Immunosuppressants were administered to six patients (one received rituximab alone)[13]. One patient was administered an angiotensin-converting enzyme inhibitor (ACEI) alone[10], and another was treated with the ARB olmesartan medoxomil combined with the traditional Chinese medicines Radix Astragali and Huangkui[11]. The treatments of the remaining three cases were not mentioned in the original articles[6,9,12]. Although treatment guidelines for PIG have not been established to date, a review of the literature above showed that corticosteroids are most widely used for PIG, followed by immunosuppressants and ACEI/ARB. Analysis of concomitant diseases showed that PIG was mostly associated with collagen diseases, such as systemic lupus erythematosus (SLE) (4/15), rheumatoid arthritis (1/15), and primary Sjögren’s syndrome (2/15). In Joh et al's report[1], 13 of the 25 cases had combined SLE. It was also seen in patients with non-collagen diseases, such as undifferentiated connective tissue diseases (2/15), Schimke immuno-osseous dysplasia (1/15), hypertension (1/15), multiple myeloma (1/15), and Hashimoto’s thyroiditis (1/15). The exact relationship between collagen diseases and PIG requires further research. In addition, in one case[2], two renal biopsies were performed; the first renal biopsy showed only PIG, but the second also suggested focal segmental glomerulosclerosis. Thus, in PIG cases, we should pay attention to follow-up, particularly when the patient’s condition does not improve for an extended period or worsens, which may indicate the need for a repeat renal biopsy. Our patient was a 26-year-old woman without systemic autoimmune conditions, based on serological findings. The mesangial area showed mild proliferation and matrix thickening on light microscopy; however, electron microscopy confirmed the diagnosis of PIG. PIG has been classified into three groups based on electron microscopy findings: Invagination of only primary podocytes in type A, invagination and microstructures in type B, and only microstructures in type C[13]. Our patient was classified as type B (Figure 1B-D). After a period of treatment with corticosteroids, she developed femoral head necrosis. Thereafter, she was treated with immunosuppressants and renin-angiotensin system inhibitors, but the results were somewhat disappointing. She came to our hospital for treatment in February 2022. Along with Western medicine treatment, we began to treat her with Chinese herbal medicine treatment based on syndrome differentiation. Her 24-h proteinuria decreased from 6370 mg to 1586 mg over the course of a year. Nonetheless, the treatment outcomes attained cannot be generalized as this is a case report. Future research in prospective studies and case series are required. accumulation of further case reports will yield the experience required to provide patients with better counseling. CONCLUSION Although PIG is a very rare glomerular disease, the combination of our case and those reported in PubMed leads us to believe that treatment with corticosteroids, immunosuppressants, and renin-angiotensin system inhibitors is effective. However, some patients cannot tolerate corticosteroids and immunosuppressants. When adverse effects occur with these treatments, clinicians should avoid making negative treatment. Doctors should actively intervene and suggest treatments to patients, among which traditional Chinese medicine is an effective treatment. ACKNOWLEDGEMENTS We thank Ya-Li Ren from Peking University First Hospital for technical assistance with electron microscopy. Informed consent statement: Informed written consent was obtained from the patient for publication of this report and any accompanying images. Conflict-of-interest statement: The authors declare that they have no conflict of interest to disclose. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016). Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: February 28, 2023 First decision: May 8, 2023 Article in press: May 31, 2023 Specialty type: Medicine, research and experimental Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): D Grade E (Poor): 0 P-Reviewer: Al-Ani RM, Iraq; Pezeshgi A, Iran S-Editor: Fan JR L-Editor: A P-Editor: Zhao S ==== Refs 1 Joh K Taguchi T Shigematsu H Kobayashi Y Sato H Nishi S Katafuchi R Nomura S Fujigaki Y Utsunomiya Y Sugiyama H Saito T Makino H Proposal of podocytic infolding glomerulopathy as a new disease entity: a review of 25 cases from nationwide research in Japan Clin Exp Nephrol 2008 12 421 431 19012046 2 Iguchi A Sohma A Yamazaki H Ito T Saeki T Ito Y Imai N Osawa Y Narita I A case of podocytic infolding glomerulopathy with focal segmental glomerulosclerosis Case Rep Nephrol Urol 2013 3 110 116 24027576 3 Harada M Kamijo Y Ehara T Shimojo H Shigematsu H Higuchi M A case of podocytic infolding glomerulopathy with multiple myeloma BMC Nephrol 2014 15 32 24528497 4 Kwon KW Jeong HJ Lee JH Podocytic infolding glomerulopathy: A case report Ultrastruct Pathol 2016 40 374 377 27661791 5 Matthai SM Mohapatra A Mathew AJ Roy S Varughese S Danda D Tamilarasi V Podocyte Infolding Glomerulopathy (PIG) in a Patient With Undifferentiated Connective Tissue Disease: A Case Report Am J Kidney Dis 2018 72 149 153 29395482 6 Fang JY Song AH Shen B Liu YL A Case of Podocytic Infolding Glomerulopathy with Primary Sjögren's Syndrome and Hashimoto's Thyroiditis Chin Med J (Engl) 2018 131 2747 2748 30425203 7 Wöstmann F Müller RU Göbel H Benzing T Becker JU Bartram MP Case report: a peculiar glomerulopathy in a patient suffering from nephrotic syndrome BMC Nephrol 2019 20 326 31438874 8 Zhang T Sun W Xue J Chen J Jiang Q Mou L Du H Podocytic infolding glomerulopathy: two new cases with connective tissue disease and literature review Clin Rheumatol 2019 38 1521 1528 30879204 9 Malvar A Davila P Ferrari M Delgado P Iscoff P Lococo B Alberton V Podocyte infolding glomerulopathy; report of the first case in Latin America and review of the literature Nefrologia (Engl Ed) 2020 40 469 473 31952852 10 Xiong S Shuai L Li X Dang X Wu X He Q Podocytic infolding in Schimke immuno-osseous dysplasia with novel SMARCAL1 mutations: a case report BMC Nephrol 2020 21 170 32393263 11 Shi J Zheng R Gao H Zhao Z Wu H Zhang Z Podocyte infolding glomerulopathy with undifferentiated connective tissue disease: a case report Ultrastruct Pathol 2020 44 245 248 32172632 12 Manabe S Sato M Kataoka H Taneda S Mochizuki T Nitta K Cell invasion in glomerular basement membrane: infolding glomerulopathy Kidney Int 2020 98 1623 33276871 13 Pandit AP Rennke HG Denker BM Podocytic Infolding Glomerulopathy in a Patient with Phospholipase A2 Receptor-Positive Membranous Nephropathy and Review of the Literature Nephron 2021 145 496 502 33965947 14 Ting JA Hung W McRae SA Barbour SJ Copland M Riazy M Podocyte Infolding Glomerulopathy, First Case Report From North America Can J Kidney Health Dis 2021 8 20543581211048357 34659774 15 Liu X Huang J Zhang K Niu Y Liu Y Cui C Yu C A case of Podocytic Infolding Glomerulopathy with SLE and literature review BMC Nephrol 2021 22 410 34895156 16 Burkholder PM Hyman LR Barber TA Extracellular clusters of spherical microparticles in glomeruli in human renal glomerular diseases Lab Invest 1973 28 415 425 4703848 17 Dales S Wallace AC Nuclear pore complexes deposited in the glomerular basement membrane are associated with autoantibodies in a case of membranous nephritis J Immunol 1985 134 1588 1593 3968428 18 Sato H Saito T Yoshinaga K Intramembranous fine deposit disease associated with collagen disorders: a new morphological entity? Virchows Arch A Pathol Anat Histopathol 1992 420 447 451 1595195

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4677 10.12998/wjcc.v11.i19.4677 Case Report Rare cause of cerebral venous sinus thrombosis: Spontaneous intracranial hypotension syndrome: A case report Huang P. SIH with CVST Huang Pan Department of Neurology, People’s Hospital of Deyang City, Deyang 618000, Sichuan Province, China. 1032857970@qq.com Author contributions: Huang P performed all the work for this paper. Corresponding author: Pan Huang, MD, Clinician, Department of Neurology, People’s Hospital of Deyang City, No. 173 Section 1, North Taishan Road, Deyang 618000, Sichuan Province, China. 1032857970@qq.com 6 7 2023 6 7 2023 11 19 46774683 16 2 2023 28 3 2023 8 5 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Spontaneous intracranial hypotension syndrome is a relatively uncommon neurological disorder of unknown etiology with a good prognosis. Cerebral venous sinus thrombosis is a specific type of cerebrovascular disease caused by multiple etiologies of cerebral venous sinus or vein thrombosis that obstructs cerebral venous return and is associated with impaired cerebrospinal fluid absorption; this entity is rarely seen clinically. Spontaneous intracranial hypotension syndrome is one of the causes of cerebral venous sinus thrombosis, and the probability of their combined occurrence is only 1%-2%. As such, it is easily overlooked clinically, thus increasing the difficulty of diagnosis and treatment. CASE SUMMARY A 29-year-old young woman presented with postural headache. Lumbar puncture suggested a pressure of 50 mmH2O (normal 80 mmH2O-180 mmH2O), and magnetic resonance imaging cerebral venography suggested thrombosis of the supratentorial sinus. These findings were considered indicative of cerebral venous sinus thrombosis due to spontaneous intracranial hypotension syndrome after ruling out immunological causes, tumor, infection, abnormal coagulation mechanism, and hypercoagulable state, etc. She was treated with rehydration and low-molecular heparin anticoagulation for 15 d, and follow-up magnetic resonance imaging cerebral venography suggested resolution of the thrombus. The patient had complete improvement of her headache symptoms. CONCLUSION Spontaneous intracranial hypotension syndrome is one of the rare causes of cerebral venous sinus thrombosis, which is frequently misdiagnosed or missed and deserves consideration by clinicians during differential diagnosis. Dehydration should be avoided in such patients, and early rehydration and anticoagulation therapy are effective treatment options. Spontaneous intracranial hypotension Cerebral venous sinus Thrombosis Rehydration Anticoagulation Case report ==== Body pmc Core Tip: In patients with cerebral venous sinus thrombosis of unknown origin, spontaneous intracranial hypotension syndrome may be considered as a rare cause. Lumbar puncture and cerebral venous sinus angiography are important diagnostic tools. INTRODUCTION Intracranial hypotension syndrome is a group of clinical syndromes characterized by postural headache and cerebrospinal fluid (CSF) pressure < 60 mmH2O. Spontaneous intracranial hypotension syndrome (SIH) may be considered in cases of intracranial hypotension with unknown etiology. The annual incidence of SIH is estimated at 4/100000[1]. Cerebral venous sinus thrombosis (CVST) refers to a special type of cerebrovascular disease caused by cerebral venous sinus or venous thrombosis. CVST may arise due to a variety of causes, and obstructs cerebral venous return and is frequently accompanied by impaired CSF absorption. CVST is rare, and occurs frequently in young and middle-aged people with an annual incidence of 0.5/100000; it is also associated with 0%-3% of all strokes. Spontaneous intracranial hypotension syndrome is a rare cause of cerebral venous sinus thrombosis, with an incidence of only 1%-2%; clinically, it is easy to miss and presents challenges in diagnosis and treatment[2,3]. A patient with spontaneous intracranial hypotension syndrome with intracranial venous sinus thrombosis was recently admitted to our unit. Given that this case is rarely seen clinically and there are no clear treatment guidelines, the clinical presentation, imaging features, and diagnostic and treatment procedures are summarized here with the goal of providing a reference for clinicians. CASE PRESENTATION Chief complaints The patient, a 29-year-old female, was admitted to the hospital with a 3-d postural headache. History of present illness Three days prior to admission, the patient had a headache after waking up in the morning. The headache was located in the bilateral temporal and occipital areas, with persistent, severe, and unbearable distending pain. The headache was accompanied by a pulling pain in the back of the neck, which worsened with sitting or standing positions, and improved in a lying position. The patient also endorsed nausea and vomiting several times. She denied loss of consciousness, fever, limb convulsions, incontinence, slurred speech, and limb weakness. History of past illness The patient had no pertinent past illness history. Personal and family history The patient had no pertinent personal or family history. Physical examination At admission, the patient’s vital signs were as follows: Temperature 36.3℃, pulse 64 beats/min, respiration rate 19 breaths/min, and blood pressure 137/79 mmHg. No significant abnormalities were seen in the heart, lungs, or abdomen. The patient was clearly conscious, fluent in speech, and had normal orientation. The pupils were round and equal in size (about 3 mm in diameter) with a sensitive light reflex and no nystagmus. There was no facial tongue palsy and the pharyngeal reflex was present. The muscle tone of the extremities was normal and the muscle strength was grade 5. Sensory examination was normal. Bilateral pathological signs were negative, meningeal stimulation signs were negative, and tendon reflexes of all 4 limbs were present and symmetrical. Laboratory examinations The following blood tests were performed: Liver function, renal function, serum electrolytes, thyroid function, coagulopathy, markers of myocardial injury, syphilis, HIV, glycosylated hemoglobin, lipids, rheumatoid factor, C-reactive protein, antistreptolysin-O, erythrocyte sedimentation rate, immune panel, and tumor markers. All laboratory values were within reference ranges. An electrocardiogram demonstrated sinus bradycardia. Imaging examinations Computed tomography of the head suggested a nodular, linear high-density shadow in the right parietal cerebral cortex, superior parietal sagittal sinus with a differential of venous embolism or vascular malformation with thrombosis (Figure 1). No abnormality was seen in brain parenchyma, and magnetic resonance imaging (MRI) with magnetic resonance venography (MRV) was recommended (Figure 2). Computed tomographic venography of the head and neck suggested a hypodense filling defect in the cortical veins of the right parietal sagittal sinus, consistent with venous thrombosis (Figure 3). Figure 1 Head computed tomography with nodular, linear hyperdense shadow in the right parietal cerebral cortex and superior sagittal sinus (white arrow). Figure 2 Head magnetic resonance imaging with magnetic resonance venography. A: A right superior sagittal sinus strip of low signal was seen on T1-weighted imaging; B: The right superior sagittal sinus strip of low signal was seen on T2-weighted imaging; C: T1-enhanced scan shows a low signal in the right superior sagittal sinus; D: The lesion is not clearly displayed on contrast-enhanced magnetic resonance venography. Figure 3 Head and neck computed tomographic venography. A hypointense filling defect was seen in the parasagittal sinus cortical vein of the right parietal lobe, and venous thrombosis was considered (white arrow). FURTHER DIAGNOSTIC WORK-UP An opening pressure of 50 mmH2O was measured by uncomplicated lumbar puncture, and the CSF was colorless with no significant laboratory abnormalities. A cranial MRI enhancement scan demonstrated a localized filling defect in the right superior sagittal sinus strip with striped low signal in weighted sequences (T1WI and T2WI), suggesting a possible focal thrombus. FINAL DIAGNOSIS Taking into consideration the clinical symptoms, absence of medical history, the physical exam findings, and the laboratory and radiographic findings, a final diagnosis of spontaneous intracranial hypotension syndrome with cerebral venous system thrombosis was made. TREATMENT The patient was admitted to the hospital as subarachnoid hemorrhage could not be excluded. Initial treatment included nimodipine to prevent vasospasm, rehydration, and symptomatic analgesia. The patient's headache symptoms were partially resolved after 2 d of treatment. After the diagnosis of spontaneous intracranial hypotension syndrome with cerebral venous system thrombosis was clear, the treatment was adjusted to include rehydration and anticoagulation with low molecular weight heparin (5000 IU every 12 h via subcutaneous injection). After 13 d of treatment, the patient's headache symptoms completely resolved, and she was discharged on oral warfarin anticoagulation and recommended to increase her water intake to 2000 mL-3000 mL per d). OUTCOME AND FOLLOW-UP Fifteen d after discharge, the patient had no further postural headache symptoms, and a follow-up head MRI with MRV suggested no residual venous sinus thrombosis (Figure 4). Three mo later, the patient was seen again in follow-up, and she again endorsed no further headache symptoms, and anticoagulant therapy was discontinued. Figure 4 Review of head magnetic resonance imaging. A: No abnormal signal shadow was seen in the right sagittal sinus on T1-weighted imaging; B: No abnormal signal shadow was seen in the right sagittal sinus on T2-weighted imaging; C: No abnormal signal shadow was seen in the right sagittal sinus on T1-enhanced scan. DISCUSSION The presenting symptom of this patient was postural change-related headache. Intracranial hypotension syndrome was considered after uncomplicated lumbar puncture and related examinations were performed. The patient had no history of infection or fever during or near the onset of symptoms, the CSF opening pressure was low, and the CSF was normal on laboratory evaluation, so infectious meningitis could be excluded. There was no nodule-like enhancement on MRI, there were no features of hypertrophic cranial pachymeningitis, and there were no abnormalities on immunological examination, so hypertrophic cranial pachymeningitis was excluded. The patient’s symptoms followed an acute course, there was no blood in the CSF, no subarachnoid hyperdensity was seen on computed tomography, no aneurysm was seen on computed tomographic angiography of the head and neck, and there was no recent history of head trauma, so subarachnoid hemorrhage was not considered. The patient denied prior head trauma, cranial surgery, lumbar puncture, myelography, subarachnoid block, history of poisoning, and history of dehydration; therefore, secondary hypocranial pressure due to these etiologies was excluded. Hematological disorders are also a cause of venous sinus thrombosis; however, after a hematological workup, this cause was also ruled out[4]. Although it was not clear whether CSF leak existed in this patient, the opening pressure on lumbar puncture was less than 60 mmH2O, which could still be considered indicative of SIH. SIH is a rare clinical neurological disorder frequently associated with CSF leakage. The typical clinical manifestation of SIH is postural headache due to reduced intracranial pressure which causes pulling on the meninges; imaging often shows dilatation of the cerebral venous system, dural enhancement, subdural effusion, and brain tissue prolapse, while myelography shows epidural CSF accumulation[5-7]. In SIH, the major etiology is a decrease in CSF volume rather than a decrease in pressure, so normal or elevated CSF pressure measurements cannot be used as a basis for excluding hypocranial pressure syndrome because CSF pressure measured during lumbar puncture in the lateral position does not reflect intracranial pressure in the upright position, nor does it provide information on CSF dynamics during postural changes[8]. In addition to SIH, further refinement of head and neck angiography in this patient suggested CVST, which is a rare and potentially fatal cerebrovascular disease with an annual incidence of 0.5/100000, accounting for 0% to 3% of all ischemic strokes[9]. The order of occurrence of spontaneous intracranial hypotension syndrome and cerebral venous thrombosis has been uncertain, however, with the advancement of science and technology, it has been confirmed that spontaneous intracranial hypotension syndrome is one of the risk factors for CVST[3]. The pathophysiological mechanism is not definitively known, but several hypotheses have been proposed. First, the Monro-Kellie theory states that due to the loss of CSF, the compensatory blood volume in the venous cavity increases, causing the cerebral venous system to dilate resulting in the slowing and stagnation of venous blood flow promoting thrombosis[10]. In support of this theory, Kranz et al[11] found that patients with low intracranial pressure had dilated cerebral venous sinuses and that their cross-sectional area was 70% larger than the corresponding section in normal subjects. Another possibility is outlined by the theory of abnormal CSF buoyancy, which suggests that the loss of CSF buoyancy results in intracranial tissue structure sagging, pulling the cerebral veins and venous sinuses leading to disruption of venous hemodynamics or even stagnation of venous blood flow[12]. A final possible mechanism to consider is that if a CSF loss occurs, this can reduce the absorption of CSF by the venous system, resulting in increased viscosity and hypercoagulability of blood in the cerebral venous sinus cavity, thus increasing the risk of thrombosis[13]. Treatment of SIH includes conservative approaches (e.g., bed rest, massive fluid replacement, oral caffeine, hormone therapy), epidural blood patching, epidural saline injection, surgical treatments (e.g., CSF leak repair or other cause-specific procedures), and various treatments for complications[3]. One study showed the success rate of conservative treatment to be approximately 24.47%, and suggested that hormone therapy may be a potential first-line treatment option. The use of hormones was proposed to significantly improve the clinical symptoms of SIH and reduce the probability of need for invasive epidural blood patching[14,15]. In addition, hormone therapy may also be effective in improving postural headache in some patients who fail epidural blood patch treatment[16]. However, the ideal duration, type, dose, and route of administration of hormones are still inconclusive; however, the majority of patients could have symptom relief within days of administration[17]. Hormones may exert their pharmacological effects by: (1) Ameliorating brain edema and inhibiting inflammation caused by brain herniation; (2) Inhibiting meningeal inflammation and that of CSF cells or proteins, thus reducing CSF leakage; (3) Inhibiting excessive absorption of CSF; and (4) Promoting reabsorption of CSF from the epidural space and increasing the overall CSF volume[18]. In cases where SIH symptoms persist or are associated with CVST, epidural hemopexy is recommended abroad to close the CSF leak before treating CVST[19,20]. Treatment of CVST includes anticoagulation and endovascular intervention, with anticoagulation being the first-line treatment option. Anticoagulation is an important treatment modality in patients with SIH with CVST. Endovascular therapy is only indicated in patients with CVST with severe thrombotic load, psychiatric abnormalities during the course of the disease, or worsening symptoms with systemic anticoagulants. CONCLUSION In conclusion, SIH can often lead to the rare complication of CVST, which is frequently misdiagnosed or missed in clinical practice. For patients with postural headache with focal neurological deficits or imaging findings of venous sinus thrombosis, clinicians should consider the possibility of coexistence of both diseases, and lumbar puncture and cerebral venous sinus angiography should be performed. Patients whose clinical symptoms and imaging improve after conservative management with rehydration and anticoagulation likely do not need epidural hemorrhage patching, but should be monitored for intracranial hemorrhage. Informed consent statement: Written informed consent was obtained from the patient for publication of this report and any accompanying images. Conflict-of-interest statement: The authors declare that they have no conflicts of interest. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016). Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: February 16, 2023 First decision: March 24, 2023 Article in press: May 8, 2023 Specialty type: Clinical neurology Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Arboix A, Spain; Sidoti A S-Editor: Ma YJ L-Editor: Filipodia P-Editor: Ma YJ ==== Refs 1 Schievink WI Maya MM Moser FG Simon P Nuño M Incidence of spontaneous intracranial hypotension in a community: Beverly Hills, California, 2006-2020 Cephalalgia 2022 42 312 316 34553617 2 Ferrante E Trimboli M Rubino F Spontaneous intracranial hypotension: review and expert opinion Acta Neurol Belg 2020 120 9 18 31215003 3 Ferrante E Trimboli M Petrecca G Allegrini F Cerebral venous thrombosis in spontaneous intracranial hypotension: A report of 8 cases and review of the literature J Neurol Sci 2021 425 117467 33894614 4 Arboix A Jiménez C Massons J Parra O Besses C Hematological disorders: a commonly unrecognized cause of acute stroke Expert Rev Hematol 2016 9 891 901 27367035 5 Schievink WI Spontaneous Intracranial Hypotension N Engl J Med 2021 385 2173 2178 34874632 6 Kranz PG Malinzak MD Amrhein TJ Gray L Update on the Diagnosis and Treatment of Spontaneous Intracranial Hypotension Curr Pain Headache Rep 2017 21 37 28755201 7 Kranz PG Tanpitukpongse TP Choudhury KR Amrhein TJ Gray L Imaging Signs in Spontaneous Intracranial Hypotension: Prevalence and Relationship to CSF Pressure AJNR Am J Neuroradiol 2016 37 1374 1378 26869465 8 D'Antona L Jaime Merchan MA Vassiliou A Watkins LD Davagnanam I Toma AK Matharu MS Clinical Presentation, Investigation Findings, and Treatment Outcomes of Spontaneous Intracranial Hypotension Syndrome: A Systematic Review and Meta-analysis JAMA Neurol 2021 78 329 337 33393980 9 Arboix A Bechich S Oliveres M García-Eroles L Massons J Targa C Ischemic stroke of unusual cause: clinical features, etiology and outcome Eur J Neurol 2001 8 133 139 11430270 10 Mokri B The Monro-Kellie hypothesis: applications in CSF volume depletion Neurology 2001 56 1746 1748 11425944 11 Kranz PG Tanpitukpongse TP Choudhury KR Amrhein TJ Gray L How common is normal cerebrospinal fluid pressure in spontaneous intracranial hypotension? Cephalalgia 2016 36 1209 1217 26682575 12 Haritanti A Karacostas D Drevelengas A Kanellopoulos V Paraskevopoulou E Lefkopoulos A Economou I Dimitriadis AS Spontaneous intracranial hypotension: clinical and neuroimaging findings in six cases with literature review Eur J Radiol 2009 69 253 259 18182266 13 Savoiardo M Armenise S Spagnolo P De Simone T Mandelli ML Marcone A Morciano G Andreula C Mea E Leone M Chiapparini L Dural sinus thrombosis in spontaneous intracranial hypotension: Hypotheses on possible mechanisms J Neurol 2006 253 1197 1202 16680559 14 Ferro JM Aguiar de Sousa D Cerebral Venous Thrombosis: an Update Curr Neurol Neurosci Rep 2019 19 74 31440838 15 Pascual LF Santos S Escalza I Iñiguez C Morales-Asín F Spontaneous intracranial hypotension: quick clinical and magnetic resonance imaging response to corticosteroids. A case report Headache 2002 42 359 361 12047337 16 Hannerz J Dahlgren G Irestedt L Meyerson B Ericson K Treatment of idiopathic intracranial hypotension: cervicothoracic and lumbar blood patch and peroral steroid treatment Headache 2006 46 508 511 16618271 17 Goto S Ohshima T Yamamoto T Shimato S Nishizawa T Kato K Successful steroid treatment of coma induced by severe spontaneous intracranial hypotension Nagoya J Med Sci 2016 78 229 236 27303109 18 Tonello S Grossi U Trincia E Zanus G First-line steroid treatment for spontaneous intracranial hypotension Eur J Neurol 2022 29 947 949 35141990 19 Zhang D Wang J Zhang Q He F Hu X Cerebral Venous Thrombosis in Spontaneous Intracranial Hypotension: A Report on 4 Cases and a Review of the Literature Headache 2018 58 1244 1255 30238694 20 Paris P Joubert M Clavelou P Moisset X Cerebral venous thrombosis due to spontaneous intracranial hypotension: Reperfusion after epidural blood patch only Rev Neurol (Paris) 2021 177 1039 1041 33648781

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4531 10.12998/wjcc.v11.i19.4531 Retrospective Study Application of cross-migration theory in limb rehabilitation of stroke patients with hemiplegia Lu YH et al. Cross-migration theory in stroke limb rehab Lu Yan-Hong Geriatric Rehabilitation Department, Shanghai Jing'an District Shibei Hospital, Shanghai 200040, China Fu Yi Geriatric Rehabilitation Department, Shanghai Jing'an District Shibei Hospital, Shanghai 200040, China Shu Jin Geriatric Rehabilitation Department, Shanghai Jing'an District Shibei Hospital, Shanghai 200040, China Yan Li-Yan Department of Nursing, Shanghai Jing'an District Shibei Hospital, Shanghai 200040, China Shen Hai-Jian Department of Nursing, Shanghai Jing'an District Shibei Hospital, Shanghai 200040, China. lemondeer2001Lyh@163.com Author contributions: Lu YH and Fu Y contributed equally to this work; Shu J designed the study; Yan LY contributed to the analysis of the manuscript; Shen HJ and Lu YH were involved in the data and writing of this article; All authors have read and approved the final manuscript. Corresponding author: Hai-Jian Shen, RN, Associate Chief Nurse, Department of Nursing, Shanghai Jing'an District Shibei Hospital, No. 4460 Gonghe Xin Road, Jing'an District, Shanghai 200040, China. lemondeer2001Lyh@163.com 6 7 2023 6 7 2023 11 19 45314543 6 4 2023 8 5 2023 22 5 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Stroke is a common cause of neurological dysfunction, often resulting in hemiplegia. Thus, rehabilitation of limb function in stroke patients is an important step towards accelerating recovery and improving quality of life. AIM To investigate whether unilateral strength training in hemiplegic stroke patients could lead to cross-migration, an increase in bilateral muscle strength, and an improvement in lower limb motor function. METHODS We randomly divided 120 patients with hemiplegic stroke into two groups: Eexperimental and control groups, with 60 patients in each group. Both groups received routine rehabilitation treatment, while the experimental group additionally received ankle dorsiflexion resistance training on the healthy side for 6 wk. We measured the maximum voluntary contract (MVC), changes in surface electromyography (EMG), and the lower limb motor function using the simplified Fugl Meyer Motor Function Assessment Scale (FMA) before and within 1 wk after training. RESULTS The FMA score in the experimental group improved significantly compared to both their pre-training score and the control group's post-training score (P < 0.05). The integrated EMG of the anterior tibialis muscle and pulmonary intestine muscle in the experimental group were significantly different after training than before (P < 0.05). Furthermore, the MVC of the anterior tibialis muscle on both the healthy and affected sides and the MVC of the pulmonary intestine muscle on both sides showed significant improvement compared with before training and the control group (P < 0.05). CONCLUSION Our findings suggest that ankle dorsiflexion resistance training on the healthy side in hemiplegic stroke patients can increase strength in the opposite tibialis anterior muscle and antagonist's muscle, indicating a cross-migration phenomenon of strength training. Furthermore, this type of training can also improve lower limb motor function, providing a new exercise method for improving early ankle dorsiflexion dysfunction. Stroke hemiplegia Cross migration Strength training Lower limb function Resistance training ==== Body pmc Core Tip: Unilateral ankle dorsiflexion resistance training on the healthy side can improve lower limb motor function and lead to cross-migration of strength in hemiplegic stroke patients. INTRODUCTION Stroke, also known as a cerebrovascular accident, refers to acute cerebral vasospasm, occlusion, or rupture caused by various reasons, resulting in neurological impairment diseases dominated by hemiplegia. The incidence of stroke is very high, and the complications are characterized by high disability and high recurrence rate. Hemiplegia is a syndrome with the main clinical manifestation of incomplete or complete loss of random movement of one side of the limbs caused by brain diseases[1]. China is the largest country in the world affected by strokes. Every year, there are two million new stroke patients, with one-third of them having obvious disabilities and 80%-90% having movement disorders. Rehabilitation of patients' limb functions is an essential step in their recovery and return to their families and society. It is the most important part of rehabilitation care. Under stable conditions, rehabilitation nursing can effectively accelerate the recovery time of patients, minimize the disability of patients, improve recovery of patients' self-care ability, and thus improve the quality of life of stroke patients. In sports physiology, there is a phenomenon of cross-migration of training effects, which means that unilateral limb movement can produce simultaneous improvement in the functions of opposite homologous parts (strength, joint range of motion, function)[2]. Short, high-intensity resistance exercises on one side of a healthy person's limb can cause the strength of the untrained limb on the opposite side to increase[3]. It has been found that training the opposite arm can offset muscle atrophy caused by limb fixation, and the phenomenon of cross-migration is unilateral, which can only be transferred from the dominant limb to the non-dominant limb[4]. Training the healthy side of the limb inhibits the flexor modes of the upper limb and the extensor modes of the lower limb, effectively establishing a normal mode. This improves the level of ADL[5]. Cross–migration could be applied in stroke patients with hemiplegia, and equal-length resistance training could be performed on the healthy side to increase the strength of the affected side without exercise and improve the motor function of lower limbs[6]. It shows that when joints and muscles of one side of a limb are injured, their injuries and dysfunction can affect both sides of the limb, leading to a decline of the healthy side function. It suggests that training uninjured limbs may be one of the important measures to prevent its occurrence. Even for stroke patients in the 1st week, muscle strength of the healthy side lower limb has decreased to varying degrees, and the state of the healthy side limb will affect the overall function of stroke patients[7]. It also shows that muscular atrophy and hypotonia in stroke patients are not limited to paralyzed limbs but also can cause disuse muscular atrophy and hypotonia in healthy limbs. At present, a consensus has been reached on the training of affected limbs, with various theories and training methods emerging endlessly. For stroke patients with hemiplegia, functional training of the affected limb is generally emphasized, while compensation and functional training of the healthy limb is easily ignored. In clinical rehabilitation training, therapists pay more attention to the recovery of the limb function of the affected side but neglect or pay less attention to functional activities of the healthy side, leading to the disuse degradation of the healthy side. Active exercise of healthy limbs can improve the tension of the central nervous system, activate the physiological function of the system, prevent complications, improve the general condition, and increase the confidence of patients in rehabilitation[8]. It has been confirmed that cross-migration refers to the concept that training one side of a limb can not only enhance the muscle strength of that side of the limb but also enhance the muscle strength of the opposite limb that has not received direct training[9]. The study found that functional exercise of healthy limbs can accelerate the establishment of ipsilateral cerebral collateral circulation, promote the recurrence of tissues around lesions, and further enhance the functional recovery of affected limbs. Bilateral exercise can promote the formation of new brain nerve channels, aid in the recovery of neural function, and trigger changes in EMG signals[10]. A study of 20 stroke patients who trained five times per week for 5 wk found that maximum wrist resistance training increased extension strength by 42% in the healthy wrist and 35% in the affected wrist. The scores of motor function assessment (FMA) of four patients increased, and longer training time resulted in a more noticeable rehabilitation effect. The rehabilitation of healthy limbs is not only effective for establishing collateral circulation but also improves daily life capacities. The rehabilitation training of healthy limbs is equally important to rehabilitation training of affected limbs in the functional recovery of hemiplegia. Stroke has become one of the global public health problems, with the highest incidence observed in people aged ≥ 41-50 years. Motor dysfunction is one of the most common dysfunctions in stroke, and early successful rehabilitation nursing can reduce muscle atrophy, joint dislocation, joint contracture deformity, foot drop, or varus, which drugs cannot replace. Resistance movement is a type of resistance movement of the limbs that uses muscle-strength exercise equipment (such as sandbags and elastic belts). It increases the diameter of muscle fibers, exercises muscles, and effectively enhances muscle strength through rhythmic weight bearing and repeated exercises. Active exercise against resistance is the best approach to enhance muscle strength and has a positive effect on enhancing bone density and bone metabolism. Current scientific consensus is that 3-6 sets of resistance exercise are optimal for strength gains. Beyond 6 sets, there is no additional benefit for strength[11]. After 12 wk of low-intensity resistance exercise, the lower limb movement ability of older people has been improved to a certain extent. Rehabilitation nursing intervention for stroke patients can improve the effect of nursing, help patients recover better, and improve patient satisfaction. Nursing intervention aims to improve the quality of life and self-management ability during the recovery period from a stroke. The resistance exercise based on cross-migration theory has a broad application prospect for the rehabilitation of limb motor function of stroke patients. However, there is little research on this model of resistance exercise worldwide. The central control mechanism of cross-migration and how to effectively use this principle in the field of rehabilitation still needs to be further explored. This study focuses on patients with unilateral hemiplegia after stroke. The research team was comprised of rehabilitation nurses, doctors, and therapists. Under the guidance of cross-migration theory, while carrying out routine rehabilitation training of the affected limb, we provided progressive resistance exercise training to the healthy limb through nursing intervention to maintain muscle strength of the healthy limb, promote the growth of muscle strength of the affected limb, prevent disuse syndrome, improve motor function, improve patient's quality of life, and improve nursing satisfaction. MATERIALS AND METHODS Research contents Between September 2020 and June 2022, we selected 120 eligible stroke hemiplegic patients hospitalized in the geriatric rehabilitation department of our hospital, randomly dividing them into control and experimental groups with 60 patients in each. Patients received conventional therapies, such as improving cerebral microcirculation, protecting nerves, and promoting metabolism. The control group received conventional rehabilitation training, while the experimental group added healthy side-limb resistance exercise to their conventional rehabilitation training. We compared the improvement of hemiplegic limb muscle strength, peak change of surface electromyography, and FMA score before the intervention, 4 wk after the intervention, and 12 wk after the intervention. Preparation before intervention We created a sports manual and videos of healthy limb resistance movement; We purchased the necessary equipment for the study, including standard sandbags weighing 0.5kg-2.5kg. Research objects Inclusion criteria: (1) The diagnosis was in line with the diagnostic criteria for stroke formulated at the 4th Academic Conference on Cerebrovascular Diseases and confirmed by head computed tomography or magnetic resonance imaging scanning; (2) The primary disease was stroke, and there was no obvious dysfunction before onset; (3) The course of the disease is within 1-6 mo, and the condition is stable; (4) The patients were aged between 50-79; and (5) One side was hemiplegic. The muscle strength of the hemiplegic side is ≤ 2 Levels, and the muscle strength of the healthy side is 4-5 Levels. Exclusion criteria: (1) People with aphasia, cognitive impairment, and other nervous system symptoms that made them unstable and unable to communicate; (2) People with severe osteoporosis, traumatic diseases, or peripheral nerve injury; (3) People with acute joint disease and injury; (4) People with severely limited joint mobility; and (5) People with serious heart, liver, kidney diseases, or bleeding diseases in the past. Sample size estimation We used PASS11 to estimate the sample size with α = 0.05 (one side), β = 0.1, a ratio of 1:1 (prevalence rate was 0.5). The results showed that at least 106 people, including 53 patients and 53 controls, should be included. Considering a 10% loss rate, we included 60 patients and 60 controls in the study. A total of 120 patients were enrolled in the control and experimental groups, respectively. We recorded the personal information of 120 subjects, including name, sex, age, diagnosis, contact address, and telephone number, in a registration book and entered it into a computer for archiving so that subjects could better cooperate with the study and follow-up. Specific Implementation Control group: We implemented routine rehabilitation training: After the rehabilitation assessment, the rehabilitation physician issued a sports prescription, and the rehabilitation therapist implemented sports therapy based on the patient's condition and limb dysfunction. The treatment consisted of Placement and conversion of antispasmodic postures; the range of motion training of hemiplegic limbs; muscle strength training of hemiplegic side; bridge sports; bedside sitting balance training; standing balance training; activities of daily living ability training. The above treatment was administered once a day, for 30 min each time, five times a week, for 6 consecutive weeks. The intensity and time of each training were properly adjusted according to the patient's movements, step by step, so as not to increase the patient's overall spasticity or cause excessive fatigue. Patients in the control group also received routine health education. The responsible nurse issued an exercise manual and instructed patients to move the healthy side of their limb autonomously. Experimental group: Progressive resistance exercise of the contralateral side was carried out as described below. The experimental group received a sports intervention consisting of rehabilitation nurses, doctors, and therapists. The head nurse acted as the team leader and coordinated the intervention and health education activities. The rehabilitation physician formulated the intervention plan and dealt with adverse events. The rehabilitation therapist was responsible for routine exercise training, evaluation, and collection of muscle strength and electromyography data. The researcher trained the team members through a PowerPoint and on-site demonstration to highlight the key points and precautions of the resistance movement. The rehabilitation nurse was mainly responsible for implementing the patient's healthy limb resistance exercise, monitoring the patient's discomfort, blood pressure, and heart rate changes during muscle strength training, and providing health education and discharge follow-up of patients and their families through WeChat management. Health education included issuing exercise manuals to patients and their families, highlighting the importance of motor rehabilitation for stroke, and instructing patients and their families on the importance of healthy side movement as well as how to resist movement of healthy side limbs. Electronic screens were used to play an operation video of the resistance movement. WeChat groups were established for intervention members and patients or family members to provide related knowledge of stroke motor function exercise and enhance motor concept of patients and family members. Sports program The movement mode for the experimental group was progressive resistance movement of the healthy side limb using sandbags to exert resistance. The standard sandbags purchased by the hospital's procurement office were ankle leggings sandbags and wrist leggings sandbag with a weight range of 0.5 kg-2.5 kg. Before the intervention, healthy side muscle strength was evaluated, and sandbags with appropriate weight were selected to apply resistance according to age and individual tolerance. The exercise intensity was medium to low and was evaluated according to Borg's subjective exertion scoring method. According to a patient's subjective feeling of exertion, a score of 6 points indicates no exertion, while a score of 20 points indicates exhaustion. If a patient's score was between 12 and 14 points, it indicated medium and low exercise intensity. If a subject experienced dizziness, asthma, dyspnea, arrhythmia, chest tightness, chest pain, and other discomfort symptoms, the exercise was stopped immediately. The exercise steps were divided into 4 wk of resistance exercise carried out 0.5 h after regular rehabilitation training, subject to completion of the specified number of times, each time for 30 min, five times a week, to avoid excessive exercise. Before and after resistance exercise, warm-up exercises were carried out, including muscle relaxation exercises and gripping palm and finger joints exercises. The maximum exercise intensity of each patient was evaluated by a rehabilitation technician every week. The weight added every week was not more than 10% of the original; the weight of sandbags or the number of exercises that the patient could tolerate. The adaptation stage lasted about 1 wk, and the heart rate response to exercise was observed. In the improvement phase, the weight of the sandbag gradually increased. In the maintenance phase, the exercise intensity was maintained until the end of the intervention. Specific sports methods Upper limb exercise method: To apply resistance on the wrist, appropriate sandbags were selected and divided into two groups of five movements each. They included: shoulder joint horizontal adduction and abduction, shoulder joint forward flexion and extension, and elbow joint flexion and extension. There was a two- minute rest between the groups. Lower limb exercise method: Appropriate sandbags were selected to apply resistance, and five movements were performed. They included: Straightening and lifting the lower limb of one side; and extending the knee joint. There was a two-minute rest between the sets. Discharge education Rehabilitation nurse education: The rehabilitation nurse educated the patients about the essential aspects of exercise training, emphasizing to both patients and their families that rehabilitation was a gradual process. The patients were encouraged to adhere to the training regime. Patients were given wrist and ankle sandbags and an exercise manual to continue functional exercise at home. Home caregivers were instructed to record the frequency and time of resistance movement of healthy side limbs in each diary. WeChat and telephone follow-up: After discharge, the patients were followed up by WeChat and telephone. Home caregivers sent daily resistance exercise videos to the WeChat group. The rehabilitation nurses supervised the implementation of the exercise plan, answered questions of patients and their families, and ensured compliance of the exercise regime. Regular outpatient visits: Patients were advised to regularly visit the geriatric rehabilitation department to evaluate their condition and motor function recovery. If necessary, the patients were re-admitted to the hospital for further rehabilitation treatment. Efficacy evaluation The patients were evaluated before the intervention, 4 wk after the intervention (after training), and 12wk after the intervention. The specific evaluation contents are described below. Free hand muscle strength assessment: Muscle strength was measured according to the internationally accepted modified MRC grading assessment standard. Based on the Lovett grading method, muscle strength was further subdivided by the degree of motion amplitude and degree of applied resistance. The muscle strength test results are divided into 0-5 grades, with level 5 indicating normal force, level 4 indicating anti-gravity and resistance active motion, level 3 indicating anti-gravity but not resistance active motion, level 2 indicating gravity-free active motion, and level 1 indicating muscular contraction. Grade 0 meant no muscle contraction. If muscle strength reached a certain level, but the range of activity could not reach the full range, "-" was added to the upper right corner of the level symbol. If at the end of the movement, it showed resistance or gravity beyond the level, "+" was added to the upper right corner of the level symbol. The higher the percentage of normal muscle strength, the better the effect. Surface electromyography: The subject's surface electromyography (sEMG) was monitored by removing oil with a 75% alcohol cotton ball and taking a supine position. The patient was required to perform isometric contraction with maximum strength, adhere to it for five seconds, and then perform voluntary contraction for 10 s after a rest of 1min. Surface EMG signals of the upper limb deltoid muscle, biceps brachii muscle, and lower limb quadriceps femoris muscle on the hemiplegic side were recorded, and EMG analysis software was used for signal processing and analysis. The ability of EMG signal is concentrated at 0-500Hz. After filtering, 3S extreme maximum voluntary contraction (MVC), integrated EMG value (IEMG), and root mean square value were selected to compare the activation intensity of EMG before and after training. The growth showed that muscle contraction was improved. Limb function: The Fugl Meyer motor function evaluation scale was used to evaluate limb function. The total score of 100 points included two dimensions of upper and lower limb motor function, including 33 items of the upper limb, 0-2 points for each item, and 66 points for the total score. The lower limbs include 17 items with 0-2 points for each item and 34 points in total. The lower the score, the more serious the motor dysfunction of the patient (Figure 1). Figure 1 Technical roadmap. FMA: Function assessment scale. RESULTS Initially, 19 patients were enrolled in the clinical trial. However, 4 patients dropped out of the study, leaving 15 patients in the treatment group and 15 in the control group. Table 1 and Figure 1 present a comparison of the general characteristics of the subjects. Independent sample T test showed that there was no statistical difference in age, course of disease, height, and weight between the two groups (P < 0.05). Similarly, the chi-square test revealed no significant differences in sex and hemiplegic side limbs between the two groups (P < 0.05), indicating that the groups were comparable at baseline. Table 1 Comparison of general data between experimental and control Parameter Experience, mean ± SD Control, mean ± SD Number 60 60 Left hemiplegia, n 44 40 Right hemiplegia, n 16 20 Male sex, n 40 36 Age in yr 64.42 ± 9.12 63.82 ± 7.77 Course of disease in d 45.45 ± 15.34 49.00 ± 14.90 Height in cm 165.42 ± 6.91 164.45 ± 7.12 Weight in kg 64.25 ± 8.05 63.62 ± 9.64 There was no significant difference between baselines of experience and control. The FMA scores improved after the training intervention, with varying degrees of improvement between the treatment and control groups. The results are shown in Table 2 and Figure 2. Specifically, the FMA scores in the experimental group increased significantly compared to both pre-training and post-training scores in control group (P < 0.05). These results suggest that the training intervention effectively improved motor function of the experimental group. Figure 2 Comparison of general data between the two groups. A-C: Age data for two groups of patients; D-F: Two groups of patients with general data α diversity analysis; G-H: Data cluster analysis heat map between two groups of patients. Table 2 Comparison of function assessment scale before and after training Indication Treatment, mean ± SD Control, mean ± SD Before training After training Before training After training FMA score, points 20.05 ± 3.06 26.55 ± 2.66 19.15 ± 3.05 23.42 ± 2.94 FMA: Function assessment scale. After the training intervention, the MVC of the anterior tibialis muscle and pulmonary intercostal muscle on both the healthy and affected sides were significantly improved compared with pre-training values (P < 0.05), as presented in Table 3 and Figure 3. Furthermore, the MVC of the anterior tibialis muscle and pulmonary intercostal muscle on both the healthy and diseased sides in the experimental group were significantly higher than those in the control group (P < 0.05) after training. These findings suggest that the training intervention improved the muscle strength, particularly in the experimental group. Figure 3 Comparison of function assessment scale values between the two groups before and after training. A-C: Two groups of patients with general data comparison; D-F: Two groups of patients before and after training function assessment scale value α diversity analysis; G-H: Data cluster analysis heat map between two groups of patients. Table 3 Comparison of maximum voluntary contract before and after training Group Cases, n MVC of tibialis anterior muscle, kg, mean ± SD MVC of gastrocnemius, kg, mean ± SD Contralateral Affected side Contralateral Affected side Treatment group Before training 60 10.43 ± 2.16 4.87 ± 2.08 11.07 ± 2.66 4.00 ± 1.36 After training 60 12.86 ± 1.75 8.21 ± 1.53 13.65 ± 2.86 7.77 ± 1.16 Control group Before training 60 9.82 ± 1.84 4.44 ± 2.03 10.91 ± 2.26 3.86 ± 1.55 After training 60 10.22 ± 1.54 6.97 ± 1.68 11.44 ± 2.34 6.87 ± 1.42 MVC: Maximum voluntary contract. After the training intervention, the IEMG of the anterior tibialis muscle and pulmonary intercostal muscle on the healthy side in the experimental group was significantly different from pre-training values (P < 0.05), as shown in Table 4 and Figure 4. Additionally, IEMG of tibialis anterior and pulmonary intercostal muscles on both the healthy and affected sides in the experimental group were significantly different from those in the control group (P < 0.05) after training. These results suggested that the training intervention influenced the muscle activation patterns, particularly in the experimental group (Figure 5). Figure 4 Comparison of maximum voluntary contract values between the two groups before and after training. A-C: Two groups of patients with general data comparison; D-F: Two groups of patients before and after training maximum voluntary contract value α diversity analysis; G-H: Data cluster analysis heat map between two groups of patients. Figure 5 Comparison of integrated electromyography values between the two groups before and after training. A-C: Two groups of patients with general data comparison; D-F: Two groups of patients before and after training integrated electromyography value α diversity analysis; G-H: Data cluster analysis heat map between two groups of patients. Table 4 Comparison of integrated electromyography values before and after training Group Cases, n Tibial anterior muscle IEMG, mV, mean ± SD Gastrocnemius IEMG, mV, mean ± SD Contralateral Affected side Contralateral Affected side Treatment groups Before training 60 60.05 ± 15.97 34.55 ± 5.76 49.71 ± 10.96 12.42 ± 6.11 After training 60 76.89 ± 8.41 58.45 ± 6.66 62.82 ± 8.76 32.45 ± 6.25 Control groups Before training 60 58.82 ± 16.07 32.71 ± 7.42 48.91 ± 10.97 12.31 ± 7.22 After training 60 62.05 ± 15.54 50.05 ± 10.35 50.65 ± 10.38 27.85 ± 7.91 IEMG: Integrated electromyography value. DISCUSSION Stroke, also known as a cerebrovascular accident or cerebrovascular disease, is a major health concern worldwide, particularly in aging populations. In China, the annual incidence rate of stroke exceeds 2.4 million with more than 70% of patients experiencing limb movement disorders in China[12]. Stroke survivors often experience functional disorders, including cognitive, sensory, language, and motor dysfunction. Motor dysfunction is the most common type, accounting for about 80% of stroke-related dysfunction[13,14]. Research has shown that up to 80% of stroke patients experience decreased upper limb motor function in the acute stage, and only 5% to 20% of patients recover full upper limb function even with rehabilitation treatment[15]. Myodynamia of hemiplegic limbs after stroke is a common problem that limits the rehabilitation of affected limb function, and the healthy side of stroke patients also suffers from myodynamia decline[16]. Currently, most of the treatment for stroke limb rehabilitation is limited to the hemiplegic side, and training of the healthy side is often ignored. Cross-migration is a training method that leverages unilateral limb training to stimulate strength development in the corresponding muscles on the opposite side of the limb. Therefore, cross-migration can be used to train the healthy side of stroke limbs to promote rehabilitation of the hemiplegic side[17]. This study investigated the effects of ankle dorsiflexion equal-length resistance training on the strength of the anterior tibialis muscle on the affected side of stroke hemiplegic patients as well as the strength of the pulmonary intestinal muscles. The results showed that after 6 wk of training, there was a significant increase in the strength of the anterior tibialis muscle of the affected side and the pulmonary intercostal muscles, compared to before training and the control group (P < 0.05). Interestingly, ankle dorsiflexion equals length resistance training on the healthy side not only increased the strength of the affected side's tibialis anterior muscle without muscle strength training but also increased the strength of the antagonist's muscle. This phenomenon of cross-migration has been reported in previous studies, with two main mechanisms proposed: myogenic and neural[18,19]. A group of 30 young people received unilateral electrical stimulation and underwent maximum voluntary contraction strength training of the right lower limb for 6 wk. The results showed that high-intensity unilateral training increased the MVC of the opposite side, but there was no evidence of hypertrophy in the leg muscles, indicating that the myogenic mechanism does not play a major role in cross-migration[20,21]. Instead, the brain and spinal cord were found to play a significant role in the occurrence of cross-migration[22,23]. Additionally, it was observed that voluntary contraction could affect the motor pathway of the contralateral homologous muscle, which is consistent with the increase of motor cortex excitability and plays an important role in the strength training process[24,25]. Unilateral strength training was shown to increase the strength of both sides of the limbs, reduce inhibitory transmission of signals from the training side of the cerebral hemisphere to the untrained side, increase the excitability of the motor cortex on the same side of the trained limb, and reduce inhibition and transmission of bilateral corticospinal tracts. In conclusion, while the mechanism of cross-migration requires further discussion and research, this study provides valuable insights into the neural mechanisms that underlie this phenomenon and its potential application in clinical rehabilitation. CONCLUSION The study concludes that ankle dorsiflexion resistance training on the healthy side can improve lower limb motor function and increase strength in the opposite tibialis anterior muscle and antagonist's muscle, suggesting a cross-migration phenomenon of strength training. This provides a new exercise method for improving early ankle dorsiflexion dysfunction in hemiplegic stroke patients. ARTICLE HIGHLIGHTS Research background Stroke-induced hemiplegia has a high incidence rate and is characterized by movement disorders. Rehabilitation of limb function is an essential step for patients' recovery, and resistance training is a promising method to improve muscle strength and motor function. Research motivation Rehabilitation care can accelerate stroke patients' recovery time, minimize disability, and improve their quality of life. This study aims to investigate the effectiveness of unilateral strength training in improving lower limb motor function and muscle strength in hemiplegic stroke patients. Research objectives The objective of this study is to investigate whether ankle dorsiflexion resistance training on the healthy side can lead to cross-migration of strength, an increase in bilateral muscle strength, and an improvement in lower limb motor function in hemiplegic stroke patients. Research methods In this study, 120 hemiplegic stroke patients were randomly divided into two groups. The experimental group received ankle dorsiflexion resistance training on the healthy side for 6 wk, in addition to routine rehabilitation treatment. Both groups were assessed before and after the training using various measures, including EMG and FMA. Statistical analysis was conducted to compare the results between the two groups. Research results Ankle training improves post-stroke strength & motor function. Cross-migration phenomenon observed. New exercise method for early dysfunction. Research conclusions Ankle training improves strength & motor function in stroke patients. Research perspectives Could investigate the long-term effects and optimal duration/frequency of ankle training for stroke patients. Data sharing statement No additional data are available. Institutional review board statement: The study was reviewed and approved by the Department of Geriatric Rehabilitation, Shibei Hospital, Jing'an District, Shanghai Institutional Review Board. Informed consent statement: All study participants, or their legal guardian, provided informed written consent prior to study enrollment. Conflict-of-interest statement: The authors declare that they have no conflicts of interest. Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: April 6, 2023 First decision: April 19, 2023 Article in press: May 22, 2023 Specialty type: Rehabilitation Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Higashi K, Japan; Nogueira ML, Brazil S-Editor: Ma YJ L-Editor: Filipodia P-Editor: Ju JL ==== Refs 1 Xu SY Zhang M Wu XW Li L Li CX The mechanisms of limb hemiplegia after ipsilateral brain hemisphere stroke Int J Clin Exp Med 13 7386 7392 2 Ling X Zhang G Xia Y Zhu Q Zhang J Li Q Niu X Hu G Yang Y Wang Y Deng Z Exosomes from human urine-derived stem cells enhanced neurogenesis via miR-26a/HDAC6 axis after ischaemic stroke J Cell Mol Med 2020 24 640 654 31667951 3 Zhang D Liu G Huang L Zhang L Gui X Tao J Zeng P Ding M Monitoring of Motor Nerve Function Rehabilitation in Patients with Upper Extremity Hemiplegia Based on Functional Magnetic Resonance Imaging J Med Imaging Health Inform 2021 11 1761 1770 4 Knoll JM Knight LR Quiroz D Popat SM Pederson TG Morton-Gonzaba N Variation in Clinical Presentations and Outcomes of Heat Stroke Victims in the Mass-Casualty Setting J Emerg Med 2019 57 866 870 31606230 5 Noh J Jung E Jung AY Lee BH Lee BS Kim EAR Kim KS A Novel COL4A1 Mutation in a Neonate with Intrauterine Intraventricular Hemorrhage and Porencephaly Neonatal Medicine 2020 27 16 20 6 Stancioiu F Makk R Post-stroke recovery of motor function with a new combination of medicines-A pilot study EJMO 2019 3 167 181 7 Xie Q Zhang X Peng S Sun J Chen X Deng Y Yi L Identification of novel biomarkers in ischemic stroke: a genome-wide integrated analysis BMC Med Genet 2020 21 66 32228489 8 Le Roux M Barth M Gueden S Desbordes de Cepoy P Aeby A Vilain C Hirsch E de Saint Martin A Portes VD Lesca G Riquet A Chaton L Villeneuve N Villard L Cances C Valton L Renaldo F Vermersch AI Altuzarra C Nguyen-Morel MA Van Gils J Angelini C Biraben A Arnaud L Riant F Van Bogaert P CACNA1A-associated epilepsy: Electroclinical findings and treatment response on seizures in 18 patients Eur J Paediatr Neurol 2021 33 75 85 34102571 9 Ullah S Bin Ayaz S Zaheer Qureshi A Samir Tantawy S Fe Flandez M Characteristics and functional outcomes of pediatric stroke survivors at a rehabilitation unit in Saudi Arabia J Clin Neurosci 2020 81 403 408 33222951 10 Ghosh MK Chakraborty D Sarkar S Bhowmik A Basu M The interrelationship between cerebral ischemic stroke and glioma: a comprehensive study of recent reports Signal Transduct Target Ther 2019 4 42 31637020 11 Prasetyanto D Yona S Meridian acupuncture in stroke rehabilitation: a literature review IJNHS 2019 2 48 54 12 Borlongan CV Concise Review: Stem Cell Therapy for Stroke Patients: Are We There Yet? 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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4553 10.12998/wjcc.v11.i19.4553 Retrospective Study Network pharmacology and molecular docking-based analyses to predict the potential mechanism of Huangqin decoction in treating colorectal cancer Li YJ et al. Huangqin decoction for CRC: Mechanism prediction Li Ying-Jie Guizhou University of Traditional Chinese Medicine, Guizhou University of Traditional Chinese Medicine, Guiyang 550005, Guizhou Province, China Tang Dong-Xin Digestive Department, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, Guizhou Province, China. tangdongxin@sina.com Yan Hong-Ting Guizhou University of Traditional Chinese Medicine, Guizhou University of Traditional Chinese Medicine, Guiyang 550001, Guizhou Province, China Yang Bing Digestive Department, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, Guizhou Province, China Yang Zhu Guizhou University of Traditional Chinese Medicine, Guizhou University of Traditional Chinese Medicine, Guiyang 550001, Guizhou Province, China Long Feng-Xi Guizhou University of Traditional Chinese Medicine, Guizhou University of Traditional Chinese Medicine, Gui Yang 550001, Guizhou Province, China Author contributions: Li YJ and Tang DX contributed equally to this work; Yan HT designed the study; Yang B contributed to the analysis of the manuscript; Yang Z and Long FX were involved in the data acquisition and writing of this article; All authors read and approved the final manuscript. Supported by National Natural Science Foundation of China, No. 82260957 ; No. 82274610; No. 81860819; No. 81860819; and No. 81960818; Guizhou Provincial Science and Technology Program (Qian Kehe Foundation-ZK[2022] General 498, Qian Kehe Foundation-ZK [2022] General 487, Qian Kehe Support [2021] General 095, Qian Kehe Platform Talent [2020]5013); National Key R&D Program Project (2019YFC1712504); Guizhou Traditional Chinese Medicine Tumor Inheritance and Science and Technology Innovation Talent Base (No. Deaf leader-[2018] No. 3); Guizhou high-level innovative talent training plan (100 levels) (No. Qian Kehe Talents [2016] No. 4032); Yang Zhu, Guizhou Province, “Traditional Chinese Medicine Oncology” Graduate Tutor Studio (No. Teaching and research GZS-[2016]08). Corresponding author: Dong-Xin Tang, MD, Doctor, Digestive Department, The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, No. 71 Baoshan North Road, Nanming District, Guiyang 550001, Guizhou Province, China. tangdongxin@sina.com 6 7 2023 6 7 2023 11 19 45534566 24 4 2023 27 5 2023 13 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND To analyze the potential action mechanism of Huangqin decoction (HQD) in colorectal cancer (CRC) treatment on the basis of network pharmacology and molecular docking. AIM To investigate the molecular mechanisms of HQD for CRC treatment by using network pharmacology and molecular docking. METHODS All HQD active ingredients were searched using the Systematic Pharmacology and Traditional Chinese Medicine Systems Pharmacology databases and the Bioinformatics Analysis Tool for Molecular Mechanisms in traditional Chinese medicine. Then, the targets of the active ingredients were screened. The abbreviations of protein targets were obtained from the UniProt database. A “drug–compound–target” network was constructed to screen for some main active ingredients. Some targets related to the therapeutic effect of CRC were obtained from the GeneCards, DisGeNET, Therapeutic Target Database, and Online Mendelian Inheritance in Man databases. The intersection of targets of Chinese herbs and CRC was taken. A Venn diagram was drawn to construct the intersection target interactions network by referring to the STRING database. Topological analysis of the protein interaction network was performed using Cytoscape 3.7.2 software to screen the core HQD targets for CRC. The core targets were imported into the DAVID 6.8 analysis website for gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses and visualization. Finally, molecular docking was performed using AutoDockTool and PyMOL for validation. RESULTS In total, 280 potential drug-active ingredients were present in HQD, including 1474 targets of the drug-active ingredients. The main active ingredients identified were betulin, tetrahydropalmatine, and quercetin. In total, 10249 CRC-related targets and 1014 drug-disease intersecting targets were identified, including 28 core targets of action such as Jun proto-oncogene, AP-1 transcription factor subunit, signal transducer and activator of transcription 3, tumor protein p53, vascular endothelial growth factor, and AKT serine/threonine kinase 1. The gene ontology enrichment functional analysis yielded 503 enrichment results, including 406 biological processes that were mainly related to the positive regulation of both gene expression and transcription and cellular response to hypoxia, etc. In total, 38 cellular components were primarily related to polymer complexes, transcription factor complexes, and platelet alpha granule lumen. Then, 59 molecular functions were closely related to the binding of enzymes, homologous proteins, and transcription factors. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis yielded 139 enrichment results, involving epidermal growth factor receptor tyrosine kinase inhibitor resistance and HIF-1 and mitogen-activated protein kinase signaling pathways. CONCLUSION HQD can play a role in CRC treatment through the “multi-component-target–pathway”. The active ingredients betulin, tetrahydropalmatine, and quercetin may act on targets such as Jun proto-oncogene, AP-1 transcription factor subunit, signal transducer and activator of transcription 3, tumor protein p53, vascular endothelial growth factor, and AKT serine/threonine kinase 1, which in turn regulate HIF-1 and mitogen-activated protein kinase signaling pathways in CRC treatment. The molecular docking junction clarified that all four key target proteins could bind strongly to the main HQD active ingredients. This indicates that HQD could slow down CRC progression by modulating multiple targets and signaling pathways. Huangqin decoction Colorectal cancer Network pharmacology ==== Body pmc Core Tip: Huangqin decoction may treat colorectal cancer through multi-component targeting and modulation of multiple signaling pathways. INTRODUCTION The incidence of colorectal cancer (CRC) has continued to increase, accounting for nearly 10% of cancer patients. CRC is the second most common cause of cancer death[1,2]. Many pathogenic factors could cause CRC such as obesity, poor diet, alcohol addiction, and genetic mutations or familial inheritance[1-4]. According to domestic statistics, CRC incidence in China has increased each year from 1990 to 2019. Using the ARIMA model, the standardized CRC incidence in China is predicted to reach 33.93/100000 in 2025, bringing a huge burden to the Chinese healthcare system[5]. Radical surgical resection, chemotherapy, radiotherapy, and targeted therapy are the current main treatment modalities for CRC[6]. However, these treatments are generally deficient as they are prone to recurrence and side effects. Therefore, finding effective drugs with few side effects in CRC treatment is valuable. CRC is classified as “viscera toxin” and “intestinal accumulation” in traditional Chinese medicine (TCM). In the early CRC stage, clearing heat, regulating qi, dispelling dampness, eliminating phlegm, removing blood stasis, detoxifying, soft, and firm dispersing, and other treatment methods are used in TCM for dispelling evil spirits[7]. Huangqin Decoction (HQD) is derived from “Treatise on Febrile Diseases: Taiyang and Shaoyang combined disease, diarrhea, treat with HQD.” The HQD recipe consists of Scutellariae Radix (3 taels), Paeonia lactiflora Pall. (2 taels), Glycyrrhiza uralensis Fisch. (2 taels), and Ziziphus jujuba Mill. (12 pieces). Scutellariae Radix for the king, bitter, and cold can clear heat. Paeonia lactiflora Pall. can relieve pain. HQD can supply the qi. The combination of various drugs can clear heat and relieve pain[8]. HQD has been proven to treat CRC clinically with a remarkable effect, but the exact mechanism has not been elucidated. TCM is based on the formulation principles of “king, minister, adjuvant, and ambassador” and incompatibility taboo. With the characteristics of the multi-component-target-pathway and synergistic treatment, TCM has great potential to improve cancer symptoms. Network pharmacology is an emerging discipline based on systems biology theories. It analyses and calculates the relationship between relevant drugs and diseases through drug-compound-target interaction networks[9]. By inferring the treatment effects and regulatory mechanisms of drugs in disease through multiple targets, it also provides new methods for TCM application, thereby facilitating the promotion of the development and heritage of Chinese herbs and compounds. Using informatics, molecular docking predicts the potential binding conformation of ligands and receptors. It also verifies the binding affinity of core components and targets. We here constructed a drug-compound-target network according to the HQD active ingredients by using network pharmacology and molecular docking and thus analyzed and calculated the molecular mechanism and regulation signal pathways of HQD for CRC treatment. The study findings also provided a certain theoretical basis for future studies. MATERIALS AND METHODS Screening of bioactive components and targets of HQD In this study, Traditional Chinese Medicine Systems Pharmacology and Bioinformatics Analysis Tool for Molecular Mechanisms in traditional Chinese medicine databases were used to search the constituent herbs of HQD and their chemical and pharmacological data. Duplicate compounds were combined and removed to gather information about the HQD active ingredients. The targets of all these active ingredients were further searched and screened by referring to Traditional Chinese Medicine Systems Pharmacology and UniProt to realize the herb targets of HQD. All database URLs in this study are shown in Table 1. Table 1 The web addresses of databases Database Address TCMSP http://www.tcmspw.com/tcmsp.php BATMAN-TCM http://bionet.ncpsb.org.cn/batman-tcm/ Genecards https://www.genecards.org TTD http://bidd.nus.edu.sg/group/ttd/ttd.asp OMIM https://omim.org/ DisGeNET https://www.disgenet.org/home/ Wayne tool http://jvenn.toulouse.inra.fr/app/example.html String https://string-db.org/ DAVID 6.8 http://david.ncifcrf.gov Omicstudio https://www.omicstudio.cn/tool PDB https://www.rcsb.org PubChem https://pubchem.ncbi.nlmnih.gov TCMSP: Traditional Chinese Medicine Systems Pharmacology; TTD: Therapeutic target Database; OMIM: Online Mendelian Inheritance in Man; BATMAN-TCM: Bioinformatics Analysis Tool for Molecular Mechanisms in traditional Chinese medicine. Construction of drug-compound-target interaction networks We analyzed the obtained components and targets and constructed a network of “drug-compound-target” by using Cytoscape 3.7.2. In this network, nodes represent drugs, compounds, and targets, and edges represent the relationship between the three nodes. We analyzed the number of connections of each node by calculating the “degree” value. Screening for CRC-related targets The term “colorectal cancer” was searched, and potential targets were screened for in GeneCards, TTD, OMIM, and DisGeNET databases. All CRC-related targets were acquired after removing duplicate targets. HQD action targets and CRC-related targets were imported into the online Wayne tool to map the intersection of HQD and CRC targets and visualize the results. Construction of the protein-protein interaction network and screening for core targets To elucidate the functional interactions between the screened proteins, the intersecting targets were put into the STRING database to construct the protein-protein interaction (PPI) network. This PPI network was then put into Cytoscape 3.7.2, and the CytoNCA plugin was applied to filter core proteins based on betweenness (BC), closeness (CNC), degree (DC), eigenvector (EC), local average connectivity (LAC)-based method, and network (NC) 2 times the median value of the core proteins. Gene ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses We obtained information about the primary biological processes and signaling pathways by importing the screened core targets into the DAVID 6.8 database for gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Then, the enrichment results were visualized using the LianChuan BioCloud platform. Molecular docking of active ingredients to core targets The three-dimensional (3D) structure of the core target proteins was first obtained from the PDB database. The MOL2 file of the core active compounds was then obtained from the PubChem platform. Chem 3D was used to optimize the total amount of core component energy. PyMOL software was used to optimize the number of core target proteins removed for water molecule hydrogenation and charge calculation. Molecular docking and binding activity analyses were performed using the AutoDock Vina program. Finally, docking sites were visualized on the PLIP platform. When the binding energy of the molecular docking conformation was lower, the binding conformation was more stable. This reflects the greater likelihood of binding between the receptor molecule and ligand. Therefore, only the highest absolute value of binding energy for each molecular docking pair was retained for the docking results. RESULTS We identified the HQD active ingredients and the potential therapeutic targets and mechanisms of HQD in CRC treatment by conducting network pharmacology and functional gene pathway analyses. Screening of bioactive ingredients and targets of HQD Among the active ingredients obtained, 46 were Scutellariae Radix, 28 were Paeonia lactiflora Pall., 160 were Glycyrrhiza uralensis Fisch., and 58 were Ziziphus jujuba Mill. Then, 280 compounds and 1474 potential targets were obtained for the action of the ingredients after removing the duplicate information. Construction of drug-compound-target network The network “drug-compound-target” was built using Cytoscape 3.7.1 software. It included 1695 nodes and 5567 edges (Figure 1), with red squares representing drug co-components. The specific information is presented in Table 2. The HQD compounds with high activity are betulin, tetrahydropalmatine, and quercetin, which may be crucial for CRC treatment. Figure 1 Drug-compound-target network. The yellow ovals represent the potential genes. The pink circles represent the active ingredients of glycyrrhiza. The light blue circles represent the active ingredients of Paeonia lactiflora Pall. The dark blue circles represent the active ingredients of Scutellariae Radix. The green circles represent the active ingredients of Ziziphus jujuba Mill. The red squares represent the active ingredients of all herbs. Table 2 Common active ingredients information for Huangqin decoction Numbers Mol ID Molecule name OB (%) DL A1 MOL000359 Sitosterol 36.91 0.75 A2 MOL000211 Mairin 55.38 0.78 A3 MOL000422 Kaempferol 41.88 0.24 A4 MOL008583 Beta-sitosterol 15.00 0.81 A5 MOL000492 (+)-Catechin 54.83 0.24 A6 MOL000096 (-)-Catechin 49.68 0.24 B1 MOL002320 γ-Sitosterol 36.91 0.75 B2 MOL002307 20-Hexadecanoylingenol 28.20 0.68 C1 MOL004349 Ruvoside 18.13 0.63 C2 MOL000098 Quercetin 46.43 0.28 OB: Oral bioavailability; DL: Drug- likeness. Screening for CRC-related targets We obtained 10249 CRC-related targets for its treatment from the disease databases of Genecards, OMIM, DisGeNET, and TTD. The targets of both CRC and the HQD active ingredients were imported into the Venn diagram editing website, and then 1014 potential targets of HQD in CRC treatment were identified (Figure 2). Figure 2 Huangqin decoction–colorectal cancer intersection target. A: Targets of Huangqin decoction; B: Colorectal cancer-related targets. CRC: Colorectal cancer; HQD: Huangqin decoction. PPI network analysis and key target screening The STRING was used to obtain key proteins for CRC treatment with HQD. The analysis data were imported into Cytoscape 3.7.2. Based on the BC, CNC, DC, EC, LAC, and NC values of topological parameters calculated using CytoNCA, 28 targets that play key roles in CRC, such as Jun proto-oncogene, AP-1 transcription factor subunit (JUN), signal transducer and activator of transcription 3 (STAT3), tumor protein p53 (TP53), vascular endothelial growth factor (VEGFA), and AKT serine/threonine kinase 1 (AKT1) were screened (Figure 3). These targets could be considered the HQD core targets in CRC treatment. Figure 3 The core target screening process. A: All potential targets of Huangqin decoction in the treatment of colorectal cancer (purple nodes); B: Core targets screened based on CytoNCA (yellow nodes); C: The top 28 targets screened by betweenness, closeness, degree, eigenvector, local average connectivity and network values of topology parameters. JUN: Jun proto-oncogene, AP-1 transcription factor subunit; STAT3: Signal transducer and activator of transcription 3; TP53: Tumor protein p53; VEGFA: Vascular endothelial growth factor; AKT1: AKT serine/threonine kinase 1 (AKT1); MAPK: Mitogen-activated protein kinase; EGFR: Epidermal growth factor receptor. GO function and KEGG pathway enrichment analyses We imported 28 core targets into the DAVID 6.8 database to identify the relevant HQD biological functions in CRC treatment by analyzing GO functional enrichment. This analysis revealed 503 enrichment results, including 406 biological processes, 38 cellular components, and 59 molecular functions. The biological processes were mainly related to the positive regulation of gene expression and transcription and cellular response to hypoxia. The cellular components were principally related to the macromolecular complex, transcription factor complex, platelet alpha granule lumen, etc. The molecular functions primarily involved the binding of enzymes, identical proteins, and transcription factors. The GO enrichment results were sorted from the smallest to the largest P value. The top 10 entries were selected to create a histogram (Figure 4). To determine the potential mechanism underlying CRC treatment with HQD, we performed the KEGG pathway enrichment analysis and obtained 139 HQD-associated signaling pathways for CRC treatment. The top 20 pathways were visualized in descending order of the P value (Figure 5). These mainly included signaling pathways such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance, HIF-1, and mitogen-activated protein kinase (MAPK), suggesting that HQD can treat CRC through these pathways. Figure 4 Gene ontology functional enrichment analysis. The purple bars represent biological processes, the blue bars represent cellular components, and the green bars represent molecular functions. GO: Gene ontology. Figure 5 Kyoto Encyclopedia of genes and genomes pathway enrichment analysis. The top 20 signaling pathways of Huangqin decoction are involved in the treatment of colorectal cancer. The size of nodes is proportional to the number of genes enriched. KEGG: Kyoto Encyclopedia of Genes and Genomes; EGFR: Epidermal growth factor receptor; MAPK: EGFR: Mitogen-activated protein kinase. Molecular docking of potential active ingredients to core target proteins Five key target proteins (JUN, STAT3, TP53, VEGFA, and AKT1) were molecularly docked to three core components (betulin, tetrahydropalmatine, and quercetin) to assess the binding capacity of the target and ligand proteins. The binding capacity was predicted using AutoDock software, with binding energies typically < -5.0 kcal·mol−1 indicating significant binding activity between the molecules. All three active components exhibited a strong binding capacity to JUN, TP53, VEGFA, and AKT1 (Table 3), with birudinol failing to bind well to STAT3. The 3D model plots of the three active ingredients docked with JUN, TP53, VEGFA, and AKT1 were visualized using PyMOL and PLIP software (Figures 6-8). Figure 6 The docking model of the core active ingredient betulinol with the target proteins. A: Docking of betulinol to Jun proto-oncogene, AP-1 transcription factor subunit; B: Docking of betulinol to vascular endothelial growth factor; C: Docking of betulinol to tumor protein p53; D: Docking of betulinol to AKT1 serine/threonine kinase 1. JUN: Jun proto-oncogene, AP-1 transcription factor subunit; VEGFA: Vascular endothelial growth factor; TP53: Tumor protein p53; AKT1: AKT serine/threonine kinase 1. Figure 7 The docking model of the key active ingredient tetrahydropalmatine to the target proteins. A: Docking of tetrahydropalmatine to Jun proto-oncogene, AP-1 transcription factor subunit; B: Docking of tetrahydropalmatine to tumor protein p53; C: Docking of tetrahydropalmatine to vascular endothelial growth factor; D: Docking of tetrahydropalmatine to AKT1 serine/threonine kinase 1. JUN: Jun proto-oncogene, AP-1 transcription factor subunit; TP53: Tumor protein p53; VEGFA: Vascular endothelial growth factor; AKT1: AKT serine/threonine kinase 1. Figure 8 The docking model of the key active ingredient quercetin to the target proteins. A: Docking of quercetin to Jun proto-oncogene, AP-1 transcription factor subunit; B: Docking of quercetin to tumor protein p53; C: Docking of tetrahydropalmatine to vascular endothelial growth factor; D: Docking of tetrahydropalmatine to AKT1 serine/threonine kinase 1. JUN: Jun proto-oncogene, AP-1 transcription factor subunit; TP53: Tumor protein p53; VEGFA: Vascular endothelial growth factor; AKT1: AKT serine/threonine kinase 1. Table 3 Molecular docking results Active ingredients Binding energy (kcal/mol) JUN TP53 VEGFA AKT1 Betulinol -8.4 -6 -8.7 -10.3 Tetraydropalmatine -7.2 -6.1 -9.4 -9.6 Quercetin -6.4 -8.3 -8.1 -8.3 JUN: Jun proto-oncogene, AP-1 transcription factor subunit; TP53: Tumor protein p53; VEGFA: Vascular endothelial growth factor; AKT1: AKT serine/threonine kinase 1. DISCUSSION According to TCM, the diseased region in CRC is the large intestine. Moreover, CRC is closely related to the spleen and stomach. “Miraculous Pivot” mentioned that “The injury of the stomach, the blood overflow in the parenteral, the parenteral cold, the juice foam and blood phase kneading, the combination of condensation cannot be dispersed, and the product into.” Several clinical studies have retrospectively investigated the TCM symptoms of CRC patients and their associated factors. These studies have found that the most common TCM classification of CRC is the syndrome of damp heat accumulation[10-12]. HQD was first mentioned by Zhang Zhongjing in “Treatise on Febrile Diseases.” According to this monograph, HQD could clear away heat and relieve pain and is a classic therapeutic formula for damp heat in Chinese medicine. We conducted a network pharmacological analysis to determine active compounds, potential targets, and action mechanisms of HQD in CRC treatment. In total, 280 core ingredients of HQD were screened. The topological analysis of the “drug-compound-target” network revealed that the top-ranked compounds were betulin, tetrahydropalmatine, and quercetin. These may be the potential compounds of HQD in CRC treatment. Among them, betulinol induced apoptosis of CRC cells through the MAPK signaling pathway and significantly inhibited malignant metastasis of CT26 cells in the lungs[13]. The therapeutic efficacy of tetrahydropalmatine has been demonstrated in various cancers[14,15], suggesting that tetrahydropalmatine can be used as a potential novel therapeutic agent for CRC patients. Quercetin, a flavanol compound, has various biological activities, such as antioxidant activity. Quercetin could interfere with CRC development in various ways, such as inhibiting growth and proliferation and affecting the cycle of CRC cells. Thus, it could induce apoptosis and inhibit metastasis and invasion of CRC[16-19]. The aforementioned results proved that the main active ingredients of HQD play a crucial role in CRC treatment, suggesting that HQD is an effective therapeutic formula for CRC treatment. On the basis of BC, CNC, DC, EC, LAC, and NC, the top five key targets, namely JUN, STAT3, TP53, VEGFA, and AKT1, were finally screened through cascading analysis of key targets using the PPI network. JUN is a transcription factor that recognizes and binds to the enhanced heptameric motif 5’-TGA[CG]TCA-3’. JUN can upregulate HuR expression by inhibiting miR-22 transcriptional regulation, which in turn leads to proliferation, migration, and tumor growth in CRC cells[20]. STAT3, a member of the STAT cytoplasmic transcription factor family, is the family member most associated with CRC development[21]. Large amounts of STAT3 are activated in CRC to promote tumor growth, invasion, migration[22], and proliferation of cancer cells. These features suggest that STAT3 can be used as a therapeutic target in CRC. TP53 encodes the tumor suppressor p53, which is the major driver gene for suppressing the development of various cancers. The TP53 mutation has been observed in up to 80% of patients with advanced metastatic CRC[23], and this mutation can lead to poor prognosis in various cancer patients, including CRC patients[24]. VEGFA, the subtype of the vascular endothelial growth factor (VEGF), has a critical role in angiogenesis. According to some studies, tumor growth, spread, and metastasis depend on angiogenesis. VEGFA is a major proangiogenic factor in CRC and is strongly related to metastasis in this cancer[25]. The receptor targeting the tyrosine kinase VEGF can inhibit the angiogenesis, proliferation, and growth of liver metastases in CRC. This anti-angiogenic therapy significantly increases endothelial and tumor cell apoptosis, thereby suggesting that VEGF has a crucial role in the tumor endothelium[26]. AKT is a member of the serine/threonine kinase proto-oncogene family that regulates different inflammatory and metabolism-related signaling pathways and is involved in cancer cell proliferation, survival, and metabolic processes[27]. Approximately 70% of CRC exhibits highly activated AKT, which is closely associated with cancer development. During molecular docking, the three core ingredients of HQD exhibited strong affinity to the four key target proteins, confirming the targeting effect of HQD in CRC treatment. The predicted results thus theoretically elucidate the ameliorative effect of HQD on CRC and help in the further investigation of the action mechanism of HQD or its bioactive compounds as an alternative treatment for CRC. The GO functional enrichment suggested that CRC treatment with HQD was mainly related to the positive regulation of gene expression and transcription as well as a cellular response to hypoxia. The KEGG pathway revealed that the pathways most closely related to CRC were EGFR tyrosine kinase inhibitor resistance, and HIF-1 and MAPK signaling pathways. EGFR is closely associated with CRC recurrence and deterioration. Studies have demonstrated that miR-323a-3p can promote the apoptosis of CRC cells by directly targeting EGFR signaling[28]. Acquired resistance to 5-fluorouracil is a clinical challenge for CRC treatment. The HIF-1α and 5-fluorouracil combination significantly enhances the antitumor effects of 5-fluorouracil[29]. Therefore, targeting HIF-1α could act as an effective therapeutic strategy for 5-fluorouracil-resistant CRC. The MAPK signaling pathway is activated by peptide growth factors, cytokines, oxidative stress, etc. This pathway then regulates tumor cell proliferation, differentiation, survival, and death[30]. Several MAPK, ERK, c-Jun, and JNK subfamilies are involved in CRC pathogenesis[31], with ERK/MAPK playing a crucial role in cell proliferation[30]. Therefore, EGFR tyrosine kinase inhibitor resistance and HIF-1 and MAPK signaling pathways are closely linked to CRC development. CONCLUSION In conclusion, by analyzing “multi-components-targets-biological pathways,” this study found that HQD could exert therapeutic effects. Our findings also provide a clue regarding the theoretical basis for the active ingredients of HQD that are most effective in CRC treatment. However, because of the deficiencies of network pharmacology and molecular docking, follow-up in vivo and in vitro experiments are warranted to verify the action mechanism of HQD in CRC treatment. ARTICLE HIGHLIGHTS Research background Colorectal cancer (CRC) is the second most common cause of cancer death in recent years. CRC is a danger to human health. Research motivation The side effects caused by radiotherapy severely hinder the treatment progress of CRC patients. Huangqin decoction (HQD) can improve the immunity of CRC patients and enhance their quality of life. Research objectives This study exploited network pharmacology and molecular docking to uncover the potential targets and mechanisms of HQD for CRC treatment. It also provided a molecular biological basis for CRC treatment with HQD in a clinical setting. Research methods This study involved preliminary exploration of the potential targets and mechanisms of HQD for CRC treatment by using network pharmacology and molecular docking. Research results The active ingredients betulin, tetrahydropalmatine, and quercetin in HQD may act on targets such as Jun proto-oncogene, AP-1 transcription factor subunit, signal transducer and activator of transcription 3, tumor protein p53, vascular endothelial growth factor, and AKT serine/threonine kinase 1, which in turn regulate HIF-1 and mitogen-activated protein kinase signaling pathways in CRC treatment. Research conclusions HQD could treat CRC by regulating HIF-1 and mitogen-activated protein kinase signaling pathways. Research perspectives At present, many shortcomings still exist in studying the action mechanisms of herbal medicine for various diseases on the basis of network pharmacology. Future research should be focused on in vivo and in vitro experiments to verify the key targets and type pathways and then combined proteomics and metabolomics to more systematically elucidate the action mechanism of HQD in CRC treatment. Data sharing statement No additional data are available. Institutional review board statement: The study was reviewed and approved by the Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou, China, Institutional Review Board. Conflict-of-interest statement: The authors declare that they have no competing interests. Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: April 24, 2023 First decision: May 8, 2023 Article in press: June 13, 2023 Specialty type: Gastroenterology and hepatology Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Batra SK, United States; Chiang SF, Taiwan S-Editor: Ma YJ L-Editor: Filipodia P-Editor: Zhao S ==== Refs 1 Siegel RL Miller KD Goding Sauer A Fedewa SA Butterly LF Anderson JC Cercek A Smith RA Jemal A Colorectal cancer statistics, 2020 CA Cancer J Clin 2020 70 145 164 32133645 2 Biller LH Schrag D Diagnosis and Treatment of Metastatic Colorectal Cancer: A Review JAMA 2021 325 669 685 33591350 3 Keum N Giovannucci E Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies Nat Rev Gastroenterol Hepatol 2019 16 713 732 31455888 4 de la Chapelle A Peltomäki P Genetics of hereditary colon cancer Annu Rev Genet 1995 29 329 348 8825478 5 Liu Y Zhang C Wang Q Wu K Sun Z Tang Z Zhang B Temporal Trends in the Disease Burden of Colorectal Cancer with Its Risk Factors at the Global and National Level from 1990 to 2019, and Projections Until 2044 Clin Epidemiol 2023 15 55 71 36659904 6 Zheng Y Fu Y Wang PP Ding ZY Neoantigen: A Promising Target for the Immunotherapy of Colorectal Cancer Dis Markers 2022 2022 8270305 35211210 7 Sun L Mao JJ Yan Y Xu Y Yang Y Patient Reported Traditional Chinese Medicine Spleen Deficiency Syndrome (TCM-SDS) Scale for Colorectal Cancer: Development and Validation in China Integr Cancer Ther 2021 20 15347354211020105 34116615 8 Xu DD Hou XY Wang O Wang D Li DT Qin SY Lv B Dai XM Zhang ZJ Wan JB Xu FG A four-component combination derived from Huang-Qin Decoction significantly enhances anticancer activity of irinotecan Chin J Nat Med 2021 19 364 375 33941341 9 Pei T Zheng C Huang C Chen X Guo Z Fu Y Liu J Wang Y Systematic understanding the mechanisms of vitiligo pathogenesis and its treatment by Qubaibabuqi formula J Ethnopharmacol 2016 190 272 287 27265513 10 Wang YN Zou M Wang D Zhang ZK Qu LP Xu J Shi CD Gao F An exploratory study on TCM syndrome differentiation in preoperative patients with colorectal cancer assisted by laboratory indicators Heliyon 2022 8 e10207 36033259 11 Lu Y Zhou C Zhu M Fu Z Shi Y Li M Wang W Zhu S Jiang B Luo Y Su S Traditional chinese medicine syndromes classification associates with tumor cell and microenvironment heterogeneity in colorectal cancer: a single cell RNA sequencing analysis Chin Med 2021 16 133 34876190 12 Wang P Ding S Sun L Feng Y Guo K Zhu Y Huang D Ruan S Characteristics and differences of gut microbiota in patients with different Traditional Chinese Medicine Syndromes of Colorectal Cancer and normal population J Cancer 2020 11 7357 7367 33193900 13 Han YH Mun JG Jeon HD Kee JY Hong SH Betulin Inhibits Lung Metastasis by Inducing Cell Cycle Arrest, Autophagy, and Apoptosis of Metastatic Colorectal Cancer Cells Nutrients 2019 12 14 Xia X He J Liu B Shao Z Xu Q Hu T Yu C Liu X Liao Y Liu N Huang H Targeting ERα degradation by L-Tetrahydropalmatine provides a novel strategy for breast cancer treatment Int J Biol Sci 2020 16 2192 2204 32549765 15 Gong J Xing C Wang LY Xie SS Xiong WD L-Tetrahydropalmatine enhances the sensitivity of human ovarian cancer cells to cisplatin via microRNA-93/PTEN/Akt cascade J BUON 2019 24 701 708 31128026 16 Yang L Liu Y Wang M Qian Y Dong X Gu H Wang H Guo S Hisamitsu T Quercetin-induced apoptosis of HT-29 colon cancer cells via inhibition of the Akt-CSN6-Myc signaling axis Mol Med Rep 2016 14 4559 4566 27748879 17 Al-Ghamdi MA Al-Enazy A Huwait EA Albukhari A Harakeh S Moselhy SS Enhancement of Annexin V in response to combination of epigallocatechin gallate and quercetin as a potent arrest the cell cycle of colorectal cancer Braz J Biol 2021 83 e248746 34495165 18 Raja SB Rajendiran V Kasinathan NK P A Venkatabalasubramanian S Murali MR Devaraj H Devaraj SN Differential cytotoxic activity of Quercetin on colonic cancer cells depends on ROS generation through COX-2 expression Food Chem Toxicol 2017 106 92 106 28479391 19 Han M Song Y Zhang X Quercetin Suppresses the Migration and Invasion in Human Colon Cancer Caco-2 Cells Through Regulating Toll-like Receptor 4/Nuclear Factor-kappa B Pathway Pharmacogn Mag 2016 12 S237 S244 27279714 20 Liu Y Chen X Cheng R Yang F Yu M Wang C Cui S Hong Y Liang H Liu M Zhao C Ding M Sun W Liu Z Sun F Zhang C Zhou Z Jiang X The Jun/miR-22/HuR regulatory axis contributes to tumourigenesis in colorectal cancer Mol Cancer 2018 17 11 29351796 21 Corvinus FM Orth C Moriggl R Tsareva SA Wagner S Pfitzner EB Baus D Kaufmann R Huber LA Zatloukal K Beug H Ohlschläger P Schütz A Halbhuber KJ Friedrich K Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth Neoplasia 2005 7 545 555 16036105 22 Xiong H Zhang ZG Tian XQ Sun DF Liang QC Zhang YJ Lu R Chen YX Fang JY Inhibition of JAK1, 2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells Neoplasia 2008 10 287 297 18320073 23 Brannon AR Vakiani E Sylvester BE Scott SN McDermott G Shah RH Kania K Viale A Oschwald DM Vacic V Emde AK Cercek A Yaeger R Kemeny NE Saltz LB Shia J D'Angelica MI Weiser MR Solit DB Berger MF Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions Genome Biol 2014 15 454 25164765 24 Olivier M Hollstein M Hainaut P TP53 mutations in human cancers: origins, consequences, and clinical use Cold Spring Harb Perspect Biol 2010 2 a001008 20182602 25 Peeters M Price T Biologic therapies in the metastatic colorectal cancer treatment continuum--applying current evidence to clinical practice Cancer Treat Rev 2012 38 397 406 21899955 26 Ellis LM Takahashi Y Liu W Shaheen RM Vascular endothelial growth factor in human colon cancer: biology and therapeutic implications Oncologist 2000 5 Suppl 1 11 15 10804085 27 Li D Wang G Jin G Yao K Zhao Z Bie L Guo Y Li N Deng W Chen X Chen B Liu Y Luo S Guo Z Resveratrol suppresses colon cancer growth by targeting the AKT/STAT3 signaling pathway Int J Mol Med 2019 43 630 640 30387805 28 Zhang Y Liang S Xiao B Hu J Pang Y Liu Y Yang J Ao J Wei L Luo X MiR-323a regulates ErbB3/EGFR and blocks gefitinib resistance acquisition in colorectal cancer Cell Death Dis 2022 13 256 35319011 29 Dong S Liang S Cheng Z Zhang X Luo L Li L Zhang W Li S Xu Q Zhong M Zhu J Zhang G Hu S ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1α-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer J Exp Clin Cancer Res 2022 41 15 34998404 30 Kim EK Choi EJ Pathological roles of MAPK signaling pathways in human diseases Biochim Biophys Acta 2010 1802 396 405 20079433 31 Fang JY Richardson BC The MAPK signalling pathways and colorectal cancer Lancet Oncol 2005 6 322 327 15863380

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4707 10.12998/wjcc.v11.i19.4707 Case Report Poststroke rehabilitation using repetitive transcranial magnetic stimulation during pregnancy: A case report Joong Ho Jo et al. Safety and effectiveness of TMS during pregnancy Jo Joongho Department of Rehabilitation Medicine, Chungbuk National University Hospital, Cheongju-si 28644, Chungbuk, South Korea Kim Hyojong Department of Rehabilitation Medicine, Chungbuk National University Hospital, Cheongjusi 28644, Chungbuk, South Korea. hyojong80@gmail.com Author contributions: Jo JH contributed to manuscript writing and editing and case selection; Kim HJ contributed to manuscript supervision; all authors have read and approved the final manuscript. Corresponding author: Hyojong Kim, MD, PhD, Professor, Department of Rehabilitation Medicine, Chungbuk National University Hospital, Department of Rehabilitation Medicine, Chungbuk National University Hospital, 776, Sunhwan-ro, Seowon-gu, Cheongju 28644, Korea, Cheongjusi 28644, Chungbuk, South Korea. hyojong80@gmail.com 6 7 2023 6 7 2023 11 19 47074712 10 4 2023 22 5 2023 9 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Repetitive transcranial magnetic stimulation (rTMS) is a form of magnetic stimulation therapy used to treat depression, migraine, and motor function impairment in patients with stroke. As there is little research on the effects of rTMS in pregnant women, it is not widely used in these patients. This case report aimed to demonstrate the safety of rTMS in pregnant patients. CASE SUMMARY After much consideration, we applied rTMS to treat recent stroke and hemiplegia in a 34-year-old pregnant woman. The patient received 45 sessions of low-frequency treatment over the course of 10 wk. We closely monitored the mother and fetus for potential side effects; the results showed significant improvement in the patient's motor function, with no harmful effects on the mother or fetus during pregnancy or after delivery. The patient’s fine motor and walking functions improved after treatment. This case is the first instance of a stroke patient treated with rTMS during pregnancy. CONCLUSION This case demonstrates that rTMS could be used to improve motor function recovery in stroke patients during pregnancy. Health Pregnancy Rehabilitation Stroke Transcranial magnetic stimulation Case report ==== Body pmc Core Tip: This case report describes the use of repetitive transcranial magnetic stimulation (rTMS) to improve the motor function of a patient after stroke. The patient was pregnant, but low-frequency rTMS was deemed to be safe and was administered for 10 wk. After treatment, the patient regained function in her hands and was able to walk without a cane, and no side effects were observed in the patient or her baby. This case demonstrates that rTMS can be used safely in pregnant patients. INTRODUCTION As society ages, the incidence and mortality rates of stroke remain high, and interest in active rehabilitation therapy has grown[1]. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive therapy that uses magnetic fields to modulate the activity of specific cortical areas, and it is widely used to aid motor function recovery in stroke patients, as well as to alleviate depression and migraines[2-4]. In cases of severe motor deficits in affected limbs after stroke, early and aggressive treatment is necessary to prevent long-term impairment[5]. Therefore, in clinical settings, rTMS is actively utilized as an early rehabilitation option for patients with hemiplegia[6]. A combination of rTMS and physical therapy was found to be more effective than physical therapy alone in stroke patients[7]. Caution is advised regarding the use of rTMS in patients with severe cerebral hemorrhage or in pregnant women, owing to the potential risks involved. However, a recent study reported a positive outcome when rTMS was applied to patients with perinatal depression. No side effects were observed in patients who underwent rTMS, and no issues were found in their newborns, indicating that rTMS could be a safe alternative for new mothers with depression[8]. Based on this previous study, we considered the use of rTMS as an early and active treatment option for a pregnant stroke patient with severe hemiplegia. After careful consideration, we decided to apply rTMS in combination with conventional rehabilitation therapy to improve the prognosis of a young mother who suffered a stroke during pregnancy. CASE PRESENTATION Chief complaints A 34-year-old pregnant woman at 24+0 wk of gestation presented to the emergency room with dysarthria and right-sided weakness. History of present illness A pregnant women at 24 wk gestation (para 0) presented to our emergency department on 28 November 2022 at approximately 1:50 am with decreased consciousness. In the emergency room, the patient complained of dysarthria and right hemiparesis, with an initial National Institutes of Health Stroke Scale (NIHSS) score of 14. History of past illness The patient had no underlying medical conditions, and all routine prenatal checks since conception were normal, including the screening for chromosomal abnormalities (at 11-14 wk and 16-18 wk gestation), nuchal translucency measurement (at 11-14 wk gestation), and fetal ultrasound (at 20-24 wk gestation). Personal and family history The patient reported a history of two intrauterine insemination cycles and five in vitro fertilization cycles with embryo transfer. She had experienced no complications (such as preeclampsia) during the current pregnancy. Physical examination The initial evaluation performed by the Department of Rehabilitation Medicine demonstrated that the patient had completely flaccid upper and lower extremity muscles on the right side; she was unable to walk and had an NIHSS score of 14. Laboratory examinations All of the following tests conducted to check for complications in the mother and fetus were normal: Blood pressure tests, blood glucose tests, other blood tests, echocardiography, 24-hour Holter monitoring, Doppler ultrasound of the leg, pulse wave velocity and ankle-brachial index measurements, transcranial Doppler ultrasound, and duplex Doppler ultrasound of the carotid arteries. In addition, all blood tests related to autoimmune diseases were normal. Imaging examinations Imaging tests conducted upon admission revealed acute infarction in the left side of the corpus callosum, thalamus, occipital lobe, pons, and midbrain (Figure 1). Figure 1 Cerebral infarction lesion shown on a brain magnetic resonance imaging image. A: Left corpus callosum and thalamus; B: Left occipital lobe; C: Left pons; D: Left midbrain. FINAL DIAGNOSIS Based on the patient’s medical history, the final diagnosis was acute infarction in the left side of the corpus callosum, thalamus, occipital lobe, pons, and midbrain. TREATMENT The patient received enoxaparin at a dose of 1 mg/kg twice daily as medical treatment for suspected hypercoagulability. In addition, she received functional electrical stimulation for right ankle dorsiflexor and mat activity as well as gait training for 20 minutes twice a day, five days a week. She received occupational therapy for the recovery of fine motor function and activities of daily living for 30 min per day, 5 d a week. She also received speech therapy for dysarthria once a week for 30 min. After much consideration, we decided to use rTMS therapy in combination with conventional rehabilitation therapy to promote the patient's motor recovery. We used the rTMS protocol reported by Kim et al[9] and administered 45 sessions of rTMS over 10 wk. OUTCOME AND FOLLOW-UP After three weeks of combined therapy, the patient was able to walk with a cane and showed significant improvement in upper extremity muscle strength and hand function (Table 1) (Video 1). At the time of delivery, she was able to raise her right hand above her head, use both hands to type on a laptop, and walk with a cane under supervision (Figure 2). During the 10 wk of rTMS therapy, the patient did not report any significant side effects, and detailed fetal monitoring did not reveal any fetal dysfunction. Intensive rehabilitation therapy and rTMS therapy continued until three days before delivery. The delivery was performed by cesarean section at 37+3 wk of gestation, and the newborn weighed 2900 g with Apgar scores of 10 at 1 and 5 minutes. Both the mother and newborn had normal vital signs and postpartum examinations. After an additional four days of monitoring and testing, they were discharged to a postpartum care center with no reported complications. Figure 2 Images of the patient after completing repetitive transcranial magnetic stimulation sessions. A: Hand elevation; B: Walking with a cane under supervision. Table 1 Timecourse of motor recovery as determined at 3, 6 and 10 weeks after the combination of repetitive transcranial magnetic stimulation and rehabilitation therapy Assessment Baseline 3 wk 6 wk 10 wk Purdue test 0/8 0/10 1/13 4/15 Grip power(kg) 0/12 6/14 12/16 12/16 JHFT 0/80 2/85 14/85 17/85 FAC 0 2 3 4 MBI 10 40 64 75 MRS 5 4 4 3 RTMS: Repetitive transcranial magnetic stimulation; JHFT: Jebsen hand function test; FAC: Functional ambulatory category; MBI: Modified barthel index; MRS: Modified rankin scale. DISCUSSION Neuroplasticity-induced cortical reorganization is a crucial mechanism for motor recovery in patients with stroke, and rTMS is commonly used as a treatment to enhance neuroplasticity[10,11]. In this case, the patient was a pregnant woman with only three months left until delivery. In addition, due to cerebral infarction, she was almost completely paralyzed on one side and was unable to walk. If this patient had not received appropriate rehabilitation treatment in a timely manner, her motor disabilities could have persisted, and the risk of fetal growth restriction or even miscarriage could have increased. Conventional rehabilitation activities; therefore, additional therapies are needed. Although no previous studies have reported the use of rTMS in pregnant patients after stroke, there have been reports in which rTMS was used safely for the treatment of perinatal depression. Therefore, based on this evidence and with the consent of the patient and her family, we decided to perform rTMS. Currently, there is no standard rTMS procedure to improve motor function recovery in patients with early stroke. However, according to a study by Du et al[12], both high-frequency (HF; > 10 Hz) rTMS over the ipsilesional primary motor cortex and low-frequency (LF; 1 Hz) rTMS over the contralesional primary motor cortex are effective in improving motor function. To minimize any potential negative effects on the mother or fetus caused by HF rTMS, we used LF rTMS, and treatment was stopped three days before delivery. The mother underwent a total of 45 treatments over 10 wk using inhibitory mode rTMS, and we actively monitored vital signs and side effects during each treatment session. We also ensured that all periodic obstetric examinations were performed, and all examinations demonstrated normal results. The patient's motor function gradually improved during treatment, and at the time of delivery, she was able to use both hands to type on a laptop, and her ability to walk with a cane (under supervision) had significantly improved. With no previously published cases, there were concerns about the safety of using rTMS in a poststroke pregnant patient; however, this case demonstrates that rTMS can be safely used in such patients and can greatly aid in motor recovery when combined with conventional rehabilitation therapy. This case has significant implications for the treatment of poststroke pregnant patients with motor deficits. CONCLUSION Stroke during pregnancy is a rare but serious condition that can cause neurological deficits. Active rehabilitation therapy is necessary for functional recovery. In this patient with severely impaired motor function, the combination of rTMS and rehabilitation therapy was effective in improving function, and there was no harm to the fetus or mother. Therefore, rTMS may be a good therapeutic tool for perinatal stroke treatment. Informed consent statement: Informed written consent was obtained from the patient for publication of this report and accompanying images. Conflict-of-interest statement: No potential conflicts of interest relevant to this article were reported. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016). Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: April 10, 2023 First decision: May 12, 2023 Article in press: June 9, 2023 Specialty type: Rehabilitation Country/Territory of origin: South Korea Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: luo W, China; Shao A, China S-Editor: Ma YJ L-Editor: A P-Editor: Ma YJ ==== Refs 1 Jung SH Stroke Rehabilitation Fact Sheet in Korea Ann Rehabil Med 2022 46 1 8 35272435 2 Medical Advisory Secretariat Repetitive transcranial magnetic stimulation for the treatment of major depressive disorder: an evidence-based analysis Ont Health Technol Assess Ser 2004 4 1 98 3 Lan L Zhang X Li X Rong X Peng Y The efficacy of transcranial magnetic stimulation on migraine: a meta-analysis of randomized controlled trails J Headache Pain 2017 18 86 28831756 4 Málly J Dinya E Recovery of motor disability and spasticity in post-stroke after repetitive transcranial magnetic stimulation (rTMS) Brain Res Bull 2008 76 388 395 18502315 5 Gao C Pu SX Zhu DY [Effects of early rehabilitation on motor function of upper and lower extremities and activities of daily, living in patients with hemiplegia after stroke] Zhongguo Kangfu Yixue Zazhi 2001 1 27 29 6 Dionísio A Duarte IC Patrício M Castelo-Branco M The Use of Repetitive Transcranial Magnetic Stimulation for Stroke Rehabilitation: A Systematic Review J Stroke Cerebrovasc Dis 2018 27 1 31 29111342 7 Barros Galvão SC Borba Costa dos Santos R Borba dos Santos P Cabral ME Monte-Silva K Efficacy of coupling repetitive transcranial magnetic stimulation and physical therapy to reduce upper-limb spasticity in patients with stroke: a randomized controlled trial Arch Phys Med Rehabil 2014 95 222 229 24239881 8 Zhang D Hu Z RTMS may be a good choice for pregnant women with depression Arch Womens Ment Health 2009 12 189 190 19238519 9 Kim JS Kim DH Kim HJ Jung KJ Hong J Kim DY Effect of Repetitive Transcranial Magnetic Stimulation in Post-stroke Patients with Severe Upper-Limb Motor Impairment Brain Neurorehabil 2020 13 e3 36744269 10 Buma F Kwakkel G Ramsey N Understanding upper limb recovery after stroke Restor Neurol Neurosci 2013 31 707 722 23963341 11 Adeyemo BO Simis M Macea DD Fregni F Systematic review of parameters of stimulation, clinical trial design characteristics, and motor outcomes in non-invasive brain stimulation in stroke Front Psychiatry 2012 3 88 23162477 12 Du J Yang F Hu J Xu Q Cong N Zhang Q Liu L Mantini D Zhang Z Lu G Liu X Effects of high- and low-frequency repetitive transcranial magnetic stimulation on motor recovery in early stroke patients: Evidence from a randomized controlled trial with clinical, neurophysiological and functional imaging assessments Neuroimage Clin 2019 21 101620 30527907

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4698 10.12998/wjcc.v11.i19.4698 Case Report With two episodes of right retromandibular angle subcutaneous emphysema during right upper molar crown preparation: A case report Bai YP et al. Subcutaneous emphysema during molar crown preparation Bai Yun-Peng Department of Prosthodontics and Implantology, Shenzhen University Affiliated Shenzhen Stomatology Hospital, Shenzhen 518001, Guangdong Province, China Sha Jing-Jing Department of Endodontics, Shenzhen University Affiliated Shenzhen Stomatology Hospital, Shenzhen 518001, Guangdong Province, China Chai Chang-Chang Department of Prosthodontics and Implantology, Shenzhen University Affiliated Shenzhen Stomatology Hospital, Shenzhen 518001, Guangdong Province, China Sun Hai-Peng Department of Prosthodontics and Implantology, Shenzhen University Affiliated Shenzhen Stomatology Hospital, Shenzhen 518001, Guangdong Province, China. shplysz@126.com Author contributions: Bai YP and Sun HP contributed to the conceptualization and visualization of the manuscript; Bai YP, Sha JJ, and Sun HP involved in the funding acquisition, original draft writing; Bai YP investigated; Bai YP, Sha JJ, and Chai CC contributed to the methodology of the manuscript; Bai YP and Sha JJ contributed to the supervision, writing-review and editing of this manuscript; and all authors have read and agreed to the published version of the manuscript. Supported by Shenzhen Science and Technology Program, No. JCYJ20220530165409022 . Corresponding author: Hai-Peng Sun, PhD, Professor, Department of Prosthodontics and Implantology, Shenzhen University Affiliated Shenzhen Stomatology Hospital, Guiyuan North Street 70th, Luohu, Shenzhen 518001, Guangdong Province, China. shplysz@126.com 6 7 2023 6 7 2023 11 19 46984706 25 3 2023 29 4 2023 6 6 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND Subcutaneous emphysema is a well-known complication of oral surgery, especially during mandibular wisdom tooth extraction. However, subcutaneous emphysema secondary to dental procedures such as crown preparation is rare. The main symptom of emphysema is swelling and crepitus on palpation. Uncontrolled emphysema may spread along the fascial planes and cause deep space infections or a pneumomediastinum. CASE SUMMARY In this paper, we report a 34-year-old female who underwent upper molar tooth preparation for crowns and subsequently developed extensive subcutaneous emphysema on the retromandibular angle on two different occasions. The treatment plan for this patient involved close observation of the airway, and administration of dexamethasone and antibiotics via intravenous drip or orally. Ice bag compression was quickly applied and medication was prescribed to alleviate discomfort and promote healing. Although the main reason is unclear, the presence of a fissure in the molar is an important clue which may contribute to the development of subcutaneous emphysema during crown preparation. It is imperative for dental professionals to recognize such pre-disposing factors in order to minimize the risk of complications. CONCLUSION This case highlights the need for prompt diagnosis and management of subcutaneous emphysema because of the risk of much more serious complications. Awareness of relatively “benign” subcutaneous emphysema during any dental procedure is critical not only for inexperienced dentists, but also for those who work in rural and remote settings as members of surgical teams. In this study, we review the clinical presentation, mechanism, and differential diagnosis of subcutaneous emphysema. Subcutaneous emphysema Dental procedures Dental prosthesis preparation Retro-mandibular space Intraoperative complications Case report ==== Body pmc Core Tip: Subcutaneous emphysema secondary to dental procedures such as crown preparation is rare. In this paper, we report a 34-year-old female who underwent upper molar tooth preparation for crowns and subsequently developed extensive subcutaneous emphysema on the retromandibular angle on two different occasions. Prompt diagnosis and management of subcutaneous emphysema is necessary for dentist. INTRODUCTION Subcutaneous emphysema is a rare but serious complication of dental and oral surgery that can arise during tooth extraction, crown preparation, and endodontic therapy[1]. High-speed air turbine drills are widely used during dental procedures such as exodontia, tooth sectioning, and dental restoration. The drills are driven by compressed air (rate of 3.5-4.0 kgf/cm2, rotation speed of 450000 rpm)[2]. The use of high-speed air turbines appears to be associated with most cases of subcutaneous emphysema; pressurised air is blown into the deep space[3]. The maxillofacial fascial spaces are close to the retropharyngeal space and mediastinum. Therefore, air and water that enter the maxillofacial spaces could trigger deep space infections with serious life-threatening complications[4]. Subcutaneous emphysema caused by dental procedures can involve the facial area, orbital region[5], neck[6], and (rarely) mediastinum[7]. One case of vision loss caused by optic nerve damage has been reported[8]. Sometimes, all of these complications develop simultaneously[9], and are then very difficult to manage[10]. Fortunately, most emphysema is self-limiting, benign, and resolves safely[11]. The most common symptom of subcutaneous emphysema is some degree of rapid swelling that generates crepitus when pressed by the fingers[4]. Most cases occur after mandible third molar extraction while using an air turbine; the deep facial and soft tissue spaces swell in such cases[1-3]. Conventional case management for subcutaneous emphysema including: (1) Immediate recognition: Quickly identify the signs and symptoms of subcutaneous emphysema, such as swelling, crepitus on palpation, and potential difficulty in breathing; (2) Airway monitoring: Closely monitor the patient’s airway to ensure there is no obstruction or compromise due to the swelling; (3) Administration of medications: Prescribe medications, such as anti-inflammatory drugs (e.g., corticosteroids like dexamethasone) and antibiotics (e.g., cefuroxime), to manage inflammation and prevent infection; and (4) Follow-up care: Schedule follow-up appointments to monitor the patient’s progress and ensure resolution of the subcutaneous emphysema and any potential complications. We here present a rare case of subcutaneous emphysema that occurred twice during crown preparation of the right upper molars (teeth #16 and #17); sudden swellings developed around the mandibular angle while the patient was being treated. Crown preparation is generally considered a low-risk procedure for subcutaneous emphysema compared to tooth extractions, particularly when involving impacted third molars. The likelihood of air being forced into the fascial spaces is lower during crown preparation compared to more invasive procedures. Repeated instances of subcutaneous emphysema in the same patient during similar dental procedures are unusual. Most patients who experience this complication once are unlikely to encounter it again, particularly if the dental professional is aware of the previous occurrence and takes extra precautions during subsequent treatments. Individual patient factors may contribute to the rarity of this occurrence[11]. Some patients might have anatomical variations or predisposing factors that make them more susceptible to subcutaneous emphysema during dental procedures. In this report, case management was also described, along with the diagnosis. The purpose of this report is to alert the dental community about the incidence of subcutaneous emphysema from a routine dental procedure, and how to recognize and manage its occurrence. CASE PRESENTATION Chief complaints A 34-year-old female with no medical history consulted us for crown restoration of the right upper molars (teeth #16 and #17) after successful endodontic treatment. The patient stated that these teeth were painful when biting even soft food. The distal surface of #16 and medial surface of #17 had undergone composite resin restoration. Both teeth were mildly painful on percussion, but were not loose. The gingiva was healthy, exhibiting a normal colour and gingival sulcus depth. There was no bleeding on probing. We obtained a panoramic radiograph and periapical films before operations (Figure 1). Figure 1 Panoramic and periapical films of teeth #16 and #17. A: Panoramic of teeth #16 and #17; B and C: Periapical films of teeth #16 and #17, both #16 and #17 had undergone successful root canal treatment. History of present illness Crown preparation proceeded smoothly until the occlusion face of #17 was ground; the patient reported a sudden transient pain at this point. The operation was stopped and we immediately examined the patient. She was alert and complained of a bump in the right retromandibular angle that gradually hardened, but was not in pain and spoke in full sentences. History of past illness There is no past illness. Personal and family history There is no personal and family history. Physical examination The tissue around the right retromandibular was obviously swollen, but was non-erythematous and non-tender. On palpation, there was a sensation of crepitation. The patient did not experience much pain, and had no difficulty with mouth-opening or breathing (Figure 2). Figure 2 Subcutaneous emphysema is clearly evident in the right retromandibular angle. The skin temperature rose and the skin became red. Mouth-opening was not restricted and the patient felt no pain in the region of swelling. Yellow arrows: Subcutaneous emphysema. A: Front view; B: Profile view; C: Profile view while mouth opening. Laboratory examinations As crepitations were noted during palpation, we diagnosed subcutaneous emphysema. The operation was immediately interrupted; the patient’s blood pressure, heart rate, respiratory rate, temperature, and oxygen saturation were normal. No deep periodontal pocket was found around the tooth (Figure 3 and Table 1). Figure 3 Panoramic imaging and gingival sulcus depth probing were performed after emphysema developed. A: Panoramic imaging was performed after emphysema developed; B and C: Gingival sulcus depth probing were performed after emphysema developed. Gingival sulcus depth pocket probing was normal (buccal lateral: #16: 2 mm; #17: 2.8 mm). No significant anomaly was found. Table 1 The gingival sulcus depth assessment result of #16 and #17 Teeth Mesio-buccal site Buccal centre Distal-buccal site Mesio-lingual site Lingual centre Distal-lingual site #16 2.8 mm 2.0 mm 2.5 mm 2.5 mm 2.2 mm 3.0 mm #17 2.7 mm 2.8 mm 2.7 mm 3.0 mm 2.5 mm 3.2 mm Imaging examinations We performed cone-beam computed tomography (CBCT) after the emphysema developed, which revealed obvious swelling and thickening of soft tissue on the masseter muscle and a discrete region of emphysema in the layer between the masseter muscle and subcutaneous fascia (Figure 4). Figure 4 Cone-beam computed tomography scans obtained after emphysema developed. A-E: Coronal views of the swelling; F-H: An air-filled fissure between the masseter muscle and soft tissue. Blue arrows: Swelling. Yellow arrows: The fissure. Treatment and follow-up The treatment plan was close observation of the airway, and dexamethasone (10 mg/d) and antibiotics (cefuroxime 1.5 g/d) for 3 d. This approach aimed to manage inflammation and reduce the risk of infection. Ice bag compression was quickly applied and medication was prescribed to alleviate discomfort and promote healing. The patient was not admitted but visited our outpatient clinic daily for intravenous drip therapy. After 3 d of treatment, the swelling had completely disappeared and there were no complications (Figure 5). Figure 5 After 3 d of intravenous drip, the swelling of the right retromandibular angle disappeared. A: Front view; B: Profile view. Second emphysema episode After 2 wk of observation, the swollen tissue in the right retromandibular angle had recovered well. As tooth preparation had not been concluded, the patient again visited our outpatient clinic to finalise the prosthodontic procedure prior to crown placement on the right upper molars. On this occasion, we proceeded with great care. When gently and slowly grinding the occlusion surface of the medial-buccal cusp of #17 with a low-speed turbine and normal water and air flow, the patient again reported a sudden pain in the right retromandibular angle. FINAL DIAGNOSIS The affected area exhibited a striking similarity to previous instances, with the skin turning noticeably red and gradually swelling up as a reaction to the underlying issue. After a few seconds, crepitation was observed, which is a crackling sound produced when air is trapped under the skin. This is a classic sign of subcutaneous emphysema, a medical condition that occurs when air infiltrates the subcutaneous tissue layer beneath the skin. Therefore, we confirmed that the patient had subcutaneous emphysema in the same area once again. TREATMENT We stopped the operation and applied an ice bag (cold compress) to the swelling. Ibuprofen and cephalosporin were immediately given orally. After 2 h of observation, the swelling was limited to a very small area and the temperature of the skin had normalised, but the swelling was light red in colour and the tissue was hard when palpated. As before, no deep periodontal pocket or gingival inflammation around the tooth were found (Figure 6). Figure 6 The symptoms were similar to those on the first occasion. A and B: The emphysema was in the retromandibular area; the yellow arrows indicate the swelling; C-E: The teeth and gingiva were normal and healthy; F and G: Optical scans acquired using an oral scanner (3Shape, Copenhagen, Denmark) also revealed very good general health. However, beginning on the occlusion surface of #17, a subfissure line was found between the tooth and resin (blue arrow). Grinding at this point triggered subcutaneous emphysema in the retromandibular area. OUTCOME AND FOLLOW-UP At the 1-wk follow-up, the symptoms had disappeared and there were no other complications. The patient then re-visited us and we placed provisional crowns. DISCUSSION Dental procedures can disrupt the mucosa of the oral cavity and introduce air into connective tissue spaces, thereby triggering subcutaneous emphysema. Although this is generally benign, there is a risk of progression to serious consequences including pneumothorax, air embolism, mediastinitis, cranial nerve palsy, and cardiac tamponade[12]. Subcutaneous emphysema spreads near sites where valves are located. When the upper teeth are involved, periorbital swelling is the most common symptom; when the lower teeth are implicated, cheek and neck bulges are more common[3]. Subcutaneous emphysema developed during crown preparation for teeth #16 and #17 is rare, especially when addressing an occlusion surface, but the emphysema affected the right retromandibular angle rather than the periorbital area (which was the most frequently affected area in previous studies). The reason for this is unclear; we offer two possible hypotheses. First, strong air flow into the gingival sulcus destroyed a loose connective tissue attachment. Then, gingival tissue was detached from the bony maxillary tubercle bone and air entered the layers between the buccinator muscle and buccal fat tissue. The gingival valve closed immediately, triggering the valvular effect described in other clinical situations[3]. The air did not travel beneath the buccinator muscle along the bone; if it had done so, emphysema would have occurred in the infraorbital area. The air spread to the masseter muscle layer along the buccinator muscle fascia to reach the posterior edge of the mandible. The reason why the swelling was confined to this area, and thus did not spread into the deeper cervical region, was because we stopped the operation immediately, and thus the air volume was limited. The other hypothesis involves the subfissure of tooth #17 (Figure 6). When preparing the occlusion surface, the turbine moved the tooth, thereby increasing the size of the subfissure, and air entered deep tissue to cause subcutaneous emphysema. The air pathway in this case would be the same as that described above. This hypothesis may be supported by the fact that both emphysema episodes started during preparation of the occlusion surface of #17, but the gingival sulcus depths of #16 and #17 were healthy and normal. The patient also reported pain in the right upper molar when biting, especially on hard food. However, the root canal treatment had been successful, so we suspect that the subfissure hypothesis may be correct. We plan further studies to test this hypothesis. As stated by Fasoulas et al[13], subcutaneous emphysema is almost twice as common in females than males. In their systematic review, emphysema was mainly reported in females (39/65) rather than males (20/65) and the age of the patients ranged from 18 to 63 years (mean = 38 years). In another study, Jeong et al[11] indicated that subcutaneous emphysema originating from maxillary teeth (n = 8; 66.7%) was two times more common than that originating from mandibular teeth (n = 4; 33.3%), meanwhile subcutaneous emphysema was more common in posterior teeth (91.7%, n = 11) than in anterior teeth (8.3%, n = 1). Approximately 18% (2 of 11 patients) of subcutaneous emphysema cases can be attributed to crown preparation[11]. Nadarajah reported a serious and huge cervical subcutaneous emphysema and pneumomediastinum occurring after extraction of a mandibular right molar by using an air turbine handpiece[10]. Although there are no documented cases in the literature, it is important for dental professionals to be aware that cervical subcutaneous emphysema caused by dental crown preparation can also potentially lead to very serious complications. Rapid diagnosis, management is critical for both patient and dentist. The rationale for antibiotic therapy is that introduced air may contain bacteria, which could trigger rapidly spreading cellulitis or necrotizing fasciitis[1]. Fasoulas et al[13] prescribed antibiotics to 40 cases for 2-10 d, depending on the severity of the condition. Βeta-lactam antibiotics such as penicillin and amoxicillin, as well as various cephalosporins (given orally or intravenously), were the most common choices. Analgesics and non-steroidal anti-inflammatory drugs, cold or hot compresses, pressure or massage of the swollen area, and ice bag treatment have been recommended by some studies. Based on our experience, when crown preparation triggers subcutaneous emphysema, immediate cessation of the operation and application of an ice bag to compress the swelling controls the emphysema very effectively; oral medicines can be added if required. Subcutaneous emphysema can also be induced by sneezing, blowing forcefully, coughing, or vomiting after a dental procedure[1]. Dentists may attribute immediate swelling after a dental operation to angioedema or an allergic reaction, and delayed symptoms to a hematoma or soft tissue infection such as cellulitis or Ludwig’s angina. Patients with subcutaneous emphysema typically exhibit painless oedema of the face and neck; the palpable crepitus is pathognomonic, which clearly distinguishing emphysema from other conditions[13]. However, dental professionals should be vigilant if a patient complains of breathing difficulties; the emphysema may have spread to the paratracheal, mediastinal, or thoracic spaces. In most cases, air re-absorption begins within 2-3 d and is frequently complete by day 7-10 after onset[11]. Oxygen inhalation via a nasal cannula accelerates this process by reducing the blood nitrogen pressure, thus enhancing air re-absorption[14]. When encountering iatrogenic air emphysema, airway restriction must be considered. CBCT is useful to determine the extent of the damage. Also, patients should be told to avoid any activity after treatment that might increase the oral cavity pressure, including coughing, smoking, nose-blowing, the use of a straw, and vomiting. CONCLUSION Subcutaneous emphysema in the posterior region of the right retromandibular angle during molar crown preparation in #16 and #17 is rare. Caution should be exercised when using air turbines. Rapid diagnosis and appropriate management reduce the risk of serious complications. Informed consent statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper. Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article. CARE Checklist (2016) statement: The authors have read the CARE Checklist (2016), and the manuscript was prepared and revised according to the CARE Checklist (2016). Provenance and peer review: Unsolicited article; Externally peer reviewed. Peer-review model: Single blind Peer-review started: March 25, 2023 First decision: April 20, 2023 Article in press: June 6, 2023 Specialty type: Medicine, research and experimental Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Ardila CM, Colombia; Rakhshan V, Iran S-Editor: Wang JJ L-Editor: A P-Editor: Cai YX ==== Refs 1 McKenzie WS Rosenberg M Iatrogenic subcutaneous emphysema of dental and surgical origin: a literature review J Oral Maxillofac Surg 2009 67 1265 1268 19446214 2 Romeo U Galanakis A Lerario F Daniele GM Tenore G Palaia G Subcutaneous emphysema during third molar surgery: a case report Braz Dent J 2011 22 83 86 21519655 3 Sarfi D Haitami S Farouk M Ben Yahya I Subcutaneous emphysema during mandibular wisdom tooth extraction: Cases series Ann Med Surg (Lond) 2021 72 103039 34815859 4 D'Agostino S Dolci M Subcutaneous Craniofacial Emphysema Following Endodontic Treatment: Case Report with Literature Review Oral 2021 5 Chang CH Lien WC Palpebral emphysema following a dental procedure Am J Emerg Med 2018 36 908.e1 908.e2 6 Singh V Ganti L Haupt JD Marshall B Iatrogenic post-pulpectomy cervicofacial subcutaneous emphysema in a paediatric patient BMJ Case Rep 2020 13 7 Rawlinson RD Negmadjanov U Rubay D Ohanisian L Waxman J Pneumomediastinum After Dental Filling: A Rare Case Presentation Cureus 2019 11 e5593 31700706 8 Rubinstein A Riddell CE Akram I Ahmado A Benjamin L Orbital emphysema leading to blindness following routine functional endoscopic sinus surgery Arch Ophthalmol 2005 123 1452 16219746 9 An GK Zats B Kunin M Orbital, mediastinal, and cervicofacial subcutaneous emphysema after endodontic retreatment of a mandibular premolar: a case report J Endod 2014 40 880 883 24862722 10 Nadarajah A Arron TY Elston T Huge surgical emphysema and pneumomediastinum as a sequela to conservative dental restoration: A case report J Case Rep Images Surg 2022 8 1 4 11 Jeong CH Yoon S Chung SW Kim JY Park KH Huh JK Subcutaneous emphysema related to dental procedures J Korean Assoc Oral Maxillofac Surg 2018 44 212 219 30402412 12 Tomasetti P Kuttenberger J Bassetti R Distinct subcutaneous emphysema following surgical wisdom tooth extraction in a patient suffering from 'Gilles de la Tourette syndrome' J Surg Case Rep 2015 2015 13 Fasoulas A Boutsioukis C Lambrianidis T Subcutaneous emphysema in patients undergoing root canal treatment: a systematic review of the factors affecting its development and management Int Endod J 2019 52 1586 1604 31271657 14 Brzycki RM Case Report: Subcutaneous Emphysema and Pneumomediastinum Following Dental Extraction Clin Pract Cases Emerg Med 2021 5 58 61 33560953

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==== Front World J Clin Cases WJCC World Journal of Clinical Cases 2307-8960 Baishideng Publishing Group Inc jWJCC.v11.i19.pg4625 10.12998/wjcc.v11.i19.4625 Systematic Reviews Combined medial patellofemoral ligament and medial patellotibial ligament reconstruction in recurrent patellar instability: A systematic review and meta-analysis Abbaszadeh A et al. MPFL MPTL reconstruction: A systematic review Abbaszadeh Ahmad Department of Orthopedic, Emam Khomeini Teaching Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz 6135715794, Iran Saeedi Mohsen Department of Orthopedic, Emam Khomeini Teaching Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz 6135715794, Iran Hoveidaei Amir Human Sports Medicine Research Center, Tehran University of Medical Sciences, Tehran 14395-578, Iran Dadgostar Haleh Department of Sports and Exercise Medicine, School of Medicine, Rasool Akram Medical Complex, Iran University of Medical Sciences, Tehran 1445613131, Iran Razi Saeed Bone and Joint Reconstruction Research Center, Department of Orthopedics, School of Medicine, Iran University of Medical Sciences, Tehran 1545913487, Iran Razi Mohammad Department of Orthopedic Surgery, Rasoul Akram Hospital, Iran University of Medical Sciences, Tehran 1445613131, Iran. mrazi@razimd.info Author contributions: Razi M, Hoveidaei AH, and Dadgostar H contributed to conception and design of the study and made critical revisions related to important intellectual content of the manuscript; Abbaszadeh A, Saeedi M, and Razi S drafted the article and performed acquisition of data and analysis and interpretation of data; all authors approved of the final version of the article to be published. Corresponding author: Mohammad Razi, MD, President, Professor, Surgeon, Department of Orthopedic Surgery, Rasoul Akram Hospital, Iran University of Medical Sciences, Department of Orthopedic Surgery, Rasoul Akram Hospital, Iran University of Medical Sciences, Shahid Hemmat Highway, Tehran 1445613131, Iran. mrazi@razimd.info 6 7 2023 6 7 2023 11 19 46254634 24 1 2023 8 5 2023 31 5 2023 ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved. 2023 https://creativecommons.org/licenses/by-nc/4.0/ This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. BACKGROUND The medial patellofemoral ligament (MPFL), along with the medial patellotibial ligament (MPTL) and medial patellomeniscal ligament, aid in the stabilization of the patellofemoral joint. Although the MPFL is the primary stabilizer and the MPTL is a secondary limiter, this ligament is critical in maintaining joint stability. There have been few studies on the combined MPFL and MPTL reconstruction and its benefits. AIM To look into the outcomes of combined MPFL and MPTL reconstruction in frequent patellar instability. METHODS By May 8, 2022, four electronic databases were searched: Medline (PubMed), Scopus, Web of Science, and Google Scholar. General keywords such as "patellar instability," "patellar dislocation," "MPFL," "medial patellofemoral ligament," "MPTL," and "medial patellotibial ligament" were co-searched to increase the sensitivity of the search. RESULTS The pooled effects of combined MPFL and MPTL reconstruction for Kujala score (12-mo follow-up) and Kujala score (24-mo follow-up) were positive and incremental, according to the findings of this meta-analysis. The mean difference between the Cincinnati scores was also positive, but not statistically significant. The combination of the two surgeries reduces pain. According to cumulative meta-analysis, the trend of pain reduction in various studies is declining over time. CONCLUSION The combined MPFL and MPTL reconstruction has good clinical results in knee function and, in addition to providing good control to maintain patellofemoral joint balance, the patient's pain level decreases over time, making it a valid surgical method for patella stabilization. Medial patellofemoral ligament reconstruction Medial patellotibial ligament patella dislocation Patella instability ==== Body pmc Core Tip: In patellar instability, combined medial patellofemoral ligament and medial patellotibial ligament reconstruction is associated with good clinical outcomes and can be considered a standard treatment in patellar instability treatment guidelines. INTRODUCTION The patella is a vital component of the knee joint that includes the extensor mechanism. The main stabilizing ligamentous structure, the medial patellofemoral ligament (MPFL), allows the patella to stabilize in conjunction with the medial patellotibial ligament (MPTL) and medial patellomeniscal ligament (MPML). During knee movements, the MPFL is important in maintaining the patella's stability and position and is thought to be the primary internal stabilizing ligament[1,2]. Although the MPFL ligament is the primary stabilizer, the MPTL and MPML ligaments also play an important role in maintaining joint stability, particularly in the final stages of stretching from 26 degrees in extension and 46 to 90 degrees in flexion[3]. Recurrent patella dislocation is associated with patella alta, a large Q angle, a hypoplastic lateral femoral condyle, and congenital ligament laxity[2,4,5]. According to a review of the literature, most patients with non-surgical treatments experience frequent instability in the injured knee and reduced activity level[6]. A large number of surgical methods for treating patellofemoral instability have been described in various studies, but the best method is still controversial[7,8]. MPFL reconstruction is widely recognized as an important component of the current treatment for recurrent patellar instability. This method can be used alone or in conjunction with bone methods such as tibial tuberosity distalization or trochleoplasty in the case of patella alta or high-grade trochlear dysplasia[9]. Another surgical method is a combined MPFL and MPTL reconstruction, which has yielded positive results in clinical trials. In a cohort study conducted by Hetsroni, the effect of combined surgery (MPFL and MPTL) was investigated during 73 mo on the patients. Their findings showed that combined surgery in young patients improved knee function better than MPFL surgery alone and maintained patella-femoral balance with more degrees of flexion, even though they did not return to pre-injury levels of activity[10,11]. Hence, according to role of MPTL reconstruction in combination of MPFL reconstruction in treatment of patella instability and the fact that many studies have not been done in this field, the present study was performed with the purpose of studying the combined MPFL and MPTL reconstruction in recurrent patellar instability. MATERIALS AND METHODS Search strategy To find relevant studies, a comprehensive literature search for English-language observational studies was performed in Medline (PubMed), Scopus, Web of Science and Google Scholar. In order to maximize the sensitivity of the search, general keywords such as "patellar instability", “patellar dislocation”, “MPFL”, “medial patellofemoral ligament”, “MPTL”, and “medial patellotibial ligament” were co-searched (Supplementary material). The results of searches were refined through checking for and removing duplicate papers. Study selection In keeping with standard meta-analysis techniques and PRISMA guidelines[12], we included studies published to May 8, 2022. Studies were independently selected for inclusion and only the original papers were included in the review. Two investigators independently applied the inclusion and exclusion criteria, which had questions about the main methodological aspects of descriptive studies, such as the sampling method, measurement of variables, objectives, and statistical analysis. To be included in this study, inclusion criteria were: (1) Studies that used both MPFL and MPTL methods simultaneously for recurrent patellar instability; and (2) Primary studies. And Exclusion criteria were: (1) Not primary studies as well as those with only poster presentation; (2) Duplicated publication (we included just one); and (3) Studies that used only one of two methods, MPFL or MPTL for recurrent patellar instability. Figure 1 shows the PRISMA flow diagram. Figure 1 Flow diagram. Flow diagram of the study selection process and including publications for the Combined medial patellofemoral ligament and medial patellotibial ligament reconstruction in recurrent patellar instability. Screening and data extraction All publications were reviewed independently by two researchers (Mohsen Saeedi and Ahmad Abbaszadeh). In case of discrepancies between investigators regarding inclusion criteria, it was resolved by the third author (Haleh Dadgostar). After the final evaluation, the selected publications were briefed by the name of first author, date of publication, country, sample size, study design, age average, Extension, Flexion, International knee Documentation Committee score, visual analogue scale (VAS), Patellar tilt, Patellar shift, Insall–Salvati ratio, Modified Insall–Salvati ratio, Caton–Deschamps Index, Tibial tuberosity-trochlear groove (TT–TG) distance, Kujala score, Cincinnati, Lysholm, range of motion (ROM) in degrees and Congruence angle. All the extracted data were then entered into Excel software. Quality assessment of studies In this study, due to the small number of studies in this field, publication bias was not investigated. Statistical analysis The "metan" command was used to apply a fixed or random effects model based on the results of Cochran's Q test or a large Higgins and Thompson’s I2 value. Standardized mean difference (SMD) estimated by Hedges' g based on sample size, mean, and standard deviation before and after intervention. Forest plots were used to describe the results graphically. In addition, the "metacum" command was used for cumulative meta-analysis to determine the trend of the Kujala score. Stata software (version 14) was used for all statistical analyses. RESULTS Five studies with a sample size of 148 knees after surgery were included in the systematic review and meta-analysis after reviewing and evaluating collected papers. These papers were published between 2013 and 2020. The highest average age was in the Maffulli study, which was 26.5 + 10.7 years. These studies were conducted in five countries. Only one study used Extension, Flexion, Caton-Deschamps Index, Tegner score, Patellar Shift, and Modified Insall-Salvati Ratio to assess the effect of combined medial patellofemoral and patellotibial ligament reconstruction. TT–TG distance and congruence angle were reported in two studies, but in one study, the value before surgery was the only one available. ROM and Lysholm were also reported in two studies, but the follow-up period was 12 mo in one study and 24 mo in another study. In two studies, the Insall–Salvati ratio was reported, but in one of them, the standard deviation was not reported. By the way, the average of VAS, patellar tilt angle, and Cincinnati were reported in two studies with a follow-up period of 24 mo. The Kujala score was also reported with a follow-up period of 12 mo in two studies and a follow-up period of 24 mo in four studies. More details about the studies are given in Tables 1-4. Table 1 Description of eligible studies evaluating combined medial patellofemoral and patellotibial ligament reconstruction in recurrent patellar dislocation regarding international knee documentation committee score, Extension and flexion degrees, and visual analogue scale ID Ref. Year1 Type of study Country N Mean age Male/female IKDC score Extension, degree Flexion, degree VAS Preop2 12 mo3 24 mo4 Preop 12 mo3 24 mo4 Preop 12 mo3 24 mo4 Preop 12 mo3 24 mo4 1 Maffulli et al[24] 2020 Prospective cohort Italy 34 26.5 ± 10.7 27:7 2 Yang et al[15] 2019 Prospective China 58 22.6 ± 4.9 25:23 51.9 ± 13.8 80 ± 19.2 85 ± 13.9 2 ± 3 2 ± 4 3 ± 4 143 ± 7 141 ± 8 142 ± 7 58 ± 11 12 ± 5 11 ± 4 3 Hetsroni et al[10] 2019 - Israel 20 18 ± 2 6:14 75.7 ± 18.1 4 Sadigursky et al[19] 2017 Case series Brazil 7 11.28 ± 1.49 4:3 5 Sobhy et al[22] 2013 Prospective Egypt 29 20.1 ± 3 21:8 63 ± 13 18 ± 9.7 1 Year of publication. 2 Preoperative. 3 12-mo follow-up. 4 24 & > 24 -mo follow-up. IKDC: International knee documentation committee; VAS: Visual analogue scale. Table 2 Description of eligible studies evaluating combined medial patellofemoral and patellotibial ligament reconstruction in recurrent patellar dislocation regarding Caton–Deschamps Index, Tibial tuberosity-trochlear groove distance, Kujala score, and Cincinnati ID Ref. Year1 Type of study Country N Mean age Male/female Caton–deschamps index TT–TG distance, mm Kujala score Cincinnati Preop2 12 mo3 24 mo4 Preop 12 mo3 24 mo4 Preop 12 mo3 24 mo4 Preop 12 mo3 24 mo4 1 Maffulli et al[24] 2020 Prospective cohort Italy 34 26.5 ± 10.7 21:8 47 ± 17 82 ± 175 51 ± 22 90 ± 19 2 Yang et al[15] 2019 Prospective China 58 22.6 ± 4.9 21:8 1.41 ± 0.21 1.32 ± 0.17 1.31 ± 0.17 21.5 ± 0.6 20.2 ± 1.5 19.9 ± 1.7 55.1 ± 15.2 82.6 ± 14.9 89.5 ± 10.2 3 Hetsroni et al[10] 2019 - Israel 20 18 ± 2 21:8 54.9 ± 15.2 86.4 ± 12.56 4 Sadigursky et al[19] 2017 Case series Brazil 7 11.28 ± 1.49 21:8 42.57 ± 8.9 88.57 ± 5.09 5 Sobhy et al[22] 2013 Prospective Egypt 29 20.1 ± 3 21:8 36.6 ± 6 90.6 ± 7 50 ± 7.1 88 ± 6 1 Year of publication. 2 Preoperative. 3 12-mo follow-up. 4 24 & > 24-mo follow-up. 5 Postoperative (the mean follow-up was 43 ± 17). 6 Postoperative (the mean follow-up was 43 ± 17). Table 3 Description of eligible studies evaluating combined medial patellofemoral and patellotibial ligament reconstruction in recurrent patellar dislocation regarding Lysholm, range of motion, congruence angle, tegner score ID Ref. Year1 Type of study Country N Mean age Male/female Lysholm ROM, degree Congruence angle Tegner score Preop2 12 mo3 24 mo4 Preop 12 mo3 24 mo4 Preop 12 mo3 24 mo4 Preop 12 mo3 24 mo4 1 Maffulli et al[24] 2020 prospective cohort Italy 34 26.5 ± 10.7 21:8 2 Yang et al[15] 2019 prospective China 58 22.6 ± 4.9 21:8 3 Hetsroni et al[10] 2019 - Israel 20 18 ± 2 21:8 4 ± 3 4.8 ± 2.45 4 Sadigursky et al[19] 2017 case series Brazil 7 11.28 ± 1.49 21:8 33.71 ± 9.6 87.71 ± 5.70 117.85 ± 8.09 148.57 ± 3.77 5 Sobhy et al[22] 2013 prospective Egypt 29 20.1 ± 3 21:8 51.9 ± 4.7 89.5 ± 5.6 112.1 ± 7.1 136.7 ± 8.5 11.93±1.85 -6.48 ± 3.8 1 Year of publication. 2 Preoperative. 3 12-mo follow-up. 4 24 & > 24-mo follow-up. 5 Postoperative (the mean follow-up was 43 ± 17). ROM: Range of motion. Table 4 Description of eligible studies evaluating combined medial patellofemoral and patellotibial ligament reconstruction in recurrent patellar dislocation regarding patellar tilt angle, patellar shift, insall–salvati ratio, and modified insall–salvati ratio ID Ref. Year1 Type of study Country N Mean age Male/female Patellar tilt angle Patellar shift, mm Insall–Salvati ratio Modified Insall–Salvati ratio Preop2 12 mo3 24 mo4 Preop 12 mo3 24 mo4 Preop 12 mo3 24 mo4 Preop 12 mo3 24 mo4 1 Maffulli et al[24] 2020 Prospective cohort Italy 34 26.5 ± 10.7 21:8 1.1 1.1 2 Yang et al[15] 2019 Prospective China 58 22.6 ± 4.9 21:8 19.1 ± 7.2 11.5 ± 5.6 11.3 ± 5.2 6.2 ± 2.1 1.2 ± 0.6 1.1 ± 0.7 1.49 ± 0.22 1.39 ± 0.23 1.37 ± 0.19 2.25 ± 0.18 1.95 ± 0.22 1.95 ± 0.25 3 Hetsroni et al[10] 2019 - Israel 20 18 ± 2 21:8 4 Sadigursky et al[19] 2017 Case series Brazil 7 11.28 ± 1.49 21:8 5 Sobhy et al[22] 2013 Prospective Egypt 29 20.1 ± 3 21:8 10.9 ± 1.7 2.45 ± 2.2 1 Year of publication. 2 Preoperative. 3 12-mo follow-up. 4 24 & > 24-mo follow-up. The effects of combined medial patellofemoral and patellotibial ligament reconstruction on different outcomes According to these studies, the pooled effects of combined medial patellofemoral and patellotibial ligament reconstruction changes for Kujala score (12 mo and 24 mo follow-up) were positive and incremental (SMD = 3.64; 95%CI: -0.38 to 7.65 and SMD = 3.53; 95%CI: 2.03 to 3.03, respectively), but they were significant only for the 24 mo follow-up. The mean difference for Cincinnati was also positive but not statistically significant (SMD = 7.74; 95%CI: -2.95 to 18.44 and SMD = 3.75; 95%CI: 0-7.5, respectively). SMD for VAS was -4.76 mm and significant 95%CI: -6.5 to -30.03, which means that the combined effect of the two treatments can reduce the amount of pain (Figure 2). Also, according to the cumulative meta-analysis, the rate of pain reduction in different studies is decreasing over time (a decrease in the SMD value from 8.17 to 2.04). Figure 2 The pooled effects of combined medial patellofemoral and patellotibial ligament reconstruction. SMD: Standardized mean difference. DISCUSSION Patellar instability is one of the most common causes of knee injury and anterior knee pain, and it is associated with frequent dislocation, which prevents most patients from returning to sports and physical activities[13,14]. The main finding of this systematic review was that the combined surgical method of MPFL and MPTL reconstruction has good clinical outcomes in terms of knee function, and that patients' pain levels decrease over time, in addition to good control of patellofemoral balance. Although MPFL surgery alone was popular in the past, studies have shown that this treatment method was ineffective in some patellar conditions such as patella alta, and reconstruction of this ligament with the MPFL method alone increased the level of knee function but was ineffective when compared to the combined method[15]. According to the findings of a study conducted by Ambra et al[16], the MPFL method alone was ineffective in treating patellar instability. The combined reconstruction (MPFL, MPTL) is typically used in patients who have at least two lateral patella dislocations, a delta patella, an increased TT-TG distance greater than 20 mm, an Insall-salvative-index greater than 1.2, multiple ligament damage, and an unstable joint[17]. The studies revealed that, while the MPFL ligament acts as the primary internal stabilizer of the patella, the MPTL is also required as a secondary lateral stabilizer to maintain patella stability and improve knee function, emphasizing the practical importance of combined surgery (MPFL, MPTL)[18]. In the current study, the Kujala score improved significantly after 12 and 24 mo of follow-up. After combined MPFL and MPTL reconstruction, the patients' performance level improved and their knee pain decreased. According to the studies reviewed, the SMD for VAS was -4.76 mm, which was statistically significant. It demonstrates that combining MPFL and MPTL reconstruction can reduce pain. Furthermore, according to a meta-analysis, the rate of pain reduction in various studies is decreasing over time. (SMD decreased from 8.17 to 2.04). This study found that combining MPFL and MPTL reconstruction can reduce knee pain over time while also increasing knee stability and function. Perhaps it is due to the method's anatomical and biomechanical similarities with the normal knee. Then, it may be an effective method of treating recurrent Patella instability. Yang et al[15] discovered that normalizing the size and height of the patella, as well as decreasing its slope, can lead to increased tendon stability and pain reduction over time. Moreover, studies show that combining MPFL and MPTL reconstruction reduces the need for procedures like Tibial Tuberosity Osteotomy (TTO) and surgical complications in comparison to MPFL Reconstruction on its own[14]. Another study found that combined reconstruction can stabilize the patella even in the presence of other factors when treating patellar instability in children with TT-TG>15 mm. In other words, the children's performance and range of motion improved, as measured by the Kujala and Lysholm scores[19]. Furthermore, patellar tilt angle was measured in two studies over a period of 24 mo. This angle decreased after surgery, but it was not statistically significant, which could be attributed to the small sample size in the studies examined. Over the last few years, there has been an increase in interest in the combined reconstruction of MPFI and MPTL in the management of recurrent patella instability. It could be because new histological, anatomical, and biomechanical studies have revealed that the MPTL is a true ligament that is important for patellofemoral stability[4,20]. In patients with moderate dysplasia, combined reconstruction of MPFL and MPTL may reduce the need for both bony procedures such as TTO and trochleoplasty[21]. Furthermore, it may improve outcomes when compared to MPFL reconstruction alone[1,20,22,23]. Therefore, combined MPFI and MPTI reconstruction is a safe method for management of recurrent Patella Dislocation and in the future, it may become a part of the algorithms used for the treatment of recurrent Patella instability. However, based on some previous studies, the conclusion stating that the combined procedure would be beneficial has to be made with caution. The study's limitations included a small number of studies and a lack of randomized controlled trials. There are no randomized controlled trials that compare this method to other surgical treatments, and no article included a control group. The current study's strength is the use of precise inclusion and exclusion criteria, as well as the use of meta-analysis, which made our study more reliable. CONCLUSION The current meta-analysis review study found that combining MPTL and MPFL is a safe method with favorable clinical results in patellar dislocation and instability, with fewer complications and the possibility of patellar recurrence or subluxation. There is a need for clinical trial studies with a control group and long-term follow-ups to know the desired results of the surgery, as well as comparing this method with the MPFL method alone, due to a lack of studies and samples in the research conducted in this field. ARTICLE HIGHLIGHTS Research background The patellofemoral joint stability is aided by the medial patellofemoral ligament (MPFL), along with the medial patellotibial ligament (MPTL) and medial patellomeniscal ligament (MPML). While the MPFL is the primary stabilizer, the MPTL plays a critical role as a secondary limiter. However, there are limited studies on the combined reconstruction of MPFL and MPTL and its advantages. Research motivation Different studies on the results of patellar instability management are published with no certain consensus. So, it was necessary to do an analysis to clarify the role of combined reconstruction of MPFL and MPTL in patellar instability management. Research objectives To find out the efficacy of combined reconstruction of MPFL and MPTL. Research methods Several databases were searched to obtain eligible randomized controlled trials. Outcomes were mechanical ventilation time, length of intensive care unit stay, and duration of postoperative hospitalization. Research results Combined MPFL and MPTL reconstruction led to positive outcomes in Kujala scores at 12 and 24-mo follow-ups. The surgeries reduced pain, but the trend of pain reduction decreased over time according to cumulative meta-analysis. Research conclusions Combined MPFL and MPTL reconstruction is a safe method with favorable clinical results in patellar dislocation and instability. Research perspectives Our conclusion needs further confirmation through the conduct of additional high-quality studies. Conflict-of-interest statement: The authors declare that they have no conflict of interest. PRISMA 2009 Checklist statement: The authors have read the PRISMA 2009 Checklist, and the manuscript was prepared and revised according to the PRISMA 2009 Checklist. Provenance and peer review: Invited article; Externally peer reviewed. Peer-review model: Single blind Corresponding Author's Membership in Professional Societies: Iranian Orthopedic Association, President; Iranian Society of Knee Surgery, Arthroscopy, and Sports Traumatology, Past President; Asian Federation of Sports Medicine, Past President. Peer-review started: January 24, 2023 First decision: April 20, 2023 Article in press: May 31, 2023 Specialty type: Orthopedics Country/Territory of origin: Iran Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Oommen AT, India S-Editor: Ma YJ L-Editor: A P-Editor: Zhao S ==== Refs 1 Ebied AM El-Kholy W Reconstruction of the medial patello-femoral and patello-tibial ligaments for treatment of patellar instability Knee Surg Sports Traumatol Arthrosc 2012 20 926 932 21935619 2 Garth WP Jr Connor GS Futch L Belarmino H Patellar subluxation at terminal knee extension: isolated deficiency of the medial patellomeniscal ligament J Bone Joint Surg Am 2011 93 954 962 21593372 3 Philippot R Boyer B Testa R Farizon F Moyen B The role of the medial ligamentous structures on patellar tracking during knee flexion Knee Surg Sports Traumatol Arthrosc 2012 20 331 336 21748394 4 Hinckel BB Gobbi RG Demange MK Pereira CAM Pécora JR Natalino RJM Miyahira L Kubota BS Camanho GL Medial Patellofemoral Ligament, Medial Patellotibial Ligament, and Medial Patellomeniscal Ligament: Anatomic, Histologic, Radiographic, and Biomechanical Study Arthroscopy 2017 33 1862 1873 28662894 5 Arendt EA Dejour D Patella instability: building bridges across the ocean a historic review Knee Surg Sports Traumatol Arthrosc 2013 21 279 293 23124628 6 Zaffagnini S Grassi A Marcheggiani Muccioli GM Luetzow WF Vaccari V Benzi A Marcacci M Medial patellotibial ligament (MPTL) reconstruction for patellar instability Knee Surg Sports Traumatol Arthrosc 2014 22 2491 2498 24196574 7 Aulisa AG Falciglia F Giordano M Savignoni P Guzzanti V Galeazzi's modified technique for recurrent patella dislocation in skeletally immature patients J Orthop Sci 2012 17 148 155 22234373 8 Sappey-Marinier E Sonnery-Cottet B O'Loughlin P Ouanezar H Reina Fernandes L Kouevidjin B Thaunat M Clinical Outcomes and Predictive Factors for Failure With Isolated MPFL Reconstruction for Recurrent Patellar Instability: A Series of 211 Reconstructions With a Minimum Follow-up of 3 Years Am J Sports Med 2019 47 1323 1330 31042437 9 Shah JN Howard JS Flanigan DC Brophy RH Carey JL Lattermann C A systematic review of complications and failures associated with medial patellofemoral ligament reconstruction for recurrent patellar dislocation Am J Sports Med 2012 40 1916 1923 22679297 10 Hetsroni I Mann G Dolev E Nyska M Combined reconstruction of the medial patellofemoral and medial patellotibial ligaments: outcomes and prognostic factors Knee Surg Sports Traumatol Arthrosc 2019 27 507 515 30238237 11 Hautamaa PV Fithian DC Kaufman KR Daniel DM Pohlmeyer AM Medial soft tissue restraints in lateral patellar instability and repair Clin Orthop Relat Res 1998 174 182 12 Moher D Liberati A Tetzlaff J Altman DG PRISMA Group Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement PLoS Med 2009 6 e1000097 19621072 13 Ambrožič B Novak S Nabergoj M Medial Patellofemoral Ligament Reconstruction Techniques. 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