bluuebunny/crossref_metadata_2025 huggingface dataset:
Pretreatment neutrophilto lynphocyte ratio in ewing sarcoma family of tumors: An analysis of 570 patients.
Authors: Biswas Bivas , Jha Akash , Batra Atul , Sengupta Moushumi , Bakhshi Sameer
Published: September 2018

e22504

Background: Ewing sarcoma family tumor (ESFT) is a systemic disease and inflammatory in nature. Inflammatory biomarkers have been identified as prognostic marker in various malignancies including sarcoma. Here we have evaluated the prognostic implication of pretreatment neutrophil to lymphocyte ratio (NLR) in ESFT. Methods: We have calculated baseline NLR from peripheral blood parameters in 570 patients of ESFT treated with uniform chemotherapy protocol in our institution from June’2003 to Feb’2015. Treatment protocol consists of neoadjuvant chemotherapy followed by surgery and/or radiotherapy as local treatment modality and adjuvant chemotherapy. NLR was dichotomized as high and low with high NLR defined as value over the median. Data was censored on 30th Jan’2016. Results: Median age was 15 years (range: 0.1-55) with male: female ratio of 40:17 and 41% (n = 236) patients had metastasis at presentation. Most common sites of tumor were long bones in 209 (37%), thorax in 108 (19%) and pelvis in 90 (16%) patients. Hundred-one (18%) had a soft tissue primary. Twenty-five percent (n = 144) patients had systemic symptoms at presentation. Median symptom duration was 4 months (range: 0.5-36) with median tumor size 8.8 cm (range: 1.6-26). Median NLR was 1.55 (range: 0.08-12.87). High NLR was associated with older age (p = 0.002), female sex (p = 0.02), low serum albumin (p = 0.003) and high lactate dehydrogenase level (p = 0.03). After median follow-up of 32 months (range: 0.8-152), 5-year event-free survival (EFS) was 25.3% & 35.8% and overall survival (OS) was 38% & 48.7%, respectively in whole cohort and in those with localized disease. In the whole cohort, high NLR ( > 1.55) emerged as independent prognostic factor predicting inferior EFS (Hazard ratio-1.36, p = 0.03) along with metastasis at presentation (p < 0.001) and tumor diameter > 8 cm (p = 0.005), in multivariate analysis. NLR couldn’t predict OS. Conclusions: High pretreatment NLR emerged as independent prognostic factor predicting inferior EFS in patients with ESFT. It is an easily available and cost effective biomarker. Prognostic nature of NLR should be evaluated in a prospective study and if proven, should be included in prognostic model to tailor therapy.
Systemic treatment preferences for patients with advanced desmoid-type fibromatosis (DF) in Europe.
Authors: Van Cann Thomas , Requilé Annelies , Schoffski Patrick
Published: September 2018

e22523

Background: The treatment of DF as a rare, benign mesenchymal tumor is poorly standardized and based on physician’s choice. Only a few clinical trials have assessed the different treatment options in DF and randomized evidence is virtually non-existing. We evaluated systemic treatment preferences for patients (pts) with advanced DF among European sarcoma experts, aiming to provide a reference for a generally acceptable control treatment for potential prospective randomized trials. Methods: A 7-item structured questionnaire was sent to physicians in Europe and Israel involved in the multidisciplinary care and experimental treatment of mesenchymal tumors, to assess their systemic treatment choices for DF. Results: The questionnaire was sent to 266 experts (117 institutions, 21 countries), 54 experts (52 institutions, 14 countries) responded. Wait and see was the most common primary approach for pts with advanced DF. (Symptomatic) Disease progression and failure or non-availability of local treatment options were cited as common reasons for considering systemic therapy. Treatment preferences for pts with sporadic vs hereditary DF were similar, with a tendency of earlier use of chemotherapy in hereditary DF. At least 28 different agents or regimens are used in DF. Tamoxifen +/- nonsteroidal anti-inflammatory agents (NSAIDs) or NSAIDs alone are most commonly used in first line, followed by methotrexate- or anthracycline-containing regimen. Tyrosine kinase inhibitors are often used in further lines; other drugs are sporadically used. Clinical trial activity in DF was restricted to only one country/one multi-centric study. Conclusions: DF pts in Europe and Israel have broad off label access to systemic agents, but only to a few clinical trials. Treatment approaches are diverse, poorly standardized and based on weak evidence. There is an urgent need for practice-defining, prospective randomized trials. Such trials should ideally select a homogeneous population of pts with well documented, progressive, symptomatic disease and/or functional impairment after wait and see and/or local treatments. NSAIDs +/- tamoxifen could be considered a generally accepted standard control treatment for clinical trials.
Low-dose chemotherapy with methotrexate and vinblastine for desmoid tumors: A single institution experience.
Authors: Souza Juliana , Lourenco Henrique Monnerat , Mendes Gelcio L. Q.
Published: September 2018

e22524

Background: Desmoid tumor (DT) is a rare and locally invasive proliferative disease. Although, the absence of metastatic potential, it has the propensity for locally invasive growth and recur. Chemotherapy may be considered in inoperable and/or recurrent disease. Methods: Patients with histological diagnosis of DT and treated with weekly low-dose chemotherapy with vinblastine and methotrexate between January 1998 and December 2015 were identified and their medical records were analyzed. Results: Of 23 patients analyzed, most of them were women (female-to-male ratio 2.8:1). The median age at presentation was 29 years (range, 18-59 years). Tumors location was: thoracoabdominal wall (n: 11, 47.8%), extremities (n: 7, 30.4%), abdominal cavity (n: 1; 4 %), and head and neck (n: 4, 17.3%). Tumor sizes were documented in 18 cases and ranged from 3 to 20 cm in largest linear dimension (median, 10 cm). Eight (34.7%) female had pregnancy history and 2 (8%) had familial adenomatous polyposis history. Eleven (47,8%) underwent surgery as first-line treatment. Five (21.7%) patients received first-line treatment with vinblastine and methotrexate, four (17.3%) patients as second-line, and 14 (60.8%) patients as third and fourth-line. Fourteen ( 60.8%) patients had stable disease, four (17.3%) had partial response, and five (21.7%) patients had progressive disease during chemotherapy treatment. After a median follow-up of 58 months, 12 patients had progression disease and 2 patients died. The median PFS was 29 months, without any progression after 32 months. Conclusions: Weekly low-dose chemotherapy with vinblastine and methotrexate appears to have significant activity. Chemotherapy could be an acceptable alternative to radical surgery in selected patients with desmoid tumors.
Multimodality treatment of synovialsarcoma: A single institution experience.
Authors: Greto Daniela , Delli Paoli Camilla , Francolini Giulio , Becherini Carlotta , Dominici Luca , Saieva Calogero , Scotti Vieri , Campanacci Domenico Andrea , Beltrami Giovanni , Scoccianti Guido , Loi Mauro , Desideri Isacco , Meattini Icro , Bonomo Pierluigi , Livi Lorenzo
Published: September 2018

e22530

Background: Synovialsarcoma (SS) is a relatively rare cancer, accounting for 8% of all Soft-Tissue Sarcomas (STS). Identifying prognostic factors could allow to improve treatment strategy for this disease. Methods: Data of 52 patients treated at University of Florence between 1999 and 2016 were retrospectively analysed. Patients and treatment features (Table 1) were correlated with outcome. Results: At a median follow-up of 8.4 years, 9 deaths, 3 local recurrences (LR) and 19 distant metastases (DM) have occurred (17,3%, 5,8% and 36,5%, respectively). Survival Kaplan-Meier analysis showed that Overall survival (OS), Local recurrence disease free survival (DFS-LR) and Distant metastases disease free survival (DFS-DM) were 74,5%, 90% and 59,3%, respectively. Size>10 cm was the only significant predictor of OS (p=0,004). Age>40, size>10 cm and G3 significantly influenced DFS-DM (p=0,043, p=0,002 and p=0,002, respectively). Cox univariate regression analysis confirmed the impact of size on OS (HR 5.8, 95% CI 1.37-24.43, p=0,017). Size (HR 5.06, 95% CI 1.73-14.79, p=0.003) and Grade (7.19, 95% CI 1.65-31.37, p=0.009) influenced DFS-DM. G3 was the only independent prognostic factor associated with DFS-DM. Conclusions: These data confirm that age, size and grade significantly influence outcome in patients affected by SS. Further studies are needed in order to develop tailored treatment strategies in this setting. [Table: see text]
Management of liposarcoma: A single institutional analysis.
Authors: Greto Daniela , Becherini Carlotta , Saieva Calogero , Francolini Giulio , Campanacci Domenico Andrea , Beltrami Giovanni , Scoccianti Guido , Meattini Icro , Loi Mauro , Bonomo Pierluigi , Desideri Isacco , Delli Paoli Camilla , Dominici Luca , Livi Lorenzo
Published: September 2018

e22531

Background: Liposarcoma (LPS) are classified into subtypes: well-differentiated (WDLPS), de-differentiated (DDLPS), myxoid (MLPS) and pleomorphic (PLS). We report a single institution cohort of patients with LPS, undergoing surgery and radiotherapy, to explore prognostic factors related to outcome and toxicity. Methods: This retrospective analysis included 186 LPS patients. Patient, tumor, and treatment variables were analyzed for local recurrence (LR-DFS) and distant metastasis (DM-DFS) disease free survival and overall survival (OS). Results: At a median follow-up of 8.6 years (range, 0.1-27.3 years), Kaplan-Meier (KM) survival analysis showed that LR, DM and OS were 75.5%, 76.6% and 48.1%, respectively. KM analysis showed that Age>56, DDLPS and lower limb localization were related to LR (p=0,001, p=0,0001 and p=0,0001, respectively). Association between LR, Age and DDLPS persisted both at univariate (p=0,003 and p=0,0001, respectively) and multivariate Cox regression (CR) analysis (p=0,024 and p=0,002). Age, tumor depth and grading influenced distant recurrence, both at KM (p=0,023, p = 0.026 and p = 0.016) and univariate CR (p=0,026, p=0,042 and p=0,012). Age and grading were confirmed at multivariate analysis (p=0,009 and p 0,017). Patients with WDLPS and wide excision had significantly better OS (p=0,001 and p=0,03, respectively), while histologic G3 and age>56 were related with worse OS (p= 0,008 and p=0,0001, respectively). Age, DDLPS and Grade were related to OS at univariate (p=0,0001, p=0,0001 and p=0,03, respectively) and multivariate CR analysis (p=0,031, p=0,0001 and p=0,001, respectively). Conclusions: Our analysis confirmed that Grade, tumor depth and histological subtype influenced survival. Further studies are needed in order to explore the influence of hystopathologic features on treatment outcomes. [Table: see text]
Oncometabolite succinate to promote angiogenesis by upregulating VEGF expression through GPR91-mediated STAT3 and ERK activation.
Authors: Mu Xianmin , Zhao Ting , Xu Che , Shi Wei , Geng Biao , Shen Jiajia , Zhang Chen , Pan Jinshun , Yang Jing , Hu Shi , Lv Yuanfang , Wen Hao , You Qiang
Published: September 2018

e23000

Background: Altered cellular metabolism is now generally acknowledged as a hallmark of cancer cells, the resultant abnormal oncometabolites cause both metabolic and nonmetabolic dysregulation and potential transformation to malignancy. A subset of cancers has been found to be associated with mutations in succinate dehydrogenase genes which result in the accumulation of succinate. However, the function of succinate in tumorigenesis remains unclear. In the present study, we aim to investigate the role of oncometabolite succinate in tumor angiogenesis. Methods: Succinate levels were measured in gastric cancer and paracancerous tissues as well as cell culture supernatants from normal cells and gastric cancer cells. Chemotactic motility, capillary structure formation and proliferation of primary human umbilical vascular endothelial cells (pHUVECs) were determined in the presence of succinate in vitro. Moreover, the vessel formation in zebrafish embryo was also used to examine the effect of succinate on angiogenesis. The activation of STAT3 and ERK signaling by succinate was checked by Western Blot. Results: Our data demonstrated the accumulation of markedly elevated succinate in gastric cancer tissues compared with that in paracancerous tissues. Moreover, succinate was able to increase the chemotactic motility, tube-like structure formation and proliferation of pHUVECs in vitro, as well as promote the blood vessel formation in transgenic zebrafish. Our mechanistic studies reveal that succinate upregulates vascular endothelial growth factor (VEGF) expression by activation of signal transducer and activator of transcription 3 (STAT3) and extracellular regulated kinase (ERK)1/2 via its receptor GPR91 in a HIF-1α independent mechanism. Conclusions: These data indicate an important role of the succinate-GPR91 axis in tumor angiogenesis, which may enable development of a novel therapeutic strategy that targets cancer metabolism.
Tenascin C to regulate the function of sHER3, an endogenous inhibitor of neuregulin signaling.
Authors: Cai Chunlin , Zhu Mojun , Maihle Nita J.
Published: September 2018

e23002

Background: HER3 expression is associated with poor prognosis in melanoma patients but the mechanistic basis has not been determined. An endogenous secreted isoform of HER3, p85-sHER3, has been shown by us to inhibit neuregulin-1 mediated, Akt-dependent breast and ovarian cancer cell growth in vitro. Here we demonstrate that p85-sHER3 also regulates neuregulin-dependent melanoma cell migration via an Akt- independent, tenascin C-dependent mechanism. Methods: Immunoblot analysis and RT-PCR were used to study HER3 and p85-sHER expression. Cell proliferation and chamber assays were conducted to determine the effect of p85-sHER3 on melanoma cell growth and migration. Mass spectrometry (MS) was performed to identify sHER3-interacting proteins. CRSPR in vitro editing methods were used to knock out tenascin C (TNC) expression. Results: HER3 and p85-sHER3 were expressed in 5 of 6 human melanoma-derived cell lines tested, with exception of SK-MEL-103 which did not express either gene product. Exogenous addition of 10 nM p85-sHER3 inhibited growth in 3 cells lines, although Akt phosphorylation was not reduced. Protein extracts from M14-MEL cell conditioned media were characterized by MS, and identified p85-sHER3 and TNC. Their association was confirmed by co-immunoprecipitation. sHER3 was further shown to inhibit neuregulin stimulated cell migration in 2 cell lines which exhibited high levels of TNC. CRSPR knockout of TNC in these cell lines (but not in control lines) eliminated sHER3 inhibition of neuregulin stimulated melanoma cell migration. Conclusions: Our results suggest that p85-sHER3, predominantly secreted by HER3-expressing melanoma cells, inhibits melanoma cell proliferation and migration via an Akt-independent mechanism, which is likely achieved through interaction(s) with TNC. TNC promotes an intermediate adhesive state favoring cell migration and this state appears to be mediated by Rho-associated kinase signaling. The Rho inhibitor, CCG-203971 has been shown to inhibit melanoma cell migration. Together, these results suggest the existence of a TNC and HER3-dependent signaling pathway in melanoma that regulates cell migration, and therefore may be correlated with poor patient survival.
Knockdown of TPPP3 to inhibit cell proliferation and invasion in human colorectal cancer.
Authors: Li Yintao , Yu Jinming
Published: September 2018

e23006

Background: Tubulin Polymerization Promoting Protein Family Member 3, TPPP3, a member of the TPPP protein family, has been reported to play important roles in initiation and progression of human cancers, such as lung cancer. However, the expression and underlying function of TPPP3 in colorectal cancer (CRC) have not yet been fully clarified. Methods: In this study, the mRNA and protein levels of TPPP3 in 96 clinical CRC specimens were determined by RT-PCR and immunohistochemistry. The relation between TPPP3 expression and clinicopathologic characteristics and overall survival (OS) were evaluated. TPPP3 was stably knockdowned by shRNA. In addition, CCK-8、Colony formation、Flow cytometric、Transwell and Angiogenesis assay were to examine the biological function of TPPP3 in CRC cells in vitro. Results: We show that TPPP3 was significantly increased in CRC tissues and associated with aggressive factors and poor patient survival. Further experiments showed that knockdown of TPPP3 inhibited cell proliferation, migration and invasion and induced cell apoptosis in vitro. In addition, TPPP3 silencing resulted in a decrease of angiogenesis and S phase fraction. And TPPP3 significantly affected the invasion and migration of CRC cells via the expression of MMP-9, Rac-1 and E-cadherin. Conclusions: Our results suggested that TPPP3 played an important role in CRC progress and might serve as novel therapeutic targets for CRC treatment.
Characteristics of proteomic patterns in patients with metastatic and non-metastatic colorectal cancer.
Authors: Vodolazhsky Dmitriy I. , Kutilin Denis S. , Kharin Leonid , Kolesnikov Evgeniy N. , Bogdanova Olesia A. , Sustretov Vyacheslav A. , Trifanov Vladimir , Kit Oleg I.
Published: September 2018

e23007

Background: Over the past 20 years, the number of deaths from colorectal cancer (CRC) increased by 46% worldwide: every year more than 1 million people are diagnosed with this pathology. Proteomic studies using protein microarrays are a very attractive strategy for the screening of new highly specific biomarkers which can improve prognosis prediction of CRC. We performed a screening proteomic study to find new informative biomarkers for metastatic and non-metastatic colorectal cancer which can be further used in routine histochemical studies. Methods: The samples of tumor and non-tumor tissues of 20 patients with histologically confirmed adenocarcinoma of the colon were studied. Extraction, labeling and hybridization of proteins on the slide containing 224 antibodies for key cellular proteins were performed using the Panorama Antibody Microarray - Cell Signaling Kit, and the GenePix 4100A scanner was used for scanning. The data were analyzed using GenePixTMPro 6.0. Results: We revealed a significant (p < 0.05) increase in expression of such proteins as C-myc, SMAD4, PCAF, Adaptin (b1 + b2), FAK Phospho (pY577), PKC g and Phospho-Pyk2 (pY579/580) by 2.1; 1.5; 1.5; 1.5; 2.7 and 1.6 times, respectively, in tumor tissue of patients with metastatic colorectal cancer compared to non-tumor colon tissues. A significant increase (p < 0.05) in expression of proteins NGFR p75 (Nerve Growth Factor Receptor) by 3.8 times and Phospho-Ta (pS199 / 202) by 1.9 times was found in tumor tissue of patients with non-metastatic CRC in comparison with non-tumor colon tissue. Conclusions: The study showed changes in proteomic profile of malignant colon tissue, and proteomic profile differed significantly in tumor tissues of patients with metastatic and non-metastatic cancer. We found changes in expression of 9 proteins, differential in each group of patients (with metastatic and non-metastatic CRC). These proteins have a high potential as biomarkers for CRC prognosis prediction.
The effects of very low doses of chemotherapeutic drugs on endothelial cells.
Authors: Sonugur Fatma Gizem , Ocal Muge , Babahan Cansu , Abdi Abgarmi Samira , Icli Fikri , Akbulut Hakan
Published: September 2018

e23014

Background: Angiogenesis is one of the key processes leading tumor progression and metastasis. Conventional chemotherapeutic drugs are usually cytotoxic to tumor cells at the therapeutic doses. The lowered doses of those drugs have been shown to induce apoptosis in endothelial cells and implemented as metronomic schedule in the clinic. We aimed to study the effects of very low doses of chemotherapeutic drugs on endothelial cell functions and gene and protein expression of pro-and anti-angiogenic factors. Methods: We first determined the IC50 doses of 5-fluorouracil, irinotecan, and oxaliplatin in a human umbilical endothelial cell line (HUVEC). Then we’ve tested the effects of the IC50, -1log, -2log and -3log of IC50 doses on endothelial cell migration and tube formation. We’ve also studied the effects of the assigned doses on expression levels of the genes VEGF-A, VEGFR2, PDGF-B, angiopoietin-2 (Ang-2), endothelin-1 (End-1), thrombospondin-1 (Tbs-1), iNOS, eNOS by real-time PCR, protein secretion levels of VEGF-A, PDGF-BB, Ang-2, End-1 and Tbs-1 by ELISA and nitrate/nitrite production by a colorimetric assay. Results: The IC50 dose levels of 5-FU, irinotecan, and oxaliplatin were 9,2 μM, 9,8 μM and 11,8 μM, respectively. We found that all the -1log, -2log and -3log of IC50 doses of the drugs tested in the study significantly decreased the migration capacity of the HUVEC cells. However, no significant changes were observed in tube formation of endothelial cells. Likewise, the expression levels of pro-angiogenic genes including Ang-2, iNOS, eNOS, PDGF-B and VEGF-A were significantly decreased at the same doses of the drugs. Accordingly, the levels of Tbs-1 were significantly increased at all 3 dose levels of the drugs. The most efficacious doses of the drugs to decrease NO levels in HUVEC cells were -1logIC50 for 5-FU, -2logIC50 for irinotecan and -3logIC50 for oxaliplatin. Conclusions: In conclusion, our results suggest that 5-FU, irinotecan, and oxaliplatin could decrease the pro-angiogenic and increase anti-angiogenic factor production in endothelial cells at the doses much lower than the usual metronomic schedules and warrant further research in animal models and clinical trials as an anti-angiogenic treatment modality.
The expression of pluripotency genes regulates properties of cancer stem cells in MCF-7 breast cancer model.
Authors: Miftakhova Regina R. , Rakhmatullina Aigul R , Mingaleeva Rimma N. , Garanina Ekaterina E , Khaiboullina Svetlana F , Rizvanov Albert A
Published: September 2018

e23018

Background: More than 1.6 million cases of breast cancer (BC) are diagnosed annually worldwide. Despite advances in diagnoses and treatment, BC remains the third leading cause of death in women. Metastases and chemotherapy resistance are the main factors contributing to BC mortality. The ability of tumor cells to overcome the drug-induced growth inhibition is now linked to a unique population of cancer initiating tumor cells, often referred as cancer stem cells (CSC). CSC represent a small fraction of tumor cells, therefore, currently used isolation protocols have a low yield and are poorly reproducible, hampering research on the role of these cells in cancer chemoresistance. We propose a novel approach to generate large quantities of CSC-like cells by genetic reprogramming of non-stem cancer cells. Methods: We postulate that CSC-enriched cell line can be developed in vitro by upregulating of proteins controlling cancer cell pluripotency including Oct4, Sox2, NANOG, KLF4 and c-Myc. Increased transcriptional and translational activity of these genes in MCF-7 cells was demonstrated by real time-PCR and Western blot. Proliferation and migration of cells overexpressing Oct4, Sox2, NANOG, KLF4 and c-Myc were analyzed as well. Also, changes in CSC population counts and their sensitivity to chemotherapy were investigated using sphere formation assay. Results: We found that cell proliferation rate was correlated with the expression level of c-Myc and Oct4. Increased CSC counts were found in cells with overexpressed Oct4 (62.7%), KLF4 (97%) and NANOG (121.3%) proteins as compared to the parental cells. Overexpression of SOX2, NANOG and KLF4 significantly increased CSC resistance to docetaxel (83,3±6,8; 93,3±4,5 and 80,3±5,0 spheres respectively) when compared to the original cells (48,3±3,0 spheres). Conclusions: We conclude that overexpression of pluripotency proteins Oct4, Sox2, NANOG, KLF4 and c-Myc changes the CSC counts and proliferation capacity of BC cells. Acknowledgements: The study was funded by RFBR, according to the research project No. 16-34-60210 mol_а_dk, and by Russian Government Program of Competitive Growth of Kazan Federal University.
Aspirin to inhibit the formation and EMT of circulating tumor cells in patients with metastatic colorectal cancer but not breast cancer.
Authors: Ni Chao , Yang Liu
Published: September 2018

e23024

Background: High circulating tumor cells (CTCS) have been acknowledged as a poor indication in malignant disease. Besides, platelets are found play crucial role in tumor cells’ epithelial – mesenchymal transition and metastasis, so here we test if disruption of platelet function with aspirin, could decrease the number and inhibit the EMT of CTCs in metastatic colorectal (MCC) or breast cancer (MBC). Methods: Patients with MCC or MBC who were not receiving cytotoxic chemotherapy currently (except for capecitabine maintenance), while endocrine therapy, bisphosphonate or molecular targeted therapy were accepted. Base line of Platelet aggregation rate (PAR), CTCs’ number and molecular phenotype were recorded. Patients whose CTCs ≥ 5 cells/7.5ml peripheral blood were included and receive aspirin 100mg/day for the following 8 weeks. Then the phlebotomy was performed at 4, 8 weeks thereafter to obtain specimen and assess PAR, CTCs’ number and phenotype. The phenotypes of CTCs were classified into epithelial (E+), mesenchymal(M+) and middle type(express both E+ and M+ markers) with fluorescence in situ hybridization assay. Results: Forty patients (19 MBC and 21 MCC patients ) were enrolled. Base line CTC numbers were 8.7± 3.4 in MBC patients, the ratio of E+ and M+ type were 48.2 ± 22.4% and 25.1 ± 14.9% respectively; the base line CTC numbers were 10.7 ± 4.8 in MCC patients, and the ratio of E+ and M+ type were 45.3 ± 27.3% and 22.0 ± 18.5% respectively. Despite adequate platelets inhibition in both groups, CTC numbers were similar in MBC patients in the following 8 weeks ( p= 0.0532 ), while the fraction of E+ or M+ CTCs were also unchanged (E+ CTCs p= 0.305; M+ CTCs p= 0.09); however, both CTC numbers and fraction of M+CTCs were markedly decreased in MCC patients (total numbers: p< 0.01; E+ CTCs p= 0.031; M+ CTCs p= 0.013) . Conclusions: Aspirin could decreased the number of CTCs and block EMT transition in MCC patients, and our results provide potential explanation of how aspirin impede the metastasis of colorectal cancer. But this effect could not be observed in MBC patients. Future studies evaluating the underlying mechanism of this differences remain of interest, and they may be informed by our results. Clinical trial information: NCT02602938.
Enumeration of circulating tumor cells and assessment of immunotherapy biomarker PD-L1 in patients with colorectal cancer.
Authors: Tseng Ju-Yu , Chen Chwen Cheng , Lin Chun-CHI , Lin Hung-Hsin , Chen Yen-Ru , Lee Chia-Ying , Wu Li-Fan , Wang Shin-Hang , Yu Hui-Min , Fang Wei-Feng , Jiang Jeng-Kai
Published: September 2018

e23029

Background: Circulating tumor cells (CTC) play a prognostic role in patients with metastatic colorectal cancer (mCRC). Studies investigating detection of CTC in peripheral blood (PB) showed limited clinical sensitivity. This study compares clinical sensitivity of CTC analysis in mesenteric venous blood (MVB) vs PB. PD-L1 expression on isolated CTCs was also characterized in various stages of CRC patients. Methods: 8mL PB and MVB samples were collected into K2EDTA-containing vacationer tubes and subsequently performed CTC enumeration and PD-L1 analysis by MiSelect R system. CTC is defined as EpCAM+, cytokeratin+ and CD45- with intact nuclei. CTC and PD-L1+ CTC number were counted and correlated to the patients’ clinical manifestations. Results: Blood samples were collected from 75 CRC patients with various stages. In MVB, CTC detection rate was 20%, 33%, 44% and 58% for stage I, II, III, and IV respectively, and the overall detection rate is 41%. In PB, CTC only detected in sporadic cases (7%). Also, the amount of CTC detected in MVB was more abundant than that in PB ( P < 0.001) In addition, preoperative serum CEA levels were significantly associated with presence of CTC in MVB ( P= 0.011). PD-L1 expression was detected in 32% of the isolated CTCs, and was more abundant in those from late stage patients. The expression level of PD-L1 showed heterogeneity among CTCs. Conclusions: CTC enumeration in MVB could potentially increase the clinical sensitivity for CTC analysis in CRC. The CTC level in MVB significantly correlated with CEA levels which could serve as an important biomarker information for post-operative management. In additions, using MiSelect R System to determine PD-L1 expression on CTC could provide a promising approach for both patient selection or therapeutic monitoring of immunotherapies for CRC.
Landscape of somatic mutations in different subtypes: Advanced breast cancer with circulating tumour DNA analysis.
Authors: Yi Zongbi , Ma Fei , Guan Yanfang , Yuan Fang Li , Sun Xiaoying , Guan Xiuwen , Li Chunxiao , Yi Xin , Qian Haili , Xu Binghe
Published: September 2018

e23039

Background: It is particularly important to provide precise therapies and understand tumor heterogeneity based on the molecular typing of gene expression. Yet the landscape of somatic mutations in different subtypes of advanced breast cancer (ABC) is largely undetermined. Methods: Overall, 100 blood samples were obtained from 100 advanced female breast cancer patients who underwent therapy at Cancer Hospital, Chinese Academy of Medical Sciences from March 2015 to September 2016. Mutations in 1021 tumor-related genes in ctDNA was assayed by gene-panel target-capture next-generation sequencing. Results: Somatic genomic alterations in ctDNA including copy number variants and point mutations were identified in 96 of 100 patients (96.0%). The number of somatic mutations varied markedly between individual patients (mean 2.9, range1-31). No difference was found between four subtypes for the number of somatic mutations. However, the mean number of somatic mutations was higher in age at 40-50 year than patients over age 60 ( p= 8.46 vs 3.88; p= 0.01). Results from multivariate analyses showed that the number of somatic mutations was increased with the number of endocrine therapy line ( p= 0.007). TP53 and PIK3CA were two most frequently mutated genes detected in ctDNA of 100 patients which were recurrently detected in 43 (43.0%) and 32 (32.0%) patients, respectively. ESR1/PIK3CA were more prevalent in HR+ cancers ( p= 0.007, 0.025 respectively). NOTCH1 are more frequently detected in HER2- group than in HER2+ patients (15.63% vs 2.94%; p= 0.033). In multiple logistic regression analysis indicated that pathological grade, tumour size at diagnosis and PR statue were positive associated with PIK3CA mutations. Multiple regression analysis also revealed that ki-67, metastatic at diagnosis, number of metastatic sites, number of endocrine line were associated with ESR1 mutations. Conclusions: The results revealed that different subtypes ABC have their own genetic alterations features. Certain gene mutations may be related to clinical treatment especially endocrine therapy.
Validation of the denaturing capillary electrophoresis (DCE) assay for non-invasive liquid biopsy in lung carcinoma: A study of concordance with the cobas EGFR mutation test v2.
Authors: Minarik Marek , Belsanova Barbora , Fiala Ondrej , Pesek Milos , Benesova Lucie
Published: September 2018

e23040

Background: The detection of circulating tumor DNA (ctDNA), referred to as “liquid biopsy” is becoming a preferred option for some patients with lung adenocarcinomas. The key for a suitable technology is high sensitivity enabling to detect small fractions of mutated alleles, the method has to be solid against false-positives. The aim of this work was to compare our method based on capillary electrophoresis under denaturing conditions (DCE) to FDA and CE IVD approved commercial kit - cobas EGFR Mutation Test v2. Methods: A total of 44 plasma samples were acquired from patients with clinicaly confirmed stage IV lung adenocarcinoma. There were samples from patients prior to receiving therapy (with a known tissue EGFR genotype) as well as several ones showing progression on a previously administered antiEGFR-therapy (with no tissue info available at the time of plasma sampling). DNA was isolated from multiple 2 mL aliquots of each plasma. The first aliquot was run on cobas z480 real-time instrument using unmodified manufacturer's protocol. Another 2ml aliquot was run by DCE assay (accredited method under ISO 15189:2012), which is based on a PCR followed by a fragment analysis on an ABI capillary analyzer (Sanger sequencer). Results: The mutual concordance was 84.1% (37/44). cobas assay identified 11 EGFR-positive plasma samples (6 x Ex19del, 3 x L858R, 1x T790M combined with Ex19del and 1x T790M combined with L858R). DCE identified 8 EGFR-positive samples (6 x Ex19del, 1 x L858R, 1xT790M combined with L858R). DCE missed 5 of cobas-positive samples (2x Ex19del, 2x L858R and 1x T790M) while in turn identifying 2x Ex19del plasma samples that were concordant with tissue, but missed by cobas. Overall concordance with tissue was 73.3% (22/30) for cobas, 70.0% (21/30) for DCE assay and 80.0% (24/30) for both combined. Conclusions: DCE approach is reliable for low levels of mutations in plasma of lung cancer patients and comparable (and complementary) to cobas. Its advantage is simplicity, low cost and a universal, straighforward adaptability to virtually any mutation at any site allowing to detect oncogenic as well as tumor supressor mutations in ctDNA. Supported by grant 17-30748A.
Circulating LncRNAs as predictive biomarkers by molecular subtypes in advanced breast tumors.
Authors: Salvador Javier , Pecero Maria Luisa , Gil Ana , Rodriguez de la Borbolla Maria , Ruiz Manuel , Montano Alvaro , Gonzalez Rosario , Benavent Viñuales Marta , Molina Sonia
Published: September 2018

e23041

Background: Breast cancer is the most common malignant tumor in women worldwide. The leading breast cancer deaths are due to advanced disease stage. For this reason, identification of novel breast cancer progression-associated biomarkers would enhance the clinical management of each patient. Long non-coding RNAs (LncRNAs) are defined as noncoding RNAs longer than 200 nucleotides and are involved in several regulatory processes, such as regulation of gene expression. Moreover, it has been described that LncRNA expression is deregulated in different human diseases such as breast cancer. However, it is unclear if circulating LncRNAs could be used as progression biomarkers of breast cancers. The purpose of this study was to evaluate the role of circulating LncRNAs in patients with advanced breast cancer. Methods: Expression of 84 LncRNAs was quantified by “RT2 IncRNA PCR Array Human Cancer PathwayFinder” (Qiagen) in plasma from 24 subjects (12 patients with advanced breast cancer and 12 healthy subjects). cDNAs were pooled into four groups, two groups containing a mixture of six patients with advanced breast cancer and another two groups containing a mixture of six cDNAs from healthy subjects. Three differentially expressed LncRNAs were selected for validation by specific real-time PCR assay. Results: Three deregulated LncRNAs were identified and validated in patients with advanced breast cancer versus healthy subjects. We found that LncRNAs GAS5 and ZFAS1 expression was decreased in the Luminal, Her2-positive and Triple Negative advance cancer groups compared to healthy subjects. In contrast, LncRNA RMRP expression was increased in Luminal and Her2-positive advance cancer groups. To our knowledge, there are no publications relating the LncRNA RMRP expression with advance breast cancer research. Conclusions: These results contribute to unreveal the role of circulating LncRNAs as biomarkers in the advanced breast cancer and provide novel non-invasive tools in this disease.
Serum metabolism during breast cancer treatment.
Authors: Giskeødegård Guro Fanneløb , Madssen Torfinn Støve , Vettukattil Riyas , Flote Vidar Gordon , Husøy Anders , Lofterød Trygve , Lundgren Steinar , Bertheussen Gro Falkener , Schlichting Ellen , Wist Erik Andreas , Thune Inger , Bathen Tone Frost
Published: September 2018

e23043

Serum metabolism during breast cancer treatment Background: Breast cancer treatment may include surgery, systemic therapy and radiation, often involving side-effects. Many patients experience weight gain during treatment, which is associated with decreased survival rates1. The purpose of this study was to describe serum metabolic alterations in breast cancer patients undergoing treatment, and relate these alterations to weight gain during treatment. Methods: This pilot study includes 60 breast cancer patients, aged 35-75 years, with histologically verified stage I/II disease. All patients underwent tumor surgery, and were treated according to national guidelines. Samples were collected before and 6 months after surgery, and analyzed by MR spectroscopy (MRS) and mass spectrometry (MS). 170 metabolites and 105 lipoprotein subfractions were quantified by combined MRS and MS analyses. Results: Multilevel PLS-DA showed significant alterations in serum metabolite profiles post-treatment, both in patients receiving (n = 35) and not receiving (n = 25) chemotherapy (classification accuracy: 86.7% and 77.0%, resp., p < 0.001). Lipoprotein profiles were also significantly altered in both groups (p < 0.001). Chemotherapy recipients had decreased levels of citrate, ornithine, and methionine after treatment, while non-recipients had increased levels of glutamate, alanine, proline and two biogenic amines, and decreased levels of acylcarnitines. 17/52 patients (32.7%) gained weight (≥ 1.5 kg) during treatment. Weight gain was predicted from pre-treatment samples with accuracy 67.0% (p = 0.020). Weight gain patients had lower levels of three acylcarnitines and 20 phosphocholines, and higher levels of lysine and isoleucine, suggesting aberrant lipid and amino acid metabolism. Weight gain was also reflected in the post-treatment samples (accuracy 66.8%, p = 0.015), with weight gain patients having higher levels of five acylcarnitines, and lower levels of glycine, isoleucine and valine. Conclusions: This study indicates that treatment induces changes in serum metabolite levels. Patients gaining weight had significantly different metabolite profiles than those not gaining weight both before and after treatment. 1. Chan et al, Ann Oncol 25: 1901-14, 2014.
Baseline mutation profiling of circulating cell-free DNA from healthy individuals to improve the detection accuracy of circulating tumor DNA in cancers.
Authors: Tian Geng , Xia Ligang , Li Zhoufang , Li Xiaohua , Xu Feiyue , Liu Chaoyu , He Jiankui
Published: September 2018

e23057

Background: Cell-free circulating tumor DNA (ctDNA) has emerged as an effective blood-based biomarker for clinical diagnostics in cancers, improving the accuracy and efficiency of cancer treatment for clinic. However, the somatic mutations originated from inheritance or normal biological metabolism in circulating cell-free DNA (cfDNA) in plasma can largely interfere with the detection of ctDNA. The baseline for ctDNA evaluation is urgently needed for accuracy identification of ctDNAs in patients. Methods: Averagely 20 ml blood samples were collected from more than 1200 individuals including healthy volunteers, lung cancer, colorectal cancer, pancreatic cancer, gastric cancer patients, etc. Genomic DNA from white blood cells and cfDNA from plasma were extracted and constructed as sequencing libraries using a panel contains 50 cancer-associated genes, respectively for each sample. Then they are subjected to ultra-deep sequencing with average depth > 40000 folds covering ~21K nucleotide regions. Results: The background somatic mutation frequencies were detected in genomic DNA and cfDNA in healthy controls. The results showed that most of mutations in cfDNA were consistent with those in genomic DNA. Using data from 1200 individuals, we generated the baseline mutation profiling of cfDNA, which was referred in the ctDNA determination in cancer samples, which significantly improved the accuracy of ctDNA detection compared with tissue biopsy. Conclusions: Our studies demonstrated the importance of sequencing both cfDNA and genomic DNA for ctDNA detection in cancers. We also determined the baseline mutation profiling of circulating cfDNA from more than 1200 healthy individuals and confirmed the value of it by comparing with DNA sequencing data in cancer tissue. Our work offers clue on how to improve the detection accuracy of circulating tumor DNA in early cancer diagnosis.
LKB1 and its importance for survival in EpCAM negative circulating tumor cells (CTC) in metastatic breast cancer.
Authors: Trapp Elisabeth Katharina , Andergassen Ulrich , Koch Julian Gregor , Majunke Leonie , Zill Beate , Kurt Ayse , Harbeck Nadia , Mahner Sven , Tzschaschel Marie Lena Johanna , Friedl Thomas W. P. , Janni Wolfgang , Rack Brigitte Kathrin , Alunni-Fabbroni Marianna
Published: September 2018

e23061

Background: To reach distant sites, CTC overcome metabolic stress and acquire a mesenchymal phenotype. So we assessed the pro survival function of liver kinase B1 (LKB1) a cellular metabolic stress controller, in combination with the EMT transcription factor Zinc finger E-box binding homeobox factor 1 (ZEB1) and stem cell markers e.g. CD44 in EpCAM negative CTC. Tumor spheroids and targeted si-RNA knockdown of LKB1 were analyzed in two breast cancer cell lines to support transcriptomic data. Methods: CTC were isolated from whole blood of 27 metastatic breast cancer patients. 17 age matched healthy donors were negative control. All patients were analyzed with CellSearch system to detect EpCAM positive CTC, which were eliminated by immunodepletion (AdnaTest BreastCancerSelect Kit, TATAA Biocenter) together with CD45 positive white blood cells (Miltenyi Biotec). RNA transcripts were quantified by qPCR (TaqMan Gene Expression Assay, ThermoFischer Scientific) and the relative expression was calculated by the 2-DCtmethod. EpCAM negative CTC and spheroids were analyzed by immunofluorescence. Data were analyzed by non-parametric statistical tests. Results: According to the CellSearch system, most patients (74%, n = 20) were CTC negative. 20% of this sub-group showed enhanced levels of ZEB1, 15% of LKB1, and 40% of stem cell markers. ZEB1 and LKB1 were never co-expressed, while there was an almost significant association between enhanced ZEB1 and stem cell marker expression (p = 0.053). Immunofluorescence analysis on single CTC confirmed the transcriptomic result. MDA-MB 231 cell lines, normally negative for LKB1 and unable to grow in suspension, showed the ability to generate spheroids only if LKB1 expression was activated. Targeted knockdown of LKB1 confirmed these results. Conclusions: CellSearch system negative patients present a heterogeneous CTC population with enhanced levels of ZEB1, CD44, or LKB1. While ZEB1 and CD44 might be highly expressed in cells that already have entered EMT, LKB1 overexpression could represent the response to metabolic stress induced in the early phase of intravasation Hence, LKB1 might represent a novel therapeutic target to chase CTC and prevent metastasis.
Serial CTC/cfDNA assessment for the identification of pathogenic mutations in patients with breast cancer.
Authors: Irie Hanna , Schmidt Paul H. , Port Elisa R. , Kinoshita Yayoi , de la Iglesia Janis , Pisapati Kereeti , Couri Ronald , Kolodka Olivia , Arib Hanane , Sebra Robert P. , Donovan Michael Joseph
Published: September 2018

e23070

Background: Serial liquid biopsy assessment provides a unique platform for detecting existing and / or emergent resistance mutations; it may be particularly beneficial when patients begin to progress on otherwise effective therapy. We aimed to determine the utility of such an approach for detection of tumor genetic alterations at multiple points during the clinical course of patients diagnosed with breast cancer. Methods: Twenty patients diagnosed with stage I-IV breast cancer (50% with triple negative disease) receiving treatment at the Dubin Center of Mount Sinai were consented for serial CTC/cfDNA collections between October 2015 and November 2016. Circulating tumor cell (CTC)/cell-free DNA (cfDNA) were isolated using Cynvenio’s Liquid Biopsy platform and sequenced utilizing the Hotspot/Oncomine panels. DNA from corresponding patient tumors, when available, was also sequenced. Results: 25% of patients had at least one known pathogenic mutation detected in either cfDNA or CTC DNA in at least one collection; all patients had stage IV disease at collection. Pathogenic mutations detected were in p53 (15%), PIK3CA (10%) and Smad4 (5%). For those patients with available tumor tissue, the same pathogenic mutation was detected in the primary tumor. Mutations were more often detected in cfDNA rather than CTC DNA. Finally, known pathogenic mutations were more consistently detected in serial collections with clinical disease progression and resistance to treatment. Conclusions: Pathogenic mutations were identified in 25% of patients, more frequently in cfDNA and not in every collected sample, supporting serial collection studies to monitor response. Presence of same pathogenic mutations in the patient’s primary tumor suggest a role for surveillance in early stage, as well as advanced disease.
Ultrastructural study of tumor-infiltrating СD4+ Т-lymphocytes in colon cancer.
Authors: Grankina Anastasia , Zlatnik Elena Yurievna , Novikova Inna Arnoldovna , Filippova Svetlana Yu. , Logvinov Aleksandr K. , Kolesnikov Vladimir
Published: September 2018

e23074

Background: The purpose of the study was to assess stress-induced metabolic changes in СD4+ T-lymphocytes among tumor-infiltrating lymphocytes (TILs) according to ultrastructural morphometric parameters. Methods: Samples (n = 12) of colon adenocarcinoma, corresponding resection line tissues and blood of patients and healthy donors were studied. After grinding the samples and PBMC isolation using the density gradient, CD4+ T-lymphocytes separation with magnetic columns was performed using CD4 MicroBeads, human (MACS Miltenyi Biotech). The pellet obtained after centrifugation was embedded in agarose and prepared for transmission electron microscopy. Mitochondria images on ultrathin sections of Т-lymphocytes were randomly selected for the analysis. The total number of mitochondria studied were 71 (tumor), 78 (normal tissue), 78 (blood of patients), 80 (blood of healthy donors). For each image the unchanged cristae area was determined as a percentage of the total area of mitochondria cross section. Results: We found no differences in the structures of mitochondria of blood lymphocytes in patients and in healthy donors. Comparison of parameters of CD4+ Т-lymphocyte mitochondria in the colon unchanged tissues and in the blood of patients did not show significant differences indicating minimal damage during TILs` isolation from the tissues. Electron microscopy of TILs derived from tumor tissue showed more frequent apoptotic and necrotic death of lymphocytes, as well as some mitochondrial swelling with changes in the shape and the number of cristae compared to the norm. Statistical analysis revealed significant decrease in the relative area of unchanged cristae: 57% in tumor against 73% in the normal tissue (p < 0.0001, χ2Test). Conclusions: The results suggest that stress-induced changes are developed in tumor-infiltrating CD4+ T-lymphocytes, and these changes apparently enhance their death as demonstrated by ultrastructural characteristics of mitochondria.