bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of next-generation sequencing methodologies and direct sequencing for the detection of EGFR mutations in non-small-cell lung carcinoma: Implications for optimal clinical testing.
Authors: Kim Hye Sook , Kim Yeul Hong , Sung Jae Sook , Kim Seung Tae , Park Kyong Hwa , Kim Jung Sun , Choi Yoon Ji 
Published: March 2020

e22025 Background: The efficacy of next-generation sequencing (NGS) for the prediction of clinical benefits from EGFR TKIs in non small cell lung cancer(NSCLC) has yet to be fully established. The purpose of the present study was to evaluate the predictive effect of EGFR-evaluating methodologies on the use of EGFR TKIs in patients with NSCLC. Methods: cTumor samples were obtained from patients with NSCLC at 5 institutions in Korea. Study cohort A consisted of surgically resected or biopsy NSCLC samples enriched with adenocarcinoma histology. Study cohort B consisted of patients who received EGFR TKI during the treatment period. EGFR status was tested with direct sequencing, Peptide nucleic acid-locked nucleic acid PCR clamp (PNA-LNA PCR Clamp) method, and Personal genome machine (PGM) sequencing using the Ion AmpliSeq Cancer Panel on the Ion Torrent. Results: EGFR mutations in cohort A were identified in 16 (28.1%), 27 (47.4%), and 29 (50.9%) of 57 patients by direct sequencing, PNA-LNA clamping, and the Ion Torrent PGM, respectively. In 2 cases of EGFR mutant detected by Ion Torrento PGM in contrast to EGFR wild type by PNA-LNA clamping, EGFR mutation was found on novel sequence other than classical EGFR mutations. In total, 33 out of 37 patients in cohort B whose frozen NSCLC tissue was available were examined. Patients with EGFR mutations tested by PNA-LNA clamping did not show a significantly higher response rate than did patients with the wild-type EGFR (P=0.607). Mutational status detected by 2 methods did not significantly predict overall survival and progression free survival. However, curves showed a greater differentiation when the mutation was detected by PNA-LNA clamp as compared to mutations detected by direct sequencing. Conclusions: Though this study was conducted in a small number of cases, the results suggest that both PNA-LNA clamping and the Ion Torrent PGM are highly sensitive procedures relative to direct sequencing, and are useful screening tools for the detection of EGFR mutations in clinical practice.
Levels of circulating natural killer T and natural killer cells in breast cancer patients.
Authors: Thein Mya Sanda , Chumsri Saranya , Eeast James , Li Junxin , Kesmodel Susan , Bao Ting , Feigenberg Steven J. , Tkaczuk Katherine Hanna , Webb Tonya 
Published: March 2020

e22034 Background: Invariant natural killer T (iNKT) cells are a unique subset of T lymphocytes that express markers characteristic of both T cells and natural killer (NK) cells. Following activation, iNKT cells rapidly produce a variety of cytokines and activate many other immune cells, thus iNKT cells are key mediators of innate immune responses (IIR) against tumors. The aim of this study was to assess the percentage (%) of iNKT, NKT-like, and NK cells in the peripheral blood of healthy volunteers (HV) and breast cancer (BC) patients. Methods: The % of circulating iNKT (Va24Ja18+CD3+), NKT-like (CD3+CD16CD56+), and NK (CD3-CD16CD56+) cells was analyzed by standardized flow cytometry in 22 newly diagnosed stage 0-3 BC patients and 27 HV. Luminal A BC was defined as ER/PR+≥1%, HER2- Ki67<20%. Differences between groups were assessed using the Mann-Whitney U test for nonparametric data and ANOVA for ≥ 3 groups. Results: iNKT cells % in BC patients (ages: 37-75) was significantly lower compared to HV (ages: 21-64) (Mean: 0.32 vs 0.12, p=0.0036). Among BC patients, NK cells were lower in patients aged ≥50 yrs (p=0.003) and with BMI ≥ 30 (p=0.0036). NK cell % was higher in Asians compared to other races. Patients with luminal A BC, which generally have the best prognosis, have a significantly higher % of NKT-like cells. Conclusions: Interestingly our data shows that older and obese BC patients have lower % of circulating NK cells, while patients with luminal A tumors have a higher % of NKT-like and a lower % of iNKT cells. Further studies on the effects of BMI and aging on IIR as well as functional studies are needed to better understand immunosenescence-related increasing incidence of cancers and IIR-mediated anti-tumor activity in BC patients. [caption]iNKT, NKT-like, and NK cells (% lymphocytes) by age, BMI, race, and pathology in BC patients.[/captions] [Table: see text]
Potential applications of circulating melanoma cells (CMCs) expressing γ-H2AX.
Authors: Rossi Elisabetta , Facchinetti Antonella , Grassi Angela , Rossi Carlo Riccardo , Stragliotto Silvia , Pigozzo Jacopo , Chiarion-Sileni Vanna , Amadori Alberto , Zanovello Paola , Zamarchi Rita 
Published: March 2020

e22036 Background: The CMC analysis was recently improved by the CellSearch platform that offers an accurate (and reproducible) assessment of tumor burden, to be used as prognosticator and predictor of treatment response.Moreover, not only decreased total circulating cells numbers but also increased fraction of apoptotic cells may represent response-related markers. To monitor changes in the balance between live and apoptotic Circulating Tumor Cells (CTCs) ( Rossi, Basso et al. 2010 ) during therapy, a novel CTCs assay with a companion algorithm (the ΔAUC) was reported to be a pharmacodynamic marker, as documented by consistent radiological findings in MBC and mRCC patients. Methods: Fluorescent anti γ-H2AX mAb (specific for the phosphorylated form of H2AX) was integrated in the standard CMC assay to assess DNA damage and to measure cell viability and apoptosis in spreading melanoma cells. The integrated test was developed using MCF-7 and SK23mel cells line and tested in melanoma patients. From June-2011 till now, basal blood samples from 64 patients (pts) in stage IV were collected; median age 62 (range: 27-85). Both considering the cost of the CMC assay and the little already known about this new method in melanoma, to evaluate the feasibility of CMC retrieval and ΔAUC calculation an interim analysis was specified in the study protocol and conducted at September 2012 on the first 40 pts. Results: At baseline, in Padova’s cohort 72.6% of pts are CMC-positive. The CMC number ranged from 1 to 94. The γ-H2AX-positive CMCs ranged from 0% to 100% (median 67%). Shorter median Progression-free survival PFS was observed for patients who had > 2 CMCs (n=26, 162 days) compared with < 2 CMCs patients (n=14, 203 days, P value = 0.0270). No significant differences were found with the ΔAUC criterion (i.e. exceeding live vs apoptotic CMCs). Furthermore, a significant association was found between CMC-positive status at baseline and disease sites at progression (visceral and CNS, Chi-square = 8,521 with 3 df, P value = 0.036). Conclusions: Accrual is ongoing. Updated data including evaluations on prognostic value of total and apoptotic CMCs and correlation with clinical stage, tumor burden, metastatic sites and survival will be available and presented at the meeting.
Screening of NSCLC samples from Chinese lung cancer patients for activating rearrangements of the ALK, RET, and ROS1 genes.
Authors: Zheng Li-Mou , Whyte David B. , Ruan Li , Song Roman , Fei Luo , Yang Nancy , Zhou Caicun 
Published: March 2020

e22066 Background: The ALK, RET, and ROS1 genes are involved in gene rearrangements in a fraction of non-small cell lung cancers. The resulting oncogenic fusion genes define molecular sub-types of NSCLC with distinct sensitivities to treatment with various kinase inhibitors. We developed real-time reverse transcriptase PCR assays to detect rearrangements of ALK, RET, and ROS1 in FFPE lung cancer tissue. Methods: mRNA from NSCLC FFPE tissue samples was reverse transcribed to cDNA. Multiplex quantitative PCR was performed to detect 9 variants of EML4-ALK fusions, 9 variants of RET fusions and 14 variants of ROS1 fusions. A total of 409 samples were analyzed: 267 were classified as adenocarcinoma, 104 as squamous cell carcinoma and 38 had undetermined histology. EGFR and KRAS mutation status is unknown. The junctions of fusion-positive samples were sequenced by Sanger sequencing. Results: Among the 409 NSCLC specimens tested the frequency was 5.4% (22/409) for EML4-ALK fusions, 1.5% (6/409) for RET fusions, and 2.2% (9/409) for ROS1 fusions. EML4-ALK fusions were more prevalent in patients that were less than 60 years old (9.1% versus 2.0%, p= 0.004). The TNM stage was not correlated with the presence of any of the fusions. The table below lists the frequencies for specific rearrangements as determined by sequencing the real-time PCR products. Conclusions: Real-time PCR assays based on cDNA from FFPE tissue can identify patients with ALK, RET and ROS1 fusion genes. The ALK, RET and ROS1 assays will allow selection of patients most likely to respond to therapies that specifically target these cancer drivers. Further clinical testing of NSCLC patients in the Chinese population will be performed to support SFDA registration of these assays in China. [Table: see text]
Dynamic changes of miRNA and cancer related proteins (CRPs) expression during neoadjuvant chemotherapy (NAC) for locally advanced breast cancer (LABC).
Authors: Al-Khanbashi Manal , Caramuta Stefano , Al-Ajmi Adel , Al-Hadabi Ibrahim , Kumar Shiyam , Dsouza Philomena charlotte , Zahid Khawaja Farhan , Malik Kamran Ahmed , Muhammad Furrukh , Burney Ikram A , Al Bahrani Bassim J. , Lui Weng-onn , Al-Moundhri Mansour S. 
Published: March 2020

e22099 Background: NAC is used in LABC to downsize the primary tumor and eradicate metastases. This study assessed the Dynamic changes of miRNA and CRP expression during NAC with aim to identify molecular biomarkers that may predict outcome. Methods: Tissue, blood and serum samples were collected at four time points from LABC patients undergoing NAC, including time of diagnosis (base line) after 1st and 4th cycle of Adriamycin/cyclophosphamide, and after 4th cycle of Docetaxel (end point). We used microRNA-microarray for tissue samples and Proximity Ligation Assay (PLA) for serum samples to quantify global miRNA expression and 92 CRPs in LABC patients. The miRNA and CRPS were correlated with clinico-pathological features including stage, grade and clinical response.Statistical analysis was performed using Significant Analysis of Microarray ( SAM), Student's t-test, and Pearson's correlation test. Significant p value was defined as p<0.05. Results: Tissue miRNA expression profiling (n=14) revealed 58 miRNA species that were differentially expressed between NAC endpoint and the baseline (FDR < 15%). Reduced miR-21 expression was associated with disease recurrence. Using PLA (n=33) we identified serumosteoprotegerin, MCP-1, Prolactin, Carbonic Anhydrase IX, CXCL10, CD30L and TGF-alpha levels were significantly associated with overall clinical response (t-test, p<0.05). Moreover, Prolactin and Erythropoietin showed significant associations with the stage (t-test, p<0.05), while MMP3, ErbB4, Midkine, Cathepsin D and Prostasin proteins were associated with the recurrence. Analysis is ongoing to determine the dynamic changes of these biomarkers during the NAC. Conclusions: Our study identified specific miRNAs and CRPs that were differentially expressed during NAC in LABC patients which may have clinical significance.
Utility of microsieve device for enumeration and miRNA expression profiling of CTCs for personalized cancer management.
Authors: Tucker Steven , Tan Karen Mei Ling , Leong Sai Mun , Wen Chua Hui , Jumaat Delly Fareda , Tan Doreen Chek Yee , Li Mo-Huang , Koay Evelyn S. C. 
Published: March 2020

e22107 Background: Although a rare peripheral cell population, circulating tumor cells (CTCs) represent a potential alternative to serial invasive biopsies as a source of tissue in monitoring clinical states of non-hematological malignancies. CTCs harvested from a single patient are a heterogeneous mixture of cells differing in their transcriptome expressional profiles, metastatic potential and drug resistance properties. Mere identification and enumeration of CTCs are unable to provide qualitative information about individual CTC biology. The presence of a few highly malignant, metastasis-capable CTCs and cancer stem cells among the other less aggressive CTCs in the circulating population will not be apparent from CTC counts alone. Methods: We developed a high-throughput size-exclusion microsieve device (CellSievo, Singapore) to isolate CTCs (defined as DAPI-positive, CD45-negative cells), regardless of the cell surface receptors expressed, from 74 breast cancer patients. Using CTCs eluted from the microsieve post-capture, we performed microRNA expression profiling to further characterize breast cancer CTCs. Results: Using CellSievo’s microsieve device, we obtained an average mean CTC count of 21.2 (+/- 31.8; range=0-112.5) per 7.5ml peripheral blood, for breast cancer patients, as opposed to 1.3 (+/-1.6, range 0-5.0) for healthy subjects. Viable CTCs from breast cancer patients were successfully harvested for downstream microRNA profiling. CTCs from breast cancer patients were routinely found to express microRNA profiles mediating epithelial-to-mesenchymal transition, tumor invasion, and regulation of chemotherapeutic drug resistance to tamoxifen, anthracyclines, taxanes, and trastuzumab. Serial monitoring of microRNA expression pattern of CTCs could identify emergence of novel tumor populations with distinct microRNA expression patterns during or after anticancer therapy (detailed microRNA expression data to be presented). Conclusions: Our results suggest that microRNA biomarkers derived from peripheral CTCs can yield highly useful information and offer prognostic and predictive applications for therapeutics and companion diagnostic development.
Clinical importance of circulating tumor cells in lung cancer patients.
Authors: Benedikova Andrea , Srovnal Josef , Klein Jiri , Skalicky Pavel , Szkorupa Marek , Cwiertka Karel , Radova Lenka , Hajduch Marian 
Published: March 2020

e22109 Background: Assessment of the circulating tumor cells (CTCs) in lung cancer patients could prevent burdensome surgery in understaged cases. Therefore we have tested the hypothesis that the presence of CTCs is a negative prognostic factor in these patients and correlates with poor survival. Methods: This was a prospective study to test the presence of CTCs in peripheral blood, pulmonary blood and bone marrow in 108 lung cancer patients (75 males, 33 females; mean age 66.2 years, ranging from 29 to 82 years) at the time of surgery using real-time RT-PCR for carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), lung-specific X protein (LUNX) and hepatocyte growth factor receptor (c-met). Results: We found a statistically significant association between c-met expression level in the pulmonary blood and clinical stage (p<0.044) and lymph node involvement (p<0.0023). C-met marker also showed significantly higher positivity ratio in the peripheral (p<0.034) and pulmonary (p<0.04) blood in patients with histologically proven lymphatic metastases than in patients with negative lymph nodes. The positivity of CTCs in peripheral blood or bone marrow using c-met was independent of clinical stage, age, sex and grading using Cox regression analysis (p=0.006, resp. 0.001). 82 patients followed up for more than 1 year were involved into the overall survival (OS) analysis. 27 of them (32.9 %) died, the OS median was 19.8 months. The patients with the presence of CTCs in the peripheral blood or the bone marrow using c-met had significantly shorter OS and higher hazard ratio (p<0.024; HR=4.39 [95% CI: 1.58-12.22], resp. p<0.008; HR=5.08 [95% CI: 1.79-14.43]). In addition, patients with the presence of CTCs detected in bone marrow using CEA and/or c-met had significantly shorter OS (p<0.025; HR=3.08 [95% CI: 1.26-7.5]). Conclusions: Our study demonstrates that the presence of CTCs is independent negative prognostic factor in lung cancer patients. The analysis of CTCs is clinically feasible, reproducible and further studies focused on early clinical stages should demonstrate its applicability in individualization of adjuvant therapy in lung cancer patients and identification of individuals who would not benefit from extensive lung surgery.
Effect of PARP inhibitors on sensitivity of lung and breast cancer cells to trans-platinum as compared to cis-platinum.
Authors: Jankovic Radmila , Radulovic Sinisa , Arandjelovic Sandra , Filipovic Lana 
Published: March 2020

e22115 Background: Poly(ADP-ribose) polymerase inhibitors (PARP-i) represent promising strategy in treatment of cisplatin resistant lung cancer and triple-negative breast cancer. We compared PARP inhibition by 3-aminobenzamide (3-AB) or NU1025, on activity of cis- and trans-platinum on NSCLC cells (A549) and breast cancer cells (MDA-MB-361), and correlated with p53 and ERCC1 status. Methods: Cell cycle distribution was determined by FACS-flow and PI-staining, while morphological changes were followed by light microscopy. ERCC1 siRNA transfection was performed by HiperFect reagent, and mRNA was analyzed by qRT-PCR. Protein expression levels were determined by Western blot. PARP-inhibition was quantified as NAD(P)H depletion using colorimetric XTT assay. Results: Trans-platinum compound with planar ammine ligands, trans-[PtCl2(4-acetylpyridine)2] showed cytotoxic activity comparable to cis-platin and exhibited enhanced action in combination with PARP-i. 3-AB enhanced the activity of trans more efficient comparing to cis-platinum in MDA-MB-361 cells. At equitoxic concentrations corresponding to IC50 values in A549 cells, cis-platin and trans-platinum compound differently affected cell survival in combination with PARP-i. XTT assay confirmed efficient inhibition of PARP activity at 200 μM NU1025 and 2 mM 3-AB in both cell lines. ERCC1 silencing in p53 proficient A549 cells, alone or in combination with NU1025, almost completely diminished resistance to trans-[PtCl2(4-acetylpyridine)2], decreasing cell survival to 50 % following 24 h action and more than 85 % following 48 h treatment, comparing to siRNA/NU1025 treated control. By contrast, sensitivity to cisplatin was much less altered. Simultaneous alteration of ERCC1 and PARP itself did not affect cell cycle progression, but prolonged sub-G1 fraction by trans-platinum as well as G2-M fraction by cis-platin. Conclusions: PARP-inhibition enhanced tumor cell sensitivity to trans-platinum drug, stronger than cis-platinum. Knockdown of ERCC1 further increases sensitivity to platinum drugs, irrelevant to p53 status. Trans-platinum complexes should be further explored as good candidates for combining with PARP-i.
Identification of real-time PCR-negative EGFR mutations by direct sequencing test.
Authors: Wang Tao-Yeuan , Chen Chi-Kuan 
Published: March 2020

e22118 Background: High percentage of patients with non-small cell lung cancer (NSCLC) has EGFR activating mutations in Taiwan and responses to EGFR inhibitors such as Tarceva (1, 2, 3). Timely molecular diagnosis is critical and molecular testing should be performed before treatment for the individual patient. It is important to develop an effective molecular diagnostic method for accurately detecting mutations and preventing false positive results. Therefore, we used a universal genetic detecting method (FemtoPath) to increase sensitivity of gene mutation detection and improve the sensitivity limitation (0.1%). Our previous reports indicated that FemtoPath/direct sequencing test is more sensitive, accurate and inexpensive than Cobas KRAS real- time PCR mutation test. Methods: In this study, we compared two molecular testing methods for the detection of EGFR mutation; the direct sequencing and the real-time PCR. Results: FemtoPath EGFR direct sequencing test identified mutations in 57 of the 73 tumors (77.03%) and Cobas EGFR mutation test identifies mutations in 53 of the 73 tumors (72.97%). Six negative results obtained by Real-Time PCR test were re-confirmed to be positive by direct sequencing test. From the results above, FemtoPath/direct sequencing test not only accurately detected all of the mutations, but also identified mutations adjacent toexons 18, 19, 20 and 21 of the EGFR gene. In 52 consensus cases of EGFR mutation obtained from both methods, there were 10 major discrepancies including two S768, four Exon 19 deletion, one L858, five T790, and one exon 20 insertion in this experiment. Conclusions: FemtoPath/direct sequencing test has lower invalid rate than Real-Time PCR test while fewer sample amount was required with increased detection efficiency of unknown mutation site.
The role of apoptosis in colorectal carcinogenesis and its prognostic value.
Authors: Alcaide Julia , Funez Rafael , Rueda Antonio , Pérez-Ruiz Elisabeth , Pereda Teresa , Rodrigo Isabel , Perez Diego , Villatoro Rosa , Nogales Esteban , Redondo Maximino 
Published: March 2020

e22131 Background: Most colorectal cancers (CRCs) progress through a multistep process from normal tissue to adenoma to carcinoma. The role of apoptosis during this tumourigenesis sequence has not yet been completely clarified. The aim of our study was to determine the influence of apoptotic variations on the carcinogenesis and prognosis of CRC. Methods: A TUNEL assay was performed on the formalin-fixed, paraffin-embedded tissues from 103 CRCs resected at the Costa del Sol Hospital (after excluding patients previously treated with chemotherapy or radiation therapy), 26 adenomas and 20 samples of normal epithelia. Clinical data were obtained from the tumour registry and hospital charts. The study was approved by Research Ethics Committee. We used the median (m) of apoptotic percentage (1%, range: 0-6%) as a threshold value for survival (sv) studies. Analysis of association between apoptosis and prognostic factors used the X2test for categorical variables and the analysis of variance test for continuous variables (natural log transformed if necessary). We estimated sv using the Kaplan-Meier method, and sv curves were compared with the log rank test. Cox’s proportional hazards sv analysis was used to determine the relative risk (RR) through multivariate analyses. Results: Median follow-up was 50 months (mo). Apoptotic index (AI) was higher in CRCs (1.09 ± 0.13) than in adenomas (0.38 ± 0.23, p=0.059) and significantly greater than in normal epithelium (0.06 ± 0.04, p=0.001). AI was greater in stage IV than in other stages (p=0.017). Higher AI was associated with lower disease-free survival (DFS) and overall survival (OS) than lower AI (Table). In multivariate analysis, AI (RR=2.18, 95% CI 1.08-4.37) and TNM stage (RR=2.41, 95% CI 1.20-4.85) were independent prognostic factors for OS. Conclusions: Our study demonstrates an increase in apoptosis during colorectal carcinogenesis and a correlation between apoptotic rates and survival. Higher AIs are associated with more aggressive tumours and a poorer prognosis in CRC. [Table: see text]