bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational analysis of circulating tumor cells in breast cancer patients by targeted clonal sequencing.
Authors: Xu Xunhai , Shen Yufeng , Dewitt William , Pandya Deep , Bischoff Farideh Z. , Crew Katherine D. , Hershman Dawn L. , Maurer Matthew A. , Parsons Ramon , Kalinsky Kevin 
Published: March 2020

10516 Background: The molecular signature of circulating tumor cells (CTCs) may serve as a surrogate marker for accurate description of the metastatic tumor of interest, especially in the setting of treatment response and selection. We present a method for mutational analysis of CTCs in metastatic breast cancer (MBC) patients by using an emulsion-formulated, semiconductor-based, targeted clonal sequencing platform. Methods: CTCs in MBC patients were enriched by a microfluidic OncoCEE device using antibodies against both epithelial and mesenchymal markers. Genomic DNA was extracted from enriched CTC samples. Emulsion-based multiplex-PCR targeted for various cancer genes was performed, after which semiconductor-based deep sequencing was completed. The read error rates were analyzed based on quality score and context of sequence including homopolymers. Statistical significance for each mutational analysis was assessed using a method based on beta-binomial distribution. Results: Of the 17 patients samples obtained, we were able to enrich CTC samples in 9 of them (CTC range 1-1063, median=12). Multiplex targeted sequencing was performed on DNA from the enriched CTC patient samples (purity range 0.3-6%). Greater than 3000-fold coverage was accomplished. Missense mutations at E545D on PIK3CA (p= 2.0e-07), F354L on STK11 (p=2.0e-04), and Q61R on NRAS (p=2.0e-07) were detected. Novel mutations of L540F and Q1033K within the hot spot regions of PIK3CA were observed (p=1.3e-04). Genomic DNA from WBCs from a healthy female was analyzed concurrently as a negative control, in which none of the mutations were observed. Conclusions: Mutational analysis of CTCs in MBC patients can be accomplished by deep sequencing. We developed a de novo protocol for clonal mutation analysis on CTCs in MBC and detected various significant and novel mutations. We anticipate reporting sequencing results on CTCs and matched WBCs, as negative controls, from 40 MBC patients. This will provide the foundation for the future studies in which we will compare the mutational profile between CTCs and primary/metastatic tumors. We intend to validate clonal mutational analysis of CTCs as a predictive blood-based biomarker in subsequent trials.
Intraoperative detection of lymph node metastasis using one-step nucleic acid amplification (OSNA) in breast cancer patients: Effect on second surgery rate and delay for adjuvant therapy.
Authors: Klingler Sophie , Marchal Frederic , Rauch Philippe , Kenouchi Ouarda , Chrétien Anne-Sophie , Genin Pascal , Leroux Agnès , Merlin Jean-Louis 
Published: March 2020

10517 Background: Sentinel lymph node (SLN) analysis is conventionally analysed using HES and CK19 immunohistochemistry. In case of SLN involvement, a second surgery is required for axillary lymph node (ALN) resection thus delaying the initiation of adjuvant therapies. Methods: 381 pts with invasive breast cancer were considered in this retrospective study. SLN were detected using combined radio-isotope and color detection. SLN involvement was analysed using OSNA for CK19 mRNA, in 100 pts (group 1) and compared to conventional histopathology in 281 pts (reference population, group 2). In all cases, control cytology was performed. Results: No significant difference was found between group 1 and 2 regarding patients characteristics, tumor localization, size, grade, steroid receptors and HER2 expression and the mean number of SLN analysed per pt, 2.4 (range 1-7) and 2.5 (range 1-8), respectively. Considering positive SLN as “++” (CK19 mRNA copy number>5000), “+” (250 < CK19 mRNA copy number < 5000) and positive by inhibition in group 1 and macro-, micrometastases and isolated tumor cells in group 2, no difference in lymph node involvement rate was found between the two groups with 29.0 and 29.9% of positive SLN, respectively. Only one discordance case was observed with negative OSNA analysis and positive cytology with isolated tumor cells. Using OSNA intraoperatively, the mean time to process SLN was 42 min (range 10-104) allowing immediate ALN resection. Significant (P<.01) reduction of re-intervention rate was observed when OSNA was used (9 vs 39%), including margins insufficiency (5 vs 13%), ALN resection (3 vs 15%) or both (1 vs 11%). Delay before adjuvant therapy was also significantly (P <.01) reduced when OSNA was used with 43 (range 20-82) vs 59 (range 14-212) days. Conclusions: Results achieved with OSNA are fully consistent with those achieved using conventional immunohistochemistry analysis. SLN analysis using OSNA avoids a second operation in most patients for ALN resection and shortens the delay for adjuvant therapy.
Reproducibility, performance, and clinical utility of a genetic risk prediction model for prostate cancer in Japanese patients.
Authors: Akamatsu Shusuke , Takata Ryo , Takahashi Atsushi , Inoue Takahiro , Kubo Michiaki , Kamatani Naoyuki , Inazawa Johji , Fujioka Tomoaki , Habuchi Tomonori , Ogawa Osamu , Nakamura Yusuke , Nakagawa Hidewaki 
Published: March 2020

10520 Background: Prostate specific antigen (PSA) is widely used as a diagnostic biomarker for prostate cancer (PC). However, due to its low predictive performance, many patients without PC suffer from the harms of unnecessary prostate needle biopsies. The present study aims to evaluate the reproducibility and performance of a genetic risk prediction model and estimate its utility as a diagnostic biomarker in a clinical scenario. Methods: We created a logistic regression model incorporating 16 SNPs that were significantly associated with PC in a genome-wide association study of the Japanese. The model was validated by two independent sets of samples comprising 3,294 cases and 6,281 controls. Various cut offs were evaluated to be used in a clinical scenario. Results: The area under a curve (AUC) of the model was 0.679, 0.655, and 0.661 for the samples used to create the model and those used for validation respectively. The AUC of the model was not significantly altered in samples with PSA 1-10 ng/ml. 24.2% and 9.7% of the patients had odds ratio <0.5 (low risk) or >2 (high risk) in the model, and assuming the overall positive rate of prostate needle biopsies to be 20% in PSA gray zone (PSA 2-10 ng/ml), the positive biopsy rates were 10.7% and 42.4% respectively for the two genetic risk groups. Conclusions: The genetic risk prediction model was highly reproducible, and its predictive performance was not influenced by PSA. The model could have a potential to affect clinical decision when it is applied to patients with gray-zone PSA, which should be confirmed in future clinical studies.
Frequency and clinical characterization of NSCLC patients harboring PIK3CA mutations identified within a regional screening network.
Authors: Gardizi Masyar , Scheffler Matthias , Heukamp Lukas Carl , Bos Marc Christiaan Allardt , Albus Kerstin , Hayn Birgit , Ko Yon-Dschun , Schlesinger Andreas , Brockmann Michael , Serke Monika Heidi , Gerigk Ulrich , Hekmat Khosro , Stoelben Erich , Reiser Marcel , Schnell Roland , Schmitz Stephan H. , Frank Konrad , Buettner Reinhard , Wolf Jurgen , Zander Thomas 
Published: March 2020

10526 Background: PIK3CA mutations are a rare oncogenic event of potential therapeutic relevance in NSCLC. Here we report frequency and characteristics of patients with PIK3CA mutated lung tumors. Methods: Patients with NSCLC and PIK3CA mutations were identified within our regional Network for Molecular Screening in Lung Cancer. We further analyzed the presence of BRAF, KRAS, EGFR mutations as well as ALK translocation, ERBB2 and FGFR1 amplifications in PIK3CA mutated samples. Clinical data on age, sex, TNM classification and tumor stage, histological type, grading, overall survival, smoking status, comorbidity, BMI and secondary malignancies were retrieved from clinical charts in accordance with the local ethics committee. Results: PIK3CA mutations were detected with a frequency of 3.7% (24% exon 20,76% exon 9) in 1000 patients. Histologically 32% were defined as squamous cell carcinoma, 48% as adenocarcinoma and 18% other histological subtypes or NSCLC-NOS. Exon 9 mutations were present in the acinar and lepidic subtype, whereas exon 20 mutations were seen in the papillary and solid subtype. Cooccuring genetic lesions were observed in 16% (mutations in KRAS=2, EGFR=1, BRAF=1; FGFR1 amplification=2). 14 were female, 23 male with a mean age of 69 years. 21 of these patients were further clinically annotated. 11 patients presented with stage IIIb/IV eligible for palliative treatment and 10 stage I – IIIa eligible for surgical therapy +/- adjuvant therapy. All but 1 patient were smokers with an average BMI of 26,2kg/m2 with a typical high load of comorbidity mainly of cardiovascular diseases, 8 of 21 patients showed prior malignancies in their medical history. The median overall survival within this population has not been reached yet. Conclusions: Screening for PIK3CA mutations is feasible. A high proportion (38%) of patients with PIK3CA mutated lung cancer have prior malignancies and show a high load of comorbidity. Furthermore PIK3CA mutations are not exclusive to KRAS, EGFR or BRAF mutations or FGFR1 amplifications. Successful identification of patients with oncogenic lesions in lung cancer in a screening network might allow future personalized treatment of these patients.
Circulating melanoma cells isolated from clinical blood samples and characterized by full-length mRNA sequencing at single-cell level.
Authors: Wang Yu-Chieh , Ramskold Daniel , Luo Shujun , Li Robin , Deng Qiaolin , Daniels Gregory A. , Khrebtukova Irina , Loring Jeanne F. , Laurent Louise C. , Schroth Gary P. , Sandberg Rickard 
Published: March 2020

10539 Background: Melanoma is the most aggressive type of skin cancer. Late-stage melanoma is highly metastatic and currently lacks effective treatment. This discouraging clinical observation highlights the need for a better understanding of the molecular mechanisms underlying melanoma initiation and progression and for developing new therapeutic approaches based on novel targets. Although genome-wide transcriptome analyses have been frequently used to study molecular alterations in clinical samples, it has been technically challenging to obtain the transcriptomic profiles at single-cell level. Methods: Using antibody-mediated magnetic activated cell separation (MACS), we isolated and individualized putative circulating melanoma cells (CMCs) from the blood samples of the melanoma patients at advance stages. The transcriptomic analysis based on a novel and robust mRNA-Seq protocol (Smart-Seq) was established and applied to the putative CMCs for single-cell profiling. Results: We have discovered distinct gene expression patterns, including new putative markers for CMCs. Meanwhile, the gene expression profiles derived of the CMC candidates isolated from the patient’s blood samples are closely-related to the expression profiles of other cells originated from human melanocytes, including normal melanocytes in primary culture and melanoma cell lines. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which provides advantage for better analyzing transcript isoforms and SNPs. Conclusions: Our results suggest that the techniques developed in this research for cell isolation and transcriptomic analyses can potentially be used for addressing many biological and clinical questions requiring genomewide transcriptome profiling in rare cells.
HER2 expression on circulating tumor cells (CTC) in patients with early HER2-positive breast cancer: Results of the German SUCCESS B trial.
Authors: Jaeger Bernadette Anna Sophia , Rack Brigitte Kathrin , Neugebauer Julia Katharina , Andergassen Ulrich , Melcher Carola Anna , Mouarrawy Doraid , Ziemendorff Gabriele , Clemens Michael , von Abel Ekkehard , Heinrich Georg , Schueller Katharina , Schneeweiss Andreas , Lichtenegger Werner , Beckmann Matthias W. , Pantel Klaus , Sommer Harald Leo , Friese Klaus , Janni Wolfgang 
Published: March 2020

10540 Background: Recent reports showed a discrepancy in the Her2-status of metastases or minimal residual disease in blood and bone marrow compared to the primary tumor in patients with breast cancer. The Her2-status of CTC was prospectively evaluated in the German multicenter SUCCESS B study. Methods: The SUCCESS B trial is a randomized Phase III study comparing FEC-Docetaxel (Doc) vs. FEC-Docetaxel-Gemcitabine (Doc-G) as well as Her2 targeted therapy with Trastuzumab as adjuvant treatment in patients with early, Her2 positive, node positive or high risk node negative primary breast cancer. As part of the translational research program, 23ml of peripheral blood were drawn before adjuvant chemotherapy. In 638 samples CTC and Her2-status were assessed using the CellSearch System (Veridex, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labeled with anti-CK8/18/19, anti-CD45 antibodies as well as a fluorescein conjugate antibody for Her2 phenotyping. Cutoff for CTC-positivity was ≥1 CTC and for Her2 ≥1 CTC with strong Her2-staining (+++). Results: 40.2% of pts (n=257) were positive for CTC (mean 4.52; range 0-1689). The number of detected CTC was distributed as follows: 1 CTC (n=112; 43.6%), 2 CTC (n=65; 25.3%), 3 CTC (n=36; 14.0%), 4 CTC (n=12; 4.7%) and ≥5 CTC (n=31; 12.1%). Her2 status on CTC was negative or weak in 12.5% (n=32) and 8.9% (n=23) of CTC positive patients respectively and therefore categorized as Her2 negative. In 21.4% (n=55) we detected moderate and in 57.2% (n=147) strong Her2-staining of ≥1 CTC per sample. Therefore 57.2% of CTC-positive samples were diagnosed as Her2 positive and 21.4% as questionable. No association was found between the detection of CTC or the Her2-status on CTC with tumor size, histopathological grading, hormone receptor status or axillary lymph node involvement. Conclusions: The data of this trial confirms the frequent discordance of Her2 expression on CTC compared to the primary tumor. Survival data within the Success B trial will give further insight into the tumor biology of Her2 positive disease and the role of the Her2-status on CTC to predict treatment efficacy.
Combined targeting of LSD1 (KDM1A) and histone deacetylases exerts superior efficacy against human AML.
Authors: Fiskus Warren , Sharma Sunil , Rao Rekha , Balusu Ramesh , Venkannagari Sreedhar , Ganguly Siddhartha , Bearss David , Bhalla Kapil N. 
Published: March 2020

10549 Background: LSD1 (KDM1A) is FAD-dependent histone H3K4Me2 demethylase. Inhibition of LSD1 increases H3K4Me3-a permissive mark for gene expression, and inhibits growth of pluripotent cancer cells. We have previously noted that treatment with the histone deacetylase (HDAC) inhibitor panobinostat (PS) depletes EZH2 (the catalytic subunit of the polycomb repressive complex 2, PRC2) and disrupts its interaction with the other PRC2 proteins, attenuates LSD1, and de-represses growth inhibitory and pro-apoptosis genes. Methods: In the present studies, we determined the chromatin effects and the pre-clinical in vitro and in vivo anti-AML activity of the novel, non-monoamine oxidase, reversible inhibitor of LSD1, HCI2509 (100 to 500 nM), alone and in combination with PS, utilizing cultured (OCI-AML3 and HL-60) and primary human AML blast progenitor cells. Results: Treatment with HCI2509 dose-dependently increased the levels of H3K4Me3, p16, p27, and CEBPα in cultured AML cells. This correlated with inhibition of cell growth and induction of morphologic differentiation in AML cells. Exposure to HCI2509 disrupted the binding of LSD1 with the co-repressor CoREST and HDAC1. Treatment with PS (10 to 50 nM) dose-dependently depleted not only LSD1 but also EZH2, SUZ12 and BMI1 in AML cells. Co-treatment with PS enhanced the chromatin modifying effects of HCI2509. The combination also synergistically induced loss of viability of cultured and primary AML cells (combination indices, CI <1.0). Following tail vein infusion and establishment of AML by OCI-AML3 cells in NOD-SCID mice, treatment with HCI2509 (25 mg/kg, b.i.w, IP, for 3 weeks) improved survival, as compared to the control mice (p <0.001). HCI2509 treatment (15 mg/kg IP) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts expressing FLT3-ITD. Survival was further improved upon co-treatment with HCI2509 and PS (5 mg/kg IP, MWF). Mice did not experience any toxicity or weight loss. Conclusions: Combined epigenetic therapy with HCI2509 and PS exerts superior pre-clinical activity, warranting further in vivo development and testing of HCI2509 against human AML.
Prognostic role of microRNA polymorphisms in advanced gastric cancer patients.
Authors: Stenholm Linnea , Stoehlmacher-Williams Jan , Al-Batran Salah-Eddin , Heussen Nicole , Akin Sema , Pauligk Claudia , Lehmann Sylvia , Senff Tina , Ehninger Gerhard , Brummendorf Tim H. , Goekkurt Eray 
Published: March 2020

10552 Background: As little is known about the impact of microRNA (mir) polymorphisms on prognosis of advanced gastric cancer (AGC) we sought to determine their impact on overall survival (OS) in these patients (pts). Methods: Pts were treated with either 5-fluorouracil, leucovorin and oxaliplatin (FLO) or with additional docetaxel (FLOT) in frame of a phase III or 3 phase II trials of the Arbeitsgemeinschaft Internistische Onkologie. All pts consented genetic analyses. DNA was extracted from whole blood samples (n=515) and polymorphisms of mir26a1 (rs7372209), mir27a (rs895819), mir100 (rs1834306), mir125a (rs12975333), mir146a (rs2910164), mir196a2 (rs11614913), mir219-1 (rs107822) and mir423 (rs6505162) were genotyped using PCR-based methods. Hetero- and homozygous variant genotypes were grouped versus homozygous wild type genotypes. Selection of clinical and genetic parameters for the final multivariate model was based on univariate and multivariate Cox-regression analyses with a cut-off value of p < 0.25 and included 498 pts. Results: Median age was 67 (range 23 to 86) years. 42% were treated with FLO and 58% with FLOT with a median OS of 14 months. Multivariate analyses revealed following associations: Genetic factors significantly associated with OS were mir26a1 variant genotypes (HR 1.3 [95%CI (1.01;1.6), p=.025]), mir27a variant genotypes (HR 1.3 [95%CI (1.01;1.6), p=.036]) and mir196a2 variant genotypes (HR 0.8 [95%CI (0.6;0.99), p=.046]). Clinical factors with significant impact on OS were ECOG 2 performance status (HR 1.9 [95%CI (1.3;2.8), p=.002]), curative surgery of advanced disease (HR 0.3 [95%CI (0.1;0.5), p<.001]) and locally advanced disease (HR 0.5 [95%CI (0.3;0.7), p<.001]). Combined analyses of the identified adverse genotypes of mir26a1, mir27a and mir196a2 resulted in a median OS of 9, 13, 14 and 17 months for pts with 0 (n=72), 1 (n=193), 2 (n=171) and 3 (n=54) adverse genotypes (p=.029), respectively. Conclusions: In addition to established clinical factors, mir26a1, mir27a and mir196a2 polymorphisms were significantly associated with OS. Our data suggest a strong impact of these mir polymorphisms on prognosis in AGC and might be of help for a better guidance of treatment decisions.
Activation of and dependence on ataxia telangiectasia mutated kinase in PTEN-deficient tumor cells.
Authors: McCabe Nuala , Walker Steven M. , Goffard Nicolas , Wikstrom Katarina , Greenan Caroline , McLean Estelle , McDyer Fionnuala A , Keating Karen E , James Iain , Harrison Timothy , Mullan Paul B , Harkin D. Paul , Chen Clark , Kennedy Richard D. 
Published: March 2020

10553 Background: Loss of PTEN function has been widely reported to cause up-regulation of the PI3K/AKT signalling pathway resulting in increased cell growth, proliferation and survival. More recently it has been reported that PTEN null cells demonstrate genomic instability and have increased production of reactive oxygen species (ROS) and oxidative stress induced DNA damage. Ataxia Telangiectasia Mutated (ATM) is the primary response kinase, which responds to stalled DNA replication and DNA double strand breaks due to oxidative DNA damage. Methods: A metagene representing ATM activation was generated from cell line data and used to perform hierarchical clustering analysis of public DNA microarray profiling datasets of breast cancer, ovarian cancer and glioblastoma with known PTEN IHC/mutation status. Furthermore, we ask if ATM activation may be therapeutically exploited in PTEN null tumours using ATM specific siRNA and compounds in 2 PTEN isogenic cell line model systems. Results: We show that PTEN null cells have elevated levels of ROS, DNA damage and have endogenous activation of ATM, an enzyme important in responding to DNA damage resulting from oxidative stress. We hypothesised that PTEN deficient tumours may rely on ATM enzyme for survival. To investigate this we generated a 189-gene list representing ATM activation and used this to perform hierarchical clustering analysis of a breast cancer DNA microarray dataset. This list was able to significantly cluster tumours with known loss of PTEN expression (p=0.004). Furthermore, this gene list was able to segregate PTEN null/mutant tumours from PTEN wild-type tumours in 2 independent datasets of glioblastoma and ovarian cancer (p=0.015 and p=0.012). In addition, we found that inhibition of ATM using the selective inhibitor KU-55933 caused DNA damage, cell cycle arrest and apoptosis specifically in PTEN deficient cells when compared to PTEN wild-type cells. Conclusions: These observations suggest that ATM may represent a therapeutic target in PTEN deficient tumours and furthermore ATM activation may be the basis of a biomarker of PTEN status in human cancers.
ALK amplification and crizotinib sensitivity in non-small cell lung cancer cell lines and patients report.
Authors: Khadija Kalai , Auger Nathalie , Lueza Beranger , Commo Frederic , Valent Alexander , Rousseau Vanessa , Dorvault Nicolas , Friboulet Luc , Olaussen Ken , Rajpar Laetitia , Planchard David , Lacroix Ludovic , Pignon Jean-Pierre , Soria Jean-Charles , Besse Benjamin 
Published: March 2020

10556 Background: ALK high copy number (HCN) seems to be a frequent event, described in 13-17% of Non-small cell lung cancer (NSCLC). The goal of this study was to describe ALK genomic aberrations on NSCLC patients and cell lines, to explore the ALK HCN response to crizotinib through in vitro assays and to report three patients case. Methods: 191 Paraffin embedded specimens from advanced NSCLC patients and 27 NSCLC cancer cell lines were screened for ALK copy number by fluorescent in situ hybridization (FISH). Crizotinib sensitivity was evaluated in 9 cell lines through WST1 assays and clonogenic tests. Three patients exhibiting ALK HCN were assessed for response to crizotinib. Results: EML4-ALK translocation was present in 22 pts (11.5%). 21 pts (11%) exhibited over 6 copies of ALK. 6 (22%) cell lines displayed more than 5 copies of ALK, 19 (70%) presented a gain of 3 or 4 ALK copy number, only one cell line exhibited normal ALK copies and one harbored EML4-ALK translocation. FISH with CEP2 revealed a polysomy of chromosome 2 in cases with ALK HCN.Out of the 9 cell lines tested, 4 ALK HCN cell lines (H661, A427, BEN, H1299) exhibited increased sensitivity for crizotinib vs. 3 low ALK copy number (LCN) cell lines (H1975, H1651, H1650) with a low sensitivity. Median IC50 with crizotinib values was1750 nM [300-2800nM] in ALK HCN cell lines vs 4500 nM [800-8000nM] in ALK LCN cell lines, p=0.35. 3 patients with ALK HCN tumor received crizotinib ( in 4th , 5th and 6th -line therapy) for 2, 3 and 5 months with stable disease as best response and clinical benefit in 2 pts. Conclusions: ALK HCN may predict sensitivity to crizotinib. A clinical study is planned in ALK HCN pts.