bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of BEAMing assays and competitive approaches in the detection of main alteration of RAS in circulating DNA of non small-cell lung cancer (NSCLC) and metastatic colon cancer. Manuscript 2016.
Authors: Garcia Jessica , Forestier Julien , Dusserre Eric , Rodriguez-Lafrasse Claire , Cheynet Valérie , Wozny Anne-Sophie , Ferraro Peyret Carole , Brengel-Pesce Karen , Guillet Marielle , Chauvenet Marion , Brevet Marie , Couraud Sebastien , Walter Thomas , Payen Lea Francoise
Published: September 2018

e23056

Background: A number of RAS mutations confer resistance to anti-EGFR therapies routinely used in the treatment of colon cancer. The objective of this study was to evaluate the pertinence of analyzing circulating-free plasma DNA (cfDNA) as an indicator of the mutational status of a tumor, in order to use liquid biopsies instead of invasive and painful tumor biopsies during tumor progression. Methods: A cohort of 24 lung and 25 colon cancer patients was constituted in the Hospices Civils of Lyon. Liquid biopsy plasma samples were collected at diagnosis (colon cancer) and during tumor progression (lung cancer) for the purpose of the current study. KRAS and NRAS somatic alterations were quantified using three different technologies: the Droplet Digital polymerase chain reaction (ddPCR) from BioRad, the BEAMing Digital PCR from Sysmex Inostics, and the NGS NextSeq 500 by Illumina with the Accel-Amplicon 56G Oncology Panel from SWIFT BIOSCIENCES. Results: We observed a high level of sensitivity and specificity with the BEAMing technology, which provided us with excellent matches, around 96% and 73%, between solid and liquid biopsies taken at diagnosis (colon cancer) or during tumor progression (lung cancer), respectively. Indeed, when examining cfDNA from patients displaying one of the KRAS or NRAS mutations, 11 of the 13 mutations were confirmed using this technology, whereas only 5-6 matched the initial NGS status, using the two other technologies. The detection threshold was estimated at 1% for samples containing at least 0.8 ngctDNA/µL for the multiplex screening ddPCR from BioRad and for the 56G Oncology Panel from SWIFT BIOSCIENCES. The threshold was lower, at 0.03%, in samples containing only 0.25 ng ctDNA/µL for the BEAMing technology, which includes a PCR pre-amplification step. Conclusions: The advantage of the Illumina NGS technology is the larger coverage of longer gene regions, and thus the detection of more genetic mutations. Finally, the BEAMing technology enabled us to follow the appearance and disappearance of somatic alterations, with a very high level of sensitivity.
Metabolomics signature in serum months to years before thoracic cancer: The HUNT study.
Authors: RÃ¸e Oluf D. , Lagani Vincenzo , Kvitvang Hans Fredrik , Markaki Maria , Tsamardinos Ioannis , Mjelle Robin
Published: September 2018

e23059

Background: The Cancer-Biomarkers in HUNTinitiative seeks to identify novel biomarkers for the early cancer diagnosis. For lung cancers and mesothelioma clinically useful early markers are not available. In the prospective HUNT study in Norway, pre-diagnostic samples ranging 0-20 years before diagnosis are available for research purposes. Here we present our first results on high-throughput metabolomics analysis in serum two months to 16 years before diagnosis. Methods: LC-MS untargeted (Amide-) metabolites (n = 1042) were profiled in serum samples from 48 future patients (12 each of adeno-, squamous cell carcinoma, small-cell lung cancer and mesothelioma) and from 48 controls that were cancer-free 5 years after blood sampling. All were active smokers. Metabolic features for (a) each cancer and (b) all cancers pooled together were analyzed with moderated t-test (R limma package). Multivariate analyses included (a) OPLS-DA and (b) signature identification through a data-analysis pipeline that includes feature selection (such as the algorithm in [1]), non-linear modelers (e.g., Random Forests) and Cross-Validation with bootstrapping [2] for optimizing algorithms and providing unbiased performance estimation. The pipeline is implemented in the Just Add Data software (Gnosis Data Analysis). Results: Univariate and OPLS-DA analyses did not identify any association between metabolites and cancer. The non-linear data analysis pipeline identified a signature containing five metabolites able to discriminate between cancer and non-cancer patients, statistically significantly better than random (AUC = 0.667, CI = [0.536, 0.784]). Conclusions: Our results indicate that metabolic profiling in serum may help in identifying subjects who are likely to be diagnosed with lung cancer/mesothelioma in a time period of several years before diagnosis. More data will be presented at the annual meeting. Further validation studies are planned for confirming the replicability of these findings. 1) Lagani V et al., 2016. arXiv:1611.03227 2) Greasidou L, 2017. Bias Correction of the Cross-Validation Performance Estimate and Speed Up of its Execution Time, MSc Thesis, University of Crete
Detecting ultra low-frequency variants and gene fusions in lung cancer patients using an amplicon-based Firefly NGS method.
Authors: Weng Li , Wang Lin , Chen Xiao , Zhang Jacey , Lin Chiahui , Wang Yingyu , Brice Tim , Huang Yi , Wang Hongyan , Tang Paul , Sun Zhaohui Johnny , Zhao Grace Q. , Lou Jiatao , Ying Kang , Faham Malek , Lin Shengrong
Published: September 2018

e23062

Background: The analysis of EGFR, KRAS, and BRAF mutations and Alk fusion is critical for guiding targeted therapy selection, detecting drug resistance, and monitoring residual disease in patients with NSCLC. Designing next-generation sequencing (NGS) assays for detecting low-frequency variants, however, is an ongoing challenge. The limited availability of cfDNA combined with the breadth of coverage necessary to create meaningful, clinically-actionable results requires a solution with multiplex capacity which, in turn, requires greater technological sensitivity and specificity. Here we aim to develop such a solution: an ultra-accurate NGS technology using concatmer-based error correction with amplicon workflow for fast detection of rare mutations including SNV and fusion. Methods: We developed an amplicon-based panel covering variants of EGFR, BRAF, and KRAS, as well as a panel to detect Alk fusion. CfDNA simulate and cfDNA from healthy individuals were used to test assay sensitivity and specificity. Further validation was performed via a comparative analysis of 64 late-stage lung cancer patients using both Firefly -Comet and ddPCR. Results: Analytical sensitivity of the EGFR-TKI 3-gene panel was 100% detection at an allele frequency of 0.1% for 20ng of cfDNA input. Similarly, analytical sensitivity of the Alk fusion panel was 75% detection at an allele frequency of 0.1% and 100% at an allele frequency of 0.25% for the same input. Among our patient cohort, 5 EGFR variants (19del, T790M, L858R, G719X, L861X) and 2 KRAS variant (G12X) were detected. Firefly-Comet demonstrated strong per-variant detection-rate concordance ( > 99%) compared to ddPCR results. The PPV is 100% and the NPV is 98.7%. Statistical analysis of reported allele frequency concordance between Firefly-Comet and ddPCR reveals R-Sq > 0.9. Conclusions: In summary, we have developed Firefly-Comet, an easy-to-use amplicon-based NGS assay capable of detecting single-digit copies of somatic mutants and gene fusions in cfDNA. The multiplex capacity of Firefly-Comet makes it well-suited for supporting targeted therapy selection, drug resistance detection, and treatment monitoring.
Identification of epitopes of cancer testis antigen cyclin A1 for targeted T-cell therapy of acute myeloid leukemia (AML) and solid tumors.
Authors: Teck Anja-Tatjana , Urban Sabrina , Quass Petra , Nelde Annika , Walz Juliane , Keilholz Ulrich , Ochsenreither Sebastian
Published: September 2018

e23080

Background: Cyclin A1 is a promising antigen for T-cell therapy being selectively expressed in ovarian cancer and AML stem cells. For adoptive T-cell therapy, a single epitope has to be selected, which should bind with high affinity to MHC class I and should be adequately processed and presented by malignant cells to trigger full activation of specific T-cells. Methods: Three algorithms were used to predict in silicopeptides of Cyclin A1 with high affinity to HLA A*02:01. Affinities were tested by HLA stabilization assay using TAP-deficient T2 cells. Immunogenicity of peptides was assessed by stimulation of T-cells with peptide pulsed dendritic cells and monocytes. Specificity of T-cells was assessed by intracellular cytokine staining. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA A*02:01-positive AML line THP-1. Data from immunopeptide analysis based on direct isolation of HLA peptides by affinity chromatography and mass spectometry of 20 primary AML samples were analyzed retrospectively to identify naturally presented epitopes of Cyclin A1. Results: Thirteen peptides nine to eleven amino acids of length were predicted. Ten showed stabilization of HLA A*02:01 on T2 cells. By repetitive stimulation of T-cells of two healthy donors with single peptides, 40 T-cell lines were generated, of which eight were specific for the respective peptides. Five peptides were immunogenic. Against two peptides (Cyclin A1 isoform c, p156-164, p410-420) T-cell lines could be generated from both donors. For T-cell clones against two peptides (p111-120, p289-297), specific lysis of THP-1 was shown indicating adequate processing. Peptide p410-420 was found by mass spectroscopy-based ligandome analysis to be presented by AML blasts in vivo. Conclusions: We identified ten peptides with adequate binding to HLA A*02:01. We describe three new HLA A*02:01–restricted epitopes as targets for T-cell therapy. Epitope p410-420 showed high affinity to HLA A*02:01 and immunogenicity in both donors. It proved to be naturally presented on AML blast, and therefore warrants further development for clinical application.
Human papillomavirus (HPV) induced malignancies and markers of immunogenicity.
Authors: Hashemi Sadraei Nooshin , Koncar Robert , Poorman Kelsey , Bahassi El Mustapha
Published: September 2018

e23083

Background: Approximately 5% of all cancers result from HPV, commonly leading to head and neck (HN) and anogenital (AG) cancers. Immune therapy is used in many cancers including HN. Positive PDL1 and high tumor mutation load (TML) have been proposed as potential markers of immunogenicity among many tumors, but little is known specifically among HPV driven cancers. Methods: A retrospective analysis of patients with HPV related cancers was performed based on tissue availability. TP53 wild type status was used as a surrogate for HPV positivity. Among oropharynx cases with TP53 wt, p16 testing was performed for confirmation. Next generation Sequencing (NGS Sanger) was used for tumor mutation load (TML) calculation. High TML cut off was defined as ≥17 mutations/megabase based on prior publications. PDL1 status was reported based on IHC ≥5%. A 2-tail Fisher’s exact test was utilized for statistical analysis. Results: A total of 737 patients with HN and AG (cervical, anal, vulvar, penile) squamous cell carcinoma were identified. Of these, 251 had complete 592 gene panel NGS data. HPV + TP53 Wt disease was detected in oropharynx (70%), cervical (88%), and anal (82%) cancer. HPV+ HNSCC had a trend to a higher PDL1 compared to HPV neg (50% vs 31%, p = 0.16) but there were no significant differences among PDL1 between HPV+ and neg patients across the entire combined disease cohorts (41% vs 36%, p = 0.56). TML-high patients showed a trend to increased PDL1 positivity (60% vs 38%, p = 0.09). However, high TML was less common in HPV+ HNSCC vs HPV neg (2.2 vs. % 9.7% p = 0.19). Unlike some other malignancies, high TML was uncommon across HPV+ tumors in general (8%). Conclusions: Despite fewer TML high cases among HPV + patients, there was no difference among PDL1 expression. It is possible that a lower threshold may need to be used when determining TML high vs. low in viral-driven cancers. It is also possible that in view of viral immunogenicity, certain genomic alterations contribute more to higher PDL1 expression rather than the sum of total genomic alterations. We are currently pursuing both lines of investigation. These findings are hypothesis generating for future trial design rather than treatment decision-making, and need to be confirmed in large prospective cohorts.
Comparative characteristics of the genes expression of cancer-testis antigens in tumor and non-tumor breast tissues.
Authors: Mogushkova Khava A. , Vodolazhsky Dmitriy I. , Kutilin Denis S. , Kit Oleg I.
Published: September 2018

e23085

Background: Breast cancer (BC) is the most common cancer among women. The development of immunotherapy which targets cancer-testis antigens (CTA) is needed for the decreasing in breast cancer mortality. Therefore, the aim of this work was the study of specific CTA of breast cancer tissue among CTA-family, role of which has been previously mentioned. Methods: The paired surgical biopsies of tumor and non-tumor breast tissue of 25 patients (50 samples) residing in the South of Russia and treated in Rostov Research Institute of Oncology were included in the present study. Total RNA was extracted from tissue samples by P. Chomczynski and N. Sacchi (2006) method. cDNA library was synthesized using reagent kit "Reverta-L» (Russia, "InterLabService"). Primers were designed using the NCBI GenBank database. The relative expression of 17 genetic loci ( MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-B1, MAGE-B2, GAGE1, GAGE3, GAGE4, MAGE-C1, BAGE, CTAG-1B, XAGE3, NY -ESO1, SSX2, SYCP1, PRAME1) was determined by Real-Time qPCR (reference gene - GAPDH) using CFX96 thermal cycler (Bio-Rad, USA). Statistical analysis was performed using nonparametric Mann-Whitney U-test. Results: The expression of CTA genes MAGE-A4, MAGE-B1, MAGE-B2 and GAGE3 was found to be significantly (p < 0.05) increased by 1.4; 9.5; 4.0 and 9.8 fold, respectively, in tumor tissue compared with non-tumor breast tissue. The expression of other CTA genes were not differed between tumor and non-tumor samples. Conclusions: Detected increasing of the expression levels of MAGE-A4, MAGE-B1, MAGE-B2 and GAGE3 genes evidences that these CTA may serve as the effective immunotherapeutic targets in breast cancer.
Diagnostic accuracy of molecular tumor profiling in determining the tissue of origin in adult patients with cancer of unknown primary: A meta-analysis.
Authors: Inocencio Rowena Torres , Kesari Kavitha , Smith Susan
Published: September 2018

e23092

Background: Appropriate cancer treatment requires determination of the primary site of origin. The standard of care for diagnosing Cancer of unknown primary (CUP) based on the 2016 NCCN Guidelines is a comprehensive clinical work-up including biopsy and immunohistochemistry (IHC). However, IHC was able to identify a primary tumor in only 25% of CUP. Molecular tumor profiling (MTP) validation studies demonstrated sensitivities of 74-89% and specificities of 95-99%. Only a few studies with small sample sizes using MTP to diagnose the primary in CUP have been performed yielding variable results. This study aims to evaluate the diagnostic accuracy of MTP in determining the tissue of origin in adult patients with CUP. Methods: Literature search included articles published in any language indexed in MEDLINE and Cochrane. A Google Scholar search and a review of all published articles’ references were performed. Unpublished studies and abstracts from conference proceedings were also reviewed. Studies involving adult patients with CUP who underwent both MTP and clinical evaluation with IHC were identified. Articles were included if they had primary data sufficient to calculate both sensitivity and specificity. Methodological qualities of the included studies were evaluated using the QUADAS-2 tool. Data was analyzed using Review Manager 5.3 and MetaDiSc 1.4. Results: Seven studies with 549 patients were analyzed. MTP was found to have a pooled sensitivity of 89% (95% CI 0.85-0.92)and pooled specificity of 74% (95% CI 0.67-0.80)with pooled positive and negative likelihood ratios of 2.97 (95% CI 1.17-7.63) and 0.20 (95% CI 0.07-0.51), respectively. Area under the curve was measured to be 0.9107. Conclusions: This meta-analysis suggests that MTP could be useful in determining the tissue of origin in adult patients with CUP. With an area under the curve of 0.9107, it is an excellent diagnostic test. The results showed considerable heterogeneity as expected in meta-analyses of diagnostic test accuracy. In this case, it may be due to variability among the MTP assays used which may reflect different cutoffs.
Copy number variation of MDM2 and p53 genes in extracellular DNA as potential marker for lung adenocarcinoma diagnostics.
Authors: Kutilin Denis S. , Enin Yaroslav S. , Vodolazhsky Dmitriy I. , Sustretov Vyacheslav A. , Ayrapetova Tamara G. , Anistratov Pavel A. , Pyltsin Sergey P. , Leyman Igor A. , Chubaryan Anna V. , Bogdanova Olesia A. , Kharin Leonid , Turkin Igor N. , Dyritsky Maxim N. , Kit Oleg I.
Published: September 2018

e23094

Background: Today, the problem of early diagnostics of lung cancer remains unsolved as every fourth patient diagnosed with the disease already has distant metastases. Studying gene copy number variation (CNV) in extracellular DNA in the blood plasma can become a basis for a new effective and low invasive method for predictive diagnostics and disease prognosis. The purpose of the study was to examine the copy number of the MDM2 and p53 genes in extracellular DNA of patients with metastatic and non-metastatic lung cancer and healthy donors. Methods: The blood samples of 30 patients with lung adenocarcinoma (collected before surgery) and 30 healthy donors (without cancer) were studied. Each sample was centrifuged to obtain blood plasma. DNA was isolated from plasma using a phenol-chloroform extraction method. Detection of relative copy number of the MDM2 and p53 genes (reference gene - GAPDH) was performed by RT-qPCR using CFX96 thermocycler (Bio-Rad, USA). The groups were compared by the Mann-Whitney U- test. Results: Reduction of the p53 gene copy number by 57% (p < 0.05), as well as increasing of the MDM2 gene copy number by 160% (p < 0.05), were found in the extracellular DNA of patients with lung adenocarcinoma compared with healthy donors. As a result, the ratio of copy number of pro-/anti-apoptotic genes p53:MDM2 in extracellular DNA of patients with lung adenocarcinoma (1:23 ratio) was different from that of healthy donors (1:4 ratio). The MDM2 gene copy number in extracellular DNA of patients with metastases exceeded the value in non-metastatic patients two-fold (p < 0.05). The p53 gene copy number in extracellular DNA of patients with metastatic and non-metastatic cancer did not differ significantly. Conclusions: CNV of the p53 and MDM2 genes in extracellular DNA has a high potential for low invasive diagnostics and prognosis of lung adenocarcinoma.
Integrated DNA, RNA and protein analyses for a better treatment of cancer patients.
Authors: Laes Jean-Francois , Cesbron Francois , Ghitti Gregori
Published: September 2018

e23099

Background: Advances in the molecular profiling of tumours, together with the expanding portfolio of targeted cancer therapies have established the terrain on which personalised cancer treatment can be conducted. This expanding area of precision medicine has the potential to be offered as a routine cancer-diagnostic service. Methods: We evaluated two molecular-profiling approaches : next-generation sequencing (NGS) and a group of several assays, termed Package Plus (PP), which have been primarily designed at identifying specific clinically-relevant alterations including protein expression/activation (by immunohistochemistry), and gene-promoter methylation, gene translocations and microsatellite instability (by PCR). The molecular profiling was conducted as diagnostic service for practising oncologists, who provided formalin-fixed paraffin-embedded tumour samples (for NGS and PP) and blood samples for circulating tumour DNA (for NGS only). A subset of oncologists who received the molecular profiling results and treatment advice was then surveyed to assess whether and how the results affected their treatment plans. Results: 980 samples from 16 different cancer types were received, out of which 914 (93%) were of sufficient quality to be included in this study. Clinically-relevant (actionable) alterations that provided treatment advice were identified for the large majority (841/914; 92%) of patients using the combination of NGS and PP data, but only for a minority of (247/912; 27%) of patients using NGS data alone. Treatment advice was adhered to by the oncologist in the majority (60%) of surveyed cases, and in the cases where the advice was not followed, reasons most often cited were treatment unavailability or cost. Conclusions: Our study demonstrates the utility of a precision-medicine service based on supervised tests (protein and RNA) in combination with NGS (DNA) profiling methods for advising oncologists on appropriate cancer-treatment plans.
Molecular sequencing and gene fusion detection in non-small cell lung cancer (NSCLC) patients: Impact of co-existing alterations.
Authors: Martinez de Castro Ana , Remon Jordi , Cedres Perez Susana , Navarro Alejandro , Martinez Marti Alexandre , Pardo Aranda Nuria , Hernando Alberto , Zeron-Medina Jorge , Aguilar-Company Juan , Scheenaard Eulalia , Recasens Sergi , Ruiz-Pace Fiorella , Villacampa Guillermo , Jimenez Jose , Dienstmann Rodrigo , Nuciforo Paolo , Felip Enriqueta , Vivancos Ana
Published: September 2018

e23103

Background: There is a growing need for accurate molecular diagnostics for proper targeted therapeutics selection in NSCLC patients (pts). We assess whether next generation techniques increase the identification of alterations in a prospective cohort of NSCLC pts, and evaluate prognostic value of different molecular alterations. Methods: Next generation sequencing (Amplicon-seq, 60 genes) and NanoString Gene fusion (20 genes) testing were conducted in NSCLC pts treated in a single center from November 2014 to May 2016. Overall survival (OS) according to the molecular profile was analysed by Kaplan-Meier method and Cox models. Results: Molecular tests were performed in 73advanced NSCLC pts.Median age was 54 year, 50% were women. Histology: 73% adenocarcinoma; 7% squamous, 20% others (Neuroendocrine, NOS). Tests were performed in biopsies from metastatic sites in 55% of pts.These techniques identified molecular alterations in 89% of cases: TP53 mutation (mut) 37/73 (51%); KRASmut 21/73 (29%); EGFRmut 11/73 (15%) ; CDKN2mut 7/73 (9%); BRAFmut (pG469R), ERBB2mut and METexon14mut in (1/73) 1.3%, respectively; ALKfusion (fus) 2/73 (2.7%); ROS1fus 1/73 (1.3%) , and RET fus 3/73 (4%). Concomitant mutations were identified in 32% of pts mainly with TP53 (TP53/EGFR: (5/11) 45% and TP53/KRAS: (7/21) 33%). Matched targeted treatment was offered in 17/73 (23%), mostly tyrosine kinase inhibitors (TKI); anti-EGFR in 11/17 (65%) and ALK/ROS TKI in 3/17 (17.5%). Three pts (17.5%) received anti-MET or anti-HER2 TKI. OS in this whole population treated with target therapies (n = 17) was 45.7 months (m) (CI95% 26.8-NA). The KRASmut group had median OS of 10 m (CI95% 7.5-NA), while the remaining population with wild type (WT) for all driver alterations had median OS of 23 m (CI95% 20-NA), (HR 1.71 for KRASmut vs WT pts, p = 0.18). The presence of coexisting TP53mut did not negatively impact on OS in the cohorts with EGFRmut and KRASmut (p > 0.1) in a multivariate model. Conclusions: Combined next generation techniques for molecular profile in NSCLC pts identifies molecular alterations that have clinically relevant impact in survival. Coexisting TP53mut alteration shows no detrimental effect in OS.
Correlation of low CT attenuation and necrotic features of tumor in contrast-enhanced CT with nivolumab response.
Authors: Ariyasu Ryo , Horiike Atsushi , Koyama Junji , Saiki Masafumi , Sonoda Tomoaki , Takano Natsuki , Kawashima Yosuke , Oguri Tomoyo , Nishikawa Shingo , Kitazono Satoru , Yanagitani Noriko , Ohyanagi Fumiyoshi , Oikado Katsunori , Nishio Makoto
Published: September 2018

e23105

Background: Anti-PD1/PD-L1 antibodies, including nivolumab, produce durable responses in non-small cell lung cancer (NSCLC), but the response rate is about 20% in unselected NSCLC. Identifying predictive markers of response is necessary. Although PD-L1 expression may be a potential biomarker, evaluating whole tumors based on a small sample is difficult. Identifying radiographic features of responders can be very useful. We hypothesized that low attenuation on computed tomography (CT), considered to represent necrosis, correlates with nivolumab response. Methods: We retrospectively evaluated contrast-enhanced CT images before treatment and tumor response in 60 patients receiving nivolumab. The largest tumor lesion was characterized in each patient. CT attenuation (CTA) of whole tumor areas was measured; the presence of radiological findings of necrosis (necrotic features), presenting cavitation or ring- or patchy-enhancement in tumors, were assessed. We evaluated the association between CT imaging characteristics and nivolumab response and that between CT imaging characteristics and PD-L1 expression in tumors. Results: Thirty-nine target lesions in lung, 12 in lymph node, and 9 in other organs were chosen. The mean CTA was 58.5 ± 15.3 HU. Necrotic feature were observed in 24 (40%) of 60 lesions.Twenty of 60 lesions (33%) shrank to > 30% with nivolumab treatment. Mean CTA in responding lesions was 48.6 HU, significantly lower than that in nonresponding lesions (63.4 HU) (p < 0.001). The response rate of lesions with/without necrotic features was 45% and 28%, respectively (p = 0.246).Systemic tumor response, assessed by RECIST v1.1, was confirmed in 20 (33%) patients. Mean CTA of target lesions was lower in responders than patients with stable disease or progressive disease (51.4 vs. 62.0 HU, p = 0.011). The systemic response rate of lesions with/without necrotic features was 50% and 25%, respectively (p = 0.081).Mean CTA of target lesions was lower in tumors with high PD-L1 expression than in tumors with low PD-L1 expression (54.8 vs. 64.4 HU, p = 0.045). Conclusions: Low CTA and necrotic features in CT may correlate with nivolumab response. Tumors with low CTA may have high PD-L1 expression.
MET aberrations across multiple tumor types: Results of a molecular screening program (MOSCATO 01).
Authors: El Dakdouki Yolla , Verlingue Loic , Massard Christophe , Rouleau Etienne , Lacroix Ludovic , Auger Nathalie , Scoazec Jean-Yves , Balogh Zsofia , ANGEVIN ERIC , Soria Jean-Charles , Hollebecque Antoine
Published: September 2018

e23110

Background: MET gene, encoding the tyrosine kinase receptor for HGF, has been shown to have an oncogenic role in several tumors. We report here the frequency, distribution and clinical characteristics of the patients (pts) found to have MET alterations in a molecular screening trial. Methods: InMOSCATO clinical trial, pts with locally advanced or metastatic solid cancers had an on-purpose tumor biopsy to perform a comprehensive molecular characterization. Results: Among the 844 adult pts who had a successful molecular portrait, 29 (3.4%) had MET aberrations distributed as follows: 18 had abnormalities at the genomic analysis level while 11 had exclusively expression of phosporylated-MET by immunohistochemistry (IHC) and/or a positive fluorescence in situ hybridization (FISH). Only one of the 74 pediatric pts included had a missense mutation. According to the primary tumor, MET alterations were found in 13/170 successfully biopsied pts with lung cancer, 8/197 with digestive tumors, 3/158 with genito-urinary tumors, 2/83 with gynecologic tumors, 2/111 with head and neck cancers, 1/135 with breast cancer and 1 pt with pediatric glioblastoma. Pathogenic variants (PV) were detected in 3 pts by targeted Next Generation Sequencing (NGS), 2 of these PV were exon 14 donor splice site mutations detected also by whole-exome sequencing (WES) in 2 lung tumors. Missense mutations were identified in 3 pts by RNA-sequencing and in one patient by WES. 8 pts had MET amplification identified by comparative genomic hybridization array (CGH) in 5 cases, by FISH in one case and by both FISH and CGH in 2 cases. One patient displayed both amplification and a missense mutation. Ten pts received an anti-MET targeted therapy. One had PV on NGS and missense mutation on WES, 3 had amplification on CGH or FISH and 6 positive IHC. One patient displaying MET exon 14 donor splice site mutation was treated with crizotinib achieving a rapid and sustained partial response. The other 9 pts were treated with SAR125844, a selective MET tyrosine kinase inhibitor, in TED11449 trial (NCT01391533). Conclusions: In this analysis, we saw that MET aberrations are rare in various types of advanced cancers. In selected patients, targeting MET is of clinical interest. Clinical trial information: NCT01566019.
Molecular profiling of advanced malignancies: A community oncology network experience.
Authors: Tayshetye Pritam , Monga Dulabh K. , Finley Gene Grant , Vishwanathan Swati , Brem Candice , Silverman Jan F
Published: September 2018

e23111

Background: Many genomic alterations have been identified that are critical to the malignant phenotype. Some of these, termed “driver mutations”, are critical for tumor proliferation and progression. The landscape of targeted therapy has expanded as well. Next-generation sequencing (NGS) of tumors reveals cancer-related genomic alterations and provides therapeutic recommendations for specific targeted therapy. We analyzed our experience with FoundationOne, a validated NGS genomic profile in a community oncology network. Methods: NGS results from May 2014 to September 2016 from a community oncology network in Western Pennsylvania were analyzed. Medical records were reviewed for primary site, stage, biopsy site, timing of testing, prior treatment, FDA-approved therapy in patient’s and other tumor types and potential clinical trials based upon mutations detected. Endpoints determined were percentage of patients with targetable mutations and those in whom treatment decisions were made based on NGS results. Results: 157 NGS results were available for analysis. 38.2% cases were tested early in the disease course (at diagnosis, during or shortly after first-line treatment) and 61.8% at progression. FDA-approved targeted agent for a specific mutation was found in 14% of the primary tumor type and 19.8% of other tumor types. Clinical trials were available for 93% cases. The NGS results were utilized in treatment decisions in 28 patients of which, 9 were tested early and 19 at disease progression. Of the 28, 11 were initiated on a targeted agent. Median duration of targeted therapy was 2.6 months. 9 patients were on appropriate targeted agent prior to testing. 8 patients were unable to start the targeted agent because of insurance denial, clinical deterioration or patient preference. Conclusions: NGS is a valuable tool for personalizing cancer care. From our experience, the actual number of patients starting a targeted agent based on NGS results is low. It nevertheless can provide additional treatment options and facilitate clinical trial enrollment. Optimal timing of testing (early vs late), financial implications of testing and using targeted therapy and survival benefit of targeted therapy need further studies.
Accurate and precise targeted NGS with BRCA MASTR Plus Dx for BRCA1 and BRCA2 mutation detection in formalin-fixed paraffin-embedded tumor tissue-derived DNA.
Authors: Boulet Gaelle , Van Barel Djana , Rotthier Annelies , Goossens Dirk , Del-Favero Jurgen
Published: September 2018

e23116

Background: Targeted next-generation sequencing (NGS) has tremendous potential in clinical diagnostics as it allows oncogenetic profiling to steer therapy. Inhibitors of poly-(ADP-ribose) polymerase (PARPi) have emerged as a new class of targeted anti-cancer drugs, specifically for tumors showing homologous recombination repair deficiency, including BRCA1- and BRCA2-mutated ovarian and breast cancers. This multicentre study evaluated the performance of BRCA Tumor MASTR Plus Dx* (Multiplicom) to routinely diagnose somatic and germline BRCA mutations in formalin-fixed paraffin-embedded (FFPE) tumor tissue-derived DNA. Methods: Three genetic centres participated in this performance evaluation study (PES) to detect single nucleotide variants (SNV) and small indels in the BRCA genes at a variant allele frequency down to 5%. The sample population comprised 54 FFPE-derived DNA extracts from 51 clinical and 3 reference samples. DNA extracts were subjected to quality control using Multiplicom’s QC plex assay. The clinical samples were characterized using an independent targeted NGS method and Integrative Genomics Viewer (IGV) analysis of the mapped raw reads. SNV calling was performed using third-party bioinformatics platforms in the BRCA coding regions +/- 2 intronic bp. Results: BRCA MASTR Plus Dx*showed a uniformity of 93.9%, i.e. the percentage of amplicons with at least 0.2x the mean amplicon coverage, and a target specificity of 99.1%. The limit of detection (LOD) proved to be as low as 1%. The diagnostic accuracy was ≥ 99.99% [95% CI ≥ 99.98%] (100% sensitivity [95% CI ≥ 99.02%] and ≥ 99.99% specificity [95% CI ≥ 99.98%]). Both repeatability and reproducibility were ≥ 99.99% [95% CI ≥ 99.98%]. Lot equivalence was 100% [95% CI ≥ 99.99%]. Conclusions: This multicentre study demonstrated that BRCA MASTR Plus Dx* can be routinely applied as an accurate and precise method with an LOD of 1%. The assay can be used to direct patients with somatic or germline BRCA mutations to PARPi therapy. Currently, a PES for BRCA MASTR Plus Dx* and Multiplicom’s MASTR Reporter software is ongoing. *Products described above are CE-IVD and not available for sale in the US
Assessment of the clinical utility of chip-based digital PCR for HER2 assessment in formalin fixed paraffin-embedded breast carcinoma tissue.
Authors: Shah Parth , Murarka Shiva , Sands Jacob , Mehta Bhavna , Joshi Anupam , Patel Khushbu , Parmar Vipal
Published: September 2018

e23120

Background: 15%-25% of breast cancer neoplasms exhibit Human epidermal growth factor receptor-2(HER2) amplification, as the driver mutation.Techniques to identify HER2 amplification include Immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH). Digital PCR (dPCR) has been increasingly explored in determining HER2 status in cases of indeterminate results on IHC, mainly in archived samples. In this study, we aim to demonstrate the clinical utility of the Quantstudio 3D Digital PCR system to evaluate HER2 levels from Formalin Fixed Paraffin Embedded (FFPE) tissue with RNaseP as a control target. Methods: 61 tissue samples were analyzed by IHC and dPCR in parallel in a double blinded manner. IHC equivocal samples were reflexed to FISH and compared to the results obtained from dPCR. Samples suboptimal for IHC or FISH were satisfactorily processed by dPCR. dPCR results were analyzed on the Thermofisher Cloud platform. The general turnaround time(TAT) was about 2 and 3 days for IHC and FISH respectively with that of dPCR being 24 hours. Results: All 9 IHC positive and 35 negative samples had similar results on dPCR using an amplification ratio threshold for a positive result of 1.8. Of 17 IHC-equivocal samples, 5 resulted as positive, 10 negative and 2 as equivocal by dPCR. There was 100% concordance between the dPCR and FISH results. Two IHC equivocal samples that were unanalyzable by FISH were negative on dPCR Conclusions: Our results demonstrate that the chip based dPCR was non-inferior for HER2 detection in FFPE samples in a clinical setting. Superior TAT's and objective results were obtained compared to more subjective techniques like FISH and IHC even with low sample input. dPCR requires controls but no standards for calibration as it gives absolute copy numbers. Further study is needed to understand dPCR interpretation in cases of chromosomal aneuploidy. [Table: see text]
Effect of MCAM on the aggressive phenotype in human prostate cancer.
Authors: Kruithof-de Julio Marianna , Zoni Eugenio , Astrologo Letizia , Melsen Janine , Klima Irena , Grosjean Joel , Van der Pluijm Gabri , Thalmann George N.
Published: September 2018

e23135

Background: Prostate Cancer (PCa) is the most common cancer in males and the second leading cause of death from cancer in men. Understanding the factors that regulate homing and survival of metastatic cancer cells in the bone is important for the identification of new therapeutic targets. High MCAM expression has been detected in the stroma of lytic and blastic lesions in preclinical models of PCa bone metastasis. The objective of this study is to characterize the role of MCAM in the maintenance of the aggressive phenotype in human PCa. Methods: We knocked and down MCAM in the lytic PC-3M-Pro4Luc2_dTomato and in the blastic C4-2B_dTomato PCa cell lines. Validation was done at both protein and RNA level. We performed functional assays such as migration and proliferation. RT-qPCR was used to test MCAM knockdown on EMT markers. The effect of the knockdown on the maintenance of cancer stem/progenitor-like cells was measured by ALDEFLUOR. Results: MCAM knockdown reduced proliferation in PC-3M-Pro4Luc2_dTomato PCa cells and resulted in increased E-Cadherin expression. Metastatic human PCa cells target the hematopoietic stem cell (HSC) niche in the bone marrow at the level of an “endosteal/osteoblast” niche and a “vascular/perivascular” niche. We set-up an in vitro model of “osteoblast niche” to study the prostate cancer cells upon co-culture with osteoblasts and to determine the effects on cancer stem/progenitor-like markers. We found that MCAM is required for the osteoblast-mediated induction of ALDH activity on PCa cells and MCAM knockdown prevented the increase in the size of the ALDHhigh subpopulation in PC-3M-Pro4Luc2_dTomato, mediated by human osteoblasts. Additionally, MCAM knockdown in PCa cells co-culture with osteoblast, prevented the induction of MCAM expression by osteoblasts. Finally, MCAM is significantly increased in the ALDHhigh cells and identifies a new subset of ALDHhigh / MCAMhigh cells which could be depleted upon MCAM knockdown. Conclusions: We detected a new subset of ALDHhigh/MCAMhigh cells and demonstrated the MCAM influences the maintenance of an aggressive-mesenchymal phenotype in human PCa. Therefore, MCAM represent an interesting target molecule to modulate the behavior of aggressive PCa cells.
Integrated genomics-based discovery of new druggable kinases as a therapeutic target and cancer biomarker for lung cancer.
Authors: Daigo Yataro , Takano Atsushi , Nakamura Yusuke
Published: September 2018

e23144

Background: Since the number of lung cancer patients responding well to current standard therapies is still small, further development of new anti-cancer agents with minimum risk of adverse effect and highly sensitive cancer biomarkers is eagerly awaited. Methods: We have been developing new molecular therapies targeting oncoproteins with their biomarkers; i) To identify up-regulated genes in 120 lung cancers by the gene expression microarray representing 27,648 genes, ii) To verify the candidate genes for their low expression in 23 normal tissues, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering 407 non-small cell lung cancers (NSCLCs), iv) To verify whether they are essential for the growth/invasion of cancer cells by siRNA and antibody assays, and v) To measure their serum protein levels by ELISA in 343 lung cancer patients. Results: We identified 50 druggable oncoproteins and selected a LSERT (lung cancer-specific receptor tyrosine kinase). Strong LSERT protein expression was associated with poor prognosis for NSCLC patients (P < 0.0001; N = 407) as confirmed by multivariate analysis. We established an ELISA to measure serum LSERT (a cleaved form of its extracellular domain) and found that the proportion of serum LSERT-positive cases was 149 (56.4%) of 264 NSCLC and 35 (44.3%) of 79 SCLC patients, while only 6 (4.7%) of 127 healthy volunteers were falsely diagnosed. A combined ELISA for both LSERT and CEA classified 77.2% of the NSCLC patients as positive, and the use of both LSERT and ProGRP increased sensitivity in the detection of SCLCs up to 77.5%, while the false positive rate was 7 - 8%. Oncogenic LSERT activity was suppressed by treatment of lung cancer cells with anti-LSERT antibody or siRNA. Induction of LSERT increased the cellular growth and invasion by directly phosphorylating oncogenic driver kinase proteins for lung cancer and enhancing their downstream signaling of MAPK, AKT, and STAT3. Conclusions: Integrated cancer genomics might facilitate the development of diagnostic and prognostic biomarkers as well as therapeutic targets for small molecules, monoclonal antibodies, nucleic acid drugs, and cancer vaccines.
In vitro functional analysis of HER2 variants in lung cancers to evaluate their oncogenic activity and predict clinical response to HER2 targeted therapies.
Authors: Offin Michael , Olah Zachary T. , Sabari Joshua K. , Tandon Nidhi , Feldman Daniel , Abou-Alfa Ghassan K. , Lok Benjamin H. , Burck Nitza , Tarcic Gabi , Miron Ben , Arcila Maria E. , Kris Mark G. , Rudin Charles M. , Li Bob T.
Published: September 2018

e23150

Background: HER2 variants are implicated as oncogenic drivers in a variety of cancers. However, not all HER2 variants found on next-generation sequencing (NGS) have been studied, raising the question if these variants of unknown significance (VUS) are oncogenic. This study explores the use of an in-vitro functional assay on HER2 and other variants found on NGS in patients (pts) with lung cancer, and its predictive value for response to HER2 targeted therapy. Methods: Pts with lung cancer at Memorial Sloan Kettering identified to have a HER2 variant by NGS on MSK IMPACT are eligible. NGS data on HER2 variants and co-mutations were analyzed. Functional status was determined by the NovellusDx FACT platform. Mutations were synthesized and transfected into HeLa cells with a fluorescently tagged signaling pathway reporter (MAPK, NFKB, JAK-STAT). Fluorescent microscopy quantified the translocation of reporters to the nucleus, inferring gain of function. Afatinib was added and its effect on pathway activation analyzed. Functional information was correlated with clinical outcomes. Results: As of 2/1/17, 13 pts were included, 9 unique HER2 variants analyzed. Known activating HER2 mutations analyzed (exon 20 insertions (A775_G776insYVMA, G778_P780insGSP, G776delinsVC), point mutations (V659E, L755P, S301F)) showed in vitro gain of function. HER2 VUS analyzed included D277Y showed no gain of function; exon 16 splice variant c.1889 showed gain of function. Afatinib was added to 4 cell lines with gain of function variants (G778_P780insGSP, V659E, L755P, exon 16 splice variant c.1889) all had inhibited pathway activation suggesting in vitro sensitivity to afatinib. Clinically, no response was seen in 2 pts treated with afatinib but more pts are planned to be treated. Objective responses to ado-trastuzumab emtansine (NCT02675829) were seen in 5 pts, all of which had HER2 variants with gain of function. Conclusions: In vitro functional analysis of HER2 variants based on NGS reports is feasible, and has the potential to identify novel actionable oncogenic drivers from VUS. Its predictive value for clinical response to targeted therapy requires further study.
Type II RAF inhibition predicts superior ERK suppression compared to type I RAF inhibition in different BRAF mutant types recurrently found in lung cancer.
Authors: Giron Philippe , Noeparast Amir , De Brakeleer Sylvia , De Ridder Ulrike , Teugels Erik , De Greve Jacques
Published: September 2018

e23154

Background: Somatic driver BRAF mutations account for 6-8% of lung cancers. As opposed to melanoma in which V600E mutant BRAF predominates, the majority of lung cancer-derived BRAF mutations are non-V600. Yet, the efficacy of RAF-inhibitors and the possible resistance mechanisms in non-V600 BRAF mutant cells remain to be uncovered. Recently, we have shown that non-V600 BRAF mutations recurrently found in lung cancer predict sensitivity to the combination of type I RAF inhibitor Dabrafenib and a MEK inhibitor Trametinib. As a single agent, Dabrafenib shows only weak suppression of mutant BRAF-induced ERK signaling; moreover it can induce ERK paradoxical activation in CRAF overexpressing cells. Methods: Several recombinant BRAF expression vectors were generated by performing site-directed mutagenesis. We compared the effects of Dabrafenib and a type II RAF inhibitor (AZD-628) at clinically relevant dose as single agents or in combination with MEK inhibitor Trametinib on ERK activity in HEK293T cells expressing several tumor-derived BRAF mutants and a non-V600 BRAF mutant lung cancer cell line (H1666). Viability and caspase3/7 activation assays were performed using the H1666 cell line model. Results: In contrast to Dabrafenib, AZD-628 does not induce paradoxical ERK activation in CRAF expressing cells. Increased CRAF expression desensitizes BRAF-mutant expressing cells to Dabrafenib but not to AZD-628. Notably, AZD-628 has superior ERK-inhibitory effect in HEK293T cells co-expressing several different BRAF-mutants with CRAF and in H1666 cells. Combination of Trametinib and AZD-628 has superior MEK-inhibitory and pro-apoptotic effect in H1666 cells compared to combined Trametinib/Dabrafenib. Moreover, upon down titration of the RAF inhibitors with a steady dose of Trametinib, AZD-628 resulted in overall stronger effect on viability compared to Dabrafenib. Conclusions: In our in vitro model, we obtained strong indications that at conventional doses, type II RAF-inhibitor AZD628 is superior to type I RAF-inhibitor Dabrafenib in combination with MEK inhibitor Trametinib for the treatment of non-V600 BRAF mutant lung cancer.