Shared Oncogenic Pathways Implicated in Both Virus-Positive and UV-Induced Merkel Cell Carcinomas

Merkel cell carcinoma (MCC) is a highly malignant neuroendocrine tumor of the skin whose molecular pathogenesis is not completely understood, despite the role that Merkel cell polyomavirus can play in 55e90% of cases. To study potential mechanisms driving this disease in clinically characterized cases, we searched for somatic mutations using whole-exome sequencing, and extrapolated our findings to study functional biomarkers reporting on the activity of the mutated pathways. Confirming previous results, Merkel cell polyomavirus-negative tumors had higher mutational loads with UV signatures and more frequent mutations in TP53 and RB compared with their Merkel cell polyomavirus-positive counterparts. Despite important genetic differences, the two Merkel cell carcinoma etiologies both exhibited nuclear accumulation of oncogenic transcription factors such as NFAT or nuclear factor of activated T cells (NFAT), P-CREB, and P-STAT3, indicating commonly deregulated pathogenic mechanisms with the potential to serve as targets for therapy. A multivariable analysis identified phosphorylated CRE-binding protein as an independent survival factor with respect to clinical variables and Merkel cell polyomavirus status in our cohort of Merkel cell carcinoma patients.This work was supported by grants from Instituto de Salud-Carlos III (ISCIII); cofinanced by the European Union; (FEDER) (PI12/00357), and a Ramón and Cajal research program (MINECO; RYC-2013-14097) to JPV, Asociación Española Contra el Cáncer and ISCIII grants (RD06/0020/0107, RD012/0036/0060) to MAP, and Coordinated Project of Excellence inter-Institutos de investigación acreditados institutes (ISCIII; PIE15/00081) to MAP. The Ramón and Cajal research program also supports IV. SD was supported by the Torres Quevedo subprogram (MICINN; PTQ-12-05391)

Identification of novel fusion genes in lung cancer using breakpoint assembly of transcriptome sequencing data

Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8

Integrative and comparative genomic analyses identify clinically relevant pulmonary carcinoid groups and unveil the supra-carcinoids

International audienceThe worldwide incidence of pulmonary carcinoids is increasing, but little is known about their molecular characteristics. Through machine learning and multi-omics factor analysis, we compare and contrast the genomic profiles of 116 pulmonary carcinoids (including 35 atypical), 75 large-cell neuroendocrine carcinomas (LCNEC), and 66 small-cell lung cancers. Here we report that the integrative analyses on 257 lung neuroendocrine neoplasms stratify atypical carcinoids into two prognostic groups with a 10-year overall survival of 88% and 27%, respectively. We identify therapeutically relevant molecular groups of pulmonary car-cinoids, suggesting DLL3 and the immune system as candidate therapeutic targets; we confirm the value of OTP expression levels for the prognosis and diagnosis of these diseases, and we unveil the group of supra-carcinoids. This group comprises samples with carcinoid-like morphology yet the molecular and clinical features of the deadly LCNEC, further supporting the previously proposed molecular link between the low-and high-grade lung neuroendocrine neoplasms

Differential Orthopedia Homeobox expression in pulmonary carcinoids is associated with changes in DNA methylation

Limited number of tumor types have been examined for Orthopedia Homeobox (OTP) expression. In pulmonary carcinoids, loss of expression is a strong indicator of poor prognosis. Here, we investigated OTP expression in 37 different tumor types, and the association between OTP expression and DNA methylation levels in lung neuroendocrine neoplasms. We analyzed publicly available multi-omics data (whole-exome-, whole-genome-, RNA sequencing and Epic 850K-methylation array) of 58 typical carcinoids, 27 atypical carcinoids, 69 large cell neuroendocrine carcinoma and 51 small cell lung cancer patients and TCGA (The Cancer Genome Atlas) data of 33 tumor types. 850K-methylation analysis was cross-validated using targeted pyrosequencing on 35 carcinoids. We report bimodality of OTP expression in carcinoids (OTPhigh vs OTPlow group, likelihood-ratio test P = 1.5 × 10−2), with the OTPhigh group specific to pulmonary carcinoids while absent from all other cohorts analyzed. Significantly different DNA methylation levels were observed between OTPhigh and OTPlow carcinoids in 12/34 OTP infinium probes (FDR .2). OTPlow carcinoids harbor high DNA methylation levels as compared to OTPhigh carcinoids. OTPlow carcinoids showed a significantly worse overall survival (log-rank test P =.0052). Gene set enrichment analysis for somatically mutated genes associated with hallmarks of cancer showed robust enrichment of three hallmarks in the OTPlow group, that is, sustaining proliferative signaling, evading growth suppressor and genome instability and mutation. Together our data suggest that high OTP expression is a unique feature of pulmonary carcinoids with a favorable prognosis and that in poor prognostic patients, OTP expression is lost, most likely due to changes in DNA methylation levels

Elucidating the mechanisms of acquired resistance in lung adenocarcinomas

Abstract In lung adenocarcinomas, targeted therapy with the EGFR tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and afatinib is associated with longer progression free survival (PFS) and higher radiographic response (RR) rates when compared to standard first-line chemotherapy. In ALK rearranged lung cancers, targeted therapy with crizotinib is associated with PFS of approximately 9,7 months and RR of 60.8%. However, despite the initial success of these agents, all patients progress with a median PFS of 7 to 16 months. Acquired resistance in EGFR mutant tumors is driven by the occurrence of a secondary EGFR mutation (T790M) in about 50% of the cases and by MET amplification in 5 to 10 % of the cases. Other mechanisms include HER2 amplification, PTEN loss, phenotypic change to small cell histology, rare mutations in BRAF and AXL activation. Resistance to crizotinib, on the other hand, is caused by secondary mutations in the ALK kinase domain, by ALK or cKIT amplification or by alterations in EGFR and KRAS. Here, we made use of next generation sequencing techniques to better understand the mechanisms that drive resistance in lung adenocarcinomas treated with erlotinib or crizotinib. For this purpose, we used transbronchial or CT-guided rebiopsies from patients that had either prolonged stable disease or partial response to therapy, and developed radiographic progression under TKI therapy. Samples were analyzed by FISH and sequenced on a benchtop Illumina platform (MiSeq) in order to evaluate the presence of known mechanisms of resistance. Samples that were negative for any of the reported mechanisms were analyzed by genome, exome or trascriptome sequencing. From the sequencing output of the pan-negative samples, filtering of mutation candidates included: absence of the mutation in the pre-treatment sample (when available), expression of the candidate gene in lung adenocarcinomas, absence of the mutation in primary lung adenocarcinomas, high impact of the mutation at protein level (Polyphen), mutant allelic fraction in the tumor higher than 10%, among other factors. After filtering, validation of mutation calls was performed by Sanger sequencing. Sequencing of the erlotinib resistant samples revealed mutations in members of a functionally wide spectrum of protein families including the proteoglycan family, the ATP-binding cassette (ABC) transporters family, an Fms-related tyrosine kinase receptor and a member of the transforming growth factor beta family of cytokines. On the other hand, crizotinib resistant samples showed mutations in a cell surface receptor for macrophage-stimulating protein with tyrosine kinase activity, in a C2H2 type zinc finger gene, a semaphorin, a mitogen-activated protein kinase and a member of the SWI/SNF family of proteins. Our results evidence the possible contribution of a wide range of cellular pathways in the process of acquired resistance to EGFR and ALK inhibitors in lung adenocarcinomas. Citation Format: Sandra Ortiz-Cuarán, Lynnette Fernandez-Cuesta, Marc Bos, Lukas Heukamp, Christine M. Lovly, Martin Peifer, Masyar Gardizi, Matthias Scheffler, Ilona Dahmen, Christian Müller, Katharina König, Kerstin Albus, Alexandra Florin, Sascha Ansén, Reinhard Buettner, Jürgen Wolf, William Pao, Roman K. Thomas. Elucidating the mechanisms of acquired resistance in lung adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 956. doi:10.1158/1538-7445.AM2014-956</jats:p