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Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR = 0.42, 95% CI: 0.21–0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR = 1.53, 95% CI: 1.21–1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features., Large scale sequencing study is of paramount importance to unravel the heterogeneity of colorectal cancer. Here, the authors sequenced 205 cancer genes in more than 2000 tumours and identified additional mutated driver genes, determined that mutational burden and specific mutations in TP53 are associated with survival odds.

Colorectal cancer (CRC) is the 3rd most common cancer in men and women, excluding skin cancer, and the 2nd leading cause of cancer death in the United States. In this biologically heterogeneous disease, a comprehensive molecular characterization is valuable for understanding tumorigenesis and studying associations with clinical, lifestyle, environmental, and germline genetic factors. The Colon Cancer Family Registry (CCFR) and Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) collected clinical, germline genetic, and epidemiologic data from over 125,000 CRC cases. In a subset of 2,500 CRC cases, we conducted targeted deep sequencing of tumor and matching normal DNA. The custom AmpliSeq sequencing panel includes 205 genes, which were prioritized from literature and analyses of existing CRC tumor whole exome sequencing data. Next-generation sequencing was conducted on Illumina Hi-Seq 2500, and mean sequence coverages of 748x and 287x were attained for tumors and normal DNA samples, respectively. So far, 1,705 cases have been analyzed. Strelka and MuTect were used to identify somatic single nucleotide variants, and VarsScan2, Verdict, and Strelka were used to call indels. Orthogonal technologies were used for validation and improving mutation calling algorithms. Approximately 18% of tumors were hypermutated, among which 70% exhibited microsatellite instability (MSI). Hypermutated tumors with MSI occurred more frequently in proximal colon compared to distal colon and rectum (OR, 10.10; 95% CI, 6.98-14.63; P CRC-specific survival was significantly more favorable among individuals with hypermutated tumors, regardless of POLE mutation status (HR=0.36, 95% CI: 0.23-0.57). This survival benefit was attenuated in analyses of overall survival (HR=0.82, 95% CI: 0.65-1.02). This large dataset is being used to study associations with clinical, lifestyle, and environmental factors. This study will provide valuable information to develop better strategies for the prevention, diagnosis, and treatment of CRC. Citation Format: Syed H. Zaidi, Amanda I. Phipps, Tabitha A. Harrison, Robert S. Steinfelder, Quang Trinh, Barbara L. Banbury, Adilya Rafikova, Megan Van Tassel, Emma Reid, Stefanie Brezina, Marios Giannakis, Charles S. Fuchs, Li Hsu, Andrea Gsur, Shuji Ogino, Steven Gallinger, Polly A. Newcomb, Peter T. Campbell, Wei Sun, Thomas J. Hudson, Ulrike Peters. Deep targeted sequencing of colorectal cancer cases to study associations of molecular subtypes with clinical, genetic, and lifestyle risk factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1598.

Colorectal cancer (CRC), a common malignancy, is a biologically heterogeneous disease. Next-generation sequencing (NGS) has enabled CRC characterization by identifying somatically mutated genes which now allow us to better define colorectal tumor subtypes (e.g. by mutated pathways). However, the relationship of such CRC subtypes to patient survival and genetic and lifestyle risk factors has not been comprehensively studied. To identify somatic mutations in CRC cases, we designed a targeted AmpliSeq panel of CRC related genes and genomic regions informed by whole exome sequencing data from ~1,200 CRC cases. The sequencing was conducted on Illumina HiSeq 2500 with a mean coverage of 740x and 240x for DNA extracted from FFPE tumor tissues and matched normal samples, respectively. Strelka, MuTect, VarDict, and Varscan2 were used to identify somatic single nucleotide variants and indels. Sanger sequencing was performed to validate a subset of variants. To date, we have sequenced ~2,400 CRC tumors and matched control tissues from four studies participating in the Colon Cancer Family Registry (CCFR) and Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). In most tumors, we identified non-silent mutations in genes belonging to the WNT (77%), p53 (44%), IGF2/PI3K (22%), RTK-RAS (47%), and TGF-beta (26%) signaling pathways. Among the 15% of tumors that could be classified as hypermutated, based on the number of mutations, 39% exhibited non-silent mutations in MLH1, MLH3, MSH2, MSH6, and PMS2 and 41% exhibited non-silent mutations in POLE and POLD1. In a subset of studies with available survival data, we used Cox regression to assess the association of hypermutation status and the presence of non-silencing mutations in key signaling pathways with overall (OS) and disease-specific (DSS) survival. OS and DSS were significantly more favorable in cases with hypermutated vs. non-hypermutated CRC (HR=0.77, 95% CI: 0.60-0.98, p=0.04 and HR=0.35, 95% CI: 0.22-0.57, p=2x10-5, respectively); these associations were most pronounced for POLE/POLD1 mutated hypermutated CRC (HR=0.69, 95% CI: 0.46-1.02, p=0.06, HR=0.21, 95% CI: 0.08-0.56, p=2x10-3, respectively). There was no significant association of mutations in WNT, p53, IGF2/PI3K, RTK-RAS, or TGF-beta pathways with survival (p>0.05). The comprehensive molecular characterization of this large panel of CRC cases will support further studies of molecular subtypes of CRC with clinical, lifestyle, and environmental factors. A better understanding of molecular mechanisms of CRC will be valuable in developing strategies for prevention, diagnosis, and treatment of this life-threatening disease. Citation Format: Syed Zaidi, Amanda Phipps, Tabitha Harrison, Catherine Grasso, Robert Steinfelder, Quang Trinh, Charles Connolly, Barbara Banbury, Adilya Rafikova, Philipp Hofer, Stefanie Brezina, Marios Giannakis, Xinmeng Jasmine Mu, Michael Quist, Charles Fuchs, Levi Garraway, Li Hsu, Lincoln Stein, Andrea Gsur, Shuji Ogino, Steven Gallinger, Polly Newcomb, Peter Campbell, Wei Sun, Thomas Hudson, Ulrike Peters. Deep targeted tumor sequencing of colorectal cancer cases to study associations of molecular subtypes with clinical, genetic, and lifestyle risk factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1224.