Whole exome sequencing identifies genomic alterations in proximal and distal colorectal cancer
Majority of colorectal cancer (CRC) patients are presented with advanced disease at diagnosis, particularly in cases of proximal CRCs. Little is known about the relationship between the genetic landscape and the anatomical location of the tumour; as well as the prognostication in CRC patients. The objectives of this study were to determine the somatic single nucleotide variants (SNV) and the cellular pathways between the proximal and distal CRCs. Whole exome sequencing was performed on the Ion Proton platform on 10 pairs of normal and CRC samples. The sequencing results were analysed using the Torrent Suite Software and the variants were annotated using ANNOVAR; followed by validation with Sanger sequencing. APC is the most frequently altered gene in both proximal and distal CRCs. KRAS and ATM genes were particularly altered in the proximal CRCs with a frequency of 60% and 40%, respectively. On the other hand, TP53 mutations did not show any CRC anatomical predominance. There were five recurrent novel variants in proximal CRCs and no recurrent variants identified in distal CRC. Wnt signalling pathway was the most frequently altered pathway in both proximal and distal CRCs whereas TGF-β and PI3K signalling pathways were predominantly altered in the proximal CRCs. We found that proximal CRCs presented with more variants and different altered pathways as compared to distal CRCs. However, further study in a larger series of samples coupled with functional studies will be required to confirm the identified variants and determine their roles in the pathogenesis of proximal and distal CRCs.
Bray F, Ferlay J, Soerjomataram I, et al. GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2018; 68(6): 394–424.
Zainal Ariffin O. and Nor Saleha IT. National Cancer Registry Report: Malaysia Cancer Statistics- Data and Figure 2007. 2011. Ministry of Health, Malaysia.
Lim GCC, Rampal S, and Halimah Y (Eds). Cancer incidence in Peninsular Malaysia 2003–2005. 2008. National Cancer Registry, Ministry of Health Malaysia.
Gonzalez EC, Roetzheim RG, Ferrante JM, et al. Predictors of proximal vs. distal colorectal cancers. Dis Colon Rectum, 2001; 44(2):251–258.
Bufill JA. Colorectal cancer: Evidence for distinct genetic categories based on proximal or distal location. Ann Intern Med, 1990; 113(10):779–788.
Minoo P, Zlobec I, Peterson M, et al. Characterization of rectal, proximal and distal colon cancers based on clinicopathological, molecular and protein profiles. Int J Oncol, 2010; 37(3):707–718.
Missiaglia E, Jacobs B, D’Ario1 G, et al. Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features. Ann Oncol, 2014; 00: 1–7.
Maus MKH, Hanna DL, Stephen CL, et al. Distinct gene expression profiles of proximal and distal colorectal cancer: Implications for cytotoxic and targeted therapy. Pharmacogenomics J, 2015; 15: 354–362.
Bogaert J. and Prenen H. Molecular genetics of colorectal cancer. Ann of Gastroenterol, 2014; 27(1): 9–14.
Benedix F, Kube R, Meyer F, et al. Comparison of 17,641 patients with right and left-sided colon cancer: Differences in epidemiology, preoperative course, histology, and survival. Dis. Colon Rectum, 2010; 53(1): 57–64.
Myer PA, Mannalithara A, Singh G, et al. Proximal and distal colorectal cancer resection rates in the United States since widespread screening by colonoscopy. Gastroenterology, 2012; 5: 1227–1236.
Li FY. and Lai MD. Colorectal cancer, one entity or three. J Zhejiang Univ Sci B, 2009; 10(3): 219–229.
Wu X, Chen VW, Martin J, et al. Comparative analysis of incidence rates subcommittee, data evaluation and publication committee, North American Association of Central Cancer Registries. Subsite-specific colorectal cancer incidence rates and stage distributions among Asians and Pacific Islanders in the United States, 1995 to 1999. Cancer Epidemiol Biomarkers Prev. 2004; 13(7): 1215–1222.
Irby K, Anderson WF, Henson DE, et al. Emerging and widening colorectal carcinoma disparities between Blacks and Whites in the United States (1975–2002). Cancer Epidemiol Biomarkers Prev, 2006; 15(4): 792–797.
Goh KL, Quek KF, Yeo GT, et al. Colorectal cancer in Asians: A demographic and anatomic survey in Malaysian patients undergoing colonoscopy. Aliment Pharmacol Ther, 2005; 22(9): 859–864.
Meldrum C, Doyle MA. and Tothill RW. Next generation sequencing for cancer diagnostics: A practical perspective. Clin Biochem Rev, 2011; 32: 177–195.
Roychowdhury S, Iyer MK, Robinson DR, et al. Personalized oncology through integrative high-throughput sequencing: A pilot study. Sci Transl Med, 2011; 3: 111–121.
Cingolani P, Platts A, Wang le L, et al. Program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in
the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin), 2012; 6(2): 80–92.
Wang K, Li M, and Hakonarson H. ANNOVAR: Functional annotation
of genetic variants from high-throughput sequencing data. Nucleic Acids Res, 2010; 38(16): e164.
O’Leary NA, Wright MW, Brister JR, et al. Reference sequence (Ref-Seq) database at NCBI: Current status, taxonomic expansion, and functional annotation. Nucleic Acids Res, 2016; 44(D1): D733–745.
Rosenbloom KR, Armstrong J, Barber GP, et al. The UCSC genome browser database: 2015 update. Nucleic Acids Res, 2015; 43: D670–681.
Flicek P, Amode MR, Barrell D, et al. Ensembl 2014. Nucleic Acids Res, 2014; 42 Database issue: D749-D755; doi:10.1093/nar/gkp972.
Siepel A, Bejerano G, Pedersen JS, et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res,
; 15, 1034–1050.
Wenqing F, Timothy DO, Goo J, et al. NHLBI Exome Sequencing Project, Joshua MA. Analysis of 6,515 exomes reveals the recent origin of
most human protein-coding variants. Nature, 2013; 493: 216–220.
Monkol L, Konrad JK, Eric VM, et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature, 2016; 536: 285–291.
Sherry ST, Ward MH, Kholodov M, et al. dbSNP: The NCBI database of genetic variation. Nucleic Acids Res, 2001; 29(1): 308–311.
Landrum MJ, Lee JM, Benson M, et al. ClinVar: Public archive of interpretations of clinically relevant variants. Nucleic Acids Res, 2015; 44(D1): D862–868.
Forbes SA, Beare D, Gunasekaran P, et al. COSMIC: Exploring the world’s knowledge of somatic mutations in human cancer. Nucleic Acids Res, 2015; 43(Database issue): D805–811.
Ng PC and Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucl Acids Res, 2003; 31 (13): 3812–3814.
Adzhubei IA, Jordan DM, and Sunyae SR. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet, 2013; 7: Unit 7.20.
Griffith M, Griffith OL, Coffman AC, et al. Mining the druggable genome. Nat Methods, 2013; 10(12): 1209–1210.
The 1000 Genomes Project Consortium. A global reference for human
genetic variation. Nature, 2015; 526: 68–74.
Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol, 2011; 29(1), 24–26.
Thorvaldsdóttir H, Robinson James T, and Mesirov JP. Integrative Genomics Viewer (IGV): High-performance genomics data visualization and exploration. Brief Bioinformatics, 2013; 14, 178–192.
Kumar P, Henikoff S, and Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc, 2009; 4(7): 1073–1081.
Altschul SF, Gish W, Miller W, et al. Basic local alignment search tool. J Mol Biol, 1990; 215: 403–410.
Wang L, Tsutsumi S, Kawaguchi T, et al. Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency. Genome Res, 2012; 22(2): 208–219.
The Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature, 2012; 487: 330–337.
DePristo MA, Banks E, Poplin R, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet, 2011; 43(5): 491–498; doi: 10.1038/ng.806.
Guo Y, Ye F, Sheng Q, et al. Three-stage quality control strategies for DNA re-sequencing data. Brief Bioinformatics. 2014; 15(6):879–889.
Perreault N, Katz JP, Sackett SD, et al. Foxl1 controls the Wnt/betacatenin pathway by modulating the expression of proteoglycans in the gut. J Biol Chem. 2001; 276: 43328–43333.
Moreno-Bueno G, Hardisson D, Sanchez C, et al. Abnormalities of the APC/beta-catenin pathway in endometrial cancer. Oncogene, 2002; 21: 7981–7990.
Segditsas S, Rowan AJ, Howarth K, et al. APC and the three-hit hypothesis. Oncogene, 2009; 28: 146–155.
Reya T, and Clevers H. Wnt signalling in stem cells and cancer. Nature, 2005; 434: 843–850.
Van Es JH, Jay P, Gregorieff A, et al. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nat Cell Biol, 2005; 7:
Femia AP, Dolara P, Giannini A, et al. Frequent mutation of Apc gene in rat colon tumours and mucindepleted foci, preneoplastic lesions in experimental colon carcinogenesis. Cancer Res, 2007; 67: 445–449.
Nagel R, le Sage C, Diosdado B, et al. Regulation of the adenomatous polyposis coli gene by the miR-135 family in colorectal cancer. Cancer Res, 2008; 68: 5795–5802.
Clevers H and Nusse R. Wnt/beta-catenin signalling and disease. Cell, 2012; 149: 1192–1205.
Diaz LA Jr, Williams RT, Wu J, et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.
Nature, 2012; 486: 537–540.
Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome
landscapes. Science, 2013; 339: 1546–1558.
Ashktorab H, Daremipouran M, Devaney P, et al. Identification of novel mutations by exome sequencing in African American colorectal cancer patients. Cancer, 2014; 121(1): 34–42.
Nikhil W, Michael FB, Matthew JD, et al. High-throughput detection
of actionable genomic alterations in clinical tumour samples by targeted, massively parallel sequencing. Cancer Discov, 2012; 2(1): 82–93.
Fang W, Radovich M, Zheng Y, et al. Druggable alterations detected by Ion Torrent in metastatic colorectal cancer patients. Oncol Lett, 2014; 7: 1761–1766.
Salem ME, Weinberg BA, Xiu J, et al. Comparative molecular analyses of left-sided colon, right-sided colon and rectal cancers. Oncotargets, 2017; 8(49): 86356–86368.
Gao J, Aksoy BA, Dogrusoz U, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal, 2013; 6(269): 11.
Birgisson H, Edlund K, Wallin U, et al. Microsatellite instability and mutations in BRAF and KRAS are significant predictors of disseminated disease in colon cancer. BMC Cancer, 2015; 15: 125.
Timmermann B, Kerick M, Roehr C, et al. Somatic mutation profiles of MSI and MSS colorectal cancer identified by Whole Exome Next Generation Sequencing and Bioinformatics Analysis. PLoS One. 2010; 5(12): 15661.
Kandoth C, McLellan MD, Vandin F, et al. Mutational landscape and significance across 12 major cancer types. Nature, 2013; 502: 333–339.
Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science, 2013; 339: 1546–1558.
Pamplona RS, Doriga AL, Brunet LP, et al. Exome sequencing reveals AMER1 as a frequently mutated gene in colorectal cancer. Clin Cancer Res, 2015; 21(20): 4709–4718.
Scholer-Dahirel A, Schlabach MR, Loo A, et al. Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent
on Wnt/β-catenin signalling. Proc Nat Acad Sci U.S.A., 2011; 108(41): 17135–17140.
Pino MS, and Chung DC. The chromosomal instability pathway in colon cancer. Gastroenterology, 2010; 138(6): 2059–2072. doi:10.1053/j.gastro.2009.12.065.
Malekzadeh R, Bishehsari F, Mahdavinia M, Ansari R. Epidemiology and molecular genetics of colorectal cancer in Iran: A review. Arch Iran Med, 2009; 12 (2): 161–169.
Mahdavinia M, Bishehsari F, Verginelli F, et al. P53 mutations in colorectal cancer from northern Iran: Relationships with site of tumour origin, microsatellite instability and K-ras mutations. J Cell Physio, 2008; 216(2): 543–550.
Manne U, Weiss HL, Myers RB, et al. Nuclear accumulation of p53 in colorectal adenocarcinoma: Prognostic importance differs with race and location of the tumour. Cancer, 1998; 83: 2456–2467.
Samowitz WS, Curtin K, Ma KN, et al. Prognostic significance of p53 mutations in colon cancer at the population level. Int J Cancer. 2002; 99: 597–602.
Soong R, Grieu F, Robbins P, et al. p53 alterations are associated with improved prognosis in distal colonic carcinomas. Clin Cancer Res, 1997; 3: 1405–1411.
Adrover E, Maestro ML, Sanz-Casla MT, et al. Expression of high p53 levels in colorectal cancer: a favorable prognostic factor. Br J Cancer, 1999; 81: 122–126.
Iacopetta B. TP53 Mutation in Colorectal Cancer. Hum Mutat, 2003; 21: 271–276.
Daniel BL, Paul DH and Patrick GJ. 5-Fluorouracil: Mechanisms of action and clinical strategies. Nat Rev Cancer, 2003; 3: 330–338.
Batey MA, Zhao Y, Kyle S, et al. Preclinical evaluation of a novel ATM inhibitor, KU59403, in vitro and in vivo in p53 functional and dysfunctional models of human cancer. Mol Cancer Ther, 2013; 12(6): 959–967.
Song H, Hollstein M, and Xu Y. p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM. Nat Cell Biol, 2007; 9: 573–580.
Reinhardt HC, Aslanian AS, Lees JA, et al. p53-deficient cells rely on ATM and ATR-mediated checkpoint signalling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell, 2007; 11: 175–189.
Rosty C, Young JP, Walsh MD, et al. Colorectal carcinomas with KRAS mutation are associated with distinctive morphological and molecular features. Mod Pathol, 2013; 26(6): 825–834.
Wenbin L, Tian Q, Wenxue Z, et al. Colorectal carcinomas with KRAS codon 12 mutations are associated with more advanced tumor stages. BMC Cancer, 2015; 15: 340.
Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med, 2013; 369(11), 1023–1034.
Misale S, Yaeger R, Hobor S, et al. Emergance of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature, 2012; 486: 532–536.
Morgan OH, Maria SD, and Silvio JG. Novel insights into G protein and G protein-coupled receptor signalling in cancer. Curr Opin Cell Biol, 2014; 27: 126–135.
Gaorav PG and Joan M. Cancer metastasis: Building a framework. Cell Press, 2006; 127(4): 679–695.
Mohd Yunos R, Ab Mutalib N, Khor SS, et al. Characterisation of genomic alterations in proximal and distal colorectal cancer patients. PeerJ Preprints, 2016; 4: e2109v1.
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