Novel Genetic and Epigenetic Biomarkers of Prognostic and Predictive Significance in Stage II/III Colorectal Cancer
Authors: Xiao-Jing Luo, Qi Zhao, Jia Liu, Jia-Bo Zheng, Miao-Zhen Qiu, Huai-Qiang Ju, Rui-Hua Xu
Journal: Molecular Therapy (2020)

The therapeutic strategies of stage II/III colorectal cancer (CRC) patients after curative surgery remain controversial. In the clinical decision-making process, oncologists need to answer questions such as whether adjuvant chemotherapy is necessary or which therapeutic regimen should be given to each patient. At present, whether adjuvant chemotherapy should be applied is primarily based on histopathological features and clinical risk factors. However, only a fraction of patients can benefit from it. More rigorous stratifying biomarkers are urgently needed to help further distinguishing these populations of patients. Recent progress in next-generation sequencing and high-throughput technologies has greatly promoted biomarker discovery as well as our understanding of the underlying mechanisms in CRC. Novel genetic and epigenetic biomarkers that are associated with prognosis or therapeutic responses have emerged. In this review, we discuss the strategies of biomarker discovery and summarize the status and assess the utility of previously published biomarkers in CRC. The therapeutic strategies of stage II/III colorectal cancer (CRC) patients after curative surgery remain controversial. In the clinical decision-making process, oncologists need to answer questions such as whether adjuvant chemotherapy is necessary or which therapeutic regimen should be given to each patient. At present, whether adjuvant chemotherapy should be applied is primarily based on histopathological features and clinical risk factors. However, only a fraction of patients can benefit from it. More rigorous stratifying biomarkers are urgently needed to help further distinguishing these populations of patients. Recent progress in next-generation sequencing and high-throughput technologies has greatly promoted biomarker discovery as well as our understanding of the underlying mechanisms in CRC. Novel genetic and epigenetic biomarkers that are associated with prognosis or therapeutic responses have emerged. In this review, we discuss the strategies of biomarker discovery and summarize the status and assess the utility of previously published biomarkers in CRC. Colorectal cancer (CRC) was estimated to be the fourth most frequently diagnosed cancer and the second most common cause of cancer deaths worldwide in 2018.1Bray F. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J. Clin. 2018; 68: 394-424Crossref PubMed Scopus (34310) Google Scholar CRC has been recognized as a disease affecting mostly developed countries. However, in recent years, the incidence of CRC has kept increasing in developing countries, such as China, possibly due to changes in lifestyle and nutritional habits.2Chen W. Zheng R. Zeng H. Zhang S. He J. Annual report on status of cancer in China, 2011.Chin. J. Cancer Res. 2015; 27: 2-12Crossref PubMed Google Scholar Early-stage CRC is mostly asymptomatic, and many patients are diagnosed at advanced stages.3Benitez-Majano S. Fowler H. Maringe C. Di Girolamo C. Rachet B. Deriving stage at diagnosis from multiple population-based sources: colorectal and lung cancer in England.Br. J. Cancer. 2016; 115: 391-400Crossref PubMed Scopus (33) Google Scholar The tumor-node-metastasis (TNM) stage remains the most potent prognostic factor in the clinical decision-making process. Based on current guidelines, most stage II CRC patients are treated surgically without adjuvant chemotherapy. However, almost 15% of these patients undergo tumor recurrence and death caused by disease progression.4Benson 3rd, A.B. Schrag D. Somerfield M.R. Cohen A.M. Figueredo A.T. Flynn P.J. Krzyzanowska M.K. Maroun J. McAllister P. Van Cutsem E. et al.American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer.J. Clin. Oncol. 2004; 22: 3408-3419Crossref PubMed Scopus (1066) Google Scholar,5Figueredo A. Coombes M.E. Mukherjee S. Adjuvant therapy for completely resected stage II colon cancer.Cochrane Database Syst. Rev. 2008; : CD005390PubMed Google Scholar Whether a stage II CRC patient should receive adjuvant chemotherapy after surgery largely depends on the recurrence risk assessed by a group of clinical factors.6Oñate-Ocaña L.F. Montesdeoca R. López-Graniel C.M. Aiello-Crocifoglio V. Mondragón-Sánchez R. Cortina-Borja M. Herrera-Goepfert R. Oros-Ovalle C. Gallardo-Rincón D. Identification of patients with high-risk lymph node-negative colorectal cancer and potential benefit from adjuvant chemotherapy.Jpn. J. Clin. Oncol. 2004; 34: 323-328Crossref PubMed Scopus (26) Google Scholar,7O'Connor E.S. Greenblatt D.Y. LoConte N.K. Gangnon R.E. Liou J.I. Heise C.P. Smith M.A. Adjuvant chemotherapy for stage II colon cancer with poor prognostic features.J. Clin. Oncol. 2011; 29: 3381-3388Crossref PubMed Scopus (291) Google Scholar Still, a considerable proportion of stage II patients who are evaluated as low-risk patients later suffer from recurrence and progression. Stage III patients routinely receive adjuvant chemotherapy after surgery.8Jonker D.J. Spithoff K. Maroun J. Gastrointestinal Cancer Disease Site Group of Cancer Care Ontario's Program in Evidence-based CareAdjuvant systemic chemotherapy for stage II and III colon cancer after complete resection: an updated practice guideline.Clin. Oncol. (R. Coll. Radiol.). 2011; 23: 314-322Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar However, evidence showed these patients did not respond equally.9Gray R. Barnwell J. McConkey C. Hills R.K. Williams N.S. Kerr D.J. Quasar Collaborative GroupAdjuvant chemotherapy versus observation in patients with colorectal cancer: a randomised study.Lancet. 2007; 370: 2020-2029Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 10Moertel C.G. Fleming T.R. Macdonald J.S. Haller D.G. Laurie J.A. Tangen C.M. Ungerleider J.S. Emerson W.A. Tormey D.C. Glick J.H. et al.Intergroup study of fluorouracil plus levamisole as adjuvant therapy for stage II/Dukes' B2 colon cancer.J. Clin. Oncol. 1995; 13: 2936-2943Crossref PubMed Scopus (314) Google Scholar, 11Schippinger W. Samonigg H. Schaberl-Moser R. Greil R. Thödtmann R. Tschmelitsch J. Jagoditsch M. Steger G.G. Jakesz R. Herbst F. et al.Austrian Breast and Colorectal Cancer Study GroupA prospective randomised phase III trial of adjuvant chemotherapy with 5-fluorouracil and leucovorin in patients with stage II colon cancer.Br. J. Cancer. 2007; 97: 1021-1027Crossref PubMed Scopus (62) Google Scholar, 12Shiovitz S. Bertagnolli M.M. Renfro L.A. Nam E. Foster N.R. Dzieciatkowski S. Luo Y. Lao V.V. Monnat Jr., R.J. Emond M.J. et al.Alliance for Clinical Trials in OncologyCpG island methylator phenotype is associated with response to adjuvant irinotecan-based therapy for stage III colon cancer.Gastroenterology. 2014; 147: 637-645Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Therefore, the need for biomarkers to more precisely identify stage II/III CRC patients that are suitable for adjuvant chemotherapy is highlighted. The biomarkers that doctors use to predict CRC patients' prognosis and therapeutic response at present, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9),13Sener S.F. Imperato J.P. Chmiel J. Fremgen A. Sylvester J. The use of cancer registry data to study preoperative carcinoembryonic antigen level as an indicator of survival in colorectal cancer.CA Cancer J. Clin. 1989; 39: 50-57Crossref PubMed Google Scholar,14Zhong W. Yu Z. Zhan J. Yu T. Lin Y. Xia Z.S. Yuan Y.H. Chen Q.K. Association of serum levels of CEA, CA199, CA125, CYFRA21-1 and CA72-4 and disease characteristics in colorectal cancer.Pathol. Oncol. Res. 2015; 21: 83-95Crossref PubMed Google Scholar have relatively low sensitivity and specificity.15Sanders D.S. Kerr M.A. Lewis blood group and CEA related antigens; coexpressed cell-cell adhesion molecules with roles in the biological progression and dissemination of tumours.Mol. Pathol. 1999; 52: 174-178Crossref PubMed Google Scholar,16Ritts Jr., R.E. Del Villano B.C. Go V.L. Herberman R.B. Klug T.L. Zurawski Jr., V.R. Initial clinical evaluation of an immunoradiometric assay for CA 19-9 using the NCI serum bank.Int. J. Cancer. 1984; 33: 339-345Crossref PubMed Google Scholar In this case, more meticulous biomarkers are needed. Thanks to the development of genomic high-throughput screening technologies, including next-generation sequencing and microarray analysis, a large number of molecular biomarkers and signatures with potential clinical prognostic and predictive values have been discovered via comprehensive association and bioinformatics analyses. Notably, several genomic-based biomarkers, such as the mismatch repair (MMR) or microsatellite instability (MSI) status, which plays a role as a predictive marker for adjuvant chemotherapy in patients with stage II CRC, have already entered into clinical practice and been validated.17Hewish M. Lord C.J. Martin S.A. Cunningham D. Ashworth A. Mismatch repair deficient colorectal cancer in the era of personalized treatment.Nat. Rev. Clin. Oncol. 2010; 7: 197-208Crossref PubMed Scopus (152) Google Scholar, 18Sinicrope F.A. DNA mismatch repair and adjuvant chemotherapy in sporadic colon cancer.Nat. Rev. Clin. Oncol. 2010; 7: 174-177Crossref PubMed Scopus (48) Google Scholar, 19Zaanan A. Shi Q. Taieb J. Alberts S.R. Meyers J.P. Smyrk T.C. Julie C. Zawadi A. Tabernero J. Mini E. et al.Role of deficient DNA mismatch repair status in patients with stage III colon cancer treated with FOLFOX adjuvant chemotherapy: a pooled analysis from 2 randomized clinical trials.JAMA Oncol. 2018; 4: 379-383Crossref PubMed Scopus (50) Google Scholar, 20Vilar E. Gruber S.B. Microsatellite instability in colorectal cancer-the stable evidence.Nat. Rev. Clin. Oncol. 2010; 7: 153-162Crossref PubMed Scopus (454) Google Scholar In this review, we summarize novel genomic and transcriptomic biomarkers as well as signatures reported recently with prognostic and predictive potentials in stage II/III CRC. We broadly examined current studies, focusing mainly on transcriptomic markers and signatures, which include newly discovered molecules such as microRNAs (miRNAs), long noncoding RNA (lncRNAs), and circular RNAs (circRNAs). We also assessed the clinical effectiveness of those already revealed and discussed their potential applications. The purpose of this review is to highlight some of the new candidate prognostic and predictive molecular markers for stage II/III CRC and assess their clinical utility for possible future applications. Currently, two major strategies are adopted by researchers to discover new molecular biomarkers: mechanism-based strategies and unbiased high-throughput screening. In the first scenario, a biomarker study often begins with candidate genes that play essential roles in tumorigenesis and cancer development with already defined molecular mechanisms that have been fully investigated both in vitro and in vivo. These genes are usually hallmark genes in biological processes such as the cell cycle, apoptosis, and drug metabolism.21Singh A. Sweeney M.F. Yu M. Burger A. Greninger P. Benes C. Haber D.A. Settleman J. TAK1 inhibition promotes apoptosis in KRAS-dependent colon cancers.Cell. 2012; 148: 639-650Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 22Whitfield M.L. George L.K. Grant G.D. Perou C.M. Common markers of proliferation.Nat. Rev. Cancer. 2006; 6: 99-106Crossref PubMed Scopus (371) Google Scholar, 23Popat S. Matakidou A. Houlston R.S. Thymidylate synthase expression and prognosis in colorectal cancer: a systematic review and meta-analysis.J. Clin. Oncol. 2004; 22: 529-536Crossref PubMed Scopus (383) Google Scholar In the case of CRC, Vogelstein et al.24Vogelstein B. Fearon E.R. Hamilton S.R. Kern S.E. Preisinger A.C. Leppert M. Nakamura Y. White R. Smits A.M. Bos J.L. Genetic alterations during colorectal-tumor development.N. Engl. J. Med. 1988; 319: 525-532Crossref PubMed Google Scholar proposed a model describing the sequential progression from adenoma to carcinoma process (Figure 1). Biomarkers developed by mechanism-based strategies include Kirsten rat sarcoma viral oncogene (KRAS), adenomatous polyposis coli (APC), and tumor protein p53 (TP53) that can be traced back to Vogelstein's model.25Wang M.T. Holderfield M. Galeas J. Delrosario R. To M.D. Balmain A. McCormick F. K-Ras promotes tumorigenicity through suppression of non-canonical Wnt signaling.Cell. 2015; 163: 1237-1251Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar,26Cancer Genome Atlas NetworkComprehensive molecular characterization of human colon and rectal cancer.Nature. 2012; 487: 330-337Crossref PubMed Scopus (5038) Google Scholar Investigators first group patients by clinical parameters (e.g., responders versus non-responders to fluorouracil-based chemotherapy), then they compare the expression levels of the candidate genes between groups and evaluate the power of these genes in distinguishing patients' grouping status. However, only discrete genes with well-known roles, but not new molecules, can be investigated and developed as biomarkers using this hypothesis-driven, mechanism-based strategy. Furthermore, this strategy might lead to unilateral conclusions considering the heterogeneity of CRC. While target-based approaches can search only a small part of the genome, unbiased high-throughput profiling has opened new avenues for marker discovery by screening the whole genome. For example, whole-transcriptome expression can be profiled comprehensively by high-throughput technologies such as transcriptomic assays and RNA sequencing. Moreover, by the use of modified library construction or capture probes, as well as other bioinformatics analysis strategies, the technology can help develop novel RNA biomarkers such as miRNAs,29Zhang J.X. Song W. Chen Z.H. Wei J.H. Liao Y.J. Lei J. Hu M. Chen G.Z. Liao B. Lu J. et al.Prognostic and predictive value of a microRNA signature in stage II colon cancer: a microRNA expression analysis.Lancet Oncol. 2013; 14: 1295-1306Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar lncRNAs,30Wang R. Du L. Yang X. Jiang X. Duan W. Yan S. Xie Y. Zhu Y. Wang Q. Wang L. et al.Identification of long noncoding RNAs as potential novel diagnosis and prognosis biomarkers in colorectal cancer.J. Cancer Res. Clin. Oncol. 2016; 142: 2291-2301Crossref PubMed Scopus (43) Google Scholar and circRNAs.31Ju H.Q. Zhao Q. Wang F. Lan P. Wang Z. Zuo Z.X. Wu Q.N. Fan X.J. Mo H.Y. Chen L. et al.A circRNA signature predicts postoperative recurrence in stage II/III colon cancer.EMBO Mol. Med. 2019; 11: e10168Crossref PubMed Scopus (42) Google Scholar Specifically, CRC can be better characterized by transcriptomic subtypes that encompass not only information within the tumor but also stromal and immune components.32Dienstmann R. Vermeulen L. Guinney J. Kopetz S. Tejpar S. Tabernero J. Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer.Nat. Rev. Cancer. 2017; 17: 79-92Crossref PubMed Scopus (347) Google Scholar Genomic and bioinformatics technologies have yielded many potential biomarkers and related signatures, which may prove useful with prognostic and predictive values. Data acquired from high-throughput technologies such as microarrays are usually enormous and disordered. Researchers use hierarchical clustering analysis to examine these data.33Oh S.C. Park Y.Y. Park E.S. Lim J.Y. Kim S.M. Kim S.B. Kim J. Kim S.C. Chu I.S. Smith J.J. et al.Prognostic gene expression signature associated with two molecularly distinct subtypes of colorectal cancer.Gut. 2012; 61: 1291-1298Crossref PubMed Scopus (0) Google Scholar Hierarchical clustering analysis is considered an unsupervised clustering method that is suitable for an exploratory analysis to determine how microarray groups cluster together according to similar features without taking experimental variables into account. Patients are sorted according to similarities in their gene expression profiles to predict different clinical outcomes. Genomic instability and oncogene mutations, tumor suppressor gene mutations, and mismatch repair genes can serve as biomarkers. Here, we summarize a few clinically approved genomic biomarkers in stage II/III CRC. CIN and MSI are two types of genomic instabilities. CIN refers to the increased rate of chromosomal gain and loss that leads to numerical or structural chromosome aberrations. MSI is defined as tumors having a defective DNA mismatch repair system due to the inactivation of either one of the following genes as the dominant genomic feature: mutL homolog 1 (MLH1), MLH3, mutS homolog 2 (MSH2), MSH3, MSH6, or PMS1 homolog 2 (PMS2).26Cancer Genome Atlas NetworkComprehensive molecular characterization of human colon and rectal cancer.Nature. 2012; 487: 330-337Crossref PubMed Scopus (5038) Google Scholar,34Grady W.M. Carethers J.M. Genomic and epigenetic instability in colorectal cancer pathogenesis.Gastroenterology. 2008; 135: 1079-1099Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar,35Boland C.R. Goel A. Microsatellite instability in colorectal cancer.Gastroenterology. 2010; 138: 2073-2087.e3Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar CIN is more commonly presented in CRC than MSI.36Boland C.R. Thibodeau S.N. Hamilton S.R. Sidransky D. Eshleman J.R. Burt R.W. Meltzer S.J. Rodriguez-Bigas M.A. Fodde R. Ranzani G.N. Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar The prognostic value of both CIN and MSI has been validated in a large meta-analysis.37Walther A. Houlston R. Tomlinson I. Association between chromosomal instability and prognosis in colorectal cancer: a meta-analysis.Gut. 2008; 57: 941-950Crossref PubMed Scopus (250) Google Scholar,38Popat S. Hubner R. Houlston R.S. Systematic review of microsatellite instability and colorectal cancer prognosis.J. Clin. Oncol. 2005; 23: 609-618Crossref PubMed Scopus (1317) Google Scholar Patients with CIN disease have a worse prognosis than those without CIN. In addition, MSI-high (MSI-H) patients have a better prognosis than microsatellite stable (MSS) patients in stage II CRCs39Petrelli F. Ghidini M. Cabiddu M. Pezzica E. Corti D. Turati L. Costanzo A. Varricchio A. Ghidini A. Barni S. Tomasello G. Microsatellite instability and survival in stage II colorectal cancer: a systematic review and meta-analysis.Anticancer Res. 2019; 39: 6431-6441Crossref PubMed Scopus (11) Google Scholar and maintain their survival advantages in the presence of 5-fluorouracil (5-FU) treatment;40Klingbiel D. Saridaki Z. Roth A.D. Bosman F.T. Delorenzi M. Tejpar S. Prognosis of stage II and III colon cancer treated with adjuvant 5-fluorouracil or FOLFIRI in relation to microsatellite status: results of the PETACC-3 trial.Ann. Oncol. 2015; 26: 126-132Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar however, MSI has no prognostic value in stage III CRCs.41Wang B. Li F. Zhou X. Ma Y. Fu W. Is microsatellite instability-high really a favorable prognostic factor for advanced colorectal cancer? A meta-analysis.World J. Surg. Oncol. 2019; 17: 169Crossref PubMed Scopus (5) Google Scholar Aberrant DNA methylation is the most broadly studied epigenetic alteration in cancer. The CIMP was first described in CRC in 1999 by Toyota et al.42Toyota M. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. CpG island methylator phenotype in colorectal cancer.Proc. Natl. Acad. Sci. USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2020) Google Scholar It was characterized as a cluster of exceptionally hypermethylated CpG dinucleotides. Weisenberger et al.43Weisenberger D.J. Siegmund K.D. Campan M. Young J. Long T.I. Faasse M.A. Kang G.H. Widschwendter M. Weener D. Buchanan D. et al.CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer.Nat. Genet. 2006; 38: 787-793Crossref PubMed Scopus (1435) Google Scholar later defined CIMP in CRC based on the methylation status of five genes: calcium channel voltage dependent alpha 1G (CACNA1G), insulin-like growth factor 2 (IGF2), neurogenin 1 (NEUROG1), RUNX family transcription factor 3 (RUNX3), and suppressor of cytokine signaling (SOCS). Multiple, extensive clinical studies have demonstrated that specific methylated DNA signatures could be developed as prognostic and predictive biomarkers in CRC. Several independent studies reported that CIMP-positive cancers were correlated with an unfavorable prognosis, including a cohort of more than 600 MSS CRC patients by Lee et al.44Lee S. Cho N.Y. Choi M. Yoo E.J. Kim J.H. Kang G.H. Clinicopathological features of CpG island methylator phenotype-positive colorectal cancer and its adverse prognosis in relation to KRAS/BRAF mutation.Pathol. Int. 2008; 58: 104-113Crossref PubMed Scopus (128) Google Scholar and a cohort of 206 stage III CRC patients by van Rijnsoever et al.45Van Rijnsoever M. Elsaleh H. Joseph D. McCaul K. Iacopetta B. CpG island methylator phenotype is an independent predictor of survival benefit from 5-fluorouracil in stage III colorectal cancer.Clin. Cancer Res. 2003; 9: 2898-2903PubMed Google Scholar This conclusion was validated later in two other cohorts of stage II/III patients.46Tan I.B. Ivanova T. Lim K.H. Ong C.W. Deng N. Lee J. Tan S.H. Wu J. Lee M.H. Ooi C.H. et al.Intrinsic subtypes of gastric cancer, based on gene expression pattern, predict survival and respond differently to chemotherapy.Gastroenterology. 2011; 141 (485.e1–11): 476-485Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar,47Min B.H. Bae J.M. Lee E.J. Yu H.S. Kim Y.H. Chang D.K. Kim H.C. Park C.K. Lee S.H. Kim K.M. Kang G.H. The CpG island methylator phenotype may confer a survival benefit in patients with stage II or III colorectal carcinomas receiving fluoropyrimidine-based adjuvant chemotherapy.BMC Cancer. 2011; 11: 344Crossref PubMed Scopus (69) Google Scholar However, the opinions differ: some studies have suggested that the prognosis of CIMP-positive CRC patients depends on the MSI status of the tumor,48Suzuki H. Yamamoto E. Maruyama R. Niinuma T. Kai M. Biological significance of the CpG island methylator phenotype.Biochem. Biophys. Res. Commun. 2014; 455: 35-42Crossref PubMed Scopus (0) Google Scholar,49Juo Y.Y. Johnston F.M. Zhang D.Y. Juo H.H. Wang H. Pappou E.P. Yu T. Easwaran H. Baylin S. van Engeland M. Ahuja N. Prognostic value of CpG island methylator phenotype among colorectal cancer patients: a systematic review and meta-analysis.Ann. Oncol. 2014; 25: 2314-2327Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar while other studies have suggested that the poor prognosis of CIMP-positive CRCs was from coexisting B-Raf proto-oncogene (BRAF) V600E mutations.44Lee S. Cho N.Y. Choi M. Yoo E.J. Kim J.H. Kang G.H. Clinicopathological features of CpG island methylator phenotype-positive colorectal cancer and its adverse prognosis in relation to KRAS/BRAF mutation.Pathol. Int. 2008; 58: 104-113Crossref PubMed Scopus (128) Google Scholar,50Ogino S. Nosho K. Kirkner G.J. Kawasaki T. Meyerhardt J.A. Loda M. Giovannucci E.L. Fuchs C.S. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer.Gut. 2009; 58: 90-96Crossref PubMed Scopus (601) Google Scholar, 51Pai R.K. Jayachandran P. Koong A.C. Chang D.T. Kwok S. Ma L. Arber D.A. Balise R.R. Tubbs R.R. Shadrach B. Pai R.K. BRAF-mutated, microsatellite-stable adenocarcinoma of the proximal colon: an aggressive adenocarcinoma with poor survival, mucinous differentiation, and adverse morphologic features.Am. J. Surg. Pathol. 2012; 36: 744-752Crossref PubMed Scopus (144) Google Scholar, 52Lochhead P. Kuchiba A. Imamura Y. Liao X. Yamauchi M. Nishihara R. Qian Z.R. Morikawa T. Shen J. Meyerhardt J.A. et al.Microsatellite instability and BRAF mutation testing in colorectal cancer prognostication.J. Natl. Cancer Inst. 2013; 105: 1151-1156Crossref PubMed Scopus (290) Google Scholar These contrary findings may be due to the heterogeneity of patient cohorts or the different CIMP criteria used in different studies. However, the CIMP status is still promising and can be further investigated as a prognostic factor. The KRAS proto-oncogene encodes a small GTPase that affects cell proliferation and differentiation.53Worthley D.L. Whitehall V.L. Spring K.J. Leggett B.A. Colorectal carcinogenesis: road maps to cancer.World J. Gastroenterol. 2007; 13: 3784-3791Crossref PubMed Scopus (95) Google Scholar KRAS mutations in codons 12, 13, and 61 impair the GTPase activity of the encoded protein, which leads to the consistent activation of RAS/RAF signaling.54Markowitz S.D. Bertagnolli M.M. Molecular origins of cancer: molecular basis of colorectal cancer.N. Engl. J. Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1262) Google Scholar KRAS mutations occur at the early stage of CRC, accounting for one-third of all cases, and a large majority of the mutations are located in codon 12 (approximately 80%), followed by codon 13.55Andreyev H.J. Norman A.R. Cunningham D. Oates J.R. Clarke P.A. Kirsten ras mutations in patients with colorectal cancer: the multicenter "RASCAL" study.J. Natl. Cancer Inst. 1998; 90: 675-684Crossref PubMed Google Scholar Many studies have evaluated KRAS as a prognostic biomarker based on its association with CRC outcomes.50Ogino S. Nosho K. Kirkner G.J. Kawasaki T. Meyerhardt J.A. Loda M. Giovannucci E.L. Fuchs C.S. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer.Gut. 2009; 58: 90-96Crossref PubMed Scopus (601) Google Scholar,55Andreyev H.J. Norman A.R. Cunningham D. Oates J.R. Clarke P.A. Kirsten ras mutations in patients with colorectal cancer: the multicenter "RASCAL" study.J. Natl. Cancer Inst. 1998; 90: 675-684Crossref PubMed Google Scholar, 56Andreyev H.J. Norman A.R. Cunningham D. Oates J. Dix B.R. Iacopetta B.J. Young J. Walsh T. Ward R. Hawkins N. et al.Kirsten ras mutations in patients with colorectal cancer: the "RASCAL II" study.Br. J. Cancer. 2001; 85: 692-696Crossref PubMed Scopus (0) Google Scholar, 57Samowitz W.S. Curtin K. Schaffer D. Robertson M. Leppert M. Slattery M.L. Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study.Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1193-1197PubMed Google Scholar, 58Ince W.L. Jubb A.M. Holden S.N. Holmgren E.B. Tobin P. Sridhar M. Hurwitz H.I. Kabbinavar F. Novotny W.F. Hillan K.J. Koeppen H. Association of k-ras, b-raf, and p53 status with the treatment effect of bevacizumab.J. Natl. Cancer Inst. 2005; 97: 981-989Crossref PubMed Scopus (291) Google Scholar The RASCAL study, a large meta-analysis, found that the glycine to valine substitution in codon 12 of KRAS was associated with a poor prognosis in stage III CRC patients.56Andreyev H.J. Norman A.R. Cunningham D. Oates J. Dix B.R. Iacopetta B.J. Young J. Walsh T. Ward R. Hawkins N. et al.Kirsten ras mutations in patients with colorectal cancer: the "RASCAL II" study.Br. J. Cancer. 2001; 85: 692-696Crossref PubMed Scopus (0) Google Scholar However, several other large studies found no association.50Ogino S. Nosho K. Kirkner G.J. Kawasaki T. Meyerhardt J.A. Loda M. Giovannucci E.L. Fuchs C.S. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer.Gut. 2009; 58: 90-96Crossref PubMed Scopus (601) Google Scholar,57Samowitz W.S. Curtin K. Schaffer D. Robertson M. Leppert M. Slattery M.L. Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study.Cancer Epidemiol. Biomarkers Prev. 2000; 9: 1193-1197PubMed Google Scholar,58Ince W.L. Jubb A.M. Holden S.N. Holmgren E.B. Tobin P. Sridhar M. Hurwitz H.I. Kabbinavar F. Novotny W.F. Hillan K.J. Koeppen H. Association of k-ras, b-raf, and p53 status with the treatment effect of bevacizumab.J. Natl. Cancer Inst. 2005; 97: 981-989Crossref PubMed Scopus (291) Google Scholar In stage II CRC, no prognostic significance was found of the KRAS mutation type.59Wang C. van Rijnsoever M. Grieu F. Bydder S. Elsaleh H. Joseph D. Harvey J. Iacopetta B. Prognostic significance of microsatellite instability and Ki-ras mutation type in stage II colorectal cancer.Oncology. 2003; 64: 259-265Crossref PubMed Scopus (57) Google Scholar BRAF is the downstream target of KRAS. Mutations in KRAS and BRAF seem to be mutually exclusive.60Rajagopalan H. Bardelli A. Lengauer C. Kinzler K.W. Vogelstein B. Velculescu V.E. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status.Nature. 2002; 418: 934Crossref PubMed Scopus (1023) Google Scholar,61Di Nicolantonio F. Martini M. Molinari F. Sartore-Bianchi A. Arena S. Saletti P. De Dosso S. Mazzucchelli L. Frattini M. Siena S. Bardelli A. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer.J. Clin. Oncol. 2008; 26: 5705-5712Crossref PubMed Scopus (1383) Google Scholar BRAF mutation was considered a prognosticator for poor survival in stage II/III CRC patients receiving adjuvant chemotherapy.62Zhu L. Dong C. Cao Y. Fang X. Zhong C. Li D. Yuan Y. Prognostic role of BRAF mutation in stage II/III colorectal cancer receiving curative resection and adjuvant chemotherapy: a meta-analysis based on randomized clinical trials.PLoS ONE. 2016; 11: e0154795Crossref PubMed Scopus (18) Google Scholar The most famous BRAF V600E mutation was also reported to