Comprehensive Biostatistical Analysis of CpG Island Methylator Phenotype in Colorectal Cancer Using a Large Population-Based Sample

Abstract

BackgroundThe CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers) using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel.Metholodology/Principal FindingsDNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers), multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and β-catenin (CTNNB1) activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status.ConclusionsOur study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern which may lead to CIMP-low tumors.

Loss of heterozygosity, aberrant methylation, BRAF mutation and KRAS mutation in colorectal signet ring cell carcinoma

“…In one study, CpG island methylation phenotype-positive status was observed in 17% of cases with signet ring cells; however, the standard definition of signet ring cell carcinoma (450% signet ring cells) was not used in this study. 43 CpG island methylation phenotype-positive status, as defined in this study, was observed in nearly half of the signet ring cell carcinomas, and was associated with BRAF V600E mutation, but did not correlate with any clinicopathologic feature or survival. This association of CpG island methylation phenotypepositive status with BRAF V600E mutation is similar to that observed in other histologic subtypes of colorectal cancer.…”

Colorectal carcinomas with KRAS mutation are associated with distinctive morphological and molecular features

“…Our exon 2 KRAS mutation rate of 28% is in agreement with previous studies when mutations in exon 2 only are considered. 10,11,16,17 Like BRAF-mutated carcinomas, KRAS-mutated carcinomas showed an increased frequency of mucinous differentiation. This finding has been previously observed by Lin et al 28 and more recently by Pai et al, 8 who reported that 32% of proximal KRAS-mutated carcinoma showed mucinous differentiation compared with 25% of null carcinomas.…”

“…1,8,9 Somatic-activating mutation in the KRAS proto-oncogene is observed in 35-40% of colorectal carcinoma. 7,10,11 The overwhelming majority of KRAS mutations (90%) occur in codons 12 and 13 of exon 2, with the most frequent alteration being a G4A transition in codon 12. 12 The clinical significance of KRAS mutation in colorectal carcinoma patients is controversial; some studies reported no association with survival, 13,14 whereas others suggested that patients with KRASmutated colorectal carcinoma have poorer outcome for any mutation subtype, 15 mutation in codon 12 only 16,17 or codon 13 only.…”

Oncogenic mutations as predictive factors in colorectal cancer

“…Although the EGFR gene is not or very rarely mutated in colorectal tumours, activating mutations of KRAS or BRAF, involved in the RAS/MAPK pathway, are present in 32-37% and in 10-17% of the cases in large population-based studies, respectively (Samowitz et al, 2000;Rajagopalan et al, 2002;Brink et al, 2003;Samowitz et al, 2005a;Nosho et al, 2008a;de Vogel et al, 2009;Ogino et al, 2009b), and mutations of PI3KCA that encodes the catalytic subunit of the PI3K protein involved in the PI3K/AKT pathway are observed in 15% of the cases (Bamford et al, 2004;Barault et al, 2008b;Nosho et al, 2008b).…”