Evaluation of Markers for CpG Island Methylator Phenotype (CIMP) in Colorectal Cancer by a Large Population-Based Sample

Abstract

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation is a distinct phenotype in colorectal cancer. However, a choice of markers for CIMP has been controversial. A recent extensive investigation has selected five methylation markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) as surrogate markers for epigenomic aberrations in tumor. The use of these markers as a CIMP-specific panel needs to be validated by an independent, large dataset. Using MethyLight assays on 920 colorectal cancers from two large prospective cohort studies, we quantified DNA methylation in eight CIMP-specific markers [the above five plus CDKN2A (p16), CRABP1, and MLH1]. A CIMP-high cutoff was set at > or = 6/8 or > or = 5/8 methylated promoters, based on tumor distribution and BRAF/KRAS mutation frequencies. All but two very specific markers [MLH1 (98% specific) and SOCS1 (93% specific)] demonstrated > or = 85% sensitivity and > or = 80% specificity, indicating overall good concordance in methylation patterns and good performance of these markers. Based on sensitivity, specificity, and false positives and negatives, the eight markers were ranked in order as: RUNX3, CACNA1G, IGF2, MLH1, NEUROG1, CRABP1, SOCS1, and CDKN2A. In conclusion, a panel of markers including at least RUNX3, CACNA1G, IGF2, and MLH1 can serve as a sensitive and specific marker panel for CIMP-high.

Methylation of the 3p22 region encompassing MLH1 is representative of the CpG island methylator phenotype in colorectal cancer

“…Regional 3p22 methylation markers may thus serve as an alternative set of markers to characterize CIMP status, providing further support for the previous recommendation that MLH1 should be included as one of the markers in the panel for the characterization of CIMP þ. 16 It was notable that CIMP þ and the BRAF V600E mutation were all independent predictors of regional 3p22 methylation in multivariate analysis, despite their strong associations with one another. This suggests that heterogeneity in genetic and epigenetic events may still underlie the development of a given subset of colorectal cancers with strong molecular and clinicopathological similarity.…”

“…40 Previous observations between the BRAF V600E mutation and methylation of multiple genes at distinct loci in conjunction with CIMP þ have been reported. 14,16,[47][48] One other study has also reported the close association between long-range epigenetic silencing of the 2q14 region and CIMP þ in sporadic colorectal cancer. 24 Taken together, the independent findings of long-range epigenetic silencing of both the 3p22 and 2q14 regions in the context of CIMP þ provide further support for a common mechanism governing epigenetic dysregulation that presents with the overlapping features of long-range epigenetic silencing and CIMP in the development of sporadic colorectal cancer.…”

“…14 Cancers were considered CIMP þ when Z3/5 (60) markers showed intensities of methylation at PMR 44.0. 16 …”

“…The CpG island methylator phenotype (CIMP) has been proposed to describe the simultaneous methylation of multiple but discrete CpG islands at distinct loci throughout the genome in the development of a subset of colorectal cancers, now referred to as CIMP þ. [13][14][15] Interestingly, the CIMP þ phenotype also correlates closely with the presence of the BRAF V600E mutation, MLH1 methylation and microsatellite instability, [13][14][15][16] although the basis for the inter-relationship between these genetic and epigenetic phenomena remains unknown. More recently, long-range epigenetic silencing, in which concordant CpG island methylation and chromatin modification across large genomic regions induces silencing of multiple contiguous genes, has also been demonstrated for specific chromosomal regions in a proportion of sporadic colorectal cancers.…”

“…24 In the present study, we analysed a cohort of 946 sporadic colorectal cancer cases to determine whether MLH1 methylation is associated with the germline c.-93G4A SNP genotype or methylation of the 3p22 region, and furthermore, if methylation of the 3p22 gene cluster correlates with the CIMP þ phenotype. As it is now well established that a small subset of tumours exist that are microsatellite stable, BRAF V600E mutant and CIMP þ, [14][15][16]25 we also sought evidence that this subtype of microsatellite stable cancers is concomitantly methylated at gene promoters within the 3p22 chromosomal region other than MLH1. Finally, to determine whether methylation of MLH1 and flanking genes, as well as the BRAF V600E mutation, might predispose to cancer development, we sought these changes in the apparently normal colorectal mucosa from a subset of the colorectal cancer cases and controls without neoplasia.…”