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.

Figure 1

BRAF and KRAS mutation frequencies according to number of methylated promoters. A: Tumors with ≥6/8 methylated promoters show high BRAF mutation rates, whereas tumors with ≤5/8 methylated promoters show high KRAS mutation rates. B: MSI-H tumors distribute bimodally, and the frequencies of KRAS and BRAF mutations clearly distinguish CIMP-high tumors from CIMP-low tumors. C: MSI-L/MSS tumors can be separated into CIMP-high (≥6/8 methylated promoters) and CIMP-low/0 (≤4/8 methylated promoters) based on the frequencies of BRAF and KRAS mutations. Tumors with 5/8 methylated promoters reside on the borderline between CIMP-high and CIMP-low.

Figure 2

Sensitivity, specificity, and cross-panel classification error rate against panel 8. Panel 1 (RUNX3 only) through panel 7 contain incrementing numbers of markers, adding one by one from CACNA1G, IGF2, MLH1, NEUROG1, CRABP1, and SOCS1. Panel 8 contains all eight markers including CDKN2A. A: Specificity generally increases with an increasing number of markers. Sensitivity depends on the number of markers and a CIMP-high cutoff. B: The classification error rate decreases with an increasing number of markers.

Figure 3

Frequencies of right-sided tumors (A), poorly differentiated tumors (B), and mucinous tumors (C) in various MSI/CIMP subtypes of colorectal cancer. Gray and open bar graphs indicate frequencies of each feature in MSI/CIMP subtypes determined by CIMP panel 4 and CIMP panel 8, respectively. Note that there were no substantial differences in the features examined (anatomical location, tumor grade, or mucinous features) between classifications determined by CIMP panel 4 and panel 8.