CpG Island Methylator Phenotype in Colorectal Cancers: Comparison of the New and Classic CpG Island Methylator Phenotype Marker Panels

Archives of Pathology & Laboratory Medicine, Oct 2008 by Lee, Sun, Cho, Nam-Yun, Yoo, Eun Joo, Kim, Jung Ho, Kang, Gyeong Hoon

Context.-CpG island methylator phenotype (CIMP) designates a subset of colorectal cancers featuring concordant hypermethylation of multiple promoter CpG islands. Little is known about the clinical outcome or histologic characteristics of CIMP-positive colorectal cancers defined by recently identified CpG island methylator phenotype panels.

Objective.-To investigate and compare the molecular and clinicopathologic features of CIMP-positive colorectal cancers defined by classic (p16, hMLH1, MINT1, MINT2, MINT31) and new (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1) CIMP panels.

Design.-We analyzed 130 colorectal cancers for hypermethylation of both panels using methylation-specific polymerase chain reaction.

Results.-With at least 2 markers methylated, both classic (39/130; 23.1%) and new (23.1%) CIMP-positive colorectal cancers were significantly associated with proximal tumor location, microsatellite instability, and BRAF mutation (all P values were less than .05). The new panel outperformed the classic panel in detecting these features. With at least 3 markers methylated, new CIMP-positive colorectal cancers (16.9%) were closely associated with proximal tumor location, low frequency of KRAS mutation, and high frequency of BRAF mutation (all P values were less than .05), whereas classic CIMP-positive colorectal cancers (18.5%) were closely associated with proximal tumor location, frequent microsatellite instability, and frequent BRAF mutation (all P values were less than .05). Analyzing a combination of CIMP and microsatellite instability status, CIMP-positive/microsatellite instability-negative colorectal cancers had the worst clinical outcomes.

Conclusions.-Whereas the classic panel outperformed in predicting clinical outcome, the new panel was superior in detecting known clinicopathologic features of CIMP but inferior in prognostication power.

(Arch Pathol Lab Med. 2008;132:1657-1665)

Promoter CpG island hypermethylation is an important epigenetic change associated with gene silencing and is now recognized as an alternative mechanism to mutations for the inactivation of tumor suppressor genes or tumor-related genes in cancer cells.1 CpG island methylator phenotype (CIMP) is a subset of colorectal carcinoma (CRC) characterized by concordant hypermethylation of multiple promoter CpG island loci2 and peculiar clinicopathologic findings, such as close association with microsatellite instability (MSI), proximal tumor location, female preponderance, high frequency of mutation of the protooncogene BRAF, and low frequency of mutation of the proto- oncogene KRAS.2-9 Furthermore, CIMP-positive (CIMP+) CRCs have been reported to be associated with poor clinical outcomes.10-12

A new CIMP marker panel comprising the genes CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1 was recently introduced by the Laird laboratory7 as superior to the original CIMP marker panel, comprising CDKN2A, MINT1, MINT2, MINT31, and MLH1,5,13,14 in that the new CIMP panel easily detected a heavily methylated subset of CRCs that encompasses almost all BRAF mutants and sporadic MSI-high CRCs. However, prognostic power was not compared between the 2 CIMP panels. Moreover, both CIMP panels were tested on the platform of real-time- based, quantitative, methylation-specific polymerase chain reaction (PCR; MethyLight technology) rather than with traditional methylation-specific PCR (MSP). Methylated alleles of most CpG island loci are heterogeneous with respect to the methylation status of individual CpG sites,15-17 and thus the MethyLight assay detects a subset of DNA alleles that are heavily methylated in all CpG sites targeted by both primers and probe.18,19 The MethyLight assay cannot, however, detect alleles that are not fully methylated in the targeted CpG sites but are sufficiently methylated to inactivate gene expression.20 Traditional MSP detects both fully methylated alleles and substantially but incompletely methylated alleles. Thus, it remains to be determined whether this new CIMP panel outperforms the original panel using traditional MSP.

In the present study, we used traditional MSP to determine the CIMP status of 130 sporadic CRCs based on both the new and classic CIMP panels and explored the differences in clinicopathologic and molecular features between CIMP+ CRCs defined by each marker panel. Moreover, both CIMP panels were compared for their prognostic power.

MATERIALS AND METHODS

Tissue Samples

Our study was approved by the Seoul National UniversityHospital's Institutional Review Board. Tumor tissues were obtained from 130 patients with sporadic CRC who had undergone curative surgery at Seoul National University Hospital, Seoul, Korea, between 2001 and 2002. Formalin-fixed, paraffin-embedded tissues were used for DNA extraction. Through light microscopic examination, we marked tumor areas where tumor cells occupied 50% or more of all cells and represented the main histology and differentiation of the tumor. Ten serial 10-µm-thick histologic slides of methanol-fixed tumor and normal tissue blocks were used for manual microdissection. Dissected tissue samples were subjected to tissue lysis using proteinase K-containing lysis buffer. Mutation analysis of KRAS codons 12 and 13 and BRAF codon 600 and MSI analysis were previously performed and determined for these 130 samples.21 Clinicopathologic information, including age, sex, histologic type, tumor location, stage, and overall survival, was obtained from all patients. Tumor staging was based on the TNM staging system of the American Joint Committee on Cancer.22