Epigenetic profiling of synchronous colorectal neoplasias by quantitative DNA methylation analysis

Transcriptional inactivation of tumor-suppressor genes by promoter CpG island methylation is thought to be an important mechanism in human carcinogenesis. The CpG island methylator phenotype (CIMP) with extensive promoter methylation appears to be a distinct epigenetic subtype of colorectal carcinoma. Most previous studies on CpG island methylation in colorectal carcinoma used methylation-specific PCR, which may detect low levels of DNA methylation with little or no biological significance. In contrast, quantitative DNA methylation assays have been shown to provide useful information beyond that which can be achieved with methylation-specific PCR. Synchronous neoplasias provide a unique model for investigators to examine molecular alterations in multistep tumorigenesis within one individual. However, no study to date has quantified DNA methylation of CIMP-specific promoters in synchronous colorectal neoplasias. Utilizing real-time PCR (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G (calcium channel, voltage-dependent, T type alpha-1G subunit), CDKN2A (p16/INK4A), CRABP1 (cellular retinoic acid binding protein-1), MLH1 and NEUROG1 (neurogenin 1)] and MGMT in six synchronous carcinoma pairs (12 carcinomas) and eight synchronous carcinoma and adenoma pairs (16 tumors). We found that while some synchronous tumor pairs showed discordant promoter methylation patterns, other tumor pairs showed similar, but not exactly identical, patterns of promoter methylation. All but two pairs showed concordant patterns of CIMP status (CIMP positive vs CIMP negative) (P = 0.05 in cancer pairs). BRAF mutations were present in only CIMP-positive tumors. A high degree of microsatellite instability (MSI-H) was observed in both CIMP-positive and CIMP-negative tumors. KRAS mutations were not concordant in any synchronous neoplasia pair. In conclusion, epigenetic alterations at CIMP-specific promoter CpG islands in synchronous colorectal neoplasias likely have both random and nonrandom components.