Lack of Aberrant Methylation in an Adjacent Area of Left-Sided Colorectal Cancer

The molecular nature and the rate-limiting step of epigenetic field defects in the evolution of left-sided colorectal cancer (LCA) remain uncertain. The methylation status of 27 candidate field defect markers, six classic CpG island methylator phenotype (CIMP) markers, and LINE-1 were determined in LCA and adjacent normal mucosas (ADJs) from 33 LCA patients and in left normal colorectal mucosa (LNM) from 33 age- and sex-matched controls. Hotspot mutation analyses in KRAS codons 12 and 13 and BRAF V600E were performed by genomic PCR and pyrosequencing using DNA extracted from endoscopically biopsied tissues. Among the 27 candidate genes tested, we confirmed 15 differentially methylated genes in cancer (15 DMGs; ER, SFRP1, MYOD1, MGMT, CD8a, SPOCK2, ABHD9, BNIP3, IGFBP3, WIF1, MAL, GDNF, ALX4, DOK5, and SLC16A12) in comparison to ADJ samples. We further compared the methylation status of 15 DMGs of ADJs to LNM and found only methylation levels of SLC16A12 in ADJs of LCA patients to be significantly higher than that in LNM (17.3% vs. 11.5%, p=0.002). Based on the CIMP, no significant differences in methylation levels of the 15 DMGs were found between ADJs in CIMP positive LCA cases and those without CIMP. In mutation analyses, no mutation was found in ADJs, while significant KRAS mutations (6/33, 18%) were noted in LCA samples. Epigenetic field defect marked by aberrant methylation is uncommon in normal-appearing ADJs of LCA, indicating the critical rate-limiting change of methylation is likely to occur with morphological alterations in the evolution of LCA.