Epigenotyping by quantitative DNA methylation analysis revealed three epigenotypes in colorectal cancer

Aberrant DNA methylation, one of the major epigenetic alterations in cancer, has been reported to accumulate in a subset of colorectal cancer (CRC), so-called CpG island methylator phenotype (CIMP), which was known to correlate with microsatellite instability (MSI)-high CRC. To select new methylation markers genome-widely and epigenotype CRC by DNA methylation comprehensively, we performed methylated DNA immunoprecipitation-on-chip analysis using MSI-high CRC cell line HCT116 and microsatellite-stable SW480, and re-expression array analysis by 5-aza-2-deoxycytidine/Trichostatin A. Methylation levels of 44 new markers selected and 16 previously reported markers were analyzed quantitatively in 149 clinical CRC samples using MALDI-TOF mass spectrometry. By unsupervised two-way hierarchical clustering, CRC was clustered into high-, intermediate-, and low-methylation epigenotypes. Methylation markers were clustered into two groups: Group-1 showing methylation in high-methylation epigenotype and including all the 11 CIMP-related markers except NEUROG1, and Group-2 showing methylation in high- and intermediate-methylation epigenotypes. Marker panel deciding methylation epigenotypes with the highest accuracy was developed: 1st-Panel consisting of Group-1 genes (CACNA1G, LOX, SLC30A10) to extract high-methylation epigenotype, and 2nd-Panel consisting of Group-2 genes (ELMO1, FBN2, THBD, HAND1) and SLC30A10 again to divide the remains into intermediate- and low-methylation epigenotypes. High-methylation epigenotype correlated significantly with BRAF mutation, MSI, proximal tumor location, and mucinous component, in concordance with reported CIMP. Intermediate- and low-methylation epigenotypes significantly correlated with KRAS-mutation(+) and KRAS-mutation(-), respectively. KRAS-mutation(+) intermediate-methylation epigenotype showed worse prognosis than KRAS-mutation(-) low-methylation epigenotype (p=0.030). These three epigenotypes with different genetic characteristics suggested different molecular CRC genesis, and the markers might be useful to predict prognosis.

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Yagi K, Akagi K, Hayashi H, Nagae G, Tsuji S, Isagawa T, Midorikawa Y, Nishimura Y, Sakamoto H, Seto Y, Aburatani H, Kaneda A