Multiple sporadic colorectal cancers display a unique methylation phenotype

Abstract

Epigenetics are thought to play a major role in the carcinogenesis of multiple sporadic colorectal cancers (CRC). Previous studies have suggested concordant DNA hypermethylation between tumor pairs. However, only a few methylation markers have been analyzed. This study was aimed at describing the epigenetic signature of multiple CRC using a genome-scale DNA methylation profiling. We analyzed 12 patients with synchronous CRC and 29 age-, sex-, and tumor location-paired patients with solitary tumors from the EPICOLON II cohort. DNA methylation profiling was performed using the Illumina Infinium HM27 DNA methylation assay. The most significant results were validated by Methylight. Tumors samples were also analyzed for the CpG Island Methylator Phenotype (CIMP); KRAS and BRAF mutations and mismatch repair deficiency status. Functional annotation clustering was performed. We identified 102 CpG sites that showed significant DNA hypermethylation in multiple tumors with respect to the solitary counterparts (difference in β value ≥0.1). Methylight assays validated the results for 4 selected genes (p = 0.0002). Eight out of 12(66.6%) multiple tumors were classified as CIMP-high, as compared to 5 out of 29(17.2%) solitary tumors (p = 0.004). Interestingly, 76 out of the 102 (74.5%) hypermethylated CpG sites found in multiple tumors were also seen in CIMP-high tumors. Functional analysis of hypermethylated genes found in multiple tumors showed enrichment of genes involved in different tumorigenic functions. In conclusion, multiple CRC are associated with a distinct methylation phenotype, with a close association between tumor multiplicity and CIMP-high. Our results may be important to unravel the underlying mechanism of tumor multiplicity.

Figure 1. Heatmap showing the 90 most significantly hypermethylated CpG sites that differentiate multiple CRCs (n = 12) with respect to solitary tumors (n = 29) based on the Infinium DNA methylation data.

The DNA methylation β-values are represented by using a color scale from red (high DNA methylation) to green (low DNA methylation). Rows represent probes and columns represent tumor samples. Clinical and molecular features (group, gender, tumor location, CIMP-H and KRAS mutational status) are represented above the heatmap with horizontal bars.

Figure 2. Technical validation of Infinium methylation data using Methylight assays.

Four genes (MAP1B, HTRA1, ALOX15, TIMP3) were selected based on strict criteria (β value in solitary tumors <0.2; β value >0.3 in multiple tumors; difference in β value between multiple versus solitary ≥0.2; and an adjusted p value<0.05). Box-plots display the Percentage Methylation Ratio (PMR) determined by Methylight. The lines inside boxes denote median, and boxes mark the interval between the 25th and 75th percentiles. Black lines denote the highest and lowest PMR value. P values for the comparison between multiple (red) and solitary (blue) tumors (Mann-Whitney test) are shown.

Figure 3. Heatmap showing the 218 most significantly hypermethylated CpG sites that differentiate CIMP-H (n = 13) and CIMP-0/L tumors (n = 28) based on the Infinium DNA methylation data.

The DNA methylation β-values are represented by using a color scale from red (high DNA methylation) to green (low DNA methylation). Rows represent probes and columns represent tumor samples. Clinical and molecular features (group, gender, tumor location, CIMP-H and KRAS mutational status) are represented above the heatmap with horizontal bars.

Figure 4. Overlap between significantly hypermethylated CpG sites in multiple and CIMP-H tumors.

Blue circle shows 102 hypermethylated CpG sites found in multiple versus solitary tumors and yellow circle shows the 301 hypermethylated CpG sites in CIMP-H versus CIMP-L/0 tumors. Remarkably, 76 out of the 102 hypermethylated genes in multiple tumors were also seen to be hypermethylated in CIMP-H tumors, and are represented as an intersection.