DNA methylation signatures associated with tumor location in early-onset colorectal cancer identified across two independent cohorts

Background: Incidence of early-onset colorectal cancer (EOCRC; ages 18-49) continues to rise globally, with rectal cancers increasing more rapidly than proximal or distal colon cancers. Germline variation does not fully explain these trends or the disproportionate rise in rectal tumors, suggesting a role for epigenetic mechanisms. However, the association of DNA methylation (DNAm) to EOCRC tumor location remains poorly understood. Methods: We used The Cancer Genome Atlas (TCGA) as the discovery cohort to identify tumor location-specific DNAm markers in EOCRC. First, we conducted an epigenome-wide association study, screening Cytosine-Phosphate-Guanine site, (CpGs) at FDR< 0.2, followed by multivariable logistic regression adjusting for age, sex, stage, and treatment. CpG sites were modeled as independent variables and tumor location (rectum [defined as rectum and rectosigmoid] vs. colon) as the outcome; significant CpGs were defined at <fdr 0.05.="" to="" validate="" these="" findings,="" we="" tested="" the="" tcga-identified="" cpgs="" in="" an="" independent="" eocrc="" cohort="" of="" patient="" tumor="" tissue="" from="" ut="" southwestern="" medical="" center="" (utsw)="" using="" same="" analytic="" framework.="" for="" replicated="" cpgs,="" evaluated="" dnam="" patterns,="" mapped="" annotated="" genes,="" and="" conducted="" pathway="" enrichment="" analysis.="" tcga="" served="" as="" discovery="" dataset,="" utsw="" dataset="" replication="" cohort. Results: Among the TCGA cohort of 67 patients, (31.3% rectum, mean age 44 years, 55.2% women, 73% White, 41.8% stage III), 766 CpG sites differed significantly between rectal and colonic tumors (FDR < 0.05); 27.7% were hypermethylated and 72.3% were hypomethylated in rectum tumors. The UTSW replication cohort included 72 patients (31.9% rectum, 45.8% women, 56.9% White, 37.5% stage III). Of the 504 CpGs available for replication testing, 266 showed the same direction of association, with 14.3% hypermethylated and 85.7% hypomethylated in rectal/rectosigmoid versus colonic tumors. Principal component analysis on replicated CpGs demonstrated separate clusters of rectum and colonic tumors. Replicated CpGs mapped to 202 genes, including SRC, IGF1R, EPHB3, and MAP4K4. Pathway enrichment analysis highlighted processes related to epithelial cell migration, cell-cell adhesion, and immune activation. Conclusion: Tumor location-specific DNAm signatures differentiate rectal from colonic tumors in EOCRC, and nearly half of TCGA-identified CpGs showed directional replication in independent patient tumor tissue samples. These findings support a biological role for DNAm in anatomic heterogeneity of EOCRC and identify a focused set of CpGs and pathways for future mechanistic investigation to uncover epigenetic drivers.