ARTICLE ABSTRACT
DNA methylation is a vital early step in carcinogenesis. Most findings of aberrant DNA methylation in head and neck squamous cell carcinomas (HNSCC) are array-based with limited coverage and resolution, and mainly explored by human papillomavirus (HPV) status, ignoring the high heterogeneity of this disease. In this study, we performed whole-genome bisulfite sequencing (WGBS) on a well-studied HNSCC cohort (n=36) and investigated the methylation changes between fine-scaled HNSCC subtypes in relation to genomic instability, repetitive elements, gene expression, and key carcinogenic pathways. The previously observed hypermethylation phenotype in HPV-positive (HPV+) tumors compared to HPV-negative tumors was robustly present in the immune-strong (IMU) HPV+ subtype but absent in the highly keratinized (KRT) HPV+ subtype. Methylation levels of IMU tumors were significantly higher in repetitive elements, and methylation showed a significant correlation with genomic stability, consistent with the IMU subtype having more genomic stability and better prognosis. Expression quantitative trait methylation (cis-eQTM) analysis revealed extensive functionally-relevant differences, and differential methylation pathway analysis recapitulated gene expression pathway differences between subtypes. Consistent with their characteristics, KRT and HPV-negative tumors had high regulatory potential for multiple regulators of keratinocyte differentiation, which positively correlated with an expression-based keratinization score. Together, our findings revealed distinct mechanisms of carcinogenesis between subtypes in HPV-positive HNSCC and uncovered previously ignored epigenomic differences and clinical implications, illustrating the importance of fine-scale subtype analysis in cancer.
