Whole-genome CpG-resolution DNA Methylation Profiling of HNSCC Reveals Distinct Mechanisms of Carcinogenesis for Fine-scale HPV+ Cancer Subtypes

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.

Tumor Subtype Classification Tool for HPV-associated Head and Neck Cancers

“…Consistently, HPV+ HNSCC tumors have been characterized as either immune strong (IMU) or highly keratinized (KRT) 13 . These subtypes have been further characterized based on mutations, CNVs, DNA methylation 18 , DNA hydroxymethylation 19 , and comprehensively reviewed in Qin et al 11 . However, previous classifications have not provided the ability to classify new tumors.…”

“…More recently, Locati et al further discriminated KRT tumors into high and low stromal subtypes 16 , and demonstrated that IMU patients have better prognosis than either high or low stromal KRT. The subtype naming convention of IMU/KRT was adopted in the Nature review, Leemans et al 17 , and these subtypes have now been characterized using additional high-throughput technologies, including DNA methylation 18 showing stronger global hypomethylation in KRT, and DNA hydroxymethylation 19 . The IMU/KRT subtypes have also been demonstrated to significantly associate with HPV E6 isoform gene expression, with KRT tending to have higher levels of the spliced E6*I isoform compared to E6 full length 20,21 .…”