Distinct epigenetic signatures define HPV-positive and HPV-negative tonsil carcinomas

Abstract

Introduction Epigenetic modifications, particularly DNA methylation, play a key role in oropharyngeal squamous cell carcinoma (OPSCC). This is the first study to apply genome-wide methylated DNA sequencing (MeD-seq) to identify differentially methylated regions (DMRs) in OPSCC. By comparing HPV-positive and HPV-negative tonsillar carcinomas, we evaluate MeD-seq’s applicability to validate known and identify novel DMRs. These findings may support the identification of potential biomarkers for risk stratification and prognostic assessment in OPSCC. Methods Methylated DNA sequencing (MeD-seq) was used to analyze genome-wide DNA methylation patterns in formalin-fixed paraffin-embedded (FFPE) tonsil samples. Gene set enrichment analysis explored the biological role of DMR associated genes. A literature search of DMR’s was performed to compare MeD-seq to other techniques. Results Thirty-one analyzed samples (10 HPV + OPSCC, 9 HPV- OPSCC, and 12 controls) revealed 4,962 DMRs. We focused on the top five promoter-region DMRs with the highest fold change per comparison (HPV + vs. HPV−; HPV + vs. controls; HPV − vs. controls). Key DMRs involved IRF4 , TCF24 , and CLSTN2 . IRF4 was hypermethylated in both OPSCC groups compared to controls, while CLSTN2 was hypermethylated in HPV + versus HPV − tumors. Enrichment analysis indicated enrichment of cell cycle–related pathways in HPV + tumors and IGF1R signaling pathways in HPV − tumors. MeD-seq confirmed known methylation patterns and identified novel OPSCC-specific DMRs. Discussion This pilot study demonstrates HPV-associated methylation profiles in OPSCC, highlighting MeD-seq as a reliable technique for tumor methylation analysis, confirming existing and identifying novel DMRs. Larger studies are needed to validate potential biomarkers for prognosis and personalized treatment.