Identifying targeted cell-free DNA methylation regions in head and neck cancer via paired methylome analysis.

Despite the critical need, progress in developing cell-free DNA (cfDNA) liquid biopsy biomarkers for the diagnosis and risk stratification of head and neck squamous cell carcinoma (HNSC) has been limited. In this study, we present a comprehensive paired-sample differential methylation region (psDMR) analysis in HNSC, aimed at identifying reliable and HNSC-specific regions for cfDNA biomarker discovery. Traditional DMR analyses often overlook paired-sample information and fail to account for the heterogeneity within HNSC tissues. Our findings reveal a substantial overlap of hypermethylated DMRs across two independent HNSC methylation datasets, demonstrating the robustness and clinical relevance of these regions for cfDNA biomarker development. Furthermore, we identified consistent DMRs in both HNSC and lung squamous cell carcinoma (LSCC), suggesting the potential for pan-squamous cell carcinoma biomarkers. Notably, gene clusters such as , , and were frequently hypermethylated, providing new insights into the shared epigenetic landscape of HNSC and LSCC. This study underscores the importance of incorporating matched normal tissues in cancer methylome analyses and establishes a foundation for advancing cfDNA-based biomarker discovery across squamous cell carcinomas.