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Clarifying the epigenetic regulation of tumor-related genes (TRGs) can provide insights into the mechanisms of tumorigenesis and the risk for disease recurrence in HPV-negative head and neck cancers, originating in the hypopharynx, larynx, and oral cavity. We analyzed the methylation status of the promoters of 30 TRGs in 178 HPV-negative head and neck cancer patients using a quantitative methylation-specific PCR. Promoter methylation was correlated with various clinical characteristics and patient survival. The mean number of methylated TRGs was 14.2 (range, 2-25). In the multivariate Cox proportional hazards analysis, the methylation of COL1A2 and VEGFR1 was associated with poor survival for hypopharyngeal cancer, with hazard ratios: 3.19; p = 0.009 and 3.07; p = 0.014, respectively. The methylation of p16 and COL1A2 were independent prognostic factors for poor survival in laryngeal cancer (hazard ratio: 4.55; p = 0.013 and 3.12; p = 0.035, respectively). In patients with oral cancer, the methylation of TAC1 and SSTR1 best correlated with poor survival (hazard ratio: 4.29; p = 0.005 and 5.38; p = 0.029, respectively). Our findings suggest that methylation status of TRGs could serve as important site-specific biomarkers for prediction of clinical outcomes in patients with HPV-negative head and neck cancer.

Analysis of Site-Specific Methylation of Tumor-Related Genes in Head and Neck Cancer: Potential Utility as Biomarkers for Prognosis