The incidence of Human Papilloma Virus (HPV) associated oropharyngeal cancer is dramatically increasing in developed countries, due to mainly HPV 16. Whereas viral infection status could affect molecular subtypes in other cancers, molecular features of HPV(+) and HPV(-) OPSCCs including epigenetic aberrations are not fully clarified. We here performed genome-wide DNA methylation analysis of OPSCC. We identified 144 candidate methylation-associated silencing genes using Infinium 450k analysis of 13 OPSCC samples, 4 normal mucosal samples and two HNSCC cell lines with/without 5Aza/TSA. Re-expression of the hypermethylated genes by 5-aza-2-deoxycytidine was confirmed by RNA-seq. Among the 144 candidate genes, 9 genes were selected and their methylation status was analyzed in 70 pharyngeal SCC cases including 55 OPSCC and 15 hypopharyngeal SCC cases using pyrosequencing. HPV(+) samples showed higher methylation level within all the samples (P=0.0002, t-test) and within OPSCC (P=0.001, t-test). While HPV infection significantly correlated with positive staining of p16 in immunohistochemistry, HPV(+) as well as p16(+) status significantly correlated with better prognosis (P = 0.005 and 0.002, log-rank test). The aberrant hypermethylation of an identified marker gene also correlated with better prognosis significantly when all the cases were analyzed (P = 0.04). Interestingly, the its hypermethylation status also correlated with better prognosis within p16(-) cases (P = 0.03). Our data suggest that aberrant DNA methylation might be involved in genesis of OPSCC, that DNA methylation might associate with HPV infection, and that methylation marker might work as prognostic marker. To elucidate the correlation between DNA methylation and OPSCC including HPV(+) and HPV(-) OPSCCs, we additionally performed genome-wide DNA methylation analysis of OPSCC using Infinium 450k analysis of 83 OPSCC samples, and integrated our data with 81 OPSCC data from the Cancer Genome Atlas (TCGA). HPV(+) and HPV(-) OPSCCs were mainly classified into two epigenotypes, respectively. Analyzing the correlation with clinicopathological data, we created a new classification strategy for OPSCC.

Citation Format: Takuya Nakagawa, Keisuke Matsusaka, Kiyoshi Misawa, Masaki Fukuyo, Kiyoko Takane, Toyoyuki Hanazawa, Hisahiro Matsubara, Yoshitaka Okamoto, Atsushi Kaneda. Frequent promoter hypermethylation correlates with better prognosis in pharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3318.