Abstract 154: Genetic and epigenetic analysis of HPV-positive and HPV-negative Oropharyngeal squamous cell carcinoma
Authors: Nakagawa Takuya , Matsusaka Keisuke , Misawa Kiyoshi , Fukuyo Masaki , Kurokawa Tomoya , Mima Masato , Matsubara Hisahiro , Hanazawa Toyoyuki , Kaneda Atsushi 
Published: September 2020

Abstract
Background: The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing in these two decades, primarily due to human papillomavirus (HPV). Whereas HPV-associated OPSCC generally correlates with better prognosis, approximately 20% of the patients do not show good prognosis or response to treatment. Appropriate molecular markers have not been available to stratify OPSCC into distinct molecular subtypes reflecting these different clinicopathological features, making treatment stratification difficult, while de-escalation strategies for better prognosis group are expected to decrease the long-term chemoradiation-related morbidities.
Methods: We performed DNA methylome analysis using Infinium 450k for 89 OPSCC cases and targeted exon sequencing for 86 OPSCC cases, and analyzed the data together with 81 cases reported by The Cancer Genome Atlas. We stratified OPSCC by hierarchical clustering analysis using methylome data. Methylation levels of classifier markers were validated quantitatively using pyrosequencing, and area under the curve (AUC) values of receiver operating characteristics (ROC) curves were calculated. Association between clinicopathological factors and DNA methylation or mutation status was statistically analyzed.
Results: OPSCC was stratified into four epigenotypes: HPV(+) high-methylation (OP1), HPV(+) intermediate-methylation (OP2), HPV(−) intermediate-methylation (OP3), and HPV(−) low-methylation (OP4). Ten methylation marker genes were generated: five to classify HPV(+) cases into OP1 and OP2, and five to classify HPV(−) cases into OP3 and OP4. AUC values of ROC curves were 0.969 and 0.952 for the two marker panels, respectively. While significantly higher TP53 mutation and CCND1 copy number gains were observed in HPV(−) than in HPV(+) groups (P&lt;0.01), no significant difference of genomic aberrations was observed between OP1 and OP2, or OP3 and OP4. The four epigenotypes showed significantly different prognosis
(P=0.0006), distinguishing the most favorable OPSCC subgroup (OP1) among generally favorable HPV(+) cases, and the most unfavorable OPSCC subgroup (OP3) among generally unfavorable HPV(−) cases. Generally favorable HPV-associated OPSCC as well as generally unfavorable HPV-negative OPSCC can be successfully subdivided into two different epigenotypes each, reflecting significantly distinct prognosis.
Conclusions: HPV(+) and HPV(−) OPSCC are further divided into distinct DNA methylation epigenotypes, showing significantly different prognosis. These findings will contribute to greater understanding of the molecular basis of OPSCC and de-escalation treatment strategies.
Citation Format: Takuya Nakagawa, Keisuke Matsusaka, Kiyoshi Misawa, Masaki Fukuyo, Tomoya Kurokawa, Masato Mima, Hisahiro Matsubara, Toyoyuki Hanazawa, Atsushi Kaneda. Genetic and epigenetic analysis of HPV-positive and HPV-negative Oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 154.