Abstract 57: Human papillomavirus-16 DNA methylation in head and neck squamous cell carcinomas.
Authors: Cole Lauren , Rozek Laura S. , Peters Edward S. , Colacino Justin A. , Virani Shama , Dolinoy Dana C. , Sartor Maureen A. , Peterson Lisa , Wolf Gregory T. , Carey Thomas E. , Walline Heather M. , Bradford Carol R. , Chepea Douglas B. , McHugh Jonathan B. 
Published: August 2013

Abstract
Human papillomavirus (HPV) is etiologically associated with head and neck squamous cell carcinomas (HNSCC), representing a distinct subset of the disease. Compared to those with no evidence of HPV in HNSCC, HPV-associated HNSCC are most often located in the tonsil and base of tongue, associated with less tobacco and alcohol exposure, and improved prognosis. Methylation of certain CpG sites in the HPV genome is associated with expression of oncogenic E6 and E7 proteins, and is an indication of viral integration in the human genome. Further, somatic methylation changes in the host genome are a hallmark of HNSCC, but the clinical significance and epidemiologic correlates of methylation within the HPV genome in HNSCC tumors has not yet been well described. Here, we measured methylation of the HPV genome in 69 HPV-16 positive tumors from patients enrolled in the University of Michigan Head and Neck Cancer Specialized Program of Research Excellence (SPORE) ongoing survivorship cohort to describe the distribution of CpG site methylation of the HPV genome and its relationship with associated clinicoepidemiologic characteristics. DNA was extracted from areas of &gt;70% cellularity from formalin-fixed paraffin-embedded tumors. Using bisulfite sequencing (pyrosequencing), we quantified methylation of 13 total CpG sites in the E6 promoter, enhancer, and long control region (LCR) of the HPV-16 genome. Epidemiologic data on several risk factors (age, sex, smoking status, pack-years, alcohol status, stage, and site) were examined in relation to methylation levels. Of the 69 tumors, there were 57 oropharynx (82.6%), 7 oral cavity (10.1%), 3 larynx (4.3%), 1 nasopharynx, and 1 hypopharynx (1.5% each). The average age at diagnosis was 55.6 (SD = 9.4), the majority were stage 4 (78.3%), and 85.5% were male. Overall, mean methylation was 12.4% of the 5 CpG sites in the E6 promoter region (range = 0 to 93.5%), 2.4% of the 4 CpG sites in the enhancer region (range = 0.4 to 10.7%), and 10% of the 4 CpG sites in the LCR region (range = 0.9 to 41.5%). CpG sites were consistently methylated within the E6 promoter and enhancer regions, while the LCR region had higher methylation in one CpG site (7455) compared to the other sites within that region. No significant differences in mean methylation levels for any of the regions in the HPV-16 genome were observed for age, smoking status (current, former or never smoker at baseline), alcohol status (current, former or never drinker at baseline), stage, or site. Within the E6 promoter region, patients with 15 pack-years or greater had a lower mean percent methylation (6.6%) than those with less than 15 pack-years (17.7%, p &lt; 0.05). Females tended to have a higher mean percent methylation than males in the E6 promoter region (24.25% vs. 10.44%, p &lt; 0.09). Of the 69 cases, 10 died or recurred within 3 years. We did not observe any association between HPV methylation and survival; however, our power to detect an association was low. Our results suggest that methylation of the HPV genome may be associated with epidemiologic risk factor characteristics and offer potential avenues for prevention and treatment of this disease.
Citation Format: Lauren Cole, Laura S. Rozek, Edward S. Peters, Justin A. Colacino, Shama Virani, Dana C. Dolinoy, Maureen A. Sartor, Lisa Peterson, Gregory T. Wolf, Thomas E. Carey, Heather M. Walline, Carol R. Bradford, Douglas B. Chepea, Jonathan B. McHugh. Human papillomavirus-16 DNA methylation in head and neck squamous cell carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 57.