HPV-16 Oncogene Expression and Disruption of the E2 Gene in Relation to Clinical Outcome in Head and Neck Squamous Cell Carcinoma

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Risk factors for developing HNSCC include tobacco use, alcohol use and human papillomavirus (HPV) infection. The predominant genotype in HPV+ HNSCC is HPV-16. Oropharyngeal squamous cell carcinoma (OPSCC) is a subset of HNSCC with a high prevalence of HPV. Although HPV+ HNSCC cases have a better prognosis than HPV- HNSCC cases the reason is unknown and the incidence of HPV+ HNSCC cases is increasing globally. The extent of heterogeneity in HPV+ HNSCCs regarding viral oncogene expression and the state of the viral genome is also not well understood. We hypothesized that comparing integrity of the viral genome and expression levels of HPV oncogenes with results of the expression of host genes and prospectively collected clinical data would allow us to identify phenotypic subsets of HPV+ HNSCC. Our goals in this thesis work were to identify HPV+ HNSCC phenotypes and to determine if these behave differently from each other according to viral expression and DNA status.;The expression of HPV mRNA in HNSCC tumors was evaluated by qRT-PCR for transcripts of HPV-16 oncogenes E6 and E7 as well as the HPV E2 gene, a transcriptional regulator of E6 and E7. The integrity of the viral genome was evaluated by examination of the disruption of the E2 gene using a PCR-based method that amplifies sequential sequences of the E2 DNA. These results were confirmed using a second PCR assay for the full length E2 gene. We found that of 31 OPSCCs tested 21 (70%) had an intact E2 gene and these had high E6, E7 and E2 mRNA expression. In the non-OP tumors, 10 of 17 tested (56%) had a disrupted E2 gene, and these had low E6, E7 and E2 expression. Additionally, samples with an intact E2 gene had higher viral loads regardless of site, determined by dot blot hybridization, indicating that these samples had higher viral copy number.;P16, a cell cycle regulator, is upregulated in HPV+ OPSCC due to the inactivation of pRb by E7. Immunohistochemistry for p16 has been used as a surrogate marker for HPV infection. The CDKN2A locus encodes p 16INK4A, and an alternative transcript, p14ARF. Expression of ARF is increased in HPV+ OPSCC tumors. Based on our results which demonstrated increased E6 and E7 expression in HPV+ OPSCCs with an intact E2 gene, we hypothesized that expression of INK4A and ARF would be high in E2 intact samples and low in E2 disrupted samples. Consistent with this hypothesis high expression of both transcripts was found in OPSCCs with an intact E2 gene and low expression in samples with a disrupted E2. There was no association with expression of these transcripts and integrity of the E2 gene in non-OP tumors.;To identify clinical phenotypes that could be associated with HPV expression and E2 disruption we analyzed E2 disruption and HPV expression data with prospectively collected clinical data. OPSCC tumors with either an intact E2 gene or high E7 expression had improved disease specific survival and lower incidence of loco- regional reoccurrence.;These results support that there are distinct subsets of HPV+ OPSCC which may be identified by viral expression levels and E2 gene disruption. Furthermore, these subsets of HPV+ OPSCCs may be phenotypically and clinically unique. Further research into expression of HPV-16 oncoproteins and integrity of the viral genome, such as disruption of the E2 gene, may help to identify patients with more aggressive tumors within the HPV+ OPSCC population.