Methylation status of HPV16 E2‐binding sites classifies subtypes of HPV‐associated oropharyngeal cancers

Abstract

BACKGROUND:The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. METHODS: Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods. RESULTS: Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P 5.06]). CONCLUSIONS: Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.

Human Papillomavirus-Related Head and Neck Cancer

“…In contrast to the classical HPV DNA integration-based idea of HPV E2 deregulation, and in particular in tumors with non-integrated HPV genomes, methylations at E2BS in the upstream regulatory region of E6 and E7 appear to regulate their expression in the presence of E2 [49,50]. DNA methylation patterns have been published for viral-driven cancers including HPV-associated neoplasms [51,52].…”

“…Based on viral DNA integration and the E2BS methylation status, distinct subgroups of HPV-associated SCC causally linked with viral oncogene expression have been identified Mirghani et al, 2016 [40] Hui et al, 2013 [43] Gao et al, 2013 [44] Lajer et al, 2012 [41] Lajer et al, 2011 [42] X X XXX = Common between 3 studies; XX = common between 2 studies; X = common between 2 studies but regulated in opposite direction. [28,49,50]. Interestingly, the clinical outcome of HNSCC patients, including HPV-related OPSCC, could be predicted depending on differentially methylated promoters of 5 host genes (ALDH1A2, OSR2, IRX4, GRIA4, and GATA4) [51,53].…”

The role of integration in oncogenic progression of HPV-associated cancers

“…However, in tumors with exclusively extrachromosomal viral DNA, the viral genome has usually acquired genetic or epigenetic changes that result in dysregulated E6/E7 gene expression [12, 21, 22]. For example, methylation of the E2 binding sites in the URR can alleviate E2-mediated repression of the viral oncogenes [22–24]. Therefore, while integration is very frequently detected in HPV-associated cancers, it is not absolutely required.…”

Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial

“…7 Although the site in the host genome in which the viral genome integrates varies, the HPV genome is most frequently disrupted in the region coding for E2, which functions normally to regulate expression of E6 and E7. 8–10 Additionally, in some HPV-associated tumours in which viral integration is incomplete, methylation of E2 has been reported. 11,12 Expression of both E6 and E7 is needed, but not sufficient, for initiation and persistence of disease, providing non-self antigenic targets for immune-based therapies.…”