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Cancer. 2015 Jun 15;121(12):1966-76. doi: 10.1002/cncr.29315. Epub 2015 Mar 2.

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

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Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.
Clinical Cooperation Unit, German Cancer Research Center, Heidelberg, Germany.
Jean-Uhrmacher Institute for Oto-Rhino-Laryngological Research, University of Cologne, Cologne, Germany.
Institute of Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany.
Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Cologne, Cologne, Germany.
Department of Pathology, Maastricht University Medical Center, Maastricht, The Netherlands.
Department of Otorhinolaryngology, Maastricht University Medical Center, Maastricht, The Netherlands.
Department of Otorhinolaryngology, Head and Neck Surgery, University of Giessen, Giessen, Germany.



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.


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.


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 = .06]).


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.


E2-binding sites; human papillomavirus (HPV); methylation; oropharyngeal cancer; upstream regulatory region

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