Host-viral epigenetic interactions in HPV16-mediated oropharyngeal cancer


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Richard Shaw, Anastasia Filia, Janet Risk, Terry Jones, Triantafillos Liloglou

University of Liverpool, UK

Abstract

Proffered paper presentation

Background
Recent evidence suggests that HPV-16 is involved in the development of oropharyngeal carcinoma (OPSCC). Crosstalk between the viral genome and the epigenetic machinery of the host may represent critical aetiological mechanisms. Regarding therapeutic HPV vaccine clinical trials, biomarkers indicating pathogenesis of viral oncoproteins would have clinical value. In this study we assess the frequency of HPV-16 in a retrospective cohort and identify the relevant methylation patterns of both the HPV-16 and host genomes.

Methods
DNA was extracted from 55 oropharynx samples. HPV-16 identification was undertaken by duplicate PCR reactions utilising HPV-16/E6 specific primers. Multiple positive and negative controls were included to eliminate false positive/negative results. Also, confirmation of HPV-16 typing was achieved by sequencing all positive PCR products. DNA methylation analysis for the HPV LCR region and the genomic LINE-1 retrotransposable element (global methylation) was undertaken by pyrosequencing.

Results
Twenty-two of 55 (40%) of the OPSCC samples examined were HPV positive. Sequencing confirmed the HPV-16 type in all cases. Hypomethylation of LINE-1 (methylation index<65%) was demonstrated in 29/55 (53.7%) samples. No significant differences in the global methylation (LINE-1) index were found between the HPV positive/negative groups. Regarding the DNA methylation status of the LCR region of the HPV genome, only 1/23 HPV-16 positive tumours showed significant methylation (MtI =84%).

Conclusion
HPV DNA is present in a substantial percentage of OPSCC, thus it may play a role in its pathogenesis. This finding is important for the HPV-vaccine based clinical trials. The presence of HPV DNA does not affect global methylation of the host. It still remains to be evaluated whether the expression of specific HPV16 genes may affect this process. The LCR region, which controls the expression of E6/E7, is not frequently methylated in OPSCC, while the status of the E2 - located CpG island remains to be examined.