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Background: Human Papilloma Virus has been considered as the main cause for cervical cancer. In this study we investigated epigenetic changes and especially methylation of specific sites of HPV genome. The main goal was to correlate methylation status with histological grade as well as to determine its accuracy in predicting the disease severity by establishing optimum methylation cutoffs. Methods : In total, sections from 145 cases genotyped as HPV16 were obtained from formalin- fixed, paraffin-embedded tissue of cervical biopsies, conization or hysterectomy specimens. Highly accurate pyrosequencing of bisulfite converted DNA, was used to quantify the methylation percentages of UTR promoter, enhancer and 5' UTR, E6 CpGs 494, 502, 506 and E7 CpGs 765, 780, 790. The samples were separated in different groupings based on the histological outcome. Statistical analysis was performed by SAS 9.4 for Windows and methylation cutoffs were identified by MATLAB programming language. Results: The most important methylation sites were at the enhancer and especially UTR 7535 and 7553 sites. Specifically for CIN3+ (i.e. HSIL or SCC) discrimination, a balanced sensitivity vs. specificity (68.1%, 66.2% respectively) with positive predictive value (PPV) and negative predictive value (NPV) (66.2%, 68.2% respectively) was achieved for UTR 7535 methylation of 6.1% cutoff with overall accuracy 67.1%, while for UTR 7553 a sensitivity 60.9%, specificity 69.0%, PPV=65.6%, NPV=64.5% and overall accuracy=65.0% at threshold 10.1% was observed. Conclusion: Viral HPV16 genome was found methylated in NF-1 binding sites of UTR in cases with high grade disease. Methylation percentages of E6 and E7 CpG sites were elevated at the cancer group.

Searching HPV genome for methylation sites involved in molecular progression to cervical precancer