Methylation of Human Papillomavirus DNA: biological significance and clinical utility

DNA methylation helps regulate transcriptional activity and is widely studied in cancer biology. This investigation aimed to establish the significance of Human Papillomavirus (HPV) DNA methylation in HPV-associated disease both in terms of basic biology and as a potential biomarker. Assays to assess DNA methylation and gene expression were developed and evaluated. Pyrosequencing was used to assess DNA methylation of four regions of the HPV16 genome (E2, L1/L2, enhancer, promoter). Gene expression was assessed using quantitative PCR with assays for E2, E6 and E7. HPV integration was assessed using Detection of Integrated Papillomavirus Sequences (DIPS). The relationship between HPV methylation, gene expression and integration was explored in vitro and in vivo using cell cultures and clinical cohorts. A variety of sample materials were used including short term and immortal cell lines, cervical cancer biopsies, cytology samples and Vulval Intraepithelial Neoplasia (VIN) biopsies. In general, hypermethylation of the HPV genome was associated with low HPV gene expression and the presence of integrated HPV genomes. To better understand the potential clinical utility of HPV DNA methylation, the relationship between HPV DNA methylation and various stages of cervical disease was determined. The HPV genome was progressively hypermethylated with increasing severity of cervical disease and certain regions of the HPV genome were more affected than others. A longitudinal study was also performed in order to determine a relationship between HPV methylation and clinical outcome. Differences in HPV methylation among patients who had persistent HPV infection and low grade disease, persistent infection and high grade disease and patients that cleared HPV infections were observed. Throughout the study the potential application of a HPV biomarker was considered and the correct biomarker design procedures were referred to. Several of the early biomarker development steps were successfully achieved