J-108 Role of DNA methylation in prediction of human papillomavirus persistence and carcinogenesis
Authors: Lorincz Attila 
Published: April 2019

High risk human papillomavirus types (HPV) account for an estimated 530,000 new cervical cancers and 270,000 deaths annually, most of which occur in developing countries. In addition to cancer of the cervix HPV causes many other kinds of epithelial malignancies, including oropharyngeal, anal and vulvar cancer. More than 25 types of HPV are transmitted through sexual contact but most infections do not cause symptoms and are cleared after a short time.

There is a well-defined premalignant phase in HPV carcinogenesis, which makes HPV infection tractable to effective cancer prevention efforts and the study of molecular mechanisms involved in precancer progression to frank invasive cancer. When HPV infection becomes persistent there is an increased chance of high grade cervical intraepithelial neoplasia (CIN3). It can take 10–15 years of persistent HPV infection to develop invasive cervical cancer. A clearer understanding of the molecular changes during HPV persistence leading to CIN3 and early cancer has provided more accurate biomarkers for diagnosis and prognosis and allows better informed consideration of preferential druggable targets for effective non-surgical elimination or stalling of persistent HPV infections.

Current modalities for elimination of persistent HPV infection invariably involve physical removal or destruction of the affected tissues. These surgical options have a large effect in reducing the risk of incident cervical cancer, albeit at the cost of considerable overtreatment and morbidity, most especially serious complications during pregnancy. Follow-up studies consistently show that molecularly detectable HPV prior to treatment becomes undetectable after treatment, with the most ultrasensitive DNA tests. However, we must not confuse lack of detectability with elimination of the persistent viral reservoir. It is not known to what extent there remain hidden inactive reservoirs of HPV which can reactivate much later and produce new disease. The age trend of HPV prevalence, where high levels of HPV DNA detectability in young women (25% or more) are followed by rather low levels of HPV prevalence in middle-aged women (5%–10%) and subsequently by significantly increased prevalence in older women, suggests that a reactivatable silent reservoir remains, which leaves older sexually inactive women at risk of cervical cancer.

The study of DNA methylation levels of candidate human genes and the HPV genome is a way to shed light on the molecular progression of precancerous cervical lesions and on issues of HPV persistence. Most often studied is methylation at the 5-position of cytosine in a CpG motif. DNA methylation is a reproducible and mitotically stable epigenetic change involved in a variety of cellular processes that plays an important role in the progression of many different cancers. With respect to HPV-related disease, numerous studies have highlighted the importance of host and viral gene methylation in the development of cervical cancer and potential for use as biomarkers to triage HPV positive patients. A strong and statistically highly significant association is observed between DNA methylation patterns and CIN of various grades in both cervical scrapes and biopsies. DNA methylation in normal cervical epithelium and low-grade disease is generally absent or low. In contrast increased methylation is observed in patients with advanced intraepithelial lesions and very high levels are seen in carcinoma. These observations have been validated in many large studies in different countries and led to the development of clinically relevant DNA methylation classifiers such as S5. A growing role of DNA methylation testing is related to the definition of HPV persistence (by means of epigenetic signatures) and to the determination of treatment options.

It was originally thought that cervical cancer evolved from HPV infected normal cervical epithelium via sequential progression through consecutive pre-cancerous grades of CIN1, CIN2, CIN3 and early invasive cancer. However, we have shown by means of methylation signatures that CIN1 is not a pre-cancer and that CIN3 can evolve directly from normal HPV-infected epithelium. We used epigenetic markers and HPV typing to investigate 2 main models of precancerous cervical disease progression in areas of cervical lesions with discrete coexisting foci of different grades. Methylation of tumor suppressor gene EPB41L3 and the viral regions of HPV16-L1/L2, HPV18-L2, HPV31-L1 and HPV33-L2 were determined by a highly accurate quantitative pyrosequencing of bisulfite converted DNA. There was a significant trend of increased methylation with disease grade comparing normal to CIN1 and CIN3 (P < 0.0001). CIN1 adjacent to CIN3 predominantly shared the same HPV types as the CIN3, however, methylation differed substantially between adjacent-CIN1 and CIN3 (P = 0.008). In contrast, diagnostically principal-CIN1 (no adjacent CIN3 detectable) had an indistinguishable methylation distribution compared to CIN1 that was adjacent to CIN3 (P > 0.1). Our results suggest that progression from normal epithelium to CIN1 or CIN3 is usually promoted by the same HPV type but occurs via distinct DNA epigenotypes.

The implications of our results are profound and suggest that cervical cancer is predominantly or perhaps solely an epigenetic disease. To further test the epigenetic origin of cancer we performed extensive exome sequencing of a large number of precancer specimens from women followed-up in the UK ARTISTIC trial. We observed progression-related SNPs of both somatic and germline origin; however, it appears from omics comparisons that the epigenetic biomarkers are far more common and much stronger than genetic biomarkers defined by Watson-Crick type changes. Despite these contrasts it remains of interest to construct multivariable classifiers composed of both epigenetic and genetic components in an attempt to further improve predictions of precancer progression. These biomarker signatures will directly impact on persistent HPV infections by allowing a more refined approach to treatment options.