Human papillomavirus (HPV) infection accounts for an estimated 530,000 cervical cancer cases and 270,000 deaths annually, with the majority (86% of cases and 88% of deaths) occurring in developing countries. Infection with high-risk HPV types can lead to mild, moderate or severe cervical intraepithelial neoplasia (CIN1, 2 or 3) with a small percentage of persistent HPV infections and CIN3 subsequently developing into cancer. Study of viral persistence and molecular pathways in precancerous lesions is critical for an understanding of cervical carcinogenesis and optimal prevention strategies.
We assessed DNA methylation levels of host and HPV genes within the microenvironment of individual discrete lesions to look for significant associations between HPV genotype, methylation, and lesion severity in cervical surgical tissues and corresponding exfoliated cell specimens.
354 CIN were macrodissected from surgical specimens provided by 127 women who underwent loop electrosurgical excision procedures (LEEP). Samples were HPV genotyped and DNA methylation of EPB41L3 and viral regions of HPV16L1 and L2, HPV18L2, HPV31L1 and HPV33L2 were measured by quantitative pyrosequencing of bisulfite converted DNA1.2.3.
Adjacent CIN of different grades usually contained the same hrHPV types. However, methylation patterns differed significantly and were much more characteristic of histopathological grade. Methylation levels in all CIN1 were the same regardless of whether a CIN1 was adjacent to CIN3 or was the highest diagnosis on the cervix. There was a significant trend of increased methylation with disease progression from normal and CIN1 to CIN3 (p<0.0001). A popular notion is that cervical carcinogenesis is a continuous progression from CIN1, CIN2, and finally CIN3 to cancer. However, experimental evidence indicates that CIN1 is not necessarily a direct precursor of CIN3, instead different grade lesions can develop directly in a simultaneous or staggered timeframe. Our results indicate that elevated methylation characteristic of CIN3 seems to be related to a discrete molecular “high methylation” switch from the normal state rather than a gradual secular increase. HPV genotype and methylation results in LEEP biopsies and corresponding exfoliated cells were similar. 99% of CIN3 and 88% of CIN1 had the same or matching HPV types. There was a significant correlation between LEEP and cervical scrapes for both EPB41L3 (Spearman r=0.2033, p=0.0253, n=121, and HPV (Spearman r=0.2156, p=0.0237, n=110).
Our study supports the use of DNA methylation testing as a prognostic biomarker of CIN3 and cancer risk and it may be used as a robust triage for hrHPV positive women .
1. Vasiljevic N, et al. J Clin Virol 2014 ;59:161-6.
2. Brentnall AR, et al. Int J Cancer 2014 ;135:1425-32.
3. Vasiljevic N, et al. Gynecol Oncol 2014 ;132:709-14.