To evaluate the predictive ability of methylation of the most common human papillomavirus(HPV16) L1 region and Long Control Region(LCR) for cervical precancer and cancer. And to assess the correlation of methylation between different CpG sites, as well as between methylation and HPV viral load.
A hospital-based case-control study consisting 27 HPV16 single positive women was designed to identify the significant CpG sites. Then 10-year population-based screening cohort conducted in cervical cancer high- incidence area (Shanxi province) was used to valid the identified CpG sites. The cohort included 1742 women followed up in 2005, 2010 and 2014 using HPV testing and cytology. Women with any positive screening result received colposcopic examination and biopsy if necessary. Based on the combination of biopsy result at 3 follow-up visits, women were classified into CIN2+ persistence/progression group and regression group to assess the longitudinal performance of previously identified CpG sites. DNA extracted from cervical cytology specimens was quantified for methylation levels at 35 CpG sites throughout HPV16 L1 and LCR. The Mann-Whitney U test was used to compare the methylation pattern between different biopsy results. And spearman-test was used to assess the relation between different CpG sites and between HPV viral load and methylation.
HPV 16 methylation increased with the grade of cervical precaner(p<0.001). The median methylation level for CIN1/normal, CIN2, CIN3+ was 11.16%, 11.54% and 23.19%, respectively. Specially, methylation of 14 CpG sites (L1:5602、5608、5611、5617、5709、5726、6367、6389、6457、6650、7034，LCR:7455、7535、7553）was significant higher among women with CIN3+ than <CIN3. Considering literature review, another 10 CpG sites were also evaluated during follow-up. 77 women diagnosed with CIN2+ were HPV16 positive in 2005. After 5 years, the methylation of 6650 was significantly higher among CIN2+ persistence/progression group, another CpG(nucleotide position 31) also showed the predictive ability for CIN2+ after 10 years. Correlation was found between many different CpG sites, especially, the correlation coefficient between 6367, 6389 and 52 were both more than 0.7. No strong relationship was identified between HPV viral load and methylation level stratified by biopsy result. The max correlation coefficient was no more than 0.4.
HPV 16 methylation of L1 and LCR can be used as biomarker for cervical precancer and cancer. The methylation of 6650 in L1 showed promising predictive ability for the progression of cervical precancer. The correlation was identified between different CpG sites, but HPV viral load was not related to methylation level.