Elevated methylation of HPV16 DNA is associated with the development of high grade cervical intraepithelial neoplasia

Abstract

We explored the association of human papillomavirus type 16 (HPV16) DNA methylation with age, viral load, viral persistence and risk of incident and prevalent high grade CIN (CIN2+) in serially collected specimens from the Guanacaste, Costa Rica cohort. 273 exfoliated cervical cell specimens (diagnostic and pre-diagnostic) were selected: (1) 92 with HPV16 DNA clearance (controls), (2) 72 with HPV16 DNA persistence (without CIN2+) and (3) 109 with CIN2+. DNA was extracted, bisulfite converted and methylation was quantified using pyrosequencing assays at 66 CpGs across the HPV genome. The Kruskal-Wallis test was used to determine significant differences among groups, and receiver operating characteristic curve analyses were used to evaluate how well methylation identified women with CIN2+. In diagnostic specimens, 88% of CpG sites had significantly higher methylation levels in CIN2+ after correction for multiple tests compared with controls. The highest area under the ROC curve (AUC) was 0.82 for CpG site 6457 in L1, and a diagnostic sensitivity of 91% corresponded to a specificity of 60% for CIN2+. Prospectively, 17% of CpG sites had significantly higher methylation in pre-diagnostic CIN2+ specimens (median time of 3 years before diagnosis) versus controls. The strongest pre-diagnostic CpG site was 6367 in L1 with an AUC of 0.76. Age-stratified analyses suggested that women older than the median age of 28 years have an increased risk of precancer associated with high methylation. Higher methylation in CIN2+ cases was not explained by higher viral load. We conclude that elevated levels of HPV16 DNA methylation may be useful to predict concurrently diagnosed as well as future CIN2+.

Figure 1

Median % methylation for the outcome groups at each CpG site for specimens collected at diagnosis (A.) and 27–72 months (median time) before diagnosis (B.). The legend indicates the color of each outcome group and the number of women in each group; women with CIN2 and CIN3 are combined because of their similarity in median % methylation. The x-axis indicates each individual CpG site by nucleotide position grouped by gene region.

Figure 2

Odds ratio estimates for the association between high methylation and CIN2-3 stratified by the median age of 28 years. Logistic regression models were used to obtain the odds ratios for CIN2-3, using the controls as the referent group, for methylation dichotomized using the second tertile based on the distribution for that site in the controls. The legend shows the color of each age group. The x-axis indicates each individual CpG site by nucleotide position grouped by gene region.

Figure 3

Mean % methylation by CpG site for 30 serial samples from 7 women collected 0–7 years prior to diagnosis of CIN3. The legend indicates the color of each time interval. The x-axis indicates each individual CpG site by nucleotide position grouped by gene region.

Figure 4

Receiver operating characteristic (ROC) curves for the CpG site at nucleotide position 6457 (A.) in L1 using diagnostic specimens and at 6367 (B.) in L1 using pre-diagnostic specimens. These are the strongest diagnostic and pre-diagnostic CpG sites with the highest AUCs. The % sensitivity, the true positive rate, is given along the y-axis versus 100-specificity %, the false positive rate, is shown along the x-axis, with a diagonal reference line. The 95% confidence intervals are shown as dotted lines for each ROC curve.