Background: The widespread introduction of Pap testing in the 1960second was followed by significant reductions in the incidence of cervical squamous cell cancer (SCC). However, the incidence of cervical adenocarcinoma (ADC) did not decrease, likely because of low Pap test sensitivity for ADC and adenocarcinoma in situ (AIS). This study assessed a novel HPV/host DNA Methylation-Score for AIS/ADC screening.
Methods: We measured methylation levels at CpG-sites in the L2/L1 open-reading-frames of HPV16, HPV18, and HPV45-as well as two human loci, DCC and HS3ST2. Specifically, we tested exfoliated cervicovaginal cells from women in the "HPV Persistence and Progression (PaP) Cohort" who were positive for one of HPV16/18/45, including: (i) N = 176 with AIS/ADC (ii) N = 353 with cervical intraepithelial neoplasia (CIN3) or SCC, and (iii) Controls who either cleared ("HPV-Clearers"; N = 579) or had persistent HPV16/18/45 infection ("HPV-Persisters"; N = 292). CpG-site-specific methylation percentages were measured using our reported next-gen methods. The Methylation-Score was the average methylation percentage across all 35 CpG-sites tested.
Findings: Each individual CpG-site had higher methylation percentages in AIS/ADC and CIN3/SCC cases relative to either control group (weakest p-value = 0.004). The Methylation-Score for AIS/ADC had a sensitivity = 74% and specificity = 89%. The multivariate odds ratio (OR) between the Methylation-Score (4th vs 1st quartile) and AIS/ADC was ORq4-q1 = 49.01 (pBenjamini-Hochberg=4.64E-12), using HPV-Clearers as controls. CIN3/SCC had similar, albeit weaker, associations with the Methylation-Score.
Interpretation: HPV16/18/45-infected women with Methylation-Scores in the highest quartile had very high odds of AIS/ADC, suggesting they may warrant careful histologic evaluation of the cervical transition zone (eg, conization or LEEP).
Keywords: CIN3; CpG sites; DNA methylation; Methylation-Score; adenocarcinoma; adenocarcinoma in situ (AIS); biomarker; cervical cancer; cervical intraepithelial neoplasia (CIN); cervical precancer; human papillomavirus (HPV); squamous cell carcinoma.
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