DNA methylation signatures of cervical pre-invasive and invasive disease: An epigenome-wide association study

Epigenetic alterations are essential in the development of cancers, while epigenome-wide exploration in cervical cancer has been limited. In this epigenome-wide association study (EWAS) we explore differential DNA methylation signatures associated with CIN (cervical intraepithelial neoplasia) grade 3 and cervical cancer to better understand potential drivers and biomarkers of cervical carcinogenesis. 247 women were recruited between 2014 and 2020 (N = 119 benign, N = 74 CIN3/CGIN [cervical glandular intraepithelial neoplasia] and N = 54 cancer). Methylation signatures were obtained from exfoliated cervical cells and sequenced using the Illumina 850 k array. Logistic regression and conditional analyses were used to test for independent associations between Cytosine-phosphate-Guanine (CpG) sites and case–control status, with adjustment for batch, chip, age, and human papillomavirus (HPV) status. 409 CpG sites were strongly associated with CIN3/cancer (p-value <5 × 10⁻⁸). Following conditional analysis, two CpG sites located in PAX1 (cg16767801) and NREP-AS1 genes (cg23642047) were independently associated with case status, yielding an area under the curve (AUC) of 0.92 (AUC = 0.97 for invasive disease). In a validation dataset (CIN3 only) PAX1/NREP-AS1 yielded a combined AUC of 0.77. Methylation markers offer promise for use in cervical screening, particularly as triage tests and self-sampling. We have identified a novel combined methylation marker that offers a high accuracy for the detection of CIN3 or worse.