Clinical performance of methylation as a biomarker for cervical carcinoma-in-situ and cancer diagnosis: a worldwide study.

The shift towards primary HPV-based screening has necessitated the search for a secondary triage test that provides sufficient sensitivity to detect high grade cervical intraepithelial neoplasia (CIN) and cancer, but also brings an improved specificity to avoid unnecessary clinical work and colposcopy referrals. We evaluated the performance of the previously described DNA-methylation test (S5) in detecting CIN3 and cancers from diverse geographic settings in high, medium and low income countries, using the cut-off of 0.80 and exploratory cut-offs of 2.62 and 3.70. Assays were performed using exfoliated cervical specimens (n=808) and formalin-fixed biopsies (n=166) from women diagnosed with cytology-negative results (n=220), CIN3 (n=204) and cancer stages I (n=245), II (n=249), III (n=28) and IV (n=22). Methylation increased proportionally with disease severity (Cuzick test for trend, p<0.0001). S5 accurately separated women with negative-histology from CIN3 or cancer (p<0.0001). At the 0.80 cut-off, 543/544 cancers were correctly identified as S5 positive (99.81%). At cut-off 3.70, S5 showed a sensitivity of 95.77% with improved specificity. The S5 odds ratios of women negative for cervical disease versus CIN3+ were significantly higher than for HPV16/18 genotyping at all cut-offs (all p<0.0001). At S5 cut-off 0.80, 96.15% of consistently hrHPV-negative cancers (tested with multiple hrHPV-genotyping assay) were positive by S5. These cancers may have been missed in current primary hrHPV-screening programmes. The S5-test can accurately detect CIN3 and malignancy irrespective of geographic context and setting. The test can be used as a screening and triage tool. Adjustment of the S5 cut-off can be performed considering the relative importance given to sensitivity versus specificity.