Investigation of DNA methylation and microbial biomarkers to improve cervical cancer triage and early diagnosis

The shift towards primary HPV-based screening roused the search for a secondary triage test that provides sufficient sensitivity to detect high-grade cervical intraepithelial neoplasia (CIN) and cancer, but also brings high specificity to avoid unnecessary clinical work and colposcopy referrals. To date, molecular biomarkers like DNA methylation and microbial signatures were investigated for molecular triage purposes as they could offer an objective, cost-effective alternative to cytology. The S5 DNA-methylation classifier based on target CpG sites of the host gene EPB41L3, and viral gene regions of HPV16/18/31/33 demonstrated improved performance for detecting CIN2+ compared to either HPV16/18 genotyping, cytology or combination. We tested the performance of the S5 in detecting CIN3 and cancer from diverse geographic settings using the cut-off of 0.80 and the exploratory cut-off of 3.70 for use in low- and middle-income countries. Assays were performed using exfoliated cervical specimens and formalin-fixed biopsies from women with cytology negative results, CIN3 and cervical cancer diagnoses. We observed that S5 can accurately detect high-grade CIN and malignancy irrespective of geographic context and setting. We also show that adjustment of the S5 cut-off can be performed considering the relative importance given to sensitivity versus specificity, thus reflecting local triage modality needed. In parallel, we investigated biomarkers from the cervicovaginal microbiota and their association with CIN3 development and the S5 DNA methylation signatures. In a pilot longitudinal study, we identified S. amnii as a consistent microbial biomarker for CIN3 development. The increase in S. amnii abundance was directly proportional to the increase of S5 classifier scores and disease severity. S. amnii abundance might play a role in sustaining the epigenetic landscape of the cervicovaginal space. We also found that higher proportions of L.helveticus, L.suntoryeus and L.vaginalis might have a potential protective role against CIN3 development in women with persistent hrHPV infections

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

The shift towards primary human papillomavirus (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 < .0001). S5 accurately separated women with negative-histology from CIN3 or cancer (P < .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 vs CIN3+ were significantly higher than for HPV16/18 genotyping at all cut-offs (all P < .0001). At S5 cut-off 0.80, 96.15% of consistently high-risk human papillomavirus (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 vs specificity