Abstract
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.
Highlights
The implementation of cervical cancer prevention programmes by systematic cytology screening[1] has contributed to a reduction in cervical cancer-associated deaths in high-income countries.[2]
Aberrant DNA methylation has been reported to increase with cervical cancer disease progression,[7] allowing this epigenetic event to be used as a temporal biomarker, with a potential to accurately predict whether hr-human papillomavirus (HPV) infection will lead to cervical intraepithelial neoplasia grade 2 or above (CIN2+) or disappear.[5,8]
We investigated the false positive rates in women with HPV(À)/Cyt(À), HPV(+)/Cyt(À), cervical intraepithelial neoplasia Grade 3 (CIN3) and cancer including stages I (CSI)-IV at the UK-predefined cut-off of 0.80, the Youden-J index cutoff based on the S5 methylation scores of cervical cancers: 2.62 and the previously explored low- and middle-income countries (LMICs) cut-off of 3.70.12 For all groups analysed, the false positive rate decreased with the increase in cut-off (A)
Summary
The implementation of cervical cancer prevention programmes by systematic cytology screening[1] has contributed to a reduction in cervical cancer-associated deaths in high-income countries.[2]. Methylation biomarkers can offer an accurate alternative to detect clinically significant infection and associated disease and can identify women who have the highest risk of progressing into invasive cervical cancer.[5,6] Aberrant DNA methylation has been reported to increase with cervical cancer disease progression,[7] allowing this epigenetic event to be used as a temporal biomarker, with a potential to accurately predict whether hr-HPV infection will lead to cervical intraepithelial neoplasia grade 2 or above (CIN2+) or disappear.[5,8]. Extensive validation of the S5 classifier will support implementation of the test in global screening and disease-management systems, especially with the rise in acceptance of screening based on self-sampling.[13,14] The main objective of the present study is to analyse the performance and consistency of S5 in detecting high-grade lesions and cervical cancers from diverse settings that reflect Asia, Europe, Africa and the Americas. The present study aims to complement previous works on the S5 DNA-methylation classifier.[6,9,11,12,15]
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