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

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.

Keywords: DNA methylation; HPV; cancer screening; cervical cancer; high-risk human papillomavirus; molecular triage; triage.

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Study design flow-chart

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S5 performance in the study group: A. Distribution of the S5 scores based on the histopathological diagnostic of the patient. Data was plotted as log10 of the S5 score according to lesion grade: HPV(−)/Cyt(−), HPV(+)/Cyt(−), CIN3 and all stages of cervical cancer (CSI-IV). The S5 classifier was significantly different between the following group comparisons: HPV(−)/Cyt(−) vs CIN3 (p < 0.0001), HPV(−)/Cyt(−) vs CSI-IV (all, p < 0.0001), HPV(+)/Cyt(−)vs CIN3 (p < 0.0001), HPV(+)/Cyt(−)vs CSI-IV (all, p < 0.0001) and CIN3 vs CSI-IV (all, p < 0.0001). Other comparisons were not significant (HPV(−)/Cyt(−)vs HPV(+)/Cyt(−), among CSI-IV). The proposed cut-offs for analysis are: 0.80 (red), 2.62 (blue) and 3.70 (green). The top of box represents the upper quartile (p75), bottom the lower quartile (p25), and the line the median (p50). The upper whisker extends to the largest point of the inter-quartile range from the upper quartile. The lower whisker extends to the smallest point of the inter- quartile range from the lower quartile. The Cuzick test for trend was highly significant (p < 0.0001). B. Cumulative S5 positivity per lesion grade at the three cut-offs: 0.80 (red), 2.62 (blue) and 3.70 (green). Median values are shown by each bar and error bars show 95% CI of the median. Abbreviations: HPV(−)/Cyt(−),HPV negative and cytology negative; HPV(+)/Cyt(−), HPV positive and cytology negative; CIN, cervical intraepithelial neoplasia (of grade 3); CSI-IV, cervical cancer stages I-IV.

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S5 component breakdown in HPV+ NEG, CIN3 and CSI-IV samples. The weight of the methylation on the S5 components: EPB41L3, HPV16, HPV18, HPV31 and HPV33 has been calculated for each group. Percentages of EPB41L3 and HPV16 weights in the classifier are noted at the top of the corresponding bars. HPV methylation becomes less important versus EPB41L3 as the lesions become more advanced, however EPB41L3 weight in the classifier does not change after stage II cancer.