Methylation estimates the risk of precancer in HPV-infected women with discrepant results between cytology and HPV16/18 genotyping

Abstract

Background Vigilant management of women with high-risk human papillomavirus (hrHPV) is necessary in cancer screening programs. To this end, we evaluated the performance of S5 (targeting DNA methylation in HPV16, HPV18, HPV31, HPV33, and human gene EPB41L3) to predict cervical intraepithelial neoplasia grade 2 or higher (CIN2+) in a sample of hrHPV-infected women referred to colposcopy in the FRIDA Study, a large screening trial in Mexico. A nested case-control sample with women referred to colposcopy either by atypical squamous cells of undetermined significance or higher (ASCUS+) in cytology and/or positive for HPV types 16 or 18 was tested by S5. Seventy-nine cases of CIN2+ were age-matched to 237 controls without a diagnosis of CIN2+ (<CIN2). DNA from exfoliated cervical cells was bisulfite converted and PCR amplified for S5 targets, and methylation was quantified at specific cytosines by pyrosequencing. Results The S5 classifier separated women with CIN2+ from <CIN2 with a highly significant area under the curve (AUC) of 0.75 (95% CI 0.69–0.82), while AUC for CIN3+ was 0.81 (95% CI 0.74–0.89). To optimize sensitivity and specificity for Mexico, an alternative S5 cutoff of 3.7 was implemented to account for overall higher methylation seen in our already triaged women. All three invasive cancers were detected by methylation or HPV16/18 but none by cytology. Sensitivity of S5 for CIN2+ was 62% (95% CI 50.4–72.7%), specificity was 73% (95% CI 66.9–78.5%), and adjusted PPV was 15.1% (95% CI 12.0–18.3%). In contrast, the crude sensitivity of HPV16/18 detection and cytology were 63.3% (95% CI 51.7–73.9%) and 57.0% (95% CI 45.3–68.1%) respectively; specificity was 29.1% (95% CI 23.4–35.3%) and 62.4% (95% CI 55.9–68.6%) respectively, while adjusted PPV was 6.4% (95% CI 4.9–8.1%) and 10.5% (95% CI 8.0–13.1%), respectively. Methylation testing could reduce colposcopy referrals by 30 to 50% with virtually no loss of sensitivity for CIN2+ and CIN3+. Conclusions S5 testing on hrHPV-positive women significantly increased diagnostic information compared to triage by HPV16/18 plus cytology and appears to have clinical utility as an additional test to substantially lessen burdens on colposcopy. Trial registration The FRIDA Study is registered in ClinicalTrials.gov, number NCT02510027.

Evaluation of a methylation classifier for predicting pre-cancer lesion among women with abnormal results between HPV16/18 and cytology

“…On the one hand, it is not crucial to detect all pre-cancer lesions in the first screening because most of pre-cancer lesions regress or progress slowly. However, cancers are not reversible, and S5 classifier detected all cancer cases in our study and three other previous studies [18][19][20]. These results indicate that S5 is safe for detecting all cancer cases and 89.1% of (His)HSIL+ cases.…”

“…high sensitivity and high specificity would provide additional information for women with discrepant results of HPV16/18 and cytology. Another encouraging finding of our study is reanalysis of the diagnostic utility of S5 reported in three earlier studies, a large screening study in the UK, a nested case-control triage study in Mexico [18], and a randomized control trial in Canada [21]. In the hrHPVbased screening study from the UK, using a cutoff of 0.8 for S5, the AUC of the 0.8 cutoff was 0.78 (95% CI 0.69-0.88) for HSIL+ but had a lower specificity of 20.9%.…”

“…DNA methylation assays targeting host and/or HPV genes may meet this requirement, as they have been shown to have higher sensitivity and similar specificity to LCT (liquid-based cytology) for identification. S5®(car-eLYFE) methylation is based on targeting regions of HPV16, HPV18, HPV31, and HPV33 combined with the promoter region of the human tumor suppressor gene EPB41L3 [18]. The PCR-based multiplex assay was followed by quantitative pyrosequencing to measure methylation levels of each assay component.…”

“…However, the S5®(careLYFE) methylation tests for the triage of cervical cancer and "≥HSIL+" among Chinese women remain elucidated. The S5 assay was developed in a colposcopy study and has been well tested in the UK, Canada, and Mexico [19][20][21][22]. As the infection rates of HPV31 and HPV33 in China are low, as well as the lower infection rates of HPV31 and HPV33 in HSIL and cancer, Care Me is a methylation classifier consisting of three regions: EPB41L3, HPV16, and HPV18.…”

DNA methylation for cervical cancer screening: A validation trial in China

“…A number of studies have explored the role of a panel that includes EPB41L3, [15][16][17][18][19][20][21][22][23][24] JAM3, [25] or both [7,[26][27][28][29][30] for the screening or triage of HSIL and/or cervical cancer. In this validation trial, the performance of DNA methylation assessment based on the EPB41L3 and JAM3 genes was similar to that of our training set, [14] and the assessment showed favorable results in identifying CIN2+ or ADC .…”