Evaluation of a validated methylation triage signature for human papillomavirus positive women in the HPV FOCAL cervical cancer screening trial

Abstract

Human papillomavirus (HPV)‐based cervical cancer screening requires triage of HPV positive women to identify those at risk of cervical intraepithelial neoplasia grade 2 (CIN2) or worse. We conducted a blinded case–control study within the HPV FOCAL randomized cervical cancer screening trial of women aged 25–65 to examine whether baseline methylation testing using the S5 classifier provided triage performance similar to an algorithm relying on cytology and HPV genotyping. Groups were randomly selected from women with known HPV/cytology results and pathology outcomes. Group 1: 104 HPV positive (HPV+), abnormal cytology (54 CIN2/3; 50 <CIN2); Group 2: 103 HPV+, normal cytology with HPV persistence at 12 mo. (53 CIN2/3; 50 <CIN2); Group 3: 50 HPV+, normal cytology with HPV clearance at 12 mo. (assumed <CIN2), total n=257. For the combined groups, S5 risk score CIN2/3 relative sensitivity, specificity and positive predictive value (PPV) were compared with other triage approaches. Methylation showed a highly significant increasing trend with disease severity. For CIN3, S5 relative sensitivity and specificity were: 93.2% (95%CI: 81.4–98.0) and 41.8% (35.2–48.8), compared to 86.4% (75.0–95.7) and 49.8% (43.1–56.6) respectively for combined abnormal cytology/HPV16/18 positivity (differences not statistically significant at 5% level); adjusted PPVs were 18.2% (16.2–20.4) and 19.3% (16.6–22.2) respectively. S5 was also positive in baseline specimens from eight cancers detected during or after trial participation. The S5 methylation score had high sensitivity and PPV for CIN3, compatible with US and European thresholds for colposcopy referral. Methylation signatures can identify most HPV positive women at increased risk of cervical cancer from their baseline screening specimens.

Methylation of HPV 16 and EPB41L3 in oral gargles: Associations with oropharyngeal cancer detection and tumor characteristics

“…This is the first study to report levels of methylation of HPV 16 genes and EPB41L3 measured in tumors of OPC cases and oral gargle specimens from cases and controls with sufficient effect size and attributable fraction as to suggest a primary epigenetic association, a reasonable assumption given that to date no meaningful predisposing mutations have been found that may account for a majority of OPC cases. In prior studies of the cervix and anal canal methylation of HPV 16 genes and EPB41L3 were significantly positively associated with increasing grade of disease and methylation levels increased with time of progression to cancer . In our study, we report three important observations: ( i ) among OPC cases, methylation levels detected in the oral gargle and tumors were correlated, indicating that the measurement of these biomarkers in the oral gargle is representative of the tumor; ( ii ) HPV 16 methylation (L1, L2 and E2 genes) was predominantly observed among OPC cases and not detectable among disease‐free men who were oral HPV 16 positive, except for methylation of E2 in one HPV 16 positive control; ( iii ) oral EPB41L3 methylation combined with HPV 16 gene methylation status had good sensitivity and high specificity for the detection of OPC.…”

“…In prior studies of the cervix 53 and anal canal 54 methylation of HPV 16 genes and EPB41L3 were significantly positively associated with increasing grade of disease and methylation levels increased with time of progression to cancer. 55 In our study, we report three important observations: (i) among OPC cases, methylation levels detected in the oral gargle and tumors were correlated, indicating that the measurement of these biomarkers in the oral gargle is representative of the tumor; (ii) HPV 16 methylation (L1, L2 and E2 genes) was predominantly observed among OPC cases and not detectable among disease-free men who were oral HPV 16 positive, except for methylation of E2 in one HPV 16 positive control; (iii) oral EPB41L3 methylation combined with HPV 16 gene methylation status had good sensitivity and high specificity for the detection of OPC. Oral gargle HPV 16 L1 and E2 methylation in p16 positive cases was observed although HPV 16 L2 methylation was not detected.…”

“…In prior studies, HPV 16 L1, L2 and E2 methylation was associated with prevalent cervical intraepithelial neoplasia (CIN3) as well as progression of HPV 16 to CIN3 and progression of HPV infection to invasive cancer. 53,55 At the anal canal, a combined methylation score across CpG sites of these HPV 16 genes was linearly associated with anal disease severity with the lowest methylation levels observed in benign disease and highest levels among cases of high grade anal intraepithelial neoplasia (AIN). 54 This pattern of HPV gene methylation associated with disease was also observed for HPV 18, HPV 31 and HPV 45 at the cervix.…”

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

“…In previous studies of S5 performance [20], the sensitivity for cancer at the suggested triage cutoff of 0.8 was 100%. In our study, the results showed that even at a cutoff of 2.85, all cancers were detected.…”

“…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.…”