Evaluation of Six Methylation Markers Derived from Genome-Wide Screens for Detection of Cervical Precancer and Cancer

Abstract

Aim: To evaluate the triage performance of six host-cell DNA methylation markers derived from two genome-wide discovery screens for detection of cervical precancer (cervical intraepithelial neoplasia 3 [CIN]) and cancer. Materials & methods: Human papillomavirus-positive cervical scrapes of controls (≤CIN1; n = 352) and women diagnosed with CIN3 (n = 175) or cervical cancer (n = 50) were analyzed for methylation of ASCL1, LHX8, ST6GALNAC5, GHSR, SST and ZIC1. Results: Methylation levels increased significantly with disease severity (all markers p < 0.001). Three markers ( ASCL1, LHX8, ZIC1) showed receiver operating characteristic curves with area under the curve >0.800 after leave-one-out cross-validation. Bi-marker panel ASCL1/LHX8 had highest area under the curve (0.882), and detected 83.4% of CIN3 and all cervical cancers at specificity of 82.4%. Conclusion: All six methylation markers showed an equivalent, high performance for the triage of human papillomavirus-positive women using cervical scrapes with complementarity between markers.

Performance of DNA methylation analysis of ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST for the triage of HPV‐positive women: Results from a Dutch primary HPV‐based screening cohort

“…In our study, the bi-marker panel ASCL1/LHX8 had a similar sensitivity as HPV16/18 genotyping but showed a markedly higher specificity. Though small in numbers, the ASCL1/LHX8 marker panel was able to detect all cervical cancer cases (n = 3), in line with Dick et al 10 The HPV genotypes in these cancers were HPV16 (n = 1), HPV18…”

“…The most important outcome of our study is the validation of host-cell DNA methylation markers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1, SST and the bi-marker panel ASCL1/LHX8, as defined in our previous study, 10 in a large and independent hrHPV-positive screening cohort, confirming their good and robust triage performance for CIN3+. Current findings underscore the potential of ASCL1/LHX8 methylation analysis as a useful alternative to cytology as a triage test in primary HPV-based screening with the advantage of being objective, less prone to training and interpretational errors, automatable and offering a full-molecular approach.…”

“…Secondary triage of hrHPV-positive women with low-grade cytology by ASCL1/LHX8 methylation might reduce the number of women referred for colposcopy with 70%, while ensuring that most women with CIN3+ receive colposcopy (CIN3+ sensitivity of 63.6%; The methylation levels of ASCL1, LHX8, ST6GALNAC5, GHSR, SST and ZIC1 in hrHPV-positive cervical screening samples displayed an increase with the severity of the underlying cervical disease, confirming previous findings. 10 Slight variations in methylation level range between the six markers may be explained technologically by target-to-target differences in the qMSP or may be related to biology as some of the markers are known to reside on chromosomal regions that can be affected by copy number alterations. In earlier work, we observed that the methylation levels of GHSR, SST and ZIC1 (located at 3q) in cervical scrapes were significantly higher in the presence of a 3q gain in the corresponding CIN2/3 lesions compared to the absence of a 3q gain.…”

“…ROC curves and corresponding AUC for CIN3+ detection for (A) single marker classifiers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST and (B) bi-marker panel ASCL1/LHX8 (black line), including point estimates of cytology (red circle), HPV16/18 genotyping (green circle) and bi-marker panel ASCL1/LHX8 with the predefined threshold calibrated at 70% clinical specificity (yellow circle). For comparison, the cross-validated ROC curve and corresponding AUC, as obtained in the hrHPV-positive training series, are projected (grey line) 10. AUC, area under the curve; CIN, cervical intraepithelial neoplasia; ROC, receiver characteristic [Color figure can be viewed at wileyonlinelibrary.com]T A B L E 1Abbreviations: CI, confidence interval; CIN, cervical intraepithelial neoplasia; n, number of; N, group size; NPV, negative predicted value; PPV, positive predicted value.…”

Classification of high‐grade cervical intraepithelial neoplasia by p16ink4a, Ki‐67, HPV E4 and FAM19A4/miR124‐2 methylation status demonstrates considerable heterogeneity with potential consequences for management

“…Methylation status was included since methylation levels increase with increasing disease severity and are extremely high in cervical carcinomas (Figure 1). 35,[39][40][41] Furthermore, when the duration of a HPV infection is taken as a proxy for CIN lesion existence, methylation positivity is higher in CIN2/3 lesions…”