DNA methylation for the triage of HPV-positive clinician-collected cervical samples 77 3 and methylation stratified by cytology, as well as estimates for endpoint CIN2+ can be found in Table 3.2. DISCUSSION The most important outcome of this 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. 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 (n = 1) and HPV35 (n = 1). A triage strategy comprising a combination of HPV16/18 genotyping and methylation resulted in an increase in sensitivity over either triage test alone, but at a marked drop in specificity. Comparison with cytology, either or not combined with HVP16/18 genotyping, must be done with caution due to the fact that the hrHPV-positive women in the IMPROVE study were managed based on cytology. The CIN3+ sensitivity (76.9%) and specificity (74.5%) achieved by the bi-marker panel ASCL1/LHX8 are highly reassuring when compared to a meta-analysis of methylation markers for the detection of CIN3+ 6. This meta-analysis reported, when restricting to studies in which specificity was set at 70%, a pooled sensitivity for CIN3+ of 71.1%. It is assumed that methylation analysis identifies the subset of CIN2/3 lesions with a high progression risk to cancer that are in need of direct treatment 5. Methylation levels of several genes were found significantly higher in CIN2/3 lesions associated with a longstanding (>5 years) hrHPV infection (i.e., so-called advanced lesions, with high copy number aberrations), compared to CIN2/3 with a more recently acquired (≤5 years) hrHPV infection (early or incident lesions, with lower copy number aberrations) 5, 18. The high sensitivity for advanced CIN as well as cervical cancer makes methylation marker analysis an attractive triage tool.
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