The ongoing TRACE prospective, multicenter study provided a thorough clinical evaluation of the POU4F3 methylation by using the CONFIDENCE Marker™ RUO test in HPV triage by comparison to cytology triage. A new version of POU4F3 methylation test, called CONFIDENCE Marker™ (IVD-CE) was developed with the intended use of triaging hrHPV positive women aged 30 years or older and giving an indication about the women's current CIN2+ risk.
CONFIDENCE Marker™ (IVD CE) measures the relative methylation level of the promoter region of the gene called POU4F3 by quantitative methylation specific real-time PCR (qMSP) technology compared to the reference gene COL2A1 (providing a so-called Methylation index). Clinical performance of the CONFIDENCE Marker™ (IVD CE) was assessed on hrHPV positive (CONFIDENCE HPV™) LBC samples (CIN2- n=187; CIN2+ n=26) selected from the TRACE study collected from subjects aged 30 years or older. The results of the CONFIDENCE Marker™ (IVD CE) for CIN2+ and CIN3+ clinical endpoints and their agreement with the CONFIDENCE Marker™ RUO test results in the TRACE study was calculated.
The CONFIDENCE Marker™ (IVD CE) achieved sensitivity of 88.5% (69.8-97.6%) with the relative sensitivity of 0.96 (0.77-1.19) and specificity of 69.6% (47.1-86.8%) with the relative specificity of 1.13 (0.79-1.59) for the histologically confirmed samples and 88.5% (69.8-97.6%) with a relative sensitivity of 0.96 (0.77-1.19) and 75.9% (69.2-81.9%) with a relative specificity of 0.99 (0.88-1.11) calculated on all samples, respectively for CIN2+ histological endpoint in the age group 30-65 of hrHPV positive women. The relative values were assessed by comparison to the CONFIDENCE Marker™ RUO test results in the TRACE study. The overall agreement of the two CONFIDENCE Marker™ test workflow results was 96.2% (80.4-99.9%) and 100% for CIN2+ and CIN3+ endpoints, respectively. The current analysis is focused on the baseline cross-sectional clinical results, the 3 years follow-up of the study is ongoing.
Based on the relative sensitivity and specificity values obtained, TRACE's clinical evaluation of the CONFIDENCE Marker™ RUO can be considered valid for the CONFIDENCE Marker™ (IVD CE) as the results do not show a significant statistical difference.
On the basis of our findings, one of the first IVD-CE validated methylation assay, the new CONFIDENCE Marker™ (IVD CE) detecting the POU4F3 methylation as a triage test of hrHPV positives appears to be a promising method. We can reasonably assume that its quantitative nature offers the potential of an objective and discriminative risk assessment tool in the prevention and diagnostics of high-grade CIN lesions and cervical cancer.