ASCL1 and LHX8 methylation in HPV-positive self-collected samples 95 4 and cervical tissue specimens. Differences in methylation levels between paired sample types were assessed using the Mann-Whitney U test. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the detection of CIN3+ were determined with Wald 95% confidence intervals (95% CI) for the following triage strategies: methylation analysis, HPV16/18 genotyping, HPV16/18 genotyping combined with methylation analysis and cytology. For comparison, relative CIN3+ sensitivity and specificity were determined together with 95% CIs. The methylation status of ASCL1 and LHX8 was labelled positive when Cq was below 40. The ASCL1/LHX8 marker panel was considered positive if at least one of the markers tested positive (“believe-the-positive”) 31. Genotyping results were available for 491 women, and HPV16/18 genotyping as triage strategy was labelled positive if HPV16 and/or HPV18 were present. HPV16/18 genotyping combined with methylation analysis was labelled positive if HPV16 and/or HPV18 were present or the ASCL1/LHX8 marker panel was positive. Cytology results of the paired clinician-collected cervical sample were available for 593 women and cytology as triage was labelled positive if baseline cytology on the paired clinician-collected cervical sample was ≥ASC-US (i.e., ≥BMD). The association between methylation and age was studied using a logistic regression analysis. Methylation data on paired self-collected and clinician-collected cervical samples were compared, by calculating overall agreement and Cohen’s kappa and by calculating relative CIN3+ sensitivity and relative specificity together with 95% CIs. Statistical analyses were performed using SPSS software for Windows (version 26.0, SPSS Inc., Chicago, Il, USA) and GraphPad Prism (version 9.1.0). RESULTS METHYLATION OF ASCL1 AND LHX8 IN HRHPV-POSITIVE SELF-COLLECTED SAMPLES A total of 593 self-collected samples from hrHPV-positive women who participated in the IMPROVE study (median age 40.0; IQR 34–49; range 29–60) were included for analysis of the DNA methylation markers ASCL1 and LHX8 (Figure 4.1). The series comprised of one woman with cervical squamous cell carcinoma, 85 women with CIN3, 45 women with CIN2, and 462 control women who had no evidence of CIN2+, including 293 women with two consecutive normal cytology results, 71 with no CIN and 98 with CIN1. The methylation levels of each marker for each disease category are shown in Figure 4.2. The methylation levels of ASCL1 and LHX8 in hrHPV-positive self-collected samples increased
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