General discussion and future perspectives 173 8 A combination of ASCL1 and LHX8 reached an AUC of 0.890 for the detection of CIN3, resulting in a cross-validated CIN3 sensitivity of 83.4% and a specificity of 82.4%. The bi-marker panel detected all 50 cervical cancers in the validation set. Evaluation of these markers in an independent hrHPV-positive cohort from a Dutch cervical screening study (IMPROVE) (Chapter 3) confirmed good performance for CIN3+ detection, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). Combined as a bi-marker panel with predefined threshold as set in Chapter 2, ASCL1/LHX8 methylation showed a sensitivity of 76.9% (95% CI 68.3-85.6%) and a specificity of 74.5% (95% CI 71.1-77.9%) for detecting CIN3+. The slightly lower performance may be explained by the utilisation of an HPV-based screening cohort in Chapter 3, as opposed to the case-control setting employed in Chapter 2. Additionally, Chapter 3 included CIN2 lesions. The bi-marker panel ASCL1/ LHX8 had a similar sensitivity as HPV16/18 genotyping but showed a markedly higher specificity. A triage strategy using 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. This is in line with previous studies evaluating a combination of methylation and genotyping 63, 64. The CIN3+ sensitivity (76.9%) and specificity (74.5%) achieved by the bi-marker panel ASCL1/LHX8 (Chapter 3) are reassuring when compared to the triage performance of other methylation markers 65, 66 although comparisons are complicated due to variations in gene panels and study designs. A meta-analysis of pooled data from various methylation markers (i.e., CADM1, MAL, miR124-2, FAM19A4, POU4F3, EPB41L3, PAX1, SOX1 and HPV16 (L1/L2)) demonstrated a sensitivity of 70.5% (95% CI 64.8-75.6%) for detecting CIN2+ in hrHPV-positive women and a specificity of 74.5% (95% CI 70.8-78.1%) 65. For the comparison of DNA methylation testing with cytology at a cut-off of ≥ASC-US, the pooled analysis revealed similar sensitivity for detecting CIN3+ (ratio 0.84, 95% CI 0.64-1.17) but higher specificity (ratio 1.37, 95% CI 1.02-1.85). Compared to HPV16/18 genotyping, methylation markers showed comparable CIN3+ sensitivity (ratio 1.19, 95% CI 0.94Figure 8.2 Overview of cervical cancer screening in the Netherlands and proposed methylationbased strategies. HPV-based cervical cancer screening with cytology triage in the Netherlands (A); Proposed strategy for the use of methylation analysis as a triage test for hrHPV-positive women (B), and as a secondary triage test for hrHPV-positive women with ASC-US/LSIL cytology (C). Abbreviations: ASC-US, atypical squamous cells of undetermined significance; DNA, deoxyribonucleic acid; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade intraepithelial lesion; 12m, twelve months; me, methylation; NILM, negative for intraepithelial lesion or malignancy; –, negative; and +, positive
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