English summary 199 9 importance of the study described in Chapter 4 is the evaluation of the triage performance in women who were offered primary HPV self-sampling for cervical cancer screening. In addition, the unique paired screen-positive design of the Dutch IMPROVE screening trial (NTR5078) allowed for direct comparison of methylation analysis on self-collected and clinician-collected samples from the same hrHPV-positive women. DNA methylation markers ASCL1 and LHX8 were tested in 593 hrHPV-positive self-collected samples. The diagnostic performance of methylation markers for CIN3+ was evaluated and compared with that of paired hrHPV-positive clinician-collected cervical samples. Methylation levels of the markers in self-collected samples increased with increasing severity of cervical disease, in line with our earlier data on clinician-collected cervical samples. Significantly higher methylation levels of ASCL1 and LHX8 were found in hrHPV-positive self-collected samples of women with CIN3+ than control women with no evidence of disease (both p values < 0.0001). The ASCL1/LHX8 marker panel exhibited a sensitivity of 73.3% (63/86; 95% CI 63.9-82.6%) and a specificity of 61.1% (310/507; 95% CI 56.9-65.4%) for detecting CIN3+. ASCL1/LHX8 marker panel performance did not differ from the performance of HPV16/18 genotyping. The relative sensitivity for detecting CIN3+ was 0.95 (95% CI 0.821.10) for self-collection versus clinician-collection, and the relative specificity was 0.82 (95% CI 0.75-0.90). Overall, these results suggest that the ASCL1/LHX8 methylation marker panel is a feasible direct triage method for detecting CIN3+ in hrHPV-positive women who participate in routine screening through self-sampling. DNA methylation analysis often involves the extraction of genomic DNA from a clinical sample, followed by treatment with sodium bisulphite before PCR amplification. During this process, unmethylated cytosines are converted to uracil, whereas methylated cytosines remain unchanged. This leads to specific changes in the DNA sequence that are dependent on the methylation status of the individual cytosines, which can be quantitatively measured using methylation-specific PCR (qMSP). In order to make the laboratory protocol for DNA methylation more efficient and implementable in a high-throughput cervical cancer screening setting, Chapter 5 investigates a direct cell conversion protocol as an alternative to the commonly used approach. A total of 120 clinician-collected cervical samples were used to compare the direct conversion protocol with a standard protocol using normalised genomic DNA as input for sodium bisulphite conversion. The success rate and analytical performance of the converted samples were compared for the ACTB control gene and the methylation of FAM19A4 and miR124-2 genes. The results of this study show that the direct cell conversion protocol had a high success rate and good analytical performance on clinician-collected cervical samples.
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