General discussion and future perspectives 177 8 referrals. Nevertheless, none of the strategies, even when combined or using extended genotyping, attained a CIN3+ risk low enough with an NPV of ≥ 98%, a threshold for return to routine screening in cervical cancer screening in the Netherlands 48. Therefore, as suggested by Polman et al. 48, it is essential to conduct follow-up testing within 12 months to detect any potentially missed CIN3 lesions. In Chapter 6, methylation analysis of both FAM19A4/miR124-2 and ASCL1/LHX8 was conducted as a risk stratification tool for hrHPV-positive women with ASC-US/LSIL cytology within a Dutch primary HPV-based screening cohort. The added value of this study over above-mentioned studies is its validation of earlier findings in a prospective HPV-based screening cohort. An incremental analysis was employed to identify strategies that optimally balance the detection of CIN3+ against the number of colposcopy referrals (marginal PPV). Methylation of FAM19A4/miR124-2 and ASCL1/LHX8 demonstrated improved PPV and NPV for CIN3+, accompanied by lower colposcopy rates compared to HPV16/18 genotyping. Three strategies with mPPV ≥ 20% were identified: 1) HPV16/18 AND FAM19A4/miR124-2 positive (mPPV = 50.0%, NPV = 90.8%); 2) HPV16/18/31/33/45 AND FAM19A4/miR124-2 positive (mPPV = 33.3%, NPV = 91.2%), and 3) FAM19A4/miR1242 positive (mPPV = 20.0%, NPV = 92.2%). Triage by ASCL1/LHX8 yielded an mPPV of 17.2% and NPV of 94.2%, while all other strategies had an mPPV ≤ 10.6% and NPV ≤ 96.7%. Although these results are promising, further assessment is necessary to determine the effectiveness of methylation analysis as a secondary triage method for hrHPV-positive women with ASC-US/LSIL cytology. The findings underscore the potential of incorporating methylation analysis as a tool for refining triage strategies and reducing unnecessary colposcopy referrals. The implication is that integrating methylation analysis, either alone or in combination with HPV genotyping, can enhance the efficiency and precision of screening approaches in HPV-based cervical screening programs. However, since none of the evaluated strategies achieved an NPV of 98%, follow-up testing for women with a negative result within 12 months is essential to ensure their long-term safety. Figure 8.2C illustrates a proposed strategy for the use of methylation analysis as a secondary triage test. 8.1.3 METHYLATION ANALYSIS IN VACCINATED WOMEN There has been significant research conducted on HPV-based screening and the role of methylation markers. However, there are still important aspects that require further investigation. With the increasing implementation of HPV vaccination programs, it is crucial to evaluate the effectiveness of methylation-based screening in vaccinated women. To
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