200 Chapter 9 This practical workflow may provide the basis for automation to aid effective highthroughput DNA methylation analysis and support a fully molecular solution to cervical cancer screening. In the current population screening program in the Netherlands, the implementation of primary HPV screening with cytology as a triage test has resulted in an increase in referrals for colposcopy. This rise is particularly noticeable among hrHPV-positive women with mild cytological abnormalities (ASC-US/LSIL cytology). Due to this increase in colposcopy referrals, there is a need for a second triage test for women with ASC-US/LSIL. Chapter 6 evaluates various molecular secondary triage methods for hrHPV-positive women with ASC-US/LSIL cytology to identify CIN3+. The study utilised 194 HPV-positive, ASC-US/LSIL clinician-collected cervical samples from the Dutch IMPROVE screening trial (NTR5078) that had been tested for FAM19A4/miR124-2 and ASCL1/LHX8 methylation, as well as had been genotyped for 14 hrHPV types. An incremental analysis was used to identify strategies that optimally weigh the number of CIN3+ detected against the number of colposcopy referrals. FAM19A4/miR124-2 methylation and ASCL1/LHX8 methylation demonstrated improved PPV and NPV for CIN3+, with lower colposcopy rates than 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%. This study underscores the potential of incorporating methylation analysis, possibly combined with HPV genotyping, as a secondary triage test for HPV-positive women with ASC-US/ LSIL cytology. The proposed strategies enhance the efficiency of cervical screening by significantly reducing the number of colposcopy referrals at baseline. However, none of the strategies had a sufficiently high NPV to warrant return to routine screening and repeat testing at 6-12 months is recommend after negative triage. HPV vaccines have been available since 2006. In the Netherlands, the HPV bivalent vaccine was incorporated into the National Immunisation Program for 12-year old girls in 2009. In 2023, the first women vaccinated against HPV will reach the cervical cancer screening age of 30. Owing to the reduced incidence of cervical cancer among vaccinated women, the predictive value of a positive screening result may be reduced and the proportion of false-positive results could increase. Hence, screening may pose a potential risk outweighing its benefits if not adjusted to HPV-vaccinated women. It is important to
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