78 Chapter 3 As cytology may remain a triage method of choice and hrHPV-positive women with lowgrade cytology generate a substantial increase in referral rate while relatively few CIN3+ are detected, it is noteworthy that additional triage of hrHPV-positive women with lowgrade cytology by ASCL1/LHX8 methylation showed good performance. Secondary triage of hrHPV-positive women with low-grade cytology by ASCL1/LHX8 methylation might reduce the number of women referred for colposcopy with 70%, while ensuring that most women with CIN3+ receive colposcopy (CIN3+ sensitivity of 63.6%; Table 3.2). Even though ASCL1/LHX8 methylation showed good performance data in hrHPV-positive women with high-grade cytology, the diagnosis of high-grade cytology among hrHPV-positives carries high risk of CIN3+ making that these women should be sent to colposcopy regardless of methylation status. The ASCL1/LHX8 methylation data stratified by cytology presented here are in line with a multi-centre study by Bonde et al. which reported on another methylation marker panel consisting of FAM19A4 and miR124-2 19. The methylation levels of ASCL1, LHX8, ST6GALNAC5, GHSR, SST and ZIC1 in hrHPV-positive cervical screening samples displayed an increase with the severity of the underlying cervical disease, confirming previous findings 10. Slight variations in methylation level range between the six markers may be explained technologically by target-to-target differences in the qMSP, or may be related to biology as some of the markers are known to reside on chromosomal regions that can be affected by copy number alterations. In earlier work we observed that the methylation levels of GHSR, SST and ZIC1 (located at 3q) in cervical scrapes were significantly higher in the presence of a 3q gain in the corresponding CIN2/3 lesions compared with the absence of a 3q gain 8. The markers used in this study were identified by genome-wide methylation profiling of cervical samples 8, 9 and have been reported in other cancer types as well. For example, ASCL1 was found to be methylated in amongst others colorectal, oral and anal cancer 20-22, and LHX8 in breast and lung cancer 23, 24. ASCL1 is a proneural, oncogenic transcription factor 25, 26 and LHX8 is a highly conserved transcription factor involved in cell fate in neurogenesis, tooth morphogenesis and oogenesis 24. The functional involvement and methylation-dependent expression regulation of these genes in cervical carcinogenesis remain to be clarified. The major strength of the current study is the evaluation of a large and independent series of hrHPV-positive clinician-collected cervical samples derived from primary HPVbased screening. The IMPROVE study was nested within a population-based screening setting and intended to reflect the new routine primary HPV-based screening programme that was introduced in 2017 in the Netherlands, suggesting that results could be scalable
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