ASCL1 and LHX8 methylation in HPV-positive self-collected samples 93 4 had a histology endpoint or two consecutive normal cytology results, and who provided consent for follow-up research (n = 780). Of these, 187 samples were excluded due to insufficient leftover material for valid methylation analysis, resulting in a final study population of 593 hrHPV-positive self-collected samples with ASCL1/LHX8 methylation results (Figure 4.1). ASCL1/LHX8 methylation data on paired hrHPV-positive cliniciancollected cervical samples were available for 485 women (Figure 4.1, subset A) 25. These women were hrHPV-positive on both their clinician-collected and their self-collected sample 5. In a subgroup of 116 women (Figure 4.1, subset B), the corresponding formalinfixed paraffin-embedded (FFPE) tissue sample was additionally available for methylation analysis. This study was approved by the Medical Ethics Committee of Amsterdam UMC, Vrije Universiteit Amsterdam (Amsterdam, The Netherlands; METC 2018/09, TcB 2018.106). The IMPROVE study was approved by the Ministry of Public Health (The Hague, The Netherlands; IMPROVE VWS no. 2014/32). DNA ISOLATION DNA from self-collected samples was isolated using the NucleoMag 96 Tissue kit (Macherey-Nagel GmbH&Co. KG, Düren, Germany) and a Microlab Star robotic system (Hamilton, Gräfelfing, Germany), according to the recommendations of the manufacturer. Whole tissue sections from the FFPE tissue blocks were prepared using the sandwich Subset B Methylation results on corresponding cervical tissue specimen n = 116 no CIN n = 6 CIN1 n = 22 CIN2 n = 20 CIN3 n = 67 Cancer n = 1 Subset A Methylation results on paired HPV-positive clinician-collected cervical samples n = 385 2x NILM n = 212 no CIN n = 64 CIN1 n = 87 CIN2 n = 42 CIN3 n = 79 Cancer n = 1 Study population Methylation results on HPV-positive self-collected samples n = 593 2x NILM n = 293 no CIN n = 71 CIN1 n = 98 CIN2 n = 45 CIN3 n = 85 Cancer n = 1 Figure 4.1 Venn diagram showing the relation between the sample series used in this study. Data analysis comprised methylation results of self-collected samples from 593 hrHPV-positive women (white ellipse; study population), methylation results of paired clinician-collected samples from 485 (out of 593) women (light pink ellipse; subset A) and methylation results of corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples of 116 (out of 485) women (dark pink ellipse; subset B). Abbreviations: HPV, human papillomavirus; n, number of; NILM, negative for intraepithelial lesion of malignancy; CIN, cervical intraepithelial neoplasia

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