ASCL1 and LHX8 methylation in HPV-positive self-collected samples 99 4 were available for 485 hrHPV-positive women (Figure 4.1, subset A), including one woman with cervical squamous cell carcinoma, 79 women with CIN3, 42 women with CIN2, and 363 controls. The overall agreement in ASCL1/LHX8 methylation outcomes between self-collected and clinician-collected samples was 64.1% (310/507), 95% CI 59.7-68.4% (Cohen’s kappa 0.250). The CIN3+ sensitivity and specificity of ASCL1/LHX8 methylation analysis were lower in self-collected samples compared to clinician-collected cervical samples, with a relative sensitivity of 0.95 (95% CI 0.82-1.10) and a relative specificity of 0.82 (95% CI 0.75-0.90). For a small subset of women (Figure 4.1, subset B, n = 116), paired cervical tissue specimens were also available for methylation analysis. The series comprised of one woman with squamous cell carcinoma, 67 women with CIN3, 20 women with CIN2, 22 women with CIN1 and 6 women with no CIN. Paired methylation data stratified for histology are visualised in Figure 4.4. A moderate correlation between the methylation levels of both ASCL1 and LHX8 in different sample types was observed (Spearman’s Rho 0 1 2 3 4 0 1 2 3 4 Methylation level ASCL1 (sqrt ΔΔCq ratio) Methylation level LHX8 (sqrt ΔΔCq ratio) S F C S F C S F C S F C S F C S F C S F C S F C no CIN / CIN1 n = 28 CIN2 n = 20 CIN3 n = 67 Cancer n = 1 no CIN / CIN1 n = 28 CIN2 n = 20 CIN3 n = 67 Cancer n = 1 Figure 4.4 Conditional scatterplots displaying the methylation levels for (A) ASCL1 and (B) LHX8 for paired self-collected samples (S), clinician-collected cervical samples (C) and cervical tissue specimens (F) stratified for histology. Differences in methylation levels of ASCL1 and LHX8 between paired sample types were assessed using the Mann-Whitney U test: self-collected samples compared to clinician-collected cervical samples, both p values < 0.0001; self-collected samples compared to cervical tissue specimens, p value = 0.134 and 0.002, respectively; and clinician-collected cervical samples compared cervical tissue specimens, p value = 0.130 and 0.179, respectively. Abbreviations: Cq, quantification cycle; CIN, cervical intraepithelial neoplasia; sqrt, square-root; S, self-collected sample; C, clinician-collected sample; F, cervical tissue specimen; n, number of
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