184 Chapter 7 Table 2: Parameter estimates of cfDNA concentration in the urine of NSCLC patients measured across the different sampling time points according to the fitted linear mixed model corrected for sex. cfDNA concentration Fixed effects Estimates 95%-CI p (Intercept) 19.51 (8.31, 45.72) day [2] -0.13 (-0.52, 0.25) 0.512 time [afternoon] 0.11 (-0.36, 0.60) 0.638 time [evening] 0.25 (-0.22, 0.76) 0.298 sex [male] -1.32 (-4.00, -0.08) 0.034 Random Effects σ2 1.27 τ00 subject 1.24 ICC 0.49 N subject 23 Observations 138 cfDNA concentration estimates are presented in ng DNA/mL urine. σ2 = within-subject variability; τ00 = between-subject variability; cfDNA = cell-free DNA; ICC = Intraclass Correlation Coefficient. NSCLC = non-small cell lung cancer. Variation in methylation levels Variation during the day and between days DNA methylation levels of CDO1, SOX17, and TAC1 were measured in all urine samples (n=138) by qMSP (Figure 3). Five urine samples were excluded from the analysis based on an ACTB Ct value of ≥32. The discriminatory power of the qMSP was verified by comparing methylation levels in 11 pairs of NSCLC and adjacent normal tissues (Supplementary Figure 1). Differences in time were assessed by a linear mixed model framework. None of the studied markers showed systematic differences in methylation levels during the day or between the two days (Table 3). Methylation levels found were independent of sex, age, weight, therapy during urine collection, survival, tumor stage, and tumor histology. For each marker, a significant association between methylation level and the cfDNA concentration was observed (p < 0.05; Wald test). Between- and within-subject variation The variation of DNA methylation levels between and within individual patients is displayed in Figure 4 and was examined within the same linear mixed model with subject as a random effect. The ICC values of the markers CDO1, SOX17, and TAC1 were 0.74, 0.57, and 0.14, respectively (Table 3). This indicated that 26% of the variation observed in CDO1 methylation levels is due to variability within patients, as opposed to 43% for SOX17 and 86% for TAC1. Model assumptions were not violated as indicated by diagnostic tests (Supplementary Material).
RkJQdWJsaXNoZXIy MjY0ODMw