Thesis

187 Dynamics of methylated cell-free DNA in the urine of non-small cell lung cancer patients Sample time point CDO1 methylation level (square root ct ratio) 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 a Sample time point SOX17 methylation level (square root ct ratio) 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 b Sample time point TAC1 methylation level (square root ct ratio) 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 c Sex Female Male day 1 day 2 m a e m a e day 1 day 2 m a e m a e day 1 day 2 m a e m a e Figure 4: Conditional scatterplots displaying the between- and within-subject variability of CDO1 (a), SOX17 (b), and TAC1 (c) methylation levels of each patient across the six sampled time points (m = morning, a = afternoon e = evening), stratified by sex (pink square = female, blue circle = male). Missing data points indicate excluded urine samples with an ACTB value of ≥ 32. Prolonged urine sampling To explore whether collecting multiple urine samples provides a more accurate test outcome, methylation levels of CDO1, SOX17, and TAC1 were also measured in urine samples of healthy controls (n=60). The control cohort had a median age of 69 (range 58-79) and 30 controls were female. The qMSP Ct values are provided in the Supplementary Data file (online). The discriminatory power of each marker was first evaluated when including six samples per patient and compared to the levels found in controls. Linear mixed models were used to correct for repeated measurements in the patient group. Significant differences in methylation levels of cases and controls were found for SOX17 (Wald test, p=0.030), and also TAC1 showed a trend toward significance (Wald test, p=0.059), both independent of age and sex (Figure 5 and Supplementary Table 2). 7

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