158 Chapter 6 The DNA methylation levels of CDO1, SOX17, TAC1, and CDO1 + SOX17 combined were visualized for each individual sample using predicted probabilities (Figure 3). Urine samples of surgical NSCLC patients were stratified per histological category, including LUAD, LUSC and NOS. Sorting the samples on predicted probabilities found for CDO1 + SOX17 combined illustrates the added value of using a marker panel, instead of individual markers. Methylation levels were highly variable among both the surgical NSCLC patients and controls. Predicted probabilities ranged from 0.13 (green) to 0.56 (red). The majority of cases showed high methylation levels (red/orange) and most controls showed low methylation levels (yellow/green) for the combined marker panel. CDO1 Methylation level SOX17 TAC1 CDO1 + SOX17 High Low Non-metastatic NSCLC (n=44) Control (n=50) LUAD LUSC NOS Figure 3: DNA methylation of CDO1, SOX17, TAC1, and CDO1+SOX17 combined in urine samples of surgical NSCLC patients per histological category and healthy controls. The methylation levels are shown per sample (column) and visualized using predicted probabilities in a three-color gradient from green (lowest predicted probability – 0.13, indicating low methylation levels) to red (highest predicted probability – 0.56, indicating high methylation levels). Samples are stratified per histological category, including LUAD, LUSC, and NOS, and sorted based on the predicted probabilities of the optimal marker panel CDO1 + SOX17. LUAD = lung adenocarcinoma, LUSC = lung squamous cell carcinoma, NOS = carcinoma not otherwise specified. DNA methylation in post-operative urine samples The primary objective of this study was to evaluate the diagnostic potential of DNA methylation analysis in urine of NSCLC patients undergoing surgery with curative intent. Upon acquisition of preliminary results thereof, postoperative urine samples were also collected from a subset of the enrolled NSCLC patients to assess the methylation levels after resection of the tumor. Postoperative urine samples were collected from 14 NSCLC patients, 6 or 7 days (n=4) or 63 to 974 days (n=10) after surgery with curative intent (Supplementary Table 1). The upper quartile value (i.e. 75% percentile) of methylation levels in control samples was used to arbitrarily define a threshold (1.2 for CDO1 and 1.3 for SOX17) for preoperatively elevated methylation levels. Since TAC1 showed no differences in urine methylation between cases and controls, defining such a threshold was not possible and therefore only methylation levels of CDO1 and SOX17 were evaluated in postoperative samples. To explore if methylation levels were altered shortly after curative intent surgery, 4 patients provided urine samples before and 6 or 7 days after surgery (Figure 4). In 3 patients with preoperatively elevated methylation levels for both markers (patient 1) or a single marker (patient 2 and 4), methylation levels were reduced in the post-operative
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