159 Detection of non-metastatic non-small cell lung cancer in urine samples. Patient 2, 3, and 4 were diagnosed with recurrent disease after 101-497 days of follow-up, and patient 1 showed no recurrence up to 979 days of follow-up. Despite the fact that no clear pattern of methylation can be seen shortly after curative intent surgery in these few individual cases, it is remarkable that the only patient in which both markers were clearly elevated preoperatively, a decrease was seen postoperatively (patient 1). To obtain an impression on whether an increase in urine DNA methylation could be indicative of the presence of disease recurrence, another subset of patients (n=10) provided a second urine sample between 63 and 974 days after surgery (Figure 5). Seven of these patients (patients 5-8 and 10-12) showed elevated methylation levels of at least one marker pre-operatively, which were all reduced in the post-operative samples. None of these patients were diagnosed with recurrent disease during followup. Since only a single patient had recurrent disease after 307 days of follow-up (patient 9) and showed low methylation levels in both pre- and post-operative urine samples no correlation between recurrence and methylation could be assessed. CDO1 SOX17 1 4 3 2 Follow up time (days) Methylation level (square root Ct ratio) 0 3 500 1000 Surgery First sample Second sample No recurrence 979 days 1 7 0 CDO1 SOX17 0 5 Surgery First sample Second sample 132 1 6 101 Recurrence Multiple skeletal metastases Death 0 CDO1 SOX17 0 3 Surgery 7 First sample Second sample 874 1 7 492 Recurrence Brain metastasis Radiotherapy 0 CDO1 SOX17 7 6 0 3 Surgery 6 First sample Second sample 389 1 6 257 Recurrence Multiple brain metastases Death 0 CDO1 SOX17 Figure 4: Pre- and postoperative methylation levels of CDO1 and SOX17 for the detection of residual disease or early distant metastasis. Methylation levels of CDO1 (circles) and SOX17 (triangles) measured before (first sample) and shortly after (second sample) surgery. Moments of sampling, presence of recurrence, treatment, and patient survival are also represented for each case. Methylation levels are depicted as square root transformed Ct ratios. Note that the methylation levels of patient 2 are represented on a different y-axis to show the high CDO1 methylation level measured in the first sample. The upper quartile value of methylation levels in control samples is visualized on the y-axes to illustrate an arbitrarily defined threshold (1.2 for CDO1 and 1.3 for SOX17) for preoperatively elevated methylation levels. 6
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