152 Chapter 6 We considered the markers CDO1, SOX17, and TAC1 as most interesting methylation marker candidates to evaluate the detection of non-metastatic NSCLC in the urine. This is based on their diagnostic potential for detecting NSCLC in sputum (17, 27), plasma (17, 19), and urine (19). Previously, high diagnostic efficacy of these methylation markers was shown in genome-wide discovery studies using both tissues from The Cancer Genome Atlas (TCGA) database (17, 19, 27) and other discovery cohorts (27). CDO1 was also specifically identified and validated as a biomarker for stage I NSCLC detection in minimally invasive samples (27). In this study, the diagnostic potential of DNA methylation analysis for non-metastatic NSCLC detection in urine was evaluated by assessing the previously described NSCLC methylation markers CDO1, SOX17 and TAC1 (17, 19, 27, 28) in preoperative urine samples. Furthermore, we explored the methylation levels of these genes in postoperative urine samples to evaluate whether methylation levels altered after surgery. MATERIAL AND METHODS Study design and population This was a single-institution prospective study from a non-screening population in The Netherlands. Eligible patients, planned for anatomical pulmonary resection for (suspected) NSCLC, were consecutively enrolled and urine was collected between March 2018 and September 2020 at the outpatient clinic of the Department of Pulmonary diseases of the Amsterdam UMC, a tertiary referral center in Amsterdam, the Netherlands. Patients were older than 18 years, diagnosed with NSCLC of any histological subtype, did not undergo any anti-cancer (induction) therapy for at least one year prior to sampling and had no diagnosis of any type of other cancer in the last 5 years preceding lung cancer diagnosis. The cancer stage was determined using the 8th edition of the TNM classification system of the International Association for the Study of Lung Cancer (IASLC) (29). For exploration purposes, also postoperative urine samples were collected during the course of the study to evaluate whether methylation levels alter after resection of the tumor. Hence, postoperative samples were only collected in a subset of patients at various time points after surgery with curative intent. Control samples were obtained from healthy volunteers through the Urine Controls (URIC) Biobank. URIC participants were selected for eligibility through a questionnaire to exclude controls with a cancer history in the previous 5 years. Furthermore, age, sex,
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