178 Chapter 7 METHODS Study population In this prospective cohort study, patients with histologically confirmed NSCLC were consecutively recruited between November 2019 and January 2020 at the outpatient clinic of the Department of Pulmonology of the Amsterdam University Medical Center, location VUmc, Amsterdam. Inclusion criteria of NSCLC patients involved being diagnosed with active disease (i.e. before anti-cancer therapy or at disease progression after therapy) without the presence or history of any other primary malignancies. The revised eighth edition of the American Joint Committee on Cancer/ Union for International Cancer Control Tumor-Node-Metastasis (TNM) Staging was used to determine tumor stage (25). Other relevant patient characteristics that were documented included sex, age, weight, tobacco use, therapy during study, survival, and histological subtype. As controls, urine samples from healthy volunteers were collected through the Urine Controls (URIC) Biobank. Inclusion criteria of controls involved not having any cancer diagnosis in the past 15 years. Sex and age were registered from each participant. Informed consent was acquired from each participating individual before urine collection. Ethical approval was obtained by the Medical Ethical Committee of the VU University Medical Center for both the DAYTIME study (No. 2017.333 and 2017.545) and the use of the URIC biobank (No. 2017.112). Urine sample collection and processing Each patient was carefully instructed to collect 30 mL of urine at three different time points for two subsequent days, adding up to a total of six samples per patient. To this end, special collection kits were designed, containing clear illustrated instructions, collection tubes, and postal envelopes. The three time points comprised morning (6:00 AM – 11:00 AM), afternoon (12:00 noon– 5:00 PM), and evening (6:00 PM – 12:00 midnight). Patients registered the time of urine collection and shipped their urine samples to the Pathology department of Amsterdam UMC, location VUmc, by regular mail. To ensure the preservation of genetic material in the urine, collection tubes contained 2 mL 0.6 M ethylenediaminetetraacetic acid (EDTA) as a preservative agent (final concentration 40 mM), and sample processing was performed within 72 h after collection. Urine samples of healthy volunteers were retrieved from the URIC biobank, which were collected once at a random time point of the day, according to the same collection protocol.
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