177 Dynamics of methylated cell-free DNA in the urine of non-small cell lung cancer patients INTRODUCTION Lung cancer remains the leading cause of cancer-related deaths worldwide (1). Screening programs have shown that cancer-related mortality could be reduced by using low-dose computed tomography (LDCT) screening in selected high-risk patients (2-4). Combining this approach with molecular marker testing in liquid biopsies could further improve the screening selection and management of positive LDCT screening tests. The analysis of methylated cell-free DNA (cfDNA) in liquid biopsies is a promising, safe, and easily applicable tool that is now being investigated for the detection of lung cancer. Methylation, an epigenetic DNA modification that regulates gene expression, is known as a critical process, involved in early lung cancer development and progression (5). Amongst liquid biopsies, blood and sputum are the most commonly reported sources of cfDNA for methylation analyses (6-14). On the other hand, urine is an upcoming means for liquid biopsy analyses in lung cancer diagnostics (10, 15, 16). Urine-based liquid biopsies are of particular interest, as the collection is completely non-invasive and can be performed at home. Moreover, large volumes can be collected regularly, which allows for repetitive sampling at frequent intervals. Despite encouraging developments of urine-based liquid biopsies for lung cancer detection, this technique is not yet ready for implementation into clinical practice. Over the past years, considerable improvements have been achieved by optimization and standardization of pre-analytical conditions (17-20). However, one of the major remaining questions regarding the yield of cfDNA is the uncertainty on whether the circadian rhythm leads to variations in the amounts of methylated DNA in urine. There is also a limited understanding of the range of biological variation of methylated cfDNA in the urine of lung cancer patients. Biological variability refers to the random fluctuation of analyte concentrations around a homeostatic set point (within-subject variability), which varies per individual (between-subject variability) (21). Previous studies have focused exclusively on the abundance of cfDNA in plasma of healthy controls and lung cancer patients, which appeared to vary greatly within (22) and between individuals (23), and during the day (24). The aim of this study, therefore, was to investigate the dynamics of methylated cfDNA in the urine of lung cancer patients to estimate both between- and within-subject variability, and to evaluate whether a preferred urine collection time and sampling frequency exist. 7
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