191 Dynamics of methylated cell-free DNA in the urine of non-small cell lung cancer patients diagnosed with chronic obstructive pulmonary disease) remains to be determined as no longitudinal sample sets of such subjects were available. This will provide essential information since biomarkers with small biological variability or even negative values in controls are clinically most useful for diagnostic and prognostic purposes. Detectable changes in such biomarkers will most likely reflect disease processes and not merely natural occurring variation (48). The strengths of the study include its relatively large sample size and the measurement of both cfDNA concentrations and DNA methylation levels of three genes at each collection time point. Moreover, the use of a standardized and reproducible urine processing protocol limited pre-analytical variance (18). Together with a sophisticated linear mixed modelling approach, this allowed an accurate estimation of the within- and between-subject variation of all analytes assessed in the current study. Furthermore, although not collected longitudinally, the inclusion of a representative control group enabled evaluation of the potential benefit of prolonged sampling. In conclusion, no clear circadian pattern of methylated cfDNA in the urine of NSCLC patients was observed, implying that no preferred time of urine collection exists. Nevertheless, the observed between- and within-patient variation indicates that single methylation marker measurements should be interpreted carefully, and that collecting multiple urine samples may increase the chance of detecting lung cancer in urine. Improved understanding of the dynamics of urinary cfDNA provides a fundamental step toward the development of urine-based biopsies and their translation into clinical practice. Acknowledgments The authors would like to thank the URIC biobank team for their assistance, and patients and their families for participating in this study. Funding This work was supported by the Cancer Center Amsterdam Foundation; Edli Foundation; and Weijerhorst Foundation. Funders only provided financial support for the conduct of the research or had no role in conducting the research and preparation of the manuscript. This research did not receive any other specific grants from funding agencies in the public or commercial sectors. Disclosure of interest RS has a minority stake in Self-screen B.V., a spin-off company of VU University Medical Center Amsterdam. SB, RS, IB, and GK are named inventors on patent applications 7
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