Dynamics of methylated cell-free DNA in the urine of non-small cell lung cancer patients

High levels of methylated DNA in urine represent an emerging biomarker for non-small cell lung cancer (NSCLC) detection and are the subject of ongoing research. This study aimed to investigate the circadian variation of urinary cell-free DNA (cfDNA) abundance and methylation levels of cancer-associated genes in NSCLC patients. In this prospective study of 23 metastatic NSCLC patients with active disease, patients were asked to collect six urine samples during the morning, afternoon, and evening of two subsequent days. Urinary cfDNA concentrations and methylation levels of CDO1, SOX17, and TAC1 were measured at each time point. Circadian variation and between- and within-subject variability were assessed using linear mixed models. Variability was estimated using the Intraclass Correlation Coefficient (ICC), representing reproducibility. No clear circadian patterns could be recognized for cfDNA concentrations or methylation levels across the different sampling time points. Significantly lower cfDNA concentrations were found in males (p=0.034). For cfDNA levels, the between- and within-subject variability were comparable, rendering an ICC of 0.49. For the methylation markers, ICCs varied considerably, ranging from 0.14 to 0.74. Test reproducibility could be improved by collecting multiple samples per patient. In conclusion, there is no preferred collection time for NSCLC detection in urine using methylation markers, but single measurements should be interpreted carefully, and serial sampling may increase test performance. This study contributes to the limited understanding of cfDNA dynamics in urine and the continued interest in urine-based liquid biopsies for cancer diagnostics.

尿液中甲基化DNA的高浓度代表了一种新兴的非小细胞肺癌（NSCLC）检测生物标志物，并成为持续研究的主题。本研究旨在探究NSCLC患者尿液中游离DNA（cfDNA）丰度和癌症相关基因甲基化水平的昼夜节律性变化。在该前瞻性研究中，针对23名具有活跃疾病的转移性NSCLC患者，患者被要求在连续两天的上午、下午和晚上收集六次尿液样本。在每一个时间点，测量了尿液中cfDNA的浓度以及CDO1、SOX17和TAC1基因的甲基化水平。利用线性混合模型评估了昼夜节律性变化以及个体间的差异和个体内的变异。通过类内相关系数（ICC）估计了变异性，反映了可重复性。在cfDNA浓度或甲基化水平上，未能识别出明确的昼夜节律模式。在男性患者中发现了显著较低的cfDNA浓度（p=0.034）。对于cfDNA水平，个体间的差异和个体内的变异相当，ICC值为0.49。对于甲基化标志物，ICC值差异较大，范围从0.14至0.74。通过每位患者收集多个样本可以提高测试的可重复性。总之，在尿液中使用甲基化标志物检测NSCLC时，不存在首选的采集时间，但应谨慎解释单一测量结果，而连续采样可能提高测试性能。本研究有助于深化对尿液中cfDNA动态变化的理解，并持续激起了基于尿液液体活检的癌症诊断的兴趣。