Analysis of the Fragmentation and Integrity of Urine Cell-Free DNA as a Diagnostic and Staging Biomarker for Bladder Cancer

Abstract

Bladder cancer (BC) is a lethal urological malignancy, with current diagnostic and follow-up methods being invasive and costly. Cell-free DNA (cfDNA) in liquid biopsies has shown promise in cancer diagnostics, but its fragmentation and integrity in urine remain underexplored in BC, becoming the aim of this study. cfDNA was isolated from the urine of 156 patients with BC of most stages and 79 matched controls without renal or bladder conditions. The amount of a large (>250-bp) and a nested small (<125-bp) fragment of ACTB, AR, MYC, BCAS1, and STOX1 was quantified by quantitative real-time PCR. Fragmentation and integrity (ratio of large/small) were analyzed with ordinal logistic regression. The increase in the ratio of large/small ACTB fragments and the small fragments of AR and MYC may represent a valuable tool to diagnose and stage BC when classified as both non-muscle-invasive and muscle-invasive BC or considering grades and stages separately. The small fragment of MYC, leading the effect observed, displayed a valuable diagnostic capacity [area under the receiver operating characteristic (ROC) curve = 0.7221; 95% CI, 0.6527-0.7915; P < 0.0001; sensitivity = 50%; specificity = 95%], particularly for muscle-invasive BC (area under the ROC curve = 0.8098; 95% CI, 0.6674-0.9523; P < 0.0001; sensitivity = 70%; specificity = 97%). Herein, the analysis of urine cfDNA fragmentation and integrity of these surrogate markers is proposed as noninvasive biomarkers to diagnose and stage BC. Once validated, the proposed biomarkers could improve patient management by reinforcing or substituting current invasive and expensive techniques.