Researchers at the Health Research Institute Hospital La Fe (IIS La Fe) in Valencia, Spain, have discovered that analyzing specific patterns of cell-free DNA (cfDNA) fragmentation in urine could be a new and effective way to diagnose and stage bladder cancer. These findings, published in The Journal of Molecular Diagnostics, describe how the small fragment of the MYC gene, which is implicated in the development and progression of many types of cancer, could indicate the presence of both non–muscle-invasive and muscle-invasive bladder cancer.
“Our most significant finding was that the small fragment of the MYC gene may represent a valuable tool to diagnose bladder cancer, as it exhibited excellent specificity (97%) and predictive value (88%) for identifying muscle-invasive bladder cancer,“ said senior author Pilar Medina, PhD, of the hemostasis, thrombosis, arteriosclerosis and vascular biology research group, at IIS La Fe.
The researchers sought new diagnostic options because current standard diagnostic methods for bladder cancer are either invasive or are plagued by low sensitivity. For example, cystoscopy remains the standard for diagnosing and monitoring bladder cancer but is invasive, expensive, and is associated with potential side effects.
“Novel noninvasive markers are needed to early diagnose and stage patients with BC, which may prevent the frequent performance of this (cystoscopy) detrimental technique, may reduce the resulting high rate of false positives and negatives, and may improve the therapeutic impact,” the researchers wrote.
Urine was chosen for this study because it is already know that bladder tumors shed DNA fragments directly into urine, providing a fertile ground for the development of a liquid biopsy.
The team analyzed urine samples from 156 patients with bladder cancer representing most stages and 79 controls without urological disease. Using quantitative real-time PCR, they measured large (>250 bp) and small (<125 bp) DNA fragments of five genes: ACTB, AR, MYC, BCAS1, and STOX1. The study built on earlier findings that cfDNA reflects patterns of apoptosis and necrosis, both of which generate DNA fragments of different sizes. Prior research had examined cfDNA integrity in serum or plasma but only a handful of studies had evaluated cfDNA found in urine for bladder cancer detection.
In addition to identifying the specific short cfDNA MYC fragment as a key biomarker of bladder cancer, the investigators also showed that increases in the ACTB large-to-small fragment ratio and the AR small fragment were associated with disease stage. These findings confirmed a hypothesis that more severe and advanced disease produces a higher proportion of necrotic tumor cell death, which leads to more abundant large cfDNA fragments. “The ratio of large/small fragments of ACTB could be an effective diagnostic and staging biomarker for BC, as its levels progressively increase from controls to the most severe forms of BC,” the researchers noted.
To validate their findings, the team used ordinal logistic regression models adjusted for age and sex and also compared their data with clinical records, including relapse data, to explore potential use of these biomarkers for patient follow-up. Because they showed that the small fragments of MYC and AR and the ACTB fragment ratio may increase with tumor relapse compared with those patients with tumor remission, these markers could be used for post-treatment monitoring for disease recurrence.
The IIS La Fe researchers are now looking to conduct confirmatory studies with larger and more diverse patient cohorts. The intent is to evaluate patients with benign urological conditions, search for additional biomarkers, and evaluate the use of higher-sensitivity detection platforms such as droplet digital PCR. If validated, a urine-based cfDNA fragmentation diagnostic could reduce reliance on cystoscopy and provide a more robust liquid biopsy approach for detecting bladder cancer.
“Our findings show that urine can tell us much more than we thought; it holds the potential to transform how we detect and manage bladder cancer,” Medina said.
