Cell-free DNA from urine samples – a valuable source for bladder cancer biomarkers?

Bladder cancer (BC) is among the most frequent cancer types in the world and is the most lethal urological malignancy. Presently, diagnostic and follow-up methods for BC are expensive and invasive. Thus, the identification of novel predictive biomarkers for diagnosis, progression, and prognosis of BC is of paramount importance. To date, several studies have evidenced that cell-free DNA (cfDNA) found in liquid biopsies such as blood and urine may play a role in the particular scenario of urologic tumors, and its analysis may improve BC diagnosis report about cancer progression or even evaluate the effectiveness of a specific treatment or anticipate whether a treatment would be useful for a specific patient depending on the tumor characteristics. In the present review, we have summarized the up-to-date studies evaluating the value of cfDNA as potential diagnostic, prognostic, or monitoring biomarker for BC in several biofluids.

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Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by Ultra-Deep Sequencing of Plasma Cell-Free DNA in Patients With Urothelial Bladder Carcinoma

Journal of Clinical Oncology ◽

10.1200/jco.18.02052 ◽

2019 ◽

Vol 37 (18) ◽

pp. 1547-1557 ◽

Cited By ~ 58

Author(s):

Emil Christensen ◽

Karin Birkenkamp-Demtröder ◽

Himanshu Sethi ◽

Svetlana Shchegrova ◽

Raheleh Salari ◽

...

Keyword(s):

Bladder Cancer ◽

Early Detection ◽

Risk Stratification ◽

Deep Sequencing ◽

Therapeutic Efficacy ◽

Patient Specific ◽

Cell Free Dna ◽

Free Dna ◽

Metastatic Relapse ◽

Ctdna Analysis

PURPOSE Novel sensitive methods for early detection of relapse and for monitoring therapeutic efficacy may have a huge impact on risk stratification, treatment, and ultimately outcome for patients with bladder cancer. We addressed the prognostic and predictive impact of ultra-deep sequencing of cell-free DNA in patients before and after cystectomy and during chemotherapy. PATIENTS AND METHODS We included 68 patients with localized advanced bladder cancer. Patient-specific somatic mutations, identified by whole-exome sequencing, were used to assess circulating tumor DNA (ctDNA) by ultra-deep sequencing (median, 105,000×) of plasma DNA. Plasma samples (n = 656) were procured at diagnosis, during chemotherapy, before cystectomy, and during surveillance. Expression profiling was performed for tumor subtype and immune signature analyses. RESULTS Presence of ctDNA was highly prognostic at diagnosis before chemotherapy (hazard ratio, 29.1; P = .001). After cystectomy, ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring (100% sensitivity, 98% specificity). A median lead time over radiographic imaging of 96 days was observed. In addition, for high-risk patients (ctDNA positive before or during treatment), the dynamics of ctDNA during chemotherapy was associated with disease recurrence ( P = .023), whereas pathologic downstaging was not. Analysis of tumor-centric biomarkers showed that mutational processes (signature 5) were associated with pathologic downstaging ( P = .024); however, no significant correlation for tumor subtypes, DNA damage response mutations, and other biomarkers was observed. Our results suggest that ctDNA analysis is better associated with treatment efficacy compared with other available methods. CONCLUSION ctDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in bladder cancer is feasible and provides a basis for clinical studies that evaluate early therapeutic interventions.

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Comparative Analysis of Urine Fractions for Optimal Bladder Cancer Detection Using DNA Methylation Markers

Cancers ◽

10.3390/cancers12040859 ◽

2020 ◽

Vol 12 (4) ◽

pp. 859 ◽

Cited By ~ 5

Author(s):

Anouk E. Hentschel ◽

Jakko A. Nieuwenhuijzen ◽

Judith Bosschieter ◽

Annina P. van Splunter ◽

Birgit I. Lissenberg-Witte ◽

...

Keyword(s):

Dna Methylation ◽

Bladder Cancer ◽

Comparative Analysis ◽

Area Under The Curve ◽

Practical Reasons ◽

Methylation Analysis ◽

Cell Free Dna ◽

Molecular Tests ◽

Free Dna ◽

Tumor Tissues

DNA methylation analysis of full void urine and urine pellet seems promising for bladder cancer (BC) detection and surveillance. Urinary cell-free DNA from urine supernatant is now gaining interest for other molecular tests in BC. This study aims to evaluate which urine fraction is preferred for BC diagnosis using methylation markers: full void urine, urine pellet or supernatant. Methylation levels of nine markers were determined in the three urine fractions and correlated with their respective tumor tissues in BC patients and compared to controls. For all markers and marker panel GHSR/MAL, diagnostic performance was determined by calculating the area under the curve (AUC) of the respective receiver operating characteristic curves. For most of the markers, there was a significant correlation between the methylation levels in each of the urine fractions and the matched tumor tissues. Urine pellet was the most representative fraction. Generally, AUCs for BC diagnosis were comparable among the fractions. The highest AUC was obtained for GHSR/MAL in urine pellet: AUC 0.87 (95% confidence interval: 0.73–1.00), corresponding to a sensitivity of 78.6% and a specificity of 91.7%. Our results demonstrate that cellular and cell-free DNA in urine can be used for BC diagnosis by urinary methylation analysis. Based on our comparative analysis and for practical reasons, we recommend the use of urine pellet.

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Urinary Cell-Free DNA IQGAP3/BMP4 Ratio as a Prognostic Marker for Non–Muscle-Invasive Bladder Cancer

Clinical Genitourinary Cancer ◽

10.1016/j.clgc.2019.04.001 ◽

2019 ◽

Vol 17 (3) ◽

pp. e704-e711

Author(s):

Yanjie Xu ◽

Ye-Hwan Kim ◽

Pildu Jeong ◽

Xuan-Mei Piao ◽

Young Joon Byun ◽

...

Keyword(s):

Bladder Cancer ◽

Prognostic Marker ◽

Invasive Bladder Cancer ◽

Muscle Invasive Bladder Cancer ◽

Cell Free Dna ◽

Free Dna ◽

Muscle Invasive

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Urinary cell-free DNA as a potential tumor marker for bladder cancer

The International Journal of Biological Markers ◽

10.5301/jbm.2008.4356 ◽

2007 ◽

Vol 22 (4) ◽

pp. 287-294 ◽

Cited By ~ 9

Author(s):

H.W. Chang ◽

K.H. Tsui ◽

L.C. Shen ◽

H.W. Huang ◽

S.N. Wang ◽

...

Keyword(s):

Bladder Cancer ◽

Tumor Marker ◽

Cell Free Dna ◽

Free Dna

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CELL-FREE DNA IN SERUM OF PATIENTS WITH BLADDER CANCER: DIAGNOSTIC AND PROGNOSTIC IMPLICATIONS

European Urology Supplements ◽

10.1016/s1569-9056(08)60543-6 ◽

2008 ◽

Vol 7 (3) ◽

pp. 207 ◽

Cited By ~ 1

Author(s):

J Ellinger ◽

N Ellinger ◽

L.C. Heukamp ◽

P Kahl ◽

F.G. Perabo ◽

...

Keyword(s):

Bladder Cancer ◽

Cell Free Dna ◽

Cancer Diagnostic ◽

Free Dna ◽

Prognostic Implications

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Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with Bladder Cancer

Urologia Internationalis ◽

10.1159/000438828 ◽

2015 ◽

Vol 96 (1) ◽

pp. 25-31 ◽

Cited By ~ 10

Author(s):

Antonin Brisuda ◽

Eva Pazourkova ◽

Viktor Soukup ◽

Ales Horinek ◽

Jan Hrb�cek ◽

...

Keyword(s):

Bladder Cancer ◽

Dna Quantification ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna

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Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples

PLoS ONE ◽

10.1371/journal.pone.0247851 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0247851

Author(s):

Amy Oreskovic ◽

Barry R. Lutz

Keyword(s):

Large Volume ◽

Magnetic Beads ◽

Limit Of Detection ◽

Extraction Methods ◽

Urine Samples ◽

Hybridization Method ◽

Analytical Methodology ◽

Cell Free Dna ◽

Double Stranded Dna ◽

Free Dna

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6–117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25–150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.

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