








































This is an open access article under the terms of a license that permits non-commercial use, provided the original work is properly cited.  
© 2023 The Authors. Société Internationale d'Urologie Journal, published by the Société Internationale d'Urologie, Canada.

SIUJ.ORG SIUJ  •  Volume 4, Number 4  •  July 2023

Key Words Competing Interests Article Information

Bladder cancer, nonmuscle-invasive bladder 
cancer, NMIBC, liquid biopsy, urine tumor DNA

None declared. Received on April 26, 2023 
Accepted on July 2, 2023 
This article has been peer reviewed.

Soc Int Urol J. 2023;4(4):301–307

DOI: 10.48083/EABM2528

301

REVIEW — LIQUID BIOPSY

Urinary Tumor DNA-Based Diagnosis and Surveillance 
for Nonmuscle-Invasive Bladder Cancer—Current 
Landscape and Future Directions

Alexander Shiang,1,2,* Cayce Nawaf,1,3,* Pradeep S. Chauhan,2 Aadel A. Chaudhuri,2,3,4,5,6  
Zachary L. Smith1,3 Gautum Agarwal7

1 Division of Urology, Department of Surgery, Washington University School of Medicine, St. Louis, United States 2 Division of Cancer Biology, Department of Radiation 
Oncology, Washington University School of Medicine, St. Louis, United States 3 Siteman Cancer Center, Barnes Jewish Hospital and Washington University School of 
Medicine, St. Louis, United States 4 Department of Computer Science and Engineering, Washington University in St. Louis, St. Louis, United States  
5 Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, United States 6 Department of Genetics, Washington University School of 
Medicine, St. Louis, United States 7 Division of Urology, David Pratt Cancer Center, Mercy Hospital, St. Louis, United States

* These authors contributed equally to this work.

Abstract

Bladder cancer has a significant impact on patients, in terms of both morbidity and financial burden. This is especially 
true for patients with nonmuscle-invasive bladder cancer, who undergo long-term surveillance via cystoscopy and 
imaging, resulting in significant costs and risks. To address this issue, urinary tumor DNA analysis, or “urinary 
liquid biopsy,” has emerged as a potential solution to reduce the testing burden and mitigate many of the costs and 
risks. Over time, urinary tumor DNA analysis has undergone several refinements. However, existing FDA-approved 
urinary biomarker assays currently lack the sensitivity and specificity to significantly impact clinical decision-
making. Subsequent iterations of these technologies have attempted to bridge this gap by improving their diagnostic 
accuracy, and ultimately, clinical utility. Here, we discuss the current role as well as future directions of urinary tumor 
DNA analysis for the detection and long-term surveillance of nonmuscle-invasive bladder cancer.

Introduction

Each year, there are over 500 000 new cases of bladder cancer diagnosed worldwide[1]. This places a substantial 
burden on patients, with estimated annual costs exceeding $100 000 per patient[2–4]. Consequently, the development 
of efficient and cost-effective methods for early disease detection is of the utmost importance. In current practice, 
diagnostic workup typically involves a combination of urinalysis, cytology, cystoscopy, and imaging[5]. These tests 
are also used in the surveillance setting after patients have local treatment with transurethral resection of bladder 
tumor (TURBT). Cystoscopy, in particular, is associated with increased costs of care and exposes patients to potential 
discomfort and risks for infection and other complications[6,7]. Compliance with invasive testing can also be an issue 
for patients, leading to increased rates of progression and recurrence. Additionally, cytology comes with significant 
limitations in the form of low sensitivity[8]. Previous meta-analyses have demonstrated overall sensitivities ranging 
from 30% to 40%, with higher-grade disease associated with greater sensitivity. Because of this, surveillance of 
nonmuscle-invasive bladder cancer (NMIBC) beyond cystoscopy poses great challenges.

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sequencing–based hybrid capture assay[14]. uCAPP-Seq 
captures single nucleotide variants (SNVs), insertions 
and deletions, and copy number alterations in genomic 
regions known to harbor common driver mutations for 
bladder cancer. The panel was based on data published 
from The Cancer Genome Atlas (TCGA)[15]. In total, it 
assays regions encompassing more than 450 genes and 
was predicted to identify a median of 7 mutations per 
bladder cancer patient.

Similarly to Springer et al., Dudley et al. tested uCAPP-
Seq in both detection and surveillance cohorts. The 
researchers employed both tumor-informed and 
tumor-naive profiling approaches to each group. In 
the detection cohort, the majority of which exhibited 
stage pTa (74%) with low-grade disease (54%), their 
tumor-naive approach achieved a sensitivity of 83% 
with a specificity of 97%. In comparison, cytology alone 
had a sensitivity of only 14% and specificity of 100%.  
As expected, their tumor-informed approach reached 
an even greater sensitivity of 93% with a specificity of 
96%. However, it is important to note that this approach 
requires prior tumor and germline sequencing, making 
it less suitable for disease detection.

In their surveillance cohort of patients who had previ-
ously received local bladder cancer treatment, their 
tumor-naive approach successfully detected disease 
recurrence in 84% of patients compared with only 
38% for cytology. When cytology and cystoscopy were 
combined, only 53% of patients who had recurrence 
were detected, significantly lower than the 84% detected 
by uCAPP-Seq positivity alone (P = 0.0057).

UroMark (Feber et al.)
Feber et al. developed UroMark, a bisulphite urine 
sediment sequencing assay, and performed a proof-
of-concept investigation[16]. This 150-loci multiplex 
assay was developed using 86 muscle-invasive bladder 
cancer (MIBC) patients and 30 healthy controls. It was 
subsequently validated with an independent cohort 
consisting of 167 bladder cancer (BC) patients and 274 
healthy controls.

The assay itself performed robustly with a sensitivity 
of 98%, a specificity of 97%, and a negative predictive 
value (NPV) of 97% for detecting primary BC. Future 
investigations will focus on accurately diagnosing 
patients presenting with hematuria within a clinical 
context.

Xiao et al.
Xiao et al. continue to advance the diagnostic landscape 
of NMIBC[17]. They devised a study to comprehensively 
characterize the entire DNA methylation landscape of 
bladder cancer to determine the relevant biomarkers for 
the diagnosis of non-invasive bladder cancer.

In this multicenter, prospective cohort study, the 
researchers collected 304 samples from 224 donors 
and performed genome-wide bisulphite sequencing for 
DNA methylation signature discovery. They developed 
a targeted sequence assay for bladder cancer–specific 
DNA methylation signatures to distinguish tumor 
tissue from normal urine or MIBC from NMIBC.

Independent validation was performed by double-
blinded targeted sequencing of urine samples to deter-
mine the clinical diagnosis and prognostic value of 
DNA methylation-based classification models. The 
study measured the concordance between pathology 
results and urinary tumor DNA (utDNA) methyla-
tion, genomic mutations, and other state-of-the-art 
diagnostic methods. The assay showed positivity in 60 
of 60 patients (100%) found to have high-grade blad-
der cancer on initial TUR BT. Additionally, 21 of 
57 patients (37%) with a positive DNA methylation 
signal experienced disease progression. A subset of 
these patients had negative pathology results on initial 
TURBT but were later found to harbor bladder cancer 
on subsequent TURBT. This result further highlights 
the potential of utDNA for early diagnosis before visi-
ble tumors are present.

Strandgaard et al.
Strandgaard et al. retrospectively analyzed 156 NMIBC 
patients who had received Bacillus Calmette-Guérin 
(BCG) treatment[18]. In all patients, urine samples were 
available for analysis both before and after treatment 
with BCG. The researchers analyzed the samples for 
the presence of 92 immuno-oncology–related proteins 
using the Olink Target 96 Immuno-Oncology Assay 
(Olink; Uppsala, Sweden). They also performed deep-
targeted sequencing of tumor-specif ic mutations 
in urine cf DNA with 3 different NGS panels (Twist 
Bioscience; San Francisco, US). The researchers found 
significantly higher tumor DNA (tDNA) levels in 
the urine of the post-BCG recurrence group, but no 
difference was observed in tDNA levels for the pre-BCG 
samples. Notably, the exhaustion status defined by CD8 
T-cells in tumor was found to be significantly associated 
with tDNA levels in both the pre- and post-BCG urine 
samples. They also reported that patients with tDNA 
clearance after BCG treatment has significantly better 
recurrence-free survival than patients without clearance.

Additionally, the investigators identified several genes 
related to cell division and immune function that were 
upregulated in patients who subsequently developed 
high-grade recurrence after BCG treatment. They were 
then able to assign scores based on these genes to predict 
the likelihood for tumor recurrence. These exciting 
new urine biomarkers allow for real-time assessment 
of a patient’s response to standard-of-care treatment, 

There has been growing interest in the application of 
urinary liquid-biopsy–based assays for the detection and 
surveillance of NMIBC. Because it is a readily available 
biofluid, urine has received most of this interest. Urine 
allows for the potential detection of both genetic mate-
rial released directly from tumor cells and malignant 
cells exfoliated into the urine[9]. There are currently 
severa l FDA-approved urinar y biomarker assays 
for the detection and surveillance of bladder cancer 
such as ImmunoCyt, NMP22, UroVysion, BTA, and 
Cxbladder[10]. However, these tests exhibit low sensi-
tivities and specificities, leading to poor utilization by 
providers within clinical contexts (Table 1).

While the first-generation urine-based assays did 
not meet the required performance levels for clinical 
applications, further efforts have focused on developing 
tests with improved sensitivities and specificities. These 
tests aim to be non-invasive, reproducible, and capa-
ble of not only detecting initial disease but also serving 
as a marker for treatment response and disease recur-
rence, providing real-time information on a patient’s 
disease status. Ideally, these tests should predict the 
presence or absence of both low-grade and more inva-
sive disease. Such improvements would aid greatly in 
alleviating the morbidity and economic hardship associ-
ated with frequent clinic visits, imaging, and cystoscopy 
surveillance.

Currently, plasma circulating tumor DNA (ctDNA) 
tests are used in the invasive and metastatic settings, 
as well as for monitoring response to immunother-
apy[11,12]. The application of urinary tumor DNA 
(utDNA) in a purely non-invasive setting has not yet 
been established. Therefore, multiple trials are exploring 
utDNA analysis and attempting to characterize tumor 
biopsy specimens on a molecular basis to improve prog-
nostication and personalize treatment options.

Here, we will highlight an array of exciting past 
and ongoing investigations related to utDNA analysis 
for NMIBC, which we consider to be at the forefront 
of the field.

Completed Studies/Trials
UroSEEK (Springer et al.)
The first generation of urinary tumor DNA assays 
faced significant limitations in both sensitivity and 
specificity, particularly regarding the detection of low-
grade tumors. Springer et al. attempted to address this 
issue through a combinatorial approach that involved 
the development of UroSEEK[13]. UroSEEK combined 
3 separate tests (10 gene multiplex, TERT singleplex, and 
aneuploidy) to detect mutations in 11 different genes 
and copy number changes on 39 chromosome arms. 
UroSEEK positivity was defined as a positive result in 
any one of these 3 tests, yielding a large increase in the 
assay’s sensitivity.

Patients were divided into 3 cohorts: a detection 
cohort, a surveillance cohort of those with confirmed 
bladder cancer, and a surveillance cohort of those with 
confirmed upper tract urothelial carcinoma. The detec-
tion cohort consisted primarily of patients who had 
presented with microscopic hematuria. It is import-
ant to emphasize that hematuria, either microscopic or 
gross, represents the most common initial presentation 
for bladder cancer. Therefore, the author’s detection 
group represents a realistic clinical application of their 
assay within a diagnostic context. Within this group, 
UroSEEK demonstrated a sensitivity of 83% and spec-
ificity of 99.5% for detecting bladder cancer. When 
combined with cytology, the sensitivity increased to 
95%. The authors estimated the cost of this UroSEEK-
cytology combination to be one-third of the cost of 
cystoscopy alone. In the surveillance cohort, UroSEEK 
was able to detect recurrence with a sensitivity of 68% 
and specificity of 80%. On average, UroSEEK positiv-
ity preceded the clinical diagnosis of recurrence by  
7 months.

uCAPP-Seq (Dudley et al.)
After the publication of the initial results from UroSEEK, 
Dudley et al. attempted to streamline the process further 
by developing uCAPP-Seq (utDNA CAncer Personalized 
Profiling by deep Sequencing), a novel high-throughput 

TABLE 1. 

Reported sensitivity and specificity of commercially available urine-based assays for detecting bladder cancer 

NMP22 BTA UroVysion ImmunoCyt Cxbladder UroMark

Sensitivity 0.69 0.65 0.63 0.78 0.82 0.98

Specificity 0.77 0.74 0.87 0.78 0.85 0.97

Company Alere Polymedco Vysis Scimedex Pacific Edge
UCL Cancer 

Center

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turn, will allow patients with negative results to be safely 
spared from the financial burden and risks associated 
with routine cystoscopy. By mitigating morbidity and 
costs associated with excess cystoscopy, streamlined and 
efficient personalized care can be delivered[2,3].

Despite early upfront costs, tailored genomic 
approaches have the potential for alleviating long-term 
financial strain[24]. A study by Gordon et al. estimated 
average costs of $300 for targeted panels and $3000 for 
whole genome sequencing[25]. These costs could easily 
be offset by the reduced need for computed tomogra-
phy (CT) scans and surveillance cystoscopies through 
the integration of genomic assays. Specifically, these 
management strategies have been previously modeled to 
yield cost reductions of up to 9% in low-, intermediate-, 
and high-risk bladder cancer patients[26].

When developing and evaluating these assays, an 
important consideration is whether urine sediment 
or supernatant should be used, as tumor DNA can be 
found in both. The DNA present in the supernatant is 
known as cell-free (cfDNA) and is thought to contain a 
higher proportion of tumor DNA compared with sedi-
ment[27–29]. However, a consensus has yet to be reached 
on which fraction is optimal.

FIGURE 1. 

A schematic illustrating the potential role for liquid biopsy in both the diagnosis and surveillance of NMIBC. 
This would allow for reductions in the quantity of CT and cystoscopy required for management. CT: computed 
tomography; TURBT: transurethral resection of bladder tumor; NMIBC: non-muscle-invasive bladder cancer.

Another potential avenue for expansion involves the 
application of personalized biomarkers derived from 
a patient’s original TURBT sample. This approach 
could greatly benefit surveillance settings, as demon-
strated by studies such as Dudley et al., which showed 
key improvements in assay sensitivities when using 
tumor-informed analyses instead of tumor-naive anal-
yses. Further development of tissue registries and inves-
tigations of genomic concordance between urine cfDNA 
and tissue samples will enable urine-based assays to play 
an expanded role in guiding clinical management and 
disease prognostication.

Currently, radical cystectomy is a common manage-
ment strategy for many patients with high-risk, treat-
ment-refractor y NMIBC[30,31]. Improvements in 
urine-based assays can allow for upfront risk stratifica-
tion and identification of these high-risk patients. This 
would enable the option of early tumor-informed cystec-
tomy for patients predicted to harbor high-risk NMIBC. 
The presence of micrometastatic disease represents an 
additional risk for these patients. While not discussed 
in this paper, the supplementation of preexisting plas-
ma-based assays could aid in early diagnosis and 
treatment[32].

enabling a more personalized approach to patient care.

Ongoing Studies/Trials
Assessment of the Concordance of Genomic 
Alterations Between Urine and Tissue in High-
Risk NMIBC Patients (Trial ID: NCT03563443)
This study from France aims to investigate whether 
analyzing urine cell-free tumor DNA can predict 
relapse in high-risk NMIBC. The eligibility criteria will 
be patients with high-risk BCG-naive NMIBC who 
are set to undergo treatment with BCG. Urine samples 
will be collected at various intervals, including before 
their initial BCG treatment, and at each subsequent 
instillation. Tumor samples will be collected upon 
initial TURBT and in the event of relapse. The primary 
outcome will be the agreement rate between urine 
cfDNA and tumor tissue mutation profile, focusing on 
the concordance rate between mutations identified in 
the tumor. The secondary outcome will be to evaluate 
the prognostic value of tumor mutational burden 
(TMB), which will be calculated in the urine cfDNA for 
each patient.

This study also aims to compare the NMIBC cohort 
to an MIBC cohort, which will have urine samples 
collected at each routine clinic visit and have their 
cf DNA analyzed against their initial diagnosis at 
TURBT.

Genomic Imprinting Testing for Diagnosis of 
Bladder Cancer (Trial ID: NCT03563443)
In recent years, increased attention has been devoted 
toward investigating epigenetic changes such as loss of 
imprinting (LOI) within cancer cells. These changes 
have been demonstrated to play key roles in cancer 
mechanisms such as the development of resistance to 
chemotherapy and radiotherapy[19]. A novel approach 
investigating these mechanisms, LisenID, is being 
developed by LISEN Imprinting Diagnostics company 
(Wilmington, US). Specifically, this trial aims to broadly 
investigate tumor LOI patterns to establish a predictive 
and diagnostic urine-based bladder cancer LOI panel.

Because of their ubiquitous nature, epigenetic 
changes such as LOI can aid in potentially overcomes 
issues related to tumor heterogeneity when relying solely 
on mutational methodologies. Furthermore, data has 
suggested that LOI often precedes many morphological 
changes within tumor cells, potentially expanding the 
window for their detection and enhancing the sensitivity 
of urine-based testing for NMIBC.

Previous studies integrating mutational and methyl-
ation changes have demonstrated significant improve-
ments in disease detection and surveillance[20–22].  
For example, a study by Cheng et al. combined copy 
number variant and methylation analyses to improve the 

sensitivity of detecting NMIBC to 91.9%[22]. When applied 
only to low-grade tumors, the sensitivity remained rela-
tively robust at 84.2%, indicating a substantial improve-
ment from the first-generation tests described above.

The Performance of Cancer Risk Genes in  
the Necessity of Secondary TURBT  
(Trial ID: NCT05112523)
Patients with a bladder mass undergo standard-of-care 
TURBT for tissue diagnosis ± postoperative intravesical 
treatment. If the patient is deemed to have NMIBC, 
depending on the stage and the grade, repeat TURBT 
is often recommended, or is considered standard-of-
care. However, there are currently some patients who 
do not need an additional procedure, which would 
reduce overtreatment, patient morbidity, and healthcare 
costs. This study seeks to investigate whether a urine 
biomarker can detect residual bladder tumor lesions and 
thus predict who will need repeat TURBT before further 
treatment.

Genetron Uro V1 is a non-invasive urinary liquid-bi-
opsy assay for assessing the risk for uroepithelial tumor 
using DNA extracted from urinary pellets. The investi-
gators will perform this test on patients’ urine samples 
before patients undergo a secondary TURBT and 
compare the test results with the final pathologic results. 
These findings will help to predict the need for second-
ary TURBT in high-risk NMIBC patients and establish 
correlations with patient recurrence-free survival and 
overall survival.

Discussion and Future Directions
Relying solely on pathologic analysis of bladder cancer 
specimens has been demonstrated to be surprisingly 
unreliable. In a study conducted by Luchey et al., 
dedicated genitourinary pathologists re-reviewed 1191 
bladder cancer biopsy specimens and found that nearly 
30% of them resulted in a change in pathology[23]. This 
significant level of interobserver discordance can lead 
to suboptimal patient care by providing treatments that 
do not ref lect the true pathologic stage. Urine-based 
biomarkers have the potential to supplement clinical 
workflows by allowing for more precise risk stratification 
and staging of bladder cancer patients (Figure 1, created 
in BioRender). However, these modalities are not perfect, 
and there lies a great deal of heterogeneity based on 
methodology used and thresholds placed on the assays 
themselves.

Future directions in this field involve integration of 
promising novel methylation-based approaches with 
preexisting single nucleotide variant and copy number–
based assays. The aim of these combinatorial techniques 
will be to maximize the sensitivity of detecting NMIBC 
for both diagnostic and surveillance purposes. This, in 

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REVIEW — LIQUID BIOPSY Urinary Tumor DNA-Based Diagnosis and Surveillance for Nonmuscle-Invasive Bladder Cancer—Current Landscape and Future Directions

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Conclusion
The current landscape of urine tumor-based testing for NMIBC shows promise and tremendous potential for future 
improvement and growth. Clinical applications, in both diagnostic and surveillance contexts, will enable providers 
to offer personalized, precision treatments and management strategies. Reducing the risk and financial toxicity 
associated with unnecessary cystoscopies will result in more efficient delivery of care, benefiting both patients and 
their providers.

11. Christensen E, Birkenkamp-Demtröder K, Sethi H, Shchegrova S, 
Salari R, Nordentoft I, et al. Early detection of metastatic relapse 
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307306 SIUJ.ORG SIUJ  •  Volume 4, Number 4  •  July 2023SIUJ  •  Volume 4, Number 4  •  July 2023 SIUJ.ORG

REVIEW — LIQUID BIOPSY Urinary Tumor DNA-Based Diagnosis and Surveillance for Nonmuscle-Invasive Bladder Cancer—Current Landscape and Future Directions

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