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MOLECULAR BIOMARKERS IN UROLOGIC ONCOLOGY: ICUD-WUOF CONSULTATION

Urinary-Based Markers for Bladder Cancer Detection
Tilman Todenhöfer,1,2 Michele Lodde,3 Kim van Kessel,4 Renate Pichler,5 Antonia Vlahou,6 Yair Lotan7

1 Studienpraxis Urology, Clinical Trial Unit, Germany, 2 Medical School, University of Tuebingen, Germany, 3 Centre Hospitalier Universitaire de Québec Research Centre, 
Université Laval, Québec, Canada, 4 Erasmus MC Cancer Institute, Rotterdam, The Netherlands, 5 Department of Urology, Medical University Innsbruck, Austria, 
6 Biomedical Research Foundation, Academy of Athens, Greece, 7 Department of Urology, University of Texas Southwestern Medical Center, Dallas, United States

Abstract

Background The use of urine markers for diagnosis and surveillance has been a topic of broad interest and ongoing 
controversies in the management of patients with bladder cancer. There has been a constant quest for markers that 
demonstrate clinical utility.

Aim In the framework of the International Consultation on Urological Diseases 2019 on Molecular Biomarkers 
in Urologic Oncology, a comprehensive review of literature on urinary biomarkers for bladder cancer has been 
performed.

Results Currently available urinary markers include protein-based markers, RNA-based markers, and DNA-based 
markers. The introduction of high-throughput analysis technologies provides the opportunity to assess multiple 
parameters within a short period of time, which is of interest for RNA-based, DNA-based, and protein-based marker 
systems. A comprehensive analysis of molecular alterations in urine samples of bladder cancer patients may be of 
interest not only for diagnosis and surveillance but also for non-invasive longitudinal assessment of molecular, 
potentially therapy-relevant, alterations. However, most systems lack prospective validation within well-designed 
trials and have not been broadly implemented in daily clinical practice.

Conclusions Because of limited data from prospective trials, the routine use of any urine marker except cytology is 
not considered as standard of care in international guidelines. There is an urgent need for prospective trials of urine 
markers to answer specific clinical questions.

Introduction

A diagnosis of bladder cancer (BC) is routinely made by cystoscopy followed by biopsy of suspicious lesions [1,2]. 
Cystoscopy represents the most important component of surveillance of BC. However, as white light cystoscopy may 
miss tumors and may be associated with discomfort for the patient, there has been a constant interest in the discovery 
of urine markers for use in diagnosis and surveillance of BC patients [3,4]. A marker with reasonable performance 
may be of interest in different clinical scenarios. These scenarios include the use of urine markers for patients 
with symptoms suggestive of BC (such as hematuria) and urine marker based surveillance (eg, with adaptation of 
cystoscopy intervals according to urine marker results) [5]. So far, the majority of studies performed in this field 
have shown that an increased sensitivity of non-cytology urine markers, compared with cytology, is often associated 
with limited specificity and that various factors (eg, infection and hematuria) frequently encountered in the patient 
population that is of interest for these markers may limit the diagnostic accuracy. A decreased specificity is also the 

Key Words Competing Interests Article Information

Urine, biomarkers, surveillance, screening, 
hematuria, urothelial carcinoma

Dr Lotan reports grants from MdxHealth, 
grants from Pacific Edge, grants from 
Cepheid, grants from Decipher Biosciences, 
personal fees from Nucleix, personal fees 
from Photocure, personal fees from Merck, 
personal fees from AstraZeneca, personal 
fees from Fergene,  during the conduct of  
the study. The remaining authors report no 
competing interests.

Received on July 7, 2020 
Accepted on July 29, 2020

Soc Int Urol J. 2020;1(1):49–61

http://www.siuj.org
mailto:todenhoefer%40studienurologie.de?subject=SIUJ


50 SIUJ  •  Volume 1, Number 1  •  October 2020 SIUJ.ORG

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main limitation of approaches based on a combined use 
of various markers to increase sensitivity. The current 
review summarizes data and potential applications of 
different types of urine markers, including cell-based 
markers, RNA markers, DNA markers, and protein 
markers. New technologies allowing quick and broad 
molecular analyses have been implemented in the 
context of urine markers to gain more information on 
tumor-associated alterations within a short period of 
time. These high-throughput technologies have a high 
potential of reaching some of the goals that have been set 
in the context of urinary diagnosis in BC.

Cell-Based Bladder Markers
Severa l cel l-based urine markers are avai lable, 
including conventional urinary cytology, ImmunoCyt/
uCyt + (Scimedx, Denville, US), f luorescence in situ 
hybridization (FISH) UroVysion (Abbott Molecular, des 
Plaines, US) and Cell Detect (Zetiq Technologies Ltd., 
Tel Aviv, Israel) (Table 1).

Urine cytology remains the gold standard and the 
only urine marker that is recommended by the European 
Association of Urolog y (EAU) and the American 

Urological Association (AUA) [1,2] for the diagnosis 
and surveillance of high-grade BC (in combination with 
cystoscopy).

Overall, the reported sensitivity ranges from 20% 
to 97.3%; specificity ranges from 74% to 99.5% [6–8]. 
Urine  cytology  has an excellent specificity with few 
false-positive cases for high-grade bladder cancer 
(HGBC) and carcinoma in situ (specificity 83% to 99%)
[1,9]. Historically, urine cytology had a high sensitivity 
for HG disease, but more contemporary series reported 
sensitivity for cytology at 40.8% and 54.3% for HG [10,11]. 
Furthermore, a low sensitivity in low-grade tumors 
represents a main downside of conventional cytology; 
therefore, it is not used to replace cystoscopy.

Immunocytology (Immunocyt [UCyt+], Scimedx, 
US) is based on immunoassays for detection of tumor-
associated cell-based antigens. The global sensitivity of 
immunocytology ranges between 78% and 90% and is 
higher than that of cytology, especially for low-grade 
cancers, whereas its specificity has been reported as 
68% to 87% and therefore tends to be lower than that of 
cytology [12–14] (Table 2).

ImmunoCyt/uCyt+ in patients whose cytology was 
atypical has been frequently discussed as a reflex test 
to avoid cystoscopy in patients with low-grade cancer 
since the test has a NPV of 83.7% [1,15]. Nevertheless, 
Immunocyt/uCyt+ has been recently removed from the 
market.

Fluorescence in situ hybridization (multi-target 
multicolor FISH – UroVysion) (Abbott Molecular, US) 
allows the detection of chromosomal abnormalities 
that are frequently observed in malignant urothelial 
cells (gains in chromosomes 3, 7, 17 or deletions of 
chromosome 9). These assays have been shown to be 
more sensitive than cytology in detecting BC at the cost 
of a lower specificity. Sensitivity ranges from 50% to 88% 
and specificity from 78% to 92% [12–14], depending on 

TABLE 1.

Summary of commercially available cell-basedt urinary markers

Marker / Test Marker / Test Description FDA status

Immunocyt /uCyt+ Scimedx, US Immunocytochemical assay for detection of expression of 
carcinoembryonic antigen and BC-associated mucins

Approved for follow-up; 
recently removed from 
the market

UroVysion Abbott Molecular, US Multicolor FISH assay for detection of numerical aberrations 
of chromosomes 3, 7, 17 and locus 9p21

Approved for diagnosis 
and follow-up

Cell Detect Zetiq Technologies, 
Israel

Platform technology comprising a proprietary plant extract 
and 3 dyes that enables color discrimination between 
malignant (red) and benign (green) cells based on specific 
metabolic alterations exclusive to the tumor

Not approved

Abbreviations 

AUA American Urological Association
BC bladder cancer
BCG Bacillus Calmette-Guérin
EAU European Association of Urology
FISH fluorescence in situ hybridization
HGBC high-grade bladder cancer
miRNA microRNA
NMIBC non-muscle invasive bladder cancer
NMP22 nuclear matrix protein 22
TERT telomerase reverse transcriptase

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51SIUJ.ORG SIUJ  •  Volume 1, Number 1  •  October 2020

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the BC prevalence in the cohort of the study (Table 3) 
and also on the criteria of positive tests. FISH sensitivity 
was reported to be twice as high as cytology for non-
muscle invasive bladder cancer (NMIBC) (50.9% 
versus 29.8%) [16,17] and triple (73% versus 24%) when 
scoring criteria have been modified. Notably the scoring 
algorithm of the manufacturer was developed for voided 
urine but was modified for bladder washings [17,18].

In patients with atypical cytology or indeterminate 
cystoscopy, UroVysion may help to identify those who 
would need further evaluation since 2 prospective 
studies found a higher likelihood of cancer in patients 
with positive markers with a reasonable positive 
predictive value [19,20]. This role has been included in 
the AUA guidelines as a potential use of urine markers. 
There is evidence from various sources that anticipatory 
false-positive results exist. Patients with positive FISH 
but no visible tumor in cystoscopy were reported to be 
at increased risk for recurrence and progression [21,22]. 
Persisting positive FISH during Bacillus Calmette-
Guérin (BCG) immunotherapy increased the risk of 
recurrence and progression [23]. Recently, 2 multicenter 
prospective studies confirmed the predictive value of 
FISH in detecting recurrence and progression in HGBC 
treated with BCG [10,25]. A positive FISH at initiation 
or completion of BCG is associated with 3-fold higher 

rate of recurrence but results in individual patients vary, 
which makes it challenging to make clinical decisions on 
the basis of FISH results [26].

The platform CellDetect (Zetiq Technologies, Israel) 
uses a proprietary plant extract and 3 dyes that enable 
color discrimination between malignant (red) and 
benign (green) cells on the basis of specific metabolic 
alterations associated with BC. In a multicenter 
validation study, the test reached a sensitivity of 84% 
(Table 3) (78% for detecting LG NMIBC). The specificity 
was 84% in patients undergoing sur veillance by 
cystoscopy [24].

DNA-Based Markers
Various types of DNA alterations can occur in BC, such 
as mutations, copy number alterations, and genomic 
rearrangements. In general, BC is known to have a 
high mutational burden [27,28]. Until now, none of the 
DNA-based urine assays that are based on detection of 
mutations have been FDA approved for use in clinic. 
Prospective validation of most urine assays is still 
awaited.

Some of the most frequently found DNA mutations 
in BC include mutations in the FGFR3, RAS, PIK3CA, 
and TERT genes. Approximately two-thirds of NMIBCs 
have activating FGFR3 mutations. The number of 

 TABLE 2. 

Performance characteristics of cell-based urine markers

Study
Patients

n

Studies 
included 

n
Context

Sensitivity
%

Specificity
%

Patients with 
tumor

n

Immuno-cytology

Chou et al.
[13]

1876 7
Primary 

diagnosis
85 

(78–90)
83 

(77–87)
401

Mowatt et al.
[14]

4199 10 Mixed
84

(77–91)
75 

(68–83)
NA

Schmitz Drager 
et al. [12]

4899 20 Mixed
81 

(Median)
75 

(Median)
1252

UroVysion

Chou et al.
[13]

651 2
Primary  

diagnosis
73 

(50–88)
95 

(87–98)
144

Mowatt et al.
[14]

3321 14 Mixed
76 

(65–8)
85 

(78–92)
NA

Schmitz-
Drager et al.

[12]
2852 21 Mixed

72 
(Median)

80 
(Median)

792

Cell Detect
Davis et al.  

[24]
217 1 Surveillance 84 84 96

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TABLE 3. 

Performance of assays that are based on detection of DNA alterations

Assay Parameters Author Year Technique
Patients 

n Population
Sens

%

Spec

%

Mutation: FGFR3, PIK3CA, RAS

Methylation:  41 CpG islands in 
23 genes

Microsatellite:  12 different 
primers 

Zuiverloon  
et al. 36]

2013

SNaPshot analysis

Methylation Specific 
multiplex ligation-
dependent probe 
amplification

136

Surveillance

• FGFR3 49 66
• Cytology 56 57
• FGFR3 + cytology 76 42
• FGFR3 + Microsatellite 71 44
• FGFR3 + Methylation 75 22

Surveillance, patients  
stratified by inclusion  
tumor

• FGFR3 66 50
• FGFR3 + PIK3CA + RAS 71 63
• Microsatellite 67 40
• FGFR3 + Microsatellite 82 82
• Methylation 68 42
• FGFR3 + Methylation 75 22

Mutation: FGFR3

Methylation: OTX1, ONECUT2, 
OSR1

Kandimalla  
et al. [37]

2013 SNaPshot analysis 301

 
Surveillance

• Methylation only 74 90
• Combined 79 77

Mutation: FGFR3

Methylation:  HS3ST2, SLIT2, 
SEPTIN9

Clinical parameters:  
Age, smoking status

Roprech  
et al. [38]

2016

Allele specific 
PCR Quantitative 
multiplex-
methylation  
specific PCR

167/158
• Hematuria 97 84
• Surveillance 90 65

Mutation: TERT, FGFR3

Methylation:  SALL3, ONECUT2, 
CCNA1, BCL2, 
EOMES, VIM

Dahmcke  
et al. [39]

2016
Droplet digital 
polymerase chain 
reaction (ddPCR)

475

Hematuria

• All 97 77
• TERT 82 84
• FGFR3 41 98
• SALL3 68 97
• ONECUT2 78 94
• CCNA1 67 97
• BCL2 63 98
• EOMES 46 97
• VIM 76 96
• TERT, FGFR3, ONECUT2, 

CCNA1 97 80

continued on page 53

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53SIUJ.ORG SIUJ  •  Volume 1, Number 1  •  October 2020

Urinary-Based Markers for Bladder Cancer Detection

TABLE 3. 

Performance of assays that are based on detection of DNA alterations

Assay Parameters Author Year Technique
Patients 

n Population
Sens

%

Spec

%

Mutation: FGFR3, TERT
Methylation: OTX1

Beukers  
et al. [40]

2017 SNaPshot analysis 977

Surveillance

• Previous high grade 72 55
• Previous low grade 57 59

Mutation: FGFR3, TERT, HRAS

Methylation:  OTX1, ONECUT2, 
TWIST1

Clinical parameter: Age 

Van Kessel  
et al. [41]

2016

SNaPshot analysis

Methylation-specific 
polymerase chain 
reaction (MSP)

154 Hematuria 97 83

Mutation: FGFR3, TERT, HRAS

Methylation:  OTX1, ONECUT2, 
TWIST1

Clinical parameter: Age

Van Kessel  
et al. [42]

2017

SNaPshot analysis

Methylation-specific 
polymerase chain 
reaction (MSP)

200 Hematuria 93 86

Mutations in 11 genes

Copy number changes on 
chromosome  39

Cytology

Springer  
et al. [43]

2018

Multiplex PCR

Singleplex PCR

Aneuploidy assays

570

Primary diagnosis
• 10 genes combined 68 NR
• TERT 57 NR
• Aneuploidy 46 NR
• UroSEEK (assays combined) 83 93

Cytology 

• UroSEEK + cytology 43 100
• UTUC 95 93
• 10 genes combined - -
• TERT 64 NR
• Aneuploidy 29 NR
• UroSEEK (assays combined) 39 NR

Surveillance 75 NR
• 10 genes combined 52 NR
• TERT 57 NR
• Aneuploidy 28 NR
• UroSEEK (assays combined) 68 80
• Cytology 25 100
• UroSEEK + cytology 71 82

Panel of 15 methylation 
markers (EpiCheck Bladder)

Witjes  
et al. [44]

2018

Quantitative 
multiplex-
methylation  
specific PCR

440 Surveillance 68.2 88.0

, Cont’d

activating mutations is much lower in MIBC [29], (only 
<15% of tumors have FGFR3 mutations); however, > 
40% of MIBCs overexpress FGFR3 [30]. Tumors with 
an FGFR3 mutation grow slowly and are less likely than 
FGFR3 wild-type tumors to progress to MIBC [30–33]. 
Several hotspot mutations in the FGFR3 gene have been 
identified as oncogenic.

In general, mutations in the KRAS gene are most 
frequent in cancer. For instance, KRAS is mutated in 90% 
of pancreatic cancers and 45% of colorectal cancers [34]. 
In BC, HRAS is the most commonly mutated RAS gene: 
HRAS mutations are present in approximately 5% of 
bladder tumors [27].

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Further, approximately 20% of BC tumors harbor a 
mutation in the PIK3CA gene [27]. PI3K can be activated 
by RTKs, or via crosstalk via the RTK-RAS-MAPK 
pathway. Finally, mutations in the telomerase reverse 
transcriptase (TERT) gene are frequent in BC: >70% of 
bladder tumors harbor a TERT promotor mutation [35]. 
The presence of a TERT mutation has been found to 
be more frequent in tumors that also harbored FGFR3 
mutations; however, it was not associated with stage or 
grade of the tumors [35]. Overall, significant overlap 
between different mutations occurs.

Epigenetic studies, such as genome-wide methylation 
ana lyses, have identif ied severa l genes t hat are 
significantly hypermethylated in BC cells compared 
with normal urothelial cells [45]. Methylation of several 
genes was found to be useful for the diagnosis of BC, 
with some markers being highly specific for BC. Gene 
hypermethylation has also been proposed in predicting 
disease progression [46,47]. The EpiCheck platform 
(Nucleix, Israel) has been designed for detection of 
DNA methylation changes associated with BC in a 
panel of 15 biomarkers. The test has been validated in 
several studies that included mainly patients who were 
under surveillance for NMIBC [39,40]. Other assays that 
were validated in several cohorts have been designed 
to combine the analysis of both BC-relevant DNA 
gene methylations and mutations [42,49]. Assay results 
and clinical parameters (type of hematuria) have been 
incorporated in multivariate models to obtain optimal 
performance.

In the literature, various DNA-based urinary markers 
have been developed. For most of these, prospective 
validation is still lacking. The urine-based markers 
suggested in the literature can be subdivided into 
markers used for detection of primary tumors (eg, in a 
patient with hematuria) and markers used for detection 
of recurrent tumors (eg, patient previously treated for 
BC). Furthermore, markers based on cell pellet DNA 
exist, as well as cell-free DNA-based markers Table 3. 
provides an overview of studies that combine various 
DNA marker assays.

RNA-Based Markers
A high number of RNA-based urinary gene panels are 

being validated, with the aim of improving diagnostic 
accuracy in BC without decreasing specificity [3]. 
T hree messenger R NA (mR NA)-based u rina r y 
biomarkers (Cxbladder assay [Pacific Edge Diagnostics, 
US], GeneXpert BC [Cepheid, US], and TaqMan 
Array, [ThermoFisher, US]) and various microRNA 
(miRNA)-based urinary targets (eg, members of the 
miRNA-200 family and miRNA-145) are currently 
tested in clinical trials for BC detection or surveillance.

In detail, 5 published studies [50–53] on different 

clinical scenarios (detection of BC in risk population, 
and sur veillance for recurrent BC) consistent ly 
confirmed promising diagnostic performances with 
a high sensitivity (even in LG tumors on surveillance) 
and NPV of the Cxbladder assay at the expense of a 
lower specificity than cytology. For BC screening and 
detection, the Cxbladder has the potential to reduce 
the frequency of diagnostic and invasive procedures in 
patients presenting with hematuria; however, further 
prospective validation studies are necessary. The 
GeneXpertBC test has shown 83% to 100% sensitivity 
for HG tumors and up to 77% sensitivity for LG tumors, 
and overall sensitivity ranged between 46.2% and 84%. 
Whereas the specificity in the BC detection population 
was very high (90% to 95%), in homogeneous data 
specificity was confirmed for BC surveillance (77% 
to 91%) [11,54–56].The training and validation study 
analyzing the diagnostic accuracy of a specific 12 + 2 
gene set (TaqMan Array) panel on bladder washings 
and voided urine samples showed consistently high 
sensitivities and specificities in BC detection (sensitivity: 
70%, 80%, and 98%; specificity: 86%, 96%, and 99%) 
and for discrimination between LG and HG tumors 
(sensitivity: 75% to 79%; specificity: 75% to 92%) [50,51]. 
These findings were confirmed in a prospective, blinded 
multicenter trial [59]. An overview of the diagnostic 
performance characteristics of the 3 mRNA-based 
urine assays for detection/surveillance of BC is shown  
in Table 4.

Moreover, miR NAs may become bioma rkers 
for BC detection and sur veillance in the future. 
Nevertheless, identified miRNA signatures were found 
to be heterogenous in published studies, with few trials 
confirming their results by independent validation 
cohorts, resulting in a low degree of reproducibility in 
the clinical setting. Most trials included only a small 
number of patients (n = 47 to 207) [60,61]. Another 
controversial issue that is discussed controversially is the 
feasibility of implementation and application of different 
analytical platforms and bioinformatics in the clinical 
setting [62].

In summar y, R NA-based urinar y markers are 
characterized by ease of handling, their brief hands-on 
sample preparation time, technical instrument systems 
that automate and integrate all complex PCR processes, 
and high-qua lit y standards including in-sample 
quality controls [55]. One of the major limitations of 
the application of RNA-based urinary techniques is 
the difficulty in obtaining sufficient quantity of “high-
quality” RNA from voided urine compared with bladder 
washings. Studies using bladder washings and optimized 
specimen collection and handling may achieve results 
that are not attainable in real world practice [57,63]. It 
has been shown that bladder washing samples yielded 
higher amounts of better RNA-quality than voided urine 

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TABLE 4. 

Diagnostic performance characteristics of mRNA-based urine markers for detection/surveillance of BC

Study Study design Indication
Patients

n
SN

%

SN for  
HG tumors

%

SP
%

NPV
%

Cxbladder
O’Sullivan  
et al. [51]

Marker-comparison 
study, prospective

Microhematuria 485 82 97 85

Lotan  
et al. [53]

Marker-comparison 
study, prospective

BC surveillance 803 91 97 – 96

G + P INDEX
Kavalieris  
et al. [50]

Cohort, prospective
Micro- and

Macrohematuria
695/ (MAH)  

45 (MIH)
95 45 98

Kavalieris  
et al. [52]

Cohort, prospective BC surveillance 763 93 97 – 97

GeneXpert BC
Van 

Valenberg  
et al. [11]

Marker-comparison 
study, prospective

BC detection + 
surveillance

239 (surveillance)/
508 (detection)

74 83
80 (surveillance)

95 (detection)
93

Wallace  
et al. [54]

Cohort, prospective
BC detection, 
surveillance

484 (training cohort) +  
450 (validation cohort)

73 –
90 (hematuria)

77 (surveillance)
–

Pichler  
et al. [55]

Marker-comparison 
study, prospective

BC surveillance 140 84 100 91 93

D’Elia  
et al. [56]

Marker-comparison 
study, prospective

BC surveillance 230 46.2 85.7 77 83

TaqMan Array 
(12 +2 gene 

panel)

Mengual  
et al. [57]

Cohort, prospective BC detection 
341/235 
(control) 

98 100
99

95

Mengual  
et al. [58)

Case–control study BC detection 
207 

(independent set)/ 
404 (training set)

80 – 86 –

Ribal  
et al. [59]

Cohort, prospective BC detection 525 81.5 – 91.3 –

BC: bladder cancer; HG: high grade; MAH: macrohematuria; MIH: microhematuria; NPV: negative predictive value; SN: sensitivity; SP: specificity.

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56 SIUJ  •  Volume 1, Number 1  •  October 2020 SIUJ.ORG

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samples [57]. Another limitation is the fact that there is 
a wide variability in the cost of RNA-based urine tests. 
For widespread use in the future, these tests should be 
available at reasonable cost. Another challenge of RNA-
based urinary markers in preanalytics is the mRNA 
instability, resulting in an advantage for commercial 
test systems (working with RNA-stabilizing tubes) 
compared with single urinary mRNA targets (CAIX or 
survivin). Standardized processes are indispensable for 
RNA analysis. According to the current EAU and AUA 
guidelines, RNA-based urinary biomarkers cannot be 
recommended for screening, detection of BC in patients 
with microscopic hematuria, or BC surveillance [2,64].

Protein Markers
In comparison with other-omics, the proteome can be 
directly linked to a phenotype, and hence represents a 
rich source of biomarkers and therapeutic targets (the 
latter, however, typically not in urine). On the downside, 
the extensive complexity and large dynamic range of 
protein components of a biological sample (in the case 
of urine, spanning at least 6 orders of magnitude) raise 
technical challenges, regularly encountered to some 
extent in proteome analysis and addressed, via the 
application of high-resolution mass spectrometry-based 
methodologies [67]. A large number of proteins have 
been reported in association with BC phenotypes, for 
disease prognosis or treatment prediction [68–71].

Of the most widely studied, nuclear matrix protein 
22 (NMP22), quantified by the point-of-care NMP22 
BladderChek, and the NMP22 ELISA immunoassay 
(Alere/Abbott), has received approval by the FDA for 
application in BC surveillance (both tests) and detection 
of the disease in high-risk or symptomatic populations 
(for the NMP22 BladderChek test only). Similarly, the 
BTA TRAK immunoassay-based and BTA STAT point-
of-care tests (Polymedco), detecting the complement 
factor H and complement factor H-related protein, have 
also been granted FDA approval for use in BC diagnosis 
and surveillance. Meta-analyses of existing studies 
demonstrate that the performance for both approved 
markers varies widely among the studies (ranging for 
the sensitivities and specificities from 47% to > 90%)
[65,68–72] (Table 5). The reported sensitivities are 
higher than those of cytology in the detection of low-
grade disease, but hematuria, infections, presence of 
stones or instrumentation are frequent confounders, 
compromising the specificity of the assays [73].

Besides the abovementioned FDA-approved tests, 
several additional exploratory protein biomarkers have 
been reported in primary diagnosis, and in cancer 
detection during surveillance and/or monitoring of 
treatment response. Among the most frequently reported 
are proteins of the extracellular and the nuclear matrix, 
apolipoproteins (apolipoprotein A-I, apolipoprotein 
A-II, apolipoprotein E) and other plasma proteins (alpha 

TABLE 5. 

Overall performance rates of FDA approved urinary protein markers, based on the most recent available meta-
analyses

Protein marker Reference Context Sample Size Sensitivity Specificity 

NMP22 (Nuclear  
Matrix Protein 22)

Chou et al. [4] Primary diagnosis
n=1313 for ELISA; 

n=1816 for POC
67 (ELISA)
47 (POC)

84 (ELISA)
93 (POC)

Mowatt et al. [65]
Diagnosis of primary 
and prevalent cancer

n=10119 -ELISA  
and POC

68 (overall) 79 (overall)

BTA (Complement factor 
H-related protein and 
complement factor H)

Chou et al. [4]
Diagnosis of primary 

cancer
n=1021 (POC) 76 78

Guo et al. [66]
Diagnosis of primary 
and prevalent cancer

n=3175 (POC) 67 75

POC: Point of care assay

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1 anti-trypsin, heparin cofactor II), including angiogenic 
factors (such as angiogenin, vascular endothelial growth 
factor A-VEGFA), as well as inflammatory factors (such 
as interleukins 2, 6, 8, 10, TNFα) [68,70]. Commercially 
available assays for the measurement of some of these 
proteins have been established; these include the 
UBC Rapid, (IDL Biotech AB) or CYFRA 21-1 (Cisbio 
International) tests measuring cytokeratin fragments—
mainly of cytokeratins 8 and 18 for the former [74] and 
cytokeratin 19 for the latter [75]—or ADXBLADDER, 
(Arquer Diagnostics Ltd), quantifying the levels of the 
mini-chromosome maintenance-MCM 5 protein [76], 
having being tested mainly in diagnostic contexts of 
use and, as essentially with all tests, exhibiting better 
performance in advanced than in early grade and stage 
tumors.

Applications in disease prognosis have also been 
described, including the recent examples of the shed 
ectodomains of epithelial cell adhesion molecule 
(EpCAM) and hepatocy te growth factor activator 
inhibitor-1 (HA-1), exhibiting a prognostic value for 
disease-specific death (in the range of 2 times increased 
risk at increased marker levels) in NMIBC [77]; or 
various interleukins (such as IL12, TRAIL, TNFα) in 
response to BCG treatment [78].

A clear consensus is emerging that combination of 
individual protein markers with clinicopathological 
information [79,80] in biomarker panels or multi-
parametric classifiers results in increased accuracy 
rates. Examples include the CyPRIT (cytokine panel for 
response to intravesical therapy) measuring the levels of 
9 cytokines, as a predictor of response to BCG treatment 
in intermediate- and high-risk NMIBC [78]; or the 
simultaneous quantification of matrix metalloproteases 
and plasma proteins v ia ELISA-based assays in 
diagnostic contexts of use [68,81] (Table 6). In addition, 

peptides mainly originating from extracellular matrix 
proteins (collagens, fibrinogen) but also plasma proteins 
quantified simultaneously via mass spectrometry-based 
assays and combined to a classifier (DiaPat) have shown 
diagnostic value in disease primary detection and 
surveillance [82].

Collectively, several urinary protein markers in 
association with BC phenoty pes and prognostic/
predictive contexts of use have been reported. Proper 
prospective validation is pending to define their added 
value (stand-alone or in combination) in disease 
management. Technology advancements allowing the 
simultaneous detection of marker panels provide a solid 
basis for future work in this direction.

Clinical Considerations for Use of Urinary 
Molecular Markers in Context of BC
The various marker tests differ regarding their potential 
applications in the diagnosis and surveillance of BC. 
Whereas FISH, immunocytology, and NMP22 have 
long been considered the most valuable alternatives 
to cytology, other DNA- and RNA-based assays have 
drawn broad attention within the last 10 years. With 
the advent of high-throughput analysis technologies 
there is the hope that performing multiple analyses in 
parallel with high resolution will improve the detection 
rate. Many of these test systems provide information 
on DNA, RNA, and protein levels. In the future, such 
information can be helpful not only in the detection of 
BC but also for the non-invasive monitoring of disease. 
One potential indication that has been investigated 
in the context of the UroVysion is the monitoring of 
patients treated with BCG. Because of the high risk of 
recurrence in these patients, a marker that provides 
evidence of response or failure of treatment would be of 
high value to prevent delay of cystectomy in patients who 

TABLE 6. 
Exploratory marker panels for protein-based BC detection

Protein marker Reference Context Sample Size Sensitivity, % Specificity, % 

Cytokine panel  
(9 cytokines; CyPRIT)

Kamat  
et al. [78]

NMIBC prediction of 
recurrence

n=130 80 77.4

10 protein panel  
(Meso Scale Diagnostic)

Shimizu 
et al. [81]

Diagnosis of primary 
cancer

n=200 85 81

CE-MS Peptide panel 
(DiaPat)

Frantzi 
et al. [82]

Diagnosis of primary 
and prevalent cancer 

(test set n=481)
n=481 (test set)

91 (primary)
87 (prevalent)

68 (primary)
51(prevalent)

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MOLECULAR BIOMARKERS IN UROLOGIC ONCOLOGY: ICUD-WUOF CONSULTATION

do not respond to BCG. The high level of complexity 
of new marker combination panels is associated 
with various challenges. One main challenge of any 
combination marker panel that is developed with the 
goal of achieving a high sensitivity will be to avoid false-
positive test results, which has been a major issue in the 
development of many markers that have been discussed 
as potential alternatives for cytology.

Any marker or test system that is supposed to be 
broadly implemented in clinical practice requires 
prospective clinical trials to address the value of the test 
in the relevant clinical context. Case–control studies 
provide preliminary information on test characteristics 
but will not be sufficient for a broad clinical use.  
A common study design used for va lidation of 
promising markers is to ana lyze urine samples 
prospectively from patients undergoing cystoscopy 
because of suspicion of BC. One of the primary goals of 
urine biomarkers in this context is to replace cystoscopy, 
but the cohorts studied are typically too heterogenous 
with respect to the indication for evaluation (gross 
hematuria, microscopic hematuria, or irritative voiding 
symptoms), so that the study results are not adequate 
to change practice. Moreover, many studies include 
both patients undergoing primary diagnostic workup 
and those under surveillance. Such heterogeneity 
potentially explains the considerable differences of test 
performances between different validation studies.  
To avoid such heterogeneity of patient groups, it makes 
sense to test the potential of urine markers in specific 
clinical settings. Both clinicians and companies should 
make every effort to validate an assay in a prospective 

trial designed in cooperation with guideline panels and 
key opinion leaders. One example of a prospective trial 
that has been set up in a specific setting with a specific 
hypothesis is the UroFollow trial [83]. In this trial, 
patients with low grade NMIBC are randomized to 
receive standard of care surveillance using cystoscopy 
versus regular measurement of urine markers (including 
cytology and UroVysion), with cystoscopies performed 
only in the case of a positive marker or clinical signs of 
recurrence. The hypothesis of the trial is that a urinary 
marker-based follow-up is non-inferior to a cystoscopy-
based follow-up with respect to detection of recurrence 
and progression.

Another clinical trial (NCT03988309) is randomizing 
patients to a marker-based approach using Cxbladder 
versus a standard evaluation for patients with hematuria. 
The goal of the trial is to determine if a risk-based 
approach with the addition of a marker is superior to 
cystoscopy for all patients.

Such specific study designs will not only allow a better 
understanding of the performance of an assay but also 
provide data on the potential use of a specific test.

Conclusions
Changing current clinical practice in bladder cancer 
workup is a very ambitious goal for any biomarker. 
There is currently no urinary marker available for which 
there is sufficient evidence to indicate it may change our 
current diagnostic workup, which includes cystoscopy 
and cytology. New markers should be considered for a 
use in more specific indications and clinical settings to 
allow the setup of properly designed trials.

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