








































This is an open access article under the terms of a license that permits non-commercial use, provided the original work is properly cited.  
© 2022 The Authors. Société Internationale d'Urologie Journal, published by the Société Internationale d'Urologie, Canada.

Key Words Competing Interests Article Information

Bladder cancer, predictive biomarker, 
urine biomarker, tissue biomarker, 
circulating tumor DNA, molecular subtype, 
immunohistochemistry

None declared. Received on September 28, 2021 
Accepted on December 4, 2021 
This article has been peer reviewed.

Soc Int Urol J. 2022;3(4):245–257

DOI: 10.48083/RVZV1144

245SIUJ.ORG SIUJ  •  Volume 3, Number 4  •  July 2022

REVIEW

Predictive Biomarkers in the Management of  
Bladder Cancer: Perspectives in an Evolving 
Therapeutic Landscape

Patrick J. Hensley,1 Niyati Lobo,1 Kelly K. Bree,1 Wei Shen Tan,2 Paolo Gontero,3  
Stephen B. Williams,4 Charles C. Guo,5 Gianluca Giannarini,6 Lars Dyrskjøt,7  
Ashish M. Kamat1

1 Department of Urology, University of Texas MD Anderson Cancer Center, Houston, United States 2 Department of Urology, London North West University Healthcare 
NHS Trust, London, United Kingdom 3 Department of Urology, Città della Salute e della Scienza, Molinette University Hospital, Turin, Italy 4 Division of Urology,  
The University of Texas Medical Branch at Galveston, Galveston, United States 5 Department of Pathology, University of Texas MD Anderson Cancer Center,  
Houston, United States 6 Urology Unit, Santa Maria della Misericordia University Hospital, Udine, Italy 7 Department of Molecular Medicine, Aarhus University  
Hospital, Aarhus, Denmark

Abstract

Bladder cancer (BC) is a heterogeneous disease with prognosis and therapeutic strategies highly dependent on tumor 
grade and stage. Predictive biomarkers of therapeutic response have been studied to guide selection of intravesical 
and/or systemic therapy. A predictive biomarker is measured before the start of treatment and provides information 
on the likelihood of response to a specific therapy.  Many candidate predictive biomarkers for BC have been 
identified, but few have been rigorously validated or distinguished from simply having treatment-agnostic prognostic 
capacity. Identifying predictive biomarkers tailored to therapeutic mechanism of action has considerable implications 
for the sequencing of therapies, as well as bladder preservation strategies in advanced disease states. We evaluate 
predictive tissue-based, urine-based, and serum-based biomarkers across the spectrum of non–muscle-invasive and 
muscle-invasive BC and preview predictive biomarkers for emerging targeted therapies.

Introduction

Biomarker development has undergone an evolution over the years, with increasing focus on predictive biomarkers in 
the field of bladder cancer. A predictive biomarker is measured before the start of treatment and provides information 
on the likelihood of response to a specific therapy. While many candidate predictive biomarkers for bladder cancer 
(BC) therapeutic response have been proposed, few have had their predictive value compared with non-treated 
cohorts to distinguish them from simply having prognostic capacity.

Predictive biomarkers have utility throughout the spectrum of disease in BC, from aiding diagnosis to guiding 
initial therapy selection, and even to prompting timely abandonment of ineffective treatment in lieu of definitive 
surgical management, radiotherapy, or other systemic therapy. Our understanding of specific therapeutic mecha-
nisms of action is key to designing predictive markers that offer insight into innate tumor biology and therapeutic 
susceptibility. Measuring the predicted or elicited response to local or systemic therapies provides an opportunity to 
tailor biomarker development to specific therapies.



In this narrative review, we discuss predictive BC 
tissue-based, urine-based, and serum-based markers 
(Figure 1), identify current limitations and unmet needs, 
and define the evolution of biomarker development in 
the landscape of targeted therapies. We summarize the 
current state of predictive biomarkers for both NMIBC 
and MIBC using a non-systematic review of published 
literature and provide expert opinion on the accuracy 
and clinical applicability of emerging biomarkers.

Predictive Tissue Biomarkers
Non–muscle-invasive bladder cancer
The most extensively studied tissue-based molecular 
ma rkers pred ic t ive of t herapeut ic response to 
intravesical therapy for non–muscle-invasive BC 
(NMIBC) to date are p53 and Ki67, both potent cell 
cycle regulators. Dysregulation of the tumor suppressor 
p53 has been correlated with BC progression, but not 
recurrence, following BCG therapy[1–3]. However, a 
prospective study failed to validate p53 as a predictive 
biomarker[4]. Ki67, a nuclear protein indicative of cell 

proliferation, has predictive ability for intravesical 
therapies. The expression of Ki67 has been correlated 
with recurrence following BCG[5] and both recurrence 
and progression following intravesical chemotherapy[6].

Table 1 summarizes other molecular biomarkers 
demonstrated to be predictive of BCG response. These 
include cell cycle regulators, apoptosis inhibitors, cell 
adhesion molecules, and proliferative markers[7]. The 
majority of these biomarkers have been evaluated only 
retrospectively in small single-center cohorts with 
non-standardized methods of measurement and without 
external validation, thus limiting our ability to derive 
definitive conclusions about their utility as predictive 
biomarkers.

Predictive biomarker panels have also been recently 
evaluated following intravesical BCG. A subgroup anal-
ysis of 2 large Nordic randomized trials performed 
by Malmström et al. analyzing expression of ezrin, 
CK20, and Ki67 failed to show a correlation between 
biomarker expression and recurrence or progression 
following BCG therapy[8]. Similarly, Park et al. eval-
uated the altered expression of 7 potential biomarkers 
(p53, pRb, PTEN, Ki67, p27, FGFR3, and CD9) and 
found no predictive value for recurrence or progression 
among high-grade T1 tumors treated with BCG[9]. 
These conflicting data underscore the need for compre-
hensive validation studies.

Functional mutations in DNA mismatch repair genes 
have also been implicated in predicting therapeutic 
response in BC. Sanguedolce et al. demonstrated that 
MutL homologue 1(MLH1) was an independent predic-
tor of progression-free survival among patients treated 
with adequate BCG[10]. In patients who received BCG, 
polymorphisms within DNA repair pathways have 
been associated with recurrence-free survival (RFS)[11]. 
Furthermore, tumor mutational burden has recently 
been shown to correlate with response to intravesi-
cal therapy[12]. These data were corroborated through  
a comprehensive gene analysis on index non–muscle- 
invasive tumors, with increased mutational burden 
noted among high-grade NMIBC[13]. Specifically, 
ARID1A mutations were predictive of shorter RFS in 
patients treated with BCG; however, no association was 
noted between RFS and other analyzed markers, includ-
ing TP53, MDM2, ERBB2, and FGFR3 following BCG  
therapy[13].

Damrauer et al. recently performed RNA-based 
profiling to identify a novel expression signature of an 
inflamed tumor microenvironment (TME) which was 
predictive of BCG response[14]. Notably, molecular 
subtyping and immune checkpoint gene expression 
were not predictive of treatment response. However, a 
pre-treatment TME enriched with CD25+ regulatory T 
cells and tumor-associated macrophages and decreased 

Abbreviations 
BC bladder cancer
BCG Bacillus Calmette-Guérin
ctDNA circulating tumor DNA 
NAC neoadjuvant chemotherapy
NMIBC non–muscle-invasive bladder cancer
MIBC muscle-invasive bladder cancer
RFS recurrence-free survival
TAM tumor-associated macrophage
TMB tumor mutational burden
TME tissue microenvironment
TURBT transurethral resection of bladder tumor

FIGURE 1.

Biomarker source material and analysis for  
NMIBC and MIBC

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continued on page 248

247SIUJ.ORG SIUJ  •  Volume 3, Number 4  •  July 2022

Predictive Biomarkers in the Management of Bladder Cancer: Perspectives in an Evolving Therapeutic Landscape

TABLE 1. 

Predictive urine biomarkers for BCG therapeutic response in patients with NMIBC 

Reference Year Marker
Study 

Populationa
Number of 

patients 
Detection 
Method(s)

Results

Palou et al.[5] 2009 Ezrin, Ki67 HG T1 92 IHC

Low ezrin expression (< 20%) associated 
with increased progression among 
patients receiving induction BCG  
(P = 0.031) b
Differential expression of Ki67 in 
patients with early recurrence  
following induction BCG (P = 0.015) b

Zachos et al.[83] 2009

Telomerase 
reverse 

transcriptase 
(hTERT)

HG T1  
NMIBC

30 IHC

hTERT nucleolar staining in >75% of 
cells was associated with worse RFS 
following induction BCG (relative risk  
of recurrence 8.85 [95% CI 1.9–41.6])b

Cormio et al. [84] 2010 pRb HG T1 27 IHC

Altered pRb expression was predictor 
of recurrence (P = 0.037) and 
progression (P = 0.018) in patients 
treated with adequate BCGc

Alvarez-Mugica  
et al.[85]

2010
Myopodin 

methylation
HG T1  
NMIBC

170 
Methylation 

analysis

Among patients treated with adequate 
BCG, myopodin methylation associated 
with increased recurrence rate  
(P = 0.011) and progression (P = 0.030)b

Shirotake  
et al.[86] 

2011
Angiotensin II 

Type 1 Receptor 
(AT1R)

NMIBC 79 IHC
Strong AT1R expression associated 
with worse 1-year RFS following  
BCG(P = 0.0012)b

Lima et al.[87] 2013
sialyl-Tn (sTn), 
sialyl-6-T(s6T)

High-risk 
NMIBC

94 IHC

High sTn and s6T expression was 
associated with BCG response  
(P = 0.024 and P < 0.0001) and  
with increased RFS (P = 0.001)c

Sen et al.[88] 2014 Nestin HG T1 63 IHC

Recurrence rates were higher in 
nestin(+) compared to nestin(-) among 
patients receiving induction BCG 
(60.6% versus 30%, P = 0.014)c

Cheng et al.[89] 2015 E2F4 NMIBC 188 
RNA 

sequencing

Treatment with BCG in E2F4  
score > 0 patients associated  
with improved progression-free 
survival (P = 0.06)

Raspollini  
et al.[90]

2016

P16, galectin-3, 
CD44, CD138, 

E-cadherin, 
survivin, HYAL-1, 

topoisomerase-liα

HG T1  
(≤ 3cm)

92 IHC

TOPO-2α predicted DFS (P = 0.029), 
surviving predicted PFS (P = 0.020), 
surviving and E-cad predicted OS  
(P = 0.006, 0.030)b

aAll studies listed were analyses of retrospective cohorts. bMultivariate analysis. cUnivariate analysis.



density of Th2-predominant CD4+ T cells was predic-
tive of poor RFS following BCG therapy[15]. Lim et al. 
also noted that TME-tissues from BCG-responders was 
enriched with active CD8+PD-1(-) T cells and non-reg-
ulatory CD4+FOXP3(-) T cells, whereas the TMEs of 
non-responders were characterized by increased levels of 
exhausted CD8+PD-1(+) T cells[16].

Among patients with carcinoma in situ (CIS) treated 
with induction BCG, lower tumor-associated macro-
phage (TAM) density was associated with improved 
recurrence-free survival compared with those with 
higher TAM density[17]. Furthermore, when subsets 
of TAMs (M1 and M2) were analyzed, a low density of 
M1-TAM and high density of M2-TAM were predic-
tors of worse disease-specific survival among patients 
treated with BCG[18]. Other components of the complex 
immune response to BCG therapy that have been predic-
tive of favorable treatment response include increased 
expression of major histocompatibility complex 1[19], 

low level of infiltration by tumor-infiltrating dendritic 
cells[20], and increased levels of natural killer cell recep-
tor ligands[21]. Taken together, these data indicate that 
the TME likely plays an important role in modulating 
the BCG therapeutic response and serves as a promising 
predictive biomarker target.

While there is a paucity of investigation into predic-
tive biomarkers for individual intravesical chemother-
apeutic agents in NMIBC, single-institution series 
suggest high FOXM1 expression[22] and tumors with 
high proliferation index (as measured by Ki67) achieve 
favorable responses to mitomycin C[6,23].

Muscle-invasive bladder cancer
Cisplatin-based neoadjuvant chemotherapy (NAC) 
before radical cystectomy confers an overall survival 
benef it for pat ients w it h M IBC[24]. Genom ic 
interrogation revealed that a significant proportion of 
MIBCs harbor mutations in DNA damage repair (DDR) 
genes. DDR pathways play a critical role in the cellular 

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, Cont’d 

TABLE 1. 

Predictive urine biomarkers for BCG therapeutic response in patients with NMIBC 

Reference Year Marker
Study 

Populationa
Number of 

patients 
Detection 
Method(s)

Results

Meeks et al.[12] 2016
Cancer-

associated  
gene panel

High-risk 
NMIBC

25 
DNA 

sequencing

Increased total mutational burden 
associated with IO response between 
non-progressors, progressors and 
metastatic tumors (15, 10.1, 5.1 
mutations/MB, respectively; P = 0.02)c

Pietzak et al.[13] 2017 ARID1A NMIBC 65 
DNA 

sequencing

ARID1A mutations associated  
with shorter RFS after BCG  
(HR 3.14, P = 0.002)b

Sanguedolce  
et al.[10]

2018 MLH1 HG T1 67 IHC
MLH1 expression was an independent 
predictor of PFS among patients 
treated with adequate BCGb

Mano et al.[91] 2018
HSP 60  
HSP 70 
HSP 90

HG T1 54 IHC

HSP70 associated with lower risk of 
recurrence (HR 0.29, P = 0.003) and 
progression (HR 0.33, P = 0.045),  
HSP 60 associated with higher risk  
of progression (HR 3.96, P = 0.012) 
among patients treated with at  
least induction BCGb

Shao et al.[92] 2021
Next generation 

sequencing
Intermediate or 
high-risk NMIBC

58
DNA  

sequencing

NEB, FGFR1, and SDHC were 
independent predictors of recurrence 
following BCG (P = 0.001, P = 0.004,  
and P = 0.017, respectively)b

aAll studies listed were analyses of retrospective cohorts. bMultivariate analysis. cUnivariate analysis.



response to platinum-based chemotherapy, providing a 
rationale for their use as predictive biomarkers.

ERCC2 encodes a DNA helicase that plays a central 
role in the nucleotide excision repair pathway, repairing 
DNA cross-linking caused by genotoxic agents such as 
platinum chemotherapies. Van Allen et al. performed 
whole-exome sequencing on pre-treatment tumor and 
germline DNA from 50 patients with MIBC receiving 
cisplatin-based NAC before cystectomy[25]. ERCC2 
was the only significantly mutated gene enriched in 
cisplatin-responders compared with non-responders. 
This finding was mechanistically confirmed in vitro, as 
expression of wild-type ERCC2 in an ERCC2-deficient 
BC cell line rescued cisplatin sensitivity. Using an inde-
pendent MIBC cohort, Liu et al. found ERCC2 alter-
ations in 8/20 responders to chemotherapy (40%) versus 
2/28 non-responders (7%) (P = 0.010, OR 8.3 [95% CI 1.4 
to 91.4])[26]. In a subsequent phase II trial of neoadju-
vant dose-dense gemcitabine and cisplatin in patients 
with MIBC, the presence of one or more alterations in 
a panel of 29 DDR genes, including ERCC2, was associ-
ated with chemosensitivity (positive predictive value for 
<pT2N0 of 89%)[27].

In addition to ERCC2, alterations in ATM, Rb1 
and FANCC have also been shown to be predictive of 
response to cisplatin-based NAC[28]. Plimack et al. 
showed that alteration in 1 or more of these 3 DNA 
repair genes was able to predict pathologic complete 
response to NAC. Additionally, missense mutations 
in the receptor tyrosine kinase ERBB2 (HER2) were 
enriched in patients with a complete response to 
platinum-based NAC (24% of complete responders 
compared with 0% in non-responders)[29]. As a result of 
these studies, several trials are investigating the ability 
of predictive markers to triage patients for bladder spar-
ing after a favorable clinical response to cisplatin-based 
NAC (Table 2). Recent studies, however, have found no 
correlation between ERCC2 mutation status and treat-
ment response[30,31], but have instead identified other 
DDR biomarkers associated with response (eg, BRCA2), 
highlighting a high level of tumor heterogeneity and 
inter-cohort variability.

Large-scale expression and sequencing analysis have 
subtyped BCs based on genomic RNA expression or 
specific genomic alterations[32,33]. Choi et al. reported 
that the basal subtype, with enriched expression of genes 
in the p63 pathway, responded favorably to neoadjuvant 
MVAC; in contrast, patients with “p53-like tumors,” 
with activated wild-type p53 gene expression signatures, 
were consistently chemoresistant. These findings were 
largely recapitulated through tumor profiling of patients 
in a phase II trial of dose-dense neoadjuvant MVAC 
and bevacizumab before cystectomy in which patients 
with basal tumors exhibited favorable survival but 

similar pathologic response rates[34]. Similarly, a large 
multicenter study that performed whole transcriptome 
profiling on transurethral resection of bladder tumor 
(TURBT) specimens from 343 patients with MIBC[35] 
concluded that patients with basal subtype tumors 
derived the most improvement in overall survival 
compared with those who had surgery alone, whereas 
patients with luminal tumors had similarly favorable 
outcomes irrespective of NAC use.

A multiomics approach to identifying molecular 
markers of cisplatin sensitivity in the neoadjuvant or 
first-line settings was recently reported[30]. Contrary to 
the findings of the aforementioned reports, patients with 
basal/squamous gene expression subtypes responded 
poorly to cisplatin-based chemotherapy, while patients 
who had tumors with immune cell infiltration and high 
PD-1 protein expression exhibited a favorable thera-
peutic response. Notable differences in cohort selection 
and primary outcomes measurements may account for 
differing conclusions on platinum-sensitivity of basal 
tumors[36]. While Seiler et al.[35] primarily compared 
survival outcomes in a NAC cohort with those of the 
TGCA cohorts, managed with variable adjuvant chemo-
therapeutic regimens, Taber et al.[30] measured thera-
peutic response through pathologic downstaging and 
response on imaging in NAC and first-line metastatic 
patients, respectively. Taken together, these data indicate 
that more research is needed in this setting to determine 
the predictive value of expression subtypes.

The role of molecular subtypes as predictive biomark-
ers for response to neoadjuvant immunotherapy have 
also been assessed. In the PURE-01 study evaluating the 
efficacy of neoadjuvant pembrolizumab, basal tumors 
had better response rates than non-basal tumors[37]. 
Tumor immunophenotypes have also been correlated 
with response to the anti-PD-L1 agent atezolizumab[38]. 
Favorable response was associated with the CD8+ T-cell 
phenotypes and high tumor mutational burden, while 
so-called “immune desert” tumors and those with high 
transforming growth factor β (TGFβ) signaling in the 
TME exhibited therapeutic resistance.

Despite promising results, molecular subtyping using 
whole transcriptome RNA expression profiling has yet to 
be adopted into clinical practice. This is primarily due to 
the technical difficulty of handling and profiling RNA as 
well as the lack of definitive evidence from clinical trials 
specifically designed for measuring biomarker accuracy. 
However, studies have suggested that immunohisto-
chemistry, which already has clinical applications, may 
be used to classify BC into molecular subtypes, thus 
facilitating their use in clinical practice[39].

Several PD-1/PD-L1 immune checkpoint inhibi-
tors (ICI) have been approved for cisplatin-ineligible 
patients and those with locally advanced or metastatic 

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BC[40–46]. The indication has since expanded, with 
several groups investigating the use of PD-1/PD-L1 
inhibitors in the neoadjuvant setting. In the PURE-01 
study, 50 patients with MIBC received 3 cycles of the 
PD-1 inhibitor pembrolizumab followed by radical 
cystectomy[47]. Pathologic complete response (pT0) 
was observed in 40% of PD-L1 positive patients (≥ 10% 
combined positive score) compared with only 16% of 
PD-L1 negative patients. Furthermore, a greater patho-
logic response to pembrolizumab was seen in patients 
with higher tumor mutational burden (TMB >15 muta-
tions/MB). In contrast to the PURE-01 study and trials 
in the metastatic setting, the neoadjuvant ABACUS 
study of the PD-L1 inhibitor atezolizumab was unable to 
show a significant association between PD-L1 expression 

(either on tumor cells or infiltrating cells) and therapeu-
tic response[48]. The lack of standardization of PD-L1 
assessment, such as different antibodies, thresholds for 
PD-L1 positivity, and immune cell quantification, is 
likely to contribute to its limited predictive ability in BC. 
Bandini et al. recently constructed a probability calcula-
tor incorporating 2 biomarkers (PD-L1 expression and 
TMB) and baseline clinical T stage to predict patho-
logic complete response after pembrolizumab[49]. This 
predictive model performed well with a concordance 
index of 0.77 (95% CI 0.68 to 0.86), highlighting the 
complexity of the tumor-immune interaction and util-
ity of predictive biomarker panels compared with single 
markers alone.

TABLE 2. 

Clinical trials investigating tissue-based biomarkers predictive of neoadjuvant therapy response in MIBC 

NCT Trial Name
Study 

Population

Biomarker(s) 
Under 

Investigation 
Intervention Primary Endpoints

02710734

Risk Enabled Therapy 
After Initiating 
Neoadjuvant 

Chemotherapy for 
Bladder Cancer 

(RETAIN)

cT2-3 N0 
bladder cancer 

Sequenced 
pre-NAC TURBT 
specimens for 
DDR mutations

TUR followed by accelerated MVACa; 
patients with complete clinical 
response and DDR mutation managed 
with bladder sparing, others treated 
with intravesical chemotherapy, 
radiation therapy or radical 
cystectomy

Metastasis-free 
survival (MFS) at  
2 years

03609216 Alliance A031701
cT2-3 N0 

bladder cancer

Sequenced 
pre-NAC TURBT 
specimens for 
DDR mutations

TUR followed by gemcitabine/
cisplatin; patients with DDR mutation 
and pathologic response (≤ycT1) 
managed with bladder sparing; others 
treated with chemoradiation or radical 
cystectomy

Event-free survival 
(MFS) at 3 years

03558087 HCRN GU 16-257
cT2-4 N0 

bladder cancer

Sequenced 
pre-NAC TURBT 
specimens for 
DDR mutations 

and tumor 
mutational burden

TUR followed by gemcitabine/
cisplatin/nivolumab; patients with 
complete clinical response managed 
with bladder sparing and maintenance 
novilumab, others treated with radical 
cystectomy 

(1) complete clinical 
response rate (2) 
ability of complete 
clinical response 
to predict 2-year 
metastasis-free 
survival 

02177695 SWOG 1314
cT2-4 N0 

bladder cancer

Co-expression 
extrapolation 
(COXEN) gene 

expression 
algorithm

TUR followed by neoadjuvant dose-
dose MVACa or gemcitabine/cisplatin 
prior to radical cystectomy

Assess whether 
COXEN profile is (1) 
prognostic of pT0 rate 
or ≤pT1 at cystectomy 
and (2) a predictive 
factor between 
chemotherapy 
regimens

amethotrexate, vinblastine, doxorubicin, cisplatin

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Predictive Urine Biomarkers
Non–muscle-invasive bladder cancer
Urine is a uniquely qualified biomarker source material, 
as it is readily available, easily collected, and has 
direct tumor contact. Urine-based biomarkers have 
been primarily studied for purposes of diagnosis and 
surveillance of BC, with relatively few having sufficient 
accuracy to predict therapeutic response. The true 
predictive biomarker capacity of the urine-based markers 
mentioned herein remain largely uncharacterized  
as many were studied exclusively in treated populations 
or measured as an elicited response after intravesical 
therapy.

BCG has proven efficacy in reducing recurrence 
and progression in intermediate and high-risk non–
muscle-invasive BC (NMIBC)[50]. Reliable biomark-
ers predictive of BCG therapeutic response could have 
tremendous implications in sequencing of therapy for 
NMIBC. Unfortunately, given the relative non-specific 
mechanism of action and elicited immune response 
by BCG, clinicopathologic factors such as tumor stage, 
grade, size, presence or absence of CIS, tumor focality 
and recurrence history remain the most reliable predic-
tors of BCG therapeutic response[51].

The rationale for several candidate preclinical 
biomarkers have employed the mechanism of BCG 
therapeutic response[52]. Interleukin (IL)-8 is one of 
the first cytokines with induced expression after BCG 
therapy. In a pilot study of 20 patients, high levels of IL-8 
expression measured 6 hours after BCG instillation had 
lower rates of recurrence and progression[53]. Addition-
ally, failure to induce expression of IL-2 and IL-18 after 
BCG has been associated with poor BCG therapeutic 
response[54].

Because BCG immunogenicity is complex and non- 
specific, single candidate markers alone may be unreli-
able predictive tools. The Cytokine Panel for Response 
to Intravesical Therapy (CyPRIT) nomogram was gener-
ated from expression profiling of 9 inducible urinary 
cytokines (IL-2, IL-6, IL-8, IL-18, IL-1ra, TRAIL, IFN-γ, 
IL-12[p70], and TNF-α) in 130 patients with NMIBC 
at the MD Anderson Cancer Center using an enzyme-
linked immunosorbent assay (ELISA) at baseline and 
at specified time points throughout BCG therapy[55]. 
This nomogram predicted the likelihood of recurrence 
with 85.5% accuracy. Additionally, baseline levels of 
pro-tumorigenic cytokines were profiled pre-treatment. 
Indeed, expression of IL-8 in urine was associated with 
recurrence in BCG-treated patients, with patients who 
had higher baseline urinary IL-8 levels experiencing a 
4-fold increased risk of tumor recurrence[56]. Inter-
estingly, higher baseline levels of IL-8 expression in 
peripheral blood leukocytes similarly correlated with 
disease recurrence, suggesting a role for this cytokine as 

a systemic marker for BCG immunogenicity and thera-
peutic response. Contrasting these results to the afore-
mentioned studies indicating that induced urinary IL-8 
expression after BCG instillation is a marker of disease 
recurrence and progression, these data highlight the 
complexity of baseline and elicited immune states, as 
well as the stability and kinetics of cytokine profiling, 
in determining their potential as predictive biomarkers.

The Oncuria urine-based assay measures the expres-
sion of cancer-associated markers in voided urine speci-
mens[57]. Using urine samples from the CyPRIT cohort, 
investigators found that pre-treatment concentrations of 
MMP9, VEGFA, CA9, SDC1, PAI1, APOE, A1AT, ANG, 
and MMP10 were increased in subjects with disease 
recurrence. A predictive model of treatment outcomes 
reached an area under the receiver operating curve of 
0.89 (95% CI 0.80 to 0.99), with a test sensitivity of 81.8% 
and a specificity of 84.9%.

While not specifically FDA approved for this indica-
tion, the fluorescence in situ hybridisation (FISH) assay, 
which detects aneuploidy in chromosomes 3, 7, and 
17 and loss of the 9p21 locus in voided urine samples 
(UroVysion), has been studied in the context of BCG 
therapeutic response. In a study of 37 patients primar-
ily receiving BCG for NMIBC, 100% of patients with 
a positive post-treatment UroVysion FISH developed 
tumor recurrence[58]. The predictive capacity of positive 
post-treatment UroVysion was independently confirmed 
in several studies with variable adjuvant intravesical 
agents for NMIBC[59–62].

A subset of “molecular BCG failure” patients based 
on mid-treatment persistence of a positive FISH assay 
was subsequently defined. In a study of 126 patients, 
those with a positive FISH test during therapy were 3 
to 5 times more likely to develop recurrence and 5 to 13 
times more likely to progress compared with patients 
with negative mid-treatment FISH[63]. This was subse-
quently validated in an independent, multicenter trial 
where FISH was predictive of recurrence and/or progres-
sion events at baseline (HR 2.59; 95% CI 1.42 to 4.73), 
before the sixth induction instillation (HR 1.94; 95% CI 
1.04 to 3.59) and at 3-month follow-up (HR 3.22; 95% CI 
1.65 to 6.27)[64]. Defined as positive FISH at 6 weeks and  
3 months after induction BCG in the setting of a negative 
cystoscopic evaluation, this molecular failure denotes a 
group at high risk of stage progression if managed with 
further BCG therapy, and who should be considered for 
enrolment into clinical trials or timely cystectomy[65].

Muscle-invasive bladder cancer
There currently exist no urine-based biomarkers to 

reliably predict therapeutic response in MIBC. However, 
broad genomic expression and mutational profiling of 
molecular targets of novel therapeutic agents, including 

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Predictive Biomarkers in the Management of Bladder Cancer: Perspectives in an Evolving Therapeutic Landscape



monoclonal antibodies and antibody-drug conjugates, 
have emerging rationale. For example, UroSEEK is a 
urine-based molecular assay which detects alterations 
in 11 commonly mutated genes which are druggable 
targets: TERT, FGFR3, PIK3CA, TP53, HRAS, KRAS, 
ERBB2, CDKN2A, MET, MLL, and VHL[66].

As sequencing technology becomes more refined, 
urine-based genetic material, including exfoliated tumor 
cells, cell-free DNA, and exosomes may prove feasible 
sources for molecular subtyping and further predictive 
biomarker development for MIBC.

Predictive Serum Biomarkers
Serum biomarkers for BC remain an area of active 

research. These liquid biopsy tests may have a role in 
cancer risk stratification, characterization of tumor 
molecular signatures, and predicting response to 
systemic treatment, as well as for cancer surveillance. 
To date, these tests have remained proof-of-concept in 
preclinical studies but have emerging clinical relevance 
to guide treatment decisions.

Circulating tumor cells (CTC) represent one of 
the first studied serum biomarkers. While they have 
a poor sensitivity of 35% for the detection of BC due 
to their scarcity in circulating blood, the presence of 
CTCs has been associated with higher histological 
stage, grade, lymph node involvement, and presence 
of metastatic disease[67,68]. In the pre-radical cystec-
tomy setting, CTCs have been shown to predict poor 
oncological outcomes, independent of clinicopatho-
logical variables[69].

Serum RNA markers such as long non-coding 
RNAs (IncRNAs) and microRNAs (miRNA) have been 
reported to have prognostic value. Zhang et al. reported 
that high serum UBC1 expression was associated with 
lower NMIBC RFS (P = 0.01) [70]. In a systematic review 
and meta-analysis of 26 studies, 6 miRNA (miR-21, 
miR-143, miR-155, miR-214, and miR-222) were identi-
fied as being predictive of early disease recurrence and 
progression[71].

More recently, there have been rapid advancements 
in circulating tumor DNA (ctDNA). Developments in 
deep-sequencing technology have allowed for the reli-
able identification of double strand DNA fragments as 
small as 150 bp. Birkenkamp-Demtröder et al. devel-
oped personalized ctDNA assays based on NGS of 
tumor tissue. They report that ctDNA was present even 
in NMIBC patients, and the presence of higher levels of 
ctDNA was associated with subsequent disease progres-
sion and metastasis[72]. In patients undergoing radical 
cystectomy, ctDNA predicted oncological outcomes in 
several settings[31]. Patients positive for ctDNA at diag-
nosis before NAC had a higher 12-month recurrence rate 

(42% versus 0%)[31]. Similarly, following NAC, patients 
positive for ctDNA had a higher rate of 12-month disease 
recurrence (75% versus 7%) than did ctDNA negative 
patients[31]. Additionally, in the surveillance setting, 
ctDNA had a median lead time of 96 days over radio-
logical imaging[31]. This lead time of ctDNA detection 
before radiologic or symptomatic clinical detection is 
allowing investigators to define a “biochemical relapse” 
to guide timely initiation of first-line atezolizumab after 
RC in a clinical trial setting (NCT04138628). The role of 
ctDNA as a predictive biomarker for atezolizumab has 
also been reported in a study where patients with ctDNA 
positivity had a significantly improved overall survival 
compared with the observational arm (HR: 0.59; 95% CI 
0.41 to 0.79)[73].

Unmet Needs in Biomarker Development
Characteristics for the ideal biomarker predictive 

of therapeutic response vary considerably by disease 
stage. For NMIBC, BCG is the gold standard intravesi-
cal treatment because of its efficacy, favorable cost, and 
tolerability. To date, biomarkers predictive of response 
to BCG have primarily focused on identifying early 
non-responders in an effort to transition them to off-la-
bel salvage intravesical chemotherapy options or timely 
radical cystectomy. However, in the era of BCG short-
ages and emerging intravesical and systemic therapies 
available in earlier disease states, we would benefit from 
predictive markers that could guide initial therapeutic 
response. With emphasis on bladder preserving strate-
gies, it will become equally important to identify predic-
tive biomarkers of salvage intravesical and systemic 
therapies after BCG failure.

Recent molecular classification of NMIBC has 
correlated candidate molecular subtypes to innate sensi-
tivity and resistance to BCG therapy, and provided ther-
apeutic rationale for upfront use of FGFR inhibitors, 
ICIs, or intravesical chemotherapy[74]. Lastly, favorable 
results have recently been reported in the Phase III trial 
of intravesical nadofaragene firadenovec (rAd-IFNa/
Syn3) for BCG unresponsive NMIBC[75]. These investi-
gators are validating a serum-based adenoviral antibody 
titer assay to evaluate immunogenicity of the gene ther-
apy and corresponding therapeutic response[76].

While guidelines support the role of NAC before 
radical cystectomy for MIBC, there remains a role for 
risk-stratified NAC patient selection. Clinicopatho-
logic risk factors have been implemented in predicting 
response to cisplatin-based NAC[77,78], but efforts are 
underway to profile tissue-based biomarkers for this 
purpose. The Southwest Oncology Group (SWOG) 1314 
trial prospectively profiled the ability of the COXEN 
tissue-based genetic classifier to predict complete patho-
logic response to cisplatin-based NAC (Table 2)[79]. 

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Recently reported results indicate limited predictive 
capacity of the genomic classier for individual treatment 
response, underscoring the importance of prospec-
tive validation of predictive markers in the clinical 
trial setting. Our group, among others, has also been 
involved in profiling immunohistochemical signatures 
in TURBT specimens predictive of response to NAC to 
improve appropriate stratification of patients for NAC 
or upfront cystectomy[80,81]. Lastly, biomarkers could 
significantly aid in the ability to accurately predict and 
assess a complete clinical response to NAC in those 
electing for bladder perseveration, a concept with 
supporting clinical data[82] and currently being evalu-
ated in 2 randomized trials (RETAIN, Alliance A031701; 
Table 2).

Conclusions
There are no currently FDA-approved predictive 

biomarkers for therapies in BC–neither for NMIBC 
nor for MIBC. The lack of clinically available predic-
tive biomarkers is likely multifactorial, including diffi-
culties in profiling intratumoral heterogeneity and 
dynamic cellular processes (ie, epithelial-mesenchy-
mal transition, cell growth and proliferation, immune 
response) as well as the lack of “fit for purpose” profil-
ing of biomarkers with mechanistic rationale. The 
ever-evolving armamentarium of therapeutic options 
further emphasizes unique unmet needs for predic-
tive biomarker development. By convention, we have 
relied on non-specific markers of therapeutic response 
to drugs with non-specific targets—ie, cytokine expres-
sion profiling as a litmus for induced immunogenicity 
after intravesical BCG or DNA mismatch repair genetic 
alterations to predict response to cytotoxic chemother-
apies. With the growth of novel therapeutic modalities 
with specific targets, including monoclonal antibodies, 
antibody-drug conjugates, and gene therapies, we expect 
that biomarkers which are highly specific for, and even 
proprietary to, the proposed mechanism of action of 
individual therapies are on the horizon.

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