ArticlePDF AvailableLiterature Review

Targeted therapies in bladder cancer: An overview of in vivo research

September 2015
Nature Reviews Urology

September 2015

DOI:10.1038/nrurol.2015.231

Authors:

Kim E. M. Van Kessel

Erasmus MC

Tahlita C M Zuiverloon

Erasmus University Rotterdam

Show all 5 authorsHide

Survival of patients with muscle-invasive bladder cancer is poor and new therapies are needed. Currently, none of the targeted agents that are approved for cancer therapy have been approved for the treatment of bladder cancer and the few clinical trials that have been performed had limited success, often owing to a lack of efficacy and toxic effects. However, many other novel targeted agents have been investigated in animal models of bladder cancer. EGFR, FGFR-3, VEGF, mTOR, STAT3, the androgen receptor and CD24 are molecular targets that could be efficiently inhibited, resulting in reduced tumour growth, and that have been investigated in multiple independent studies. Several other targets, for example COX-2, IL-12, Bcl-xL, livin and choline kinase α, have also been observed to inhibit tumour growth, but these findings have not been replicated to date. Limitations of several studies include the use of cell lines with mutations downstream of the target, providing resistance to the tested therapy. Furthermore, certain technologies, such as interfering RNAs, although effective in vitro, are not yet ready for clinical applications. Further preclinical research is needed to discover and evaluate other possible targets, but several validated targets are now available to be studied in clinical trials.

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Department of

Pathology (K.E.M.v.K.,

E.C.Z.), Department

ofUrology (T.C.M.Z.,

A.R.A., J.L.B.),

ErasmusMC,

POBox2040, 3000 CA,

Rotterdam,

Netherlands.

Correspondence to:

E.C.Z.

e.zwarthoff@

erasmusmc.nl

Targeted therapies in bladder cancer:

anoverview of invivo research

Kim E.M. van Kessel, Tahlita C.M. Zuiverloon, Arnout R. Alberts, Joost L. Boormans

andEllenC.Zwarthoff

Abstract | Survival of patients with muscle-invasive bladder cancer is poor and new therapies are needed.

Currently, none of the targeted agents that are approved for cancer therapy have been approved for the

treatment of bladder cancer and the few clinical trials that have been performed had limited success, often

owing to a lack of efficacy and toxic effects. However, many other novel targeted agents have been investigated

in animal models of bladder cancer. EGFR, FGFR-3, VEGF, mTOR, STAT3, the androgen receptor and CD24 are

molecular targets that could be efficiently inhibited, resulting in reduced tumour growth, and that have been

investigated in multiple independent studies. Several other targets, for example COX-2, IL-12, Bcl-xL, livin

and choline kinaseα, have also been observed to inhibit tumour growth, but these findings have not been

replicated to date. Limitations of several studies include the use of cell lines with mutations downstream of

the target, providing resistance to the tested therapy. Furthermore, certain technologies, such as interfering

RNAs, although effective invitro, are not yet ready for clinical applications. Further preclinical research is

needed to discover and evaluate other possible targets, but several validated targets are now available to be

studied in clinical trials.

van Kessel, K.E.M. etal. Nat. Rev. Urol. 12, 681–694 (2015); published online 22 September 2015; doi:10.1038/nrurol.2015.231

Introduction

Bladder tumours can be classified as either non-

muscle-invasive bladder cancer (NMIBC) or muscle-

invasive bladder cancer (MIBC). At diagnosis, 75%

of patients present with NMIBC (stageTa, Tis or T1)

and 25%present with MIBC (stage≥T2).1,2 In patients

with NMIBC, although the 5-year survival is >90%, the

recurrence rate is high (>50%), which necessitates costly

long-term surveillance with invasive cystoscopies.1,3–5 By

contrast, MIBC has a poor outcome: the 5-year overall

survival after radical cystectomy and lymph node dissec-

tion ranges from 49% for stage T3–4N0 disease to 74%

for stage T2N0 disease.6 In patients with MIBC, cispla-

tin-based neoadjuvant chemotherapy preceding radical

surgery results in an overall long-term survival benefit of

6%.7 However, overall, no improvement in the survival

of patients with MIBC has been accomplished over the

past 20years.8,9 The high incidence and recurrence rate,

together with the poor survival, make bladder cancer a

serious public health problem.10 Therefore, a clear clini-

cal need for new effective therapies in both NMIBC and

MIBC exists.

In the 1990s, targeted molecular therapy was intro-

duced as a novel treatment strategy in oncology.11 These

therapies aim to interfere with cellular processes that are

essential to cancer cell survival at the molecular level—

for example, by blocking proteins involved in tumour

cell proliferation or tumour cell metabolism, or by

delivering toxic compounds to tumour cells. Anumber

of targeted therapies are now well established for the

treatment of different cancers: imatinib in leukaemia

and gastro intestinal stromal tumours, cetuximab in

colorectal cancer and bevacizumab in kidney cancer.

However, none of the registered targeted therapies have

been approved for the treatment of bladder cancer and

the small number of clinical trials that have been per-

formed showed disappointing effects.12 Patients included

in these phaseI and phaseII clinical trials were mostly

not selected based on their molecular tumour profile,

which might explain the lack of treatment effect.12 In

addition, resistance to therapy occurs quickly, prob-

ably owing to molecular heterogeneity of the tumour.13

This molecular heterogeneity contributes to the clonal

evolution and selection of resistant subclones leading

to therapy resistance.14 Lastly, some trials investigating

therapy with sunitinib or gefitinib were stopped early

because of s ubstantial toxic effects of the treatment.12,15,16

This Review provides a comprehensive overview of

targeted therapies for bladder cancer that have been

investi gated in animal models, some of which have

potential for clinical application. We provide insights

into both the challenges and the promises of the pre-

clinical develop ment of the targeted agents, highlight-

ing the most promis ing studies (Table1), grouped by

the mechanisms and pathways that the investigated

targets are involved in. Summaries of all studies included

in this Review are p rovided in the Supplementary

informationonline.

Competing interests

The authors declare no competing interests.

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Overview of preclinical animal models

In the development of novel targeted therapies, invitro

and invivo studies precede clinical trials. Only few of the

potential novel therapies will eventually be evaluated in

clinical studies. In bladder cancer research, three types

of animal models are typically used in invivo research:

syngeneic mouse or rat models, and heterotopic or

o rthotopic xenogeneic mouse or rat models (Box1).

Syngeneic tumour models have the advantage of an

intact tumour host environment including an active

immune system. However, obvious drawbacks of these

models exist, as mouse tumours are intrinsically differ-

ent from human tumours. In addition, only few tumour

types are available.17 Xenogeneic tumour models more

closely resemble the tumours of bladder cancer patients;

however, the stroma and vasculature is murine and the

animals are immunodeficient to allow growth of human

cancer cells.17 Furthermore, tumours grownfrominjected

cell lines no longer resemble the tumour from which the

cell line originated, owing to years of cultivation, passag-

ing and expansion. Xenografts that are derived from fresh

patient material represent a possibility to achieve better

resemblance of the true hetero geneous tumour architec-

ture. In 2015, the first study describing a method to create

patient-derived xenografts for bladder cancer studies was

published.18

In studies published since 2009, the heterotopic and

orthotopic animal models were generated using many dif-

ferent bladder cancer cell lines and several of these have

been genotyped, revealing specific oncogenic mutations

(Table2). These mutations are important to consider

when choosing which tumour model to use in invivo

studies, particularly when the target of the tested agent

is upstream of these mutations in the signalling pathway,

as downstream mutations might activate the signal-

ling cascade despite inhibition of the upstream target.

However, for correct interpretation of the treatment

effect, awareness of all potential mutations—downstream

or upstream of the target—that could influence the study

result is required.

Furthermore, in 2013, a study showed that KU-7 bladder

cancer cells are actually HeLa cervical carcinoma cells, as

the original KU-7 cell line had been contaminated with

HeLa cells at the source site at some point before 1984.19

Consequently, KU-7 cells should be considered cervical

cancer cells and not bladder cancer cells. Invivo studies

using KU-7 animal models are listed in Supplementary

Table1 online, but owing to their n onurological origin

they are not being discussed in thisReview.

Receptor tyrosine kinases

Since 2009, many invivo studies have investigated agents

targeted at receptor tyrosine kinases (RTKs). An overview

of these studies is given in Supplementary Table2 online.

RTKs, such as the epidermal growth factor receptor

(EGFR), the fibroblast growth factor receptors (FGFRs)

and the vascular endothelial growth factor receptors

(VEGFRs), are essential for the communication between

cells and their environment. Specific growth factors (for

example, cytokines or hormones) bind to the extracellular

domain of an RTK, thereby activating the intracellular

kinase domain of the receptor. The activation results in

the stimulation of downstream signalling cascades that

affect cell proliferation and cell growth.20 The RTK–

Ras–MAPK pathway and the RTK–PI3K–Akt pathway

are among the most frequently affected signalling path-

ways in cancer (Figure1, Figure2).21,22 In bladder cancer,

RTKs are often overexpressed or mutated, resulting in

o verstimulation of downstream signalling pathways.

EGFR

EGFR is overexpressed in many bladder tumours

andEGFR overexpression correlates with a poor progno-

sis.23,24 Examples of agents targeting EGFR are gefitinib,

a small-molecule inhibitor used for the treatment of

chemotherapy- refractory non-small-cell lung cancer, and

cetuximab, an antibody used for the treatment of head

andneck cancer and advanced colorectal cancer.25,26 The

effect of inhibiting EGFR in bladder cancer was investi-

gated in five mouse studies (Figure2).27–31 Cetuximab was

used in three of these studies and researchers found that

growth of MGH and T24 xenografts was inhibited by this

antibody.27,30,31 In two studies, cetuximab was combined

with photodynamic therapy, which enhanced the inhibi-

tory effect in one study and was synergistic in the other.27,30

Similarly, combining afatinib (an inhibitor of EGFR and

erbB-2) with cetuximab was more effective than cetux-

imab alone.31 Addition of bevacizumab (an antibody

against VEGF-A) to cetuximab and p hotodynamic

therapy resulted in regression of tumourvessels.30

Caution must be taken in interpreting the results of four

of these studies, which used T24 cells28,31 or MGH cells,27,30

as these cell lines carry activating mutations downstream

of the treatment target. Molecular profiling has shown

that T24 cells have a mutation in HRAS, which activates

the Ras–MAPK and PI3K–Akt pathways downstream of

EGFR.32,33 One study in colorectal tumour samples dem-

onstrated that activating mutations in another Ras protein

(GTPase KRas, encoded by KRAS) render the tumour cells

insensitive to cetuximab.34 In one of the two studies using

T24 cells, the authors describe a cetuximab resistance

mechanism but the influence of the HRAS mutation in

this resistance mechanism remains unknown.31 In the

other study using T24 cells, LRIG1 was introduced into

these cells that were then used in a xenograft model.28

Key points

■Over the past 20years, survival of patients with muscle-invasive bladder cancer

has not improved

■None of the targeted therapies that are approved for other cancers have been

approved for the treatment of bladder cancer

■The small number of clinical trials that have been performed in patients with

bladder cancer had limited success owing to several limitations

■Specific oncogenic mutations in different cell lines used in invivo

research might render these cell lines insensitive to the therapy that is

beinginvestigated

■Inhibition of EGFR, FGFR-3, VEGF, mTOR, STAT3, the androgen receptor and

CD24 resulted in inhibition of tumour growth in multiple invivo studies

■Careful patient selection in clinical trials based on the molecular profile of the

tumour will be essential in demonstrating benefit of new targeted therapies

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The protein leucine-rich repeats and immunoglobulin-

like domains protein1, which is expressed from LRIG1,

inhibits EGFR through direct interaction;28 however, its

effect on the Ras–MAPK and PI3K–Akt pathways is most

likely nulli fied by the presence of the HRAS mutation in

T24 cells. In addition, the cell line MGH, which was used

in the two studies by Bhuvaneswari and colleagues,27,30 has

a mutation in AKT1; the corresponding protein PKB is

an effector for the PI3K–Akt pathway.32,33 MGH cells also

carry an FGFR3 mutation that might activate the Ras–

MAPK pathway, independently of EGFR.32,33 Although a

beneficial effect of targeting EGFR was observed, the effect

could have been more convincing if a different cell line

had been used to establish the treated mouse xenografts.

Furthermore, the observation that EGFR was down-

regulated does not imply that the downstream signalling

pathways were also downregulated.27,30 By contrast, in the

study of Rebouissou etal.29 in which EGFR was inhibited

by erlotinib, resulting in tumour growth inhibition or

delayed tumour detection by ultrasono graphy, the results

are convincing. The cell lines used (BFTC-905, JMSU1,

KK47, L1207, UMUC6, VMCUB1) were selected using

Table 1 | Promising invivo research*

Target Pathway Tumour type (n)Intervention Study

EGFR Ras–MAPK Orthotopic (40)

Heterotopic (144;

2per mouse)

EGFR inhibitor (erlotinib) 29

FGFR-3 Ras–MAPK Heterotopic (NR) FGFR inhibitor (PD173074) 48

FGFR-3 Ras–MAPK Heterotopic (80) mAb against FGFR-3 (R3Mab) 49

FGFR-3 Ras–MAPK Orthotopic (128) mAb against FGFR-3 (R3Mab) 50

FGFR-3 Ras–MAPK Heterotopic (72) FGFR inhibitor (PD173074) 51

FGFR-3

EGFR

Ras–MAPK Experiment1:

Heterotopic (30)

Experiment2: NR

Experiment1: FGFR inhibitor (PD173074) and/or EGFR inhibitor (getinib)

Experiment2: FGFR inhibitor (PD173074) and/or mAb against EGFR

(cetuximab)

VEGFRs Ras–MAPK Metastatic (NR) Tyrosine kinase receptor inhibitor (sunitinib) and/or chemotherapy

(epirubicin)

149

VEGF-A

VEGF-C

VEGFR-3

Ras–MAPK Heterotopic (22) siRNA against VEGF-A or siRNAs against VEGF-C and VEGFR-3 59

VEGF-A

EphB4

Ras–MAPK Heterotopic (32) EphB4 inhibitor (sEphB4-HSA) and/or mAb against VEGF (bevacizumab) 150

mTOR PI3K–Akt Heterotopic (37) Knockdown of p53 and PTEN and mTOR inhibitor (rapamycin) 74

mTOR PI3K–Akt Heterotopic (40) mTOR inhibitor (rapamycin) and/or chemotherapy (cisplatin) 75

mTOR PI3K–Akt Heterotopic (40) mTOR inhibitor (rapamycin) and/or PI3K inhibitor (wortmannin) 76

STAT 3

mTOR

MAPK

JAK

PI3K–Akt

Ras-MAPK

Orthotopic (84) mTOR inhibitor (rapamycin) and/or MAPK inhibitor (UO126) and/or STAT3

inhibitor (S3I-201)

STAT 3

Survivin

JAK

Apoptosis

Heterotopic (35) siRNAs against STAT3, survivin or both STAT3 and survivin 151

AR JAK

PI3K–Akt

Ras–MAPK

Heterotopic (18) siRNA against AR 93

AR JAK

PI3K–Akt

Ras–MAPK

Heterotopic (10) Androgen deprivation (surgical castration) 92

AR JAK

PI3K–MAPK

Ras–Akt

Heterotopic (18) siRNA against AR 94

CD24

Metastasis

JAK

PI3K–Akt

Ras–MAPK

Orthotopic (190)

Heterotopic (20)

CD24-knockout mice or androgen deprivation (surgical castration) 98

CD24 Metastasis Heterotopic (16)

Metastatic (44)

mAb against CD24 (ALB9) 95

CD24

HIF-1α

Metastasis

Angiogenesis

Heterotopic (40)

Metastatic (32)

shRNA against CD24 or HIF-1α128

*Additional details can be found in the Supplementary Information online. Abbreviations: AR, androgen receptor; CD24, signal transducer CD24;

EGFR,epidermal growth factor receptor; EphB4, ephrin type-B receptor4; FGFR, fibroblast growth factor receptor; HIF-1α, hypoxia-inducible factor1α;

mAb,monoclonalantibody; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; shRNA, small hairpin RNA; siRNA, small interfering

RNA; STAT3, signal transducer and activator of transcription3; VEGFR, vascular endothelial growth factor receptor.

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a gene expression classifier, discriminating between high

EGFR expression for a basal-like phenotype or low EGFR

expression for a non-basal-like phenotype, and thoroughly

examined for downstream mutations (Table1).29

Several EGFR-targeted therapies have been evaluated in

clinical trials and a number of clinical trials are ongoing.12

For instance, erlotinib monotherapy was studied in the

neo adjuvant setting in 20patients with MIBC.35 The

authors reported a pathological response rate with down-

staging to <T2 in 35% of patients. Gefitinib and cetux-

imab combined with chemotherapy were studied in the

metastatic setting.36,37 Median overall survival times were

15.1months and 14.3months for gefitinib and cetuximab,

respectively. Gefitinib was also studied as a single-agent,

second-line therapy in the meta static setting: only one of

31 treated patients responded to treatment.38 Nevertheless,

we believe that treatments targeting the EGFR can still

have a role as therapies for patients with bladder cancer in

the future. The disappointing trial results and data from

invivo research clearly highlight the need to carefully

select patients for therapy based onthemolecular profile

of their tumour.

In addition, agents targeting erbB-2 are being investi-

gated in phaseII trials in patients with bladder cancer

but most results are still pending. Examples include a

number of trials in which trastuzumab or lapatinib are

being combined with chemotherapy or given as single

agents.39–44 Promising results are expected, particularly

in the light of new data reported by The Cancer Genome

Atlas (TCGA) Research Network21 and Groenendijk and

co-workers.45 The TCGA study found that ERBB2 was

mutated or amplified in 9% of samples, demonstrating

the importance of ERBB2 mutations in the pathogenesis

of bladder cancer.21 Groenendijk etal.45 found that ERBB2

mutations were associated with a very good response to

neoadjuvant chemotherapy: 24% of complete responders

but none of the nonresponders had ERBB2mutations.

FGFRs

In addition to EGFR overexpression, approximately two-

thirds of all NMIBCs have activating FGFR3 mutations.46

In MIBC, <15% of tumours have FGFR3 m utations,

although >40% of MIBCs overexpress FGFR-3.47 Seven

invivo studies reported the effects of targeting FGFR-3.48–54

In most cases, the cell lines used either carried a mutant

FGFR3 gene or a FGFR3 fusion gene, both leading to acti-

vation of downstream pathways. The FGFR-3 was inhib-

ited by the small-molecule inhibitor PD173074 in four

studies,48,51,52,54 and by the mono clonal antibody R3Mab

in two studies (Figure2).49,50 One study evaluated the effect

of the pan-FGFR inhibitor BGJ-398.53

Five studies reported growth inhibition, reduced cell

proliferation or both.48–51,54 Induction of apoptosis—

which demonstrates cytotoxic effects of the treatment

—was only reported in one study.48 The MGH cell line

used in the study by Lamont etal.52 has an AKT1 muta-

tion, leading to activation of the PI3K pathway regardless

of inhibition of the FGFR; nevertheless, the investiga-

tors did observe a cytostatic response to treatment with

PD173074. The cell line UMUC3 used in the study by

Cheng etal.53 has a KRAS mutation and a deletion in the

PTEN gene, resulting in constitutive activation of both

Ras–MAPK and PI3K–Akt pathways. In this study, block-

age of tumour cell extravasation was reported but not

growth inhibition. In another study, inhibition of FGFR-3

and EGFR was combined in a mouse model using RT112

cells, which have no known downstream mutations in

Ras–MAPK and PI3K–Akt pathways.54 Combination

therapy with the FGFR-3 inhibitor PD173074 and cetux-

imab gave the best result regarding tumour growth

inhibition compared with treatment with either agent

alone, and also led to sustained tumour growth control.54

Overall, these mouse studies provided compelling evi-

dence that targeting FGFR-3 in human bladder tumours

is potentially effective (Table1).

In clinical trials, however, the strategy of targeting

FGFR-3 has not yielded the expected positive results.

The small-molecule inhibitor dovitinib was investigated

as a single-agent, second-line treatment in patients with

metastatic urothelial carcinoma.55 The results were

disappointing and the study was terminated owing

to a lack of beneficial treatment effects. In a phaseIb

study of dovitinib plus gemcitabine combined with cis-

platin or carbop latin in patients with advanced solid

tumours, which included two bladder cancer patients,

no treatment effect was observed.56 Furthermore, com-

bined treatment was poorly tolerated owing to myelo-

suppression. A phaseII trial of dovitinib in patients

with NMIBC who did not respond to BCG tr eatment is

ongoing and reports of the results are pending.57

In summary, studies of the inhibition of RTKs in cell-

line-derived animal models should be performed after

molecular profiling of the chosen cell line to exclude

the possibility of activating mutations downstream

ofthetarget RTK. In human bladder cancer tumours,

mutations in the Ras family of genes are rare (for example,

HRAS mutations exist in 5% of patients)21 and mutations

in the PI3K–Akt pathway occur in approximately 20%

of bladder cancer tumours.21 For these subgroups of

patients, tumour models harbouring mutations in these

pathways will be useful in therapy evaluation. However,

Box 1 | Main types of animal models used in preclinical bladder cancer research

Invivo tumour models can be classified according to the genetic background

oftumour and host and according to tumour location.

Genetic background

■Syngeneic model: the tumour arises from tissue of the host animal and has

the same genetic background as the animal; examples include spontaneously

developing cancers, BBN-induced tumours and mice that are genetically

modified to develop tumours

■Xenogeneic model: tissue or cells from another species are transplanted into

the host animal

Tumour location

■Orthotopic model: the tumour develops in the same anatomical location as the

source cell line or tissue, for example when bladder cancer cells are injected

into the bladder wall

■Heterotopic model: the tumour develops in a different anatomical location

than the source cell line or tissue, for example when bladder cancer cells

aresubcutaneously injected into the flank, or results from metastasis

Abbreviation: BBN, N-butyl-N-(4-hydroxybutyl) nitrosamine.

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RTK inhibition therapy will probably lack effectivity and

therapies not involving these pathways could be b eneficial

for this patient category.

The data reviewed here suggest that inhibition of RTKs

after stratification of patients based on molecular analy-

sis of the tumour does have potential in bladder cancer

therapy for the majority of patients. In addition, combi-

nation of treatments might enhance treatment effects;

however, some treatment combinations might also result

in increased toxic effects.

VEGFRs and angiogenesis

As tumours become larger, hypoxia will occur unless the

tumour is able to grow new blood vessels. Most tumours

stimulate neoangiogenesis by secreting VEGF. VEGF

binds to its cognate receptor on endothelial cells and

stimulates the formation of new blood vessels.58

In one study, Feng and co-workers59 treated mice

that had heterotopic mouse bladder carcinomas with

intratumoural injections of synthetic small interfering

RNAs (siRNAs) against Veg f a, Ve g fc and Veg f r3 . The

siRNAs specifically inhibited synthesis of the correspond-

ing proteins (VEGF-A, VEGF-C and VEGFR-3, respec-

tively). VEGF-A and VEGF-C are two types of ligands

that can bind to VEGFRs. Interestingly, all mice in the

control group and in the group that received siRNAs

against Veg f c and Ve g f r3 developed lung metastases but

37.5% of mice in the group treated with siRNA against

Ve g fa had no lung metastases. In addition, no animals

in the group treated with siRNA against Veg f a had liver

metastases compared with 50% and 25% of mice in the

control group and the group receiving siRNAs against

Ve g fc and Veg f r3 , respectively.59 These data suggest

that inhibition of VEGF-A expression reduces the

d evelopment of metastases.

Three of the studies on angiogenesis inhibition used

technologies that cannot yet be implemented in clinical

practice (for example, viral vehicles).59–61 Two of these

Table 2 | Bladder cancer cell lines and known oncogenic mutations and gene fusions relevant to this Review

Cell line

[alternative name]

Origin Oncogenic mutations

andgene fusions*

Invivo studies‡

253J-BV [253JBV, 253J_B-V] Human Unknown 156

5637 Human ERBB2, TP53, RB1 110,150,157

AY-27 Rat Unknown 158–160

BFTC-905 [BFTC905] Human NRAS, TP53 29

BIU-87 Human Unknown 161,162

BTT739 Human Unknown 59,61,90

EJ [MGH-U1] Human Unknown 91,98,130

HT-1197 [HT1197] Human NRAS, PIK3CA 163

HT-1376 [HT1376] Human TP53, RB1 163

J82 Human FGFR3, ERBB2, TP53 51,164,165

JMSU1 Human Unknown 29

KK47 Human Unknown 29

KU-19-19 [KU1919] Human NRAS, AKT1 88

KU-7 Human NA§166–175

L1207 Human Unknown 29

MB49 Mouse Unknown 175–178

MBT-2 [MBT2] Mouse Unknown 60,120,123,124,129,149,179

MGH [MGH-U3, MGHU3, RN] Human FGFR3, AKT1, CDKN2A 27,30,48,52

RT112 [RT-112] Human TP53, FGFR3–TACC3 fusion 49,50,52,54,180,181

RT4 [RT-4] Human RHOA 51,74,152,182

SW780 [SW-780] Human FGFR3–BIAIAP2L1 fusion 51,52,119,183

T24 [T-24] Human HRAS, TP53 28,31,93,94,99,112,113,127,151,163,178,184–191

TSU-pr1 Human NA|| 143

UMUC1 [UM-UC-1] Human Unknown 50

UMUC3 [UM-UC-3] Human PTEN, KRAS, TP53 53,75,76,87,89,92,95,98,108,111,125,128,152,192–194

UMUC6 [UM-UC-6] Human FGFR3 29

UMUC14 [UM-UC-14] Human FGFR3 48–50,81,119

VMCUB1 [VM-CUB-1] Human TP53, ERBB2, PIK3CA, CDKN2A 29

YTS-1 Human Unknown 144

*Only mutations registered in COSMIC databases are listed.32 ‡Only studies in which the cell lines have been used to create xenografts are listed. §Cervical

carcinoma cell line. ||TSU-pr1 cells have been derived from T24 cells. Abbreviation: NA, not applicable.

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studies reported tumour growth inhibition,59,61 but one

study did not find an effect on tumour growth.60 All

three studies reported a combination of either reduced

a ngiogenesis, reduction in lymphatic vessels or reduction

of metastasis.59–61

Many clinical trials of agents targeting VEGFs and

VEGFRs in patients with bladder cancer have already

been reported.12 Four studies investigated the use of

bevacizumab (targeting VEGF-A) or sunitinib (targeting

VEGFRs) combined with chemotherapy in the neoadju-

vant setting (Figure2).62–65 Pathological response rates

with downstaging to <T2 ranged from 22% to 53%. One

study of sunitinib was terminated early owing to substan-

tial toxic effects.65 Combining bevacizumab with chemo-

therapy has also been studied as adjuvant treatment in

two studies.64,66 The reported median progression-free

survival was 6.5months in one study64 and 8.2months in

the other study.66 In both studies, the aspired study goal

of 50% improved progression-free survival was not met.

Other studies have analysed monotherapy of sunitinib,

aflibercept (a fusion protein that binds VEGFs) or pazo-

panib (which targets VEGFRs) as second-line treatment

strategies in phaseII trials in patients with advanced

urothelial cancer.67–70 Two studies reported no beneficial

effect of aflibercept and pazopanib,68,69 and the other two

studies reported a partial response in 7% and 17.1% of

patients for sunitinib and pazopanib, respectively.67,70 At

present, three clinical trials investigating anti-VEGF ther-

apies (bevacizumab, pazopanib and sunitinib) in patients

with bladder cancer areongoing.71–73

Overall, VEGF-targeted therapies show promise for

clinical application. However, sunitinib treatment resulted

in high toxicity in clinical trials,12,16 and a more selective

VEGF inhibitor or antibody will be more likely to be well

tolerated. Again, selection of patients based on the down-

stream mutations in their tumour will be n ecessary to

achieve the highest benefits.

Signal transduction

Several studies have identified potential new targets for

bladder cancer therapy in different signal transduction

pathways. Signals coming from cell membrane receptors

must be communicated to downstream effector mol-

ecules. This signal transduction occurs through kinases,

such as MAP kinases and PI3 kinases, or GTPase activity,

for example of Ras proteins (Figure1, Figure2).

The signal transduction cascades offer many possi-

ble new therapeutic targets. As a consequence, inhibi-

tors have not been generated for all potential targets. In

cell culture and animal models, inhibition of a target by

inhibiting translation of its mRNA by siRNAs usually

gives the first insight into whether the target is suit-

able for further study. An overview of invivo studies in

which targeting of signal transduction pathways has been

i nvestigated is provided in Supplementary Table3 online.

The protein mTOR acts in the PI3K–Akt pathway and

is targeted by rapamycin. Several rapamycin analogues,

such as temsirolimus and everolimus, have been devel-

oped and are in use for the treatment of advanced renal

cell carcinoma. In three studies that used hetero topic

xenograft models and one study that used an orthotopic

mouse model, mTOR was targeted via intraperitoneal

injection of rapamycin (Figure2).74–77 All studies showed

that rapamycin reduced tumour growth. In addition,

Makhlin etal.75 showed that the addition of cisplatin

to rapamycin treatment resulted in increased median

survival and a lower proliferation index (decreased

expression of the proliferation marker Ki-67) in com-

parison with rapamycin alone. Similarly, Zhou etal.77

demonstrated that combining rapamycin with inhibi-

tors of MAPK and signal transducer and activator of

transcription3 (STAT3) significantly prolonged sur-

vival in a bladder cancer model in transgenic mice in

comparison with rapamycin alone. Seront etal.76 found

that rapa mycin was less effective in UMUC3 cells in

comparison with UMUC14 cells (UMUC3 cells have a

homozygous deletion in PTEN, whereas UMUC14 cells

carry wild-type PTEN). The researchers also found that

combining rapamycin with a PI3K inhibitor was required

Nature Reviews | Urology

Receptor tyrosine kinases

Signal transduction

Protein synthesis and cell metabolism

Cell cycle proteins and apoptosis

Metastasis, immune system and others

DNA

Transcription

RNA

Protein

synthesis

G2 M

SG1

Oncogene

p53

Apoptosis

Cell

metabolism

Stress

Virus

T cell

CD24

EGFR

FGFRs

VEGFRs

Hormones and

growth factors

Cell

membrane

Figure 1 | Cellular processes involved in cancer development. External stimuli,

such as hormones and growth factors, stimulate receptors on the cellular

membrane, leading to activation of downstream pathways. These pathways lead

toactivation of transcription factors and, thus, gene expression. RNA is either

translated to functional proteins or can have regulatory functions on its own.

Incancer cells, many processes eventually lead to increased cellular proliferation

by amplifying cell growth, evading cell cycle arrest and evading apoptosis.

Abbreviations: EGFR, epidermal growth factor receptor; FGFR, fibroblast growth

factor receptor; p53, cellular tumour antigen p53; VEGFR, vascular endothelial

growth factor receptor.

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to inhibit UMUC3 cell tumour growth.76 This finding,

however, was not confirmed by Puzio-Kuter etal.74 in

a mouse model of conditional knockout of PTEN and

Makhlin etal.75 in a xenograft model using UMUC3

cells. In summary, invivo studies investigating rapa-

mycin treatment of bladder cancer have demonstrated

that this inhibitor is promising, displaying effective ness

in human xenografted cell lines and models of mouse

bladder cancer (Table1).

Examples of phaseII clinical trials targeting mTOR

and the PI3K–Akt pathway in patients with bladder

cancer include several trials that combined temsiroli-

mus or everolimus with chemotherapy.78–80 The results

of these studies are not yet available. Other trials have

evaluated a single-agent strategy of temsirolimus or

everolimus (Figure2).81–83 One study was terminated

because of futility.82 Although most of the patients in

these trials did not respond to the single-agent treatment,

some patients had stable disease or partial response. For

example, upon treatment with everolimus, eight and

two out of 37 patients in one study81 and 12 and two

out of 45patients in another study83 had stable disease

or partial response, respectively, i ndicating a beneficial

effect in selected groups of patients.

A single-agent strategy with the PI3K inhibitor

BKM120 (also known as buparlisib) is being investigated

in an ongoing trial but study results are still pending

(Figure2).84 BKM120 has previously shown partial

responses in the treatment of patients with breast cancer.85

Furthermore, one trial evaluating the effect of everoli-

mus in patients with NMIBC is currently underway;86

however, the tumours of the patients enrolled in this trial

have not been molecularly characterized. Hence, the

eventual treatment effect might differ between patients

depending on the molecular profile of their tumour,

which could result in an underestimation of the overall

treatment effect. If enough patients are included, strati-

fied analysis based on the molecular tumour profile might

indicate which patients benefit more (or less) than others.

Other targets in signal transduction cascades include

transcription factors (for example, STAT3, NF-κB, zinc

finger protein224 and the androgen receptor [AR])

NF-κB

Nature Reviews | Urology

Growth factors

Cell survival Cell growthApoptosis

Cell cycle Angiogenesis

Cell cycle

p21 (14%) BAX

p53 (49%)

MDM2 (9%)‡

ARF

CP-31398,

ALT-801 Balomycin A1

Alisertib

Ramucirab,

pazopanib,

bevacizumab,

sunitinib,

aibercept,

siRNA

mTOR

Rapamycin,

temsirolimus,

everolimus

Akt (12%)

VEGF

STAT

JAK-1

E2F

MAPK

MEK

B-raf

Ras (2–5%)*

RTKs (6–15%)* PI3K (17%)

BKM120

Getinib,

cetuximab, afatinib,

erlotinib, trastuzumab,

lapatinib, PD173074,

R3Mab, dovitinib

RPS6

Apoptosis pathway

Ras–MAPK pathway

Activation

Inhibition

Targeting

JAK–STAT pathway

PI3K–Akt pathway

Figure 2 | Cellular pathways affected in bladder cancer and targeting therapies. Multiple small-molecule inhibitors and

antibodies against different receptors and pathways have been tested in preclinical and clinical studies. Agents targeting

protein translation and cell metabolism affect the molecules at the end of signalling pathways and are not shown. Pathways

and targets are located at various sites in and around the tumour cell (Figure1). The percentage of bladder cancer samples

harbouring mutations are shown for targets that have been screened by the TCGA.21 *Percentage depends on the RTK

analysed. ‡Percentage harbouring a copy number variation. Abbreviations: ARF, tumour suppressor ARF; Akt, serine/

threonine-protein kinases; B-raf, serine/threonine-protein kinase B-raf; BAX, apoptosis regulator BAX; E2F, transcription

factors of the E2F family; MAPK, mitogen-activated protein kinases; MDM2, E3 ubiquitin-protein ligase Mdm2; MEK, dual

specificity MAPK kinases1 and2; NF-κB, nuclear factor κB; p21, cyclin-dependent kinase inhibitor1; p53, cellular tumour

antigen p53; RPS6, 40S ribosomal protein S6; RTK, receptor tyrosine kinase; STAT, signal transducer and activator of

transcription1 and3; TCGA, The Cancer Genome Atlas Research Network; VEGF, vascular endothelial growth factor.

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or proteins that regulate transcription (for example,

histone deacetylases).77,87–96 Inhibition of these transcrip-

tion factors resulted in tumour growth inhibition in all

studies included in this Review.77,87–93,95,96

Testosterone and the AR might have a role in the

increased incidence of bladder cancer in men compared

with women.97 Hence, surgical castration was explored

as therapy in two animal studies: Overdevest etal.98

observed a reduction in tumour growth of orthotopic

and heterotopic xenografts in mice using this approach,

whereas Shiota etal.92 did not find a significant inhibi-

tion of tumour growth in their heterotopic xenograft

model. Inaddition, two studies that investigated siRNAs

targeting the AR in subcutaneous xenografts observed

statistically significant tumour growth inhibition.93,94

Considering these findings, inhibition of the AR might

be a possible therapeutic strategy for men with bladder

cancer (Table1); however, none of the possible tran-

scription factor targets have been evaluated in clinical

studiesyet.

Cell cycle proteins and apoptosis

Cell division occurs after the cell has gone through the

four cell cycle phases G1, S, G2 and M (Figure1). Cell

cycle checkpoint proteins, which regulate progression

through the cell cycle, are often mutated or bypassed in

malignant cells and are therefore considered potential

targets for therapy. Studies that have investigated target-

ing cell cycle control and apoptosis pathways invivo are

listed in Supplementary Table4 online.

In the Mphase, the accurate formation of the mitotic

spindle is controlled during a quality check. Aurora

kinaseA is a protein that is involved in mitotic spindle

formation and function that was evaluated as a poten-

tial therapeutic target for bladder cancer by Zhou etal.99

(Figure2). The researchers found that aurora kinaseA

expression was increased in clinical bladder cancer

tumour samples and that inhibition of aurora kinaseA

with the inhibitor MLN8237 (also known as alisertib) in

T24 bladder cancer xenografts in mice resulted in sup-

pressed tumour growth. A phaseII clinical trial evalu-

ating alisertib combined with paclitaxel in advanced

bladder cancer was registered in 2014.100

The apoptosis pathway can become activated during

the cell cycle, for example when DNA damage occurs.

In addition, apoptosis can be induced by activation

oftheRas–MAPK pathway and subsequent activation of

the tumour suppressor p53, which is encoded by TP53, a

gene that is often mutated in cancer cells. Mutations that

change an amino acid in the DNA-binding domain of p53

inhibit its function as a transcription factor and, as a con-

sequence, expression of the proapoptotic apoptosis regu-

lator BAX. Deletions of the gene likewise lead to loss of

function.101,102 In one study, CP-31398, a compound that

stabilizes the conformation of the DNA-binding domain

of p53 (Figure2), was administered to SV40T-transgenic

mice.103 The SV40 large Tprotein binds and inhibits

both p53 and the retinoblastoma-associated protein

and thereby induces bladder cancer.104 Administration

of CP-31398 resulted in decreased tumour weight and

inhibition of invasive tumour growth with an increase

in apoptosis.73 In clinical trials, p53-targeted therapy is

being evaluated using ALT-801, a fusion protein of IL-2

and a T-cell receptor domain.105 The efficacy of ALT-801

is being investi gated as a combination treatment with

gemcitabine, with or without cisplatin, in patients

with MIBC and NMIBC.106,107 Patient recruitment is

currentlyongoing.

Bcl-xL (Bcl-2-like protein1 expressed from BCL2L1)

is an antiapoptotic protein of the Bcl2 family. One study

investigated the clinicopathological significance of

Bcl-xL expression in patients with upper urinary tract

urothelial carcinoma (Figure2).108 Using a subcutane-

ous UMUC3 xenograft model in mice, the researchers

evaluated the therapeutic effect of the V-ATPase inhibi-

tor bafilomycinA1 against Bcl-xL and observed reduced

tumour growth with signs of apoptosis (confirmed by

terminal deoxynucleotidyl transferase dUTP nick end

labelling [TUNEL] assay, which is used to detect DNA

fragmentation caused by apoptosis). Another target in

this category is livin, which belongs to the inhibitors of

apoptosis proteins (IAP) family and was shown to predict

disease relapse in bladder cancer.109 Inhibiting livin in

an invivo heterotopic xenograft model led to tumour

inhibition and increased apoptosis.110

Targeting and inducing the apoptosis pathway is an

especially interesting strategy in cancer treatment, as the

occurrence of apoptosis shifts the treatment effect from

cytostatic to cytotoxic. This shift could, in theory, lead to

tumour regression rather than only reduced cell prolif-

eration. However, most of the targeted therapies that are

currently used clinically are cytostatic rather than cyto-

toxic. With the exception of the study investigating the

p53 stabilizer CP-31398,103 the number of animals used

in experiments in this category of targets is small and

none of the studies have been replicated to date.

Protein synthesis and cell metabolism

Protein synthesis

During the G1 phase of the cell cycle, many proteins

that are required for subsequent cell division are being

synthesized (translation). As the step of protein trans-

lation is essential to cell proliferation, the participat-

ing proteins are potential targets for anticancer agents.

Supplementary Table4 online provides an overview of

the relevant invivo studies.

Eukaryotic translation initiation factor3B (eIF3b) is a

subunit of the translation initiation factor complex eIF3.

Immunohistochemical assessment of 143bladder cancer

samples showed that eIF3b overexpression was corre-

lated with a high tumour stage and short disease-specific

survival in humans.111 The researchers then used siRNAs

to inhibit eIF3b formation in UMUC3 cells, which

resulted in reduced tumour growth of s ubcutaneous

xenograftsinmice.111

Some bacterial toxins target the function of proteins

during translation. One group used diphtheria toxin

subunitA to inhibit translation of elongation factor2.112,113

Expression of the toxin was controlled by the promoter of

the insulin-like growth factor2 gene (IGF2) and/or the

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promoter of the long noncoding RNA gene H19. The

products of IGF2 and H19 are highly expressed in many

tumours but not in normal cells. In two studies using

orthotopic T24 human bladder cancer xenograft models

in mice, treatment inhibited tumour growth, result-

ing in tumours that were 86% and 83% smaller than

incontrols.112,113

Targeting the translational process has also been

investi gated in other types of cancer, for example by tar-

geting eukaryotic translation initiation factor 4E (eIF4e).

After multiple promising preclinical studies, a phaseI

trial114 using an antisense oligonucleotide to block expres-

sion of eIF4e was completed in 2010.115–118 Patients with

stage4 cancer for which no proven therapy existed were

included, regardless of pathological diagnosis. Included

cancer types were mesothelioma, malignant melanoma,

non-small-cell lung carcinoma and several others. The

expression levels of eIF4e in tumour biopsy samples taken

after treatment were lower than those in pre-treatment

samples. Unfortunately, the study did not show any clini-

cal response, which again demonstrates the difficulty of

translating preclinical results to the bedside.118

Cell metabolism

In cancer, cell metabolism is often unbalanced.

Metabolism and catabolism are mediated and controlled

by a multitude of different enzymes, which are there-

fore other potential targets for bladder cancer therapy.

Invivo studies that investigated this strategy are listed

in Supplementary Table4 online. For example, stearoyl-

CoA desaturase1 is a rate-limiting enzyme in fatty acid

desaturation that was targeted by siRNA knockdown or

by an oral small-molecule inhibitor, both combined with

doxycycline therapy. Both strategies led to a significant

reduction in tumour growth in subcutaneous bladder

cancer cell xenografts in mice.119

Choline kinaseα (ChoKα) is a metabolic enzyme

responsible for the generation of phosphorylcholine

(the hydrophilic polar head group of phospholipids).120

Inhibition of this enzyme slowed tumour growth in both

subcutaneous as well as orthotopic xenografts in mice

and prolonged animal survival in the orthotopic mouse

model from a median of 34days to 42days.120 ChoKα is

also overexpressed in lung, breast, prostate, colon, ovary

and haematological malignancies.121 In 2014, a phaseI

trial testing a ChoKα inhibitor in patients with solid

tumours, including bladder cancer, finalized recruitment

but results have not been published yet.122

Two other studies have tested two other drugs that

target metabolic pathways.123,124 Treatment with a com-

bination of α-lipoic acid, which inhibits glycolysis, and

hydroxycitrate, which inhibits lipid synthesis, was able

to improve survival of mice with MBT-2 bladder tumour

xenografts. Addition of octreotide or cisplatin boosted

survival evenfurther.123,124

Overall, targeting protein translation seems to be a

promising strategy for the treatment of bladder cancer,

even though the study targeting eIF4e showed the dif-

ficulty of reproducing favourable preclinical results in a

phaseI clinical trial.

Metastasis, immune system and others

Other processes that involve potential molecular targets

for the treatment of patients with bladder cancer include

the formation of metastases and the immune response.

In addition, novel treatment strategies, such as oncolytic

viruses, are used to exploit tumour-cell-specific molecu-

lar mutations. A summary of studies investigating these

strategies is provided in Supplementary Table5 online.

Potential targets include proteins that interact with the

extracellular matrix, for example urokinase-type plas-

minogen activator (uPA), plasminogen activator inhibi-

tor1 (PAI-1) and versican core protein. These targets

are thought to be important in reducing the metastatic

potential of bladder cancer cells and, hence, might

be employed in preventive treatments.125–127 Another

promising target is the CD antigen CD24, a cell surface

protein and cancer stem cell marker that is involved in

metastatic progression in many cancers. In three pre-

clinical studies, one group showed the relevance of CD24

in bladder cancer development and metastasis.95,98,128

Inhibition of CD24 with an antibody or siRNAs reduced

tumour growth in mice with subcutaneous xenografts and

reduced metastatic load in mice inoculated with tumour

cells via their tail vein.95,98,128 Also, fewer tumours devel-

oped in CD24-knockout animals upon administration of

N-butyl-N-(4-hydroxybutyl)-nitrosamine, which induces

bladder tumours.98 Furthermore, the researchers found

that androgens stimulated CD24 expression in tumour

tissue and surgical castration of male mice inhibited

tumour growth in subcutaneous xenografts.98 As inhibi-

tion of CD24 reduced tumour growth in multiple studies,

we consider this strategy promising as a targeted therapy

in bladder cancer (Table1).

Stimulation of the immune system is another way to

target cancer cells. If the immune system is able to rec-

ognize tumour cells as foreign, the immune response

might destroy the malignant cells.129 One approach

investigated the use of IL-12 in the treatment of bladder

cancer, using transfection of EJ bladder cancer cells with a

plasmid encoding IL-12 to force secretion of IL-12.130 The

hypothesis was that secretion of IL-12 would stimulate

Tlympho cytes and natural killer cells, which should result

in increased tumour cell killing. Mice were injected sub-

cutaneously with nontransfected EJ cells or transfected EJ

cells.130 In addition, some mice were treated with injections

of pirarubicin, a doxorubicin analogue. Combination treat-

ment resulted in significantly smaller tumours compared

with single treatment or no treatment. Although these

results are positive, the need to introduce a gene into a high

percentage of tumour cells in a solid tumour makes this

approach unlikely to be translated into clinical application.

Other bladder cancer treatment strategies targeting the

immune system are currently under investigation in clini-

cal trials.131 These agents have so far not been analysed in

animal models of bladder cancer, but have proven their

efficacy in clinical trials in other types of cancer. Immune

checkpoint inhibitors, such as MPDL3280A (atezoli-

zumab), pembrolizumab, ipilimumab and nivolumab,

are examples of such promising therapies targeting the

immune system. MPDL3280A, an antibody against

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programmed cell death1 ligand1 (PD-L1), was investi-

gated as a single-agent, second-line therapy in a phaseI

trial in 68 patients with bladder cancer.132 The objective

response rate was 43% in patients with tumours positive for

PD-L1 expression upon immunohistochemical analysis.

Reported overall toxicity was low. In 2014, the FDA granted

MPDL3280A the breakthrough status for bladder cancer,

which will expedite the development and review of the

drug by the FDA.133,134 Many clinical trials of MPDL3280A

are currently ongoing and results arepending.135–142

Finally, three invivo studies employed oncolytic adeno-

viruses.130,143,144 The viruses were genetically modified

in such a way that they specifically attach to a tumour-

associated membrane protein or the extracellular matrix

and then enter and lyse the tumour cells without damag-

ing normal cells. Although this strategy leads to a reduc-

tion of tumour growth, the infection efficiency of the

used viruses is probably not high enough for all tumour

cells to be targeted.145 To achieve extensive effectiveness,

infection should not only result in direct tumour cell

killing, but also antigen release and immune stimula-

tion.146 Thus, as oncolytic viruses have shown suboptimal

potency as monotherapies, combination treatments with,

for example, immune check point inhibitors or immune-

stimulatory molecules should be considered.146,147 For

example, in 2012, a phaseI trial investigating the efficacy

of intravesical administration of an oncolytic adenovirus

expressing granulocyte–macrophage colony-stimulating

factor demonstrated tolerable safety and a complete

response rate of 48.6%.148 However, at the moment, we do

not consider treatment strategies using oncolytic viruses

ready for implementation into the clinic.

Conclusions

Although targeted therapies have proven effective in

other cancer types, none of the registered therapies have

been approved for the treatment of bladder cancer. The

small number of clinical trials that included patients

with this disease had limited success.12 With this Review,

which explores all animal studies investigating targeted

therapeutic interventions for bladder cancer that were

published since 2009, we aimed to provide a current,

comprehensive overview of this exciting field of research.

We found several animal studies that reported tumour

growth inhibition or reduced angiogenesis and were

replicated by different groups or validated by the same

group in separate studies (Table1). Inhibition of FGFR3

seemed very successful using the small-molecule inhibi-

tor PD173074,48,51,52,54 as well as via the monoclonal anti-

body R3Mab49,50 and in combination with inhibition of

EGFR.54 These treatments resulted in tumour growth

inhibition, reduced angiogenesis and increased apoptosis.

Inhibition of EGFR alone using erlotinib was also success-

ful, resulting in tumour growth inhibition in some hetero-

topic xenografts and prolonged survival of mice with

orthotopic xenografts.29 However, the results of clinical

trials investigating therapeutic targeting of FGFR3 and

EGFR have been disappointing,12 most likely owing to the

lack of patient stratification according to the downstream

mutations in the pathways of these receptors.

Inhibition of angiogenesis through blocking VEGF

or its receptor reduced tumour growth in animal

models.30,59–61,149,150 Similarly, inhibition of the cancer stem

cell marker CD24 inhibited tumour growth,95,98,128 which

was also the case for treatments targeting mTOR and the

PI3K–Akt pathway,75–77 S TAT3 77,90,151 and the AR.92–95 Most

experiments showed a cytostatic treatment effect, which

suggests that tumour growth probably resumes when

treatment is stopped. Combining targeted treatment with

chemo therapy usually enhanced the therapeutic effi-

cacy. In clinical trials, combination treatments have been

attempted but have not demonstrated an improvement

in overall survival and sometimes resulted in increased

t oxicity in patients.12

EGFR inhibitors and antibodies have proven to be

effective as targeted therapies in humans with non-small-

cell lung cancer and colorectal cancer.25,26 This work dem-

onstrates that activating mutations in the Ras–MAPK and

PI3K–Akt pathways downstream of EGFR render the

tumour resistant to EGFR inhibition. We have identified

four studies on EGFR inhibition in human bladder cancer

cell line xenografts that had such mutations in one or both

pathways.27,28,30,31 In another study, the researchers care-

fully screened the EGFR downstream pathways of the cell

lines before attempting to target EGFR.29

One limitation of the studies discussed in this Review

is the use of cell lines to create xenografts. Cell lines

might not reflect the entire spectrum of tumour types,

especially because relatively few bladder cancer cell lines

are available. In addition, cell-line-derived tumours

probably lack the tumour heterogeneity observed in

most cancers. Thus, xenografts established directly from

resected tumours might present a better model than

cell lines. Such tumour models also have human stroma

and engage murine stromal cells, which is likely to be

important in cell–cell interactions and cell prolifera-

tion.18 Furthermore, small-molecule inhibitors, inhibi-

tion of cell surface proteins by monoclonal antibodies

and antibodies against ligands of cell surface receptors

have proven their efficacy in the clinic. Inhibition or

knockdown of targets using small, modified RNAs (for

example, siRNAs), transfection of genes and the use of

viral vehicles are efficient invitro but their effectiveness

in delivery is still not good enough for systemic treatment

of tumours and metastases.

Overall, our Review shows that a multitude of differ-

ent targets has been investigated and that most of the

studies have not been replicated to date. Often, relatively

few animals were used to register an effect. In addi-

tion, almost all studies reported inhibition of tumour

growth but not increased apoptosis. Also, only five

studies reported that the investigated treatment had no

effect.53,60,96,125,152 This small number suggests a possible

publication bias. In some papers, the number of treated

animals was not reported. In2010, the ARRIVE (animals

in research: reporting invivo experiments) guidelines—

developed to improve the design, analysis and report-

ing of animal experiments—were published.153,154 We

suggest that s cientists and s cientific journals adhere to

these guidelines in future studies.

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Furthermore, bladder cancer research is consistently

underfunded, considering the number of life years lost

and the costs to health-care systems generated by patients

with bladder cancer.155 This lack of funding is the major

reason why research on novel therapies in bladder cancer

lags behind that in other tumour types. The community

of bladder cancer researchers has to invest in reaching out

to the different policy makers and grant organizations to

improve funding.

In conclusion, EGFR, FGFR-3, VEGF, mTOR, STAT3,

the AR and CD24 represent promising and validated

targets for the inhibition of bladder cancer growth in

animal studies. Treatment strategies that combine differ-

ent modalities and/or targets seem to be most promising

and clinical studies should continue to pursue these find-

ings. Careful patient selection in these clinical studies will

be essential in order to demonstrate true benefit. Many of

the other targets that are under preclinical investigation

still need further validation.

Review criteria

The Embase, MEDLINE, OvidSP, Web of Science,

PubMed, Cochrane CENTRAL and Google Scholar

databases were systematically searched (date of

search: December 2014). The full search strategy

conducted in Embase was: (‘molecularly targeted

therapy’/de OR ‘molecular therapy’/exp OR (target*

NEAR/3 (molecul* OR therap* OR antibod* OR inhibit*

OR mirna* OR microrna* OR treat* OR intervent*OR

chemo*)):ab,ti) AND (‘bladder tumor’/exp OR ‘transitional

cell carcinoma’/de OR (‘carcinoma’/de AND ‘bladder’/

de) OR ((bladder OR urothel*) NEAR/3 (tumo* OR

cancer* OR neoplas* OR carcin* OR metasta*

ORtransitional)):ab,ti). After the literature search was

completed, all reviews, expert opinions, editorials,

seminar articles, congress abstracts, articles published

before 2009 and records in languages other than

English or Dutch were excluded. In total, 93 published

animal studies of targeting molecular targets in bladder

cancer were eligible for review.

1. Kompier, L.C. etal. The development of multiple

bladder tumour recurrences in relation to the

FGFR3 mutation status of the primary tumour.

J.Pathol. 218, 104–112 (2009).

2. Babjuk, M. etal. EAU guidelines on non-

muscle-invasive urothelial carcinoma of the

bladder: update 2013. Eur. Urol. 64, 639–653

(2013).

3. Kiemeney, L.A., Witjes, J.A., Heijbroek, R.P.,

Verbeek, A.L. & Debruyne, F.M. Predictability of

recurrent and progressive disease in individual

patients with primary superficial bladder cancer.

J. Urol. 150, 60–64 (1993).

4. Sylvester, R.J. etal. Predicting recurrence and

progression in individual patients with stage Ta

T1 bladder cancer using EORTC risk tables:

acombined analysis of 2596 patients from

seven EORTC trials. Eur. Urol. 49, 466–477

(2006).

5. Surveillance, Epidemiology, and End Results

Program (SEER). seer.cancer.gov [online], http://

seer.cancer.gov/statfacts/html/urinb.html

(2015).

6. Ploussard, G. etal. Conditional survival after

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Acknowledgements

We would like to acknowledge W.M. Bramer for his

contribution to the literature search and database

management. This study was funded by an Erasmus

MC 2012 grant.

Author contributions

E.C.Z., K.E.M.v.K., T.C.M.Z. and A.R.A. researched

data for the article. E.C.Z., K.E.M.v.K., T.C.M.Z. and

J.L.B. substantially contributed to discussion of the

content. E.C.Z., K.E.M.v.K. and J.L.B. wrote article.

Allauthors contributed to review and editing of

themanuscript before submission.

Supplementary information is linked to the online

version of the paper at www.nature.com/nrurol.

REVIEWS

RNA-seq reveals novel mechanistic targets of Livin in bladder cancer

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Feb 2023
BMC Urol

Background Bladder cancer is a very common malignancy with a high recurrence rate. The survival of patients with muscle-invasive bladder cancer is poor, and new therapies are needed. Livin has been reported to be upregulated in bladder cancer and influence the proliferation of cancer cells. Materials and methods The Livin gene in human bladder cancer cell line T24 was knocked out, and the differentially expressed genes were identified by RNA-seq and qPCR. Results Livin knockdown affects gene expression and has strong negative effects on some cancer-promoting pathways. Furthermore, combined with bladder cancer clinical sample data downloaded from TCGA and GEO, 2 co-up-regulated genes and 58 co-down-regulated genes were identified and validated, which were associated with cancer proliferation and invasion. Conclusion All these results suggest that Livin plays an important role in bladder cancer and could be a potential anticancer target in clinical therapy.

The Expression and Prognostic Significance of P3H1 in Bladder Cancer: A Bioinformatics Analysis and In Vitro Experiments

Preprint

Full-text available

Jan 2024

Background Bladder cancer (BLCA) is associated with poor prognosis and the identification of new prognostic markers is needed. This study aimed to investigate the significance of Prolyl 3-hydroxylase 1 (P3H1) as a potential prognostic biomarker for BLCA. Methods The expression levels of P3H1 in BLCA and normal bladder tissues were analyzed using TCGA database, and P3H1's prognostic value was determined using Kaplan-Meier survival analysis. GSEA analysis was performed to investigate its biological functions. Assessment of the tumor microenvironment was conducted using the CIBERSORT algorithm, and the results were verified through RT-PCR and western blotting. Functional analysis of P3H1 was carried out using Colony-formation, CCK-8, wound healing, transwell, and flow cytometry assays. Results The study found that P3H1 was upregulated in BLCA tissues, and elevated expression was linked to an unfavorable prognosis. GSEA analysis revealed putative involvement of P3H1 in the JAK-STAT and WNT signaling pathways. P3H1 expression was also strongly associated with the infiltration of diverse immune cells and immune checkpoints. Conclusion Finally, knocking down P3H1 expression effectively suppressed the ability of BLCA cells to proliferate and migrate, while promoting apoptosis. In conclusion, P3H1 has the potential to serve as a prognostic biomarker for BLCA.

Phase II trial of afatinib in patients with advanced urothelial carcinoma with genetic alterations in ERBB1-3 (LUX-Bladder 1)

Article

Dec 2023

Background: Preclinical and early clinical data suggest that the irreversible ErbB family blocker afatinib may be effective in urothelial cancers harbouring ERBB mutations. Methods: This open-label, phase II, single-arm trial (LUX-Bladder 1, NCT02780687) assessed the efficacy and safety of second-line afatinib 40 mg/d in patients with metastatic urothelial carcinoma with ERBB1-3 alterations. The primary endpoint was 6-month progression-free survival rate (PFS6) (cohort A); other endpoints included ORR, PFS, OS, DCR and safety (cohorts A and B). Cohort A was planned to have two stages: stage 2 enrolment was based on observed antitumour activity. Results: Thirty-four patients were enroled into cohort A and eight into cohort B. In cohorts A/B, PFS6 was 11.8%/12.5%, ORR was 5.9%/12.5%, DCR was 50.0%/25.0%, median PFS was 9.8/7.8 weeks and median OS was 30.1/29.6 weeks. Three patients (two ERBB2-amplified [cohort A]; one EGFR-amplified [cohort B]) achieved partial responses. Stage 2 for cohort A did not proceed. All patients experienced adverse events (AEs), most commonly (any/grade 3) diarrhoea (76.2%/9.5%). Two patients (4.8%) discontinued due to AEs and one fatal AE was observed (acute coronary syndrome; not considered treatment-related). Conclusions: An exploratory biomarker analysis suggested that basal-squamous tumours and ERBB2 amplification were associated with superior response to afatinib. Clinical trial registration: NCT02780687.

PHB promotes bladder cancer cell epithelial-mesenchymal transition via the Wnt/β-catenin signaling pathway

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May 2023
Pathol Res Pract

As a member of PHB (prohibitin1) family, PHB plays important roles in many cancers, but its property in bladder carcinoma aggressiveness is unknown. This research was to explore the function and potential mechanism of PHB in bladder carcinoma in vivo and in vitro. The invasive abilities of cancer cell were determined by transwell and wound-healing assays. The function of PHB was confirmed by gene knockdown and overexpression methods. Further in vivo confirmation was performed in a nude mouse model with lung metastasis. The relationship of PHB and β-catenin was confirmed by immunoprecipitation and immunofluorescence staining assays. The protein expression of epithelial-mescenchymal transition (EMT) and Wnt/β-catenin signaling pathway was tested by immunofluorescence staining and western blotting assay. The depletion of PHB prevented bladder cancer cell invasiveness and inhibited EMT. Contrarily，the abilities of bladder carcinoma cells migration and invasion in vitro as well as metastasis in vivo were enhanced when the PHB overexpressed unnormally. Importantly, the β-catenin was identified to be bound by PHB and β-catenin knockdown reduced the cancer cell migration, invasion and EMT in PHB overexpressing cells. In addition, PHB stabilized β-catenin by inhibiting its ubiqutin-mediated degradation thus leading to increased Wnt/β-catenin signaling. These observations indicate that PHB could promote bladder cancer aggressiveness by binding with β-catenin to prevent the degradation of β-catenin and the localized invasive bladder cancer patients with PHB overexpression should take more aggressive postsurgical adjuvant anticancer therapies.

Urinary exosomes: Diagnostic impact with a bioinformatic approach

Chapter

Nov 2022
ADV CLIN CHEM

Exosomes are tiny membrane-enveloped vesicles of endosomal origin, typically 40–120nm in diameter, produced by most cells in both normal and pathological situations. These exosomes can be isolated from all biofluids, including urine. In this context, many researchers have focused on the analysis of urinary exosomes because urine can be collected in large quantities, regularly, and with minimal effort. Exosomes contain phospholipids, cholesterol, proteins, glycoconjugates, nucleic acids, and metabolites. Because all organs and tissues produce exosomes, their molecular cargo can provide first-hand information about the physiological and biological state of the site of origin. Many potential disease biomarker candidates have already been identified in urinary exosomes. In this chapter, we performed a bibliometric analysis of the keywords “exosome(s)” and “urine” to identify related terms, diseases and molecular/biological processes, and other related terms. This yielded interesting results suggesting that exosomes in urine may play a role in the pathogenesis of various diseases. Moreover, this chapter discusses exosomes isolation and characterization methodologies and highlight the importance of urinary exosomes and their role in the diagnosis, prognosis and therapy of various diseases. We offer a bibliometric approach and an in-depth analysis on several exosomes' isolation techniques, diagnostic potential for urogenital and specific non-urogenital diseases, as well as an overview of miRNAs significance on urinary exosomes, conferring a more complete status to this review, something that was still lacking in the current literature.

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Oct 2022

Bladder cancer (BC) recurrence is one of the primary clinical problems encountered by patients following chemotherapy. However, the mechanisms underlying their resistance to chemotherapy remain unclear. Alteration in the pattern of membrane proteins (MPs) is thought to be associated with this recurrence outcome, often leading to cell dysfunction. Since MPs are found throughout the cell membrane, they have become the focus of attention for cancer diagnosis and treatment. Identifying specific and sensitive biomarkers for BC, therefore, requires a major collaborative effort. This review describes studies on membrane proteins as potential biomarkers to facilitate personalised medicine. It aims to introduce and discuss the types and significant functions of membrane proteins as potential biomarkers for future medicine. Other types of biomarkers such as DNA-, RNA- or metabolite-based biomarkers are not included in this review, but the focus is mainly on cell membrane surface protein-based biomarkers.

Photoactivatable Targeting Methods

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Dec 2019

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Jul 2023
COORDIN CHEM REV

Risk Classification of Bladder Cancer by Gene Expression and Molecular Subtype

Article

Full-text available

Apr 2023

Simple Summary Our study used NanoString technology, a high-throughput platform measuring gene expression at the mRNA level to identify a set of genes predictive of clinical outcomes in bladder cancer patients. Twenty-seven differentially expressed genes were correlated with clinicopathological variables including molecular subtypes (luminal, basal, null/double-negative), histological subtypes (conventional urothelial carcinoma or carcinoma with variant histology), clinical subtype (NMIBC and MIBC), tumor stage category (Ta, T1 and T2-4), tumor grade, PD-L1 expression (high vs. low expression), and clinical risk categories (low, intermediate, high, and very high). Then, two risk models integrating the molecular subtypes and the level of expression of TP53, CCND1 and MKI67 were developed. These models provided a score ranging from 0 (best prognosis) to 7 (worst prognosis) that could be used to predict patient’ outcome and guide treatment decisions in bladder cancer. Abstract This study evaluated a panel including the molecular taxonomy subtype and the expression of 27 genes as a diagnostic tool to stratify bladder cancer patients at risk of aggressive behavior, using a well-characterized series of non-muscle invasive bladder cancer (NMIBC) as well as muscle-invasive bladder cancer (MIBC). The study was conducted using the novel NanoString nCounter gene expression analysis. This technology allowed us to identify the molecular subtype and to analyze the gene expression of 27 bladder-cancer-related genes selected through a recent literature search. The differential gene expression was correlated with clinicopathological variables, such as the molecular subtypes (luminal, basal, null/double negative), histological subtype (conventional urothelial carcinoma, or carcinoma with variant histology), clinical subtype (NMIBC and MIBC), tumor stage category (Ta, T1, and T2–4), tumor grade, PD-L1 expression (high vs. low expression), and clinical risk categories (low, intermediate, high and very high). The multivariate analysis of the 19 genes significant for cancer-specific survival in our cohort study series identified TP53 (p = 0.0001), CCND1 (p = 0.0001), MKI67 (p < 0.0001), and molecular subtype (p = 0.005) as independent predictors. A scoring system based on the molecular subtype and the gene expression signature of TP53, CCND1, or MKI67 was used for risk assessment. A score ranging from 0 (best prognosis) to 7 (worst prognosis) was obtained and used to stratify our patients into two (low [score 0–2] vs. high [score 3–7], model A) or three (low [score 0–2] vs. intermediate [score 3–4] vs. high [score 5–7], model B) risk categories with different survival characteristics. Mean cancer-specific survival was longer (122 + 2.7 months) in low-risk than intermediate-risk (79.4 + 9.4 months) or high-risk (6.2 + 0.9 months) categories (p < 0.0001; model A); and was longer (122 + 2.7 months) in low-risk than high-risk (58 + 8.3 months) (p < 0.0001; model B). In conclusion, the molecular risk assessment model, as reported here, might be used better to select the appropriate management for patients with bladder cancer.

RNA-seq reveals novel mechanistic targets of Livin in bladder cancer

Preprint

Full-text available

Oct 2022

Background Bladder cancer is a very common malignancy with a high recurrence rate. The survival of patients with muscle-invasive bladder cancer is poor, and new therapies are needed. Livin has been reported to be upregulated in bladder cancer and influence the proliferation of cancer cells. Materials & Methods The Livin gene in human bladder cancer cell line T24 was knocked out, and the differentially expressed genes were identified by RNA-seq and qPCR. Results Livin knockdown affects gene expression and has strong negative effects on some cancer-promoting pathways. Furthermore, combined with bladder cancer clinical sample data downloaded from TCGA and GEO, 2 co-up-regulated genes and 28 co-down-regulated genes were identified and validated, which were associated with cancer proliferation and invasion. Conclusion All these results suggest that Livin plays an important role in bladder cancer and could be a potential anticancer target in clinical therapy.

Thrombomodulin expression regulates tumorigenesis in bladder cancer

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Jun 2015

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Jun 2010

Combined use of anti-VEGF and Anti-EGFR monoclonal antibodies with Photodynamic therapy suppresses tumor growth in an in vivo tumor model

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Feb 2013

Photodynamic therapy (PDT) is a promising treatment option for only cancer. It can be used to treat drugresistant and inoperable tumors. However, one of the unsolved problems in PDT is tumor recurrence. The aim of this study is to suppress tumor regrowth by including angiogenesis inhibitors that target vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) pathways, to the PDT protocol. Human bladder cancer (MGH cell line) xenografts were induced in Balb/c nude mice. The animals treated with PDT received an intravenous injection of hypericin followed by irradiation after 6 hours, with a broadband light source through a 560-640 NM band-pass filter. A light dosage of 120 J/cm2 and 100 mW/cm2 was administered. Angiogenesis inhibitors, bevacizumab and cetuximab were administered at a dose of 10 mg/kg. Treatment efficacy was assessed by monitoring the tumor growth inhibition of xenograft tumors. Immunohistochemistry was performed to evaluate the expression of VEGF and EGFR. Expression of the major proteins in the VEGF and EGFR pathways was investigated by immunoblotting. One-way ANOVA with Bonferroni correction was performed to analyze the data. Tumors treated with the combination of PDT and inhibitors exhibited significantly greater treatment response compared to control and PDT groups. Downregulation of VEGF and EGFR was observed in tumors treated with PDT + bevacizumab and cetuximab. Our results show that blocking VEGF or/and EGFR pathways along with PDT can effectively suppress tumor growth.

Patient-derived bladder cancer xenografts in the preclinical development of novel targeted therapies

Article

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May 2015

Optimal animal models of muscle invasive bladder cancer (MIBC) are necessary to overcome the current lack of novel targeted therapies for this malignancy. Here we report on the establishment and characterization of patient-derived primary xenografts (PDX). Patient tumors were grafted under the renal capsule of mice and subsequently transplanted over multiple generations. Patient tumor and PDX were processed for analysis of copy number variations by aCGH, gene expression by microarray, and expression of target pathways by immunohistochemistry (IHC). One PDX harbouring an FGFR3 mutation was treated with an inhibitory monoclonal antibody targeting FGFR3. Five PDX were successfully established. Tumor doubling time ranged from 5 to 11 days. Array CGH revealed shared chromosomal aberrations in the patient tumors and PDX. Gene expression microarray and IHC confirmed that PDXs maintain similar patterns to the parental tumors. Tumor growth in the PDX with an FGFR3 mutation was inhibited by the FGFR3 inhibitor. PDXs recapitulate the tumor biology of the patients' primary tumors from which they are derived. Investigations related to tumor biology and drug testing in these models are therefore more likely to be relevant to the disease state in patients. They represent a valuable tool for developing precision therapy in MIBC.

Predictability of Recurrent and Progressive Disease in Individual Patients with Primary Superficial Bladder Cancer

Article

Jul 1993

The ultimate goal of prognostic assessment is optimization of individual counseling. Often, however, studies on prognostic factors focus on discriminating between high risk and low risk subgroups without considering the relevance of 1 or more factors for predicting disease outcome in individual patients. We quantified the accuracy of prediction of future recurrences and disease progression in individual patients with primary superficial bladder cancer. The study cohort consisted of 1,674 patients who were followed prospectively between 1983 and 1991 in the Netherlands. By analyzing half of the patients with proportional hazards regression, we computed relative risks of recurrence and progression. A prognostic index score based on these relative risks was then applied to the other half of the patients to determine whether group outcome could be predicted accurately. To assess the accuracy of prediction in individuals we used a method similar to the construction of receiver operating characteristic curves in diagnostic test assessment.

Phase II trial of neoadjuvant cisplatin, gemcitabine, and bevacizumab followed by radical cystectomy (RC) in patients with muscle-invasive transitional cell carcinoma (TCC) of the bladder.

Article

Mar 2011

276 Background: Cisplatin-based neoadjuvant chemotherapy has shown survival advantage in patients (pts) with muscle-invasive bladder cancer. This benefit is strongly associated with downstaging of tumor to pT0. It was hypothesized that the addition of antiangiogenic therapy bevacizumab (Avastin [A]) to cisplatin (C) and gemcitabine (G) chemotherapy may offer improved outcomes in these pts. Methods: Patients with clinically localized, muscle-invasive, chemotherapy-naive TCC of bladder (T2-T4a), ECOG PS 0-1 received cisplatin 70 mg/m ² IV day 1, gemcitabine 1000 mg/m ² IV day 1 and 8, bevacizumab 15 mg/kg IV day 1 on a 21- day cycle for up to 4 cycles. After completion of neoadjuvant therapy all pts underwent RC. Those pts not achieving pT0 status received paclitaxel 175 mg/m2 IV day 1 and bevacizumab 15 mg/m ² IV day 1 on a 21-day cycle for up to 3 cycles. The primary objective of the trial was to achieve pT0 status. Results: By October 2010, 15 pts were enrolled, and 13 are evaluable for response. Median age was 64 (range 47-71), 9 (70%) males, 4 (30%) females. 2/13 (15%) developed PD after neo-adjuvant therapy and were taken off protocol.11/13 (85%) underwent RC. 4/13 (31%) pts were downstaged, 3/11 (27%) achieved pTis status and none achieved pT0 status. 2/11 (18%) pts received adjuvant paclitaxel chemotherapy with bevacizumab. Dose reduction or delays were noted in 12/15 (80%) pts. Grade 3 or 4 hematologic toxicity was observed in 5/15 (33%) pts. Grade 3 or 4 nonhematologic toxicity was observed in 3/15 (20%) pts: (renal dysfunction 2 pts, pulmonary embolism 1 pt). Postoperative complications were seen in 5/12 (42%) pts: enterovesicular fistula (1), delayed wound healing (1), prolonged ileus (2), and pelvic abscess (1). With a median follow-up time of 12 months (range 3-45), 8/11 (72%) pts have remained disease free. Conclusions: Neoadjuvant CGA is feasible in pts with clinically localized, muscle-invasive TCC of the bladder. The surgical complications rate appears rather high and close monitoring is needed. Accrual to the trial continuous and updated results will be presented. No significant financial relationships to disclose.

GLOBOCAN 2012 v1.0, cancer incidence and mortality worldwide: IARC cancer base no. 11 [Internet]. International Agency for Research on Cancer, Lyon

Article

Jan 2014

EAU Guidelines on Non–Muscle-invasive Urothelial Carcinoma of the Bladder: Update 2016

Article

Jun 2016

Context: The European Association of Urology (EAU) panel on Non-muscle-invasive Bladder Cancer (NMIBC) released an updated version of the guidelines on Non-muscle-invasive Bladder Cancer. Objective: To present the 2016 EAU guidelines on NMIBC. Evidence acquisition: A broad and comprehensive scoping exercise covering all areas of the NMIBC guidelines published between April 1, 2014, and May 31, 2015, was performed. Databases covered by the search included Medline, Embase, and the Cochrane Libraries. Previous guidelines were updated, and levels of evidence and grades of recommendation were assigned. Evidence synthesis: Tumours staged as TaT1 or carcinoma in situ (CIS) are grouped as NMIBC. Diagnosis depends on cystoscopy and histologic evaluation of the tissue obtained by transurethral resection of the bladder (TURB) in papillary tumours or by multiple bladder biopsies in CIS. In papillary lesions, a complete TURB is essential for the patient's prognosis. If the initial resection is incomplete, there is no muscle in the specimen, or a high-grade or T1 tumour is detected, a second TURB should be performed within 2-6 wk. The risks of both recurrence and progression may be estimated for individual patients using the European Organisation for Research and Treatment of Cancer (EORTC) scoring system and risk tables. The stratification of patients into low-, intermediate-, and high-risk groups is pivotal to recommending adjuvant treatment. For patients with a low-risk tumour and intermediate-risk patients at a lower risk of recurrence, one immediate instillation of chemotherapy is recommended. Patients with an intermediate-risk tumour should receive 1 yr of full-dose bacillus Calmette-Guérin (BCG) intravesical immunotherapy or instillations of chemotherapy for a maximum of 1 yr. In patients with high-risk tumours, full-dose intravesical BCG for 1-3 yr is indicated. In patients at highest risk of tumour progression, immediate radical cystectomy (RC) should be considered. RC is recommended in BCG-refractory tumours. The long version of the guidelines is available at the EAU Web site (www.uroweb.org/guidelines). Conclusions: These abridged EAU guidelines present updated information on the diagnosis and treatment of NMIBC for incorporation into clinical practice. Patient summary: The European Association of Urology has released updated guidelines on Non-muscle-invasive Bladder Cancer (NMIBC). Stratification of patients into low-, intermediate-, and high-risk groups is essential for decisions about adjuvant intravesical instillations. Risk tables can be used to estimate risks of recurrence and progression. Radical cystectomy should be considered only in case of failure of instillations or in NMIBC with the highest risk of progression.

Neoadjuvant chemotherapy with DD-MVAC and bevacizumab in high-risk urothelial cancer: Results from a phase II trial at the M. D. Anderson Cancer Center

Article

Feb 2012

261 Background: DD-MVAC has shown an improved 5-year survival in metastatic urothelial cancer; however, there is no prospective data on its use in the neoadjuvant setting. Previous work suggested that over-expression of VEGF was associated with a high risk of relapse in our neoadjuvant patients, leading to the hypothesis that combining a VEGFR inhibitor with chemotherapy may improve patient outcomes. Methods: Between 8/07 and 12/10, 60 patients with urothelial carcinoma of the bladder or upper tract tumor were enrolled on a prospective phase II clinical trial. Eligibility requirements included at least one of the following: 3-D mass on EUA (cT3b disease), LVI, hydronephrosis, micropapillary features, tumor in a diverticula, and for upper tract tumors a high grade tumor or radiographically measurable sessile mass. The primary endpoint was pathologic down-staging to <=pT1N0M0. Results: Forty-four patients with bladder/urethral tumors and 16 patients with upper tract tumors were enrolled. Pathologic down-staging to <= pT1N0M0 occurred in 53% of patients overall (bladder/urethra 45%, upper tract 75%), and to <= pT0N0M0 in 38% overall (bladder/urethra 39%, upper tract 38%). At a median follow-up of 21 months, the 2-year OS and DSS was 78% and 85%, respectively (bladder 2-yr OS and DSS 75%, 82%; upper tract 93%, 93%). The median OS and DSS have not yet been reached. The most common grade 3 or greater toxicity was neutropenia in 27% of patients, followed by fatigue in 10%. The following grade 3 toxicities were observed in < 10% of patients: mucositis, DVT/PE, hypertension, nausea/vomiting, thrombocytopenia. One patient experienced cardiac ischemia. Conclusions: Neoadjuvant chemotherapy leads to pathologic down-staging in 45% of patients with bladder cancer. DD-MVAC appears an acceptable alternative to traditional M-VAC in the neoadjuvant setting. Although bevacizumab did not impact down-staging based upon historical expectations, determining the effect on recurrence requires longer follow-up.

From virotherapy to oncolytic immunotherapy: Where are we now?

Article

Jun 2015

Robert Coffin

Viruses have been suggested to be useful as anti-cancer agents since the early 20th century, although following the advent of chemotherapy and radiotherapy work largely stopped until the 1990s when a number of groups began to explore the use of engineered viruses. This overview summarizes the development of the field from the 1990s to the present day, an era when oncolytic viruses have now demonstrated clear clinical benefit to patients. The hurdles and challenges which needed to be overcome are discussed, and in particular the importance of the immune component in achieving a therapeutic effect is highlighted. Today, oncolytic therapy is generally thought of as an immunotherapy, the term 'oncolytic immunotherapy' having been widely adopted. With the advent of immuno-oncology drugs based on immune checkpoint blockade, a clear rationale for synergy between the two approaches, and initial pre-clinical and clinical data suggesting this to be the case, it might be expected that oncolytic immunotherapy combined with checkpoint blockade will provide a cornerstone of future cancer treatment. Copyright © 2015. Published by Elsevier B.V.

Targeted therapies in bladder cancer: An overview of in vivo research

Abstract

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