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Bull Cancer 2015; 102:1020–1035

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Molecular alterations during bladder

urothelial carcinogenesis

Geraldine Pignot1, Constance le Goux2, Ivan Bieche2

Received March 16, 2015
Accepted October 8, 2015
Available on the internet:
November 23, 2015
1. Institut Paoli-Calmettes, urological surgery department, 13009 Marseille, France
2. Paris Descartes University, Curie Institute, Genetics Department,
Pharmacogenomics Unit, 75005 Paris, France
Correspondence:
Geraldine Pignot,Paris Descartes University, Curie Institute, genetics department,
pharmacogenomics unit, 232, boulevard de Sainte-Marguerite,
13009 Marseille, France.
pignotg@ipc.unicancer.fr

Keywords Summary
Bladder tumor
Carcinogenesis Bladder tumors represent the sixth cause of cancer mortality in France and the prognosis
Molecular markers for infiltrative forms remains very poor due to the limited effectiveness of conventional
Targeted therapies treatments. Recent advances in molecular biology applied to tumors and the results of
recent genome-wide studies have allowed a better understanding of bladder urothelial
carcinogenesis. The main molecular alterations concernFGFR3, TP53AndHER2,and now
make it possible to differentiate three subgroups of tumors, with different molecular
profiles and prognoses. This article aims to provide an update on the different genetic
and epigenetic alterations observed in bladder cancers, their potential role as theranostic
markers in clinical oncology and new targeted therapeutic approaches according to the
concept of personalized medicine.

Keywords Summary
Bladder cancer
Carcinogenesis Recent advances in bladder urothelial carcinogenesis
Molecular markers
Bladder cancer is the sixth cause of cancer mortality in France and prognosis of muscle-invasive
Targeted therapies
tumors remains poor due to lack of effective treatments. Recent advances in molecular biology
applied to tumors and results of recent genome-wide studies have brought an important impact on
the understanding of bladder carcinogenesis. Main molecular alterations concernedFGFR3, TP53 and
HER2,and it is now possible to distinguish three subgroups of tumors according to molecular profile.
This paper proposes a review of different genetic and epigenetic alterations in bladder cancer, their
potential role as theranostic markers in clinical oncology and new targeted therapies according to the
concept of personalized medicine.
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http://dx.doi.org/10.1016/j.bulcan.2015.10.004
©2015 French Cancer Society. Published by Elsevier Masson SAS. All rights reserved.
Molecular alterations during bladder urothelial carcinogenesis
Introduction
With nearly 150,000 deaths per year worldwide, bladder
cancer represents the sixth cause of cancer mortality in France
(i.e. 4% of all cancer deaths in men and 1.9% in wife)[1].
Urothelial carcinoma is the most common form of bladder
cancer and clearly predominates in men (sex ratio close to 5).
The prognosis remains largely correlated with the stage and
grade of differentiation of these tumors. We classically
distinguish 2 types: bladder tumors not infiltrating the muscle
(TVNIM) and bladder tumors infiltrating the muscle (TVIM). It is
generally accepted that 70% of new cases of bladder tumor
are diagnosed at the non-infiltrative stage (50% pTa, 20% pT1).
These tumors that do not infiltrate the muscle pose two
different progressive risks: the risk of recurrence in the same
non-infiltrating mode and the risk of progression towards
muscle infiltration. The majority of NIMVT is well
differentiated, low grade, and amenable to conservative
treatment. The prognosis is then favorable with a specific risk
of death at 10 years of around 5 to 12%. High grade (G3) and
high stage (pT1) tumors, or carcinoma in situ (pTis) have a
worse prognosis. The risk of progression at 5 years is then
45% and the specific risk of death at 10 years is 36%.[2].
Tumors that do not infiltrate the muscle can be contrasted
with tumors that infiltrate the bladder (30% of cases at
diagnosis). These can result from the evolution towards
infiltration of a non-infiltrating tumor, but, most often, they
are tumors that are immediately infiltrating. The 5-year
survival is around 80% if the tumor does not extend beyond
the muscular plane (- pT2b). It drops to 30% in the event of
damage to the perivesical fat (pT3) and to less than 5% when
there is lymph node invasion and/or distant metastatic
locations.
[3]. Their possibly pejorative evolution requires aggressive
management.
The natural history of treated bladder tumors reveals a
progressive risk that is sometimes different within the
same stage. This highlights the insufficiency of currently
recognized prognostic factors such as tumor stage and
differentiation grade. At the individual level, they provide
little information on the evolutionary potential of a tumor.
The identification of new prognostic markers would make
it possible to isolate tumors which, at the same stage and
grade, would have a different evolutionary potential
justifying increased surveillance or early and/or more
aggressive adjuvant treatment. However, despite the
numerous lines of research developed by different teams
seeking to identify new prognostic markers in bladder
cancer, none has so far been able to replace the usual
histo-prognostic criteria. The management of bladder
tumors has therefore changed little in recent years.
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The development of molecular biology applied to tumors
has enabled the identification of somatic genetic (and
epigenetic) alterations in bladder tumors and has been an
essential step towards understanding the molecular
mechanisms of bladder carcinogenesis. 10 years ago, the
model of bladder urothelial carcinogenesis was based on
two distinct pathways of genetic alterations: the pathway
of papillary hyperplasia and non-infiltrative, low-grade
tumors (with predominant mutations in genesHRAS And
FGFR3),and the dysplasia and muscle-infiltrating tumor
pathway (with structural and functional abnormalities of
tumor suppressor genesTP53and orRB1)[4]. These two
molecular pathways need to be reconsidered with recent
results from genome-wide studies identifying new genes
involved in bladder urothelial carcinogenesis. [5.6]. These
alterations can also potentially be used as markers in
clinical oncology. Indeed, some can be useful for
diagnostic and prognostic evaluations, for assessing the
response to treatment and are currently opening the way
to new therapeutic approaches.
Genetic alterations
Genetic alterations are responsible for the abnormal activation
of oncogenes (gain of function) or the inactivation of tumor
suppressor genes (loss of function). These two classes of
genes are distinguished by their mechanism of action. The
mode of action of oncogenes is considered dominant; it is
enough for only one of the two alleles to be activated for a
positive effect on the tumor to be observed. Conversely, the
mode of action of tumor suppressor genes is considered
recessive. Their inactivation requires the alteration of their two
alleles. The gap that existed between the resolving power of
cytogenetic and molecular analysis has been considerably
reduced in recent years thanks to the development of new
hybridization techniques: comparative genomic hybridization
(CGH forcomparative genomic hybridization,then CGH-array)
and fluorescent in situ hybridization (FISH technique). At the
beginning of the 2000s, CGH on metaphase chromosomes
made it possible to increase the resolution and avoid cell
culture prior to cytogenetic analysis, a source of selective bias
for tumor cells with a high mitotic index. CGH (10 Mb
resolution) made it possible to highlight, at the chromosomal
level, the amplified or lost regions of the genome with which
oncogenes or tumor suppressor genes should be associated
respectively. CGH was quickly supplanted by CGH-techniques.
arraywhich are increasingly resolving (1 Mb resolution forBAC-
arrayto a few Kb foroligonucleotide-array)and which made it
possible to highlight new amplified or lost regions, of smaller
size, in the tumors. The development of technologies resulting
from DNA chips (microarrays)allowed us to change
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scale of work by carrying out exhaustive analyzes of all the
genes in the human genome (genome-wide analyses),
making it possible to identify a considerable number of
new molecular, genetic and epigenetic events in tumors.
A large number of these genetic alterations very probably
correspond to so-called transient alterations, having no
oncogenic power. Only part of these alterations would play
a causal role during tumor progression and could have a
direct impact on several fundamental mechanisms
common to different types of cancer and listed under the
name “Hallmarks of cancer”.[7], such as cell proliferation,
escape from cell cycle arrest, or apoptosis.
Exhaustive analysis of tumor DNA and RNA using
technologiesmicroarraysand especially by sequencing of
exomes or entire genomes (1 bp resolution) should quickly
make it possible to clarify the extent to which certain
alterations play an important role in the development of
cancer. Despite the significant progress brought by next-
generation sequencing technologies, these techniques still
remain too expensive to regularly consider large-scale
sequencing of the entire genome (3 Gb). An alternative,
used over the last five years, is the analysis of the exome
sequence, corresponding to all the coding parts of the
genome (1% of the genome, 28 Mb). In bladder cancer,
recent studies focusing on the exome have very recently
identified new genomic alterations concerning genes
involved in bladder urothelial carcinogenesis.[8]. Finally,
more recently, a genome-wide analysis was carried out as
part of The Cancer Genome Atlas project.
[9]including 131 bladder tumors, all inflammatory and high
grade[9,10]. The number of genomic alterations was
particularly high, with bladder tumors coming in third in
terms of mutation rate (around 7.7 per Megabase) after
melanomas (10/Mb) and lung cancers (8/Mb).[11]. This
study made it possible to identify a certain number of
mutations, deletions and amplifications involving genes
previously never reported as altered in bladder tumors,
and which could constitute new potential therapeutic
targets.
Deletions and inactivating mutations/tumor
suppressor genes (TSGs)
According to the Knudson hypothesis, genes whose normal
function is to suppress cell growth are usually inactivated
through a two-step mechanism: one copy of the gene is
inactivated (mutation or modification of the methylation
profile) and the second is deleted. The presence of areas of
chromosomal deletions and inactivating mutations suggests
the existence of tumor suppressor genes which are the targets
of these deletions. Unlike other types of cancer
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like hematopoietic tumors and sarcomas, whose molecular
pathogenesis involves the activation of protooncogenes by
chromosomal translocation, losses of genetic material
seem important in urothelial carcinomas, suggesting the
importance of the inactivation of numerous tumor
suppressor genes[12].
According to the recent analysis of the Cancer Genome Atlas
[9], 30 regions were identified as carrying focal deletions.
Among these, deletions on chromosome 9 are the most
frequent genetic rearrangements in bladder tumors and are
highlighted from the early stages and grades of urothelial
carcinomas. At the long arm of chromosome 9, several regions
(9q21, 9q22, 9q31, 9q33, 9q34) are candidates for containing
tumor suppressor genes involved in bladder oncogenesis. The
9q34 region for example contains the tumor suppressor gene
TSC1 (tuberous sclerosis gene 1)which negatively regulates
the anti-apoptotic Akt/mTOR pathway. The short arm of
chromosome 9 and more particularly the 9p21 locus delimited
by the markersIFNA (interferon alpha gene) andD9S171is also
an important region. Indeed, it contains genes involved in the
regulation of the cell cycle, in particular genesCDKN2AAnd
CDKN2Bwhich encode cell cycle inhibitory proteins (p14, p15
and p16). This 9p21 locus is deleted in nearly 47% of
infiltrating bladder tumors, according to recent data from the
Cancer Genome Atlas[9].
Among the other frequently deleted regions, we can note:
- the short arm of chromosome 8 (8p), with several
potentially candidate genes: the geneEXTL3 (exostoses
like 3 gene),the genePRLTS (PDGF receptor beta like
tumor suppressor),and the geneWRN (Werner syndrome
gene), coding for a helicase and whose germline
mutations are responsible for Werner syndrome,
characterized by accelerated aging and predisposition to
numerous cancers;
- the 13q14.2 region containing the geneRB1;
- several regions containing tumor suppressor genes
involved in the regulation of chromatin remodeling: the
1p36.11 region with the geneARID1A,the 16p13.3 region
with the geneCREBBP,the 17p12 region with the gene
NCOR1;
- the 2q22.1 regions (containing the geneLRP1B),2q34
(containing the geneIKZF2),3p14.2 (containing the gene
FHIT),or even 6p25.3 (containing the geneFOXQ1).
The tumor suppressor genes most frequently implicated in
urothelial carcinomas are listed in thetable I [9.13].
According to data from the Cancer Genome Atlas
[9], these genes can be divided into different categories:
genes involved in the regulation of the cell cycle, genes
involved in epigenetic regulatory mechanisms, and in
particular the regulation of chromatin, and genes involved
in the Redox system.
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TTABLEI is inactive and has a prolonged half-life, which induces
Main GSTs described as mutated in urothelial carcinomas accumulation of the abnormal form in the nucleus. The
frequency of mutations is greater in high-grade tumors
(more than 50% of cases) compared to low-grade tumors
Genoa Data Gui et al. Ross et al. TCGA (only 20%).[15]. However, although frequently altered, p53
COSMIC Nat Genet Mod Pathol Nature did not bring the expected hopes in terms of prognostic
[13] 2011[8] 2014[16] 2014[9]
significance or marker of aggression. Several authors have
TP53 44% 21% 54% 49% suggested that p53 protein overexpression is associated
RB1 26% 11% 17% 13% with prognosis and risk of progression independent of
grade, presence of vascular invasion, or presence of
P16/CDKN2A 17% – 23% 5% (47%1)
carcinoma in situ.
PTEN 3% – – 3% (13%1) [14]. These data have not been confirmed and remain
APC 3% – – 4% controversial. At the same time, approximately 50% of
tumors that metastasize do not have p53 alteration,
NF1 – 7% 6% 8%
implying the involvement of alternative signaling
TSC1 – – 6% 8% pathways. There is also a phenomenon of mutual
ATM – – – 15% exclusion between TP53AndMDM2,encoding ubiquitinated
E3 and having anti-apoptotic activity through negative
P21/CDKN1A – – – 14%
regulation of p53[9]. The gene RB1,also involved in the cell
STAG2 – – – 11% cycle, is located on chromosome 13 and codes for a
FBXW7 – – – 10% nuclear phosphoprotein (pRB) with a molecular weight of
110 Kdaltons. Gene mutationsRB1were described in 13% of
NFE2L2 – – – 8%
cases[9]. It seems that these alterations have a cooperative
TXNIP – – – 7% effect with the anomalies ofCDKN2A (p16),the alterations
Genoa of these two genes being mutually exclusive and
involved appearing more important in infiltrative and high-grade
in the bladder tumors. Other genes involved in the cell cycle have
mechanisms
also been described as altered: this is the case ofCDKN1A
epigenetics
(p21)and D'ATMwith deletions found in 14% and 15% of
MLL – 7% – 14%
cases respectively[9].
MLL2 – – – 27% More recently, analyzes by sequencing of exomes or whole
MLL3 – 5% – 22% genomes have made it possible to identify new tumor
suppressor genes mainly involved in epigenetic regulatory
ARID1A – 13% 20% 25%
mechanisms, and in particular in chromatin remodeling,
KDM6A/UTX – 21% 29% 24% and previously unknown in the bladder urothelial
EP300 – 13% – 15% carcinogenesis: KDM6A (UTX), CREBBP, EP300, ARID1A,but
alsoCHD6, MLL, NCOR1, MLL2And MLL3[8.9].
CREBBP – 13% – 12%

NCOR1 – 6% – 8% (25%1) Certain molecular alterations also seem to concern genes

1Rateof described deletions (specified when significantly different from the rate of
mutations).
Among the genes involved in cell cycle regulation,
suppressor gene alterationsTP53constitute the most
frequent somatic genetic event in tumors in humans since
found in almost half of cases [9,14]. It is located on the
short (p) arm of chromosome 17 at locus 17p13.1, a region
prone to allelic loss. Gene mutationTP53gives abnormal
p53 protein detectable by immunohistochemistry. The
mutated p53 protein
involved in the Redox system, which plays a major role in
maintaining cellular homeostasis and regulating cell
survival by fighting against oxidative stress and the
accumulation of free radicals. This is particularly the case
for genesNFE2L2AndTXNIP,mutated in 8 and 7% of cases
respectively[9].
Finally, recent genome-wide analyzes have made it possible to
identify new tumor suppressor genes previously never
described in bladder carcinogenesis.[9]. This is particularly the
case for the geneNF1,located at 17q11.2 and encoding a
protein inhibiting RAS proteins, with inactivating mutations
found in 6 to 8% of bladder tumors [8,9,16]. This is also the
case for the geneTSC1,located in
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9q34 and negatively regulating the Akt/mTOR pathway TTABLEII

involved in cell proliferation and apoptosis, with Main oncogenes described as mutated in urothelial carcinomas
inactivating mutations found in 5 to 16% of cases
depending on the series [15–18]. It seems likeTSC2is also
involved as a tumor suppressor gene but the mutations Genoa Data Gui et al. Ross et al. TCGA
described are rarer (2 to 3% of urothelial carcinomas)[9.15] COSMIC Nat Genet Mod Pathol Nature
[13] 2011[8] 2014[16] 2014[9]
. Likewise, the geneSTAG2,encoding a subunit of the
cohesin complex that regulates chromatid cohesion and FGFR3 46% 9% 11% 12%
segregationœurs during the cell cycle, presents PIK3CA 22% – 15% 20%
inactivating mutations in 11% of cases[9.19]. It appears
HRAS 7% 10% 5% 5%
that these mutations are more frequent in non-infiltrating
papillary tumors, where they have been described in more KRAS 4% 6% – 0%
than a third of cases.[20], representing a new potential NRAS 1% – – 2%
therapeutic avenue.
AKT1 3% – – 0%

CTNNB1 2% – – 2%
Material gains and activating/oncogenic EGFR – – 6% 0% (11%1)
mutations
ERBB2 – – 6% 5% (7%1)
Contrary to what was previously thought, focal and
recurrent gene amplifications are not rare events in ERBB3 – 8% – 11% (2%1)

urothelial bladder carcinomas. Indeed, data from the FGFR1 – – 14% –

Cancer Genome Atlas made it possible to identify 27
LAMA4 – 7% – –
amplified regions[9].
Among the gene amplifications highlighted in bladder CCND1 – – 14% 0%
carcinomas, those affecting chromosome 7 are of CCND3 – – 11% 4%
particular importance because they target the oncogene
MDM2 – – 11% 0%
ERBB1 encoding the EGF receptor. This receptor is
sometimes overexpressed in bladder cancers and the gene MCL1 – – 11% –
is found to be amplified in 11% of cases.[9.16]. Likewise, at ERCC2 – – – 12%
chromosome 17, one of the candidate genes is the
RXRA – – – 9%
oncogene ERBB2coding for a transmembrane receptor
with tyrosine kinase activity related to EGF receptors. This 1Rate of focal amplifications described (specified when significantly different from the
rate of activating mutations).
gene located in the 17q12 band is amplified in several
cancers, particularly in breast and bladder cancer.[21]. If
amplifications are found in 7% of cases, there also seem to
be other activation mechanisms such as “gain of function” Most of these are genes involved in key signaling
mutations found in 5% of cases and rare translocations pathways of carcinogenesis.
also involvingDIP2B (4%). Finally, activating mutations of The geneFGFR3 (fibroblast growth factor receptor 3)
ERBB3are also described in 8 to 11% of bladder cancers belongs to a family of receptors with tyrosine kinase
[8.9], opening new therapeutic perspectives. activity encoded by 4 different genes (FGFR1-4). The most
frequent mutations concern the extracellular and
The other most frequently observed material gains and/or transmembrane domains of the receptor (R248C, S249C,
amplifications are located at 1q23.3 (containing the gene G370C, and Y373C) and lead to activation of the receptor
PVRL4),in 3p25.2 (containing the genePPARG),in 4p16.3 by stabilization of the dimeric form, independently of the
(containing the geneFGFR3),in 6p22.3 (containing in particular ligand. Other mutations may involve the tyrosine kinase
the genesE2F3AndSOX4),at 8p11.23 (containing the gene domain (K650E, K650M) leading to a conformational
ZNF703),in 8q22.2 (containing the genesDORFIN, POLR2K, change and activation of the receptor. In urothelial
PABPC1, YWHAZAndSPAG1),in 11q13 (containing the genes carcinomas, the frequency of mutations ofFGFR3varies
CCND1AndFGF3),in 12q15 (containing the genesMDM2And greatly depending on the type of tumors analyzed[22].
FRS2)and in 19q12 (containing the geneCCNE1)[9]. Indeed, the frequency of mutations seems very high in pTa
The main oncogenes showing activating mutations in and low-grade tumors (around 60 to 80%) and much lower
bladder tumors are listed in thetable II. He in pT1 tumors (20 to 40%) or
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Molecular alterations during bladder urothelial carcinogenesis
pT2–pT4 and high grade (only 12% in the TCGA having mainly
included infiltrative and high grade tumors)[9.15].FGFR3is
therefore a gene preferentially mutated in bladder tumors
with a good prognosis. Recently, Van Rhijn et al. proposed a
determination of the grade based on molecular criteria
including in particular the statusFGFR3 as an alternative to
histological grade in a multicenter series of 230 patients with
non-muscle-infiltrating bladder tumors. The presence of
mutations ofFGFR3was significantly associated with good
prognostic parameters and low EORTC score. Furthermore,
“molecular grade” was an independent prognostic factor in
multivariate analysis and seemed more reproducible than
histological grade.[23]. In the subgroup of tumors with an
activating mutation ofFGFR3,however, it seems that the
existence of a deletion concerning the tumor suppressor gene
CDKN2Ais associated with an increased risk of progression
towards muscular infiltration of these tumors classically
considered to have a good prognosis[24]. Finally, gene
translocationsFGFR3with the geneTACC3 (transforming acidic
coiled coil)have also recently been identified in a subgroup of
infiltrative tumors; the componentFGFR3then loses its final
exon, including the binding site for PLCg,which leads to a loss
of negative regulation and over-expression of the receptor
[9.25]. Mutations of genes involved in the PI3K/AKT/mTOR
pathway are also described in urothelial tumors. The
frequency of activating mutations concerning the oncogene
PIK3CAis of the order of 20% in the different series, the other
genes of the signaling pathway being more rarely observed
[9,15,18]. Activation of the pathway appears to be observed
independently of stage and does not appear to correlate with
expression ofPTEN[26].
Gene mutationsRASare not considered a major molecular
event in bladder urothelial carcinogenesis; in fact,
mutations are found in less than 5% of bladder tumors,
without any correlation to tumor grade or stage[8.9].
Gain of function mutations concerning the gene promoter
TERThave also been described in more than two thirds of
urothelial carcinomas and appear to appear early in
bladder carcinogenesis[27–29]. Bladder cancer is one of
the most frequently affected by activating mutations of
TERT[27]. These are responsible for an increase in the
expression of this gene and the activity of telomerase,
compensating for the erosion of telomeres through the
synthesis of telomeric DNA, thus allowing cancer cells to
avoid senescence. . It appears that these mutations are
significantly more frequent in tumors mutated for FGFR3[
30]. Finally, the detection of mutations ofTERTin the urine
could be associated with the risk of recurrence, even if this
prognostic role remains controversial[28.30]. Activating
mutations ofTERThave the particularity of not being
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detectable by exome analyzes because they are located at the
level of the gene promoter. This is also the case for other non-
coding mutations located on upstream regulatory sequences,
such asPLEKHS1, WDR74AndSDHD[31].
There are also rare gene mutationsATRX (1% only in
urothelial carcinomas), involved in the maintenance of
telomeres independently of the action of telomerase, with
a phenomenon of mutual exclusion with TERT,suggesting
that these two genetic mechanisms confer equivalent
advantages in terms of selective growth
[27].
Finally, recent data from the pan-genome analysis carried
out as part of the Cancer Genome Atlas (TCGA) have made
it possible to highlight new oncogenes potentially involved
in bladder carcinogenesis since they present activating
mutations [ERCC2 (12%),RXRA (9%),ELF3 (8%),KLF5 (8%),
CCND3 (4%)] or recurrent amplifications [CCNE1 (12%),
CCND1 (10%),MDM2 (8%),PVRL4 (19%),PPARG (17%),
BCL2L1 (11%),ZNF703 (10%)][9]. Ultimately, the integrated
analysis of the different genetic alterations described
within the Cancer Genome Atlas made it possible to
identify three main signaling pathways deregulated in
bladder tumors, and potential preferred therapeutic
targets. Deregulations affecting the cell cycle are found in
93% of cases; those affecting the PIK3/AKT/mTOR pathway
in 72% of cases; and finally alterations of genes involved in
chromatin remodeling in 64% of cases[9].
THEclusteringof the different genetic alterations observed
made it possible to highlight three groups of tumors[9]:
- the group of tumors presenting a mutation in the
epigenetic regulatory geneMLL2;
- the group of tumors showing loss ofCDKN2Aand
activating mutations ofFGFR3,rather associated with a
papillary profile on histology;
- the group of tumors presenting inactivating mutations of
TP53,and ofRB1and amplifications ofE2F3AndCCNE1.
Instability of microsatellite markers and DNA
mismatch repair genes
Inactivation of DNA mismatch repair genes, ormismatch
repair genes (MSH2, MLH1, MSH6),is the cause of an
accumulation of genetic alterations in the form of multiple
repetitive sequences (microsatellite instability, MSI)
characteristic of Lynch syndrome tumors. Somatic
mutations of these genes are also found with variable
frequency in certain sporadic tumors. These microsatellite
instabilities are rare in urothelial carcinomas of the
bladder (<5%), but more common for urothelial
carcinomas of the upper excretory tract. These, when they
exist, are found in the early stages of the disease[32]. It
should be noted that in the
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familial forms linked to Lynch syndrome, tumors of the
urinary system are in third place after colonic and
endometrial locations. In this syndrome, germline
mutations affect 60% of the geneMSH2,in 30% of cases the
geneMLH1and more rarelyMSH6.Mutations of the latter
would, on the other hand, be more particularly associated
with low-grade and non-invasive tumors.[33].
Potential role of viruses in bladder
carcinogenesis
While viral infections are involved in 16% of cancers (more
particularly HPV and cervical cancer, EBV and
nasopharyngeal cancer, HHV8 and Kaposi's sarcoma,
HTLV-1 and T-cell lymphoma, hepatitis B and C viruses and
hepatocarcinoma)[34], no formal data existed in bladder
tumors until recent data from the Cancer Genome Atlas. In
fact, it seems that viral expression is found in 7.3% of
cases, involving certain HPV viruses (HPV16, HPV45,
HPV56, HPV6b) or CMV/HHV5, with, in the majority of
cases, an integration mechanism. viral genetic material
within the genome[35]. However, it is difficult to precisely
assess the role of these oncogenic viruses and to establish
the link between viral infection and the appearance of
cancer due to the importance of potential cofactors.
Epigenetic regulatory mechanisms
Numerous articles, published over the last 10 years,
suggest the major importance of epigenetic alterations
(DNA methylation, chromatin remodeling by biochemical
modification of histones, regulation by non-coding RNAs)
which can modify the expression of coding genes. proteins
in human tumors, and particularly in urothelial carcinomas
[36.37].
The word “epigenetics” refers to phenomena that
modulate the activity of the genome without changing its
sequence. These mechanisms are dynamic and reversible
and respond to events, such as embryogenesis or
environmental factors. These phenomena have been
demonstrated for several cancer suppressor genes, which
in certain tumors showed a total absence of transcriptional
or protein expression while being free from any alteration
at the DNA level.
Histone modification
The NH2-terminal region of histones is accessible outside
the nucleosome. Histone modifications in mammals take
place in this region. These modifications (acetylation, but
also ubiquitination, phosphorylation, etc.) determine the
structure of chromatin and therefore the expression or
repression of genes. Some of these alterations could be
associated with the aggressiveness and prognosis of
bladder tumors[38].
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Recently, genetic aberrations involving chromatin
remodeling genes (KDM6A, CREBBP, EP300, ARID1A)were
found with a frequency of more than 10%, suggesting a
major role for chromatin regulation in bladder urothelial
carcinogenesis.[8.9]. Some of them appear to have
prognostic value; this is the case ofARID1Aloss of
expression of which is associated with more aggressive
disease and increased risk of recurrence[39]. Finally,
current data from whole genome analysis as part of the
Cancer Genome Atlas have made it possible to confirm the
major importance of mutations concerning chromatin
regulation genes in bladder urothelial carcinogenesis,
mutations which seem to be more frequent in the 131
bladder tumors analyzed than in the other types of cancers
studied. Indeed, 76% of bladder tumors presented at least
one mutation and 41% two mutations among chromatin
remodeling genes.[9]. Similarly, the SWI/SNF chomatin
remodeling complex, which usually participates in the
regulation of variant exon inclusion by reducing the rate of
RNA polymerase II elongation, also appears altered in 64%
of cases. . This could indicate the possibility of developing
new therapies targeting epigenetic regulatory pathways.
DNA methylation
In the basal state, approximately 5 to 10% of cytosines are
methylated. This methylation negatively regulates gene
transcription. Two types of DNA methylation modifications
are observed in tumor cells, either hypomethylation by
global decrease in the level of 5-methylcytosines inducing
an exacerbation of transcription mechanisms, or local
hypermethylation of certain regions of the genome.
Hypermethylation in region 50of the gene promoter is
associated with transcriptional silencing and is therefore
an alternative mechanism for repressing the expression of
tumor suppressor genes other than by deletion or
inactivating mutation. Aberrant methylation of the
promoters of several genes has been described in
urothelial carcinomas:CDH1coding for E-Cadherin,
RASSF1A, SOX9, DAP kinase, RUNX3,and the genesLOXL1
And LOXL4,potential tumor suppressor genes by inhibition
of the Ras pathway[40–42].
Techniques for global analysis of gene methylation by
microarray (“methylome") from urine samples also seems
promising. Reinert et al. were thus able to identify new
methylated genes in bladder tumors:ZNF154, HOXA9,
POU4F2, EOMES, ACOT11, PCDHGA12, CA3, PTGDRAnd
TBX4[36]. Furthermore, aberrant methylation of promoter
regions appears to be associated with tumor stage and
risk of bladder tumor progression.[41]. More recently,
Marsit et al. identified, from blood samples, a DNA
methylation profile associated with the risk of
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Molecular alterations during bladder urothelial carcinogenesis

development of bladder cancer[43]. Likewise, the Cancer
Genome Atlas made it possible to associate the expression of
CIMP (CpG island methylator phenotype)with smoking status
[9]. It should be noted that in the short term, the analysis of
"methylomes" will be carried out simultaneously with the
analysis of genomes using "third generation" sequencing
methods allowing both the uninterrupted reading of
thousands of nucleotide bases and detection of the
methylation state of cytosines.
miRNAs
Gene expression is also regulated through epigenetic
silencing mechanisms involving small non-coding RNAs of
21–24 nucleotides (miRNAs), leading mainly to repression
of translation. There are more than 1000 genes encoding
microRNAs in the human genome, and each microRNA has
the capacity to regulate the expression of a large number
of target genes.
Synthesis
(around 200). MicroRNAs are involved in many
physiological processes such as proliferation, apoptosis,
differentiation, development and cellular metabolism.
MicroRNAs have also been implicated in cancers. Certain
genes, coding for these microRNAs, are located in regions
showing chromosomal translocations and/or deletions in
tumors. MicroRNAs can function as oncogenes or tumor
suppressor genes.
Several large-scale studies have made it possible to identify
expression profiles associated with bladder tumors, and
varying depending on the degree of infiltration and/or tumor
grade.[44]. THETable IIIindicates the microRNAs most
frequently reported as altered in bladder cancer, their main
target genes and their functions[44–57].
The deregulations described in bladder tumors are
essentially under-expressions, reflecting the probable
tumor suppressor effect of these miRNAs, and concern

TTABLEIII
List of miRNAs described in the literature as altered in bladder cancer, with their target messenger RNA and their function

miRNA Deregulation target mRNA Function References)

miR-1 Down LASP1, TAGLN2 Cytoskeleton, differentiation [44,45]

miR-21 Up TP53, PTEN, Bcl-2, MSH2, E2F3 Cell cycle control, apoptosis [46,47]

miR-24 Down FOXM1 Proliferation [45]

miR-31 Down FGFR3 Proliferation [48]

miR-99a/100 Down FGFR3 Proliferation [46.49]

miR-101 Down EZH2 Gene expression [50]

miR-125b Down E2F3 Apoptosis and proliferation [45.51]

miR-129 Up SOX4, GALNT1 Signal transduction, expression [47]

protein
miR-133a Down KRT7, TAGLN2, EGFR Differentiation, proliferation [44.51]

miR-143 Down HK2, MMP13, PRKCE, ERK5, Proliferation, migration [44,45]

MAPK7, AKT

miR-145 Down CBFB, PPP3CA, CLINT1 Signal transduction, apoptosis [44,45,51]

miR-183 family Up miR-96, 182 FOXO1, FOXO3 Proliferation, migration [44,45,52]

(miR-96, miR-182 and Down miR-183 EZR
miR-183)

[53]

miR-195 Down CDK-4, GLUT3 Cell cycle control, proliferation

miR-200 family Down ERRFl-1 Epithelial-mesenchymal transition [44.54–56]
(miR-200a, miR-200b, ZEB1, ZEB2
miR-200c, miR-141 and
miR-429)
miR-205 Down ACSL4, PTEN Cell cycle control, apoptosis [55]

miR-221 Up TRAIL Apoptosis [57]

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 G. Pignot, C. le Goux, I. Bieche
Synthesis
notably miR-145, involved in apoptosis, the miR-200 family,
involved in the epithelial-mesenchymal transition of high-
grade tumors, and miR-99a/100[45]. The latter is known to
target FGFR3; thus, the underexpression of miR-99a/100
would be responsible for an overexpression of FGFR3 in
low-grade tumors[46]. Some microRNA overexpressions
are also described but are more frequently observed in
infiltrating tumors. This is the case for miR-21,
overexpression of which would be associated with
inhibition of the p53 and PTEN pathway.[46,47]. Few
studies have so far reported a correlation between miRNA
expression and the prognosis of bladder tumors.[47]. The
molecular signature associating miRNAsmiR-9, miR-182
AndmiR-200bwas recently identified as associated with the
aggressiveness of bladder tumors[44].
Alterations in gene expression
If the nature of the gene alterations is variable (gene
amplifications, mutations, loss of alleles, hypermethylations,
etc.), these alterations mainly result in a quantitative
modification of the expression of the genes in question, which
can be measured both at the level of the transcript than of the
protein.
Alterations in gene expression at the messenger
RNA level: the transcriptome
At the messenger RNA level, changes in gene expression at
the individual level were initially highlighted by Northern
Blot. This method, cumbersome and requiring large
quantities of biological material, was quickly supplanted by
real-time quantitative RT-PCR. The introduction of
technologycDNA microarraysthen made it possible to
analyze gene expression and/or the expression of
microRNAs, no longer at the individual level, but at the
genome level (transcriptome) in tumor samples and to
highlight new clinical phenotypes urothelial tumors. The
ideal would be to be able to identify tumors with different
evolutionary potential in order to be able to adapt the
therapeutic strategy in a personalized manner. For NIMVT,
several studies have sought to identify gene expression
signatures to predict the risk of recurrence or progression
to an infiltrating tumor. However, there is currently no
reliable prognostic marker for NIMVT. For IMVT, on the
other hand, certain genes and molecular signatures seem
to be associated with tumor aggressiveness, risk of
progression and survival.
This is the case, for example, of molecules involved in the
angiogenesis pathway, whose role during urothelial-
bladder carcinogenesis has been particularly studied in
recent years, like most tumor pathologies. Among the
angiogenic factors, vascular endothelial growth factor
(VEGF) is a particularly interesting molecule.
1028
since its crucial angiogenic activity, in vitro and in vivo, makes
it the preferred target of current therapies. There is extensive
literature on the role of the VEGF pathway in different cancers,
with more recent interest in urothelial tumors. The different
series seem to indicate that VEGF is mainly involved in the
early stages of carcinogenesis and that its expression
decreases with the progression of the disease. [58.59].
However, its prognostic value, particularly concerning the risk
of recurrence or progression, remains very controversial.
[59.60].
The next decade will be even more prolific in terms of discoveries
with the advent of high-throughput sequencing (NGS) techniques.
next-generation sequencing)making it possible to obtain, quickly
and at low cost, all of the RNA sequences of a tumor (RNAseq)at
the quantitative (transcriptome) and qualitative (mutations, fusion
transcripts) level.
This is particularly the case of recent data obtained as part of
the Cancer Genome Atlas (TCGA) which made it possible to
highlight different molecular expression profiles defining new
tumor phenotypes within urothelial carcinomas with potential
prognostic and therapeutic implications.[9]. These tumor
profiles are also observed at the microRNA sequencing level
and at the protein expression level. Among infiltrative tumors,
we can thus distinguish the “papillary-like” or “luminal”
subtype, which corresponds to tumors of papillary
architecture, with strong expression of FGFR3, associated with
mutations or amplifications of the gene.FGFR3.We also note a
decrease in the expression of microRNAs regulating FGFR3,
and in particular miR-99a and miR-100, as well as miR-145 and
miR-125b. Conversely, the “basal/squamous-like” subtype is
characterized by the expression of epithelial or stem cell
differentiation genes, such as cytokeratins (KRT14, KRT5,
KRT6A),as well as an activation ofp63and theEGFR.We find the
same “basal-like” expression profile for certain breast cancers
or for squamous tumors of the lung or head and neck. These
tumors would be associated with a more aggressive urothelial
disease at diagnosis and a poor prognosis. Finally, “p53-like”
tumors would be characterized by resistance to conventional
chemotherapies, but a potential response to
immunotherapies.[61].
Alterations in gene expression at the protein
level: proteome
To meaningfully understand the somatic alterations of a
tumor cell, it is now essential to look at the analysis of the
proteins it contains. Indeed, proteins are the main
molecules involved in the functioning of the cellular
machinery, while genes are only information carriers. The
level of expression of messenger RNA does not necessarily
reflect the abundance, nor the nature, of the proteins
which will be found in
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Molecular alterations during bladder urothelial carcinogenesis
the tumor cell. Indeed, the translation of messenger RNAs into
proteins is subject to a large number of regulations, in
particular to post-transcriptional (alternative splicing,
microRNAs, etc.) and post-translational (glycosylation,
phosphorylation, acetylation, degradation, etc.)
modifications. .) whose role is determining for their
functionality (or their oncogenic power). These post-
translational modifications are not detected by analysis of
DNA or messenger RNA. Proteomics (study of the proteome)
owes its current growth in oncology to the emergence of high-
performance analytical technologies making it possible to
analyze thousands of proteins simultaneously: two-
dimensional electrophoresis and especially Maldi-Tof or Seldi-
Tof type mass spectrometry. Finally, more recent techniques
for analyzing several hundred candidate proteins from a small
quantity of biological material by RPPA (Reverse Phase Protein
Array) seem promising. As part of the Cancer Genome Atlas,
these RPPA techniques made it possible to analyze 130
proteins, phosphorylated or not, and thus to identify protein
expression profiles associated with certain tumor subtypes,
alongside RNA expression profiles. previously described
[9]. Thus, “papillary-like” tumors seem associated with high
protein expression of HER2, but also of ESR2 (estrogen
receptor beta),suggesting new therapeutic possibilities
such as hormone therapies (tamoxifen, raloxifene).
New therapeutic strategies
Advances in the knowledge of the molecular mechanisms
of urothelial cancers are currently leading to new
therapeutic strategies. This is particularly important for
bladder cancer since conventional chemotherapies (based
on platinum salts) have only a modest effect on overall
survival, with median survival not exceeding 15 months in
the metastatic stage.
Targeted therapies aim to specifically target certain genes
involved in carcinogenesis (or their products) with the aim of
“re-educating” these tumor cells or selectively eliminating
them. The notion of therapeutic target in oncology has always
existed. The presence of the tumor thus constituted the first
target, for which the associated therapeutic procedure was
primarily surgical excision. Then, the identification of the
uncontrolled proliferation capacities of cancer cells led to the
development of treatments targeting this property, so-called
conventional cytotoxic chemotherapies. The notions of target
and targeted therapy have naturally evolved over time due to
a better understanding of the molecular mechanisms of
carcinogenesis, in particular linked to major technological
advances. The identification of key alterations inherent to the
tumor phenotype served as support for the research and
selection of relevant targets in oncology, at the origin of the
development of
volume 102 > n812 > December 2015
Synthesis
new targeted molecular therapies which have truly
revolutionized the therapeutic management of cancer
patients. Among the cellular targets, the most used
currently are membrane receptors (mainly with tyrosine
kinase activity). More rarely, these will be cytoplasmic
targets (signaling pathway proteins) or nuclear targets
(transcription factors). The targets of the tumor
environment mainly revolve around the phenomenon of
angiogenesis, a major element for the survival of the
tumor, its development and its dissemination, including in
urothelial tumors. More recently, the concept of
immunotherapy has broadened the spectrum of new
targeted therapies. The identification of a molecular target
involved in the tumor process is necessary as a starting
point for targeted therapy but is not sufficient. Indeed, this
target must be “druggable”,that is to say that the chemical
properties of the target biomolecule must in particular
allow binding of the candidate drug with sufficient affinity.
Potential therapeutic targets are therefore at the
intersection between the genome “druggable »and key
genes in carcinogenesis. Recent genome-wide data from
the Cancer Genome Atlas showed that 69% of bladder
tumors harbor potential therapeutic targets, and most of
them are “druggable”,either with already existing
treatments or with molecules currently under
development[9]. For example, potential targets were
identified in 42% of cases within the PI3K/AKT/mTOR
pathway and in 45% of cases within the MAP kinase
pathway.
Among the potential molecular targets that may have
therapeutic applications in bladder cancer, surface
receptors with tyrosine kinase activity have been
particularly studied: EGFR and ErbB2, VEGFR, FGFR3. . .
Another avenue of research is to target proteins
controlling the cell cycle, either by modulating the activity
of cyclins and cyclin-dependent kinases, or by restoring
p53 and/or RB functions to stimulate apoptosis. Finally, it is
also possible to target chromatin modifications thanks to
the recent development of agents that bind to acetyl-lysine
binding motifs (bromodomains). All these innovative
drugs, having an original mechanism of action and rapid
release to the market, however, have the major
disadvantage of being extremely expensive, out of all
proportion to the standard treatments known to date.
Each drug newly released to the market is likely to
exponentially increase the spending curve in healthcare
establishments. It will therefore be essential to evaluate
the optimal conditions of use and the expected benefits for
each of these drugs. Thus, it is necessary to standardize
molecular diagnostic methods and to develop “companion”
tests allowing, from
1029
 G. Pignot, C. le Goux, I. Bieche
Synthesis
the analysis of theranostic biomarkers, to target patients
likely to benefit the most from different treatments.
Anti-angiogenic therapies
Tumor neo-angiogenesis is currently at the cœheart of the
development of new targeted therapies in oncology. The
idea is to be able, by inhibiting tumor neovascularization,
to prevent the supply of oxygen and nutrients within the
tumor and thus induce hypoxia and ischemic necrosis of
cancer cells. However, it seems unlikely that an anti-
angiogenic agent alone would be sufficient for disease
control. Use in combination with standard chemotherapies
could be more relevant, in the absence of toxicity.
These molecules have been evaluated in phase II and III
clinical trials with good results and now have a validated
indication as first or second line treatment of several
cancers. In bladder cancer, several clinical trials have been
conducted with controversial results. The first phase II
results concerning an anti-VEGF, bevacizumab (in
combination with gemcitabine–cisplatin chemotherapy in
first line treatment of metastatic bladder cancer) were
encouraging with 58% of patients in partial or complete
response, but at a price significant toxicity, particularly in
terms of thromboembolic (21%) and hemorrhagic (7%)
complications[62]. Trials conducted with tyrosine kinase
inhibitors, sunitinib[63], sorafenib[64]and pazopanib[65],
in the first or second line, gave disappointing results.
Other trials are underway with vandetanib, which acts on
both the VEGF and EGFR pathway. The effectiveness of
anti-angiogenic therapies may be limited by the fact that
spatio-temporal differences in tumor neovascularization
are likely to exist, in relation to the extreme heterogeneity
of bladder tumors. This heterogeneity can be observed
within the bladder tumor itself, but also between the
primary tumor and the metastatic locations. More recently,
the concept of angiogenic regulation has been discussed.
Rather than blocking the mechanisms of tumor
neoangiogenesis, the idea would be to reorganize and use
these vascular networks to improve the bioavailability of
administered cytotoxic treatments, through modulation of
vascularization at the tumor level. Anti-angiogenic
treatments have an effect of normalizing tumor
vascularization, at least at the start of their administration,
creating a therapeutic window during which tumor
diffusion of cytotoxic drugs is improved.
[66]. Tumor neovascularization ensures heterogeneous tumor
blood flow, leaving regions that are probably hypoxic, poorly
accessible to cytotoxic agents and radioresistant. Anti-
angiogenic treatment makes it possible to homogenize the
tumor vascularization through a vascular remodeling effect,
1030
in particular by blocking the VEGF pathway on which tumor
vessels are dependent, and improving the oxygenation of
tumor cells; this action can lead to a potentiation of the
effect of cytotoxic chemotherapy during the first weeks of
treatment, as has been shown in bronchial cancers. The
combination of the two therapeutic strategies has only
given disappointing results and exposes us to a sometimes
significant risk of cumulative toxicity, particularly in fragile
patients representing a significant proportion of patients
with bladder cancer.[67].
Therapies targeting the PI3K/AKT/mTOR pathway
The PI3K/AKT/mTOR pathway is an essential signaling
pathway in signal transduction within the cancer cell. It is
notably involved in the regulation of cell proliferation,
apoptosis and angiogenesis. It is frequently dysregulated
in many cancers. In bladder cancer, recent TCGA data
suggest a more important role than initially assumed for
this signaling pathway in urothelial carcinogenesis.
[9]. It could therefore be an interesting indication for
inhibitors of the PI3K/AKT/mTOR pathway, more
particularly in the event of activation of certain
components of the pathway. Everolimus, for example, has
shown effectiveness in patients with metastatic urothelial
carcinomas and presenting a TSC1 mutation (found in 8%
of cases)[68]. Conversely, the existence of an inactivating
mutation or a deletion of PTENcould be associated with
resistance to treatment[69]. However, the results of the
first phase II trials with everolimus are disappointing and
other inhibitors of the PI3K/AKT/mTOR pathway are still
being evaluated.[70,71]. The use of “companion” tests to
guide indications based on the analysis of theranostic
biomarkers should also allow for more rational use by
restricting the use of these treatments to the subgroup of
patients with activation of the pathway ( up to 20% for
PI3KCA mutations in Cancer Genome Atlas analysis[9]).
Therapies targeting the ErbB family
Data published in breast cancer suggest that the benefit of
anti-Her2 therapies is only observed in the event of Her2
amplification and that this targeted therapeutic approach
should therefore only concern rigorously selected
candidates. In bladder cancer, this has not been clearly
established[72]. Currently, these therapies are only offered
to patients with clear overexpression (2+ or 3+) of HER2 on
immunohistochemistry. In this limited indication, the
response rates seem very satisfactory. In a phase II study,
trastuzumab, a humanized anti-ErbB2 monoclonal
antibody, in combination with conventional
chemotherapies (paclitaxel, gemcitabine, carboplatin)
showed a response rate of around 70% in
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Molecular alterations during bladder urothelial carcinogenesis
patients selected for HER2 positivity in
immunohistochemistry[73]. We do not currently have
phase III data on the response rates and possible survival
benefits of these anti-Her2 therapies. The problem of
patient selection must also be taken into consideration
and probably often remains inappropriate in trials since
they are not based on FISH analysis data. However, the low
proportion of tumors overexpressing HER2 and the low
correlation between IHC data and FISH techniques make it
a targeted therapy that is poorly developed in clinical
practice in bladder cancer.[72].
More recently, anti-EGFR targeted therapies have been
evaluated in urothelial carcinomas. Cetuximab has been
studied in combination with conventional chemotherapy in
metastatic and locally advanced bladder tumors and has
not shown a benefit in progression-free survival compared
to chemotherapy alone.[74]. However, the administration
of cetuximab was not conditioned by the search for
molecular markers potentially predictive of response to
treatment. Indeed, in certain cancers such as non-small
cell lung cancer, the overexpression or amplification of
EGFRis used as a marker of sensitivity to targeted
therapies (erlotinib) and now determines the choice of
first-line metastatic treatment[75]. In bladder cancer, it
would be interesting to develop clinical trials focusing on
the 11% of infiltrating tumors having amplifications of
EGFR,in order to adapt therapies at the individual level.
The current trend is towards the use of pan-HER
molecules, acting on several ErbB receptors, amplifications
and/or mutations may indeed concern EGFR, HER2, but
also ErbB3. Lapatinib, a dual tyrosine kinase inhibitor
acting on both EGFR and HER2, has notably been
evaluated in bladder cancer as second-line treatment in
metastatic patients; the study was considered negative
because there was no significant increase in overall
survival in the total patient population included. However,
in the subpopulation of patients with overexpression of
EGFR and/or HER2, the results were encouraging with a
significant impact on overall survival.[76]. Dacomitinib, an
irreversible pan-ErbB inhibitor, is currently being evaluated
in pre-clinical studies and phase II trials[77].
Therapies targeting FGFR3
There are currently no clinical trial data available for anti-
FGFR3 targeted therapies in urothelial carcinomas. Since
mutations are mainly observed in non-infiltrative and low-
grade tumors (with more than 45% of mutations
described), it is difficult to envisage launching a trial in this
subgroup with a good prognosis or risk. progression
towards infiltration is low.
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However, it appears that a subpopulation of infiltrating
tumors presents mutations inFGFR3 (12% according to the
Cancer Genome Atlas analysis) or translocationsFGFR3/
TACC3 (5%), suggesting a potential benefit of targeted
anti-FGFR3 therapy in certain well-selected patients[78].
Antagonistic antibodies specific for FGFR3 generally act by
interrupting dimerization, and therefore activation of the
receptor for R248C and S249C mutants. Preclinical studies
seem to suggest the effectiveness of anti-FGFR3 in terms
of reducing cell proliferation and tumor growth.[79]. These
data deserve to be confirmed in prospective clinical trials
in well-selected patients, that is to say those carrying
activating alterations ofFGFR3.
Immunotherapy
Beyond the genetic and epigenetic regulatory systems, there is an
interaction between the immune system and tumor development,
according to the concept of immunosurveillance.
Bladder cancer remains one of the rare examples of
effectiveness of immunotherapy, even if the exact
mechanism of action of the response to BCG therapy in
NIMVT has not yet been clearly established. Dysfunctions
of the immune response have been specifically highlighted
in bladder cancer[80]. This immunosuppression could be
explained by the synthesis of molecules inhibiting the
activity of T lymphocytes by the tumor cells themselves.
These co-regulatory molecules generate a reduction in the
immune response through anergy or apoptosis of T
lymphocytes. This is the case in particular of PD-L1, a
surface glycoprotein which acts as a co-regulatory ligand
by binding to the transmembrane receptor PD-1. Its
expression appears aberrant in many cancers, suggesting
the benefit of blocking this pathway with monoclonal
antibodies.[81,82]. Furthermore, the overexpression of
other inhibitory costimulatory molecules, such as CTLA-4,
has been reported in other cancers, again with a potential
therapeutic implication.[83].
Immunotherapy should soon find a new place in the
therapeutic management of bladder cancers, since there
appears to be protein overexpression of PD-L1 correlated
with tumor stage and grade.[84]with a controversial
prognostic role particularly in IMVT[85,86]. Finally, it would
appear that tumors with microsatellite instability (MSI)
present a particular immune microenvironment with
overexpression of PD-1, PD-L1, CTLA-4 and LAG-3
molecules which may contribute to their cellular
development by escaping control. immune
[87]. Initially highlighted in colorectal cancers, this
information could prove useful to optimize the
management of MSI+ urothelial carcinomas, particularly in
the upper excretory tract.
1031
 G. Pignot, C. le Goux, I. Bieche
Synthesis

Other therapeutic avenues
The new molecular alterations highlighted thanks to
recent genome-wide analyzes suggest the potential place
of new targeted treatments in bladder tumors. We can in
particular mention:
- MDM2 inhibitors, which could be of interest in the 12% of
patients with an alteration of the geneMDM2,and
provided thatTP53is not mutated to direct tumor cells
towards apoptosis[9];
- bromodomain agents that target chromatin
modifications (76% of bladder tumors with at least one
mutation in a chromatin remodeling gene
[9]) ;
- telomerase inhibitors, acting by inhibiting the catalytic subunit
hTERT (the gene of which is mutated in more than two thirds of
urothelial carcinomas[27]) or chaperone proteins, by blocking
the substrate RNA with antisense oligonucleotides, or by
modifying the structure of telomeric DNA;
- MEK inhibitors, such as selumetinib, targeting the Ras/
RAF/MEK/ERK pathway, have pro-apoptotic anti-tumor
activity which seems interesting as monotherapy or in
combination with conventional chemotherapies[88]. In
bladder tumors, the existence of mutations ofNF1in 8%
and genesRASin almost 10% of cases suggests a
potential therapeutic avenue for future therapeutic trials
[9].
conclusion and perspectives
This next decade will see the emergence of numerous clinical
applications for a patient suffering from bladder cancer based
on the molecular characteristics of their tumor.
The integrative exploitation of all the “omes” (genome,
transcriptome, microRNAome, proteome but also
spliceome, metabolome, secretome, interactome, etc.) will
make it possible to have a global vision of the
pathobiological phenomena within a tumor. Tumor
sequencing will make it possible, through the identification
of mutated genes in the primary tumor then in the
resistant cellular subclones responsible for metastases, to
reliably offer the most appropriate targeted therapy for
each tumor using theranostic biomarkers. “companions”
allowing personalized patient care.
The identification of expression profiles with prognostic
implications could help guide therapeutic indications at
the individual level, as is the case in other cancers. This
rigorous selection of potentially responding patients
guarantees reasoned prescription of these new expensive
treatments on the basis of molecular rationality, according
to the concept of personalized medicine.
The future of systemic treatments for bladder cancer will
undoubtedly be a combination of conventional
chemotherapy, anti-angiogenic treatments,
immunotherapy and targeted therapies directed
specifically against certain signaling pathways, based on
the genetic and epigenetic profile of each patient.
Acquiring these therapeutic advances requires including
patients with bladder cancer in clinical trials, in order to be
able to quickly offer new molecules in the therapeutic
arsenal against this disease with a still poor prognosis.
Declaration of links of interest:the authors declare that they have no conflicts of
interest.

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