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Molecular Pathogenesis and


Diagnostics of Bladder Cancer
Anirban P. Mitra1 and Richard J. Cote1,2
Departments of 1 Pathology and 2 Urology, Keck School of Medicine and Norris
Comprehensive Cancer Center, University of Southern California, Los Angeles,
California 90033; email: amitra@usc.edu; cote r@ccnt.usc.edu

Annu. Rev. Pathol. Mech. Dis. 2009. 4:25185

Key Words

First published online as a Review in Advance on


October 7, 2008

urothelial carcinoma, risk factors, cellular pathways, prognosis,


combined marker analysis

The Annual Review of Pathology: Mechanisms of


Disease is online at pathmechdis.annualreviews.org
This articles doi:
10.1146/annurev.pathol.4.110807.092230
c 2009 by Annual Reviews.
Copyright 
All rights reserved
1553-4006/09/0228-0251$20.00

Abstract
Despite elaborate characterization of the risk factors, bladder cancer is
still a major epidemiological problem whose incidence continues to rise
each year. Urothelial carcinoma is now recognized as a disease of alterations in several cellular processes. The more prevalent, less aggressive,
recurrent, noninvasive tumors are characterized by constitutive activation of the Ras-MAPK pathway. The less common but more aggressive
invasive tumors, which have a higher mortality rate, are characterized
by alterations in the p53 and retinoblastoma pathways. Several diagnostic tests have attempted to identify these molecular alterations in
tumor cells exfoliated in the urine, whereas prognostic tests have tried
to identify aberrations so as to predict tumor behavior and identify therapeutic targets. The future of bladder cancer patient management will
rely on the use of molecular tests to reliably diagnose the presence of disease, predict individual tumor behavior, and suggest potential targeted
therapeutics.

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252

Mitra

Cote

or a n gi o ge nes

m
Tu

Gene
regulation
Signal
transduction

Cell
growth

on

The incidence of UC is three to four times


higher in males than in females (2). The increased incidence of UC in men has been
attributed to environmental and dietary exposures, innate sexual characteristics (e.g.,

Cell
death

r ce ll i nv asi

RISK FACTORS FOR


BLADDER CANCER

Cell-cycle
regulation

mo

Relative risk: ratio of


the risk of disease
among the exposed
population to the risk
among the unexposed

Cancers of the urinary bladder present as


urothelial carcinoma (UC) or as transitional
cell carcinoma, squamous cell carcinoma, adenocarcinoma, and certain other rare subtypes
(1). Global estimates suggest that in 2002,
approximately 357,000 bladder cancer cases
were diagnosed and that approximately 145,000
patients succumbed to the disease (2). Based
on 20022004 estimates, 2.35% of the individuals born in the United States today will be
diagnosed with UC during their lifetime (3).
UC is the fth most expensive cancer to treat,
accounting for $3.7 billion in direct costs (4).
There is strong evidence that the process
of malignant transformation of the bladder
urothelium is due to alterations in molecular
pathways that are otherwise responsible for the
maintenance of cellular homeostasis (5). Several key molecules and pathways that regulate
critical cellular processes have been identied
as important in the course of urothelial tumorigenesis and progression (Figure 1). These include ve intrinsic processes that can respond
to external carcinogenic cues or become internally deregulated due to genetic alterations:
cell-cycle regulation, cell death, cell growth,
signal transduction, and gene regulation. Also
important are two extrinsic processes that
contribute to tumor maintenance and progression by interacting with stromal elements and
adjoining cells: angiogenesis and tumor cell invasion. Here we review the various risk factors
that can alter these processes, the impact of
these genetic and molecular alterations on tumor progression and prognosis, and how identication of these alterations can help in the
early diagnosis of UC.

Tu

UC: urothelial
carcinoma

is

INTRODUCTION

Figure 1
Aberrant cellular processes contributing to bladder
tumorigenesis. Malignant transformation of the
bladder urothelium involves alterations in ve
intrinsic cellular processes (central pie) that can
respond to external carcinogenic cues or that can be
affected by genetic alterations. Tumor maintenance,
progression, and metastasis also depend on two
extrinsic processes, angiogenesis and invasion,
which regulate tumor interaction with stromal
elements and adjacent cells.

anatomic differences), urination habits, and


hormonal factors (1). High exposure to tobacco
smoke and occupational exposure to aromatic
amines, the two major environmental risk factors, also contribute to this effect. The disease
is more common in Caucasians than in African
Americans, Hispanics, and Asians (3). The risk
increases with advancing age, and most cases
are diagnosed in individuals between 65 and 84
years of age (6).
A variety of lifestyle choices, occupations,
dietary factors, drugs, urologic pathologies,
family histories, and genetic polymorphisms increase UC risk (Table 1). In the United States,
tobacco smoking is the most important risk
factor. The relative risk of UC development
in smokers is two to four times that of nonsmokers, and it increases with the number of
cigarettes smoked and the duration of smoking (7). Former smokers generally have a lower
risk of UC than do current smokers. Tobacco
smoke is rich in aromatic amines and other

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Table 1

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Risk factors for bladder cancer

Risk factor

Mechanism of carcinogenesis

Primary cellular
process(es) altered

Strength of
association

Reference(s)

Lifestyle
Tobacco smoking

Exposure to carcinogens in tobacco


smoke, including aromatic amines,
hydrocarbons, and tar

Cell-cycle regulation,
gene regulation

Strong

Hair dye use

Exposure to aromatic amines

Cell-cycle regulation

Weak

10

Dyestuff manufacturing

Exposure to aromatic amines and aniline


dyes

Cell-cycle regulation,
gene regulation

Strong

Rubber manufacturing

Exposure to aromatic amines, aniline,


and o-toluidine

Cell-cycle regulation

Strong

14

Painting

Exposure to aromatic amines and aniline


dyes

Cell-cycle regulation,
gene regulation

Moderate

15

Leather processing

Exposure to aromatic amines

Cell-cycle regulation

Moderate

16

Printing

Exposure to aromatic amines and aniline


dyes

Cell-cycle regulation,
gene regulation

Weak

16

Hairdressing

Exposure to aromatic amines from hair


dyes and gels

Cell-cycle regulation

Weak

10

Aluminum smelting

Exposure to polycyclic aromatic


hydrocarbons

Cell-cycle regulation

Strong

17, 18

Asphalt paving

Exposure to polycyclic aromatic


hydrocarbons

Cell-cycle regulation

Inadequate

19

Fireghting

Exposure to aromatic amines and


polycyclic aromatic hydrocarbons

Cell-cycle regulation

Weak

20

Truck driving

Exposure to diesel exhaust

Cell-cycle regulation

Moderate

21

Chlorine and chlorination


by-products (in drinking
water)

Direct carcinogenic effect

Unconrmed

Moderate

22

Arsenic (in drinking water)

Direct carcinogenic effect

Cell-cycle regulation,
signal transduction,
gene regulation

Strong

24

Coffee

Carcinogenic metabolites from caffeine


in the urine

Unconrmed

Inadequate

27

Articial sweeteners

Unknown in humans

Unconrmed

Inadequate

29

Induction of DNA fragmentation

Gene regulation

Moderate

1, 30

Schistosoma haematobium

Exposure to toxins and N-nitrosamines

Gene regulation

Strong

32

Cystitis or other urinary


tract infection

Chronic inammation

Cell-cycle regulation, cell


death, gene regulation

Moderate

Urinary calculi

Chronic inammation

Cell-cycle regulation, cell


death, gene regulation

Weak

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Occupation

Diet

Drugs and therapies


Phenacetin,
cyclophosphamide,
pelvic irradiation
Urologic pathologies

(Continued )
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Table 1

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(Continued )

Risk factor

Mechanism of carcinogenesis

Primary cellular
process(es) altered

Strength of
association

Reference(s)

Ancestry and genetics


Genetic predisposition

Depends on the genetic


alteration(s)

Strong

33

NAT2 polymorphism

Inefcient detoxication of aromatic


amines

Gene regulation

Strong

35

NAT1 polymorphism

Promotion of formation of DNA adducts


of aromatic amines

Gene regulation

Inadequate

37

GSTM1 polymorphism

Inefcient detoxication of carcinogens

Gene regulation

Weak

38

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Family history

carcinogens that can form highly reactive


species and DNA adducts (8). Defects in genes
that can repair DNA damage can lead to further
genetic deregulations and alterations in cellular
homeostasis.
Mutagenic compounds with chemical structures similar to aromatic amines were used in
hair dyes in the early 1970s. Inactivation of the
p53 protein has been suggested to be a plausible
mechanism by which hair dyes alter normal cellular processes (9). However, the association of
personal hair dye use and UC risk is relatively
weak (10). It is possible that the risk is smaller
with the newer formulations of hair dyes, as is
suggested by the decreasing trend in UC incidence among hairdressers in recent decades
(11).
UC incidence has increased with industrialization. Initial evidence indicates that workers
who manufactured aniline dyes used for coloring fabrics were at increased risk (1). These
dyes belong to a class of chemicals known
as arylamines, which include 2-naphthylamine
and aminobiphenyl. An increased risk has been
reported among workers involved in dyestuff
manufacturing involving aromatic amines, production of which has been dramatically reduced
since the 1950s. These aniline dyes and aromatic amines form DNA adducts (12) and reduce the DNA-repair capacity of the cell (13),
thereby making cells more susceptible to DNA
damage. A number of other occupations with
related exposures to aromatic amines have been
associated with increased risk of UC, including rubber manufacturing (14), painting (15),
254

Mitra

Cote

leather processing, and printing (16). Increased


risk is also observed in occupations that entail exposure to polycyclic aromatic hydrocarbons, such as aluminum smelting (17, 18) and
asphalt paving (19). The mechanisms by which
polycyclic aromatic hydrocarbons cause cellular alterations are similar to those of aromatic
amines. Fireghters are exposed to both aromatic amines and polycyclic aromatic hydrocarbons. However, their risk of UC is lower than
in previous decades, primarily as a result of better safety equipment (20). Commercial truck
drivers are also at higher risk, as they are exposed to carcinogens in diesel exhaust (21).
Long-term consumption of water containing chlorine and chlorination by-products can
increase the risk of UC (22). Although their
genotoxicity is equivocal, nongenotoxic mechanisms such as peroxisome proliferation and hypomethylation of DNA may contribute to tumor development (23). High arsenic levels in
drinking water, especially in some regions of
Bangladesh, Taiwan, Argentina, and Chile, are
another major bladder carcinogen (24). Arsenic
exposure has been associated with RASSF1A
promoter methylation (25) and with gains and
deletions of several chromosomes, especially
deletion of part or all of chromosome 17p
(26). Although the data on tea consumption are
largely negative for risk of UC, they are more
controversial for coffee consumption. Studies
have shown a marginally elevated relative risk of
UC in coffee drinkers (27), but no trends in relation to dose or duration of consumption have
been established (24). Finally, although large

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quantities of saccharine do have a carcinogenic


effect in rats (28), the association between articial sweeteners and UC in humans has largely
been inconclusive (29).
Heavy consumption of phenacetincontaining analgesics has been linked to an
increased risk of UC, whereas nonsteroidal
anti-inammatory drugs may lower the risk
(30). Phenacetin has been shown to cause
single-strand DNA breaks, thereby exerting
a genotoxic effect (31). Cyclophosphamide
chemotherapy also increases the risk (1) via the
same carcinogenic mechanism as phenacetin
(31). Pelvic irradiation is also another risk
factor, and UC is an important consideration
in patients with a radiated pelvis who present
with hematuria (1).
In Egypt and parts of the Middle East,
squamous cell carcinoma is the most common
form of bladder cancer. These are Schistosoma
haematobiumendemic areas, and chronic infection with this parasite produces toxins, inammation, and N-nitrosamines, resulting in
DNA damage (32). Chronic bladder inammation secondary to urinary tract infection or to
an indwelling foreign body, such as a stone or a
catheter, is also related to UC development (1).
UC risk is increased by approximately 50%
to 100% in rst-degree relatives of UC patients (33). Acetyltransferases detoxify aromatic
amines by N-acetylation and O-acetylation of
their N-hydroxy derivatives, although they can
also activate DNA-binding metabolites such
as aryldiamine benzidine (34). Human Nacetyltransferase (NAT) activity is encoded by
two genes, NAT1 and NAT2. The NAT2 enzyme is polymorphic, and in slow acetylators where its activity is reduced, the risk of
UC increases (35). A variant polyadenylation
signal of the NAT1 gene is associated with increased enzyme activity (36) and can promote
the formation of DNA-binding metabolites of
aromatic amines within the urinary bladder.
However, the NAT1 genotype has not been
associated with increased UC risk (37). Homozygous deletion of the GSTM1 gene that
encodes glutathione S-transferase M1, an enzyme involved in detoxifying various carcino-

gens, has been suggested to increase the risk


of UC, although the evidence is not conclusive
(38).

NONINVASIVE AND INVASIVE


BLADDER CANCERS
There are two forms of noninvasive bladder
cancer. The papillary carcinoma (Ta) phenotype has a tendency to recur locally, but it rarely
invades the basement membrane or metastasizes. However, the at carcinoma in situ (CIS)
is a dangerous lesion with a high tendency for
invasion and metastasis. The genesis of Ta tumors follows a molecular pathway that is usually distinct from CIS and the invasive (T1T4)
cancers (39), although these pathways may not
always be mutually exclusive (40) (Figure 2).
Low-grade papillary tumors usually have a constitutively active receptor tyrosine kinaseRas
pathway, exhibiting activating mutations in the
Harvey rat sarcoma viral oncogene homolog
gene (HRAS ) and in the broblast growth factor receptor 3 (FGFR3) gene. Approximately
70% of low-grade Ta tumors harbor FGFR3
mutations, compared with 10% to 20% of invasive tumors (4143) (Figure 3). High-grade
Ta tumors are often characterized by homozygous deletion of the p16INK4a gene (44). In contrast, CIS and invasive tumors show frequent
alterations in the TP53 and RB genes and pathways (45). Loss of heterozygosity of chromosome 9q is usually more prevalent in low-grade
Ta tumors (46), although Hartmann et al. (47)
found chromosome-9 deletions in both dysplastic urothelium and CIS lesions. When the
occasional papillary tumor does transform to
an invasive phenotype, it is usually a result of
the accumulation of additional alterations in
molecules in the p53 pathway (45). p16 alterations have also been identied in invasive tumors (48). Alterations in cadherins; matrix metalloproteinases (MMPs); vascular endothelial
growth factor (VEGF); and thrombospondin1 (TSP-1), which can remodel the extracellular
matrix (ECM) and promote tumor angiogenesis, are seen more commonly in the muscleinvasive (T2T4) tumors (39).
www.annualreviews.org Bladder Cancer

FGFR: broblast
growth factor receptor
Loss of
heterozygosity: loss
of an allele from a
heterozygous cell
locus; can result in
tumor-suppressorgene inactivation if the
other allele is mutated
MMP: matrix
metalloproteinase
VEGF: vascular
endothelial growth
factor
ECM: extracellular
matrix

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9Rb
p16
p53

Rb
CIS

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p16

p53

?
Ta
(HG
)

T1

p21

T2a

p16INK4a

G)
Ta (L

T2b

al
rm ium
No thel
o
ur

9qHRAS

T3a

Lamina
propria

T3b

Epithelium

T4a

3
FGFR3

Musculariss
propria
Perivesical fat

Pros
ostate

Urethra
ret
ECM
remodeling
genes

Figure 2
Composite model for urothelial tumorigenesis and progression. Noninvasive and invasive tumors are
characterized by distinct molecular alterations. Although noninvasive tumors have constitutive activation of
the Ras-MAPK (mitogen-activated protein kinase) pathway, at carcinoma in situ (CIS) and invasive lesions
have alterations in p53 and other cell-cycle-regulatory molecules. Loss of heterozygosity of 9q is more
common in low-grade papillary carcinomas (Ta), although deletions of chromosome 9 are also seen in
progressive CIS. Locations of molecules indicate characteristic alterations that pose a risk for progression of
a particular phenotype. The thickness of the arrows is approximately proportionate to the relative frequency
of occurrence. Locations of the arrow tails and heads correspond to the tumor stage(s) before and after
alteration(s) of the denoted molecule(s), respectively. Abbreviations: ECM, extracellular matrix; FGFR3,
broblast growth factor receptor 3; HG, high-grade; HRAS, protein of the Harvey rat sarcoma viral
oncogene homolog gene; LG, low-grade; Rb, retinoblastoma protein.

In addition to the differences in molecular alterations between noninvasive and invasive UC, there are also marked contrasts in
epidemiology, chromosomal alterations, clinical outcome, and therapeutic management be256

Mitra

Cote

tween these two clinical phenotypes (Figure 3).


Smokers have a much higher risk of developing invasive tumors than noninvasive tumors,
and this is especially true in current smokers (as
opposed to ex-smokers) (49). Invasive tumors

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also have more chromosomal aberrations than


Ta tumors (50). Although the risk of local recurrence is very high in Ta tumors, it decreases
with invasion (51). However, the risks of disease progression and death increase with invasion. Consequently, transurethral tumor resection and intravesical Bacille Calmette-Guerin
are generally recommended in supercial (Ta
T1) tumors, and radical cystectomy is generally
advocated in T1T3 tumors (51).

PROGNOSTIC IMPACT OF
FUNCTIONAL ALTERATIONS IN
BLADDER CANCER
Tumorigenesis involves multiple alterations in
several tightly controlled pathways that otherwise interact normally within a molecular circuitry to maintain cellular homeostasis
(Figure 4). Alterations within many of these
molecular pathways have been identied in UC.
However, it is their net effect on the deregulation of key cellular processes that establishes
the tumor and controls its fate. More importantly, alterations in key molecular markers are
important predictors of outcome and therapeutic response (Table 2), and they may also act as
druggable targets (52).

Alterations in Cell-Cycle Regulation


Alterations in the cell cycle are probably the
most extensively investigated aspects of the
molecular characterization of UC. The cell
cycle is primarily controlled by the p53 and
retinoblastoma (Rb) pathways, which, in turn,
are closely associated with the apoptotic, signal transduction, and gene regulation processes
(Figure 4).
Located on chromosome 17p13.1, the TP53
tumor-suppressor gene encodes for p53, the
central protein of the p53 pathway (53). p53 inhibits cell-cycle progression at the G1 -S transition and mediates its control through the transcriptional activation of p21WAF1/CIP1 . Although
most UCs exhibit loss of a single 17p allele,
mutation in the remaining allele can inactivate
TP53, leading to loss of its tumor-suppressor

function (54, 55). However, loss of heterozygosity on chromosome 17 occurs during the later
stages of UC and is usually associated with a
more aggressive phenotype (56).
Normally, the short half-life of p53 (630
min) prevents its accumulation in the nucleus
(57). However, TP53 mutations result in an altered protein that is resistant to normal regulatory ubiquitin-mediated degradation. This
causes increased intranuclear accumulation of
the protein, which can be detected by immunohistochemistry (58). We and others have
shown that p53 nuclear immunoreactivity is
predictive of outcome particularly for patients
with invasive, organ-conned, node-negative
(T12bN0) UC (5961). From a therapeutic
standpoint, although conventional chemotherapy has limited benets in UC patients, and although cisplatin-based combination therapies
show mixed to modest benets in the adjuvant
and neoadjuvant settings (45), evidence suggests that patients with locally advanced UC
who harbor p53 alterations respond benecially
to adjuvant chemotherapy that contains DNAdamaging agents such as cisplatin (62). The
plausible explanation is that DNA damage to
p53-altered urothelial cells might cause an uncoupling of the S and M phases of the cell cycle,
resulting in apoptosis (63). This led to the institution of the rst international multicenter clinical trial in UC that targeted molecular lesions,
to identify organ-conned invasive UC patients
with the greatest risk of progression (i.e., patients with p53-altered tumors) who would respond best to cisplatin-containing chemotherapy (64).
Although p53 nuclear accumulation is correlated with TP53 mutations (65, 66), a significant discordance does exist. Our studies examining p53 immunoreactivity with the corresponding TP53 mutations in primary UC suggest that although both nuclear accumulation
and gene mutations are independent prognostic indicators, those tumors with a mutated
TP53 and an altered protein phenotype exhibit
the worst prognosis and those with a wild-type
gene and an unaltered protein perform the best
(Figure 5) (67). Furthermore, it appears that
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Superficial

Invasive

Noninvasive
Relative risk of bladder cancer
(compared to nonsmokers)

7
6
5
4
3
2
1
0

Average frequency
of alterations

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Ta

16
14
12
10
8
6
4
2
0

Genetic alterations (% cases)

With respect to the muscularis propria

Muscle-invasive

T1

T2

T3

With respect to the basement membrane

T4

Ex-smoker (<20 cigarettes per day)


Ex-smoker (>20 cigarettes per day)
Current smoker (<20 cigarettes per day)
Current smoker (20-39 cigarettes per day)
Current smoker (>40 cigarettes per day)

Chromosomal loss
Chromosomal gain
Chromosomal amplification

80
70
60
50

HRAS-activating mutation
FGFR3-activating mutation
p53 alteration
p21 alteration
Rb alteration
p16 alteration

40
30
20
10
0

Relative risk

High
Moderate
Low
Recurrence
Progression
Mortality

Minimal
None

Mitra

Recommended therapy

258

Tumor stage

Cote

Yes

Extensive comorbid disease/


poor performance status

Transurethral tumor resection

Intravesical Bacille Calmette-Gurin

Yes

Yes

Palliative
(select
T4aN0)

Radical cystectomy

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the site of TP53 mutation may also be an important prognostic factor.


Located on chromosome 6p21, the
p21WAF1/CIP1 gene encodes for p21, a cyclindependent kinase inhibitor (CDKI) that is
transcriptionally regulated by p53, although
it can also be regulated in a p53-independent
manner (Figure 4). Loss of p21 expression is a
potential mechanism by which p53 alterations
inuence tumor progression. We have shown
that loss of p21 expression is an independent
predictor of UC progression and that the
maintenance of expression appears to abrogate
the deleterious effects of altered p53 (68).
Encoded on chromosome 12q14.3-q15, the
Mdm2 protein is involved in an autoregulatory feedback loop with p53, thus controlling
its activity (Figure 4) (69). Increased p53 levels
transactivate the MDM2 promoter, causing its
upregulation. The translated protein then mediates the proteasomal degradation of p53. The
resultant lowered p53 levels then reduce the
levels of Mdm2. MDM2 amplication has been
observed in UC, and its frequency increases
with increasing tumor stage and grade (70).
Also, a single nucleotide polymorphism (SNP)
in the MDM2 promoter region, SNP309, has
been reported to predict younger age at disease
onset and poorer survival, and it can provide an
enhanced prognostic value when coupled with
TP53 mutation status (71).
MDM2 is transcriptionally inhibited by
p14. The protein is encoded by p14ARF , one

of the two splice variant transcripts derived


from the single CDKN2A locus, which is situated on chromosome 9p21. Because p14ARF
is induced by the transcription factor E2F, it
forms the biochemical link between the Rb
and p53 pathways (72). p14ARF may be inactivated by homozygous deletion (73) or by
varying degrees of methylation of the promoter region (see Sidebar and Supplemental
Table 1; follow the Supplemental Material
link from the Annual Reviews home page
at http://www.annualreviews.org). The other
splice variant, p16INK4a , encodes for p16, which
acts as a CDKI. A study showed that homozygous p16INK4a deletions in supercial UC had a
higher recurrence rate, but it concluded that
only those deletions affecting both p16 and
p14, which deregulate both Rb and p53 pathways, correlated with the worst prognosis (44).
Berggren et al. (73) noted that p14ARF inactivation usually occurs by homozygous deletion,
although p16INK4a is the hotspot for hypermethylation in UC; however, other groups have observed higher methylation rates for p14ARF than
p16INK4a (74).
The retinoblastoma gene (RB), located on
chromosome 13q14, encodes the Rb protein,
which interacts with multiple regulatory proteins involved in the G1 -S transition. Active,
dephosphorylated Rb binds and sequesters the
transcription factor E2F. Upon phosphorylation of the protein (pRb) by cyclin-dependent
kinases (CDKs), E2F is released, resulting in

CDKI: cyclindependent kinase


inhibitor
Single nucleotide
polymorphism
(SNP): genomic
DNA sequence
variation due to
differences in a single
nucleotide; present in
>1% of the population

Supplemental Material

Figure 3
Differences between noninvasive and invasive bladder tumors. Supercial bladder tumors are lesions that do
not invade the muscularis propria (Ta, T1). However, noninvasive tumors refer to those that do not invade
the basement membrane (Ta). (a) The relative risk of developing invasive bladder cancer is higher in smokers
than in nonsmokers. (b) Invasive tumors also have a higher frequency of chromosomal losses, gains, and
amplications. (c) Although mutations in HRAS and FGFR3 decrease with invasion, the opposite is true for
p53, p21, Rb, and p16 alterations. (d ) Invasive tumors have a lower chance for recurrence, but they are more
prone to progression and increased risk of death. (e) Transurethral tumor resection and intravesical Bacille
Calmette-Guerin instillation are generally recommended for supercial tumors (TaT1), and radical
cystectomy is advocated for the invasive (T1T3) tumors. However, transurethral resection may also be
employed in patients with muscle-invasive tumors with extensive comorbid disease or poor performance
status (orange bar), and palliative radical cystectomy may be performed in select T4a patients without
metastatic lymph nodes who are responsive to chemotherapy ( pink bar). Abbreviations: FGFR3, broblast
growth factor receptor 3 gene; HRAS, Harvey rat sarcoma viral oncogene homolog gene; Rb, retinoblastoma
protein.
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EGFR

ErbB-2

VEGFR2

EGFR

Src

that a signicant proportion of Rb-expressing


tumors show clinical consequences of loss of Rb
function (76). Patients with such tumors have
high Rb expression but have a clinical outcome
similar to that of patients with no Rb expression. These tumors with the highest Rb expression levels demonstrate constitutively hyperphosphorylated Rb, which results from loss of
p16 expression and/or cyclin D1 overexpression

Grb2
Sos
Ras

VEGFR2

PIP3
PI3K

JAK

Cytoplasm

RASSF1a

PKC Raf

PLC

PTEN

E-cadherin

PDK1

MEK

Akt

ERK

-catenin

DAPK
Cytokine
receptor

STAT1
STAT3

STAT1
STAT3

ERK

Nucleus

MSK1
RSK

Bad

CDKN2A
Cyclin E
p27 CDK2
Rb
E2F

p21 p53

Mdm2 p14

E2F
pRb

c-Fos

Caspase-6
Apaf-1
Caspase-9
Caspase-7
Caspase-10
Caspase-8
Caspase-3
FADD
ASK1

MKK7

JNK

TRADD
FADD

Gene expression changes

Apoptosis

Bid

TNFR

Cyclin D1
CDK4

Bax

Cytochrome c

Fas

p16

c-Myc

Cdc25a

Bcl-2

Bcl-X L

Mitochondrion

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the transcription of genes required for DNA


synthesis. Inactivating RB mutations that result in loss of protein expression have been observed across all tumor stages and grades of UC
(75). Although it was originally believed that the
presence of Rb nuclear immunoreactivity indicated the presence of an intact functional gene
and that the loss of protein expression indicated
RB mutations, results from our group indicate

FGFR3

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c--Jun
c-Jun
c-Ju
c-J
Jun

HIF

cIAP

TRAF2 NIK IKK IB NF-B


B

NF-B
HIF VEGF

TP
MMP-2
MMP-9

Hyp
ypoxia

Tumor blood
vessell

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uPA plasmiin
n bFGF
bFGF
aFGF
SF

64
integrin

TSP-1 IL-8

ECM

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(77). This provides the biologic basis for constitutive Rb inactivation in the presence of an
intact gene through Rb hyperphosphorylation.
Rb phosphorylation is facilitated by the cyclin/CDK complexes. The specic complexes
that phosphorylate Rb are cyclin D1/CDK4/6
and cyclin E/CDK2. Although a rare event in
UC, the frequency of CDK4 amplication is
signicantly linked to higher grade and invasive potential (70). Negative regulation of the
CDKs is achieved by CDKIs such as p21, p16,
and p27, which act as tumor suppressors. Low
p27 levels have been associated with shortened disease-free and overall survival in UC
(78).
Studies investigating the combined effect of
p53 and Rb pathway alterations on UC prognosis have also shown promising results. Early
studies on combined Rb and p53 expression revealed that abnormal expression of either or
both proteins was signicantly associated with
an increased risk of progression in T1 tumors
(79). Our studies examining the combined effects of p53, p21, and Rb alterations have shown
that an incremental increase in the number
of altered markers is associated with poorer
recurrence-free and overall survival (80). Other
studies have conducted similar analyses using
various marker combinations of p53, p21, Rb,
p16, and Mdm2 to generate prognostic panels
(8185) and have demonstrated a synergism between these markers in bladder tumorigenesis,
progression, and outcome prediction.

ROLE OF METHYLATION
DNA methylation exerts its effects on the human genome
through the mutational burden of 5-methylcytosine, epigenetic
effects of promoter methylation, and induction of chromosomal
instability by DNA hypomethylation. The covalent addition of a
methyl group to cytosine at CpG palindromes by DNA methyltransferase results in the formation of 5-methylcytosine. This increases the probability of C T transition mutations by a deamination reaction; when this occurs in the coding regions of genes,
it can result in a nonfunctional protein. Further, aberrant hypermethylation of CpG islands in the promoter regions of tumorsuppressor genes is a frequent, heritable, yet potentially reversible
mechanism of transcriptional suppression in UC (Supplemental Table 1; follow the Supplemental Material link from the
Annual Reviews home page at http://www.annualreviews.org).
However, variations in the prevalence of methylation levels of
genes reported by various studies in UC hinder their applicability as potential prognostic indicators. This is notable in the case
of DAPK (death-associated protein kinase), which encodes for the
protein that prevents ERK (extracellular signalregulated kinase)
translocation from the cytoplasm to the nucleus, thereby inhibiting signaling via the Ras-MAPK pathway. A similar scenario is
observed with methylation reports on p14ARF and p16INK4a . However, RASSF1A, which encodes for a protein that can inhibit Ras
function, has shown more consistent methylation levels across
studies and holds promise as a reliable prognostic marker.

Alterations in Cell Death Pathways


Supplemental Material

The series of cell death events that occur throughout normal development and in

Figure 4
Intra- and intercellular circuitry contributing to bladder tumorigenesis. A complex network of molecular signaling is involved in
malignant transformation and tumor progression. Mitogenic signals from growth receptors ( gray) on the cell surface are conducted
along signal transduction pathways (molecules in black) to affect cell-cycle regulation (molecules in purple) and apoptosis (molecules in blue).
This leads to gene-expression changes that are controlled by key transcription factors ( yellow). The tumor cell also interacts with factors
controlling angiogenesis ( green) and invasion (orange). Abbreviations: aFGF, acidic broblast growth factor; ASK1, activator of S phase
kinase 1; bFGF, basic broblast growth factor; DAPK, death-associated protein kinase; ECM, extracellular matrix; ERK, extracellular
signalregulated kinase; FADD, Fas-associated protein with death domain; HIF, hypoxia-inducible factor; JAK, Janus kinase; JNK,
c-Jun N-terminal kinase; MEK, mitogen-activated protein kinase (MAPK)/ERK kinase; MMP, matrix metalloproteinase; MSK1,
mitogen- and stress-activated kinase 1; PDK, 3 -phosphoinositide-dependent kinase; PI3K, phosphatidylinositol 3kinase; PIP3 ,
phosphatidylinositol (3,4,5)trisphosphate; PKC, protein kinase C; PLC, phospholipase C; pRb, phosphorylated retinoblastoma
protein; PTEN, phosphatase and tensin homolog deleted on chromosome 10; RSK, ribosomal S6 kinase; SF, scatter factor; TRADD,
tumor necrosis factor (TNF) receptorassociated death domain; TRAF, TNF receptorassociated factor; TSP, thrombospondin; uPA,
urokinase-type plasminogen activator; VEGFR2, vascular endothelial growth factor receptor 2.
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Molecules and processes contributing to urothelial turmorigenesis

Marker/
expression
in urothelial
carcinoma

Molecular
pathway(s)
involved

Normal function

Prognostic impact

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Cell-cycle regulation
p53a

Inhibits G1 -S progression

p53

Increased recurrence; decreased survival;


amenable to cisplatin chemotherapy

p21b

Cyclin-dependent kinase inhibitor

p53

Increased recurrence; decreased survival

Mdm2c

Mediates the proteasomal degradation of p53

p53

Increased with tumor stage and grade

p14b

Inhibits MDM2

p53

Decreased survival

p16b

Cyclin-dependent kinase inhibitor

Rb

Increased recurrence; decreased survival

Rbd

Sequesters E2F; inhibits cell-cycle


progression

Rb

Increased recurrence; decreased survival

CDK4c

Complexes with cyclin D1; involved in the


G1 -S transition

Rb

Increased with tumor stage and grade

p27b

Cyclin-dependent kinase inhibitor

Rb

Decreased survival

Fasb

Activation signals formation of


death-inducing signaling complex; promotes
apoptosis

Extrinsic apoptotic

Decreased cause-specic survival

Bcl-2c

Inhibits caspase activation

Intrinsic apoptotic

Decreased survival; poor prognosis with


adjuvant therapy

Baxb

Releases cytochrome c from mitochondria;


promotes apoptosis

Intrinsic apoptotic

Poor prognosis; decreased overall survival

Caspase-3b

Promotes apoptosis

Common apoptosis
effector

Increased recurrence

FGFR3e

Receptor for broblast growth factor;


transmits growth signals

Ras-MAPK

Increased recurrence

EGFRc

Receptor for epidermal growth factor;


transmits growth signals

Ras-MAPK,
PI3K-Akt

Increased progression; decreased survival

ErbB-2c

Receptor for epidermal growth factor;


transmits growth signals

Ras-MAPK,
PI3K-Akt

Decreased survival

VEGFR2c

Receptor for vascular endothelial growth


factor; transmits angiogenic signals

Ras-MAPK,
PI3K-Akt

Increased with disease stage, invasion, nodal


metastasis

Cell death

Cell growth

Signal transduction
HRASc

Activates Raf and PI3K

Ras-MAPK

Increased in nonprogressing Ta tumors

PKCf

Activates Raf, c-Fos, NF-B; inhibits Bad

PLC/PKC

Increased recurrence

PTENb

Dephosphorylates PIP3 ; antagonizes PI3K


signaling

PI3K-Akt

Decreased with tumor stage and grade

Gene regulation
STAT3c

Regulates gene expression; increases Bcl-2,


Bcl-XL expression

JAK-STAT

Increased recurrence; decreased survival

NF-Bg

Regulates gene expression

NF-B

Increased recurrence with homozygous


insertion

c-Fosc

Regulates gene expression

MAPK

Increased with tumor grade

c-Junc

Regulates gene expression

MAPK

Increased recurrence; decreased survival

262

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(Continued )

Marker/
expression in
urothelial
carcinoma

Molecular
pathway(s)
involved

Normal function

Prognostic impact

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Tumor angiogenesis
HIFc

Transcribes genes responsible for angiogenesis

VEGFc

Promotes angiogenesis through nitric oxide


synthase

TPc

Promotes VEGF and interleukin-8 secretion;


induces MMP

uPAc

Degrades extracellular matrix

bFGFc

Growth factor stimulating angiogenesis

Ras-MAPK

Increased risk of local recurrence

aFGFc

Growth factor stimulating angiogenesis

Ras-MAPK

Increased with increasing stage

SFc

Growth factor stimulating angiogenesis

TSP-1b

Inhibits angiogenesis

p53

Increased recurrence; decreased survival

E-cadherinb

Mediates intercellular adhesion

Cadherin

Increased recurrence and progression;


decreased survival

-cateninb

Links cadherins to the actin cytoskeleton

Wnt/-catenin

Increased progression; decreased survival

64
integrinh

Links collagen VII to the actin cytoskeleton;


transduces regulatory signals

Cytoskeletal
signaling

Decreased survival

MMP-2c

Degrades extracellular matrix

Increased recurrence; decreased survival

MMP-9c

Degrades extracellular matrix

Increased with tumor stage and grade

TIMP-2i

Antagonizes MMP function

Increased recurrence; decreased survival (?)

Increased recurrence; decreased survival


Ras-MAPK,
PI3K-Akt

Increased recurrence and progression;


decreased survival
Increased recurrence
Increased progression; decreased survival

Increased compared to normal controls

Invasion

Altered.
Underexpressed/lost.
c
Overexpressed.
d
Lost/hyperphosphorylated.
e
Overactivated.
f
Overexpressed in membrane.
g
Polymorphic insertion/deletion in promoter region.
h
Lost/overexpressed.
i
Uncertain.
Abbreviations: aFGF, acidic broblast growth factor; bFGF, basic broblast growth factor; CDK, cyclin-dependent kinase; EGFR, epidermal growth
factor receptor; FGFR3, broblast growth factor receptor 3; HIF, hypoxia-inducible factor; HRAS, protein of the Harvey rat sarcoma viral oncogene
homolog gene; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF-B, nuclear factorkappa B; PI3K,
phosphatidylinositol 3-kinase; PIP3 , phosphatidylinositol trisphosphate; PKC, protein kinase C; PLC, phospholipase C; PTEN, phosphatase and tensin
homolog deleted on chromosome 10; Rb, retinoblastoma protein; SF, scatter factor; STAT, signal transducer and activator of transcription; TIMP-2, tissue
inhibitor of metalloproteinase 2; TP, thymidine phosphorylase; TSP-1, thrombospondin-1; uPA, urokinase-type plasminogen activator; VEGF, vascular
endothelial growth factor; VEGFR2, VEGF receptor 2.
b

response to a variety of initiation stimuli is referred to as apoptosis. It is quantied by the


apoptotic index, the ratio of apoptotic cells to
the total number of tumor cells, and has been
correlated to tumor progression as well as to
recurrence-free and overall survival of patients
with supercial tumors (86).

Apoptosis can be initiated by two alternative pathways. The extrinsic pathway involves
activation of death receptors on the cell surface, whereas the intrinsic pathway is mediated
by mitochondria (Figure 4). Both pathways activate caspases that cleave cellular substrates
and lead to the characteristic biochemical
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Estimated probability of not recurring

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1.00
Wt gene, wt protein (n=85)
0.75

Mut gene, wt protein (n=11)

0.50

Wt gene, alt protein (n=28)


0.25
Mut gene, alt protein (n=26)
P < 0.001
0.00

10

15

20

Years since cystectomy


Figure 5
Prognostic relationship between TP53 mutations and p53 protein status.
Patients with bladder tumors with a wild-type TP53 gene and negative p53
immunoreactivity have the best prognosis, whereas those with a mutated TP53
gene and positive p53 immunoreactivity (altered protein) perform the worst.
Tumors in patients with either a mutated gene or an altered protein have
intermediate probabilities of not recurring (logrank P < 0.001). Abbreviations:
alt, altered; mut, mutated; wt, wild-type. Reproduced with permission from
Reference 67. Copyright 2008, American Society of Clinical Oncology.

and morphological changes. Members of the


TNFR (tumor necrosis factor receptor) superfamily include death receptors such as Fas. UC
have been shown to acquire mechanisms to
escape Fas-mediated apoptosis in the course
of malignant transformation (87). A possible
mechanism could be due to the presence of
circulating soluble Fas that antagonizes cellsurface Fas function (88), a nding that has
been corroborated in the urine of supercial
UC patients (89). Decreased Fas immunoreactivity in UC has been associated with a higher
stage and grade, as well as with poorer prognosis (90). Interaction of death receptors with
their respective ligands allows the formation
of a death-inducing signaling complex, which
includes the FADD (Fas-associated death domain) protein. The complex recruits caspase-8
and -10, which function as initiator caspases.
As expected, in vitro tumor-specic caspase8 expression has been shown to induce apoptosis in UC cell lines (91). Initiator caspases
may directly activate effector caspase-3, -6,
and -7, which results in apoptosis. Alternatively,
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they may activate Bid, which can activate the


proapoptotic protein Bax.
The Bcl-2 family of proteins plays a crucial
role in the intrinsic apoptotic pathway; it includes antiapoptotic members such as Bcl-2 and
Bcl-XL as well as proapoptotic members such
as Bax, Bid, and Bad (Bcl-XL /Bcl-2-associated
death promoter). The BCL2 gene, identied
at the chromosomal breakpoint of t(14;18)bearing human follicular B cell lymphomas
(92), encodes the antiapoptotic Bcl-2 protein,
which inhibits caspase activation. Localized on
the outer mitochondrial membrane, it regulates
the formation of megapores that control ionic
ux and maintain potential across the mitochondrial membrane (53). Bcl-2 overexpression
correlates with poor prognosis in UC patients
treated with radiotherapy (93) or synchronous
chemoradiotherapy (94). Bcl-2 can also identify patients with advanced UC undergoing
radiotherapy who may benet from neoadjuvant chemotherapy (95). Bcl-2 immunoreactivity has been associated with decreased tumorfree survival in T1G3 tumors (96) and can be
a good indicator of mortality in supercial UC
in combination with p53 (97). Maluf et al. (98)
established a prognostic index using Mdm-2,
p53, and Bcl-2 where normal expression of all
markers was signicantly correlated with the
best survival probability and where aberrations
in all three markers corresponded to the worst
survival probability. Patients with aberrant expression of either one or two markers had an
intermediate probability of survival. Bcl-XL interacts and works with Bcl-2 in the progression
of low-stage UC (99), and antisense downregulation of this protein in bladder cancer cell lines
sensitizes them to cytotoxic agents (100). Bad
inhibits Bcl-XL and possibly Bcl-2, but there is
little evidence to suggest that alterations in this
molecule contribute to bladder tumorigenesis
(101).
Cellular stress causes rapid opening and
closing of the mitochondrial megapores, which
direct Bax to the site. This alters the mitochondrial membrane potential, causing release of
stored calcium and cytochrome c and thereby
activating effector caspases and leading to

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apoptosis (53). Bax expression is controlled by


p53 and JNK (c-Jun N-terminal kinase). Bax
is an independent predictor of a more favorable prognosis in invasive UC (97, 102, 103).
In the cytoplasm, cytochrome c binds to Apaf1, thereby activating it (53). This complex then
binds to ATP, forming the apoptosome that in
turn activates procaspase-9 to active caspase9. This leads to the activation of the downstream effector caspases that drive apoptosis.
Decreased caspase-3 expression has been associated with a higher probability of disease
recurrence in cystectomy patients (104). The
inhibitor of apoptosis (IAP) proteins, such as
cIAP, bind to and inhibit caspases and may
also promote their ubiquitin-mediated degradation. cIAP-2 has been identied as an important apoptotic regulator of UC cells in vitro, and
its overexpression may make tumors less susceptible to apoptosis-inducing therapies (105).

Deregulation in Cell Growth Signaling


Several peptide growth factors and their associated tyrosine kinase receptors are responsible for modulating growth signals from external
cues and transmitting them via signal transduction pathways into the nuclei of urothelial cells.
Aberrations in these growth factor receptors
and/or the signals transmitted by them can result in an abnormal increase in the rate of transduction of growth signals, thereby leading to
uncontrolled cellular proliferation and tumor
formation.
The FGFR family consists of four active members (FGFRs 14) of high-afnity
cell-surface receptors. Activating mutations of
FGFR3 are the most extensively studied alterations in this receptor family. As mentioned
above, approximately 70% of low-grade Ta tumors harbor FGFR3 mutations, and this gene
is strongly associated with the genesis of lowgrade papillary tumors (4143). One of the
predicted effects of FGFR3 mutation is activation of the Rasmitogen-activated protein kinase (MAPK) pathway (Figure 4). Mutations
in the FGFR3 and Ras genes are mutually exclusive (106), which probably reects activation

of the same pathway by either event. Approximately 82% of grade 1 tumors and Ta tumors
have mutations in either a Ras gene or FGFR3,
suggesting that activation of the MAPK pathway may be an obligate event in most of these
cases.
The epidermal growth factor receptor
(EGFR) family consists of four closely related
receptors that homo- or heterodimerize after
ligand activation and transmit signals via the
Ras-MAPK or phosphatidylinositol 3kinase
(PI3K)-Akt signal transduction pathways, regulating cell-cycle progression, mitogenic signaling, and other processes crucial to cancer progression (Figure 4). Among the best-studied
receptors in this family are EGFR (ErbB-1)
and ErbB-2 (Her2/neu). Unlike FGFR3, however, these receptors are also overexpressed in
invasive tumors (107, 108). Increased EGFR
expression has been associated with increased
probability of progression and death (109111).
Similarly, increased ErbB-2 expression has been
associated with worse disease-specic survival
(108, 112, 113). Interestingly, the combined
expression prole of EGFR and ErbB-2 has
been suggested to be a better predictor of outcome than each individual marker alone (114),
although this nding was not supported by another study (115).
VEGF is an important signaling protein involved in both vasculogenesis (formation of the
embryonic circulatory system) and angiogenesis (growth of blood vessels from preexisting
vasculature). All VEGF family members stimulate cellular responses by binding to VEGF
receptors (VEGFRs). VEGFR2 (KDR/Flk-1)
mediates most of the known cellular responses
to VEGF. VEGFR2 expression has been correlated with increasing disease stage and tumor
invasion into the muscle (116). Our studies have
also shown that VEGFR2 may be an important
determinant for prediction of nodal metastasis
in UC patients (117).

MAPK: mitogenactivated protein


kinase

Alterations in Signal Transduction


A variety of pathways are involved in transducing signals from cell-surface receptors to
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transcription factors in the nuclei. Deregulations in these pathways can alter signal conduction, thereby leading to abnormal regulation of genes. In UC, the most important signaling pathways identied to date include, but
may not be limited to, the Ras-MAPK pathway, the phospholipase C (PLC)protein kinase
C (PKC) signaling cascade, the Janus kinase
( JAK)signal transducer and activator of transcription (STAT) pathway, the PI3K-Akt pathway, and the nuclear factorkappa B (NF-B)
pathway.
Most low-grade papillary tumors show constitutive activation of the Ras-MAPK pathway, generally through activating FGFR3 mutations (45). Activating Ras mutations are found
in approximately 13% of Ta tumors and are
not as common as FGFR3 mutations in this
tumor stage (106). Nevertheless, constitutive
Ras-MAPK pathway activation does represent
a dominant alteration in noninvasive tumors.
HRAS mutations have been observed in exfoliated cells in the urine of patients with low-grade
bladder tumors (118). Recent studies from our
group show that HRAS is signicantly overexpressed in nonprogressing Ta tumors, compared with those that progress to an invasive
phenotype (119). Transduction of mitogenic
signals along the pathway induces MYC, a gene
that encodes the transcription factor c-Myc
(Figure 4) (45). c-Myc regulates cyclin expression and inhibits the activity of CDKIs, thereby
controlling the cell cycle. To date, however, cMyc has not proved to be a good prognostic
indicator for UC (120, 121).
PKC,
a
ubiquitous,
phospholipiddependent enzyme, is involved in signal
transduction associated with cell proliferation,
differentiation, and apoptosis. The binding
of a ligand to a tyrosine kinase receptor
activates PLC and diacylglycerol, subsequently
leading to PKC activation (122). In addition to
regulating transcription factors such as c-Fos
and NF-B, PKC can inhibit Bad activity
and promote Raf, a molecule upstream in the
Ras-MAPK pathway (Figure 4). Studies have
reported that the expressions of PKC isozymes
and decrease with increasing UC grade

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and that the levels of isozymes , , and show


the opposite pattern (123, 124). The ratio of
PKC expression in the membrane to that in
cytosol has been reported to be greater in UC
tissues and higher-grade tumors than in normal urothelium, and patients with supercial
UC with a greater membrane/cytosol PKC
ratio have a higher risk of recurrence after
chemotherapy (125).
The JAK family constitutes a distinct group
of tyrosine kinases that is activated by cytokine
and growth receptors and mediates multiple
signaling pathways in normal cells. Increased
preoperative plasma levels of interleukin-6, a
key ligand for the corresponding cytokine receptor, presumably increase JAK signaling and
are an independent predictor of recurrence
and survival in UC patients (126). Following
JAK activation, the best-characterized molecular events are tyrosine phosphorylation and activation of STATs. In addition, JAKs mediate
the recruitment of other signaling molecules,
including PI3Ks. Certain PI3Ks can also be activated by receptor tyrosine kinase signaling or
by Ras (Figure 4). Use of a PI3K inhibitor
demonstrated increased radiosensitivity in nude
mice bearing bladder tumor xenografts (127).
Once activated and localized to the membrane, PI3K phosphorylates phosphoinositol
lipids generating phosphatidylinositol trisphosphate (PIP3 ). Located on chromosome 10q23,
the PTEN (phosphatase and tensin homolog
deleted on chromosome 10) tumor-suppressor
gene encodes for PTEN, which can dephosphorylate PIP3 and thereby antagonize PI3K
signaling. PTEN can suppress growth and restore chemosensitivity in UC cell lines (128),
and expression of this protein is repressed with
increasing UC stage and grade (129). PIP3 recruits PDK1 and Akt to the plasma membrane.
PDK1 then phosphorylates and activates Akt.
In turn, Akt activates several proteins in the
cell-cycle-regulation process, including Mdm2,
p21, and p27, while inhibiting Bad and Raf activity (Figure 4). Akt is thus a key regulator of
cell proliferation and survival and is often activated in UC (130). Interestingly, epigallocatechin gallate, an antioxidant polyphenol found in

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green tea, has been shown to inhibit PI3K-Akt


activation in bladder cancer cell lines, which results in modulation of the Bcl-2 protein family
and enhances apoptosis (131).
The NF-B pathway controls the activity
of the transcription factor by its tightly regulated interaction with inhibitory IB proteins
(132). IB sequesters NF-B in the cytoplasm
of most resting cells. Growth factors, cytokines,
TNF, and other signals can activate and translocate NF-B into the nucleus by activating IB
kinase proteins that phosphorylate and consequently degrade IB. The NF-B protein is an
important transcription factor that plays a role
in cellular gene regulation (discussed below).

Alterations in Gene Regulation


Changes in cellular homeostasis are ultimately
effected at the gene level, and deregulations
in growth signals and signal transduction affect the functions of key transcription factors
that act in the nucleus. Apart from E2F and cMyc (discussed above), several other transcription factors regulate the expression of key genes
in urothelial cells. Although a comprehensive
discussion of all the genes regulated by important transcription factors is beyond the scope
of this review, certain key factors are especially
important in controlling the cellular processes
and have major implications in urothelial tumorigenesis.
The JAK-STAT pathway results in the activation of STATs, which control the transcription of several important genes, including those
that encode for the proapoptotic proteins Bcl2 and Bcl-XL (Figure 4) (133, 134). Although
the mechanism underlying regulation of the nal target genes is not entirely clear, Stephanou
et al. (133) have shown that STAT1 can reduce
the expression of Bcl-2 and Bcl-XL and that
STAT3 has the opposite effect. Recent reports
from our group indicate that increased STAT3
expression, in combination with other markers,
can predict increased risk of recurrence and decreased survival in UC patients (135).
NF-B is another important transcription
factor that plays an important role in in-

ammation, autoimmune response, cell proliferation, and apoptosis by regulating the expression of genes involved in these processes.
Bacille Calmette-Guerininduced interleukin6 expression by UC occurs as an immediateearly gene pathway that requires NF-B (136).
A study examining a functional insertion/
deletion polymorphism in the promoter region
of NFKB1 showed that supercial UC patients
with the homozygous deletion had a higher risk
of recurrence than those with the homozygous
insertion (137).
c-Fos and c-Jun are a pair of transcription factors that, in combination with other
related proteins, form the AP-1 (activating
protein 1) transcription factor complex (138).
c-Fos and c-Jun are strong transactivators that
control basal and inducible transcription of several genes involved in proliferation, differentiation, apoptosis, and transformation. c-Fos
expression has been correlated with increasing tumor grade (139), whereas c-Jun expression has been correlated with increasing tumor
stage in UC (140). Our recent studies also indicate that increased JUN expression is associated
with poorer recurrence-free and overall survival
in UC (135).

Microvessel density
(MVD): mean
number of blood
vessels over a number
of randomly selected
areas or in the densest
areas of
neovascularization
(known as hot spots)

Tumor Angiogenesis
Angiogenesis involves production of factors
by tumor cells that interact with stromal elements to recruit endothelial cells to the site
of malignancy and establish a vascular supply,
which can provide the required nutrients for
the rapid clonal expansion of the cancer cells.
Angiogenesis is measured histologically by microvessel density (MVD) estimations. We have
shown that MVD is signicantly associated with
disease-free and overall survival in UC (141).
The hypoxia-inducible factors (HIF-1 and
HIF-2) are heterodimeric transcription factors
that are regulated by oxygen concentrations and
that bind to DNA-upregulating neighboring
genes (53). Although hypoxia itself does not
upregulate the transcription of the protein, it
inhibits the oxygen-dependent degradation of
the subunits. HIF-1 overexpression has been
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signicantly correlated with poor prognosis in


UC, especially in conjunction with altered p53
expression (142, 143). HIF-1 has also been
shown to be signicantly correlated with recurrence and survival in supercial UC (144). Recent evidence also suggests that patients who
harbor two SNPs, P582S and A588T, in the
HIF-1 gene have signicantly worse diseasefree and disease-specic survival than those
without a variant allele (145).
HIF induces VEGF transcription downstream. VEGF in turn stimulates nitric oxide
synthase, which stimulates nitric oxide formation and tumor vascularization. VEGF overexpression in supercial UC is associated with
early recurrence and progression to a more invasive phenotype (146). High serum VEGF levels have also been associated with high UC stage
and grade, vascular invasion, CIS, metastases,
and poor disease-free survival (147).
The enzyme thymidine phosphorylase (TP)
promotes the production of interleukin-8 and
the MMPs (148). Expression of TP messenger RNA in invasive UC is 33-fold higher than
in supercial tumors and 260-fold higher than
in normal bladder (149), and the corresponding protein levels in invasive tumors are 8-fold
higher than in supercial tumors and 15-fold
higher than in normal bladder tissue (150). Increased TP nuclear reactivity has been associated with a higher risk of recurrence in supercial UC (151, 152). Experimental evidence
also suggests that interleukin-8 is mitogenic and
chemotactic for endothelial cells and that it enhances angiogenic activity and UC cell line invasion (153).
The MMPs activate basic and acidic broblast growth factors (bFGF and aFGF, respectively), which in turn restimulate the MMPs
to bring about endothelial cell migration
(Figure 4) (53). MMPs also stimulate scatter factor (SF), which stimulates angiogenesis. Additionally, VEGF induces the formation
of urokinase-type plasminogen activator (uPA),
which degrades ECM, facilitating endothelial
cell migration and invasion. uPA generates plasmin that stimulates bFGF, aFGF, and SF downstream. Preoperative plasma uPA levels are sig-

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nicantly associated with disease progression


and death from UC (154). Urine bFGF levels have been shown to correlate with tumor
stage (155) and with local recurrence in UC
patients (156). Urinary aFGF levels in invasive
UC patients also show a strong correlation with
disease stage (157). Also, urinary SF levels are
signicantly elevated in UC patients compared
with other control cohorts (158).
In addition to its role in cell-cycle regulation, p53 plays a key role in angiogenesis.
Our studies have shown an association between
p53 status and the degree of angiogenesis as
measured by MVD; MVD provided additional
prognostic information in patients with p53altered tumors (159). p53 upregulates TSP1, a potent angiogenesis inhibitor. We have
shown that tumors with p53 alterations are associated with low TSP-1 expression and that
these tumors demonstrate higher MVD (160).
Underexpression of TSP-1 is associated with
decreased probabilities of recurrence-free and
overall survival in UC.

Increase in Invasive Potential


The process of invasion is a key feature of
malignant growths. In UC, this process contributes toward the invasion of tumor cells into
the vasculature and lymphatics, as well as to
their spreading to adjacent and distant sites. Although an in-depth analysis of the pathways involved is beyond the scope of this review and has
been covered elsewhere (161), we do mention
some of the key molecules involved.
Ubiquitous in all tissues, cadherins are prime
mediators of intercellular adhesion. They are
localized to adherens junctions and bind to
their counterparts on adjoining cells as homodimers. E-cadherin is the prototypic member of the classical cadherin family, and it
plays a critical role in epithelial cell-cell
adhesion. E-cadherins cytoplasmic domain
is linked to the actin cytoskeleton through
critical interactions with catenins. Decreased
E-cadherin expression has been signicantly
correlated with increased risk of tumor recurrence and progression, as well as with shorter

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survival in UC patients (162165). Furthermore, loss of immunoreactivity of both Ecadherin and -catenin is a strong predictor
of poor progression-free and overall survival in
UC (166).
Integrins are transmembrane glycoprotein
heterodimers that regulate cellular processes,
which, if altered, can promote tumor progression, invasion, and metastasis. They are receptors for ECM proteins such as laminin and
collagen, and they maintain normal tissue architecture (167). The intracellular domains also
connect to the actin cytoskeleton, thus linking
the cytoskeleton to the ECM. The 64 integrin is the most commonly studied in bladder
tumorigenesis. In normal urothelial cells, the
64 integrin has a close relationship to collagen VII, a component of the hemidesmosomal
anchoring complex, and it restricts cell migration by anchoring contacts with laminin. Loss
of polarity of 64 expression has been noted in
supercial UC, and muscle-invasive UCs show
either a loss of 64 and/or collagen VII expression or a lack of colocalization of the two
proteins (168). Patients with tumors that exhibit
weak 64 immunoreactivity perform signicantly better than do those with either no expression or strong overexpression (169).
The tumors ability to degrade the matrix
and invade the basement membrane is facilitated by the actions of several protease families, especially the uPAs and MMPs. High levels
of MMP-2 and MMP-9 have been associated
with increasing stage and grade of UC (170,
171), and MMP-2 overexpression can predict
poor relapse-free and disease-specic survival
(172). Additionally, the MMP-9:E-cadherin ratio is prognostic for disease-specic survival
(173). Tissue inhibitors of metalloproteinases
(TIMPs), proteins produced by the host or
the tumor itself, antagonize MMP function,
thereby inhibiting tumor cell invasion. Although studies have reported poor prognosis
in patients with high MMP-2:TIMP-2 ratios
(174, 175), this nding is not consistent (176).
In fact, increased TIMP-2 expression has also
been associated with worse prognosis (177). Although such paradoxical TIMP behavior may

be explained as a response to elevated MMP


levels (161), it also argues that TIMPs may not
be reliable prognostic indicators. Nevertheless,
molecular markers of invasion are generally robust predictors of patient outcome in UC.

MOLECULAR DIAGNOSTIC
TESTS FOR BLADDER CANCER
Although most of the above discussion focuses
on the identication and use of markers for disease prognosis, molecular diagnostics have only
recently forayed into the eld of UC detection. This has stemmed from the need to provide rapid, reliable, noninvasive, and inexpensive diagnostic tests for UC, as hematuria detection lacks specicity, and routine cystoscopies
are both invasive and expensive (6). Although
development of prognostic markers is based
on a pathway- and process-specic approach,
diagnostic markers can identify more general
molecular characteristics of tumors; however,
these need to be very sensitive and specic in
detecting UC.

Advantages of Urine
as a Marker Source
Diagnosis of UC should preferably be made
using noninvasive procedures, and urine functions as an excellent source of surrogate molecular markers for the disease because very few
organs are exposed to it (as opposed to blood).
Furthermore, although the harsh urinary pH
degrades most proteases, those proteins that do
survive are very specic for the urologic tract,
and many are exclusive to UC. The collection
of exfoliated cells from voided urine that can
identify tumor markers is the basis for cytologic
examination and for various diagnostic molecular assays.

Molecular Tests That Identify Cancer


Markers in Urine
Molecular diagnostic tests for UC essentially
identify tumor-associated antigens or genetic
alterations in tumor cells exfoliated in urine.
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ELISA: enzymelinked immunosorbent


assay

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These include point-of-care tests that can be


performed in the urologists ofce and require
minimal to no training, as well as specialized
tests that are conducted in reference laboratories and that may require microscopic image analysis, enzyme-linked immunosorbent
assays (ELISAs), or polymerase chain reactions
(PCRs) (Table 3).
The bladder tumor antigen (BTA) assays detect human complement factor Hrelated protein (hCFHrp) in the urine that is similar in
composition, structure, and function to human
complement factor H (hCFH). BTA stat is
a point-of-care qualitative immunoassay, and
BTA TRAK is a quantitative ELISA. Urinary
hCFHrp inhibits the detection of hCFH by
the tests, and this combination of positive and
negative signals regulates their sensitivity. Although the reported sensitivity of BTA stat is
extremely variable (9.3%89%), BTA TRAK
has a sensitivity between 52% and 83% (6).
BTA TRAK is reportedly more sensitive in
detecting bilharzial-related UC (178). Urologic
pathologies that can cause hematuria often give
false positive results due to high hCFH concentrations in the serum.
Nuclear matrix protein 22 (NMP22) serves
as a nuclear scaffold and is an important mitotic regulator that is overexpressed in UC. The
NMP22 BladderChek test is a qualitative
point-of-care immunoassay that detects urine
NMP22, and the NMP22 test kit is a quantitative ELISA. The reported sensitivity and specicity of NMP22 BladderChek in UC detection are 55.7% and 85.7%, respectively (179).
The combination of cystoscopy with NMP22
BladderChek is more sensitive in detecting recurrent UC than either test or cytology individually, as well as cystoscopy and cytology combined (180). Although the reported sensitivity
of the NMP22 test kit is more variable than the
specicity (Table 3), different studies have used
different cut-off limits (3.612 U ml1 ; manufacturer cut-off 10U ml1 ), and it is expected
that lowering the cut-off limit will increase sensitivity at the cost of specicity (6). Like BTA
TRAK, the NMP22 test kit is more sensitive in detecting UC with a bilharzial etiology
Mitra

Cote

(178, 181). As NMP22 is released from dead


and dying urothelial cells and is also abundantly
present in leukocytes, many benign urologic
pathologies such as inammation, urolithiasis,
and bowel interposition can cause a false positive reading (182, 183).
BLCA-4, another protein component of the
nuclear matrix, is found throughout the urothelium (both tumor and adjacent normal) in UC
patients exclusively (6). Variations of the ELISA
have been used to detect the presence of BLCA4 in the urine. Studies have reported sensitivities between 80% and 96.4% and specicities
between 87% and 100% (6).
High VEGF levels in UC cells increase vascular permeability, which results in leakage of
serum proteins including plasminogen, brinogen, and clotting factors. Clotting factors convert plasminogen to plasmin and brinogen to
brin. Plasmin can further degrade brin to brin degradation products (FDPs) (184). Urinary
brin and FDP are measured by Accu-Dx (formerly known as AuraTek FDP), a single-step,
point-of-care gold-dye particle immunoassay.
Because brinogen is present in blood, the test
may be falsely positive in individuals with benign urologic pathologies that cause hematuria.
Cytokeratins are intermediate lament proteins that are components of the epithelial cellular cytoskeleton. Identication of urotheliumspecic cytokeratins in the urine can therefore
serve as surrogate markers for UC. Urinary levels of cytokeratins 8 and 18 are measured by
two urinary bladder cancer (UBC) quantitative
assays: UBCTM II ELISA and UBCTM IRMA.
The sensitivity of the UBCTM tests varies from
48.7% to 70% (185), but it could be as low as
25% and 13% for low-stage and low-grade UC,
respectively (186).
Hyaluronic acid (HA) is a nonsulfated glycosaminoglycan that regulates cell adhesion,
migration, and proliferation and promotes tumor progression and metastasis. Hyaluronidase
(HAase) cleaves HA into small fragments that
promote tumor angiogenesis. Urinary levels of
HA and HAase are measured by two similar
ELISA-like assays. The combined sensitivity
for both tests (collectively, the HA-HAase test)

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is higher than the individual sensitivities for


each test (187). Schroeder et al. (188) also noted
that the sensitivity and specicity for the HAHAase test were equal to or better than those for
cytology, BTA stat, hematuria detection and
UBCTM .
Telomerase, a ribonuclease-protein complex
that adds telomeres to the 5 chromosomal
ends, thereby imparting cellular immortality,
represents yet another surrogate UC marker.
Telomerase activity is measured by the telomeric repeat amplication protocol assay, which
involves (a) PCR amplication of a telomeric
template by telomerase present in exfoliated
cells and (b) analysis by a telomerase PCR
ELISA kit or real-time PCR (6). A related assay
measures the messenger RNA levels of human
telomerase reverse transcriptase, the catalytic
subunit of telomerase, by reverse-transcriptase
PCR. The sensitivity of both assays is between
70% and 100% (186). Because telomerase is expressed in proliferating cells and leukocytes, benign conditions such as urinary tract infections
and severe inammation can cause false positive
results.
Microsatellite alterations in UC are detected by PCR, wherein a panel of multiple
microsatellite markers (1520 loci on different
chromosomes) are tested on exfoliated UC cells
and compared with corresponding sequences in
peripheral lymphocytes. The detection of microsatellite alterations requires a ratio of tumor DNA to contaminating normal DNA of
more than 0.5% to 25% (186). However, the
use of dissimilar microsatellite markers in different studies limits the comparison of the test
across studies, thereby limiting the clinical application of this diagnostic tool.
The UroVysionTM test is a multitarget, multicolor uorescent in situ hybridization assay
that uses pericentromeric enumeration probes
to detect aneuploidy for chromosomes 3, 7, and
17; it additionally uses a locus specic identier
to detect loss of the 9p21 locus. The criteria
for detecting UC are (a) 5 cells with a gain of
2 chromosomes, (b) 10 cells with a gain of 1
chromosome, or (c) 20 cells with a loss of the
9p21 locus. A potential advantage of this test

is its apparent ability to detect occult tumors


that are not initially visible via cystoscopy (6).
Chromosomal abnormalities detected in exfoliated cells in urine have preceded cystoscopically identiable UCs by 0.25 to 1 year in 41%
to 89% of patients under surveillance.
Lewis X is the most commonly studied UCassociated glycoprotein antigen. It is visualized
in exfoliated tumor cells by an immunocytochemical assay using antiLewis X monoclonal
antibody. At least 100 cells are required for evaluation, and slides showing more than 5% positive cells for Lewis X are considered positive.
Lewis X immunocytology of two consecutive
urine samples instead of one can increase the
sensitivity by almost 15% (189, 190). Although
some reports have indicated that the test has
higher sensitivity than BTA stat, NMP22,
and UroVysionTM , its specicity is lower (191,
192).
ImmunoCytTM is an immunocytouorescence test that uses monoclonal antibodies to
detect a glycosylated form of carcinoembryonic antigen and mucin glycoproteins on exfoliated UC cells. A minimum evaluation of
500 epithelial cells is required, and presence of
one uorescent cell constitutes a positive test.
Combining ImmunoCytTM in standard urinary
cytology protocol can increase sensitivity (to
approximately 86% to 90%) without signicant
loss in specicity (6). However, a steep learning
curve, interobserver variability, need for constant quality control, and a relatively high testfailure rate due to inadequate specimen cellularity need to be overcome to improve general
acceptance of this test.
As with prognostic markers, studies have
concluded that using diagnostic marker panels (or markers in conjunction with cytology
or cystoscopy) can increase the sensitivity versus using single markers alone. For example,
Hautmann et al. (193) noted that although the
individual sensitivities of the HA-HAase test,
ImmunoCytTM , and cytology for UC detection were 83.3%, 63.3%, and 73.3%, respectively, the combination of the HA-HAase test
with ImmunoCytTM resulted in a sensitivity of
93.3%. When both tests were combined with
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Qualitative immunoassay;
detects NMP22

Qualitative immunoassay;
detects brin and FDP

NMP22
BladderCheka,b
(Matritech, Inc.,
Newton, MA)

Cote

Accu-Dxa
(PerImmune Inc.,
Rockville, MD)

Quantitative ELISA;
detects hCFHrp

Quantitative microplate
sandwich ELISA; detects
NMP22
Immunoblot, quantitative
ELISA
Quantitative solid-phase
sandwich monoclonal
assay
Quantitative ELISA;
detects HA and HAase

BTA TRAKa,b
(Polymedco, Inc.,
Cortlandt Manor,
NY)

NMP22a,b
(Matritech, Inc.,
Newton, MA)

BLCA-4

UBC II ELISAb ,
UBC IRMAb
(IDL Biotech,
Bromma, Sweden)

HA-HAase

91100

48.770

8096.4

48100

5283

6870

55.7

9.389

7088.8

7295

87100

7091

5090

6886

85.7

5090

Specificity
(%)

Sensitive in detecting both


low- and high-grade/
-stage tumors

No urine stabilization
required

Sensitive in detecting
high-stage and
bilharzial-related tumors

Sensitive in detecting
high-grade/-stage and
bilharzial-related tumors

Sensitive in detecting
high-stage and
bilharzial-related tumors

Sensitive in detecting
high-grade/-stage
tumors

Advantages

UTI

UTI, calculi, bowel


interposition

UTI, calculi, BPH

Microhematuria,
bladder inammation,
radiation cystitis,
urogenital
tuberculosis

UTI, calculi, bowel


interposition

UTI, calculi, BPH

False positives

Poor detection sensitivity


for low-stage/-grade
tumors

Requires urine
stabilization before
transportation

Antibodies cross-react
with hCFH

Test spot may darken


upon drying, giving a
false positive result

Lacks quantitation

Antibodies cross-react
with hCFH

Disadvantages

9 December 2008

Specialized

Qualitative immunoassay;
detects hCFHrp

BTA stata,b
(Polymedco, Inc.,
Cortlandt Manor,
NY)

Point-of-care

Principle

Detection
Sensitivity
(%)

Molecular diagnostic tests for bladder cancer

ARI

Test

Table 3

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Telomerase PCR ELISA


kit or real-time PCR;
involves PCR
amplication of a
telomeric template by
telomerase

RT-PCR; measures
hTERT mRNA levels

PCR

Multitarget, multicolor
uorescent in situ
hybridization assay
Immunocytochemistry;
detects Lewis X antigen
Immunocytouorescence;
detects a glycosylated
form of
carcinoembryonic
antigen and mucin
glycoproteins

TRAP

hTERT

Microsatellite
analysis

UroVysiona,b
(Abbott Molecular,
Inc., Des Plaines,
IL)

Lewis X

ImmunoCyta,b
(DiagnoCure, Inc.,
Quebec, Canada)

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6070

36.986.4

8996

80100

Increased sensitivity with


cytology without
signicant loss of
specicity

Sensitive in detecting
high-grade/-stage
tumors

Sensitive in detecting CIS,


high-grade, and occult
tumors

Advantages

False positives

Microhematuria, UTI,
BPH

Reactive urothelial
lesions

UTI, BPH

UTI, severe urinary


tract inammation

UTI, severe urinary


tract inammation

Disadvantages

Bladder washings cannot


be used; urine xation
required

Antigen also expressed by


benign umbrella cells of
urothelium

Lower sensitivity for


low-grade/-stage tumors

Requires highly trained


personnel and
sophisticated equipment

Sensitivity affected by
hTERT degradation in
urine and by sample
collection and processing
errors

Needs at least 50
telomerase-expressing
cells to yield a positive
result. Sensitivity
affected by TRAP
degradation in urine and
by sample collection and
processing errors

Approved by the U.S. Food and Drug Administration for bladder cancer diagnosis/management.
Commercially available.
Abbreviations: BPH, benign prostatic hyperplasia; ELISA, enzyme-linked immunosorbent assay; FDP, brin degradation product; HA, hyaluronic acid; HAase, hyaluronidase; hCFH, human
complement factor H; hCFHrp, hCFH-related protein; hTERT, human telomerase reverse transcriptase; RT-PCR, reverse transcriptase polymerase chain reaction; TRAP, telomeric repeat
amplication protocol; UTI, urinary tract infection.

7080

79.894.4

6987

7297

6070

6070

Specificity
(%)

9 December 2008

70100

70100

Sensitivity
(%)

ARI

Principle

Test

Detection

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cytology, the sensitivity increased to 96.7%.


However, this increase in sensitivity may occur at the cost of specicity, thereby leading to
more false positive results. For instance, Tetu
et al. (194) reported that while the sensitivity
of routine cytology and ImmunoCytTM in detecting recurrent UC increased from their individual levels of 29% and 74%, respectively,
to 84% in combination, their respective specicities dropped from 98% and 62% to 61% in
combination.

CONCLUSION
Scientists and clinicians now have an increased
understanding of the risk factors and molecular
alterations that contribute to bladder tumorigenesis and progression. This has led to the
identication and characterization of individ-

ual markers and marker panels that can diagnose UC and predict prognosis. Recent studies have adopted pathway-specic approaches
to identify alterations in cellular processes in
UC that, in turn, can predict clinical outcome in
individual patients. The availability of sophisticated genomic, proteomic, computational, and
statistical tools now offer us the possibility of assimilating existing data to establish diagnostic
and prognostic marker panels that can then be
validated in retrospective and prospective studies. Although we have tried to review the most
signicant pathways that control critical cellular process in urothelial cells, there are several
other lesser-known and uncharacterized cellular pathways that may also contribute towards
UC development. Future studies will aim to
identify these molecules and pathways and their
impact on tumor behavior and prognosis.

SUMMARY POINTS
1. UC is the most common type of bladder tumor, and it is as much of an economic problem
as it is a public health problem.
2. Exposure to tobacco smoke, aromatic amines, and aniline dyes are the most important
risk factors for UC. Arsenic exposure and Schistosoma haematobium infestation are also
important factors in endemic areas.
3. Noninvasive bladder tumors generally have a more distinct set of molecular alterations
and clinical behavior than the more aggressive invasive tumors.
4. Interaction of molecular pathways involved in the ve intrinsic cellular processes
cell-cycle regulation, cell death, cell growth, signal transduction, and gene regulationis
important in maintaining homeostasis of urothelial cells. Aberrations in one or more of
these processes are responsible for malignant transformation of the urothelium.
5. The extrinsic processes of angiogenesis and tumor cell invasion are also responsible
for tumor maintenance and progression.
6. In addition to the individually prognostic markers that have been identied in several
pathways involved in bladder tumorigenesis, recent studies have identied prognostic
panels comprising markers across multiple tumorigenic pathways.
7. Molecular diagnostic tests for UC are able to detect tumors using urine as a noninvasive
marker source. However, reaching optimal levels of sensitivity and specicity for most
clinically approved tests is still a challenge, thus necessitating intense and often expensive
surveillance and patient follow-up.

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FUTURE ISSUES

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1. Although many individual prognostic markers have been identied in UC, there is still a
lack of consensus on which markers can be employed clinically. Reasons include differences in study designs and patient cohorts, inconsistent immunohistochemical staining
criteria, different cut-off points for determination of positivity, and variable outcome parameters. Future studies need to validate the markers identied thus far in large prospective clinical cohorts by employing standardized study and analysis protocols.
2. Studies have already started identifying prognostic panels that consist of multiple markers. However, efforts must made to generate concise prognostic marker panels that are
pathway based, rather than combining markers without biologic rationale. Such panels will serve as robust clinical predictors while also providing further insight into the
biologic mechanisms behind UC progression.
3. Molecular tests currently employed for UC detection either lack portability or suffer
from lack of sensitivity and specicity. Concerted efforts must be made to identify reliable
diagnostic markers that can be assessed by rapid, economical, and noninvasive point-ofcare tests.
4. Identication of new diagnostic and prognostic markers is concomitantly generating a
vast library of druggable targets in UC, many of which are already targets for novel
therapeutics. This provides an impetus for initiation of clinical trials targeting patients
with tumors that harbor specic molecular lesions with such therapeutics, and will spur
discovery of other novel compounds that can target additional molecular alterations.

DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of this
review.

ACKNOWLEDGMENTS
Research on bladder cancer in the authors laboratory is supported by National Institutes
of Health/National Cancer Institute grant numbers CA86871, CA7192109, CA123027, and
EB008275; the L.K. Whittier Foundation; the Dhont Family Foundation; and the Candy Foundation. The authors apologize to the investigators whose studies could not be appropriately cited
due to space limitations.

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21
www.annualreviews.org Bladder Cancer

35. Meta-analysis
documenting that
NAT2 slow acetylators
have approximately 40%
increased risk of bladder
cancer compared to
rapid acetylators.

45. Reviews the


Ras-MAPK, p53,
retinoblastoma, and
angiogenesis pathways;
bladder cancer
epigenetics; and
potential for novel
targeted therapeutics.

49. Identifies various


risk factors for bladder
cancer using data on
1860 cases and 3934
population-based
controls.

277

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59. Demonstrates that


p53 nuclear
immunoreactivity
predicts a poor
prognosis in bladder
cancer patients.

62. Shows that p53altered bladder tumors


that include DNAdamaging agents such
as cisplatin are more
sensitive to adjuvant
chemotherapy.

67. Shows that the


combination of p53
protein and gene
statuses is a more
effective outcome
predictor than either
one alone.

278

15:4

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www.annualreviews.org Bladder Cancer

80. Examines the


combined effects of
alterations in specific
molecules in the p53
and retinoblastoma
pathways on bladder
cancer outcome.

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generate gene
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Contents

Annual Review of
Pathology:
Mechanisms of
Disease
Volume 4, 2009

The First Fifty Years in Research


Peter A. Ward p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Graft Vascular Disease: Immune Response Meets the Vessel Wall
Richard N. Mitchell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p19
Molecular Pathology of Head and Neck Cancer: Implications for
Diagnosis, Prognosis, and Treatment
Sara I. Pai and William H. Westra p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p49
Mechanisms of Endothelial Dysfunction, Injury, and Death
Jordan S. Pober, Wang Min, and John R. Bradley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p71
The Pathogenesis of Pituitary Tumors
Sylvia L. Asa and Shereen Ezzat p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p97
PTEN and the PI3-Kinase Pathway in Cancer
Nader Chalhoub and Suzanne J. Baker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 127
Pathogenesis of Classical and Lymphocyte-Predominant
Hodgkin Lymphoma
Roland Schmitz, Jens Stanelle, Martin-Leo Hansmann, and Ralf Kppers p p p p p p p p p p p p p 151
Molecular Genetics of Acute Lymphoblastic Leukemia
Michael A. Teitell and Pier Paolo Pandol p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 175
MicroRNAs in Cancer
Yong Sun Lee and Anindya Dutta p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 199
Epigenetic Changes in Cancer
Christine A. Iacobuzio-Donahue p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 229
Molecular Pathogenesis and Diagnostics of Bladder Cancer
Anirban P. Mitra and Richard J. Cote p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 251
Ovarian Cancer
Kathleen R. Cho and Ie-Ming Shih p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 287

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Drosophila Models of Neurodegenerative Diseases


Bingwei Lu and Hannes Vogel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 315
Serrated Polyps and Colorectal Cancer: New Pathway to Malignancy
Amy E. Noffsinger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 343
Nod-Like Receptors: Role in Innate Immunity and Inammatory
Disease
Grace Chen, Michael H. Shaw, Yun-Gi Kim, and Gabriel Nunez
p p p p p p p p p p p p p p p p p p p p p p p 365
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Tumor Suppressors, Chromosomal Instability, and Hepatitis C


VirusAssociated Liver Cancer
David R. McGivern and Stanley M. Lemon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 399
The Immunopathogenesis of Rheumatoid Arthritis
John B. Imboden p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 417
The Pathology of Chronic Obstructive Pulmonary Disease
James C. Hogg and Wim Timens p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 435
Linking the Cellular Functions of BRCA Genes to Cancer
Pathogenesis and Treatment
Ashok R. Venkitaraman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 461
Regulation of Hepcidin and Iron-Overload Disease
Pauline L. Lee and Ernest Beutler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 489
The Brainstem and Serotonin in the Sudden Infant Death Syndrome
Hannah C. Kinney, George B. Richerson, Susan M. Dymecki, Robert A. Darnall,
and Eugene E. Nattie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 517
Molecular Pathogenesis of Cutaneous Melanocytic Neoplasms
Nageatte Ibrahim and Frank G. Haluska p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 551
Indexes
Cumulative Index of Contributing Authors, Volumes 14 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 581
Cumulative Index of Chapter Titles, Volumes 14 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 583
Errata
An online log of corrections to Annual Review of Pathology, Mechanisms of Disease articles
may be found at http://pathol.annualreviews.org

vi

Contents

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