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Bladder Cancer
Protein Expression Patterns of Ezrin Are Predictors of
Progression in T1G3 Bladder Tumours Treated with
Nonmaintenance Bacillus Calmette-Guerin
Joan Palou a, Ferran Algaba a, Irene Vera b, Oscar Rodriguez a, Humberto Villavicencio a,Marta Sanchez-Carbayo b,*
a Fundacio Puigvert, Barcelona, Spainb Tumor Markers Group, Spanish National Cancer Research Center, Madrid, Spain
E U R O P E A N U R O L O G Y 5 6 ( 2 0 0 9 ) 8 2 9 – 8 3 6
ava i lable at www.sciencedirect .com
journal homepage: www.europeanurology.com
Article info
Article history:
Accepted September 30, 2008Published online ahead ofprint on October 9, 2008
Keywords:
Ezrin
BCG
Bladder cancer
Immunohistochemistry
Tissue arrays
Abstract
Background: Bacillus Calmette-Guerin (BCG) is a standard treatment for reducing tumour
recurrence and delaying progression of high-risk, non–muscle-invasive bladder tumours.
However, it is not clear yet which patients are more likely to be responders to BCG.
Objective: To evaluate the role of ezrin expression in bladder cancer (BCa) progression in
T1G3 bladder tumours treated with BCG.
Design, setting, and participants: Ezrin protein expression patterns were analysed on
tumour specimens belonging to 92 patients with T1G3 non–muscle-invasive BCa
undergoing nonmaintenance BCG treatment. Re-resection was not performed. The
median follow-up was 90.5 mo (range: 3.0–173.0). A specific tissue array was created
containing three representative cores of each of the tumour specimens belonging to
these patients.
Measurements: Ezrin protein expression patterns were assessed by immunohistochem-
istry on this tissue array. Proliferation rates were assessed by means of Ki67 staining.
Recurrence, progression into muscle-invasive tumours, and disease-specific overall sur-
vival (OS) rates were analysed using univariate and multivariate tests.
Results and limitations: Among the 92 patients analysed, 40 recurred (43.5%), 17
progressed (18.5%), and 14 died of the disease (15.2%). Log-rank survival analyses
revealed that an ezrin membrane expression <20% was significantly associated with
increased progression ( p = 0.009) and shorter disease-specific OS ( p = 0.006). Multi-
variate analyses showed that ezrin was an independent prognostic marker of progres-
sion ( p = 0.031) and disease-specific survival ( p = 0.035). Interestingly, the low ezrin
membrane expression correlated with high proliferation rates ( p = 0.033).
Conclusions: Immunohistochemistry analyses revealed that the membrane expression
of ezrin is associated with the clinical outcome of patients with T1G3 tumours under-
going BCG treatment. Protein expression patterns of ezrin were associated with tumour
progression in T1G3 disease. The differential expression of ezrin distinguished patients
responding to BCG from those who may require a more aggressive therapeutic approach.
# 2009 Published by Elsevier B.V. on behalf of European Association of Urology.
* Corresponding author. Tumor Markers Group, 308A, Centro Nacional de Investigaciones Oncologicas,Melchor Fernandez Almagro 3, E-28029 Madrid, Spain. Tel. +34 91 732 8053; Fax: +34 91 224 6972.E-mail address: [email protected] (M. Sanchez-Carbayo).
0302-2838/$ – see back matter # 2009 Published by Elsevier B.V. on behalf of European Association of Urology. doi:10.1016/j.eururo.2008.09.062
E U R O P E A N U R O L O G Y 5 6 ( 2 0 0 9 ) 8 2 9 – 8 3 6830
1. Introduction
Bladder cancer (BCa) is the fourth most frequent neoplasia
in men, clinically characterised by high recurrent rates and
poor prognosis when tumours invade the muscularis propia
[1,2]. The intravesical bacillus Calmette-Guerin (BCG)
immunotherapy represents a highly successful therapy
for patients with non–muscle-invasive BCa [3–6]. Despite
the superior efficacy of BCG over transurethral resection
(TUR) alone or TUR plus intravesical chemotherapy,
eventually, >50% of non–muscle-invasive BCas either
persist or recur. This is a particularly acute problem for
patients with high-risk disease, such as those with
carcinoma in situ (CIS), submucosa invasion (stage T1),
and high-grade papillary disease, because the risk of
progression increases proportionally with a narrowing
window of opportunity for conservative bladder therapy
before cystectomy is mandatory. A significant portion of
these patients fails to respond to BCG therapy; their
tumours not only persist or recur but may also become
invasive or metastatic [6,7]. None of the current tumour
biomarkers evaluated to date has provided sufficient
sensitivity and specificity to predict response to BCG
immunotherapy in any of these three subtypes of high-
risk BCa patients in clinical routine practice [2,6,7].
Improved prognostic biomarkers are required to ultimately
distinguish indolent cancers from those that are potentially
lethal so that therapeutic procedures could be tailored to
each individual patient [2–11].
The ezrin, radixin, and moesin (ERM) and merlin proteins
are closely related members of the band 4.1 superfamily of
proteins that, when activated, interact with both membrane
proteins and the actin cytoskeleton [12–15]. By organising
membrane–cytoskeleton-associated complexes and creat-
ing specialized membrane domains, the ERM proteins
regulate cellular activities such as survival, adhesion,
migration, and invasion, all of which are important during
tumour progression [12–16]. The membrane-linking pro-
tein ezrin is expressed in several types of human cancers,
and correlations of its immunoreactivity and clinicohisto-
pathologic data as well as patient outcome have previously
been shown [17–25]. However, to the best of our knowl-
edge, ezrin expression has not been reported to date in
bladder tissues. In this study, immunohistochemical
analyses were performed in T1G3 bladder tumours treated
with nonmaintenance BCG, aiming to evaluate the role of
ezrin expression in BCa progression and as a therapeutic
predictive marker of BCG response in this type of patient.
2. Methods
2.1. Patient population
Between 1989 and 1996, 92 patients were treated for primary stage T1
grade 3 urothelial carcinoma with complete transurethral resection
(TUR) at the Fundacio Puigvert. Of those, 83 were male (90.2%) and 9
were female (9.8%). The median age was 67.5 yr, ranging from 25 to 81
yr. Among these cases, 55 had concomitant CIS (59.8%), and 45 had
multifocal disease (48.9%). T1 substaging was defined following
previously reported criteria [26]. Primary bladder tumours were
collected following the guidelines for the protection of human subjects
and recruited under Institutional Review Board (IRB)–approved proto-
cols at the Fundacio Puigvert. They all received a 6-wk course of BCG
without maintenance therapy. Criteria for inclusion were that patients
had no prior intravesical treatment, histologic diagnosis (T1G3) with a
wide and deep primary resection including muscle in the specimen, and
paraffin-embedded tissue material adequate for analysis. Patients
having presented previously a tumour of the upper urinary tract were
excluded from the study. Overall, 92 patients were identified and
selected for the present analysis. Second-look TUR was not performed.
Follow-up consisted of cystoscopy with cytology every 3 mo for the first
2 yr and every 6 mo thereafter. Progression was defined as muscular
invasion (stage T2 or higher) or metastatic disease. Patients with
recurrence were treated either with another course of BCG or with
cystectomy when the disease progressed.
2.2. Tissue microarrays
Paraffin-embedded tissues belonging to these 92 patients were used for
the construction of the tissue microarray, as previously described
[27,28]. One section was stained with haematoxylin and eosin to
evaluate the presence of the tumour by light microscopy. After carefully
choosing the morphologically representative region on the paraffin-
embedded blocks (donor blocks), a core tissue biopsy specimen of
0.6 mm was punched and transferred to the donor paraffin-wax–
embedded block (recipient block). The tissue array included three cores
representative of each of the whole tumour for a total of 92 primary
T1G3 non–muscle-invasive bladder tumours that underwent BCG
treatment. Clinicopathologic and annotated follow-up information
available for BCG clinical outcome predictive analyses of the tumours
spotted onto the tissue microarray allowed the evaluation of the
histopathologic and clinical properties as well as the outcome
assessment of the protein expression patterns of ezrin, Ki67, and p53.
For progression analyses, only patients with available follow-up (either
‘‘progressing into invasive disease’’ or ‘‘alive with no evidence of
disease’’) were considered. For overall survival (OS) analyses, only
patients with available follow-up (either ‘‘dead as a result of disease’’ or
‘‘alive with no evidence of disease’’) were included. Cases with unknown
follow-up were excluded from these analyses.
2.3. Immunohistochemistry
The protein expression patterns of ezrin were assessed at the
microanatomic level using both cytospins from cancer cell lines (data
not shown) and the tissue microarrays. Standard avidin-biotin
immunoperoxidase procedures were applied for immunohistochemistry
[28]. Antigen retrieval methods (0.01% citric acid for 15 min under
microwave treatment) were used prior to incubation with primary
antibodies overnight at 4 8C. Ezrin staining was assessed using a mouse
monoclonal antibody diluted at 1:2000 (Sigma; Saint Louis, MO, USA).
P53 was assessed using a mouse monoclonal antibody at 1:50 dilution
(clone D07; Novocastra, Newcastle, UK). Ki67 was assessed using a
mouse monoclonal antibody diluted at 1:100 (clone MIB-1; DAKO,
Glostrup, Denmark). Secondary biotinylated antimouse antibodies
(Vector Laboratories, Burlingame, CA, USA) were used at 1:500 dilution.
The absence of primary antibody was used as a negative control.
Diaminobenzidine was used as the final chromogen and haematoxylin as
the nuclear counterstain. Ezrin immunoreactivity was evaluated in the
membrane and in the cytoplasm. Membrane staining was scored based
on the number of cancer cells presenting this protein sublocalisation.
Cytoplasmic staining was scored 0–3 (absent, weak, moderate, or strong
intensity). Scores were recorded for membrane stainings of ezrin, using
normal brain as positive control. P53 and Ki67 immunoreactivity was
E U R O P E A N U R O L O G Y 5 6 ( 2 0 0 9 ) 8 2 9 – 8 3 6 831
evaluated in the nucleus, as previously reported [9,10,28]. The individual
scores were reviewed, and the agreement between two independent
observers was calculated. Whenever a discrepancy was noted between
the first and the second interpretations, the pathologist decided on the
final scoring.
2.4. Statistical analysis
All cases (n = 92) were used for the analysis of association among ezrin,
Ki67, and p53 with clinicopathologic variables. The consensus (mean)
value of the three representative cores from each tumour sample arrayed
was used for statistical analyses. The association of the expression of
ezrin, Ki67, and p53 with age, sex, and histopathologic variables such as
T1 substaging, tumour size, multifocality, or the presence of associated
CIS was evaluated using the nonparametric Mann-Whitney and
Kruskall-Wallis tests [27]. The distribution of the protein expression
patterns of ezrin, Ki67, and p53 depending on these variables was
described by means of their median and range values. There is no
consensus on the cut-off of the immunohistochemical expression of
ezrin. Thus, the cut-off value for weak- and strong-expressing cases was
specified at the median percentage score of positive membrane tumour
cells, resulting in a value of 20%. Ezrin was then analysed continuously,
taking the cut-off of 20% when considered as a categoric variable [29].
The associations of membrane protein expression patterns of ezrin with
OS were evaluated using this set of 92 cases for which follow-up was
available. OS time was defined as the years elapsed between TUR and
death as a result of disease (or the last follow-up date). Patients who
were alive at the last follow-up or lost to follow-up were censored. The
association of ezrin expression levels with OS was analysed using the
log-rank test to examine its relationship, taking the 20% cut-off
mentioned above [29]. The survival curves were plotted using the
standard Kaplan-Meier methodology. Multivariate Cox regression
analyses were performed to evaluate how ezrin behaves compared
with other clinical and pathologic factors such as sex, age, the presence
of CIS, Ki67, p53, multifocality, tumour size, and T1 substaging [29]. The
simultaneous analysis of the status of Ki67 and p53 in these specimens
allowed comparison of the clinical relevance of the associations of ezrin
Table 1 – Distribution of the protein expression of ezrin, Ki67, and p53on tissue arrays.
Characteristic Ezrin % median (range)
Age p = 0.459
Cut-off �65 (n = 36) 32.5 (0.0–95.0)
Cut-off >65 (n = 56) 0.0 (0.0–100.0)
Sex p = 0.778
Male (n = 83) 12.1 (0.0–100.0)
Female (n = 9) 16.2 (0.0–85.0)
Substaging p = 0.356
1a (n = 44) 0.0 (0.0–95.0)
1b (n = 20) 42.5 (0.0–85.0)
1c (n = 28) 0.0 (0.0–95.0)
Tumour size p = 0.374
<3 (n = 61) 37.5 (0.0–95.0)
>3 (n = 31) 0.0 (0.0–100.0)
Focality p = 0.674
1 (n = 48) 25.0 (0.0–95.0)
2 (n = 17) 22.5 (0.0–100.0)
3 (n = 3) 0.0 (0.0–85.0)
Multiple (n = 24) 1.65 (0.0–95.0)
CIS presence p = 0.847
Yes (n = 55) 17.5 (0.0–100.0)
No (n = 37) 11.7 (0.0–95.0)
with these proteins previously described altered along BCa progression
[28]. Associations among ezrin with Ki67 and p53 were analysed using
Kendall tß test [29]. Only p values <0.05 were considered statistically
significant. Statistical analyses were performed using the SPSS v.11.0
(SPSS Inc, Chicago, IL, USA).
3. Results
3.1. Clinical follow-up of the cases under analyses: overall
results
At the last follow-up, of the 92 patients analysed, 40
recurred (43.5%), 17 progressed (18.5%), and 38 died
(39.1%); 14 of these cases died of BCa (15.2%). Fifty-two
of the patients analysed were alive at the end of the study
(56.5%). Two patients who experienced recurrence over the
time of the study were lost at the last follow-up. Patients
who recurred had recurrence at a median follow-up time of
15.5 mo (range: 1.0–116.0). Patients who progressed had
the progression at a median follow-up time of 17.0 mo
(range: 2.0–126.0). The median time at which patients died
of BCa was 33.5 mo (range: 7.0–136.0). The median time at
which patients died of causes other than BCa was 45.5 mo
(range: 6.0–147.0). The median follow-up time of cases free
of disease was 106.5 mo (recurrence-free survival; range:
51.0–173.0). The median follow-up time considering all the
cases under analyses was 90.5 mo (range: 3.0–173.0).
3.2. Ezrin, p53, and Ki67 expression: clinicopathologic
associations
The cellular expression patterns of ezrin were found in the
cytoplasm and in the membrane in T1G3 bladder tumours.
The immunoreactivity of ezrin in the cytoplasm was
, depending on the clinical characteristics of T1G3 patients spotted
Ki67% median (range) p53 median (range)
p = 0.271 p = 0.884
20.0 (0.0–75.0) 9.2 (0.0–90.0)
25.0 (2.5–65.0) 15.0 (0.0–96.7)
p = 0.007 p = 0.014
20.0 (0.0–75.0) 9.0 (0.0–95.0)
39.8 (25.0–60.0) 55.0 (3.3–96.7)
p = 0.817 p = 0.235
26.3 (0.0–95.0) 7.5 (4.2–75.0)
27.5 (2.5–75.0) 60.0 (0.0–96.7)
25.4 (5.0–60.0) 17.5 (0.0–95.0)
p = 0.465 p = 0.469
24.4 (0.0–95.0) 7.5 (0.0–75.0)
25.8 (3.3–75.0) 15.8 (0.0–95.0)
p = 0.321 p = 0.926
29.2 (0.0–75.0) 15.8 (0.0–96.7)
10.0 (4.2–55.0) 5.0 (0.0–95.0)
20.0 (20.0–35.0) 26.3 (0.0–85.0)
15.8 (2.5–75.0) 8.7 (0.0–85.0)
p = 0.535 p = 0.297
21.2 (4.2–75.0) 8.3 (0.0–90.0)
29.6 (0.0–75.0) 17.5 (0.0–96.7)
Table 2 – Summary of the associations among the proteinexpression patterns of ezrin, Ki67, and p53 in the cases understudy.
Association Kendall tß p value
Ezrin–Ki67 �0.172 0.033
Ezrin–p53 �0.007 0.935
Ki67–p53 0.249 0.001
Fig. 1 – Differential protein expression patterns of ezrin and Ki67 in PT1G3 blamembrane protein expression patterns of ezrin by immunohistochemistry onrepresentative immunostainings of Ki67 by immunohistochemistry on tissue aabove; (E, F) representative immunostainings of p53 by immunohistochemistrtumours shown above.
E U R O P E A N U R O L O G Y 5 6 ( 2 0 0 9 ) 8 2 9 – 8 3 6832
detected in all the cases under study, with various
intensities that did not differ in a statistically significant
manner regarding any of the clinicopathologic variables
under study. Ezrin expression in the membrane differed
among cases, with a wide range from only a few cells
positive from ezrin to nearly 100% of positive tumours cells.
The cut-off value for weak and strong expressing cases was
specified at the median percentage score of positive
dder tumours; (A, B) representative immunostainings of the differentialtissue arrays in non–muscle-invasive (T1G3) bladder tumours; (C, D)rrays in the paired non–muscle-invasive (T1G3) bladder tumours showny on tissue arrays in the paired non–muscle-invasive (T1G3) bladder
Fig. 2 – Ezrin membrane expression was associated with bladder cancerprogression in PT1G3 primary bladder tumours, with Kaplan-Meiercurve survival analysis indicating that a membrane protein expression ofezrin <20% measured by immunohistochemistry on tissue arrays wasassociated with high progression rate into muscle-invasive disease (logrank, p = 0.009).
Fig. 3 – Ezrin membrane expression was associated with poor survival inPT1G3 primary bladder tumours, with Kaplan-Meier curve survivalanalysis indicating that a membrane protein expression of ezrin <20%measured by immunohistochemistry on tissue arrays was associatedwith shorter overall survival (log rank, p = 0.006).
E U R O P E A N U R O L O G Y 5 6 ( 2 0 0 9 ) 8 2 9 – 8 3 6 833
membrane tumour cells, resulting in a value of 20%. Tumour
stroma was always negative for ezrin staining. A lack of
significant correlations was observed among p53, Ki67, and
ezrin expression, with any of the clinicopathologic variables
mentioned above, such as histopathologic T1 substaging,
tumour size, multifocality, or the presence of associated CIS
(Table 1). The statistical association found between the
expression of Ki67 and p53 regarding sex should be
mentioned, although its clinical implications is limited
because of the low number of females included in this
study. Interestingly, the low membrane protein expression
patterns of ezrin correlated with high proliferation rates, as
measured by Ki67 staining in a significant manner
(tß = �0.172, p = 0.033). The correlations among the protein
expression patterns of these proteins are summarised in
Table 2. Thus, although tumour cells showed strong
immunoreactivity in their cytoplasmic patterns, they
showed loss of membrane ezrin reactivity in highly
proliferative cases. Representing immunostainings of ezrin,
Ki67, and p53 are shown in Fig. 1.
3.3. Protein expression patterns of ezrin are associated with
progression into muscle-invasive disease and OS
The progression rate was increased for patients with low
ezrin membrane-expressing tumours compared with those
with strong membrane expression. Log-rank survival
analyses revealed that an ezrin membrane expression
<20% was significantly associated with an increased
progression into muscle-invasive disease (log-rank,
p = 0.009; Fig. 2). Moreover, a shorter disease-specific OS
was observed in patients with low membrane expression
patterns of ezrin (log-rank, p = 0.006). The 5-yr survival rate
was decreased for patients with low ezrin membrane-
expressing tumours compared with those with strong
membrane expression (Fig. 3). Cox regression analyses
revealed that ezrin ( p = 0.031), p53 ( p = 0.018), T1 substa-
ging ( p = 0.018), the presence of concomitant CIS
( p = 0.017), tumour size ( p = 0.015), and sex ( p = 0.014)
were independent prognostic factors for progression into
invasive disease. Multivariate analysis revealed that ezrin
expression was the only variable with an independent
prognostic effect on disease-specific survival ( p = 0.035)
compared with other clinical and pathologic factors,
including sex, age, concomitant CIS, Ki67, p53, multifocality,
tumour size, and T1 substaging.
3.4. Protein expression patterns of ezrin are predictive
biomarkers of BCG immunotherapy
The membrane protein expression of ezrin and Ki67
staining was found to be differentially expressed between
early recurrent patients from those who were free of disease
after BCG immunotherapy ( p = 0.041 and p = 0.015, respec-
tively). Both Ki67 and the ezrin membrane expression
served to discriminate patients who experienced recur-
rence at 3 mo after BCG intravesical immunotherapy from
those who were free of disease as confirmed by cystoscopy.
Interestingly, a nearly significant association was found
between membrane ezrin expression and disease recur-
rence along the overall follow-up of the cases in this study
( p = 0.06).
4. Discussion
Ezrin is a member of the ERM cytoskeleton-associated
protein family, which was first described as links between
membrane proteins and actin filaments [12,13]. Following
the initial description, these proteins have become impli-
cated in several important cellular complexes and processes.
Ezrin has been reported to be involved in the regulation of
critical events associated with tumour progression, such as
cell survival, proliferation, and migration [12–15]. Altera-
tions of ezrin expression can mediate many changes in the
E U R O P E A N U R O L O G Y 5 6 ( 2 0 0 9 ) 8 2 9 – 8 3 6834
cell surface signals and intracellular signalling cascade that
confer the invasive capability in tumour cells. Therefore, it is
conceivable that ezrin deregulation could contribute to the
invasive behaviour of tumours. Evidence from both animal
models and prospective human studies showing correlations
between ezrin expression levels and tumour progression are
consistent with a crucial role for ezrin in tumour invasion and
dissemination [16–23]. The results presented in this report
are consistent with these observations in the context of the
progression of non–muscle-invasive tumours into invasive
disease.
Ezrin is physiologically expressed in a variety of
epithelial tissue, including intestine, lung, and kidney,
among others. Within these tissues, ezrin has been
suggested to play an essential role in controlling cell
motility [14–16]. The membrane-linking protein ezrin is
highly expressed in several types of human cancers, and
correlations of its immunoreactivity and histopathologic
data as well as patient outcome have previously been
shown [17–25]. The presence of diverged protein expres-
sion patterns along tumour progression and prognosis has
been described for ERM proteins. Although high ezrin
expression has been reported to be associated with poor
prognosis, low expression has also been related to adverse
outcome [17–25]. Even within the same tumour type, such
as in ovarian cancer, low [19] and high [20] ezrin expression
has been associated with poor prognosis. Our observations
with ezrin expression are in line with our previous findings
reporting that the low expression of moesin—another of the
ERM proteins—was associated with poor outcome in BCa
[28]. Supporting our results is the association of weak or
negative ezrin immunoreactivity with poor outcome in
serous ovarian carcinoma [19]. To the best of our knowl-
edge, neither the potential differential expression in
bladder tissues nor the biological role of ezrin in BCa has
been reported to date. The loss of membrane ezrin protein
expression was associated with increased progression of
T1G3 tumours into invasive disease and poorer survival.
Although we focused on T1G3 disease, not covering other
non–muscle-invasive or advanced PT2+ tumours, our
results are consistent with the described biologic role of
ezrin in cancer cells. Positioned at the submembranal-
cytoskeletal interface, ezrin is believed to play a central role
in the regulation of invasion in several cancer cell types [17–
23]. The data presented above support the role of ezrin in
the progression of non–muscle-invasive T1G3 lesions into
invasive disease. Our study also showed a poorer response
to BCG in patients with low ezrin expression. Only adequate
in vitro and in vivo experiments exposing BCa cells and
derived tumours with different controlled ezrin levels to
BCG would be able to assess the biological mechanisms by
which ezrin expression and subsequent cytoskeleton
rearrangements affect BCG immunotherapeutic response.
The cytoplasmic and membrane protein expression
identified in bladder tumours is consistent with the dual
localisation reported for this protein. The N-terminal
binding domain of ezrin mediates membrane attachment
by binding the cytoplasmic tail of CD44, CD43, or
intercellular adhesion molecules. On the other end, ezrin
through its C-terminal domain associates with F-actin and
contributes to its microfilament cytoplasmic organisation.
It is well known that ezrin is exchanged between cytoplasm
and membrane. Cytoplasmic ezrin exists in a ‘‘closed’’
conformation based on intra- or intermolecular interactions
between the N- and C-terminus. Threonine and tyrosine
phosphorylation induces an ‘‘open’’ conformation. In this
state, ezrin is localised towards the membrane, where it
modulates F-actin dynamics and tethers the microfilament
system to the cytoplasmic face of cell adhesion sites [23].
Disruption of actin filaments and a decrease in focal
adhesion are common features of epithelial–mesenchymal
transition, which is associated with the onset of invasion.
Besides a structural role, ezrin may act as a signalling or
scaffold molecule. It is believed that ezrin is involved in cell
adhesion functions through interactions with the Rho-
associated signal transduction [16] and the Akt-mediated
apoptotic pathway [30]. These observations suggest that in
the presence of low ezrin expression, the subsequent
altered organisation of the cytoskeleton, the impaired focal
adhesion, and signalling deregulation would lead to an
increased invasion of BCa cells. These cellular changes
would affect disease progression, a poorer therapeutic
response to BCG, and clinical outcome. Further studies are
warranted to dissect the specific signalling events regulated
by ezrin using in vitro and in vivo models in BCa.
The results presented in this report are clinically relevant
for the patients affected with bladder tumours. It is known
that urothelial carcinomas presenting T1G3 non–muscle-
invasive lesions are high-risk tumours because of the
possibility of progression to muscle-invasive or metastatic
disease [7]. The difficulty remains in deciding which
patients should be treated under a conservative approach
using intravesical treatment with BCG and which should
undergo radical cystectomy [5]. Critical clinical variables,
such as the presence of an associated CIS or an early BCG
failure [4–8], have been defined as factors related to
progression and must be considered before making such a
decision [4–8]. Lacking reliable prognostic and predictive
tumour markers, laboratory research is warranted to
identify candidate biomarkers of clinical behaviour. In this
regard, p53 has been widely studied, with results not
conclusive enough to make clinical decisions in T1G3
disease [9,10,31], similar to our findings. In our series, ezrin
and Ki67 stainings showed a prognostic ability to dis-
criminate patients more likely to progress into muscle-
invasive disease. In this report, several issues related to the
wide heterogeneity among BCG failures have been
addressed. The loss of ezrin membrane expression was
associated with early recurrence, progression into muscle-
invasive disease, and OS. Despite the promising clinical
relevance of these findings, which suggest the adjunct value
as a prognostic and predictive biomarker, our results cannot
conclude whether the urologist could decide to undertake a
radical treatment based on the membrane protein expres-
sion of ezrin. In a multivariate survival analysis, ezrin
immunoreactivity had an independent effect on tumour
progression and disease-specific survival when controlling
for sex, age, concomitant CIS, Ki67, p53, multifocality,
E U R O P E A N U R O L O G Y 5 6 ( 2 0 0 9 ) 8 2 9 – 8 3 6 835
tumour size, and T1 substaging. This observation is relevant
because it was found in a limited number of cases analysed
(n = 92) and controlling its prognostic effect for a high
number of clinical and pathologic variables (n = 9). Further,
larger and prospective studies are warranted to define the
adjunct value of ezrin measurement in stratifying the
clinical outcome of patients with T1G3 disease undergoing
BCG immunotherapy and to decide upon tailored, more
aggressive interventions.
5. Conclusions
In summary, immunohistochemistry analyses revealed that
the membrane expression of ezrin is associated with the
clinical outcome of patients with T1G3 tumours undergoing
BCG treatment. Protein expression patterns of ezrin were
associated with tumour progression in T1G3 disease. Thus,
the differential expression of ezrin discriminated patients
responding to BCG from those who may require a more
aggressive therapeutic approach. These findings warrant
further elucidation in prospective trials to evaluate whether
ezrin immunohistochemical evaluation could be an adjunct
tool for therapeutic decisions.
Author contributions: Marta Sanchez-Carbayo had full access to all the data
in the study and takes responsibility for the integrity of the data and the
accuracy of the data analysis.
Study concept and design: Sanchez-Carbayo.
Acquisition of data: Sanchez-Carbayo, Palou, Algaba, Vera.
Analysis and interpretation of data: Sanchez-Carbayo, Palou, Algaba.
Drafting of the manuscript: Sanchez-Carbayo, Palou, Algaba.
Critical revision of the manuscript for important intellectual content: Sanchez-
Carbayo, Palou, Algaba.
Statistical analysis: Sanchez-Carbayo, Palou.
Obtaining funding: Sanchez-Carbayo.
Administrative, technical, or material support: Rodriguez, Villavicencio.
Supervision: Sanchez-Carbayo.
Other (specify): None.
Financial disclosures: I certify that all conflicts of interest, including specific
financial interests and relationships and affiliations relevant to the subject
matter or materials discussed in the manuscript (eg, employment/affilia-
tion, grants or funding, consultancies, honoraria, stock ownership or
options, expert testimony, royalties, or patents filed, received, or pending),
are the following: None.
Funding/Support and role of the sponsor: None.
Acknowledgement statement: The authors acknowledge all members of the
laboratory of Dr Sanchez-Carbayo at the Tumor Markers Group at the CNIO
for their technical support and constructive suggestions in the preparation
of this manuscript. We would like to thank all the members of our clinical
collaborators at the Fundacio Puigvert involved in this study for their
support in facilitating the tumour specimens as well as the clinical fol-
low-up of the BCa cases analysed in this study.
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Editorial Comment on: Protein Expression Patterns of
Ezrin Are Predictors of Progression in T1G3 Bladder
Tumours Treated with Nonmaintenance Bacillus
Calmette-Guerin
Ofer N. Gofrit
Department of Urology, Hadassah Hebrew
University Hospital, Jerusalem, Israel
Palou and colleagues’ [1] deal with one of the most
difficult issues in urologic oncology: the management of
patients with T1G3 urothelial carcinoma. Despite high risk
of recurrence, muscle invasion, metastases, and disease-
related death, many of these patients do well with
endoscopic resection and bacillus Calmette-Guerin
(BCG) immunotherapy alone. Can molecular markers
predict the biologic potential of this unpredictable tumor?
Can they improve our understanding of the biology of
bladder cancer?
The authors studied the expression of ezrin, a cytoplas-
mic membrane protein that acts as a link between the
membrane and the actin cytoskeleton, in tissue blocks from
92 patients given BCG immunotherapy for T1G3 tumors [1].
As expected, after a median follow-up of 90.5 mo, 17
patients (18.5%) progressed to muscle-invasive disease and
14 (15.2%) died of the disease. Multivariate analysis showed
that ezrin membrane expression in<20% of the tumor cells
is significantly associated with progression and survival.
So, can ezrin expression be used as a prognostic
marker? Can it teach us anything about the pathogenesis
of T1G3 bladder cancer? Unfortunately, the answer to both
questions is no. A valuable tumor marker must prove its
predictive capacity beyond the standard clinical and
pathologic parameters in a prospective way, not only on
archival material. Perhaps the most disappointing mole-
cule in this respect is p53. Initial results were promising:
Disease progression of 20.5% per year was found in
patients with T1 disease expressing nuclear p53 in>20% of
the tumor cells, compared with 2.5% in patients with lower
[28] Sanchez-Carbayo M, Socci ND, Charytonowicz E, et al. Molecular
profiling of bladder cancer using cDNA microarrays: defining
histogenesis and biological phenotypes. Cancer Res 2002;62:
6973–80.
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microfilament linker, signals cell survival through the phosphati-
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96:7300–5.
[31] Schmidt-Drager BJ, Goebell PJ, Ebert T, Fradet Y. p53
immunohistochemistry as a prognostic marker in bladder
cancer: playground for urology scientists? Eur Urol 2000;38:
691–700.
levels of p53 [2]. Further studies, however, including a
meta-analysis, could not confirm this finding [3–5]. After
15 yr of intense research, neither p53 nor any other
molecular marker has joined our diagnostic armamentar-
ium [6] and none is recommended in the European
Association of Urology (EAU) guidelines. Immunohisto-
chemistry studies are also very limited in their ability to
provide valuable information regarding the biologic role of
the molecule studied. Much more complicated studies at
the molecular level are needed for this purpose.
Nevertheless, ezrin is an interesting molecule that is
certainly involved in the pathogenesis of bladder cancer
and is definitely worth more investigation.
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DOI: 10.1016/j.eururo.2008.09.063
DOI of original article: 10.1016/j.eururo.2008.09.062