Group Title: CytoJournal 2006, 3:18
Title: Immunohistochemical detection of hTERT in urothelial lesions: a potential adjunct to urine cytology
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Title: Immunohistochemical detection of hTERT in urothelial lesions: a potential adjunct to urine cytology
Series Title: CytoJournal 2006, 3:18
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Creator: Khalbuss W
Goodison S
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Immunohistochemical detection of hTERT in urothelial lesions: a
potential adjunct to urine cytology
Walid KhalbussI and Steve Goodison*2


Address: 'Dept. of Pathology, University of Florida, Jacksonville, FL, USA and 2Dept. of Surgery, University of Florida, Jacksonville, FL, USA
Email: Walid Khalbuss khalbuss@yahoo.com; Steve Goodison* steve.goodison@jax.ufl.edu
* Corresponding author


Published: 10 August 2006
Cytojournal 2006, 3:18 doi: 10.1 186/1742-6413-3-18


Received: 25 April 2006
Accepted: 10 August 2006


This article is available from: http://www.cytojournal.com/content/3/1/18
2006 Khalbuss and Goodison; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Urine cytology has a critical role in evaluation for bladder carcinoma. Due to the
low sensitivity of this technique, ancillary modalities such as the detection of markers of malignancy
by immunochemistry are desirable. Promising factors in this context are components of the human
telomerase enzyme complex. Telomerase repairs and extend telomeres, which when eroded
beyond a critical limit trigger a senescence checkpoint. Accordingly, while absent in normal somatic
cells, telomerase activity has been detected in the great majority of malignant tumor specimens
tested, and so has potential value for the recognition of malignant cells in clinical specimens.
Methods: In this study, we investigated whether the immunohistochemical detection of the
catalytic subunit of telomerase (hTERT) can aid cytology in the diagnosis of bladder lesions. Findings
from the retrospective evaluation of over 100 cell blocks, including urine sediments from confirmed
malignant and benign conditions, were compared with routine urine cytology data.
Results: The presence of hTERT protein was indicative of the transformation of urothelia to a
malignant phenotype. Nucleolar hTERT was expressed in 27 (93%) of 29 samples obtained from
patients with confirmed primary bladder cancer. Conversely, hTERT was detectable in only 3
(0.8%) of 39 samples from benign conditions. The hTERT assay showed higher diagnostic sensitivity
(84.8%) than published urine cytology data (-65%) for confirmed bladder carcinoma, however, the
hTERT assay was less specific than cytology (65.2% vs. -95% respectively).
Conclusion: As a highly sensitive marker, immunohistochemical hTERT detection in urine
sediments represents a reliable adjunct to cytology in the accurate diagnosis of urothelial
neoplasms.


Background
The diagnosis of suspicious bladder lesions is, in part,
dependent on the demonstration of atypical cells in the
cytological examination of voided urine or bladder wash-
ings. However, the relatively low diagnostic sensitivity of
urinary cytology warrants the development of improved
non-invasive diagnostic techniques [1]. Furthermore, one


of the major problems in daily cytology practice is to dis-
tinguish benign/reactive cells from malignant cells. Addi-
tional techniques, such as immunocytochemistry and
flow cytometry, may provide significant help in this differ-
ential diagnosis. A number of antibodies directed against
specific cell type antigens have been used in urine cytol-
ogy to enhance the cytological diagnosis, including


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NMP22, cytokeratin 20 and human complement factors,
but the results with cytological preparations have been
conflicting [2-4]. No single diagnostic technique alone is
sufficient to establish the diagnosis in all cases, and the
search for an accurate tumor marker that reliably confirms
urothelial malignancy remains a challenge. Promising fac-
tors in this context are components of the human telom-
erase enzyme complex. The human telomerase reverse
transcriptase (hTERT) protein is the catalytic subunit of
the telomerase holoenzyme which maintains chromo-
somal telomeres [5]. Telomeres are the non-coding ter-
mini ofeukaryotic chromosomes and function to stabilize
and maintain chromosomal structure. However, telom-
eric DNA is lost at each cell division as a result of the ina-
bility of DNA polymerases to replicate the 5' end of linear
DNA [6], and erosion of these sequences beyond a critical
point is thought to signal cell cycle arrest and entry into
cellular senescence [7]. The major mechanism of telomere
repair or maintenance is mediated by the enzyme telom-
erase [5]. A close association between the activation of the
telomerase enzyme and cellular immortality has been
established, and the presence of functional telomerase
enables cells to be capable of extended proliferation or to
become immortal, and in concordance with this hypoth-
esis, telomerase activity has been detected in the great
majority of malignant tumor specimens tested [8,9]. The
enzyme is undetectable in normal somatic cells; therefore,
the detection of telomerase activity in human tissue sam-
ples has value for the recognition of malignant cells in
clinical specimens [10].

For detecting telomerase activity in a tissue specimen the
TRAP assay is a relatively sensitive and specific method,
but it can be used only on fresh tissue extracts and offers
no information at the cellular level [11]. Expression of
hTERT mRNA is very closely associated with telomerase
activity in human tumors and can be detected by RT-PCR
[12]. However, this approach also does not offer any
information at the level of the individual cell and so cor-
relative comparison of molecular data with cellular mor-
phology is not attainable. Therefore,
immunohistochemical (IHC) methods of hTERT protein
evaluation, which can both detect and localize cellular tel-
omerase expression in human tissue would be optimal for
the differential diagnosis of cellular material such as
serious effusions. Investigators have recently tested com-
mercially available anti-hTERT antibodies in formalin-
fixed and paraffin-embedded human tissues by IHC
[13,31]. One monoclonal antibody (NCL-hTERT;
Novacastra) was sufficiently specific for further investiga-
tion in clinical specimens.

In this study, we applied the hTERT antibody to urine sed-
iment cytology samples prepared as paraffin sections of
cell blocks. We evaluated over one hundred cell blocks for


hTERT immunoreactivity, and compared the findings
with available conventional cytology and biopsy pathol-
ogy information. When present, the expression of hTERT
protein was localized to the nucleoli of urothelial cells,
and hTERT expression positively correlated with urothe-
lial cell neoplasia. Whilst conventional cytology plays a
pivotal role in the diagnosis of bladder cancer, for difficult
cases, in which ancillary information is necessary, the use
of immunohistochemical detection of the telomerase
component hTERT may significantly improve diagnostic
accuracy.

Methods
Patient specimens
In total, 101 cell blocks that contained various bladder tis-
sue specimens were identified in the archives of the
Shands & University of Florida Hospital, Jacksonville, FL.
Cell blocks were prepared from urinary sediments col-
lected from patients under investigation for bladder
lesions of various types. The urinary sediments were proc-
essed into cell blocks using the plasma/thrombin tech-
nique [14]. Evaluated specimens were selected according
to the cytological diagnosis and included 29 malignant
cases, 39 non-malignant cases, and 33 cases of cytological
atypia. Tissue biopsy confirmation of malignant and
benign conditions was available in 56 cases.

Urine cytology
Smears of urine specimens from each patient were exam-
ined cytologically by standard Papanicolaou staining. All
slides were evaluated routinely by an experienced
cytopathologist without any prior knowledge of the
immunohistochemical findings. Urothelial carcinoma
grading and staging were performed according to the
World Health Organization criteria.

Immunohistochemistry
The specificity of the antibody has been reported in previ-
ous reports [13,31]. Antibody-mediated detection of
hTERT was performed using the standard streptavidin-
biotin peroxidase complex method. All steps were per-
formed on a Ventana Benchmark XT-BTS automatic
immunostainer (Tucson, Arizona). Tissue sections from
representative blocks were deparaffinized in xylene and
alcohols, and were then placed in 3% hydrogen peroxide/
methanol for 5 minutes to block nonspecific background
staining due to endogenous peroxidase activity. Antigen
epitopes were retrieved (Ventana Benchmark CC1
extended program) by heating to 1000C and reducing
heat slowly over a 90 min period at pH 8.0. The primary
antibody (NCL-hTERT IgG 2a, Batch # 147508, from
Novocastra, Newcastle-upon-Tyne, UK) was diluted 1:25
and applied to the slides for 32 min at 370C. Secondary
antibody incubation, washes and chromogen (3,3'-diami-
nobenzidine) development were performed at room tem-


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Table I: Characteristics of cases evaluated in the hTERT
immunoassay


Sex M/F


53/48


Voided urine specimens
Instrumented specimens
Positive for malignancy
Urothelial carcinoma
Squamous cell carcinoma
Small cell carcinoma
Negative for malignancy
Atypical/undetermined
Biopsy correlation available


61.4%
38.6%
28.7%



38.6%
32.7%
55.5%


perature. Slides were counterstained with hematoxylin,
dehydrated, and mounted for microscopical examination.
Optimization of conditions was performed with sections
of normal colon (hTERT-positive at the base [13] of the
crypts) (Figure 1A) and tissue bladder biopsies with
urothelial carcinoma (Figure 1B). Negative control slides
included a blank control and omission of primary anti-
body.

Assessment of hTERT immunostaining
The entire hTERT stained slide was examined for immu-
nostaining of hTERT. Positivity for hTERT expression was
evaluated by two independent observers (WK, SG). Nucle-
olar urothelial cell positivity was used for evaluation of
utility in diagnosis. Positivity in non-urothelial cells such
as inflammatory cells, lymphocytes, microorganisms, or
contaminating debris was duly noted and recorded. The
availability of corresponding tissue bladder biopsy mate-
rial, which is the gold standard for diagnosis or exclusion
of urothelial carcinoma, enabled the determination of the
sensitivity and specificity of hTERT immunostaining for
the detection of bladder cancer. The positive and negative
predictive values were determined as follows: positive pre-
dictive value is equal to the number of truly positive cases
identified/total number of cases that tested positive x
100%, negative predictive value is equal to the number of
truly negative cases identified/total number of cases that
tested negative x 100%.


Table 2: Comparison of hTERT
cytological diagnosis.


Cytological diagnosis

Positive
Negative
Atypical
Total


Results
Optimization of the hTERT immunostaining technique,
as described in Materials and Methods, revealed that spe-
cific hTERT expression was nucleolar in both prolifera-
tively active normal cells, and in tumor cells. In normal
colon crypts, staining intensity was concentrated in the
base of the crypt (Figure 1A), as previously observed [13],
where the basal cells with proliferative capacity reside. In
excised bladder tumor tissue, the expression of hTERT was
clearly evident in the multiple nucleoli of neoplastic cells
(Figure 1B). Notably, some morphologically normal
urothelia adjacent to the tumor border also expressed
hTERT, but the immunoreactivity diminished and was
lost in normal cells more distal to the tumor (Figure 1B).
Diffuse nuclear staining accompanied nucleolar positivity
in a few examples, but we did not observe any cytoplasmic
staining. Lymphocytes were reactive to hTERT antibody,
as were yeast when present (Figure 1C). Yeast is a com-
mon contaminant in clinical urine sampling and is
known to contain the telomerase enzyme, so the presence
of yeast can pose a serious confounding problem in urine-
based assays. Using the advantage of histological identifi-
cation facilitated using a hematoxylin counterstain, we
classified patient samples as positive when the presence of
specific nucleolar staining was evident in urothelial cells.
Patient demographics and tumor characteristics of the
samples evaluated are presented in Table 1. The 101 cases
came from 53 male patients and 48 female patients. The
average age of the patients were 62 years (ranges: 30-91
years). The majority of the specimens were voided urine
specimens (61.4%). The majority of the urothelial carci-
noma cases were grade III (13 cases) and grade II (8 cases),
and one case grade I. The 39 non-malignant cases
included diagnoses ofurolithiasis, cystitis glandularis and
cases with no detectable lesion. We also evaluated 33
cases classified as atypical by cytological examination. Tis-
sue bladder biopsies were available in 56 cases for histo-
logical and cytological correlation.

All types of urothelial cancer expressed hTERT, including
transitional cell carcinomas of low grade, high grade, car-
cinoma in situ (CIS), squamous cell carcinoma (Figures


1D-1G), small cell carcinoma and metastatic adenocarci-
noma. Of the 29 cases cytologically diagnosed as malig-
nant and confirmed by biopsy, 27 (93%) exhibited
immunoassay results with
nucleolar hTERT expression (Table 2). One case of high-
grade urothelial carcinoma and one case of squamous cell
hTERT carcinoma did not display hTERT expression. Among the
cytologically classified non-malignant cases, transitional
Positive Negative Total cell hTERT expression was detectable in only 3 (<1%) of
the 39 samples evaluated. One of the 3 false-positive cases
27 2 29 was from a patient with confirmed diagnosis of urolithia-
3 36 39
19 1 33 sis (Figure 1J). The other 2 cases have no corroborative
49 52 101 evidence of bladder disease to date. There were 56 cases
(malignant, benign and atypical cytologically) on which


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,,,, g4


C
'WI


rF., H


Figure I
Expression of hTERT protein in human urological tissue. hTERT immunostaining was localized in the nucleolus of urothelial
cells. Formalin-fixed, paraffin-embedded bladder tissue biopsy (A, B) and urinary sediments (C-K) were analyzed as described in
'materials and methods' (original magnification x200): (A), hTERT expression was generally confined to the lower half of nor-
mal colon crypts; (B, inset at higher magnification), bladder tumor biopsy, in which it can be seen that neoplastic cells and prox-
imal, morphologically normal urothelia react with the antibody; (C), example of immunoreactive yeast present in urinary
sample; (D), transitional cell carcinoma (low grade); (E), transitional cell carcinoma (high grade); (F), squamous cell carcinoma;
(G), carcinoma in situ (CIS); (H), cystitis glandularis; (J), urolithiasis; (K), umbrella cells (left panel shows negative example, right
panel shows an immunoreactive example).



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Table 3: Evaluation of hTERT immunoreactivity in biopsy confirmed malignant and non-malignant specimens


Malignant cases


hTERT


Positive


Cytological diagnosis


Positive
Atypical


Negative

2
3


TOTAL


TOTAL


Non-malignant cases



Cytological diagnosis


Negative/Reactive
Atypical


we had confirmatory information from bladder tissue
biopsy evaluation. Histological examination of tissue
bladder biopsy is considered the gold standard for the
diagnosis of bladder malignancy. The biopsy correlation
data are shown in Table 3. The availability of this data
enabled the calculation of the diagnostic accuracy of the
hTERT immunoassay. Overall, the hTERT immunoassay
demonstrated a positive predictive value of 77.8% and a
negative predictive value of 75% in this study. No correla-
tion of hTERT expression was observed with the mode of
specimen collection i.e. urine collection through voiding
or instrumentation. Of 4 cytologically atypical cases sub-
sequently proven to carry a bladder tumor burden
through cystoscopy and biopsy, one case expressed hTERT
(Table 3). Thus, in this case hTERT detection may have
aided diagnosis, however, of 12 cytologically atypical
cases with no evidence of a bladder lesion at the time of
sampling, 7 cases exhibited hTERT expression. Notably, 4
of these 7 positive atypical cases were from patients who
had a history of bladder cancer, indicative of a possible
'field-effect' which alters the phenotype of the entire blad-
der urothelia without resulting in an actual focal lesion.
Finally, it is noteworthy that umbrella cells were occasion-
ally positive for hTERT expression (Figure 1K). These
superficial cells were commonly identified in urine sedi-
ments and exhibited hTERT in seven cases, all of which
had negative reactive urothelial cells. In this study, cases
were only considered positive if urothelia other than
umbrella cells exhibited nucleolar hTERT staining.

Discussion
Carcinoma of the urinary bladder is the most common
malignant tumor of the urinary tract and, after prostatic
carcinoma, the second most common malignancy of the
urogenital system. Furthermore, bladder carcinoma
shows a very high recurrence rate (50-70%) and recurrent


disease, when it does occur, is associated in 15-25% of
patients with progression to a more advanced tumor
stage. Thus, careful and frequent follow-up is of prime
importance. Due to factors such as size or localization, the
great majority of carcinomas of the urinary bladder are
either undetectable with standard imaging techniques, or
cannot be definitively differentiated from non-malignant,
reactive processes. Thus, the invasive method of instru-
mental cystoscopy remains the standard detection mode
in the clinic. A non-invasive method of comparable diag-
nostic quality would significantly facilitate the initial
diagnosis of carcinoma of the urinary bladder and greatly
simplify the follow-up of treated patients. The commonly
utilized cytological examination of urine sediments is spe-
cific and readily available, but it is not sufficiently sensi-
tive for the detection of the subtle morphologic changes
that occur in well-differentiated carcinomas of the urinary
bladder. Furthermore, inter-observer variability can create
additional diagnostic pitfalls, thus, the need for non-inva-
sive techniques for the diagnosis and follow-up of carci-
noma of the urinary bladder remains.

Recent evidence has suggested that the presence of the
enzyme telomerase in urine is a potentially useful marker
for the early detection of urothelial neoplasia [15-19].
Comparative analyses of non-invasive methods for the
diagnosis of bladder cancer have shown that telomerase
has the highest combination of sensitivity and specificity
with respect to urine cytology and other biochemical
marker determinations [16,18-25]. The detection of tel-
omerase activity and the expression of the associated
genes has been customarily achieved through the tel-
omere repeat amplification protocol (TRAP) and RT-PCR
methodologies respectively. Sanchini et al recently dem-
onstrated the ability of telomerase activity levels in uri-
nary sediments to accurately detect the presence of


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hTERT


Positive


Negative


TOTAL


TOTAL


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bladder tumors. A sensitivity of 90% and specificity of
88% was achieved for bladder cancer detection by apply-
ing relative cut-off points to a quantitative TRAP assay
[26]. Telomerase activity determination by the TRAP assay
enables quantitative evaluations to be made, but is vul-
nerable to contamination by telomerase-positive, non-
malignant cell types, such as proliferative stem cells and
inflammatory elements [27,28]. Furthermore, because the
TRAP assay detects enzyme activity and not simply the
presence of the protein, valid findings require the pres-
ence of living cells. In native urine, suspended cells are
exposed for various lengths of time to destructive sub-
stances and conditions including proteases, urea, and
acidic pH values [10]. The alternative to the TRAP assay is
detection of the structural or encoding RNA components
of the telomerase enzyme, primarily the human telomer-
ase RNA component (hTR) and hTERT mRNA
[10,21,27,28]. Compared to other urine-based markers,
quantitative detection of human telomere components by
RT-PCR, particularly the RNA component (hTR), shows
acceptable diagnostic accuracy with sensitivities of up to
77% (29). Clearly, telomerase is a promising marker for
urothelial neoplasia, but these tests remain several steps
removed from becoming a routine procedure which
replaces cytology. In contrast, an in situ analysis would
enable the morphological identification of hTERT-posi-
tive cells, and if used as an adjunct to cytology such anal-
yses could be rapidly brought into practice. Good
monoclonal antibodies that specifically recognize hTERT
have been isolated only recently so few studies have
focused on the immunohistochemical evaluation of
hTERT in human tissues. In the current study, we demon-
strated the feasibility of an hTERT immunoexpression
assay on urine sediment samples, and investigated its util-
ity as an adjunct to conventional urine cytology in the
diagnosis of bladder carcinoma.

Our study has shown that through the application of a
monoclonal antibody, hTERT can reliably be detected in
paraffin-embedded solid tumor specimens and in urine
sediments archived as cell blocks. In agreement with the
few previous reports describing hTERT protein localiza-
tion, we found hTERT to be localized to the nucleolus
[13,30,31]. It is logical that hTERT is localized predomi-
nantly in the nucleolus, the site of nucleoprotein complex
assembly, because hTERT is a subunit of the ribonucleo-
protein telomerase enzyme [10]. In bladder tumor biopsy
material we observed hTERT expression in cells adjacent
to the tumor and expression was seen to diminish more
distal to the primary lesion. This spatially associated
hTERT positivity in apparently normal cells has been
observed in other organs, including the colon [13], pros-
tate [30] and melanocytic lesions [31]. The development
of a reliable hTERT antibody has revealed this phenome-
non and has the potential to better determine exactly


where hTERT is expressed at the cellular level. The expres-
sion in normal cells could result from a number of possi-
bilities, but the spatial relationship suggests that factors
secreted from the tumor can induce hTERT transcription/
translation in non-neoplastic cells.

Our goal was to evaluate the potential utility of hTERT
immunodetection in urine sediments as an adjunct to
cytology. Given the nature of the sample, which is often
composed of scanty, dispersed cells, we did not attempt to
estimate staining intensity or percentage of immunoreac-
tive cells within the sample or block section. In this study,
the presence (>3 cells observed) or absence of hTERT, spe-
cifically in transitional urothelial cells, was noted. The
expression of hTERT was observed in 85% of the histolog-
ically confirmed bladder cancer cases, which represents a
high rate of diagnostic sensitivity by examination of
voided urine. Overall, the hTERT immunoassay demon-
strated a positive predictive value of 77.8% and a negative
predictive value of 75% in this study. The hTERT assay
showed higher diagnostic sensitivity (84.8%) than pub-
lished urine cytology data (~65%) for confirmed bladder
carcinoma, however, the specificity of the hTERT immu-
noassay was 80%, lower than that obtained by cytologic
evaluation (~90%). The specificity data do not support
the use of hTERT immunodetection as a replacement for
cytology, but the improved sensitivity of the assay sug-
gests potential utility as an adjunct to cytology. It was
notable that the majority of false-positive cases were
found in the subset classified as cytologically atypical, but
which have no evidence of disease to date. It will be inter-
esting to see whether these patients will subsequently
develop bladder malignancy in which case hTERT detec-
tion may have potential as an early detection or even pre-
dictive marker. However, specimens from patients with a
history of bladder cancer are often deemed atypical cyto-
logically, due to a 'field-effect', and in this study several
cases were immunoreactive for hTERT. In agreement with
telomerase activity assays in such patients [28], we did
find hTERT-positive cells in patients who had no evidence
of urothelial lesion at the time of sampling, but who did
have a history of bladder cancer. Thus, it seems that hTERT
detection may not be reliable in the monitoring of recur-
rent bladder cancer.

Our findings are in agreement with the previous telomer-
ase-based studies in urological cancers, in that telomerase
expression correlates with malignancy, but the use of
immunohistochemistry adds specific advantages in the
context of diagnosis. Although assays such as the TRAP
assay or mRNA measurement can be designed to be semi-
or fully quantitative, they do not provide information
regarding the source of the protein. Immunohistochemi-
cal studies are beginning to reveal that normal cells can
express hTERT under certain circumstances. In our study,


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normal urothelium adjacent to tumors expressed hTERT,
as did apparently normal umbrella cells in some cases.
Given the common presence of yeast in urine samples, it
is easy to see how telomerase assays without morpholog-
ical information could be misleading. Furthermore, the
technical sensitivity of molecular assays may result in mis-
labeling samples which have only a few positive cells in a
background of negative ones. With slide-based immuno-
detection, a single positive cell can be identified regardless
of background content. These considerations are particu-
larly relevant in samples where few cells may be present,
such as urine sediments. When combined with cytology,
the immunohistochemical detection of hTERT provides
additional information upon which the observer can act.
Bladder cancer detection based on the use of antibodies
against tumor-associated antigens expressed by exfoliated
urothelial cells reflects an attractive approach, because it is
readily applicable using specimens obtained in a non-
invasive manner and involves tissue processing and inter-
pretation skills that are routinely available in a cytopa-
thology laboratory.

Conclusion
In conclusion, the detection of hTERT protein coupled
with cytology has the potential to aid the diagnosis of
benign and malignant bladder lesions in urine cytology
specimens. Although the number of cytologic cases in this
study was small, the results demonstrate the applicability
of hTERT staining to fixed cytologic cell block samples. A
larger study of the usefulness of hTERT applied to cyto-
logic samples, including smear preparations, is in progress
in our laboratory and will expand on these findings.
Beyond that it may also be a useful marker to identify
bladder carcinoma or other lesions that would be amena-
ble to therapies that would involve interference of tumor
proliferation through telomerase inhibition.

Competing interests
The authors) declare that they have no competing inter-
ests.

Authors' contributions
WK performed cytology and pathology, co-designed the
study and correlated clinical information with immuno-
histochemical data for all cases. SG conceived and
designed the study, evaluated immunohistochemistry and
drafted the manuscript.

Acknowledgements
We thank Jerry Santiago for technical expertise and performing immuno-
histochemical procedures.

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