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. 2009 Feb 12;135(9):1169–1175. doi: 10.1007/s00432-009-0557-9

Predictive value of telomerase reverse transcriptase expression in patients with high risk superficial bladder cancer treated with adjuvant BCG immunotherapy

Ioannis Zachos 1, Panagiotis A Konstantinopoulos 2,3, Gerasimos P Vandoros 4, Michalis V Karamouzis 2, Athanasios G Papatsoris 2,5, Thomas Podimatas 6, Antonios Papachristodoulou 7, Michael Chrisofos 5, Charalambos Deliveliotis 5, Athanasios G Papavassiliou 2,✉
PMCID: PMC12160213  PMID: 19214569

Abstract

Purpose

We conducted a prospective study to determine whether expression of telomerase reverse transcriptase (hTERT) is associated with recurrence-free-survival (RFS) or development of invasive disease in patients with high risk superficial bladder cancer (SBC) that received adjuvant BCG immunotherapy.

Methods

Thirty patients with high-grade T1 tumors were evaluated. Pre-BCG TURBT and post-BCG specimens were analyzed for hTERT nucleolar expression by immunohistochemistry.

Results

Post-BCG hTERT expression was statistically significantly lower than pre-BCG hTERT expression. Pre-BCG hTERT nucleolar staining in more than 75% of cells was associated with worse RFS (9 months vs. not yet reached, P = 0.05), while post-BCG hTERT nucleolar staining in more than 50% of the cells was associated with worse RFS (6 months vs. not yet reached, P = 0.001) and development of invasive disease. In multivariate analysis, post-BCG hTERT expression was independently associated with RFS and development of invasive disease.

Conclusions

Immunohistochemical evaluation of hTERT may help define a subset of high risk SBC patients that will eventually fail BCG and may therefore benefit from early salvage cystectomy.

Keywords: Superficial bladder cancer, Telomerase reverse transcriptase, BCG, Recurrence, Multivariate analysis

Introduction

Superficial bladder cancer (SBC) accounts for approximately 70% of all new cases of urothelial bladder cancer (Kirkali et al. 2005). The initial treatment of SBC is transurethral resection of all visible bladder tumors (TURBT) followed by adjuvant intravesical immunotherapy, most commonly with Bacillus Calmette-Guerin (BCG), which is offered to patients at high risk for tumor recurrence and/or development of invasive disease. BCG therapy has been shown to delay or prevent tumor recurrence and stage progression, decrease the need for subsequent cystectomy, and improve overall survival (Herr et al. 1989; Herr et al. 1995). However, a significant proportion of patients do not respond to intravesical BCG therapy; their tumors persist or recur early after BCG and may become invasive and/or metastatic. Cystectomy is an effective salvage therapy for patients in whom BCG therapy fails, as it has been shown to prevent stage progression and death (Lee et al. 2007; Herr and Sogani 2001). In that regard, a tool to predict the response to intravesical BCG therapy would be invaluable, as it could define a subset of patients that may benefit from early salvage cystectomy.

Although BCG therapy is known to trigger a variety of local immune responses that correlate with anticancer activity, the exact mechanism of its antitumor action remains to be elucidated. Previous studies in a number of in vitro models (including bladder cancer cell lines) have shown that telomerase activity was decreased in BCG-treated cells compared to vehicle-control (Reed et al. 2004; Saitoh et al. 2002). Nevertheless, the in vivo expression of telomerase prior and after BCG therapy has never been evaluated and its potential association with response to adjuvant BCG immunotherapy has never been formally assessed.

We conducted a prospective study to determine the correlation between expression of telomerase reverse transcriptase (hTERT)—the catalytic subunit of telomerase—before and after BCG therapy with the clinical outcome of high risk SBC patients treated with TURBT and adjuvant intravesical BCG.

Materials and methods

Study design

Thirty patients with high grade T1 (as per the American Joint Committee on Cancer staging system) SBC, who underwent TURBT followed by adjuvant BCG immunotherapy were enrolled prospectively in this study. Patients with previous history of bladder tumor or BCG treatment were excluded from the study. Baseline (pre-BCG) TURBT tumor specimens (at least three tumor samples per patient) as well as random biopsies from normal bladder mucosa (one sample per patient) were obtained from all patients for hTERT immunohistochemistry. Excision of all visible tumors was achieved in all cases and the muscularis propria was assessable in all specimens. After TURBT, all patients underwent adjuvant BCG treatment comprising six weekly instillations of BCG [81 mg (10.5 ± 8.7 × 108 colony-forming units) weekly for 6 weeks]. Four weeks after the last dose of BCG, all patients underwent follow-up (post-BCG) cystoscopy and biopsy of the sites where bladder tumors were excised during TURBT (at least three samples per patient), and these post-BCG specimens were also processed for hTERT immunostaining. Patients were then followed with routine cystoscopy and urinary cytology at 3-month intervals until recurrence.

Immunohistochemistry

Serial, 4 μm sections were obtained for staining with hematoxylin and eosin and for immunohistochemistry. hTERT was detected by immunohistochemistry on paraffin-embedded sections using the standard avidin–biotin-peroxidase (ABC) technique. Microwave irradiation in citric buffer (pH 6.0) was employed as antigen retrieval method. The primary mouse monoclonal antibody against hTERT (clone 44F12, Vector Laboratories, Burlingame, USA) was diluted 1:50 with phosphate-buffered saline (PBS) and applied on tissue sections for 30 min at room temperature. Antigen binding was detected using the UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen, as per the manufacturer’s recommendations. Sections from bladder tumors in which the primary monoclonal antibody was omitted, were used as negative controls.

Evaluation of hTERT expression

The immunostained sections were evaluated and scored by two investigators (I.Z. and G.P.V) independently and without prior knowledge of the patients’ clinicopathological characteristics. Since nucleolar hTERT correlates well with telomerase activity (Etheridge et al. 2002; Yang et al. 2002), we estimated the number of neoplastic cells with nucleolar hTERT staining in representative high-power fields of the tumor divided by the total number of neoplastic cells in those fields, and the result was expressed as a percentage. In agreement with previous studies (Domont et al. 2005; Smith et al. 2004), cases displaying nucleolar immunostaining in equal or more than 50% of tumor cells were regarded as positive, while cases with less than 50% tumor cells positive at the nucleolar level were considered negative. Cases with interobserver differences were re-assessed by simultaneous examination of the specimens by the two investigators in a double-headed light microscope.

Statistical analyses

The correlation of the hTERT-positive and hTERT-negative pre- and post-BCG samples with the clinical and pathological features of the cases was performed using the Fisher’s exact test. Associations between clinicopathological factors and development of invasive disease were evaluated using the Fisher’s exact test. Logistic regression was performed for multivariate analysis using development of invasive disease as the dependent variable. Recurrence free survival rates were estimated by the Kaplan-Meier method and compared using the log-rank test. Cox proportional hazards models were used for multivariate analysis with forward stepwise selection of the variables. The statistical software package SPSS 13.0 was employed for data management and analysis.

Results

Patient characteristics and follow-up data

There were 26 men and 4 women included in the study. Mean age was 73 years, range (61–85 years). Follow-up was calculated from the time of the last BCG instillation. Mean follow-up was 10 months, with a range of 1–24 months. Twelve patients (40%) developed recurrent disease during the study and five patients (16.7%) developed invasive recurrent disease. Three patients were found to have recurrent disease 4 weeks after the last BCG instillation during the planned post-BCG cystoscopy and biopsy. Patient’s clinical characteristics as well as hTERT staining results are presented in Table 1.

Table 1.

Patient clinical characteristics and hTERT staining results

Patients Total number of patients hTERT positive hTERT negative
PreBCG PostBCG PreBCG PostBCG
Gender
 Male 26 19 11 7 15
 Female 4 2 2 2 2
Age
 <70 11 8 4 3 7
 >70 19 13 9 6 10
Pre-BCG (all) 30 21 9
Post-BCG (all) 30 13 17
Recurrence
 Yes 12 11 10 1 2
 No 18 10 3 8 15
Development of invasive disease
 Yes 5 5 5 0 0
 No 25 16 8 9 17
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hTERT immunopositivity was present in the majority of resected bladder tumors prior to BCG

hTERT immunopositivity (nucleolar staining in more than 50% of the tumor cells) was detected in the bladder tumors resected during TURBT (prior to adjuvant BCG) in 21 out of 30 patients (70%), Fig. 1a. On the contrary, random biopsies of normal bladder mucosa obtained from these patients were negative for hTERT in all cases. Telomerase expression as assessed by immunohistochemistry was highly correlated between tumor samples from the same patients (Pearson correlation coefficient 0.95, P < 0.001).

Fig. 1.

Fig. 1

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hTERT positive (a) and negative (b) samples obtained from the same patient pre- and post-BCG, respectively

Pre-BCG hTERT expression in resected bladder tumors did not show any correlation with stage or age of the patients. Furthermore, pre-BCG hTERT immunopositivity was not associated with recurrence free survival in univariate or multivariate analysis and was not associated with development of invasive disease.

However, if we assumed a positivity cut-off of 75% (instead of 50%), patients with tumors that were hTERT positive (i.e. nucleolar staining in more than 75% of tumor cells) had a worse recurrence free survival compared to patients with tumors that hTERT negative (i.e. nucleolar staining in less than 75% of tumor cells) (median recurrence free survival 9 months vs. not yet reached respectively, log rank P = 0.05, Fig. 2). Furthermore, although there was a trend for tumors with hTERT staining in more than 75% of cells to be associated with development of invasive disease, this did not reach statistical significance (Fisher’s test P = 0.286).

Fig. 2.

Fig. 2

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Association between pre-BCG hTERT positivity (based on a cut-off of 75%) and recurrence free survival: pre-BCG hTERT positive patients had a worse recurrence free survival compared to patients with tumors that hTERT negative (median recurrence free survival 9 months vs. not yet reached respectively, log rank P = 0.05)

hTERT immunopositivity after BCG was associated with worse recurrence free survival and development of invasive disease

Four weeks after the last dose of BCG, all study patients underwent cystoscopy and biopsies of the sites where bladder tumors were previously excised (i.e. during TURBT). Three patients were found to have recurrent disease at that point (i.e. 4 weeks after last dose of BCG). The remaining 27 patients had an apparently normal bladder mucosa at the sites where bladder tumors were previously excised.

We performed immunohistochemistry to evaluate hTERT positivity in these post-BCG biopsy specimens. In 13 patients, post-BCG biopsies of the bladder mucosa at the sites where tumors were previously excised (during TURBT) were positive for hTERT (i.e. nucleolar staining present in more than 50% the cells) while hTERT was negative in 17 patients (Fig. 1b). hTERT expression in post-BCG specimens was statistically significantly decreased compared to pre-BCG specimens (P < 0.001). Presence of hTERT immunopositivity 4 weeks after BCG was associated with worse recurrence free survival compared to no hTERT positivity (6 months vs. not yet reached respectively, log rank P = 0.001, Fig. 3). Furthermore, post-BCG hTERT immunopositivity was associated with development of invasive disease (Fisher’s test P = 0.009). Specifically, none of the 17 patients with post-BCG negative hTERT developed invasive disease, whereas 5 out of 13 post-BCG hTERT positive patients developed invasive disease while on study.

Fig. 3.

Fig. 3

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Association between post-BCG hTERT positivity and recurrence free survival: post-BCG hTERT positive patients had a worse recurrence free survival compared to patients with tumors that hTERT negative (median recurrence free survival 6 months vs. not yet reached respectively, log rank P = 0.001)

Post-BCG hTERT positivity was independently associated with worse recurrence free survival and development of invasive disease

On multivariate analysis that included age, gender, pre-BCG hTERT positivity (either using a cut-off of 50% or a cut-off of 75% positive tumor cells) and post-BCG hTERT positivity, only post-BCG positivity remained independent predictor of poor recurrence-free survival. Patients with post-BCG positive hTERT had a relative risk of recurrence of 8.85 (as defined by Cox model, 95% confidence interval 1.9–41.6).

Forward stepwise logistic regression that included development of invasive disease as dependent variable and age, gender, pre-BCG hTERT positivity and post-BCG hTERT positivity as covariates revealed that only post-BCG hTERT immunopositivity was independently associated with development of invasive disease.

Discussion

Although adjuvant BCG immunotherapy has been shown to improve the clinical outcome of SBC in terms of tumor recurrence, development of invasive disease, need for subsequent cystectomy, and prolongation of overall survival, a significant proportion of patients fail to respond to it (Herr et al. 1989, 1995). Identification of the subset of patients that are going to develop recurrent disease or stage progression after BCG therapy (BCG non-responders) is very important, since aggressive therapy with early salvage cystectomy has been shown to improve overall survival (Herr and Sogani 2001; Lee et al. 2007). Previous studies have reported several candidate predictive factors of response to adjuvant BCG but a practical marker with good independent predictive value has not been identified (Saint et al. 2003). In that regard, traditional pathological tumor characteristics (Van Der Meijden et al. 2000), molecular markers such as p53 (Zlotta et al. 1999) and immunological markers such as PPD skin reaction (Shinka et al. 1990; Lamm et al. 1991) do not appear to have a consistent predictive value.

We decided to investigate whether immunohistochemical expression of hTERT would be a good predictor of BCG response for three reasons. Firstly, BCG has been documented to induce downregulation of telomerase activity in a variety of in vitro models (including bladder cancer cell lines) raising the possibility that telomerase may be a marker of response to BCG (Reed et al. 2004; Saitoh et al. 2002). Secondly, immunohistochemistry is routinely performed in pathology laboratories, and represents an ideal tool for screening paraffin-embedded and formalin-fixed resection specimens. Furthermore, in the case of hTERT, the association between nucleolar immunohistochemical staining and telomerase activity and hTERT mRNA expression has been well documented and validated (Etheridge et al. 2002; Yang et al. 2002). Thirdly, hTERT activity has been shown to provide prognostic and predictive information in a variety of epithelial neoplasms including gastric, renal, hepatocellular, lung and colon cancers (Domont et al. 2005; Smith et al. 2004; Usselmann et al. 2001; Fujioka et al. 2000; Chen et al. 2006; Kobayashi et al. 2001). In brief, telomerase is a ribonucleoprotein reverse transcriptase that uses its internal RNA moiety as a template for elongation of telomeres (Blackburn 1991). The telomerase holoenzyme has two functional components, a reverse transcriptase (telomerase reverse transcriptase–hTERT) which is expressed only during cellular proliferation, and a telomerase RNA molecule (telomerase RNA–TR) which is expressed constitutively and contains the template for the synthesis of new telomeric repeats. While in somatic cells telomerase expression is repressed and telomere length is progressively shortened with each cell division, in cancer cells telomerase is expressed resulting in stabilized telomere length and cell immortalization (Shay and Wright 1996).

In this study, we evaluated 30 patients with high risk SBC (all T1G3 tumors) who underwent TURBT followed by adjuvant BCG immunotherapy. We assessed immunohistochemical hTERT expression at baseline pre-BCG TURBT specimens as well as random biopsies from normal bladder mucosa obtained at the same time. Furthermore, contrary to previous studies which only evaluated baseline, pre-BCG tumor specimens, a novel characteristic of our study was that we also assessed immunohistochemical hTERT expression after completion of adjuvant BCG. Specifically, 4 weeks after the last dose of BCG, all patients underwent follow-up (post-BCG) cystoscopy and biopsy of the sites where bladder tumors were previously excised (i.e. during TURBT), and these specimens were also evaluated for immunohistochemical expression of hTERT. The primary antibody used in the present study (clone 44F12) has been widely used in the literature, in more than ten published immunohistochemical studies of hTERT (Brustmann 2005; Dalerba et al. 2005; Domont et al. 2005; Dutu et al. 2005; Elkak et al. 2005; Falchetti et al. 2003; Kraemer et al. 2003; Lantuejoul et al. 2005, 2004; Uziel et al. 2005). However, it is important to note that, after initiation of our study, Wu and colleagues raised the possibility that this antibody may recognize nucleolin instead of hTERT (Wu et al. 2006), and this remains a controversial issue.

Our study showed that the vast majority of baseline pre-BCG bladder tumor specimens were immunopositive for hTERT, while normal bladder mucosa was negative for hTERT in all cases. Furthermore, when using an hTERT positivity cut-off of 75% of tumor cells, patients with hTERT positive tumors had a worse recurrence free survival compared to patients with hTERT negative tumors. Baseline pre-BCG expression of hTERT was not associated with subsequent development of invasive disease after BCG.

Immunohistochemical assessment of hTERT in post-BCG (4 weeks after the last dose of BCG) specimens yielded some very interesting findings. Firstly, expression of hTERT was statistically significantly decreased in post-BCG specimens compared to pre-BCG specimens. This partly reflects the fact that patients underwent TURBT (i.e. resection of all visible bladder tumors) but it may also reflect an effect of BCG therapy itself on hTERT expression. Secondly, although 27 patients had not developed recurrent disease at that point (only three patients had developed recurrent disease) and had a cystoscopically normal-appearing bladder mucosa at the sites where bladder tumors were previously excised (during TURBT), almost half of them (13 out of 27) had hTERT positive biopsies suggesting that the cystoscopically normal-appearing bladder mucosa at these sites (where bladder tumors were previously excised) was not ‘normal’ at a molecular level. Importantly, presence of hTERT immunopositivity was associated with worse recurrence free survival and with development of invasive disease. Furthermore, multivariate analysis including age, gender and pre- and post-BCG hTERT expression revealed that only post-BCG hTERT immunopositivity was independently associated with worse recurrence free survival and development of invasive disease.

Our study suggests that immunohistochemical evaluation of hTERT protein in paraffin-embedded and formalin-fixed resection specimens from patients with high risk superficial bladder cancer can provide predictive information of response to BCG. It also suggests that examination of post-BCG (4 weeks after last dose of BCG) biopsy specimens from the sites where bladder tumors had been previously resected may provide additional information with prognostic implications. In this case, it was hTERT expression in the post-BCG specimens and not in the pre-BCG baseline specimens that was independently associated with recurrence free survival as well as development of invasive disease in multivariate analysis. Immunohistochemical evaluation of hTERT expression could be a practical and widely applicable marker of BCG response, as it has been well validated in other malignancies, immunohistochemistry is routinely performed in pathology laboratories and assessment of nucleolar staining (percent of tumor cells with positive nucleolar staining) provides an objective and unbiased way of evaluation of immunohistochemical hTERT expression. In that regard, assessment of immunohistochemical hTERT positivity in post-BCG biopsies of the sites where bladder tumors had been resected can be used as part of a treatment decision algorithm that identifies a subset of patients who will eventually fail BCG and would therefore be candidates for early salvage cystectomy. Finally, future studies looking at predictive markers of BCG response may incorporate immunohistochemical expression of hTERT as well as examination of post-BCG biopsy specimens in determining their final predictive models of BCG response.

Conflict of interest statement

The authors declare no conflict of interest.

Footnotes

I. Zachos and P. A. Konstantinopoulos contributed equally to this work.

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