Transforming growth factor (TGF) β, a potent growth inhibitor of proliferation in most cells, usually exerts its effects through an interaction with membrane receptors, type I (TβR-I) and type II (TβR-II). In the present study, the expression of TGF-β receptors was correlated with tumor grade, pathological stage, and probability of progression and survival in patients with bladder transitional cell carcinoma (TCC). To this end, immunohistochemistry was carried out in specimens obtained from 59 patients who underwent either radical cystectomy or transurethral resection of bladder tumor. Among these patients, 18 (30.5%) had loss of TβR-I expression, whereas 27 (44.0%) had loss of TβR-II expression. There was a correlation between the loss of expression of TβR-I and TβR-II and the tumor grade (P = 0.041 and P = 0.026, respectively). In addition, both pathological and lymph node status also were associated with the loss of TβR-I and TβR-II expression (P = 0.025 and P = 0.004, respectively). Interestingly though, only the loss of expression of TβR-I was associated with an increased probability of tumor progression and a decreased probability of survival (P = 0.0046 and P = 0.0022, respectively). These results suggest that the status of TβR-I expression may be a potential prognostic marker in patients with bladder TCC.

Bladder cancer is the second most common urological malignancy in the United States. There were ∼54,500 new cases and 11,700 deaths in 1997 (1). Approximately 90% of bladder cancers in the United States are TCCs.3 Radical cystectomy is widely used as definitive therapy for muscle invasive bladder cancer and for superficial bladder cancer that is refractory to chemo- or immunotherapy. For deep-muscle invasive and lymph node-positive cancers, adjuvant and neoadjuvant chemotherapy may improve cancer control rates and long-term survival (2, 3, 4). With such varying treatment options, identification of patients at high risk for tumor progression after the initial treatment will permit a more appropriate selection of patients for early cystectomy, while providing a more conservative approach in patients whose tumors are less likely to progress. In this regard, molecular markers may aid in identifying bladder cancer patients who have higher risk of tumor progression after the definitive treatment. One such group of potential molecular markers are TβRs.

TGF-β is a Mr 25,000 pleiotropic growth factor that is expressed by many cell lines and tissue types. There are three ubiquitously expressed TβRs: types I, II, and III (TβR-I, TβR-II, and TβR-III, respectively). TβR-III is a membrane proteoglycan that has a very short cytoplasmic tail and lacks any signaling motif, whereas TβR-I and TβR-II are serine/threonine kinases (5, 6). The present theory concerning the mechanism of action of TβRs states that both TβR-I and TβR-II are required for TGF-β signal transduction (7, 8).

TGF-β usually acts as a potent growth inhibitor in most cells, especially those of the epithelial lineage (reviewed in Ref. 9). However, malignant cells are frequently resistant to the growth-inhibitory effects of TGF-β (10, 11, 12, 13). An accumulating body of evidence suggests that alteration in the expression of TβRs may play a critical role in rendering malignant cells resistant to TGF-β (14, 15, 16, 17). Specifically, loss of expression of TβRs has been demonstrated in prostate, colon, and stomach cancer (13, 14, 15). Moreover, TβR-II has been suggested as a tumor suppressor in a subset of colon cancers (15, 16).

In bladder cancer, it has been demonstrated that the serum level of TGF-β is elevated in TCC patients (18). Among bladder cancer cell lines, loss of TβR-I has been reported (19). Moreover, transfection of TβR-I into these cells that lack TβR-I expression results in a decreased tumorigenic potential. These results suggest that the loss of TβR expression may be an important event during bladder carcinogenesis. Therefore, the prognostic value of the expression of TβR-I and TβR-II in 59 patients with bladder TCCs was investigated in the present study. We observed that the loss of TβR-I expression was associated with increased risk of progression and death in these patients.

Patient Population.

Fifty-nine patients with bladder TCCs who underwent radical cystectomy (n = 55) or transurethral resection of the bladder tumor were included in the study. Table 1 shows the tumor grade, pathological stage, lymph node status, and surgical treatment in these patients. Patients who had received radiotherapy or systemic chemotherapy previously were excluded from this study. Of the 59 patients, 46 were men and 13 were women; ages ranged from 39 to 94 years (median age, 66 years). The pathological stage was determined according to the 1997 American Joint Committee on Cancer classification (20). No patient had evidence of distant metastatic disease at the time of the surgery. The median follow-up was 41.6 months (range, 1.8–132.1) for patients who were alive at the time of analysis.

Immunohistochemistry.

All H&E-stained slides were reviewed by one pathologist (T. M. W.) to confirm stage and grade. Five μm sections were cut from paraffin-embedded tissue blocks and mounted on poly-l-lysine-coated slides. These slides were stored at 4°C until staining. The sections were dewaxed in xylene and rehydrated with graded alcohols. Endogenous peroxidase activity was inactivated by incubating in 3% H2O2 for 25 min. The antigen retrieval procedure using a microwave technique was used (21). The tissue sections were immersed in 0.1 m citrate buffer (pH 6.0) and microwaved at 800W for 10 min. Subsequently, the sections were incubated with primary antibodies in a humidified chamber for 24 h at 4°C. Anti-TβR-I and anti-TβR-II antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used at a concentration of 2 μg/ml. The specificity of this antibody was demonstrated previously (22, 23). The following day, the sections were washed three times in fresh PBS, and the secondary antibody was applied at a dilution of 1:400. Visualization of the reactivity was accomplished with an avidin-biotin complex immunoperoxidase system (Vector, Burlingame, CA), according to the manufacturer’s recommendations. Diaminobenzidine (0.03%) was used as a chromogen. The sections were counterstained with Mayer’s Hematoxylin (Sigma Diagnostics).

Negative control sections were processed in an identical manner by substitution of primary antibody with a normal rabbit IgG. No negative control sections showed any color reactions. Because immunohistochemistry is not quantitative, all sections were categorized as either positive or negative staining for TβRs. Specimens were classified as negative if the staining level was comparable with that of the negative control slides. All negative cases were confirmed with at least two indepen-dent staining experiments. All slides were reviewed indepen-dently by two individuals.

Statistics.

Survival was calculated on the basis of the date of death due to bladder TCC or the date of the last follow-up contact (either a clinical visit or information provided by the patient’s referring physician). Time to progression was calculated from the date of surgery to the date of the first documented clinical recurrence or the last follow-up. Patients whose disease never recurred and died of other causes were censored at their death. Times to progression and survival were analyzed using the Kaplan-Meier method, and the log-rank test was used to assess the difference between the negative and positive groups. The χ2 test was used to evaluate the association between the expression of TβRs and histological grade, pathological stage, and the lymph node status. For multivariate analysis, a Cox proportional hazard model was used. P < 0.05 was considered statistically significant. Statistical analyses were performed using the STATA statistics package (Stata Corp., College Station, TX).

Expression of TβRs in Normal Bladder and Bladder TCC.

A representative result of the immunohistochemistry for TβR-I and TβR-II in normal and malignant bladder tissues is shown in Figs. 1 and 2, respectively. As shown, the expression of TβR-I and TβR-II was predominantly localized in the epithelial cells in normal bladder tissues. In bladder cancer cells though, the expression of TβRs was frequently decreased with increasing tumor grade. In the 59 cases investigated in this study, 18 (30.5%) had loss of TβR-I expression, and 27 (44.0%) had loss of TβR-II expression.

Expression of TβRs and Tumor Grade and Pathological Stage.

The χ2 test showed a statistically significant relationship between TβR-I expression and tumor grade (Table 2). TβR-I expression was detectable in 100 and 87% of grades 1 and 2 tumors, respectively. In contrast, only 60% of grade 3 tumors were positive for TβR-I expression (P = 0.041). Similarly, the expression of TβR-II also correlated with tumor grade (Table 3). In grade 1 tumors, 100% had positive TβR-II expression, whereas 50 and 46% of grades 2 and 3 tumors had detectable levels of TβR-II expression, respectively (P = 0.026). To investigate the relationship between expression of TβRs and pathological stage, patients were classified into three groups: (a) those with positive lymph node metastasis; (b) those with organ-confined tumors with no evidence of lymph node metastasis; and (c) those with extravesical tumor with no evidence of lymph node metastasis. The results revealed that the expression status of both TβR-I and TβR-II was associated with tumor stage (P = 0.025 and P = 0.004, respectively; Tables 2 and 3).

TβR Expression and Probability of Progression.

As an initial attempt to determine the potential role of the expression of TβRs as a prognostic marker in bladder TCC patients, we investigated the relationship between the expression of TβRs and the probability of progression. Of 59 patients, 22 patients had progressed, with the median time to progression of 39.1 months. As shown in Fig. 3,A, the rate of progression after surgery for patients with loss of TβR-I expression was significantly higher than for those with normal expression (P = 0.0046). A multivariate analysis also demonstrated that the loss of TβR-I expression was significantly associated with tumor progression (Table 4). There was no statistically significant association between TβR-II expression and probability of progression (Fig. 3 B).

TβR Expression and Probability of Survival.

To further study the role of the expression of TβR status as a potential prognostic indicator, we investigated the relationship between the expression of TβRs and the probability of survival after surgery. Of 59 patients, 30 were dead at the time of analysis. Of these patients, 21 died of metastatic bladder TCCs, and 9 died without evidence of recurrence. As shown in Fig. 4,A, the loss of TβR-I expression was associated with the decreased probability of cancer specific survival (P = 0.0022). A multivariate analysis also demonstrated that the loss of TβR-I expression was significantly associated with tumor recurrence (Table 5). There was no statistically significant association between TβR-II expression and probability of survival (Fig. 4 B).

The results of the present study have demonstrated that the expression of TβR-I is associated with tumor grade, pathological stage, increased probability of progression, and decreased probability of survival after surgery. In contrast, the status of TβR-II expression was associated with only the tumor grade and pathological stage and not with the probability of progression and survival. Taken together, these observations suggest that the status of TβR-I expression is a potential prognostic marker in bladder TCC patients.

TGF-β is pleiotropic growth factor that regulates cellular proliferation, angiogenesis, and chemotaxis (24, 25). In cells of epithelial lineage, TGF-β is a potent growth inhibitor. Nevertheless, it has been demonstrated that malignant epithelial cells frequently have elevated levels of TGF-β expression and a loss of its receptor expression. Because overexpression of TGF-β renders survival advantage by suppressing immune response and enhancing angiogenesis, the loss of expression of TβRs may be a critical event during carcinogenesis because resistance to the growth-inhibitory effect of TGF-β likely will enable overexpression of TGF-β. This concept is consistent with the results of the present study in which the loss of TβRs was seen most frequently in high-grade tumors. In addition, loss of expression of TβRs was associated with higher pathological stage. With respect to probability of tumor progression and survival, only the loss of TβR-I expression showed a significant association by univariate analysis and was independently associated with both clinical end points by multivariate analysis. These results suggest that the loss of expression of TβRs, especially of TβR-I, may be an important event during bladder TCC carcinogenesis.

Because both TβR-I and TβR-II are required for TGF-β signaling, the loss of TβR-I should have the same biological effects as the loss of TβR-II. Yet, in the present study, only TβR-I expression status was significantly associated with prognosis. It is possible that the sample size in the present study may not have been large enough to demonstrate a correlation between the loss of TβR-II expression and prognosis. However, the possibility exists that in bladder cancer cells, the biological consequence of the loss of TβR-I expression may not be the same as the loss of TβR-II expression. Interestingly, TβR-I is mapped to human chromosome 9q23-24 (26); loss of heterozygosity of chromosome 9q has been shown in >50% of bladder cancer (27). Therefore, TβR-I may be a tumor suppressor in bladder cancer. Further work is under way to verify this concept.

In conclusion, results of the present study have demonstrated that the status of TβR-I expression is a potential prognostic marker in bladder TCC patients. In the future, the specific role of TβR-I in bladder TCC cells will be investigated.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

This work was supported in part by Hankamer Research Fund.

                
3

The abbreviations used are: TCC, transitional cell carcinoma; TGF, transforming growth factor; TβR, TGF-β receptor.

Fig. 1.

Immunohistochemistry for TβR-I in normal and malignant bladder tissues. Note the positive brown staining cells. TβR-I was predominantly expressed by epithelial cells. In addition, there was a significant decrease in the level of expression of TβR-I with increasing grade of bladder cancer

Fig. 1.

Immunohistochemistry for TβR-I in normal and malignant bladder tissues. Note the positive brown staining cells. TβR-I was predominantly expressed by epithelial cells. In addition, there was a significant decrease in the level of expression of TβR-I with increasing grade of bladder cancer

Close modal
Fig. 2.

Immunohistochemistry for TβR-II in normal and malignant bladder tissues. Note the positive brown staining cells. TβR-II was predominantly expressed by epithelial cells. In addition, there was a significant decrease in the level of expression of TβR-II with increasing grade of bladder cancer

Fig. 2.

Immunohistochemistry for TβR-II in normal and malignant bladder tissues. Note the positive brown staining cells. TβR-II was predominantly expressed by epithelial cells. In addition, there was a significant decrease in the level of expression of TβR-II with increasing grade of bladder cancer

Close modal
Fig. 3.

Kaplan-Meier estimated probability of progression related to expression of TβRs in bladder cancer tissues. A, TβR-I (P = 0.0046); B, TβR-II (P = 0.1931)

Fig. 3.

Kaplan-Meier estimated probability of progression related to expression of TβRs in bladder cancer tissues. A, TβR-I (P = 0.0046); B, TβR-II (P = 0.1931)

Close modal
Fig. 4.

Kaplan-Meier estimated cancer-specific survival related to expression of TβRs in bladder cancer tissues. A, TβR-I (P = 0.0022); B, TβR-II (P = 0.0912)

Fig. 4.

Kaplan-Meier estimated cancer-specific survival related to expression of TβRs in bladder cancer tissues. A, TβR-I (P = 0.0022); B, TβR-II (P = 0.0912)

Close modal
Table 1

Tumor grade, pathological stage, lymph node status, and surgical treatment in 59 patients with bladder TCC

No.(%)
Tumor grade   
 I 13.5 
 II 13.5 
 III 43 73.0 
Pathological stage   
 PIS 10.2 
 P1 12 20.3 
 P2a 15.3 
 P2b 10.2 
 P3 18 30.5 
 P4a 13.5 
Lymph node status   
 LN− 41 69.5 
 LN+ 18 30.5 
Surgery   
 TUR-BTa 6.8 
 Radical cystectomy 55 93.2 
No.(%)
Tumor grade   
 I 13.5 
 II 13.5 
 III 43 73.0 
Pathological stage   
 PIS 10.2 
 P1 12 20.3 
 P2a 15.3 
 P2b 10.2 
 P3 18 30.5 
 P4a 13.5 
Lymph node status   
 LN− 41 69.5 
 LN+ 18 30.5 
Surgery   
 TUR-BTa 6.8 
 Radical cystectomy 55 93.2 
a

TUR-BT, transurethral resection of the bladder tumor.

Table 2

TβR-I expression, tumor grade, and pathological stage in bladder TCC patients

TβR-I
GradeNo.Normal expression n (%)Aberrant expression n (%)
8 (100) 0 (0)  
II 7 (87) 1 (13) P = 0.041 
III 43 26 (60) 17 (40)  
LN−     
 PIS–P2b 28 24 (85) 4 (15)  
 P3–P4a 13 6 (46) 7 (54) P = 0.025 
LN+ 18 11 (61) 7 (39)  
TβR-I
GradeNo.Normal expression n (%)Aberrant expression n (%)
8 (100) 0 (0)  
II 7 (87) 1 (13) P = 0.041 
III 43 26 (60) 17 (40)  
LN−     
 PIS–P2b 28 24 (85) 4 (15)  
 P3–P4a 13 6 (46) 7 (54) P = 0.025 
LN+ 18 11 (61) 7 (39)  
Table 3

TβR-II expression, tumor grade, and pathological stage in bladder TCC patients

TβR-II
GradeNo.Normal expression n (%)Aberrant expression n (%)
8 (100) 0 (0)  
II 4 (50) 5 (50) P = 0.026 
III 43 20 (46) 23 (54)  
LN−     
 PIS–P2b 28 22 (79) 6 (21)  
 P3–P4a 13 4 (30) 9 (70) P = 0.004 
LN+ 18 7 (39) 11 (61)  
TβR-II
GradeNo.Normal expression n (%)Aberrant expression n (%)
8 (100) 0 (0)  
II 4 (50) 5 (50) P = 0.026 
III 43 20 (46) 23 (54)  
LN−     
 PIS–P2b 28 22 (79) 6 (21)  
 P3–P4a 13 4 (30) 9 (70) P = 0.004 
LN+ 18 7 (39) 11 (61)  
Table 4

Cox proportional model for progression

Hazard ratioP
Stages 1.1228 0.451 
Nodes 2.0816 0.136 
TβR-I 2.5493 0.050 
TβR-II 0.8744 0.781 
Hazard ratioP
Stages 1.1228 0.451 
Nodes 2.0816 0.136 
TβR-I 2.5493 0.050 
TβR-II 0.8744 0.781 
Table 5

Cox proportional model for cancer-specific death

Hazard ratioP
Stages 1.2693 0.157 
Nodes 2.5383 0.061 
TβR-I 2.8880 0.034 
TβR-II 0.9382 0.902 
Hazard ratioP
Stages 1.2693 0.157 
Nodes 2.5383 0.061 
TβR-I 2.8880 0.034 
TβR-II 0.9382 0.902 
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