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Keywords:

  • CD34;
  • immunohistochemistry;
  • platelet-derived endothelial cell growth factor/thymidine phosphorylase;
  • pT1 G3 bladder cancer

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Abstract Background: The management of patients with pT1 G3 bladder cancer remains controversial because of the high incidence of recurrence with muscle invasion. Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity. The aim of this study was to determine whether the expression of PD-ECGF/dThdPase in bladder cancer tissue was associated with tumor progression and recurrence in patients with pT1 G3 bladder cancer.

Methods: Fifteen patients who were pathologically diagnosed as having pT1 G3 transitional cell carcinoma of the bladder were treated with transurethral resection. Sections of paraffin-embedded bladder tissue were immunohistochemically stained with either mAb654–1, a monoclonal antibody against human PD-ECGF or anti-CD34 monoclonal antibody, respectively. When more than 10% of tumor cells were positively stained with mAb654–1, this section was defined as positive in this study.

Results: Eight of 15 sections from patients with pT1 G3 bladder cancer (53%) were positive with PD-ECGF/dThdPase. During follow up, patients in the negative group had no disease progression and only two patients had local recurrence. In contrast, seven of eight positives had recurrence (P < 0.05) and progression was also observed in four recurrent patients. However, there was no statistical relationship between PD-ECGF and CD34 expression in any of the patients.

Conclusion: The expression of PD-ECGF/dThdPase appears to be an important prognostic factor of pT1 G3 bladder cancer and did not show any significant relationship between PD-ECGF/dThdPase expression and vascular density.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Transitional cell carcinoma (TCC) of the urinary bladder consists of superficial and muscle invading tumors. At the first presentation, about 75–80% of bladder cancers are superficial and the remainder invade the muscle layers.1 Superficial bladder tumors that are localized at the vesical mucosa or lamina propria are usually managed by transurethral resection (TUR) and the prognosis is relatively good. However, if the tumor contains a grade 3 (G3) component, the incidence of recurrence with muscle invasion significantly increases.2 Because of this feature, the management of patients with stage T1 G3 bladder cancer remains controversial and some physicians advocate early cystectomy.3 Initial cystectomy would cure almost all patients of pT1 G3 bladder cancer, but is accompanied by severe deterioration of the quality of life. However, intravesical immunotherapy by bacillus Calmette-Guerin (BCG) reduces the risk of recurrence of T1 G3 bladder cancer after TUR.4,5 Suitable markers that can predict disease progression are necessary for the selection of appropriate therapy.

Several clinical and histopathological features have been identified as prognostic factors that influence the progression and recurrence of high-grade superficial bladder cancer, for example, squamous cell carcinoma (SCC) antigen,6 p53 expression,7 and DNA ploidy.8

Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity.9 The level of PD-ECGF/dThdPase expression in several cancerous tissues is higher than that in surrounding normal tissue and dThdPase seems to have an important role in tumor angiogenesis.10 The relationship between PD-ECGF/dThdPase expression and tumor grade or stage in bladder cancer has been studied.11,12 O’Brien et al. suggested that this angiogenic factor might facilitate the progression of superficial tumors to invasive disease.13 Immunohistochemical analysis has shown that dThdPase expression in bladder cancer is correlated to tumor grade and stage.14

Based on these findings, the relationship between PD-ECGF/dThdPase expression and the prognosis of patients with pT1 G3 TCC of the urinary bladder was examined.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients

Fifteen patients (13 men and two women) were diagnosed as having superficial (T1) G3 TCC of the urinary bladder at Nagasaki University Hospital between 1988 and 1995. The mean age of the patients was 73.9 years (range, 58–86 years). No patient was diagnosed with carcinoma in situ at that time and no adjuvant therapy was administered before specimens were obtained. The clinical stage of each bladder tumor was determined according to TNM criteria as described,14 and all patients underwent TUR and intravesical chemotherapy (anthracyclin). The patients were followed up with cystoscopy and urine cytology every 3 months during the first year, then every 4 months. The mean follow-up period was 40 months (range, 3–56 months; Table 1). Informed consent was obtained from each patient before this research.

Table 1.  Characteristics of patients
  1. F, female; M, male.

No. patients15
Age73.9 ± 7.5
Follow up period (months)40 ± 22
Sex (M/F)13/2
Recurrence9 (66%)
Progression4 (26%)
Prognosis
Death due to cancer2
Death due to other disease3
Radical cystectomy2

Histology

All histological analysis was performed on hematoxylin and eosin stained sections of tissue from both biopsy and TUR and diagnosed as pT1 G3 TCC of the urinary bladder.

Antibody

Mouse monoclonal antibody mAb654–1, which recognizes PD-ECGF/dThdPase, was supplied by Nippon Roche, Tokyo, Japan.15 A mouse monoclonal antibody that recognizes human hematopoietic CD34 class 1 progenitor cells, was purchased from DAKO, Copenhagen, Denmark.16

Immunohistochemical analysis

Paraffin-embedded tissues were sectioned and placed on saline-coated glass slides. Deparaffinized sections were placed in 0.1 mol/L citrate buffers (pH 7.0) and heated at 95°C‚ for 40 min. After washing with phosphate-buffered saline (PBS), sections were incubated with 2% bovine serum albumin (BSA), 10% normal goat serum in SP2001 blocking kit (Vector, Burlingame, CA, USA) at room temperature to block non-specific binding for 60 min. The primary antibody mAb654–1 or anti-CD34 antibody was applied to the sections, which were then incubated overnight at 4°C. After washing with 0.05% Tween-20 in PBS, sections were incubated with biotinylated antimouse immunoglobulin G (IgG; Zymed, South San Francisco, CA, USA) for 1 h at room temperature. After being washed with 0.05% Tween-20 in PBS, the sections were incubated with avitinized alkaline phosphatase in PBS (Vectastain sk5000 kit, Vector), washing with 0.5 mol/L NaCl in 10 mmol/L Tris-HCl (pH 7.5) incubated with alkaline phosphatase substrate in PBS (Vectastain sk5100 kit, Vector). The slides were counter-stained with 2% methyl green and mounted. When more than 10% of the tumor cells were stained, this section was defined as positive and all others were considered negative among over a thousand cells examined for dThdPase expression. Samples were considered CD34 positive when immunostained endothelial cells or clusters were clearly separated from tumor cells and other connective tissue elements.

Statistics

The relationship between PD-ECGF/dThdPase expression and categorical variables were evaluated using the χ2 analysis. A P-value < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Immunohistochemistry

Immunohistochemical staining of sections of tumor tissue with mAb654–1 was carried out to examine the expression of PD-ECGF/dThdPase. Figure 1 shows that more than 50% of tumor cells were positively stained in this section, as well as some interstitial cells. More than 10% of tumor cells were positively stained for PD-ECGF/dThdPase in eight of 15 (53%) patients with pT1 G3. PD-ECGF/dThdPase was expressed both in the cytoplasm and in the nucleus,14 but not in normal epithelium of the urinary bladder (data not shown). CD34-positive cells were detected in the endothelium of small vessels (Fig. 2).

image

Figure 1. Immunohistochemical staining of PD-ECGF in pT1 G3 bladder cancer. Sections were stained with mAb654–1, which recognizes PD-ECGF/dThdPase (original magnification × 200).

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image

Figure 2. Immunohistochemical staining of CD34 in pT1 G3 bladder cancer. Only vascular endothelium was positively stained (original magnification × 200).

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PD-ECGF/dThdPase expression and postoperative tumor progression in patients with pT1 G3 bladder cancer

Characteristics of patients with pT1 G3 bladder cancer examined in the present study are summarized in Table 1. During follow up, nine patients showed recurrence (66%) and four patients demonstrated increased clinical stage. Patients were separated into either positive (n = 8) or negative (n = 7) groups for the expression of PD-ECGF/dThdPase. There were no significant differences in the history of both groups (Table 2). However, patients without PD-ECGF/dThdPase expression showed no history of disease progression and two had local recurrence. In contrast, seven patients in the positive group (87.5%) had recurrence (P < 0.05; compared with negative group) and disease progressed in four of these patients (Table 2). A relationship between CD34 expression (vascular density) and PD-ECGF/dThdPase expression was examined, but no correlation was observed (Table 3).

Table 2.  Summary of patients with PD-ECGF/dThdPase positive and negative pT1 G3 bladder cancer
PD-ECGF expressionPositiveNegative
  • *

    P < 0.05; F, female; M, male.

No. patients87
Age (mean ± SD)73.8 ± 6.074.0 ± 9.5
Sex (M/F)6/27/0
Recurrence*72
Progression40
Death due to cancer20
Table 3.  Relationship between PD-ECGF/dThdPase and CD34 expression in bladder cancer tissues
CD34PD-ECGF
 PositiveNegativeTotal
Positive6410
Negative235
Total8715

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this study, 53% of pT1 G3 bladder cancers were positive for PD-ECGF/dThdPase expression, which was defined as more than 10% of cancer cells expressing PD-ECGF/dThdPase.14 In addition, 87.5% of positive patients had recurrence and 57.1% of these patients showed disease progression. Our results suggest that expression of PD-ECGF/dThdPase correlates with a poor prognosis for patients with pT1 G3 bladder cancer.

To extrapolate the correlation of PD-ECGF/dThdPase expression with tumor neovascularization, we examined CD34 expression in pT1 G3 bladder cancer. We found no relationship between CD34 expression and PD-ECGF/dThdPase expression. Our results indicated that PD-ECGF/dThdPase does not seem to be involved in the angiogenesis of bladder cancer. O’Brien et al. reported that PD-ECGF/dThdPase expression in the neoplastic epithelium of 27% of tumor tissues did not correlate with vascular density. Expression of PD-ECGF/dThdPase in tumor cells correlated with tumor grade, but not tumor stage.17 These findings suggest that the high expression of dThdPase may reflect the biological characteristics of bladder cancer cells. The effect of PD-ECGF/dThdPase on the cellular responses of bladder tumor cells may help to answer this question.

The management of patients with stage pT1 G3 bladder cancer remains problematic because this tumor is frequently associated with a high rate of recurrence and progression.18 A progression rate of about 50% of poorly differentiated T1 tumor can be expected after TUR.2 However, the progression rate in this study was 26.7%. Bono et al. reported disease progression in 23.4% of patients with primary pT1 G3 tumors treated with doxorubicin.19 The lower progression rate in this study might be due to adjuvant intravesical chemotherapy. Recently, intravesical immunotherapy with BCG was found to reduce the risk of recurrence and progression of pT1 G3 bladder cancer.4 However, Catalona et al. demonstrated that some patients failed two courses of BCG therapy.20 In these patients, the risk of invasive cancer or metastases developing exceeded the likelihood of eradicating the superficial tumor. Therefore, other prognostic markers will be required for such patients.

Bacillus Calmette-Guerin immunotherapy induces various cytokines and growth factors, including interleukin-1 (IL-1), tumor necrosis factor (TNF) and interferon-γ, which can induce PD-ECGF/dThdPase activity21 but also modulate angiogenesis. PD-ECGF/dThdPase has been investigated as a modulator of chemotherapeutic drugs such as doxifluridine, which is converted into 5-fluorouracil by dThdPase in tissues.13 Therefore, BCG immunotherapy combined with doxifluridine might be more relevant to some pT1 G3 bladder cancers.

In conclusion, 53% of patients with pT1 G3 bladder cancer positively expressed dThdPase in tumor cells. Immunohistochemical detection of the dThdPase will allow us to predict disease progression and recurrence of pT1 G3 bladder cancer. However, more cases will be required to confirm the significance of the dThdPase expression.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Thanks to Takumi Shimogama and Etsuji Taguchi for technical assistance and Dr Tsutomu Sakuragi and Dr Shyuji Yamashita for helpful advice. Nippon Roche Co. Ltd, Tokyo, Japan provided the mouse monoclonal antibody mAb 654–1. This work was supported in part by a Grant-in-Aid (11671560) for Scientific Research from the Ministry of Education, Science and Culture of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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