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Keywords:

  • thymidylate synthase;
  • bladder cancer;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

OBJECTIVE

To investigate the expression of thymidylate synthase (TS), a key enzyme in DNA synthesis that is over-expressed in several cancer cells, in bladder cancer and its association with patient prognosis and the response to adjuvant therapy.

PATIENTS AND METHODS

In all, 67 bladder tissue specimens were obtained from patients who had undergone transurethral resection (TUR). TS expression in bladder cancer and normal bladder tissue was analysed by immunohistochemistry.

RESULTS

Of the 67 bladder tissue specimens, 47 (70%) and 10 (15%) had positive expression for TS in cancer and normal tissues, respectively. TS expression was greater in patients with Grade 3 (16/17, 94%) than in Grade 1 and 2 (31/50, 64%; P = 0.002). It was also greater in Stage T1 (14/14) than in Stage Ta (33/53, 62%; P = 0.001). Furthermore, patients with negative TS expression had a longer postoperative recurrence-free survival (RFS) than those with positive expression during the 5 year follow-up (P = 0.028). In the patients with positive TS-expressing tumours, adjuvant therapy significantly improved RFS (P < 0.001).

CONCLUSIONS

High TS expression might be a marker of poor prognosis for patients with bladder cancer. In addition, patients with high TS expression might also be benefit from adjuvant therapy.


Abbreviations
TUR

transurethral resection

TS

thymidylate synthase

RFS

recurrence-free survival

dUMP

deoxyuridine monophosphate

dTMP

deoxythymidine monophosphate

5-FU

5-fluorouracil

RR

relative risk.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

In China TCC of the urinary bladder is the most common cancer of the genitourinary tract [1]. While it is mostly nonmuscle-invasive at initial presentation, up to 70% of patients have recurrent disease and up to 15% will progress to muscle-invasive bladder cancer [2]. Although nonmuscle-invasive bladder cancer can be treated successfully, tumour recurrence is a serious clinical problem after transurethral resection (TUR). Recently, bladder cancer studies have mainly focused on the search for early diagnosis and noninvasive tumour markers but there are no reports of patients with superficial bladder cancer recurrence and 5-fluorouracil (5-FU) chemotherapy sensitivity [3].

Thymidylate synthase (TS) is a key enzyme that catalyses the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), which is an important step in the process of DNA synthesis [4]. 5-FU is one of the widely used anticancer chemotherapeutic agents for the treatment of various cancers including bladder cancer [5]. 5-FU itself is inactive and requires its intracellular conversion to FdUMP. FdUMP exerts its cytotoxic activity through the formation of a ternary complex with TS and 5, 10-methylene-tetrahydrofolate, resulting in inhibition of TS and blockade of the DNA synthetic process. Previous studies performed on several cancers have shown that the levels of TS expression predicted the response to 5-FU-based chemotherapy [6,7]. This is also true for other solid tumours such as breast, gastric and neck cancer, in which 5-FU is part of the chemotherapy treatment. These studies suggest that the ability to predict response and outcome based on TS expression may provide the opportunity to select those patients most likely to benefit from TS-directed therapy [8]. Furthermore, immunohistochemical staining results for TS predicted the response to 5-FU [9,10].

Our previous studies on prostate cancer showed that TS expression was greater in cancerous tissue than in normal tissue. TS expression is a significant prognostic marker in patients with prostate cancer and RCC [11,12]. Reported data on TS expression in bladder cancer are limited, and little is known about the significance of TS in the biology of bladder cancer. The aim of this study was to define whether TS expression is a prognostic marker for patients with bladder cancer. Furthermore, the effect of TS expression on the response to adjuvant therapy was also examined.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

In all, 67 TUR specimens containing bladder tumours and normal bladder mucosal tissue were obtained from the surgical pathology files of Mudanjiang Medical College (Department of Urology, Hongqi Hospital, Heilongjiang, China) from July 2001 to May 2007. After surgery all of the resected primary tumours were histologically confirmed as pTa or pT1 TCC. Any patient who received preoperative therapy (neoadjuvant chemotherapy and radiation therapy) was excluded. Formalin-fixed paraffin-embedded samples with bladder cancer and adjuvant normal mucosal tissue were collected. Central pathology review for standardization is preferred. The mean (range) age of the patients at the time of surgery was 65 (40–83) years. Histological diagnosis confirmed that the patients had TCC of the bladder. Histological classification and staging was according to the 2002 Union Internationale Contre le Cancer classification system, and were as follows: 52 lesions were superficial papillary tumours (pTa) and 15 tumours invaded the lamina propria (pT1). Also, 50 tumours were classified as low malignant potential papillary neoplasms (grade 1 and 2) and 17 tumours as high-grade papillary carcinoma (grade 3). Thirty patients received 5-FU-based adjuvant therapy after TUR.

Clinical information was obtained from a review of hospital and physician charts. Patient follow-up data were also obtained from physician records and directly from the patients. Cystoscopy during follow-up was performed at 3, 6, 9 and 12 months in the first year and every 6 months thereafter. The median (range) follow-up was 30 (1–83) months. The times to first recurrence and progression were recorded. In all, 18 patients had intravesical recurrence during the first year (27%), while another 30 patients had recurrence within 2 years (21%) and 3 years (24%), respectively. This study was performed after approval by the local Human Investigations Committee and informed consent was obtained from every patient.

TS expression was examined by immunohistochemistry, as previously described [7]. Briefly, antigen retrieval was performed by microwave heating sections in 10 mm sodium citrate buffer (pH 6) for 10 min After endogenous peroxidase activity was quenched and nonspecific binding was blocked, the slides were incubated at 4 °C overnight with monoclonal antibody TS106 anti-TS (1:100, dilution, DAKO Carpinteria, CA, USA). The secondary antibody was biotinylated rabbit anti-mouse antibody (DAKO) used at a dilution of 1:200 for 30 min at 37 °C. After further washing with Tris-buffered saline, sections were incubated with Complex/horseradish peroxidase for 30 min at 37 °C. Immunolocalization was performed by immersion in 0.05% 3,3′-diaminobenzidine tetrahydrochloride as the chromagen. Slides were counterstained with haematoxylin before dehydration and mounting.

The intensity of the immunoreactivity for TS was evaluated in normal bladder and bladder cancer tissue from the same slide in each case. At least 10 high-power fields (×400) were chosen randomly, and >1000 cancer cells were counted for each section. Two pathologists (H.D and X.S.) who were unaware of the clinicopathological data and clinical outcomes of the patients examined the TS staining results. A numeric intensity score was set from 0 to 3 (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining). Cases with >25% positive tumour cells (moderate and strong staining) in a section were regarded as positive expression as previously described [7,13,14]. Figure 1 shows representative examples; Fig. 1a shows a TS-negative staining in a normal bladder specimen and Fig. 1b TS-positive staining in a bladder cancer specimen.

image

Figure 1. Immunohistochemical staining for TS in normal bladder tissue and bladder cancer. Representative images of tissue samples with absent and strong TS expression were examined. TS expression was confined to the cytoplasm of cells as shown by immunohistochemistry. Original magnification × 400. a, Negative TS staining in normal bladder tissue. b, Positive TS staining in cytoplasm of bladder cancer tissue.

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Clinicopathological and demographic characteristics were compared using the chi-square test or the Student’s t-test. Ranked categories were assessed using the Mann–Whitney U-test. The Cox proportional hazards model was used to test the association of TS expression with clinicopathological variables for the prediction of recurrence. For the evaluation of tumour recurrence, patients were surveyed at the time of their last tumour-free clinical follow-up appointment. Postoperative recurrence-free survival (RFS) was determined using the Kaplan–Meier method and P < 0.05 was considered to indicate statistical significance.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

TS EXPRESSION IN BLADDER CANCER AND NORMAL BLADDER TISSUES

TS was expressed in the cytoplasm of both normal and cancerous bladder cells. TS expression was detected in 70% (47/67) of the bladder cancer samples (Fig. 2a). In contrast, TS expression was detected in 15% (10/67) of the normal bladder tissue specimens. These results are consistent with previous reports, which showed that TS expression in cancerous tissue was higher than that in normal tissue [15,16]. In addition, the intensity of staining in cells that reacted with the TS antibodies was significantly greater in the bladder cancer specimens than that in the normal bladder samples (P < 0.001; Table 1). Notably, none of the normal bladder cells reacted strongly with the TS antibody.

image

Figure 2. Expression of TS in normal bladder tissue and bladder cancer. The percentage of TS expression detected by immunohistochemical assay as described in the Material and methods section. a, *P < 0.001 vs normal bladder tissue; b, *P = 0.002 vs Grade 1 and 2 disease; c, *P = 0.001 vs Stage Ta.

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Table 1.  Intensity of immunohistochemical staining for TS in normal tissue and bladder cancer
Cell type, n (%)Staining intensity grade
0123
  • *

    TS staining intensity in bladder cancer was significantly higher than in normal bladder tissue, P < 0.001 (Mann–Whitney U-test).

Normal bladder21 (31)36 (54)10 (15) 0
Bladder cancer* 4 (6)16 (24)29 (43)18 (27)

The staining percentage of TS expression was greater in patients with Grade 3 disease (16/17, 94%) than that for patients with Grade 1 and 2 (31/50, 64%) disease (Fig. 2b), and it was also greater in Stage T1 tumours (14/14) than in Stage Ta (33/53, 62%) tumours (Fig. 2C).

Clinical and pathological characteristics are given in Table 2. There was a significant correlation between TS expression and histological grade, stage and multiplicity. There was no significant association between TS expression and age or gender.

Table 2.  TS expression and clinicopathological variables
VariablesTotal, nTS expression, n (%)P*
PositiveNegative
  • *

    chi-square test.

No. of patients6747 (70)20 (30) 
Mean (sd) age, years 64 (11)66 (11)0.63
Gender    
 Female14 9 50.11
 Male5338 (72)15 (28) 
Histological grade    
 G1 and G25031 (62)19 (38)0.01
 G31716 (94) 1 (6) 
Stage    
 Ta5232 (62)20 (38)0.004
 T11515 0 
Multiplicity:    
 Single4427 (61)17 (39)0.03
 Multiple2320 (87) 3 (13) 

PROGNOSTIC IMPLICATIONS OF TS EXPRESSION IN BLADDER CANCER

Patients were evaluated to determine the postoperative clinical course. From these results, patients with bladder cancer were divided into two groups, those with positive TS expression and those with negative TS expression. At 7 years of follow-up, patients with negative TS expression had a greater RFS than those with positive TS expression (Fig. 3a; P = 0.028). Patients who did not receive 5-FU-based adjuvant therapy with negative TS expression also had a greater RFS than those with positive TS expression (Fig. 3b; P = 0.003). The role of TS and other clinical and pathological variables in predicting prognosis in bladder cancer were evaluated using single variable Cox proportional hazard regression analysis (Table 3). RFS was significantly increased in patients receiving adjuvant therapy (relative risk, RR, 0.29; 95% CI 0.10–0.86; P = 0.02). Age, gender and tumour stage status did not significantly influence recurrence in this group of patients.

image

Figure 3. a, RFS for patients with bladder cancer (n = 67). RFS in patients with negative TS-expressing bladder cancer (n = 20) was significantly (P = 0.028) longer than that in patients with positive TS-expressing tumours (n = 47). b, RFS for patients with bladder cancer who did not receive 5-FU-based adjuvant therapy (n = 37). RFS in patients with negative TS-expressing bladder cancer (n = 10) was significantly (P = 0.003) longer than that in patients with positive TS-expressing tumours (n = 27).

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Table 3.  Univariate and multivariate analyses of prognostic factors in patients with bladder cancer evaluated for TS expression
VariableNRR (95% CI)P*
  • *

    Cox proportional hazards regression analysis.

Univariate analysis
Age, years:   
 ≤67331.00.94
 >67341.03 (0.36–2.91) 
Gender:   
 Female141.00.99
 Male530.99 (0.31–3.10) 
Histological grade:   
 G1 and G2501.00.56
 G3171.34 (0.49–3.65) 
T stage:   
 Ta531.00.85
 T1140.89 (0.25–3.15) 
TS expression:   
 Negative471.00.04
 Positive204.66 (1.05–20.71) 
Adjuvant therapy:   
 No461.00.02
 Yes210.29 (0.10–0.86) 
Multivariate analysis
Received adjuvant therapy 0.18 (0.05–0.63)0.007
TS positive expression 7.15 (1.49–34.27)0.014

A Cox proportional hazards model was used for multivariate survival analysis to determine whether TS expression independently predicted recurrence in patients with bladder cancer (Table 3). Pathological grade, gender, tumour stage, adjuvant therapy, and TS expression were included in the stepwise model selection process. Adjuvant therapy and TS expression were predictive of patient outcome. With the use of the Cox model, positive TS expression was a significant (P = 0.014) independent predictor of recurrence in bladder cancer (RR 7.15; 95% CI 1.49–34.27).

TS EXPRESSION AND RESPONSE TO ADJUVANT THERAPY

The influence of TS expression on the response to adjuvant therapy was further analysed in bladder cancer. In patients with positive TS-expressing tumours, adjuvant therapy significantly improved RFS (P < 0.001). The RFS at 1 and 2 years after TUR in patients with positive TS-expressing tumours was 89% and 47%, respectively. The RFS with adjuvant therapy vs no adjuvant therapy was 56% and 14%, respectively, (Fig. 4a). In patients with negative TS-expressing tumours, there was no significant difference in RFS survival among patients managed with TUR vs TUR plus adjuvant therapy (Fig. 4b).

image

Figure 4. a, The effect of adjuvant therapy in patients with positive TS-expressing bladder cancer. Adjuvant therapy was associated with a significant (P < 0.001) increase in RFS vs patients treated with TUR alone. b, Effect of adjuvant therapy in patients with negative TS-expressing bladder cancer. There was no statistically significant difference in RFS among patients managed with TUR alone and patients managed with TUR plus adjuvant therapy.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

For various cancers TS has been shown to be a promising prognostic biomarker [12,17]. In the present study, we examined TS expression in bladder cancer tissues and its association with patient prognosis and the response to adjuvant therapy. The present study showed that TS was expressed at greater levels in bladder cancer than in normal bladder tissue. The levels of TS expression correlated with the increase of the stage and grade of bladder cancer. Also, patients with negative TS expression had a longer postoperative RFS than those with positive expression during the 7 years of follow-up. Finally, adjuvant therapy significantly improved RFS in patients with positive TS-expressing tumours (P < 0.001). These findings suggest that TS might play important role in regulating the malignant potential of bladder cancer and might be a promising prognostic marker for this disease.

TS expression as a prognostic factor has been most widely studied in colorectal cancer. Johnston et al. [7] determined the levels of TS protein expression immunohistochemically in primary rectal cancers of 294 patients enrolled in National Surgical Adjuvant Breast and bowel Project Protocol R-01. High TS expression was associated with a significant decrease in overall and disease-free recurrence independent of Dukes’ stage. Using a polyclonal anti-TS antibody, Takamura et al. [18] reported improved recurrence in patients with tumours with high TS-expression. The monoclonal antibody used in the present study has been used in the vast majority of the studies that have shown the negative prognostic effect of elevated TS expression [19,20]. A close correlation between TS mRNA expression measured by reverse transcription-PCR, the level of TS protein detected by Western blot, and the results of immunohistochemistry using this antibody (TS106) has been reported previously [21]. The present results suggest that TS expression in patients with bladder cancer might provide additional prognostic information beyond the orthodox clinical and pathological prognostic markers.

There are several reports that TS might have a prognostic role for the outcome of 5-FU-based therapy together with proliferation markers such as Ki-67 and p53 [22]. Ki-67 is an established marker of cell proliferation, present during the G1, S, G2 and M stages of the cell cycle [23]. Recently, Huang et al. [24] also reported that the Ki-67 proliferation index was significantly greater in TS-positive tumours than in TS-negative tumours, suggesting that TS-negative tumours might have a low rate of cell proliferation. The TS level has also been shown to correlate with the activity of a cell cycle-regulatory protein (p21), which may also influence responses to chemotherapy in bladder cancer [25,26]. p53 mutant colorectal cancer also express higher levels of TS mRNA [27]. An in vitro study reported that pancreatic cancer lacking functional p53 also had enhanced radiosensitivity with 5-FU [28]. These results indicate that TS expression is related to cell proliferation, and any neoplastic cells with greater DNA ploidy would have increased expression of enzymes involving nucleic acid metabolism.

Although TS plays an important role in pyrimidine nucleotide synthesis and represents an important chemotherapeutic target for 5-FU, the ability of TS to predict therapeutic responses to 5-FU-based chemotherapy is far less clear [29]. Several studies in patients with measurable advanced colorectal and neck cancer treated with 5-FU-based chemotherapy have shown greater response rates in patients with low TS activity [21,30]. However, in those studies, all patients received chemotherapy, making it impossible to define whether TS expression was able to predict patients that would have a survival benefit with 5-FU. TS expression has also been used to evaluate survival in patients receiving 5-FU in the adjuvant setting. In the National Surgical Adjuvant Breast and Bowel Project R-01 trial of surgery vs surgery plus lomustine, 5-FU, and vincristine for rectal cancer, both disease-free (17% to 38%) and overall survival (31% to 54%) were significantly improved in patients with high TS levels who received chemotherapy when compared with patients managed with surgery alone [7]. In the present study, patients with negative TS expression had a longer postoperative RFS than those with positive expression during the 7 years of follow-up. Furthermore, 5-FU-based adjuvant therapy significantly improved RFS in patients with positive TS-expressing tumours. The present results are consistent with other studies suggesting that the benefit of adjuvant therapy is limited to patients with high TS-expressing tumours, although the basis for this observation remains unclear. These results strongly suggest that TS might be a promising prognostic marker for bladder cancer.

In addition, it should be noted that TS expression was detected in 15% of normal bladder tissues obtained from patients with bladder cancer. Further studies are needed to clarify whether the molecular changes (possibly including increased TS expression) precede morphological changes in the normal bladder mucosal tissues.

In summary, the present results have clearly shown that TS expression was significantly greater in the cancerous bladder tissue, and that positive TS expression was associated with a worse prognosis. The present results also suggest that fluoropyrimidines will continue to play a role in the treatment of bladder cancer, and that improved understanding of fluoropyrimidine metabolism and the sensitivity of bladder cancer to these agents is critical. The present study suggests that 5-FU-based adjuvant therapy is most beneficial in patients with high TS-expressing bladder cancer. Additional prospective studies are warranted to define the role of TS in selecting patients for adjuvant therapy for bladder cancer.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

This work was supported by Natural Scinece Foundation of Heilongjiang Province of China (Grant No. D200799).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES