Thymidine Phosphorylase Expression in Human Bladder Urothelial ...

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Objective: To clarify the impact of thymidine phosphorylase (TPase) expression in patients with urothelial carcinoma of the urinary bladder (UB-UC), we ...

Urol Sci 2011;22(1):32−37

O R I G I N A L A RT I C L E

Thymidine Phosphorylase Expression in Human Bladder Urothelial Carcinoma Victor C. Lin1,4*, Jacky C. Wang1,7, Kevin Lu1, See-Tong Pang6, Kuo-Chuan Hung2, Hui-Ching Weng5, Chao-Tien Hsu3, Tsan-Jung Yu1,4 Departments of 1Urology, 2Anesthesia and 3Pathology, E-Da Hospital, Kaohsiung City, Taiwan 4 Departments of Healthcare Administration and 5Health Management, I-Shou University, Kaohsiung City, Taiwan 6 Division of Urology, Department of Surgery, Chang Gung Memorial Hospital, Chang Gung University, Taoyuan, Taiwan 7 Division of Urology, Department of Surgery, Pao-Chien Hospital, Pingtung, Taiwan

Objective: To clarify the impact of thymidine phosphorylase (TPase) expression in patients with urothelial carcinoma of the urinary bladder (UB-UC), we investigated the expression profiles of TPase and its role in the clinical behavior of UB-UC in patients with this disease. Materials and Methods: A total of 32 patients with UB-UC were enrolled in this study. The expression intensity and pattern of TPase were examined by immunohistochemical analysis. The results were correlated with clinicopathological parameters. Results: Tumor cells were significantly immunoreactive to TPase staining, but showed stronger staining in adjacent non-tumorous stromal tissues. The staining intensity and patterns were correlated with the stage (p = 0.032, p = 0.010, respectively). Conclusions: Both the intensity and pattern of expression of TPase were associated with tumor stage in UB-UC in this cohort. There were also high expression levels of TPase in most bladder cancer cells and adjacent stromal tissues. These findings suggest that tumor and stromal TPase expressions may cooperatively promote tumor progression and angiogenesis.

Received: January 18, 2010 Revised: March 19, 2010 Accepted: July 20, 2010

KEY WORDS: angiogenesis; bladder cancer; platelet-derived endothelial cell growth factor; thymidine phosphorylase; urothelial carcinoma

*Corresponding author. Department of Urology, E-Da Hospital, Department of Nursing, I-Shou University, 1 E-Da Road, Jiau-Shu Village, Yan-Chau Township, Kaohsiung City, Taiwan. E-mail: [email protected]

1. Introduction Urothelial carcinoma of the urinary bladder (UB-UC) is the fourth most common cancer in men and the tenth most common one in women in the US, accounting for an estimated 6% and 2% of new cases, respectively.1 Because of long-term consumption of groundwater contaminated by arsenic in some parts of southern Taiwan several decades previously, UB-UC is also an important cause of cancer deaths in Taiwan accounting for 3.42% of all cancer deaths in men and 1.59% in women.2 32

Similar to Western countries, the incidence rate and mortality rate in Taiwan are increasing at a stable pace. Determining how to predict biological behaviors of bladder cancers is a critical contemporary clinical issue. It is well known that angiogenesis plays important roles in tumorigenesis and invasiveness. Several angiogenic factors are involved in the process of tumor angiogenesis. Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TPase/PD-ECGF) is implicated in the process of angiogenesis. Recently, several study groups demonstrated that the expression patterns of TPase were ©2011 Taiwan Urological Association. Published by Elsevier Taiwan LLC.

TPase expression in bladder cancer associated with clinical outcomes and therapeutic responses in several cancer types, including lung cancer and breast cancer.3–10 However, it is not well understood whether TPase also plays an important role in clinical outcomes and therapeutic responses in patients with UB-UC. Previous studies on the possible role of TPase in patients with UB-UC are conflicting, which may be due to different methodologies and methods of sample collection.11–18 To clarify the impact of TPase expression in patients with UB-UC in Taiwan, we investigated the expression patterns of TPase and its role in the clinical behavior of UB-UC in patients with this disease.

2. Materials and Methods 2.1. Patients and tumor samples All studies were performed after approval and institute oversight of the Institutional Review Board for the Protection of Human Subjects at E-Da Hospital. Written informed consent was obtained from each patient or their family, as appropriate. All 47 consecutive patients admitted to our institution for treatment of bladder cancer by partial/radical cystectomy and bilateral lymphadenectomy during the period from October 2004 to September 2007 were potential candidates for these analyses. Of these 47 patients, adequate archival tissues were only available in 32 patients for immunohistochemical analysis. Among these 32 patients, 23 patients underwent a radical cystectomy, seven underwent a partial cystectomy, and two patients underwent transurethral resection of the bladder tumor. Tumors were graded according to the World Health Organization 2004 grading criteria and staged according to the American Joint Committee on Cancer staging system 2002. The clinical data were retrospectively collected by chart review, and the length of follow-up was calculated from the date of surgery. Disease recurrence was defined as any evidence of a tumor in a retained urinary bladder or previous surgical site. When patients died, the cause of death was determined by the treating physicians, by chart review corroborated by the death certificate, or by the death certificate alone. Perioperative mortality (death within 30 days of surgery) was censored at the time of death for the bladder cancer-specific survival analyses. Patients were generally followed-up postoperatively at least every 3 months for the first 2 years, semiannually for the 3rd and 4th years, and annually thereafter. Follow-up visits generally consisted of a physical examination and serum chemistry evaluation, along with liver function tests and alkaline phosphorylase when clinically indicated. Diagnostic imaging of the upper urinary tract such as ultrasonography and/or intravenous urography and chest radiography were performed at least annually or when clinically indicated. A bone scan and/or computed tomography were performed at the discretion of the Vol. 22, 32–37, March 2011

treating physician. The median follow-up was 19.94 months (range, 2–50).

2.2. Immunohistochemical staining Tissue samples in paraffin blocks were sectioned into 4 μm slices and placed on glass slides. After deparaffinization and blocking of endogenous peroxidase activity, sections were placed in 0.1 mol/L citrate buffer (pH 6.0) and heated in a microwave oven for 10 minutes. After being washed with phosphate-buffered saline (PBS) four times, sections were treated with horse normal serum in PBS (1:10) at room temperature for 40 minutes and incubated with primary anti-TPase monoclonal antibody (MoAb 654-1) (P-GE.44C; Calbiochem, San Diego, CA, USA) diluted in PBS (1:500) overnight at 4°C. After another washing with PBS, sections were further incubated with secondary biotinylated antimouse IgG (Vectarstain ABC kit; Vector Laboratories, INC., Burlingame, CA, USA) for 40 minutes at room temperature. After being washed again with PBS, sections were incubated with avidinbiotin complex (Vectastain ABC Kit; Vector Laboratories) for 40 minutes at room temperature and developed with 1 mg/mL 3,3-diaminobenzidine tetrahydrochloride in Tris-HCl buffer (pH 7.6) containing 0.03% hydrogen peroxide. Sections were also counterstained with hematoxylin and mounted. For staining detection, the Dako REAL EnVision Detection System (Dako, Hamburg, Germany) was used according to the manufacturer’s recommendations. In each experiment, a negative control was included in which non-immune antiserum was used instead of the primary antibody. We obtained the mean, maximum, range, and standard deviation of the staining intensity, and the percent positive area measurements using 10 random hot spots within each specimen as judged by two or three observers (VCL, KL, and CTH) through an open discussion. The mean of duplicate scores was calculated for data analysis. The intensity scores of immunohistochemical staining were graded from 0 to 3 (0, no staining; 1, weak staining; 2, strong staining; 3, very strong staining). The pattern scores of immunohistochemical staining ranged from 0–4 (0, no staining; 1, 0–10% positive; 2, 10–25% positive; 3, 25–50% positive; and 4, > 50% positive). Intensity scores of 0 and 1 were considered slightly immunoreactive, and those of 2 and 3 were considered highly immunoreactive. Pattern scores of 0, 1, and 2 were considered focal expression, and those of 3 and 4 were considered diffuse expression.

2.3. Statistical analysis Continuous data in the two groups were evaluated by Mann-Whitney U-tests to compare the means. Categorical data were analyzed using the chi-squared test with Fisher’s exact probability test, as appropriate. A p value of < 0.05 was considered statistically significant. Group 33

V.C. Lin, et al data are presented as the mean ± standard deviation, number, or percentage. The statistics program used for the analysis was SPSS 13.0 (SPSS, Chicago, IL, USA) for Windows.

3. Results 3.1. Patient characteristics Clinical and demographic characteristics of the patients in this study are shown in Table 1. Our study cohort consisted of 21 men and 11 women with a median age of 65.9 years (range, 42–83 years). Most patients with a high-grade tumor were younger than 65 years. All except four patients lived and patients were progressionfree during the follow-up period. Among these four patients, two died of non-cancerous causes. One patient developed pneumonia with septic shock after adjuvant chemotherapy, and the other died of respiratory failure due to a chronic obstructive pulmonary disorder with acute exacerbation. The mean follow-up time was 19.9 months (range, 2–50 months).

Table 1 Patient demographics and clinicopathological characteristics

3.2. Expression of TPase The anti-TPase antibodies significantly stained the cytoplasm and some nuclei of tumor cells, and stronger staining was also observed in adjacent non-tumorous stromal tissues. The percentage of nuclear staining in advanced stages (T2–T4) was higher than that in low stages (Ta–T1) (40% vs. 14.3%). The intensities and patterns of immunohistochemical staining were arbitrarily scored, and 65.6% of UB-UC specimens showed a moderate reaction and 40.6% showed a strong reaction. The staining intensity and patterns were correlated with the stage (p = 0.068 and p = 0.019, respectively). There were no statistically significant correlations between the degree of TPase expression and other tumor variables (Tables 2 and 3). In addition, adjacent urothelial cells and distant stromal tissues were negatively or weakly stained by anti-TPase antibodies, compared with strongly stained tumor cells and neighboring stromal tissues. However, when evaluating the postoperative recurrence after excluding the T1 subpopulation, the expression of TPase activity was correlated with disease recurrence (intensity, p = 0.027; pattern, p = 0.067). The percentage of nuclear staining was higher in advanced stages (T2–T4) than that in low stages (Ta–T1) and normal tissues (p = 0.150). Lymphovascular invasion also seems to be higher in advanced stages, however no statistical significance can be demonstrated (p = 0.069) (Figure 1).

n Sex Male Female

21 11

Median age (yr) Male Female

67.9 (42–83) 61.1 (50–79)

Follow-up period (mo)

19.9 (2–50)

Grade Low High

4 28

Lymphovascular invasion Yes No ND

12 15 5

pT category pTa pT1 pTis pT2 (2a/2b) pT3 (3a/3b) pT4 (4a/4b) pN category pN0 > pN1 pNx

1 6 0 9 (7/2) 14 (11/3) 2 (1/1) 16 4 12

Data presented as median (range) or n. ND = not determined.

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4. Discussion With recent advances in molecular biology and biotechnology, a plethora of biomarkers and therapeutic targets for cancer treatment are being explored and identified. Ongoing research into the molecular biology underlying tumorigenesis has enabled the development and clinical application of novel therapies that target key components of signaling pathways mediating angiogenesis, a process integral to tumor growth and metastasis. Among angiogenic factors implicated in angiogenesis, TPase/ PD-ECGF is a well-known, potent angiogenic molecule, which induces endothelial cell migration and proliferation, and is also a unique enzyme involved in 5-flourouracil (5-FU) metabolism. TPase/PD-ECGF is also a biomarker that predicts tumor response to 5-FU-based therapy. The expression of TPase in several types of malignant tumors is associated with increased angiogenesis, aggressiveness, and a poor prognosis.3–10 O’Brien et al. characterized the expression of TPase in 105 primary bladder cancer patients treated by transurethral resection, and they found that its expression was significantly upregulated in bladder tumors compared with that in normal bladder tissues.15 However, only the tumor grade correlated with the expression of TPase in their report. Tanaka et al also reported no correlation between the expression of TPase and the number of Vol. 22, 32–37, March 2011

TPase expression in bladder cancer Table 2 Relationship between thymidine phosphorylase (TPase) expression intensity in bladder cancer and clinicopathological characteristics* Total no.

High TPase (n = 21) n (%)

Low TPase (n = 11) n (%)

Total no. stained

64

42 (68)

22 (32)

Sex Male Female

42 22

29 (45) 13 (20)

13 (20) 9 (15)

Grade Low High

8 56

2 (3) 40 (63)

6 (9) 16 (25)

Lymphovascular invasion Yes No ND

24 30 10

18 (33) 14 (26) 10

6 (11) 16 (30) 0

pT category pTa–T1 pT2–4

14 50

4 (6) 40 (62)

10 (16) 10 (16)

Characteristics

p

0.442

0.106

0.239

0.032

*Data presented as n or n (%). TPase staining was performed twice in each patient. Staining intensity scores of 0 and 1 are defined as low intensity, while staining intensity scores of 2 and 3 are defined as high intensity. ND = not detected.

Table 3 Relationship between thymidine phosphorylase (TPase) expression patterns in bladder cancer and clinicopathological characteristics* Total no.

Diffuse TPase (n = 13) n (%)

Focal TPase (n = 19) n (%)

Total no. stained

64

26 (41)

38 (59)

Sex Male Female

42 22

18 (28) 8 (12)

24 (38) 14 (22)

Grade Low High

8 56

2 (3) 24 (38)

6 (9) 32 (50)

Lymphovascular invasion Yes No ND

24 30 10

14 (33) 6 (26) 8

10 (11) 24 (30) 2

pT category pTa–T1 pT2–4

14 50

4 (6) 40 (62)

10 (16) 10 (16)

Characteristics

p

0.712

0.613

0.570

0.010

*Data presented as n or n (%). A TPase staining pattern score of < 25% was defined as focal, whereas that of > 25% was defined as diffuse. ND = not detected.

tumors, tumor configuration, lymph node involvement, venous invasion, or overall survival.18 Furthermore, they detected no significant serum level of TPase in any patient. In contrast to the results from Tanaka et al., Arima et al. demonstrated significant correlations between TPase expression and histological grade, the infiltration pattern, local invasion, and lymph node metastasis.11 They concluded that TPase overexpression is an independent Vol. 22, 32–37, March 2011

prognostic factor, and TPase activity and its level of expression influences the progression of UC and the prognosis of bladder cancer. Subsequent studies showed that the expression of TPase is correlated with the recurrencefree rate among superficial bladder tumors.13–17 In our study, we found that TPase was not only expressed in tumor cells but was also highly expressed in stromal cells adjacent to bladder cancer. In addition, the 35

V.C. Lin, et al A

B

C

D

Figure 1 Immunohistochemical expression of thymidine phosphorylase (TPase) in urothelial cancer of the urinary bladder. (A) Absent to weak expression of TPase in normal urothelial cells (original magnification, 400×). (B) Strong expression of TPase in urothelial cancer cells (400×). (C) Strong TPase immunoreactivity in the infiltrating front of tumors (black arrow) and in areas of necrosis (white arrow, 100×). (D) Nuclear staining of TPase, as indicated by the white arrow (400×).

infiltrating front of tumors and areas of necrosis were immunoreactive to TPase as evidenced by strong staining. Tanaka et al. and Sivridis et al. reported prominent TPase expression at the invading tumor periphery (invasive front) in endometrial cancer, and these findings highlight the importance of this area where tumor angiogenesis is most active.19,20 This unique phenomenon may explain how angiogenic factors are released in this area and thereby promote subsequent angiogenesis.19,20 Our findings suggest that TPase may play an important role in modulating angiogenesis in the microenvironment between the tumor and host tissues. On the other hand, TPase immunoreactivity in tumor cells is heterogeneous, in both nuclear and cytoplasmic components in some cases, whereas in other tumors, the immunoreactivity is almost completely confined to the cytoplasm. In our study, we found that the percentage of nuclear staining was higher in advanced stages (T2–T4) than that in low stages (Ta–T1) and normal tissues (40% vs. 14.3%). Future studies need to investigate the role of nuclear TPase in tumor progression. 36

We showed no correlation between TPase expression of tumor cells and other tumor variables in our Taiwanese patients compared with findings in previous unpublished data. There were also no correlations between TPase expression and other established prognostic parameters in bladder cancer except for tumor stage. Nevertheless, high expression levels of TPase were observed in most bladder cancers and neighboring stromal tissues. These findings may provide valuable information for managing bladder cancer. Tumor tissues and also normal tissues abutting bladder cancer have the potential for angiogenesis for tumor development. In addition, a number of thymidine analogues are metabolized by TPase. Capecitabine, a prodrug of 5-FU, is converted to 5-FU by TPase. Different expression profiles of TPase in bladder cancers may indicate that capecitabine might be differently activated in tumor tissues. Tumors in which TPase activity is overexpressed could degrade salvaged thymidine and might be more sensitive to thymidylate synthase inhibition.15 Therefore, detection of TPase expression patterns might indicate the likely response to 5-FU or thymidylate synthase Vol. 22, 32–37, March 2011

TPase expression in bladder cancer inhibition such as with methotrexate-based chemotherapies. Further investigations are necessary to determine the role of TPase in chemotherapeutic responses.21 Our study has some limitations. First, the numbers in our cohort were limited. Second, our study was retrospective; therefore, some clinicopathological parameters were not adequately recorded or analyzed. A prospective large cohort study needs to be designed to deduce the actual role of TPase in human bladder cancer.

7.

8.

9.

10.

5. Conclusions The expression of TPase was only associated with tumor stage in our Taiwanese patients. In addition, there were high expression levels of TPase in most bladder cancers and adjacent stromal tissues. These findings suggest that tumor and stromal TPase expression may cooperatively promote tumor progression and angiogenesis.

11

12.

13.

Acknowledgments 14.

We would like to acknowledge E-Da Hospital for supporting this project (EDAH95-007).

15.

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