UROLOGICAL ONCOLOGY The Prevalence and Prognostic Significance of Polyomavirus Infection in Patients with Urothelial Carcinoma of the Bladder Sheng-Wen Wu,1,2* Jia-Hung Liou,3,4,5 Kun-Tu Yeh,3,5 Tung-Wei Hung,1,2 Horng-Rong Chang1,5* Purpose: Human polyomaviruses (PV) has been associated with oncogenicity; however, the association between human bladder cancer and PV remains inconclusive. Moreover, whether PV has the interaction with p53 in tumor- igenesis and their prognostic significance on human bladder cancer has yet to be determined. Materials and Methods: Bladder tumor specimens and clinical parameters from 74 patients with urothelial carci- noma were collected. Immunohistochemical analysis using monoclonal antibodies specific to PV large tumor an- tigen (TAg) and p53 protein was performed to investigate the involvement of PV in human bladder tumorigenesis and the prognostic significance of TAg and p53 expressions using Cox proportional hazards model. Results: The mean age of the 74 patients at diagnosis was 64 years and 61 (82.4%) were male. The expression of PV TAg protein was found in 45 (60.8%) tumor samples, but was not correlated with the expression of p53 (P = .280). The detection of PV TAg was significantly associated with tumor stage (P = .001) but not decreased overall survival (OS) or cancer-specific survival (CSS) (P = .661 and .738, respectively). However, the p53 overexpres- sion was significantly associated with decreased CSS (P = .028). In multivariate Cox proportional hazards analysis, age and p53 overexpression were predictors of OS (P = .026) independently of tumor stage and CSS (P = .042), respectively. Conclusion: We found that PV, which was detected in a significant percentage of tumor specimens, may be an im- portant co-factor in the tumorigenesis of the bladder in humans. However, only p53 overexpression was associated with predicting CSS independently of tumor stage. Keywords: BK virus; large T Antigen; oncogenicity; polyomavirus; protein p53; survival; urinary bladder neo- plasms INTRODUCTION Most humans become infected with human poly-omaviruses (PV) during childhood, which then establishes a life-long latent infection, particularly in the kidneys and urinary tract.(1) Reactivation of the PV infection can occur in people with a compromised immune system, such as patients undergoing organ transplantation, which can lead to diseases like hem- orrhagic cystitis and polyomavirus nephropathy.(2) PV, and mainly the BK virus (BKV), have been reported to be oncogenic viruses in many cell and animal studies. (3-5) The early region of PV encodes two known onco- proteins, large tumor antigen (TAg) and small tumor antigen (tAg), which may lead to transformation by interacting with cellular tumor suppression proteins, such as p53 and inhibiting protein phosphatase 2A, respectively.(6,7) Although an increasing number of re- cent studies have investigated the potential association between PV and various human tumors, the results are still inconclusive.(8) Bladder cancer is the most com- mon malignancy involving the urinary system in de- veloped countries, with urothelial carcinoma (UCC) being the predominant histologic type.(9) However, there is wide variation in the reported incidence in dif- ferent regions.(10) Furthermore, the incidence of bladder UCC is significantly higher in patients with chronic kidney disease and those undergoing renal transplants, particularly in Asian countries.(11,12) These findings imply that the contributory factors for bladder tumor- igenesis are complex. Considering that PV is known to persist in the urinary tract and can be reactivated in immunocompromised people, it is reasonable to hy- pothesize that urinary tract carcinomas are likely to be associated with PV, particularly in Asian countries. To date, few studies with a small number of patients have discussed the association between PV and the development of human bladder UCC and the interac- tion with p53 in tumorigenesis. In addition, it is un- clear whether PV infection has an impact on tumor grade, tumor stage and clinical prognosis of bladder cancer. Therefore, the aim of this study was to inves- tigate the involvement of PV in human bladder tumor- igenesis and clarify the prognostic significance of PV 1 School of Medicine, Chung Shan Medical University, Taichung, Taiwan. 2 Division of Nephrology, Chung Shan Medical Hospital, Taichung, Taiwan. 3 Departments of Pathology, Changhua Christian Hospital, Changhua, Taiwan. 4 Department of Medical Technology, Jen-Teh Junior College, Miaoli, Taiwan. 5 The Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan. *Correspondence: The School of Medicine, Chung Shan Medical University, Taichung, Taiwan. Tel: +886 4 24739595. Fax: +886 4 24739220. E-mail: s41111.tw@yahoo.com.tw. Received January 2016 & Accepted June 2016 Urological Oncology 2773 Vol 13 No 04 July-August 2016 2774 TAg and p53 expressions on human bladder cancer. MATERIALS AND METHODS Study Population A total of 74 formalin-fixed and paraffin-embedded tumor samples were obtained from 74 patients with a diagnosis of UCC of the bladder at Chung Shan Med- ical University and Changhua Christian Hospital after the local institutional review board approved this study (CS11140). All tumors were retrospectively re-graded and re-staged according to the 2010 American Joint Committee on Cancer (AJCC) staging system by an experienced pathologist.(13) The study included 61 male and 13 female patients with a mean age of 64.5 years. The mean follow-up period was 53 months. Procedures Paraffin-embedded tumor tissue sections (4-µm) on poly-1-lysine-coated slides were deparaffinized. After treatment with 3% H 2 O 2 in methanol, the sections were hydrated with gradient alcohol and PBS, incubated in 10 mM citrate buffer and finally heated at 100 °C for 20 minutes in PBS. The immunohistochemical proce- dure for the monoclonal antibody to SV40 TAg (On- cogen Research Products, Cambridge, MA) followed standard immunoalkaline phosphatase methods and was preceded by pressure-cooked antigen retrieval for 5 min in Ventana retrieval buffer (pH 10.0; Ventana Medical Systems, Tucson, AZ). Diaminobenzidine tet- rahydrochloride was used as a chromogen. The mono- clonal antibody was used to detect epitopes unique to PV TAg and shared by SV40, BK, and JC viruses. For p53 detection, the sections were heated in a microwave oven twice for 5 min in citrate buffer (pH 6.0), and then incubated with a monoclonal antihuman p53 antibody (DAKO, DO7; at a dilution of 1:250) for 60 min at 25°C. The conventional streptavidin peroxidase method (DAKO, LSAB Kit K675) was used to develop signals, and the cells were counterstained with hematoxylin. Evaluations The intensities of signals were evaluated independent- ly by two observers. Negative immunostaining for p53 protein was defined as 0% to 10% positive nuclei and >10% positive nuclei was defined as positive for immu- nostaining (overexpression).(14) In addition, any posi- tive nuclear reaction to PV TAg was defined as positive staining. Positive control slides for p53 protein detec- tion were purchased from DAKO and renal allograft tissues with BK virus infection were used as positive controls for PV TAg. A neutralizing peptide was used to replace antibodies and served as a negative control. Statistical Analysis Given a type I error (α) level of 0.05, type II error (β) level of 0.20, the prevalence of human PV among pa- tients with UCC of 0.17,(15) the prevalence of human P53 Polyomavirus and Urothelial Carcinoma-Wu et al. Table 1. Relationships between PV TAg immunostaining, p53 expression and clinicopathological parameters in patients with urothelial carcinoma of the bladder. PV TAg Immunostaining Variablesa Total Positive Negative P Value (N = 74) (N = 45) (N = 29) Age at diagnosis (years) 64.5 ± 14.8 66.1 ± 12.1 62.2 ± 18.4 .325 Gender Male 61 (82.4%) 38 (84.4%) 23 (79.3%) .571 Female 13 (17.6%) 7 (15.6%) 6 (20.7%) Ever-smokerb 37 (54.4%) 23 (51.1%) 14 (48.2%) .812 Tumor grade Low 2 (2.8%) 0 (0.0%) 2 (6.9%) .074 High 72 (97.2%) 45 (100.0%) 27 (93.1%) Tumor stage 0a or 0is 13 (17.5%) 4 (8.9%) 9 (31.0%) .001 I 33 (44.6%) 22 (48.9%) 11 (37.9%) II 10 (13.5%) 3 (6.7%) 7 (24.1%) III 14 (18.9%) 14 (31.1%) 0 (0.0%) IV 4 (5.5%) 2 (4.4%) 2 (6.9%) p53 immunostaining Positive 56 (76%) 36 (80%) 20 (69%) .28 Negative 18 (24%) 9 (20%) 9 (31%) Abbreviations: PV TAg, polyomaviruses large tumor antigen aData are shown as numbers with percentage or means ± standard deviation bData in six patients were missing among patients with UCC of 0.37,(14) detectable relative risks of 3.5, the minimum sample size required for each group was calculated to be 30. Additional subjects (near- ly 20%) were recruited to avoid the loss to follow-up. Finally, we recruited a total of 74 patients in this study. The patients’ age at diagnosis, gender, and smoking sta- tus were recorded. Overall survival (OS) was calculated as the period from the date of diagnosis to the date of death or the date of last follow-up. Cancer-specific sur- vival (CSS) was defined as death attributable to bladder UCC. For categorical and continuous variables, Pearson chi-square and ANOVA tests were used to determine association between PV TAg expression and variables of interest. Kaplan-Meier analysis and the log-rank test/ Gray’s test were used to evaluate the associations be- tween the expression of PV TAg and p53 with OS and CSS. A Cox proportional hazards model was used to evaluate the variables of interest in predicting OS and CSS. The combined effect of human PV TAg and p53 expressions on the survival of UCC patients was also evaluated. Interaction was further assessed using the likelihood ratio test to calculate X2 and P values. In the test for interaction, the conditional logistic regression model with only main effects was compared to that with both main effect terms and interaction term. Val- ues were expressed as mean ± SD. P-values of less than 0.05 were considered to indicate statistical significance. All analyses were performed using SPSS software for Windows version 12.0 (SPSS Inc., Chicago, IL). RESULTS Patients’ Characteristics and Clinical Parameters The patients’ characteristics are shown in Table 1. Of the 74 patients, 25 (34%) received radial cystectomy with lymph node dissection. The final pathology of these 25 patients revealed that two had positive sur- gical margins, lymphovascular invasion, and positive lymph nodes; two had positive lymphovascular in- vasion and lymph node status; one had positive sur- gical margin; and one had positive lymphovascular invasion. At the end of study, 29 patients had died, of whom 20 had died from cancer-related deaths. PV TAg Immunohistochemistry and Association with Clinical Parameters Of the 74 bladder tumors, 45 (60.8%) had positive nu- clear reactivity to PV TAg, which was only expressed in the tumor cells as well as a few adjacent normal lymphocytes in the tumor tissues (Figure 1). The re- lationships between the expression of PV TAg and clinical parameters of the patients with bladder UCC are shown in Table 1. The tumors that expressed PV TAg tended to be of a higher tumor stage than those that did not (P = .001). However, neither tumor grade nor p53 protein expression was associated with the ex- pression of PV TAg (P = .258 and .280, respectively). The Kaplan-Meier curves for OS and CSS, defined by the status of PV TAg expression, are shown in Fig- ure 2a and 2b. The OS and CSS between the patients with and without TAg expression in their bladder tum- ors were not statistically different in the Kaplan-Mei- er analysis (P = .661 and .738, respectively). P53 Immunohistochemistry and Association with Clinical parameters Urological Oncology 2775 Figure 1. Immunohistochemical analysis of the PV TAg protein, using monoclonal antibody to SV40 TAg, in urothelial cell carcino- ma of the bladder and adjacent normal tissues. (a) Negative results of PV TAg immunostaining in high-grade tumor cells (×100); (b) PV TAg protein expressed in renal allograft with PV nephropathy as a positive control (×100); (c) PV TAg protein expressed focally in high-grade tumor cells (×200); (d) PV TAg protein expressed diffusively in high-grade tumor cells (×200); (e) PV TAg protein expressed in few adjacent normal lymphocytes in high-grade tu- mor tissues (×200); (f) negative control of tumor tissues using anti- body dilution buffer to replace the antibodies (×200). Figure 2. Kaplan–Meier post diagnostic survival curves. Overall survival (a) and cancer- specific survival (b) among the patients with urothelial carcinoma of the bladder, defined by the status of PV TAg expression. Polyomavirus and Urothelial Carcinoma-Wu et al. Vol 13 No 04 July-August 2016 2776 Although 56(76%) of the 74 bladder tumors demon- strated nuclear accumulation of p53 protein (Figure 3), neither tumor grade nor tumor stage was associat- ed with the expression of p53 (P = .568 and .539, re- spectively). The Kaplan-Meier curves with Log-rank test for OS and CSS, defined by the status of p53 ex- pression, are shown in Figure 4a and 4b. The patients with bladder UCC with p53 overexpression had sig- nificantly and borderline significantly worse CSS and OS (P = .028 and .096, respectively). Furthermore, the Gray’s test was also used to compare the OS among the PV TAg-negative and PV TAg-positive patients and we found the two curves to be no significantly differ- ent (Gray’s test result: χ2 = 0.032, df = 1, P = .859). A similar result was also found among the p53-negative and p53-positive group (χ2 = 0.511, df = 1, P = .612). The Prognostic Significance of Variables of In- terest in the Cox Proportional Hazards Model The Cox proportional hazards model for predicting OS and CSS was adjusted for age, gender, tumor grade, tu- mor stage, smoking status, and the status of PV TAg and p53 expressions. The two independent factors pre- dicting OS were age (95% confidence interval [CI]: 1.11 to 5.80, hazard ratio [HR] = 2.54, P = .026) and tu- mor stage (95% CI: 1.81 to 8.04, HR = 3.81, P < .001). The two independent factors predicting CSS were p53 overexpression (95% CI: 1.08 to 61.93, HR = 8.16, P = .042) and tumor stage (95% CI: 2.26 to 16.03, HR = 6.01, P < .001). Furthermore, no significant interaction between human PV TAg and P53 expressions on the OS of UCC patients was observed (χ2 = 0.23, P = .063). DISCUSSION Bladder cancer has a significant impact on health and medical costs because of its high incidence and prev- alence in Chinese populations.(11,12) There are many es- tablished risk factors for bladder UCC, including male gender, smoking, exposure to various chemical carcino- gens, and genetic factors.(16-20) Some infections, includ- ing chronic cystitis and human papillomavirus (HPV), have been associated with bladder cancer, although the findings are not consistent across the studies.(21,22) Even though PV is regarded to be an oncogenic virus because of its transformative behavior in vitro and in animal studies, few studies have reported an associa- tion between PV infection and the development of hu- man bladder UCC.(15,23-30) In one Italian study, Monini et al.(24) reported that BKV DNA was detected in 15 (58%) of 26 bladder tumor tissues using the polymerase chain reactions (PCR). However, the prevalence of PV observed in a study from the U.S. with a larger sample size (76 patients), also using PCR, was only 5%.(29) In contrast to these studies using PCR, another study in the US using the immunohistochemical study reported a prevalence of PV in the 24 patients of nearly 17%.(15) The current study is the largest to date to investigate PV in bladder tumor samples, using immunohistochemis- try (IHC), and we found PV TAg- positive rate in the bladder tumor samples of nearly 60%. The reason for this inconsistency in results between studies is unclear. In general, PCR has a greater degree of sensitivity than Figure 3. Immunohistochemical detection of p53 nuclear reactiv- ity, using anti-p53 monoclonal antibodies in urothelial cell carci- noma of the bladder. (a) Diffuse positive staining (> 50%) in high- grade urothelial cell carcinoma (×100); (b) heterogeneous positive staining (10 to 50%) in high-grade invasive urothelial cell carci- noma (×100); (c) scattered positive staining in high-grade tumor cells (<10%) (×100); (d) non-tumor sample with scattered positive staining as a normal control (×100) Figure 4. Kaplan-Meier post-diagnostic survival curves. Overall survival (a) and cancer-specific survival (b) among patients with urothelial carcinoma of the bladder, defined by the status of p53 expression Polyomavirus and Urothelial Carcinoma-Wu et al. IHC; however, it can also overestimate the prevalence of PV due to laboratory contamination. On the other hand, PCR may lead to the underestimation of the prevalence of PV due to the “hit-and-run” phenomenon. Another important issue is that even though we used a newer immunohistochemical technique (a streptavidin-bio- tin system, less non-specific background staining than with the conventional avidin-biotin complex method) with good quality control, we cannot exclude the pos- sibility that the high prevalence of PV TAg positivity was caused, at least in part, by background staining. Although methodological differences and small sample sizes most likely explain the inconsistencies in the re- ported prevalence rates of PV in bladder tumor tissues, it is possible that ethnic and genetic variations in the susceptibility to PV carcinogenesis may be another pos- sible explanation. In light of the high prevalence of PV in our bladder tumors, PV may be an important cofactor in human bladder tumorigenesis in Chinese patients. Previous studies have reported that PV TAg may exert its transformative activity by interacting with and func- tionally inactivating cellular p53 at a molecular level. (31,32) To elucidate whether TAg affects the expression of p53, we determined the expression of p53 in bladder tu- mor tissues using IHC. We found the expression of TAg was not associated with p53 expression, which implies that TAg probably inactivates p53 by binding directly to p53 without down-regulating its expression in bladder tumor tissues. Furthermore, we could not exclude the possibility that PV TAg mediates bladder tumorigene- sis through other mechanisms, such as inactivating ret- inoblastoma susceptibility protein (pRb) or activating the insulin-like growth factor-I signaling pathway.(33,34) Recently, Alexiev et al. proposed that in the dysplastic background of p53 or pRb inactivation in BKV-infected urothelium, a “time lapse” may play an important role in tumorigenesis of bladder urothelial , which is consist- ent with the concept of multiple carcinogenesis casade. (35) To the best of our knowledge, this is the first study to report the relationships between the expression of PV TAg and clinical parameters of bladder UCC. The find- ing that the detection of PV TAg was significantly asso- ciated with tumor stage implies that PV infection affects the aggressiveness of a tumor, although further studies are needed to elucidate the mechanism. However, it is somewhat surprising that no associations were found between the expression of PV TAg and OS and CSS in Kaplan-Meir analysis. It may be attributable to inad- equate power due to an insufficient sample size or het- erogeneity of the study population. Further studies are needed to investigate the reasons for this discrepancy. It has been postulated that the overexpression of p53 implies a missense mutation of the p53 gene with a prolonged half-life, leading to nuclear accumulation of the mutant p53 protein, which can then be used as a prognostic predictor of bladder cancer.(14,36,37) Consist- ent with this hypothesis, we found that the overexpres- sion of p53 was significantly associated with decreased CSS and borderline significantly with decreased OS in the Kaplan-Meir analysis. In the multivariate Cox proportional hazards model, p53 overexpression was an independent predictor of CSS. However, it is in- teresting to note that age rather than p53 overexpres- sion was an independent predictor of OS. We think this may be explained by multiple comorbidities and poor performance in the elderly. Therefore, in treat- ing patients with bladder UCC, it is important to con- sider age when choosing the therapeutic strategy. 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