Association Between Human Papillomavirus and Transitional Cell Carcinoma of the Bladder Pourya Abdollahzadeh1, Seyed Hamid Madani2*, Sedigheh Khazaei2, Soraya Sajadimajd2, Babak Izadi2, Farid Najafi1 Purpose: Bladder carcinoma is one of the most common malignancies in worldwide. Among several risk factors, Human Papilloma Virus (HPV) have been presumed to play a causative role in the etiology of bladder cell carcino- ma. The aim of this study was to evaluate the involvement of HPV infection in biopsy specimens of patients with transitional cell carcinoma at the west of Iran. Materials and methods: In this study, 97 biopsy specimens including 67 patients with transitional cell carcinoma (TCC) of bladder and 30 cases of control group with the mean age of 63 years were studied using immunohisto- chemistry to identify HPV. Results: 22.4% of patients with TCC of bladder and 3.3% of control group were positive for HPV with a meaning- ful relation (P=.019). The prevalence of HPV was 4.3 fold higher in men than women. Most TCC patients belonged to grades II and III. Conclusion: Considering the higher incidence of HPV positivity in patients with TCC of bladder compared to control group, it seems to be a meaningful association between HPV infection and TCC of bladder, at least in the west of Iran. Keywords: biopsy; bladder carcinoma; immunohistochemistry; HPV; transitional cell carcinoma. 1Kermanshah University of Medical Sciences, Kermanshah, Iran. 2Molecular Pathology Research Center, Imam Reza University Hospital, Kermanshah University of Medical Sciences, Kermanshah, Iran. *Correspondence: Molecular Pathology Research Center, Imam Reza Hospital, Kermanshah university of Medi- cal Sciences, Kermanshah, Iran. Tel:(98)9181324029. Fax:(98)831-7235149. E-mail: shmmadani@yahoo.com. Received October 2016 & Accepted September 2017 INTRODUCTION Bladder carcinoma as a leading cause of death in worldwide, arises from urothelial epithelium(1-3). Transitional cell carcinoma (TCC) is the most com- mon type of bladder carcinoma which represents 90% of bladder malignancies and is commonly associated with cancers of other parts of the urinary tracts such as kidney, ureters and urethra. Bladder cancer is the fourth most common in men, but is less common in women(4,5). Several risk factors have been considered to be important in development of bladder carcinoma including smoking, certain industrial exposure, arsenic in drinking water, chronic irritation as well as bacterial and viral infection (6,7). Among them, the causative role of human papilloma virus (HPV) infection has been ev- luated in several studies(8-12). It has been suggested that HPV is the most important risk factor for development of carcinoma in urogenital system(13-15). The oncogenic types of HPV, HPV16 and HPV18, have been identified in invasive carcinoma of genital areas. Since the prolif- erative cycle of HPV is dependent to sequential phases of differentiation of epithelial cells, HPV possesses a particular tendency for epithelial cells with the expres- sion of its oncogenes and pathogenesis(16,17). In addition, neoplastic alterations in urothelium have several stages in which carcinogenic initiator agent via modification of DNA leads to malignancy. In this line, several stud- ies have suggested the involvement of HPV infection in the development of bladder carcinoma, especially tran- sitional cell carcinoma(18-21). Given that HPV is not able to grow in cellular cultures and also antibody tests are only available in research laboratories, several methods including PCR, in situ hybridization and appropriative immunohistochemical (IHC) staining have been developed to confirm HPV infection in bladder carcinoma tissues. In this study, we aimed to evaluate HPV infection in samples related to patients with TCC using immunohistochemical staining of paraffin embedded blocks to find the interplay be- tween HPV and TCC. MATERIALS AND METHODS Clinical samples Specimens were retrieved from paraffin embedded bladder carcinoma tissues stored in laboratory of Imam Reza Hospital medical center, including 67 cases with TCC diagnosis and 30 cases from normal biopsies from 2008 to 2011. The slides prepared from paraffin em- bedded specimens were stained with the conventional hematoxylin and eosin method. All specimens were re- viewed and original diagnoses were confirmed. Immunohistochemistry Immunohistochemical evaluation was performed on 5 μm formalin fixed paraffin embedded tissue sections. In brief, an anti-human HPV mouse monoclonal antibody (clone K1h8, DAKO) was used as the primary antibody at a dilution of 1/ 30000. Then, secondary antibody con- UROLOGICAL ONCOLOGY Urological Oncology 5047 jugated with horseradish peroxidase and diaminoben- ziden chromogen were added to identify the presence of HPV in samples. Candyloma specimen was used as a positive control. Nuclear staining of tumoral cells in TCC specimens and urothelial cells in normal speci- mens were considered as positive samples. Statistical analysis Statistical analysis was performed by SPSS statistical software package version 16, using T-test and chi- square tests. RESULTS In this study, immunohistochemistry was performed on paraffin embedded bladder TCC tissues to distinguish the involvement of HPV infection in development of carcinogenicity of bladder (Figure 1). From the pop- ulation of 67 TCC specimens and 30 normal biopsy specimens, 15 and 1 cases, respectively, were positive for HPV infection. There was a meaningful relation between TCC carcinogenicity and HPV infection (P = .019) (Figure 2). Among TCC patients, 86.6% (58 cases) were male and 13.4% (9 cases) were female. In control group, 56.7% (17 cases) were male and 43.3% (13 cases) were fe- male. As shown in Figure 3, there was not meaningful relation between gender and HPV infection (P = .933). The histological grading of tumor specimens indicated that 1 case with grade 1, 24 cases with grade 2, 32 cases with grade 3 and 10 cases with grade 4 were found in 67 TCC specimens according to WHO/ISOP classification (Figure 4A). Among them, immunohitochemical anal- ysis showed that 5 cases of grade 2, 4 cases of grade 3 and 6 cases of grade 4 were positive for HPV nuclear staining. According to Figure 4B, there was no mean- ingful relation between the grade of TCC and HPV in- fection (P = .718). DISCUSSION The involvement of human papillomavirus in bladder carcinoma has been reported by several studies(18,22). In this line, the high prevalence of HPV infection in pa- tients with TCC indicated the possible association of HPV and carcinogenesis of transitional cell carcinoma of bladder(23-26). In this study, we aimed to investigate the meaningful relationship between HPV and patients with transitional cell carcinoma. We studied the ex- pression of HPV in 67 patients with TCC and 30 nor- mal samples as control using immunohistochemistry. 15 cases (22.4%) of TCC patients’ group and 1 case (3.3%) of control group were found to be positive for HPV infection. The first report about the association of HPV and blad- der carcinoma was reported by Kitamura based on southern blot analysis in 1988(27). After that, several HPV in bladder carcinoma-Abdollahzadeh et al. Figure 1. Immunohistochemical staining for human papilimavirus (HPV) in bladder cell carcinoma. Candyloma section (Positive-HPV) as control with magnifications of 20X and 40X (A; B), HPV negative in TCC with magnifications of 20X and 40X (C; D) and HPV positive in TCC with magnifications of 20X and 40X (E; F) indicated by nuclear and cytoplasmic staining of tumor tissues. Figure 2. Infection pattern of HPV in bladder biopsy specimens among patients and control groups. Figure 3. The incidence of sex in bladder biopsy specimens among patients and control groups. Vol 14 No 06 November-December 2017 5048 contradictory studies have been reported about the in- volvement of HPV in carcinoma of bladder. Lopez-Bel- tran et al. assessed the role of HPV in TCC using IHC and ISH methods. They identified 25 cases with HPV infection using IHC and 12 cases with positive HPV- DNA positive using PCR in 76 patients with TCC(28). In another study, Youshya and co-workers studied the involvement of HPV in 76 patients with TCC using PCR and IHC methods. Interestingly, different results were acquired from two methods. 47 cases were posi- tive for HPV according to IHC while none were posi- tive for HPV–DNA by PCR. Therefore, they suggested that there were no possible association between HPV infection and carcinogenesis of TCC(9). Shigehara et al. evaluated the etiologic role of HPV infection in bladder carcinoma. They studied the involvement of HPV in 244 cases of patients with bladder cancer during 1997 to 2007. They analyzed the expression of HPV-DNA in frozen specimens of TUR bladder tissue with PCR and HPV-L1 capsid protein of HPV using IHC. Therefore, it seems that high risk types of HPV leads to carcino- genesis of low grade bladder cancer in juvenile patients (19). Another controversial study about the involvement of HPV in urothelial cancer was performed by Yavuzer et al. They studied 70 cases of urothelial carcinoma to find HPV using PCR with respect to 18 cases of pa- tients with cervical carcinoma as control. They found that there was no association between HPV infection and bladder urothelial carcinoma(29). In another study by Barghi et al., 58 cases of TCC specimens and 20 normal cases as control was evaluated using PCR to identify HPV-DNA. They found that the possibility of HPV in- volvement in TCC is related to geographical regions(15). Selma et al indicated the interplay of HPV and TCC in Tunisia. They examined 119 patients with TCC, 5 cases with squamous cell carcinoma and one case with adenocarcinoma by PCR based method to distinguish different subtypes of HPV. Their results showed that anogenital HPV infection seems not to play a signifi- cant role in pathogenesis of bladder carcinoma(30). Eslami et al. considered 147 cases of TCC patients and 39 cases of normal specimens as control to study the relation between HPV infection and TCC. They used PCR to identify HPV-DNA in patients. They found that there is no meaningful association between HPV in- fection and grade of bladder tumor; while it seems that genital HPV infection especially subtype 18 augmented the risk of bladder carcinoma in Iranian population(31). CONCLUSIONS Taken together, our present study indicated that there was a meaningful association between HPV infection and transitional cell carcinoma. However, we could not found any meaningful relation between grade of TCC, age and sex of patients and the presence of HPV. ACKNOWLEDGMENTS The authors appreciate the financial support of this investigation by Kermanshah University of Medical Sciences, and the archived file center of Imam Reza hospital and Taleghani Hospital. CONFLICT OF INTEREST The authors declare that they have no conflict of inter- est. REFERENCES 1. Oosterlinck W, Lobel B, Jakse G, et al. Guidelines on bladder cancer. Eur Urol. 2002;41:105-12. 2. Silverberg E. Statistical and epidemiologic data on urologic cancer. Cancer. 1987;60:692- 717. 3. Kaufman DS, Shipley WU, Feldman AS. Bladder cancer. Lancet. 2009;374:239-49. 4. Young R. Usual variants of primary bladder lesions and secondary tumours of the bladder. Pathology of bladder cancer. Baltimore: Williams and Wilkins; 1996:326–37. 5. Eble JL, Sauter G, Epstein JI. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs. Lyon: IARC Press; 2004: 221-49. 6. Burger M, Catto JW, Dalbagni G, et al. Epidemiology and risk factors of urothelial bladder cancer. Eur Urol. 2013;63:234-41. 7. Freedman ND, Silverman DT, Hollenbeck AR, Schatzkin A, Abnet CC. Association between smoking and risk of bladder cancer among men and women. Jama. 2011;306:737- 45. 8. Noel JC, Thiry L, Verhest A, et al. Transitional cell carcinoma of the bladder: evaluation of Figure 4. Histological grading of urinary bladder carcinoma in bladder biopsy specimens among patients (A). Relation between HPV positivity and histological grading of TCC patients(B). HPV in bladder carcinoma-Abdollahzadeh et al. Urological Oncology 5049 the role of human papillomaviruses. Urology. 1994;44:671-5. 9. Youshya S, Purdie K, Breuer J, et al. Does human papillomavirus play a role in the development of bladder transitional cell carcinoma? A comparison of PCR and immunohistochemical analysis. J Clin Pathol. 2005;58:207-10. 10. Wiener JS, Walther PJ. A high association of oncogenic human papillomaviruses with carcinomas of the female urethra: polymerase chain reaction-based analysis of multiple histological types. J Urol. 1994;151:49-53. 11. zur Hausen H. Papillomaviruses in anogenital cancer as a model to understand the role of viruses in human cancers. Cancer Res. 1989;49:4677-81. 12. Gutierrez J, Jimenez A, de Dios Luna J, Soto MJ, Sorlozano A. Meta-analysis of studies analyzing the relationship between bladder cancer and infection by human papillomavirus. J Urol. 2006;176:2474-81; discussion 81. 13. Griffiths TR, Mellon JK. Human papillomavirus and urological tumours: II. Role in bladder, prostate, renal and testicular cancer. BJU Int. 2000;85:211-7. 14. Shigehara K, Sasagawa T, Kawaguchi S, et al. Prevalence of human papillomavirus infection in the urinary tract of men with urethritis. Int J Urol. 2010;17:563-8. 15. Barghi MR, Hajimohammadmehdiarbab A, Moghaddam SM, Kazemi B. Correlation between human papillomavirus infection and bladder transitional cell carcinoma. BMC Infect Dis. 2005;5:102. 16. Alexander RE, Wang L, Lopez-Beltran A, et al. Human papillomavirus (HPV)-induced neoplasia in the urinary bladder: a missing link? Histol Histopathol. 2016;31:595-600. 17. Shigehara K, Sasagawa T, Namiki M. Human papillomavirus infection and pathogenesis in urothelial cells: a mini-review. J Infect Chemother. 2014;20:741-7. 18. De Gaetani C, Ferrari G, Righi E, et al. Detection of human papillomavirus DNA in urinary bladder carcinoma by in situ hybridisation. J Clin Pathol. 1999;52:103-6. 19. Shigehara K, Sasagawa T, Kawaguchi S, et al. Etiologic role of human papillomavirus infection in bladder carcinoma. Cancer. 2011;117:2067-76. 20. Anwar K, Naiki H, Nakakuki K, Inuzuka M. High frequency of human papillomavirus infection in carcinoma of the urinary bladder. Cancer. 1992;70:1967-73. 21. Li N, Yang L, Zhang Y, Zhao P, Zheng T, Dai M. Human papillomavirus infection and bladder cancer risk: a meta-analysis. J Infect Dis. 2011;204:217-23. 22. Furihata M, Inoue K, Ohtsuki Y, Hashimoto H, Terao N, Fujita Y. High-risk human papillomavirus infections and overexpression of p53 protein as prognostic indicators in transitional cell carcinoma of the urinary bladder. Cancer Res. 1993;53:4823-7. 23. Agliano AM, Gradilone A, Gazzaniga P, et al. High frequency of human papillomavirus detection in urinary bladder cancer. Urol Int. 1994;53:125-9. 24. LaRue H, Simoneau M, Fradet Y. Human papillomavirus in transitional cell carcinoma of the urinary bladder. Clin Cancer Res. 1995;1:435-40. 25. Chen T, Kong Q, Cao H. [The study on relation of human papillomavirus and P53 expression with bladder transitional cell carcinoma]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2000;14:345-8. 26. Wiwanitkit V. Urinary bladder carcinoma and human papilloma virus infection, an appraisal of risk. Asian Pac J Cancer Prev. 2005;6:217- 8. 27. Kitamura T, Yogo Y, Ueki T, Murakami S, Aso Y. Presence of human papillomavirus type 16 genome in bladder carcinoma in situ of a patient with mild immunodeficiency. Cancer Res. 1988;48:7207-11. 28. Lopez-Beltran A, Escudero AL, Carrasco-Aznar JC, Vicioso-Recio L. Human papillomavirus infection and transitional cell carcinoma of the bladder. Immunohistochemistry and in situ hybridization. Pathol Res Pract. 1996;192:154- 9. 29. Yavuzer D, Karadayi N, Salepci T, Baloglu H, Bilici A, Sakirahmet D. Role of human papillomavirus in the development of urothelial carcinoma. Med Oncol. 2011;28:919-23. 30. Ben Selma W, Ziadi S, Ben Gacem R, et al. Investigation of human papillomavirus in bladder cancer in a series of Tunisian patients. Pathol Res Pract. 2010;206:740-3. 31. Eslami G, Golshani M, Rakhshon M, Fallah F, Goudarzi H. The study on relation of Human Papillomavirus with bladder transitional cell carcinoma. Cancer Therapy. 2008;6:355-60. HPV in bladder carcinoma-Abdollahzadeh et al. Vol 14 No 06 November-December 2017 5050