Immunohistochemical characteristics of bladder cancer in patients with virus-positive tumors

Background. Viral infection is a major factor in virus-associated carcinogenesis.

Objective: to evaluate the expression of growth factors and markers of apoptosis, proliferative activity, and angiogenesis in patients with viral DNA-positive bladder cancer.

Materials and methods. The study included 100 bladder cancer patients (72 males and 28 females) aged between 38 and 90 years (mean age 65 ± 10). Tumor tissue samples were tested by polymerase chain reaction to detect DNA of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2 respectively), high-risk human papillomavirus (HPV), cytomegalovirus (CMV), and Epstein—Barr virus (EBV). Immunohistochemical analysis was performed in 32 patients and included the following markers: proliferation marker Ki-67, p63, apoptosis regulator Bcl-2, p53, angiogenesis marker CD31, adhesion protein CD44, and epidermal growth factor receptor (EGFR).

Results. Viral DNA in tumor tissue was detected in 34 patients; of them, 50 % had poorly-differentiated tumors. Twenty-seven patients were found to have EBV DNA in their tumor tissue; 6 patients had CMV DNA; 5 patients had high-risk HPV DNA (types 16, 39, 45, 52, and 59); 1 patient had HSV1 and HSV2 DNA. Four out of 34 participants had mixed infections (1 case of HPV 59 + EBV; 2 cases of EBV + CMV; 1 case of CMV + EBV + HPV 31 and 52). We observed a strong correlation between the presence of EBV DNA and levels of CD31 and Ki-67 expression as well as between high-risk HPV DNA and Bcl-2 expression. High levels of antibodies against EBV capsid antigen were associated with EGFR and Ki-67 expression, whereas the level of antibodies against EBV nuclear antigen correlated with CD44 expression. We evaluated specific characteristics of expression of the markers analyzed depending on the tumor stage, grade of anaplasia, and recurrence. We also assessed morphological characteristics of changes in lymphocytic and plasma cell infiltrates.

Conclusion. We found a correlation between the presence of viral DNA in bladder cancer tissue and markers of proliferative activity, angiogenesis, and apoptosis. Viral infection is likely to increase proliferative activity and suppression of apoptosis, which may cause tumor progression. Further studies are needed to assess this correlation.

1. Eble J.N., Sauter G., Epstein J.I., Sesterhenn I.A. Tumors of the Urinary system and Male Genital Organs. Lyon press, 2004.

2. Brauers A., Jakse G. Epidemiology and biology of human urinary bladder cancer. J Cancer Res Clin Oncol 2000;126 (10):575–83. PMID: 11043394.

3. Theodorescu D. Molecular pathogenesis of urothelial bladder cancer. Histol Histopathol 2003;18(1):259–74. DOI: 10.14670/HH-18.259. PMID: 12507305.

4. Babjuk M., Osterlinck W., Sylvester R. et al. EAU Guidelines on Ta, T1 (non-muscle-invasive) Bladder Cancer. In: EAU Guidelines. Edition presented at the EAU Annual Congress Milan, 2008.

5. Coussens L.M., Werb Z. Inflammation and cancer. Nature 2002;420 (6917): 860–7. DOI: 10.1038/nature01322. PMID: 12490959.

6. Kawanishi S., Hiraku Y. Oxidative and nitrative DNA damage as biomarker for carcinogenesis with special reference to inflammation. Antioxid Redox Signal 2006;8 (5–6):1047–58. DOI: 10.1089/ars.2006.8.1047. PMID: 16771694.

7. Murata M., Thanan R., Ma N., Kawanishi S. Role of nitrative and oxidative DNA damage in inflammation-related carcinogenesis. J Biomed Biotechnol 2012;2012:623019. DOI: 10.1155/2012/623019. PMID: 22363173.

8. IARC. Chronic infectionsin. In: World Cancer Report. Eds.: B.W. Stewart, P. Kleihues. Lyon, France: IARC Press, 2008. Pp. 128–135.

9. Hussain S.P., Harris C.C. Inflammation and cancer: an ancient link with novel potentials. Int J Cancer 2007;121(11):2373– 80. DOI: 10.1002/ijc.23173. PMID: 17893866.

10. Андреева Ю.Ю. Морфологические и молекулярно-биологические факторы прогноза рака мочевого пузыря. Дис. … д-ра мед. наук. М., 2008 [Andreeva Yu.Yu. Morphological and molecularbiological prognostic factors of bladder cancer. Thesis … of doctor of medical sciences. Moscow, 2008. (In Russ.)].

11. Wang L., Feng C., Ding G. et al. Ki-67 and TP53 expressions predict recurrence of non-muscle-invasive bladder cancer. Tumour Biol 2014;35(4):2989–95. DOI: 10.1007/s13277-013-1384-9. PMID: 24241960.

12. Krause F.S., Feil G., Bichler K.H. Immunohistochemical examinations (Ki-67, p53, nm23) and DNA cytophotometry in bladder cancer. Anticancer Res 2000;20 (6D):5023–8. PMID: 11326661.

13. Ding W., Gou Y., Sun C. et al. Ki-67 is an independent indicator in non-muscle invasive bladder cancer (NMIBC); combination of EORTC risk scores and Ki-67 expression could improve the risk stratification of NMIBC. Urol Oncol 2014;32(1):42.e13–9. DOI: 10.1016/j.urolonc.2013.05.004. PMID: 24360660.

14. Kovylina M.V., Prilepskaya E.A., Tupikina N.V., Reva I.A. Prognostic role of Ki-67 expression in determination of risk of non-muscle invasive bladder cancer recurrence. Onkourologiya = Cancer Urology 2017;13(1):67–73. (In Russ.].

15. Woodburn J.R. The epidermal growth factor receptor and its inhibition in cancer therapy. Pharmacol Ther 1999;82(2– 3):241–50. PMID: 10454201.

16. Gulley M.L. Genomic assays for Epstein— Barr virus-positive gastric adenocarcinoma. Exp Mol Med 2015;47:e134. DOI: 10.1038/emm.2014.93. PMID: 25613731.

17. Liang Q., Yao X., Tang S. et al. Integrative identification of Epstein—Barr virus-associated mutations and epigenetic alterations in gastric cancer. Gastroenterology 2014;147(6):1350–62. DOI: 10.1053/j.gastro.2014.08.036. PMID: 25173755.

18. Chuang K.L., Pang S.T., Liao S.K. et al. Epstein—Barr virus DNA load in tumour tissues correlates with poor differentiation status in non-muscle invasive urothelial carcinomas. BJU Int 2011;107(1):150–4. DOI: 10.1111/j.1464-410X.2010.09474.x. PMID: 20735392.

19. Breyer J., Wirtz R.M., Otto W. et al. Predictive value of molecular subtyping in NMIBC by RT-qPCR of ERBB2, ESR1, PGR and MKI67 from formalin fixed TUR biopsies. Oncotarget 2017;8 (40):67684–95. DOI: 10.18632/oncotarget.18804. PMID: 28978063.

20. Vetterlein M.W., Roschinski J., Gild P. et al. Impact of the Ki-67 labeling index and p53 expression status on disease-free survival in pT1 urothelial carcinoma of the bladder. Transl Androl Urol 2017;6 (6):1018–26. DOI: 10.21037/tau.2017.11.10. PMID: 29354488.

21. Yoshizaki T., Kondo S., Endo K. et al. Modulation of the tumor microenvironment by Epstein—Barr virus latent membrane protein 1 in nasopharyngeal carcinoma. Cancer Sci 2018;109(2):272–8. DOI: 10.1111/cas.13473. PMID: 29247573.

22. Okoye J.O., Erinle C., Ngokere A.A., Jimoh A. Low CD4 cells and viral co-infection increase the risk of VaIN: use of SCCA1 and Ki-67 as diagno-prognostic biomarkers. Pathophysiology 2018;25 (1):51–6. DOI: 10.1016/j.pathophys 2017.09.004. PMID: 29269193.

23. Yahia R., Zaoui C., Derbale W. et al. Epstein—Barr virus and invasive mammary carcinomas: EBNA, EBERs and molecular profile in a population of West Algeria. Ann Biol Clin (Paris) 2018;76 (1):75–80. DOI: 10.1684/abc.2017.1312. PMID: 29336321.

24. Ng S.B., Ohshima K., Selvarajan V. et al. Prognostic implication of morphology, cyclinE2 and proliferation in EBV-associated T/NK lymphoproliferative disease in nonimmunocompromised hosts. Orphanet J Rare Dis 2014;9:165. DOI: 10.1186/s13023-014-0165-x. PMID: 25475054.

25. Yang C.F., Yang G.D., Huang T.J. et al. EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngeal carcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop. Oncogene 2016;35 (26):3419– 31. DOI: 10.1038/onc.2015.402. PMID: 26568302.

26. Ma N., Kawanishi M., Hiraku Y. et al. Reactive nitrogen species-dependent DNA damage in EBV-associated nasopharyngeal carcinoma: the relation to STAT3 activation and EGFR expression. Int J Cancer 2008;122(11):2517–25. DOI: 10.1002/ijc.23415. PMID: 18307254.

27. Rebouissou S., Bernard-Pierrot I., de Reynies A. et al. EGFR as a potential therapeutic target for a subset of muscleinvasive bladder cancers presenting a basal-like phenotype. Sci Transl Med 2014;6 (244):244ra91. DOI: 10.1126/scitrans-lmed.3008970. PMID: 25009231.

28. Zhang L., Xu J., Yang G. et al. miR-202 inhibits cell proliferation, migration, and invasion by targeting EGFR in human bladder cancer. Oncol Res 2018. DOI: 10.3727/096504018X15149787144385. PMID: 29298735.

29. Petersson F. Nasopharyngeal carcinoma: a review. Semin Diagn Pathol 2015;32 (1):54–73. DOI: 10.1053/j. semdp. 2015.02.021. PMID: 25769204.

30. Koh Y.W., Han J.H., Yoon D.H. et al. Epstein—Barr virus positivity is associated with angiogenesis in, and poorer survival of, patients receiving standard treatment for classical Hodgkin’s lymphoma. Hematol Oncol 2017;36(1):182–8. DOI: 10.1002/hon.2468. PMID: 28744882.

31. Koh Y.W., Han J.H., Yoon D.H. et al. PD–L1 expression correlates with VEGF and microvessel density in patients with uniformly treated classical Hodgkin lymphoma. Ann Hematol 2017;96(11):1883– 90. DOI: 10.1007/s00277-017-3115-6. PMID: 28842748.

32. Loran O.B., Sinyakova L.A., Gundorova L.V. et al. Impact of latent viral infection on the course and prognosis of bladder cancer. Oral presentation at the 17th Congress of the Russian Society of Urology; November 8–10, 2017. (In Russ.).

33. Bracarda S., Altavilla A., Hamzaj A. et al. Immunologic checkpoints blockade in renal cell, prostate, and urothelial malignancies. Semin Oncol 2015;42(3):495–505. DOI: 10.1053/j.seminoncol.2015.02.004. PMID: 25965369.

34. Seo A.N., Kang B.W., Kwon O.K. et al. Intratumoural PD–L1 expression is associated with worse survival of patients with Epstein–Barr virus-associated gastric cancer. Br J Cancer 2017;117(12):1753–60. DOI: 10.1038/bjc.2017.369. PMID: 29073638.

35. Wankowicz S.A. M., Werner L., Orsola A. et al. Differential expression of PD–L1 in high grade T1 vs muscle invasive bladder carcinoma and its prognostic implications. J Urol 2017;198(4):817–23. DOI: 10.1016/j.juro.2017.04.102. PMID: 28487100.

36. Chen B.J., Chapuy B., Ouyang J. et al. PD–L1 expression is characteristic of a subset of aggressive B-cell lymphomas and virus-associated malignancies. Clin Cancer Res 2013;19(13):3462–73. DOI: 10.1158/1078–0432.CCR-13–0855. PMID: 23674495.