Epstein-Barr virus infection in urothelal transitional cell carcinoma tissues as demonstrated by the presence of EBV-encoded RNA and its possible implications in the pathogenesis of this malignancy

S-K Liao PhD^a, C-K Chuang MD PhD^a,b, M-J Liu BSc^b, T-R Chang BSc^a

^aGraduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan 333, Taiwan, ^bDivision of Urology, Department of Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan

Aim: EBV has been implicated in the pathogenesis of a number of lymphopoliferative disorders and epithelial neoplasms. We in this study sought to explore a possible correlation of EBV infection with urothelial transitional cell carcinoma (TCC) tissues. Methods: One hundred and four (104) surgical TCC specimens obtained from urology operation rooms in Chang Gung Memorial Hospital were fixed in formalin and embedded in paraffin. The tissue blocks were processed for in situ hybridization (ISH) using DNA probes in order to localize the signal of EBV-encoded RNA (EBER). Immunostaining with monoclonal antibodies to CD20 and EBV-encoded late membrane protein-1 (LMP-1) was also performed on sections of TCC tumor specimens. For analysis of mutation and deletion in the carboxyl terminal end of the LMP-1 gene, DNA was extracted and PCR amplificaton was performed using a pair of primers. PCR product was then analyzed by an ABI 377 automated sequencer. Results: Thirty-one tumor tissues were positive for EBER hybridization out of 100 specimens that could be adequately evaluated . Of these 31 positive specimens, 21 were found positive in both TCC tumor cells and tumor-infiltrating B-lymphocytes, 3 were only positive in TCC tumor cells, and 7 were only positive in B-lymphocytes. Eighty four percent EBER-positive tumor tissues (26/31) showed the expression of LMP-1, suggesting that EBER was more sensitive than LMP-1 in the detection of EBV infection. In one case where TCC tumor cells and B lymphocytes from the TCC tumor tissue were isolated, sequence analysis revealed a 30 bp deletion and 7 point mutations on the LMP-1 gene in both cell types; however, only 4 out of the 7 point mutations shared with B cells isolated from peripheral blood of the same patient in a disease-free after surgical removal of the tumor. Conclusions: Our study has demonstrated for the first time a strong association between EBV infection and a significant proportion of primary urothelial TCC tumors in Taiwanese population. Whether such an association has anything to do with malignant transformation of urothelial transitional cells and/or progression of TCC is one of the many questions that will be answered by the molecular approaches already established for nasopharyngeal carcinoma and gastric cancer. (Supported by grants from the National Science Council of Taiwan and the Chang Gung Medical Research Fund)

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 993 (Molecular pathology - Part II).