No direct role for Epstein-Barr virus in oral carcinogenesis: a study at the DNA, RNA and protein levels.

Reports on the association of EBV with oral squamous-cell carcinomas (OSCCs) are scarce and inconclusive. To determine the potential role of EBV in oral carcinogenesis, we investigated 36 EBV DNA PCR-positive OSCCs for the expression of EBV transcripts and proteins. From these EBV DNA-positive OSCCs, 13 were analysed for the presence of EBV products, either at RNA and/or protein level. EBER transcripts were investigated by RNA in situ hybridisation. EBNA-1, EBNA-2, LMP-1, LMP-2, BHRF1 and BARF0 transcripts were investigated by RT-PCR and/or NASBA. EBNA-1, LMP-1 and ZEBRA protein expressions were investigated by immunohistochemistry. All 36 OSCCs were positive for EBV DNA, using the highly sensitive BamHI W PCR, and 18 of these (50%) were positive using the less-sensitive PCR, which targets BNLF-1. However, virtually all OSCCs tested failed to reveal EBV transcripts, including EBERs and EBNA-1 transcripts. No ZEBRA and LMP-1 proteins were found in the neoplastic or any other cells of the OSCCs investigated. Immunohistochemistry using a monoclonal antibody (MAb) raised against EBNA-1 (2B4) resulted in positive staining in some cases of OSCCs, but these results were non-specific, since EBV-negative epithelial tissues showed extensive non-specific staining and no EBNA-1-specific transcripts were detected by RT-PCR or NASBA. The absence of expression of EBV encoded transcripts and proteins indicate that, with the present knowledge on EBV, an active role in oral carcinogenesis for this virus is unlikely.