Is there a role for Epstein-Barr virus in oral carcinogenesis? A study at DNA, RNA and protein levels.

I Cruz, PhD a,b, AJC van den Brule, PhD b, PJF Snijders, PhD b, I van der Waal, PhD a, CJLM Meijer, PhD b

a Department of Oral and Maxillofacial Surgery/Oral Pathology / Academic Center for Dentistry Amsterdam ACTA b Section of Molecular Pathology, Department of Pathology University Hospital Vrije Universiteit, Amsterdam, North Holland, The Netherlands

AIM: To determine if EBV plays a role in oral carcinogenesis. METHODS: 36 oral squamous cell carcinomas (OSCCs) were investigated for EBV DNA using PCR with two primer sets, which amplify a fragment within BamHI W and BNLF-1 regions, respectively. 13 of the EBV DNA positive OSCCs were analyzed for the presence of EBV RNA and/or protein. EBER transcripts were investigated by RNA in situ hybridization. EBNA-1, EBNA-2, LMP-1, LMP-2, BHRF-1 and BARF0 transcripts were investigated by RT-PCR and/or NASBA. Immunohistochemistry was used to detect EBNA-1, LMP-1 and ZEBRA proteins. Positive and negative controls were included in each experiment. RESULTS: All OSCCs were positive for EBV DNA using the highly sensitive BamHI W PCR, and 18 of these (50%) were positive using the less sensitive BNLF-1 PCR. However, virtually all OSCCs tested failed to reveal EBV transcripts and no ZEBRA and LMP-1 proteins were found in the neoplastic or any other cells. Immunohistochemistry using a monoclonal antibody raised against EBNA-1 (2B4) resulted in positive staining in some OSCCs, but these results were considered 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. CONCLUSIONS: The absence of EBV encoded transcripts and proteins in OSCCs indicate that with the present knowledge on EBV, an active role for this virus in oral carcinogenesis is unlikely. The EBV DNA detected by PCR in OSCC samples is likely to reflect EBV shedding in the saliva of OSCC patients due to impairment in their immune system.

KEY WORDS: oral squamous cell carcinoma, in situ hybridization, immunohistochemistry.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Viral Oncogenesis.