Detection of Epstein–Barr virus nucleic acid sequences and protein in nodal T‐cell lymphomas: relation between latent membrane protein‐1 positivity and clinical course

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Abstract
Forty‐six nodal T‐cell lymphomas, classified according to the updated Kiel classification, were investigated for the presence of Epstein–Barr virus (EBV) DNA by polymerase chain reaction (PCR), EBER 1 and 2 (EBER 1/2) and latent membrane protein‐1 (LMP‐1) expression. A combination of RNA in situ hybridization and immunohistochemistry was used to establish the phenotype of the Epstein–Barr virus harbouring cells. In 21 of 45 cases Epstein–Barr virus DNA sequences could be detected with the polymerase chain reaction. In 15 cases (14 of 21 EBV PCR positive cases), EBER 1/2 positive cells could be demonstrated. As judged by morphology, EBER 1/2 expression was found in non neoplastic and neoplastic lymphoid cells. Double staining revealed that more than 80% of the EBER 1/2 harbouring cells, lacked B‐, T‐ or histiocytic markers, suggesting down regulation of T‐ and B‐cell markers by Epstein–Barr virus. In eight of 15 cases some EBER 1/2 positive T‐cells (CD3, CD45RO, CD43) morphologically resembling tumour cells were found. In nine of 14 cases tested EBER 1/2 positive non‐neoplastic B‐cells (CD20) were seen. Based on in situ hybridization results, four patterns of EBER 1/2 positive cells were found, i.e. single cells (≤1 per medium power field (mpf), n= 3), scattered (1–25/mpf, n= 4), clustered (26–100/mpf, n= 5) and diffuse (≥100/mpf, n= 3). In eight of 15 cases a clustered or diffuse pattern of EBER 1/2 positive cells was found and these lymphomas were therefore considered to be strongly associated with Epstein–Barr virus. In these lymphomas LMP‐1 expression was found to be associated with an aggressive clinical course and hepatosplenomegaly.