Analysis of single EBER-positive and negative tumour cells in EBV-harbouring B-cell non-Hodgkin lymphomas

In situ hybridization for Epstein-Barr virus (EBV) encoded small nuclear RNAs (EBERs) is the method of choice for the detection of EBV infection at the single cell level. With the application of this technique it was shown that non-Hodgkin's lymphomas of B-cell type may be associated with an EBV infection of tumour cells. Interestingly, in many EBV-positive cases, only a proportion of the tumour cell population has been found to be EBER-positive. To clarify whether EBV is absent or whether EBER gene expression is downregulated in EBER-negative tumour cells, single EBER-positive and negative tumour cells were isolated from paraffin sections from four B-cell non-Hodgkin's lymphoma cases with partial EBER expression in the neoplastic cells. These single cells were then screened for the presence of EBV DNA by a nested single cell PCR. EBV DNA was undetectable in all but one of the 86 EBER-negative cells, whereas in the EBER-positive cells EBV-specific DNA amplificates could be generated following single cell PCR. This finding prompts the conclusion that the inability to detect EBERs in a proportion of tumour cells is not due to a down-regulation of gene expression but to a real absence of EBV from these cells. This partial absence of EBV is thought to be caused by a loss of the EBV episomes during cell division, rather than by infection of only a proportion of the tumour cells.