Detection of Epstein-Barr virus in CNS lymphomas by in-situ hybridization

We used an optimized in-situ hybridization technique employing a biotinylated Epstein-Barr (EB) virus sequence, BamH1V (3.1 kb), to detect this sequence in 2 EB virus-infected cell lines (B95-8 and Namalwa) and 8 CNS lymphomas. We obtained a good hybridization signal from cytospins of B95-8 (EB virus productively infected) and Namalwa (EB virus latently infected, 1 copy per cell) cell lines. We were able to detect signal from both cell lines after overnight fixation in 10% formalin and paraffin embedding, but development time in the detection chromogen required longer incubation and the signal intensity was lower than in cytospin cells. We then used the technique to examine formalin-fixed, paraffin-embedded primary CNS lymphoma tissue from 4 patients who were immunocompromised (1 renal transplant, 3 acquired immune deficiency syndrome) and 4 patients who were not. All 4 CNS lymphomas from immunocompromised patients hybridized well with BamH1V, exhibiting a pattern of staining similar to Namalwa cells and nonlytically infected B95-8 cells. There was no relationship between the intensity and degree of reaction and the patients' survival. None of the 4 CNS lymphomas in immunocompetent patients or uninvolved brain showed any reactivity with BamH1V. We suggest that low-abundance targets are detectable in paraffin-embedded tissue by in-situ hybridization using biotinylated probes. Detection of EB viral sequences in CNS lymphomas in immunocompromised patients suggests a role for the virus in the pathogenesis of this tumor.