THE PROGNOSTIC ROLE OF EBV IN PERIPHERAL BLOOD OF PATIENTS WITH DIFFUSE LARGE B CELL LYMPHOMA

Background: The Epstein-Barr virus (EBV) belongs to the herpes virus family and infects more than 90% of the human population establishing persistent latent infection in the host. Although EBV infection is benign in most individuals, it has been linked to the etiology of a number of lymphoproliferative diseases. ‘EBV-positive diffuse large B-cell lymphoma of the elderly’ was included as a provisional entity of DLBCL in the revised 2008 WHO classification. We recently showed that the plasma EBV-DNA load at HL diagnosis is an indicator of disease activity and biological characteristics associated with negative prognosis (Hohaus et al,Clin Cancer Res,2011;17(9); 2885–92). Aims: We studied the role of EBV-DNA copy number in different blood compartments (whole blood, mononuclear cell fraction, and plasma) in patients with DLBCL at diagnosis, as a potential predictive indicator for the presence of EBV in lymphoma cells and as a prognostic marker in patients treated with immunochemotherapy (R-CHOP). Methods: We analysed 136 patients with DLBCL (median age 62 years, range 15-92 years; 60 females and 76 males). EBV was detected using a commercial real-time PCR kit, amplifying a 191 bp region of the EBNA-1 gene (BioQuant EBV, Biodiversity, Brescia, Italy) in peripheral blood compartments (whole blood n=133, plasma n=55, and mononuclear cells n=52). Lymph node samples from 61 DLBCL patients were analyzed for EBV infection through in situ hybridization for EBV-encoded small RNAs (EBER). Results: EBV was frequently detected in peripheral blood: 35 of 133 whole blood samples (26%) resulted positive. The copy number varied between 200 and 196000 copies. The presence and copy number of EBV in whole blood and mononuclear cells were correlated (P<0.05, P<0.01, respectively), while there was no correlation to the detection of EBV in plasma. We did not find any association between the presence or viral load of EBV-DNA in any blood compartment and the presence of EBV in the lymphoma cells of 61 patients studied with EBER–ISH (11 patients EBER pos). The presence and viral load of EBV in PB was not related to age or gender, and other disease characteristics as LDH level, stage, and IPI. In univariate analysis on 133 patients treated with R-CHOP, the presence of EBV-DNA in peripheral blood was associated with a significantly shorter event-free survival (EFS): 60% versus 79% at 2 years, P<0.04). As well, the EBV copy number was correlated with a worse outcome (hazard ratio of 1.86 for each logarithmic increase; 95% C.I, 1.17-2.97; P<0.009). Correcting for IPI in a multivariate Cox analysis, the presence of EBV-DNA in peripheral blood retained its prognostic significance (hazard ratio 2.02; 95% C.I., 1.03-3.95; P<0.04). Summary / Conclusion: Our findings suggest that EBV can be frequently detected in peripheral blood at DLBCL diagnosis, which does not reflect the EBV status of the lymphoma cells, but associates with a worse outcome following standard immunochemotherapy. Further studies are needed to explore the mechanisms that permit the expansion of EBV-positive cells in peripheral blood of patients with DLBCL.