Expression of Epstein-Barr Virus in Cell of Classical Hodgkin's Lymphoma Tumor
Authors: Neto Abrahão Elias Hallack , Mayrink Graziela Toledo Costa , Costa Luciano J. , Santos Kelli Borges dos 
Published: October 2019

Abstract
Introduction: The association between classical Hodgkin's Lymphoma (cHL) and tumor Epstein-Barr virus (EBV) status is well established. However, the presence of EBV within Hodgkin/Reed-Sternberg (HRS) cells and its prognosis remains controversial, with conflicting findings from studies of various regions of the world. It is considered essential to deepen the understanding of the pathogenic role of EBV in cHL and its impact in prognosis. Methods: We assessed the correlation between EBV presence in HRS and outcomes in a cohort of Brazilian patients with cHL. EBV positivity was determined by in situ hybridization (ISH) for EBV-encoded RNA (EBER) and immunohistochemistry (IMH) for viral latent membrane protein (LMP-1). All cases were histologically confirmed by an expert hematopathologist who also performed the assays for EBV identification. We examined the prognostic impact of EBV status in 29 patients with cHL. The prognostic factors by IPS (International Prognostic Score) for patients with advanced stage and the risk factors by GHSG (German Hodgkin Study Group) for patients with limited stage were correlated with EBV status tumor cells. For associations between the presence of EBV and other categorical variables, we applied Chi-square or Fisher's exact tests. For describe the effect size (ES) measures for chi-square, we used Cramér's V (V) and odds ratios (OR) with the respective 95% Confidence Intervals (CIs). To evaluate the correlation between all methods of identification of EBV status and among evaluators in histological classification, we applied the Kappa test (K), which measures the degree of agreement these assessments. Differences in OS (overall survival) and EFS (event-free survival) Kaplan-Meier survival curves between EBV-positive and EBV-negative patients were compared statistically using the log-rank test. To evaluate the impact of EBV status on event-free survival controlling for prognostic factors and unfavorable risks, we applied Cox proportional hazards regression to determine hazards ratios (HR) and associated the respective 95% CIs. Multivariate analyses included variables significant at p ≤ 0.15 in univariate models. Results: The mean age at diagnosis was 33 years. Sixty-five percent of the patients had the Nodular Sclerosis histologic subtype and 62,1% had Ann Arbor stage I or II disease at diagnosis. According to GHSG, 88,3% of early-stage patients were classified with unfavorable risk (at least one risk factor) at diagnosis. Compared to advanced-stage patients, 81,9% were considered with favorable IPS (< 4 prognostic factors) at diagnosis. HRS cells were EBV-positive in 37.9% of cases. EBV-positive cHL cases were more frequent in patients ≥ 45 years (71,4% vs. 27,3%, p =0,07). Mixed cellularity (MC) histology subtype was more common in EBV-related tumor cells (p= 0,02) and its effect-size index was medium. The correlation between all methods of identification of EBV status was 96,5% (p< 0,001; K=0.93). The correlation among evaluators in histological classification was 89,6% (p< 0,001; K=0.79). In univariate analysis, age, stage, histologic subtype, nodal involvement, extranodal disease, sex, bulky disease, laboratory data were not associated with adverse EFS (p>0,05). EBV-positive HL seemed to have better EFS than EBV-negative HL (log-rank test, p = 0,07). Cox proportional hazards model confirmed that EBV-positive tumor status and prognosis factors did not impact HL outcome. Conclusions: Despite EBV status in HRS cells not being associated with adverse prognostic factors and not influencing the overall and event-free survivals, the presence of EBV was linked to MC subtype, showing possible implication in histological subtype and worse prognosis.

Disclosures
Costa: Sanofi: Honoraria, Research Funding.