Prevalence, clinical characteristics and prognosis of EBV‐positive follicular lymphoma
Authors: Nicholas Mackrides, Jennifer Chapman, Melissa C. Larson, Juan C. Ramos, Ngoc Toomey, Pei Lin, Matthew J. Maurer, Myra Rafaelle, Yaohong Tan, Offiong Ikpatt, Sergei Syrbu, Stephen M. Ansell, Thomas M. Habermann, Brian K. Link, Andrew L. Feldman, Izidore S. Lossos, James R. Cerhan, Francisco Vega
Journal: American Journal of Hematology (2018)

EBV is associated with a diverse group of tumors of lymphoid and epithelial origin, and is commonly associated with B-cell and T/NK-cell lymphoproliferative disorders.1 These disorders represent a spectrum of entities which range from indolent conditions to highly aggressive lymphomas.2 EBV-positive follicular lymphoma (FL) is an uncommon and poorly characterized disease. We previously reported the largest series of EBV-positive FL, occurring with a prevalence of 2.6% in an unselected cohort of 382 FL patients.3 Other than that study, the association between EBV and FL is not well recognized, with only few sporadic cases documented in the literature. In our prior study, the majority of patients with EBV-positive FL progressed to either a higher grade FL or to diffuse large B-cell lymphoma (DLBCL). There is a biological rationale supporting a potential contribution of EBV proteins to genomic instability in B-cell lymphomas, possibly resulting in a more aggressive clinical course.4 This generated the hypothesis that EBV-positive FL represents a more aggressive FL subgroup. A large number of cases with well annotated clinical data were needed to test this hypothesis. The goal of the current study was to confirm the prevalence of EBV in a cohort of prospectively enrolled patients with FL, and to correlate EBV status with clinicopathological features and prognosis. Patients with FL were enrolled in the University of Iowa/Mayo Clinic Specialized Programs of Research Excellence (SPORE) Molecular Epidemiology Resource (MER) cohort study from 2002 to 2015. Tissue microarrays (n = 25) were assembled using tissue from diagnostic biopsies from 488 cases of FL. Patients with HIV and patients diagnosed with composite lymphomas or concurrent DLBCL were not included in the study. Tissue microarrays were stained for EBER by in situ hybridization, and for LMP1 and EBNA2 by immunohistochemistry, as previously described.3 EBV-positive FL were those with tumor cells positive for one or more of these markers. Cases with staining of only "bystander" lymphocytes (small lymphocytes outside of neoplastic follicles) were considered negative for EBV. Patient blood/plasma samples were not available for EBV assays. Event-free survival (EFS) was defined as time from diagnosis to relapse/progression, retreatment, or death due to any cause. Early events, which are more aggressive, were defined as failing to achieve EFS at 12 months (EFS12).5 We estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for EFS and overall survival from Cox models, adjusted for age and sex. There were 488 patients with newly diagnosed FL included in the study; 2.5% (12 of 488) were EBV-positive (Supporting Information Table S1). LMP1 was expressed by 92% (11 of 12) of EBV-positive FL. EBNA2 was negative in all cases. The proportion of lymphoma cells which expressed EBER and LMP1 varied between cases, ranging from 10% to 90%, and in many cases there was staining of centrocytes and centroblasts (Supporting Information Figure S1). Immunofluorescence studies were performed on 2 EBV-positive cases, as described in the prior study,3 using antibodies against CD20, BCL2, BCL6, and LMP1. The findings further supported that the neoplastic cells were EBV-positive by demonstrating LMP1 positive B-cells within neoplastic follicles, with coexpression of BCL2 and BCL6. A subset of EBV-negative FL cases (n = 24) were confirmed negative by PCR for EBV viral genes (EBNA1, BZLF1, EBV-TK, LMP1, EBER1). In 5 EBV-negative cases, PCR detected EBV DNA, however, EBER in situ hybridization performed on whole tissue sections showed staining of only "bystander" lymphocytes in those cases. Patients with EBV-positive FL were somewhat older, more likely to have grade 3 disease, have >4 nodal sites, and have a FL international prognostic index (FLIPI) score of 3 or higher, although none of these differences were statistically significant (Supporting Information Table S2). We did not have data on immunodeficiency, although HIV/AIDS was an exclusion criteria. Through 2017, there were 273 events and 104 deaths. EBV status was not associated with EFS (HR = 1.12, 95% CI 0.55-2.28, P = 0.75; Figure 1A) or achieving EFS12 (82% for EBV+ vs. 75% for EBV−, P = 0.81). However, patients with EBV-positive FL had inferior overall survival when adjusted for age and sex (HR = 2.59, 95% CI 1.13-5.94, P = 0.049; Figure 1B), which only slightly attenuated after further adjustment for FLIPI score (P = 0.064). None of the patients with EBV-positive FL had transformation to DLBCL, and of the 6 deaths in the EBV-positive group, 3 patients died of lymphoma. In summary, in this study we analyzed a large cohort of community-based patients with FL to determine the prevalence, clinicopathological features, and prognosis of EBV-positive FL. We confirmed that EBV-positive FL was uncommon, being identified in only 2.5% of 488 cases, mostly with a type II latency program (92%, 11 of 12), rarely with latency I (8%, 1 of 12), and none with latency III (all cases were negative for EBNA2). EBV-positive FL trended toward more aggressive features, including higher FLIPI score. While there was no association of EBV status with EFS or transformation to DLBCL, there was a decreased overall survival in the EBV-positive FL group, with the caveat that this was based on a total of six deaths, only three due to lymphoma. Interestingly, the three patients who died of EBV-positive FL had failed to reach EFS12. EBV-induced upregulation of activation-induced cytidine deaminase (AID) in EBV infected B-cells has been experimentally shown to contribute to genomic instability. This could potentially contribute to lymphomagenesis, and may provide a biologic rational for increased aggressiveness in EBV-positive B-cell lymphomas.4 Future studies should assess AID expression in EBV-positive FL to evaluate for a relationship with prognosis. Considering the low incidence of EBV-positive FL, and the small number of patients who died of disease, it is difficult to draw definitive conclusions about prognosis based on this and our prior study, and much larger FL cohorts will be needed to definitively assess the impact of EBV on clinical course. However, based on the current data, EBV status has limited clinical implications for the diagnosis and management of FL patients in North America. The authors have no conflicts of interest to disclose. NM, JC, ALF, ISL, and FV wrote and edited the manuscript; SMA, AF, SS, TMH, JRC, and BKL provided study material or patients; MCL, MJM, SS, SMA, YT, OI, TMH, BKL, ALF, and JRC analyzed and interpreted the data; JCR, NT, PL and MA provided vital technical support; all authors reviewed and approved the manuscript. This work was supported by research funds from Sylvester Comprehensive Cancer Center/University of Miami, and by the NCI awards (P50 CA97274 and U01 CA195568) (to the lymphoma Epidemiology of Outcomes Cohort Study). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.