Presence of EBV antigens detected by a sensitive method in pediatric and adult Diffuse Large B‐cell lymphomas

Abstract

In 2017, the World Health Organization (WHO) confirmed a new entity, Epstein Barr virus (EBV) + Diffuse large B cell lymphoma (DLBCL), not otherwise specified (NOS). Traces of EBV transcripts were described in lymphomas, including DLBCL, that were diagnosed as EBV negative by conventional methods. The aim of this study was to detect viral genome by qPCR, as well as LMP1 and EBNA2 transcripts, with a more sensitive method in DLBCL cases from Argentina. Fourteen cases originally considered as EBV negative expressed LMP1 and/or EBNA2 transcripts. In addition, LMP1 and/or EBNA2 transcripts were also observed in bystander cells. However, EBERs+ cells cases by conventional ISH showed higher numbers of cells with LMP1 transcripts and LMP1 protein. In the cases that were EBERS− in tumor cells but with expression of LMP1 and/or EBNA2 transcripts, the viral load was below the limit of detection. This study provides further evidence that EBV could be detected in tumor cells by more sensitive methods. However, higher expression of the most important oncogenic protein, LMP1, as well as increased viral load, are only observed in cases with EBERs+ cells by conventional ISH, suggesting that traces of EBV might not display a key role in DLBCL pathogenesis.

Presence of Epstein–Barr virus (EBV) antigens detected by sensitive methods has no influence on local immune environment in diffuse large B cell lymphoma

“…Since traces of EBV infection were detected by sensitive methods in lymphomas [ 3 , 4 , 6 ], but its involvement in lymphomagenesis is still under discussion, double ISH to detect single copies of viral LMP1 and EBNA2 transcripts was assessed in 36 cases with good quality material. Of those 36 cases, 32 cases (89%) were defined as EBV− (< 20% EBERs + cells), whereas 4 cases (11%) were EBV+ (≥ 20% EBERs + cells) by ISH.…”

“…A total of 48 DLBCL patients, previously characterized [ 6 ], were enrolled in this study, 26 pediatrics and 22 adults, collected retrospectively, based on the availability of sufficient material, from the archives at Pathology Division, at the Ricardo Gutierrez Children’s Hospital, and at the Marie Curie Hospital in Buenos Aires, Argentina. The samples were reviewed independently by 4 different pathologists: DDSM, GL, CS, and DME, according to the WHO classification for lymphoid neoplasms criteria [ 20 ].…”

“…Double in situ hybridization with ViewRNA ISH Tissue 2-Plex Assay (Thermo Fisher, formerly Affymetrix) was performed in 36 FFPE DLBCL cases with good quality material, as previously reported [ 6 ]. Custom-specific probes sets against LMP1 (probe type 1) and EBNA2 (probe type 6) transcripts were customized by the manufacturer (Thermo Fisher, formerly Affymetrix), based on LMP1 and EBNA2 mRNA sequences.…”

“…Given the fact that the EBV traces may remain, some studies suggest that the virus could be involved in the pathogenesis of these lymphomas [ 3 – 5 ]. Recently, our group also proved the presence of traces of EBV in patients with DLBCL from Argentina, but suggested, since higher expression of the most important oncogenic protein, as well as increased viral load, was observed in cases with EBERs + cells by conventional ISH, that traces of EBV could not display a key role in DLBCL pathogenesis [ 6 ].…”

“…As described above, the role of TME is still under discussion in this new entity, EBV+ DLBCL, NOS, and it is still unexplored if the presence of EBV could trigger alterations at the TME, inducing a first “hit”, followed by the “run” after triggering, for example, a tolerogenic environment, as proposed by the hit-and-run theory. Under this hypothesis, once the alteration has been established, traces of EBV could be detected [ 5 , 6 ]. In addition, in Argentina EBV-associated lymphomas have a higher incidence in children < 10 years, and almost 90% of patients are seropositive by the age of 3 years [ 20 ].…”