Abstract P049

Detection of traces of EBNA2 in the Microenvironment of Pediatric Classical Hodgkin Lymphoma Reveals Its Immunomodulatory Role

The detection of Epstein–Barr virus (EBV) traces using ultrasensitive techniques in B-cell lymphomas previously classified as EBV-negative by conventional methods suggests a broader role for the virus in lymphomagenesis. Its contribution in classical Hodgkin lymphoma (cHL) remains unexplored. This study aimed to characterize viral transcripts expression by sensitive methods.

A total of 48 pediatric cHL cases were analyzed. EBV status was evaluated with the conventional approach: EBER in situ hybridization (ISH). LMP1 and EBNA2 transcripts were assessed using highly sensitive dual ISH combined with CD30 immunohistochemistry (IHC) to distinguish tumor cells from cells of the tumor microenvironment (TME). IHC evaluated protein expression for LMP1, CD4, CD8, CD56, CD68, CD163, PD-1, PD-L1 (TME and tumor cells), IL-10, FOXP3, Granzyme B (GrB), TGF-β, and T-bet. Event-free survival was analyzed using the Kaplan-Meier method.

Among the 32 EBERS+ cases, 32 expressed LMP1 transcripts in both tumor cells (LMP1t+) and the TME (LMP1m+), none expressed EBNA2 transcripts in tumor cells (EBNA2t-), and 23 expressed EBNA2 transcripts in the microenvironment (EBNA2m+). In the 16 EBERs-, 10 cases displayed LMP1t+ cells, 11 cases LMP1m+ and 6 EBNA2m+. Remarkably, EBNA2m were exclusively observed in the TME. 5 cases did not express any of the analyzed transcripts.

PD-L1 expression in the TME was significantly higher in cases expressing EBNA2 transcripts compared to those without EBNA2 expression (p=0.0373, Mann–Whitney test). Similarly, GrB expression was also significantly increased in EBNA2m+ cases (p=0.0012, Mann–Whitney test) (Figure 1). The count of IL10+ cells in the groups defined by EBNA2 transcript expression showed a trend (p=0.0768, Mann–Whitney test). Only cases expressing traces of LMP1 in both tumor cells and the TME exhibited a predominant M1 profile (CD68+cells/CD163+ cells > 1.5) (p=0.0339, p=0.0096, Fisher ́s exact test). No significant differences in event-free survival were observed according to viral transcript expression (p>0.05).

EBNA2 expression in the TME, even in EBV- cases, might be associated with immune checkpoint regulation and cytotoxic responses, highlighting a previously underrecognized role of EBV and its viral traces in shaping the cHL microenvironment. Additionally, LMP1 expression in tumor cells and the TME could contribute to the M1-polarized profile previously described in pediatric EBV- associated cHL.