DLBCL is a heterogeneous disease, and EBV DLBCL is one of the several subtypes of DLBCL with dismal prognosis [1]. EBV infection is believed to play a critical role in the pathogenesis of EBV DLBCL. Various latent genes of EBV can mediate B-cell transformation and induce B-cell lymphoma development in various ways [18-20]. Immune senescence with aging has been suggested to play a role in the development of EBV DLBCL in elderly patients. However, the incidence of EBV DLBCL in younger patients may not be associated with immune senescence. Therefore, the pathogenesis of EBV DLBCL may differ between younger and elderly patients. In this study, we tried to investigate these differences and determine whether there are differences in the microenvironment and molecular profiles of EBV DLBCL cases caused by immune senescence with aging and immunodeficiency of various causes. We found that the clinical presentation of EBV DLBCL did not differ between young, immunodeficiency-associated, and elderly patients; however, the mutational profile of elderly group was significantly different from that of the younger and immunodeficiency-associated patients. The most important and novel finding of this study was that elderly patients with EBV DLBCL had a higher frequency of TET2 and LILRB1 mutations.
TET2 aberrations were observed in 50% (10/20) of the elderly patients, but not in the other two patient groups. Another characteristic finding was that loss-of-function mutations including nonsense mutation, frameshift deletion, and deletion were detected in 8 out of 10 cases where TET2 mutation was observed. These were significantly different from the results of our validation cohort in terms of the frequency of mutations by age group and type of mutation. Tet2 is an enzyme that converts 5-mythylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by demethylation. When the function of the TET2 gene is lost, 5mC is generally accumulated in the cell [21, 22]. Loss-of-function mutations in TET2 are commonly observed in myeloid neoplasms [23, 24] and T-cell lymphomas [24-26], but can also be observed in B-cell lymphoma [27]. An increase in the frequency of TET2 mutations in EBV DLBCL was previously reported in one paper [28], but the results were not reproduced in other studies [29, 30]. Nonetheless, the exact role of TET2 mutations in EBV-induced B-cell lymphomagenesis remains unknown. TET2 is involved in demethylation of the viral genome in host cells and in the host genome [31]. TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected naïve B cells or activated type B cells from latency type I to latency type III [32]. From this point of view, it is expected that EBNA2 is activated in TET2-wild type tumors, and the latency type moves from I to III, contributing to the development of lymphoma. However, our analysis strongly suggests that the loss of TET2 function is associated with the development of EBV DLBCL. A study has also shown that the activity of TET2 and the degree of methylation are not necessarily related in one direction. In a study by Ko et al., hypomethylation, but not hypermethylation, was observed in the CpG region of cases with low 5hmC levels due to TET2 mutations in myeloid cancer [33]. In addition, a previous study suggested that the loss of TET2 activity in nasopharyngeal cancer, a representative carcinoma caused by EBV infection, can trigger cellular hypermethylation and alter EBV gene regulation [34]. Therapeutic strategies using TET2 as a target in leukemia patients are being studied [35, 36], and this strategy has the potential to be applied to lymphoma with TET2 mutations. Further studies are required to elucidate the relationship between loss-of-function mutations in TET2 and EBV transcriptional regulation and EBV DLBCL pathogenesis.
Mutations in LILRB1 were found in 10 cases, of which 8 were in elderly patients. LILRB1 is expressed in immune cells and is known to play a role in inhibiting the immune response by binding to major histocompatibility (MHC) class I antigen presenting cells [37, 38]. In one study, accumulation of EBV-specific cytotoxic T-cells expressing LILRB1 was observed in patients with persistent EBV infection, and the expression level of LILRB1 was associated with a decrease in the response capacity of EBV-specific CD8-positive T-cells to antigen [39]. Although the expression level of LILRB1 was not measured in this study, if the expression of LILRB1 is abnormally activated in EBV-positive tumor cells due to somatic mutations of LILRB1, it may contribute to the survival of tumor cells through immune evasion. In previous studies, PD-L1 expression through structural variation of the CD274 gene was suggested as a major mechanism by which EBV DLBCL evades host immunity [10, 11, 28, 40] and LILRB1 may also play an important role in immune evasion. Several recent studies have shown the potential of anti-LILRB1 blockers as immunotherapeutic agents [41, 42]. LILRB1 expression levels in EBV DLBCL tissues also need to be evaluated in future studies.
Other than NGS detecting more frequent mutations of TET2 and LILRB1 in elderly patients, the three groups of EBV DLBCL showed no other critical differences in their clinical and pathological features and tumor immune microenvironment evaluated by infiltration of cytotoxic T-cells and macrophages. In contrast to previous studies showing that nodal disease is predominant in young patients [10, 13, 14], the proportion of extranodal involution of tumors was high in our patient group. One of the characteristics of our cohort was that the proportion of monomorphic histology was higher than that in previous studies [10, 13]. Since this study was conducted on consecutively collected cases from a single institute in Korea, an EBV-endemic region, it can be said that the selection bias was minimized. Unlike previous studies, the young patients in our cohort had more monomorphic types of EBV DLBCL and higher prevalence of extranodal disease. Moreover, they did not show a significantly better prognosis than elderly patients. Further studies on the clinicopathological characteristics of young patients with EBV DLBCL in larger cohorts from more diverse regional groups are needed.
In contrast to elderly patients, no particularly frequent genetic mutations were observed in EBV DLBCL cases in young and immunodeficiency-associated patients. This finding is presumably consistent with the hypothesis that EBV-driven pathogenesis requires fewer driver mutations or oncogenic events. The fact that the MYD88L265P and CD79B mutations were mutually exclusive with the presence of EBV [28, 43] was also confirmed in all three groups in our study.
Our study had several limitations. First, since participants were only recruited from a single institution, there were not enough patients in each group, especially in the young and immunodeficiency-associated patient groups. Second, NGS was performed for molecular analysis to investigate the mutations; however, the expression levels of specific genes and degree of methylation were not evaluated. This needs to be supplemented by subsequent multicenter studies.
In conclusion, the clinicopathological characteristics of EBV DLBCL occurring in three distinct patient groups were similar, and there was no significant difference in the tumor immune microenvironment according to the immune status. However, the frequency of mutations in TET2 and LILRB1 was higher in EBV DLBCL cases among elderly patients than among young and immunodeficiency-associated patients. It is possible that TET2 and LILRB1 mutations, along with immune senescence, play critical roles in the development of EBV DLBCL in elderly patients. This finding can be used to develop therapeutic targets for immunotherapy in elderly patients with EBV DLBCL who have a dismal prognosis and may contribute to improving the prognosis of patients with EBV DLBCL.