We found that 31% of our DLBCL cases were positive for EBV infection. This is consistent with previously reported prevalence of EBV+ DLBCL tumors in the cART era(5
). We also found that EBV+ tumor was associated with expression of several of the tumor markers examined, including a positive association with expression of BLIMP1 and CD30, and negative association with BCL6 and LMO2. BLIMP1 is a transcription factor that regulates the differentiation of mature B-cells into antibody-secreting plasma cells(25
). BLIMP1 acts in an autoregulatory feedback loop that controls p53 activity through repression of p53 transcription(26
). The activity of BLIMP1 hence inhibits apoptosis, and deletion of BLIMP1 in lymphocytes induces apoptosis(26
). The positive association between EBV infection and BLIMP1 expression suggested that it may play a role in EBV-induced lymphoproliferation. CD30 is a transmembrane protein that is part of the tumor necrosis factor (TNF) receptor family. When stimulated by CD30 ligand, CD30 interacts with TNF receptor associated factors (TRAF2 and TRAF5), mediating signal transduction that leads to the activation of the NF-κB pathway(27
), which has been linked to cellular activation and carcinogenesis. This finding is consistent with an EBV-associated carcinogenic mechanism operating through the NF-κB pathway. EBV LMP1 expression is known to mimic the activity of ligated CD40, another molecule that is a member of the TNF receptor family, which in turn stimulates the NF-κB and stress activated kinase pathways. In our study sample, EBV+ DLBCL, with or without LMP1 expression, expressed CD30. However, CD30 expression was more common in LMP1+ tumors (88% vs. 23% in the EBV+/LMP1−), despite lack of statistical significance.
BCL6 and LMO2, on the other hand, are suspected favorable prognostic factors. BCL6 is a transcription repressor that is commonly translocated in lymphomas. BCL6 represses B-cell receptor signals(28
) and plays a central role in inducing the germinal center phenotype in both B and T cells(29
). Lack of BCL6 function thus enhances proliferation and inhibits differentiation(28
). To this end, BLIMP1 is a target protein repressed by BCL6(28
). LMO2 is a transcription factor that critically regulates erythropoiesis, angiogenesis, and embryogenesis(31
). LMO2 is associated with the GC phenotype, and has been reported as a favorable prognostic factor in DLBCL by previous studies(35
). The inverse relationship between EBV infection and expression of BCL6 and LMO2 suggested that these two transcription factors may be further repressed in EBV induced lymphomagenesis when compared to other lymphomagenic mechanisms that do not involve EBV.
As noted previously, EBV is thought to contribute to the development of B cell cancers by infecting cells and expressing EBV-encoded transforming proteins which in turn enhances genetic instability through mutation, translocation and aberrant expression of proto-oncogenes(18
). LMP1, a viral gene product of EBV, is known to constitutively activate the NF-κB, Jun N-terminal kinase and p38 kinase pathways(38
)as well as protect cells from p53 induced apoptosis(9
). LMP1 may also contributes to the immortalization of B cells by increasing the expression of anti-apoptotic proteins BCL2 and A20 as well as cell cycle regulator p27(9
). Vrzalikova et al reported down-regulation of BLIMP1 by EBV infection, specifically, LMP1, in lymphoblastoid cell lines established from GC B cells(39
). This seemly contrasting finding may be due to the fact in our study, most EBV+ tumors are the non-GC type. As a result, the effects of EBV seen in GC cells therefore may not be present in post-GC cells. In our exploratory exercise, no consistent pattern of elevation for markers linked to cancer development was observed in LMP1-positive tumors, although the small sample size of LMP1-positive tumors precludes an informative analysis in this study.
EBV also may up-regulate the receptor CD21, thereby protecting cells from self-destruction(40
).While our results provided some support with patient level data for these previously proposed carcinogenic mechanisms of EBV, we did not find association between tumor EBV infection status and expression of p53, BCL2, p27 or CD21. It is possible that these tumor markers were important for all lymphomagenic pathways, regardless of involvement of EBV.
We also found that detecting tumor EBV infection may have independent prognostic utility for survival among patients with HIV-related DLBCL beyond clinical prognostic factors, including IPI and CD4 cell count at diagnosis(41
). This contrasts with the findings of Chadburn et al(42
), who reported that EBV status was not associated with overall or event-free survival among 78 patients with HIV-related DLBCL. They also did not find any association between EBV status and expression of FOXP1 and BLIMP1. However, patients in the study were enrolled in a clinical trial investigating the efficacy of rituximab in HIV-infected DLBCL patients, which may have limited generalizability to HIV-related DLBCL patients at large. Two other studies in non HIV-related DLBCL patients also reported tumor EBV infection status to be an adverse prognostic factor(6
). The utility of EBV status as a prognostic marker in DLBCL should be confirmed in larger studies.
There are several potential limitations of this study. First, a large proportion of patients were excluded from the tumor marker analysis due to lack of an adequate tumor tissue for TMA construction. However, no important differences in demographic and clinical characteristics were found between those with vs. without adequate tumor specimen, suggesting this was not a significant source of bias. Also, our sample size precluded other potentially informative analyses, such as comparing expressions of LMP1 and other selected tumor markers or clinical characteristics with sufficient statistical power, which should be examined in future study to further inform the mechanism of the prognostic effect for EBV. Furthermore, we did not measure other EBV latent proteins nor define the various latent stages of the EBV infection. Despite these limitations, our study is based on a well-defined, representative cohort of HIV-related DLBCL, with comprehensive clinical information and measurement of a large number of tumor markers. To our knowledge, this study is also among the few that have examined the prognostic role of EBV status in HIV-related DLBCL.
In conclusion, we found that EBV infection status in DLBCL is associated with expression of several tumor markers that are involved in the NF-κB pathway. These factors were likely mediated by EBV and contribute to the EBV-related lymphomagenesis through activation of this pathway, as well as inhibition of apoptosis. Our results also suggest that tumor EBV status may have independent prognostic utility beyond conventional clinical prognostic scores such as the IPI, and may be used for risk stratification of patients diagnosed with HIV-related DLBCL.