Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas

Abstract

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. A predominant type-A EBV variant and a LMP1 30 bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer.

Contribution of the Epstein-Barr virus to the oncogenesis of mature T-cell lymphoproliferative neoplasms

“…In consonance, EBV-infected T cells demonstrate in vitro a potential oncogenic distinct from those observed in LCLs, including different viral gene expression profile ( 111 ). Furthermore, previous studies in vitro demonstrated that T cells are possibly more permissive for the expression of immediate early viral genes than EBV-infected B cells ( 112 , 113 ), with some evidence of this in EBV-associated neoplasia of T cell origin ( 114 ).…”

“…In a small cohort of patients from Hong Kong, it was demonstrated that all cases presented the del30 of LMP1 and the majority of cases carried type 1 EBV ( 114 ). Results like this need to be interpreted with caution.…”

“…EBV is also present in virtually all cases of NKTCL and should be detected by EBER-ISH ( 11 ). The EBV latency pattern in this lymphoma is not firmly established and only few studies have dealt with this, using only molecular techniques (as RT-PCR) ( 89 , 114 ).…”

“…Apparently, the majority of cases displays latency II with expression of EBNA1 , BART , LMP1 , LMP2A and LMP2B ( 89 , 114 ). A small proportion of cases exhibits latency III with additional expression of EBNA2 ( 89 , 114 ).…”

“…Apparently, the majority of cases displays latency II with expression of EBNA1 , BART , LMP1 , LMP2A and LMP2B ( 89 , 114 ). A small proportion of cases exhibits latency III with additional expression of EBNA2 ( 89 , 114 ). One study demonstrated that the expression of the early lytic genes BZLF1 was not accompanied by the expression of the late lytic genes BHRF1 and BLLF1 ( 114 ), suggesting an abortive lytic cycle.…”