EBV-associated Lymphoid Malignancies Research Articles

CircEAF2 counteracts Epstein-Barr virus-positive diffuse large B-cell lymphoma progression via miR-BART19-3p/APC/\u03b2-catenin axis

BackgroundEpstein-Barr virus (EBV) represents an important pathogenic factor of lymphoma and is significantly associated with poor clinical outcome of diffuse large B-cell lymphoma (DLBCL). Circular RNAs (circRNAs) play an essential role in lymphoma progression. However, the underlying mechanism of circRNA on DLBCL progression related to EBV remains largely unknown.MethodsCircRNA was screened by high-throughput sequencing in tumor samples of 12 patients with DLBCL according to EBV infection status. Expression of circEAF2, as well as the relationship with clinical characteristics and prognosis, were further analyzed in tumor samples of 100 DLBCL patients using quantitative real-time PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of circEAF2 both in vitro and in vivo. The underlying mechanism of circRNA on DLBCL progression were further determined by RNA sequencing, RNA pull down assay, dual-luciferase reporter assay, rescue experiments and western blotting.ResultsWe identified a novel circRNA circEAF2, which was downregulated in EBV + DLBCL and negatively correlated with EBV infection and DLBCL progression. In EBV-positive B lymphoma cells, circEAF2 overexpression induced lymphoma cell apoptosis and sensitized lymphoma cells to epirubicin. As mechanism of action, circEAF2 specifically targeted EBV-encoded miR-BART19-3p, upregulated APC, and suppressed downstream β-catenin expression, resulting in inactivation of Wnt signaling pathway and inhibition of EBV + DLBCL cell proliferation. In EBV-positive B-lymphoma murine models, xenografted tumors with circEAF2 overexpression presented decreased Ki-67 positivity, increased cell apoptosis and retarded tumor growth.ConclusionsCircEAF2 counteracted EBV + DLBCL progression via miR-BART19-3p/APC/β-catenin axis, referring circEAF2 as a potential prognostic biomarker. Therapeutic targeting EBV-encoded miRNA may be a promising strategy in treating EBV-associated lymphoid malignancies.

Genetic Perturbation of CD70/CD27 Co-Stimulation Promotes the Development of Bcl6-Driven Diffuse Large B-Cell Lymphoma

Multiple co-stimulatory and co-inhibitory pathways modulate T-cell dependent anti-tumor immune responses in lymphoid malignancies. We recently defined the recurrent genetic alterations and associated substructure of diffuse large B-cell lymphoma (DLBCL), including five distinct clusters (1-5), and identified potential genetic bases for immune evasion [Nature Medicine 2018; 24:679-690]. In our series, 26% of tumors had inactivating somatic mutations or copy loss of CD70 and likely disruption of CD70/CD27 co-stimulation.CD70 and CD27 are homotrimer type II and homodimer type I transmembrane proteins and members of the TNF and TNF receptor superfamilies, respectively. CD70 is transiently expressed on certain normal B-cell and dendritic cell populations upon activation and constitutively expressed on multiple B-cell tumors. CD70 activation of CD27 + T cells promotes antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Patients with germline deficiencies of either CD70 or CD27 have an increased incidence of EBV-associated lymphoid malignancies, underscoring the importance of this co-stimulatory pathway.In our series, CD70 alterations were most common in Cluster 1 DLBCLs, which also exhibited recurrent BCL6 chromosomal translocations. The co-occurrence of CD70 and BCL6 genetic alterations was noteworthy because of the established role of CD8 + T-cell dependent immune surveillance in murine models of Bcl6-driven DLBCL [Nature Medicine 2014; 20:283-290]. To assess the consequences of Cd70 deficiency and perturbed CD70/CD27 co-stimulation on Bcl6-driven lymphomagenesis, we crossed the previously described Bcl6tg/+ and Cd70-/- mice to generate Cd70-/-; Bcl6tg/+ animals. In the aging cohorts, Cd70-/-; Bcl6tg/+ mice were more likely than Bcl6tg/+ animals (or the Cd70-/- or wild-type [WT] groups) to be euthanized for symptoms before the study endpoint (18 months [mo]) (5 of 18 Cd70-/-; Bcl6tg/+ euthanized for symptoms prior to the first of 9 Bcl6tg/+). Additionally, significantly greater total numbers and percentages of Cd70-/-; Bcl6tg/+ mice, in comparison to Bcl6tg/+ or Cd70-/- animals, were euthanized for symptoms (64% Cd70-/-; Bcl6tg/+ vs. 29% Bcl6tg/+, p=0.005 and 64% Cd70-/-; Bcl6tg/+ vs. 11% Cd70-/-, p=0.002). Almost all euthanized Cd70-/; Bcl6tg/+ and Bcl6tg/+ micehad massively enlarged spleens infiltrated with histopathologically confirmed DLBCLs characterized by clonal Ig gene rearrangements. Our findings indicate that genetic perturbation of Cd70 accelerates the onset and significantly increases the incidence of Bcl6-driven DLBCL.To characterize potential differences in the anti-tumor immune responses in Cd70-/-; Bcl6tg/+ and Bcl6tg/+ mice (and Cd70-/- and WT controls), we also harvested spleens from 6 animals in each of the aging cohorts at predetermined intervals (2, 6, 14 and 18 mo). We analyzed the composition of splenic-cell suspensions by flow cytometry and evaluated the intact splenic architecture and morphology with expert hematopathologists (VS, KW and SR). At 14 and 18 mo, spleens from WT and Cd70-/- animals were largely normal in appearance and size. In contrast, spleens from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ mice exhibited disordered architecture and abnormal pre-malignant lymphoid proliferation with expanded white pulp including morphologically and immunophenotypically aberrant B cells of small to large size and increased infiltrating T-cells. Additionally, significantly higher fractions of splenic CD8 + T cells from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ animals expressed the CD69 activation marker and exhibited terminal differentiation, consistent with an ongoing anti-tumor immune response. Interestingly, Bcl6tg/+ animals had significantly higher percentages of splenic TCRβ + T cells at the earlier time point (14 mo) with delayed-onset splenomegaly (at 18 mo), which is in marked contrast to the Cd70-/-; Bcl6tg/+ mice that had significantly lower percentages of splenic TCRβ + T cells at the earlier time point (14 mo) and early-onset splenomegaly (14 mo). These findings suggest that the initial T-cell mediated immune response was more effective in Bcl6tg/+ than Cd70-/-; Bcl6tg/+ animals. Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective anti-tumor immune response in Bcl6tg/+-driven DLBCL. DisclosuresNeuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Rodig: Affimed: Research Funding; Bristol-Myers-Squibb: Research Funding; Merck: Research Funding; Immunitas: Membership on an entity's Board of Directors or advisory committees; KITE/Gilead: Research Funding. Shipp: Bristol Myers Squibb: Research Funding; Immunitas Therapeutics: Consultancy; AstraZeneca: Consultancy, Research Funding; Merck: Research Funding; Abbvie: Other: Institution: Research Grant/Funding; Bayer: Other: Institution: Research Grant/Funding.

Effects of targeting sumoylation processes during latent and induced Epstein-Barr virus infections using the small molecule inhibitor ML-792

As the second leading cause of death in the United States, cancer has a considerable impact on society, and one cellular process that is commonly dysregulated in many cancers is the post-translational modification of proteins by the Small Ubiquitin-like Modifier (SUMO; sumoylation). We documented that sumoylation processes are up-regulated in lymphoma tissues in the presence of Latent Membrane Protein-1 (LMP1), the principal oncoprotein of Epstein-Barr virus (EBV). LMP1-mediated dysregulation of cellular sumoylation processes contributes to oncogenesis, modulates innate immune responses, and aids the maintenance of viral latency. Manipulation of protein sumoylation has been proposed for anti-cancer and anti-viral therapies; however, known inhibitors of sumoylation do not only target sumoylation processes. Recently, a specific and selective small-molecule inhibitor of sumoylation (ML-792) was identified; however, nothing is known about the effect of ML-792 on LMP1-mediated dysregulation of cellular sumoylation or the EBV life-cycle. We hypothesized that ML-792 modulates viral replication and the oncogenic potential of EBV LMP1 by inhibiting protein sumoylation. Results showed that ML-792 inhibited sumoylation processes in multiple EBV-positive B cell lines and EBV-positive nasopharyngeal carcinoma cell lines but not in their EBV-negative counterparts. Focusing on its effect on B cells, ML-792 inhibited B-cell growth and promoted cell death at very low doses. ML-792 also modulated LMP1-induced cell migration and cell adhesion, which suggests the abrogation of the oncogenic potential of LMP1. Finally, while higher concentrations of ML-792 were sufficient to induce low levels EBV spontaneous reactivation, they decreased the production of new infectious virus following an induced reactivation and the infection of new cells, suggesting that ML-792 has anti-viral potential. Together, these findings suggest that ML-792 may be a potential therapeutic drug to treat EBV-associated lymphoid malignancies by targeting oncogenesis and the EBV life-cycle.

ABCL-283: A Phase I/II Trial of Brentuximab Vedotin (BV) Plus Rituximab (R) as Frontline Therapy for Patients with Immunosuppression-Associated CD30+ and/or EBV+ Lymphomas

Background: Chronic immunosuppression significantly increases the risk of developing a variety of lymphoid malignancies. Up to 80% of cases are driven by EBV-associated mechanisms of tumorigenesis. Approximately 70% will express CD30 and over 80% will express CD20. Standard-of-care rituximab (R) monotherapy followed by a response-stratified treatment approach shows poor response, with ∼75% patients requiring subsequent chemotherapy exposure; 2-year OS for the population as a whole was ∼70%. We hypothesized that a combination of brentuximab vedotin (BV) and R would yield improved breadth and depth of response compared with R monotherapy, would spare patients subsequent exposure to CIT, and result in favorable OS. Methods: Induction consisted of R 375 mg/m2 given days 1, 8, 15, and 22 and BV 1.2 mg/kg given days 1, 8, and 15 of a 28-day cycle, followed by restaging. Those with progression were removed from the study. Patients with stable disease were offered discontinuation or completion of one consolidation cycle followed by repeat assessment; consolidation was identical to induction. Patients with partial response or complete response could receive either consolidation followed by maintenance therapy (MT) or move directly to MT, which consisted of BV 1.8 mg/kg every 3 weeks and R 375 mg/m2 every 6 weeks for up to 1 year of therapy. Toxicity was defined using CTCAE 4.0, and response was assessed at the end of induction, consolidation (if given), and after cycles 4 and 7 of BV. Results: Toxicity and response data are available for 20 patients. Overall response rate was 70%, including a CR rate of 60%. Median follow-up of 26.1 months; 2-year PFS was 75%, and 2-year OS was 90%. Median time to best response was 28 days. Eighteen patients (92%) experienced toxicity at any grade with the most frequent grade 3/4 toxicities being peripheral neuropathy, neutropenia, infection, and pancreatitis. Conclusions: The combination of BV + R is effective in achieving rapid early remissions as frontline therapy for immunosuppression-related lymphomas. High toxicity rates suggest poor long-term tolerance and a different schedule or shorter treatment courses may be warranted. Further studies are needed to confirm these efficacy results and to determine optimal BV+R dosing regimens and duration.

A Phase I/II Trial of Brentuximab Vedotin (BV) Plus Rituximab (R) As Frontline Therapy for Patients with Immunosuppression-Associated CD30+ and/or EBV+ Lymphomas

A Phase I/II Trial of Brentuximab Vedotin (BV) Plus Rituximab (R) As Frontline Therapy for Patients with Immunosuppression-Associated CD30+ and/or EBV+ Lymphomas

Using glycyrrhizic acid to target sumoylation processes during Epstein-Barr virus latency.

Cellular sumoylation processes are proposed targets for anti-viral and anti-cancer therapies. We reported that Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) dysregulates cellular sumoylation processes, contributing to its oncogenic potential in EBV-associated malignancies. Ginkgolic acid and anacardic acid, known inhibitors of sumoylation, inhibit LMP1-induced protein sumoylation; however, both drugs have adverse effects in hosts. Here we test the effects of glycyrrhizic acid, a medicinal botanical extract with anti-inflammatory, anti-carcinogenic, and anti-viral properties, on cellular sumoylation processes. While glycyrrhizic acid is known to inhibit EBV penetration, its affect on cellular sumoylation processes remains to be documented. We hypothesized that glycyrrhizic acid inhibits cellular sumoylation processes and may be a viable treatment for Epstein-Barr virus-associated malignancies. Results showed that glycyrrhizic acid inhibited sumoylation processes (without affecting ubiquitination processes), limited cell growth, and induced apoptosis in multiple cell lines. Similar to ginkgolic acid; glycyrrhizic acid targeted the first step of the sumoylation process and resulted in low levels of spontaneous EBV reactivation. Glycyrrhizic acid did not affect induced reactivation of the virus, but the presence of the extract did reduce the ability of the produced virus to infect additional cells. Therefore, we propose that glycyrrhizic acid may be a potential therapeutic drug to augment the treatment of EBV-associated lymphoid malignancies.

MiR155 sensitized B-lymphoma cells to anti-PD-L1 antibody via PD-1/PD-L1-mediated lymphoma cell interaction with CD8+T cells

BackgroundMicroRNAs (miRs) are involved in lymphoma progression by regulating tumor cell interaction with microenvironment. MiR155 is overexpressed in diffuse large B-cell lymphoma (DLBCL) and its biological effect on tumor microenvironment needs to be futher investigated.MethodsMiR155 was detected by quantitative real-time PCR in patients with newly diagnosed DLBCL. The mechanism of action of miR155 on lymphoma progression and tumor microenvironment was examined in vitro in B-lymphoma cell lines and in vivo in a murine xenograft model.ResultsSerum miR155 was significantly elevated, correlated with tumor miR155 expression, and indicated poor disease outcome in DLBCL. MiR155 overexpression was associated with decreased peripheral blood CD8+T cells and inhibition of T-cell receptor signaling. Of note, EBV-positive patients showed higher serum miR155 than EBV-negative patients. In co-culture systems of B-lymphoma cells with immune cells, miR155 induced Fas-mediated apoptosis of CD8+T cells, which could be targeted by anti-PD-1 and anti-PD-L1 antibodies. Moreover, miR155 enhanced lymphoma cell PD-L1 expression, recruited CD8+T cells by PD-1/PD-L1 interaction and inhibited CD8+T cell function via dephosphorylating AKT and ERK. MiR155-induced AKT/ERK inactivation was more obvious in CD8+T cells co-cultured with EBV-infected B-lymphoma cells. In vivo in a murine xenograft model established with subcutaneous injection of A20 cells, PD-L1 blockade particularly retarded miR155-overexpressing tumor growth, consistent with maintenance of CD8+T cells and their function.ConclusionsAs a oncogenic biomarker of B-cell lymphoma, serum miR155 was related to lymphoma progression through modulating PD-1/PD-L1-mediated interaction with CD8+T cells of tumor microenvironment, indicating the sensitivity of B-cell lymphoma to PD-L1 blockade. Also CD8+T cells could be a therapeutic mediator of immune checkpoint inhibitors in treating EBV-associated lymphoid malignancies.

EBV Viral Loads in Diagnosis, Monitoring, and Response Assessment.

The quantification of circulating Epstein Barr virus (EBV) DNA loads has played an important role in the diagnosis and management of EBV-associated lymphoid malignancies. Viral load measurement is particularly useful for monitoring EBV-DNA in hematopoietic stem cell transplant patients, and for assessing the prognosis or response to therapy of EBV-associated intractable lymphomas like extranodal NK/T-cell lymphoma, nasal type. Cell-free EBV-DNA in plasma can be used as a biomarker for estimating the severity or prognosis of these lymphomas. In addition to plasma, whole blood has been used for the management of transplant patients. Although measuring EBV-DNA has been useful, there is a lack of standardization and the optimal specimens for measuring viral loads are unknown. This can be attributed to the different forms of EBV-DNA that exist in peripheral blood and the different pathologies that result from diverse EBV disease states. As a result, guidelines for EBV diagnosis or the initiation of treatment are unclear. However, the newly established World Health Organization standard for EBV quantification will encourage collaborative studies across institutions and countries to establish proper guidelines for EBV diagnosis and the initiation of treatment.

Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers Using Proteasome Inhibitors.

Epstein-Barr virus (EBV) is closely associated with several lymphomas (endemic Burkitt lymphoma, Hodgkin lymphoma and nasal NK/T-cell lymphoma) and epithelial cancers (nasopharyngeal carcinoma and gastric carcinoma). To maintain its persistence in the host cells, the virus manipulates the ubiquitin-proteasome system to regulate viral lytic reactivation, modify cell cycle checkpoints, prevent apoptosis and evade immune surveillance. In this review, we aim to provide an overview of the mechanisms by which the virus manipulates the ubiquitin-proteasome system in EBV-associated lymphoid and epithelial malignancies, to evaluate the efficacy of proteasome inhibitors on the treatment of these cancers and discuss potential novel viral-targeted treatment strategies against the EBV-associated cancers.

The Possible Involvement of Epstein -Barr virus In the Etiology of Leukemia

Background: Although considerable brainstorm has been known into Epstein-Barr virus EBV as an important etiologic factor in various tumors, virtually little is known about the relationship between EBV genes and leukemia. The actual cause of Leukemia, which is a serious cancer in Sudan, is still under scrutiny. Controversial hypotheses were proposed suggesting the role of physical as well as chemical and even biological factors as being responsible for Leukemia incidents. We hypothesized that EBV could be involved in the etiology of leukemia. We describe here the results of our attempt to find a possible link between leukemia and EBV. Methods: Real-time Polymerase Chain Reaction assay (q-PCR) has recently been used widely for detection of Cell-free EBVDNA in the plasma of patients with leukemia. To determine the possible correlation between plasma cell-free EBV DNA levels and the leukemia, we studied the plasma EBV DNA levels in patients with leukemia that were presented at Radiation and Isotope Centre Khartoum (RICK), Sudan during therapy. The concentrations of plasma cell-free EBV DNA of 128 leukemic patients, 17 patients with EBV-associated lymphoid malignancies during the course of therapy Burkitt's lymphoma, Hodgkin's disease, Nasopharyngeal carcinoma (NPC) as control positive and 15 healthy controls were determined by using the (q-PCR) assay with the PrimerDesign™ genesig quantification kit. Results: The results revealed that EBV DNA was detectable in a wide range of leukemia patients. Plasma EBV DNA was detected in 33/88 Acute lymphoblastic leukemia (ALL) patients 28/40 Chronic myelogenous leukemia (CML),15/17 patients with EBV-associated lymphoid malignancies, but not in any of 15 healthy control subjects. The median concentration of EBV DNA in leukemia and healthy control groups was 6561.00 and 0.00 copies/ml, respectively. Conclusion: Our findings provided evidence of the involvement of EBV in patients with leukemia. The results suggested that EBV DNA genome encoding the non-glycosylated membrane protein BNRF1 pl43 was observed in a significant proportion of patients with ALL. However, we could not exclude a correlation between these viral infections and later leukemogenesis in childhood ALL in Sudan.

IS8-1 - Current Understanding of the Role of Epstein-Barr Virus in Lymphomagenesis

Ubiquitous EBV infects not only B cells but also T and NK cells, and is associated with various lymphoid malignancies. Since EBV was first isolated from Burkitt lymphoma in 1964, the spectrum of EBV-associated lymphoid malignancies is expanding from Burkitt lymphoma to EBV-positive T- and NK-cell lymphoproliferative disease/lymphoma. The extent of the association to EBV varies with each disease. Some are 100% associated, but others are associated only in 1/4 to 1/3 of patients. This fact suggests that EBV is not absolutely necessary for the establishment of a disease or for its maintenance. Although EBV can immortalize B cells in vitro, its in vivo role in lymphomagenesis has not been fully clarified. Also, its role in the lymphomagenesis of T or NK cells is unclear. Interestingly, a variety of populations are at high risk for EBV-associated malignancies. This suggests that genetic backgrounds are related with the pathogenesis of these diseases, although one cannot rule out the possibility that specific EBV strains are prevalent in certain areas. In this symposium, I summarize the lymphomagenesis and clinical features of EBV-associated lymphoid malignancies.

The interplay between Epstein–Barr virus and B lymphocytes: implications for infection, immunity, and disease

Human B cells are the primary targets of Epstein-Barr virus (EBV) infection. In most cases, EBV infection is asymptomatic because of a highly effective host immune response, but some individuals develop self-limiting infectious mononucleosis, while others develop EBV-associated lymphoid or epithelial malignancies. The viral and immune factors that determine the outcome of infection are not understood. The EBV life cycle includes a lytic phase, culminating in the production of new viral particles, and a latent phase, during which the virus remains largely silent for the lifetime of the host in memory B cells. Thus, in healthy individuals, there is a tightly orchestrated interplay between EBV and the host that allows the virus to persist. To promote viral persistence, EBV has evolved a variety of strategies to modulate the host immune response including inhibition of immune cell function, blunting of apoptotic pathways, and interfering with antigen processing and presentation pathways. In this article, we focus on mechanisms by which dysregulation of the host B cell and immune modulation by the virus can contribute to development of EBV+ B cell lymphomas.

Abstract MS01-2: Artificial receptors and the cellular therapy of breast cancer

Abstract It is clear that targeting therapy to the specific molecular lesions of breast cancer affords the best opportunity to obtain effective treatment without adverse effects on normal tissues. The immune system remains one of the most effective targeting systems known, and tumor-directed monoclonal antibodies have already shown significant success in treating this and other malignant disorders. Cellular therapies based on T lymphocytes, by contrast, have not yet made an equivalent impact, even though T cell-based immune responses to tumor have many unique characteristics that should make them highly effective. In this presentation I will discuss one historic success story for cellular immunotherapy -the treatment of EBV-associated lymphoid and epithelial malignancies by tumor specific T cells -and show how the lessons learned from analyzing the mechanisms underlying this success can be implemented to treat other solid tumors including breast cancer Four components are required for effective activity;-. 1) The infused T cells must target strong and unique antigens, presented with ample accessory signals and co-stimulation; 2) The T cells should contain a polyclonal and multispecific memory population directed to tumor; 3) The T cells must be able to overcome potent immune escape mechanisms 4) The environment should favor (homeostatic) lymphoid expansion. We and others are developing a systematic approach to meet these requirements by introducing novel artificial receptors (Chimeric Antigen Receptors – CARs) and other cell engineering components into T lymphocytes. As an increasing level of success becomes documented in hematological malignancy and solid tumors, we can be hopeful that these therapies will become part of the standard of care for breast cancer as well. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr MS01-2.

Epstein-Barr virus-associated lymphoid malignancies: the expanding spectrum of hematopoietic neoplasms.

Ubiquitous Epstein-Barr virus (EBV) infects not only B cells but also T and NK cells, and is associated with various lymphoid malignancies. The spectrum of EBV-associated lymphoid malignancies is expanding from Burkitt lymphoma to the newly defined systemic EBV+ T cell lymphoproliferative disease of childhood and hydroa vacciniforme-like lymphoma. However, some EBV-associated malignancies are not defined well and overlap other diseases. Furthermore, the role of EBV in tumorigenesis of lymphoid malignancies is still not clear. Further studies are necessary to clarify the pathogenesis of EBV-associated lymphoid malignancies for a better classification of each disease and for the establishment of effective treatment.

Short-Term Exposure to Arginine Butyrate, in Combination with Ganciclovir, Is as Effective as Continuous Exposure for Virus-Targeted Therapy of EBV-Positive Lymphomas.

Abstract 4754Antiviral drugs alone have been unsuccessful in the treatment of Epstein-Barr virus (EBV)-associated malignancies because the virus maintains a latent state of replication in these tumors. In recent years, we developed a novel therapeutic approach wherein the early lytic phase of the virus is induced prior to the use of cytotoxic antiviral drugs. Butyrate derivatives induce an early lytic pattern of EBV gene expression in patient-derived EBV-positive lymphoblastoid cell lines and, together with the nucleoside analog ganciclovir (GCV), effectively reduce or eliminate tumor growth in humans. In a completed Phase 1/2 trial in which patients with relapsed or refractory EBV-associated lymphoid malignancies were treated with 3 weeks of Arginine Butyrate combined with GCV, tumor responses (CR and PR) occurred in 10 of 15 patients. Butyrate requires administration by prolonged intravenous infusion, which is challenging over long time periods. In the Phase 1/2 clinical trial, tumor responses were observed within a few days of starting therapy. We therefore investigated whether brief or discontinuous exposure to Butyrate is also capable of initiating early lytic-phase gene expression and thymidine kinase induction, and sensitizing EBV-positive lymphoma cells to ganciclovir-mediated cell growth arrest and apoptosis. Multiple daily 6-hr exposures of the EBV-positive Burkitt's lymphoma cell line P3HR1 to butyrate induced sustained expression of the EBV TK and lytic-phase protein BMRF. Discontinuous exposure to butyrate in combination with ganciclovir also induced a similar level of tumor cell death as did continuous treatment, as measured by serial enumeration of viable cells, MTT cell proliferation assays, and measurement of cellular DNA content. Based on these observations, we have initiated a new clinical trial utilizing a 5-day infusion of Arginine Butyrate and 21 days of GCV/valganciclovir for treatment of patients with EBV-positive lymphoid malignancies. The first patient enrolled, with Rituximab-refractory EBV-positive PTLD following a cord stem cell transplant for Hodgkin's Disease, has been treated on this protocol. The therapy was well-tolerated and resulted in rapid resolution of fever and cough of several weeks duration, and a rapid decrease of markedly elevated LDH levels to the normal range. At the end of the first cycle, 4 of 6 target lesions resolved completely, and two additional lesions decreased in size. High EBV, CMV and HHV6 viral loads became undetectable. This response has been durable for 2 months. These findings together suggest that a shorter, more patient-accessible regimen of this virus-targeted therapeutic strategy may be efficacious, and the clinical trial is continuing. Disclosures:Faller:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Off Label Use: The use of Ganciclovir, administered in combination with Arginine Butyrate as an inducer of viral TK,to induce apoptosis in EBV-lymphoma and EBV lymphoproliferative disease. Ghosh:HemaQuest Pharmaceuticals: Research Funding. Lerner:HemaQuest Pharmaceuticals: Consultancy. Berenson:HemaQuest Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Perrine:HemaQuest Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

EBV LMP2A affects LMP1-mediated NF-κB signaling and survival of lymphoma cells by regulating TRAF2 expression

AbstractA mechanism used by Epstein-Barr virus (EBV) for in vitro transformation of B cells into lymphoblastoid cell lines (LCLs) is activation of the NF-κB pathway, which is largely mediated by the EBV latent membrane protein 1 (LMP1). LMP1 is coexpressed with LMP2A in many EBV-associated lymphoid malignancies. Since inhibition of NF-κB leads to apoptosis of EBV-infected LCLs and lymphoma cell lines, we sought to determine whether LMP1 alone, or in combination with other viral proteins, is responsible for initiating NF-κB activation in these cells, thereby playing a role in cell survival. We found that suppression of LMP1 by RNA interference results in inhibition of basal NF-κB and induction of apoptosis. Unexpectedly, knockdown of LMP2A also resulted in comparable decrease of NF-κB activity and apoptosis. We report that LMP2A protein controls the expression of TRAF2 mRNA, which in turn is necessary for signaling by LMP1. Our data contrast with previous studies showing that transfected LMP1 can signal in the absence of LMP2A or TRAF2, and demonstrate that both LMP2A and TRAF2 are required for survival in naturally infected lymphoma cells and LCLs. These results also support LMP1, LMP2A, and TRAF2 as potential therapeutic targets in a subset of EBV-associated lymphoid malignancies.

EBV-Associated Tumors: Pathogenetic Insights for Improved Disease Monitoring and Treatment

Epstein-Barr virus (EBV) is a ubiquitous γ-herpes virus that is a well-established risk factor for Hodgkins lymphoma, Burkitts lymphoma, a subset of T cell lymphomas and various tumors of epithelial cell origin with nasopharyngeal carcinoma as the most representative. Furthermore, EBV-associated lymphoproliferative disorders are a well recognized major complication of immune suppression. EBV generally establishes a life-long asymptomatic infection in memory B lymphocytes, by mimicking cellular signaling pathways that regulate antigen-dependent B cell differentiation. In the long-lived memory B lymphocyte pool, the virus is almost completely silent or expresses a very restricted set of latent proteins, evading thus the immune control by EBV-specific effectors. EBV-associated lymphoid and epithelial malignancies are characterized by a peculiar geographic distribution and distinctive epidemiologic and histopathologic and histogenetic features. Moreover, new histogenetic and molecular features have contributed to the recognition and the diagnosis of specific disease categories. The ability of the virus to establish life-long persistent infections in humans and to contribute to cell transformation is related to the biologic properties of a set of EBV-encoded proteins, differently expressed in both normal and transformed cells. In the last decade, our understanding of the latency programs activated by EBV in cellular backgrounds and the function of EBV-encoded proteins has considerably improved. The emerging picture indicates that EBV proteins are able to hijack critical cellular pathways to promote the proliferation and survival of infected cells, while impairing anti-viral immune responses. The recent advances in the elucidation of the mechanisms underlying EBV-induced cell transformation and immune evasion help to design novel treatment approaches for EBV-related malignancies. Moreover, it has been clearly shown that the quantification of circulating EBV DNA is a valuable tool in the diagnosis, clinical monitoring and prognosis of some EBV-associated tumors. Keywords: epstein-barr virus, lymphoma, histogenesis, dna load, immune suppression, aids

Infectious mononucleosis and Epstein-Barr virus

Epstein-Barr virus (EBV) is a gamma-herpesvirus that infects over 90% of the human population worldwide. It is usually transmitted between individuals in saliva, and establishes replicative infection within the oropharynx as well as life-long latent infection of B cells. Primary EBV infection generally occurs during early childhood and is asymptomatic. If delayed until adolescence or later, it can be associated with the clinical syndrome of infectious mononucleosis (also known as glandular fever or 'mono'), an illness characterised by fevers, pharyngitis, lymphadenopathy and malaise. EBV infection is also associated with the development of EBV-associated lymphoid or epithelial cell malignancies in a small proportion of individuals. This review focuses on primary EBV infection in individuals suffering from infectious mononucleosis. It discusses the mechanism by which EBV establishes infection within its human host and the primary immune response that it elicits. It describes the spectrum of clinical disease that can accompany primary infection and summarises studies that are leading to the development of a vaccine designed to prevent infectious mononucleosis.

Circulating Cell‐Free Epstein‐Barr Virus DNA Levels in Patients with EBV‐Associated Lymphoid Malignancies

Epstein-Barr virus (EBV) is a human lymphotrophic herpesvirus implicated in the development of several lymphoid malignancies. Recently, detection of cell-free EBV DNA in sera or plasma from patients with EBV-associated malignancies has been reported. However, most published studies are qualitative in nature, with only a few providing quantitative data. We have described the temporal changes of plasma EBV DNA levels in patients with EBV-associated lymphoid malignancies during therapy using quantitative real-time PCR. A close correlation between plasma EBV DNA levels and therapeutic response was observed. Our aim was to update our results and to assess the role of the plasma EBV DNA levels as a molecular marker for disease monitoring in patients with EBV-associated lymphoid malignancies.

Quantitative analysis of circulating cell-free Epstein-Barr virus (EBV) DNA levels in patients with EBV-associated lymphoid malignancies.

Cell-free Epstein-Barr virus (EBV) DNA has recently been detected in the plasma and serum of patients with Hodgkin's disease, post-transplant lymphoproliferative disease (PTLD) and acquired immunodeficiency syndrome-related lymphoma. However, no data are available on the temporal variation of plasma/serum EBV DNA levels in patients with EBV-associated lymphoid malignancies during the course of therapy. Using a real-time quantitative polymerase chain reaction assay, we studied the plasma EBV DNA levels in 13 patients with EBV-associated lymphoid malignancies (six patients with Hodgkin's disease, four with nasal natural killer/T-cell lymphoma, two cases of PTLD and one patient with Burkitt's lymphoma) at presentation and during therapy. Plasma EBV DNA was detected in 12 of the 13 patients (median 2,266 copies/ml; interquartile range 181-8,379 copies/ml), but not in any of 35 healthy control subjects (P < 0.0001). The EBV status in tumour cells was also examined in 12 of these patients using in situ hybridization for EBV-encoded small RNAs (EBERs). EBER positivity was observed in 11 patients, all of whom had EBV DNA detectable in plasma. The one patient who had no detectable plasma EBV DNA was also negative for EBERs in tumour tissue. Serial measurements of plasma EBV DNA levels were performed in nine of the patients during the course of therapy. All patients who responded to therapy demonstrated a significant reduction of plasma EBV DNA to low or undetectable levels, whereas in two patients with ineffective therapy, disease progression was associated with a rapid increase in plasma EBV DNA levels. We concluded that plasma EBV DNA is detectable in a wide range of EBV-associated lymphoid malignancies. As plasma EBV DNA levels correlate well with the therapeutic response, such analysis may be a valuable tool for monitoring clinical progress.