EBV-associated Neoplasms Research Articles

Disparities in the Levels of Whole-Blood Epstein-Barr Virus between the Cancer and Non-Cancer Populations in Zhejiang, China

ObjectiveThis study aimed to investigate the prevalence of Epstein-Barr virus (EBV) infection in patients with and without cancer. MethodsA total of 26,648 participants who underwent whole-blood EBV DNA (WBEBV) assays between January 1, 2020, and August 31, 2023, were included. The chi-square test was used for categorical data analysis, and R software was used to analyze the differences in EBV DNA load levels and the diagnostic capabilities of WBEBV. ResultsPositive rates were 10.2% and 25.4% for healthy controls (HC) and patients, respectively. The positivity rate for EBV-associated neoplasms (EN) was the highest at 7.53%, followed by leukemia (Le) at 5.49%. The subgroup analysis showed that the positivity rate for abnormal proliferation or hyperplasia (APH) was 31.9%, followed by 30.5% for Le. The WBEBV of patients with transplants (TP), especially living-related transplants (LT), was the highest among all subgroups. WBEBV at diagnosis was used to differentiate between infectious mononucleosis (IM) and chronic active Epstein-Barr virus (CAEBV), with a sensitivity of 67.4% (95% confidence interval [CI]: 57.6–75.8) and specificity of 72% (95% CI: 63.3–79.3). We conclude that the prevalence of EBV infection is low in the healthy population in this region and that a high EBV load at baseline is more common in LT, IM, and Lymphocyte Leukemia (LL). ConclusionThis study used a large-sample survey to characterize the prevalence of whole-blood EBV levels in various diseases, including the stages and subtypes. The EBV detection rate was higher in patients with malignant disease than in those with benign disease. Our study provides clinicians with baseline information regarding EBV-associated diseases.

Ksd-101 in Patients with EBV-Associated Hematologic Neoplasms: Results from an Ongoing Phase I Clinical Study

Background: The Epstein-Barr Virus (EBV), which has been confirmed by WHO as the first human tumor-associated virus, is a lifelong carrier once infected. EBV-associated Hematologic Neoplasms seriously threats human health, with rapid disease progression, poor prognosis, easy to relapse, and high mortality rate. KSD-101 is a First-in-class autologous dendritic cell (DC) vaccine which is developed to control EBV-associated hematological diseases. Autologous DCs were loaded with the EBV-transformed B-LCL (B-Lymphoblastoid Cell Line) lysates which contains a broad-spectrum EBV antigen to prepare KSD-101 for the treatment of EBV-associated diseases. Methods: In this study, patients (pts) with EBV-associated Hematologic Neoplasms who fail to respond to or relapse after conventional treatment will be recruited and undergo leukapheresis to collect autologous peripheral blood mononuclear cell (PBMC). The enriched monocytes will be induced to differentiate into DCs and prepared as KSD-101 after loading with EBV-transformed B-LCL. Pts will undergo monotherapy with KSD-101, without the need for pre-treatment of lymphodepletion or prophylactic medication. Administration route of KSD-101 is subcutaneous injection, once every 2 weeks for 3-5 vaccinations in total. The study used a 3+3 dose-escalation design. Safety assessment is conducted according to NCI-CTCAE 5.0 grading criteria. The primary objective is to assess the tolerability and safety of KSD-101 in the treatment of EBV-associated Hematologic Neoplasms, explore the dose-limiting toxicities (DLT) and the maximum tolerated dose (MTD). The secondary objective is to explore the clinical efficacy and immune responses of KSD-101 in the patients with EBV-associated Hematologic Neoplasms. Results: As of July 2023, 9pts were enrolled in the study and 8 pts DC vaccine preparations were completed. Three pts were injected 5.0 × 10 6 cells/dose without exploring any DLT. The first two pts in the second dose level were injected 7.5 × 10 6 cells/dose. However, the third pt in second dose level was injected 5.0 × 10 6 cells/dose due to insufficient apheresis collection. Based on the safety and preliminary efficacy data, we decided not to proceed with dose-escalation to the third dose group. In the expansion phase, all pts were injected with a dose of 5.0 × 10 6 cells. Currently observed KSD-101-related toxicities were pyrexia (grade ≤ 2), injection site reactions (grade 1), and lymphadenopathy (grade 1). In all the pts who were injected, neither DLT nor MTD were explored. Based on the comprehensive considerations of safety, efficacy and easibility, we propose that the most appropriate dose is 5.0 × 10 6 cells. Of all pts enrolled, one was excluded due to hemophagocytic lymphohistiocytosis (HLH), among whom 4 pts completed the 12-week follow-up after the first injection for clinical and laboratory assessments. The results showed an overall response rate (ORR):100%, complete response (CR):100%, including 1 case of AITL (EBV infected T, B, NK),1case of CAEBV (EBV infected T, NK),2cases of NK-T cell lymphoma (1 EBV infected B, NK, and the other EBV infected NK). After vaccinations, the total number of lymphocytes of these four pts increased by 1.5 to3.4 times, including 1.6 to 4.2 times of T cells, 2.1 to 6.5 times of activated T cells (CD3 + HLA -DR +), 1.4 to 5.0 times of CD8 + T cells, 1.2 to 3.7 times of CD4 + T cells, 1.7 to 2.0 times of NK cells, and 1.2 to 32.4 times of B cells; meanwhile, the proportion of EBV-specific CTLs increased significantly (ranging from a 2.51% to 4.16% increase), and the proportion of Tregs significantly decreased compared with the baseline (ranging from a 32.49% to 73.81% reduction). Conclusions：Our current study shows that KSD-101 has excellent safety and good preliminary efficacy against EBV-associated Hematologic Neoplasms with efficient induction of immune responses. The results indicate that KSD-101 is a promising therapeutic reagent for the treatment of EBV-associated Hematologic Neoplasms. Larger prospective studies and longer follow-up time is needed to be conducted.

Epstein-Barr virus in Adygeans and Slavs in Russia: virus types, <i>LMP1</i> variants, and malignant tumors

Introduction. It is known that the structural features of the Epstein-Barr virus (EBV) affect the manifestation of its biological properties. Based on differences in the sequences of the EBNA2, EBNA3A, -B, and -C genes, two types of the virus, EBV-1 and EBV-2, have been identified that have different ability to transform B cells in vitro and possibly playing certain role in the development of EBV-associated neoplasms.Aim. To study the prevalence of EBV-1 and EBV-2 in two ethnic groups, Аdygeans and Slavs, as well as the contribution of EBV-associated tumors to the overall incidence of malignant neoplasms certain organs and tissues.Materials and methods. DNA samples were extracted from 59 oral lavages of ethnic Аdygeans from Republic of Adygea and 40 such from oral cavity of ethnic Slavs of Moscow city. These samples were used for amplification of EBV DNA, determination of the concentration of viral DNA copies per 1 cell washout, as well as for amplification of EBV LMP1 followed by sequencing of the resulting gene samples and determination of their protein variant (LMP1).Results. Studies have shown that among the representatives of the Аdygeans the 2nd EBV type prevails, and among the Slavs, the 1st one. Epstein-Barr virus isolates in representatives of the two ethnic groups also differed in the structure of LMP1. Among the Slavs, a set of its LMP1 variants (B95.8/A, China, Med- and NC) was identified. However, among the Adygeans, the only variant - B95.8 and its subtype - B95.8/A was identified. EBV-1, which prevails among the representatives of the Slavs and has the ability to transform B-cells, was projected onto a higher incidence of tumors of the pharynx, stomach, Hodgkin's and non-Hodgkin's lymphomas (where EBV-associated cases cam occur) in the population of Moscow than in the population of the Republic of Adygea. However, the differences between incidence rates for these neoplasms (with the exception for the stomach tumors) were not statistically significant (p >0.5). A higher and statistically significantly different incidence rate of stomach cancer in residents of Moscow city, compared with that in residents of the Republic of Adygea, in our opinion, is not due to EBV-1 type and/or LMP1 variants, but rather is associated with a genetic predisposition the population of Moscow city to this tumor.Conclusion. The fact that two ethnic groups of Russia were found to be prevails by different types of EBV raises the question of their ethno-geographical association and their role in the induction of EBV-associated tumors. To resolve this issue additional studies in other geographical regions of Russia among representatives of different ethnic groups are required.

EBV-associated NK and T-cell lymphoid neoplasms.

Epstein-Barr virus (EBV)-associated neoplasms derived from natural killer (NK) or T cells comprise a group of clinically and biologically heterogenous disorders affecting children and adults, which are overall rare but more prevalent in Asia and South America. This review focuses on neoplasms presenting in the adulthood, addressing recent genomic discoveries as well as therapeutic developments in these highly aggressive disorders. Distinct molecular subtypes of extranodal NK/T-cell lymphomas (ENKTCLs) have been described, with differences in cell of origin, EBV pattern, genomic alterations, clinical characteristics, response to asparaginase-based therapies and to more recent approaches targeting molecular aberrations of the lymphoma. For the last two decades, progress in the clinical management of ENKTCL was based on L-asapraginase containing combinations and the incoroperation of radiotherapy. A subset of cases with PDL1-2 structural alterations may be more responsive to treatment with immune checkpoint inhibitors. Primary nodal EBV+ lymphomas derived from T or NK cells have distinctive features separating them from both peripheral T-cell lymphoma not otherwise specified and ENKTCL. Treatment algorithms correspond to those for advanced ENKTCL. With better understanding of lymphomagenesis, genomic landscape and immunologic aspects of the diseases, future treatment options will include targeted therapies including immune checkpoint inhibitors and novel antibodies.

Comparison of Multiplex Real-Time PCR and Droplet Digital PCR for Detection of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma

Plasma Epstein-Barr Virus (EBV) DNA is an established biomarker for endemic nasopharyngeal carcinoma (NPC) and EBV-associated lymphoproliferative disorders. The ongoing NRG-HN001 randomized trial is testing one biomarker-directed treatment approach using real-time PCR (qPCR). Existing qPCR assays are limited by inter-laboratory imprecision secondary to external calibrators, and development of a standardized assay would facilitate reproducible quantitation. We hypothesized that droplet digital PCR (ddPCR) would have similar analytical sensitivity, precision, and accuracy relative to qPCR.We designed a multiplex ddPCR assay to detect two single-copy EBV targets (EBNA-1, EBER), a variable-repeat EBV target (BamHI-W), and a human internal control (RNaseP) in plasma across two reactions. This was compared against the NRG-HN001 qPCR assay, which detects EBNA-1, BamHI-W, and an internal control across two reactions. Quantitation for the ddPCR assay was based on Poisson statistics and automated threshold determination without external calibrators. Quantitation for the qPCR assay was based on two external calibrators traceable to the WHO EBV standard. Assays were compared by measuring sensitivity, specificity, 95% lower limit of detection (LLOD), lower limit of quantitation (LLOQ), accuracy (difference from nominal concentration), and precision (reproducibility). The WHO EBV standard was used as control material. Clinical specimens sent for routine plasma EBV testing were collected from patients with EBV-associated neoplasms. For clinical specimens, a composite reference standard was defined as positivity by at least two targets.The 95% LLOD for BamHI-W, EBNA-1, and EBER ddPCR were 1.49, 13.26, and 9.77 international units (IU) per well, respectively. Analytical sensitivity was not significantly different than BamHI-W qPCR (1.75 IU/well, P = 0.99) or EBNA-1 qPCR (7.58 IU/well, P = 0.60). The LLOQ for both assays was 100 IU/mL. Across the range of prognostic cutoffs for plasma EBV DNA copy number (1,500-40,000 IU/mL), within-laboratory precision was highest with BamHI-W qPCR (0.04-0.06) and ddPCR (0.04-0.08) and lower with EBNA-1 qPCR (0.04-0.14). Across this range, ddPCR had modestly improved accuracy (absolute mean difference from nominal, BamHI-W: 1104 IU/mL; EBER: 1206 IU/mL) relative to qPCR (BamHI-W: 1565 IU/mL; EBNA-1: 1874 IU/mL, P = 0.77 across platforms). Sensitivity (100% vs. 95%, P = 0.50) and specificity (100% vs. 100%) were similar between BamHI-W ddPCR and BamHI-W qPCR for clinical specimens. Sensitivity was lower for all single-copy targets (80-85%).A multiplex ddPCR amplifying single- and variable-copy EBV targets in plasma offers similar analytical performance as the existing NRG-HN001 qPCR assay without the need for external calibrators. Further research will inform whether inter-laboratory precision is superior with ddPCR, which may facilitate standardization of EBV DNA quantitation.

The role of EBV in haematolymphoid proliferations: emerging concepts relevant to diagnosis and treatment.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus with >90% of the adult population worldwide harbouring latent infection. A small subset of those infected develop EBV-associated neoplasms, including a range of lymphoproliferative disorders (LPD). The diagnostic distinction of these entities appears increasingly relevant as our understanding of EBV-host interactions and mechanisms of EBV-driven lymphomagenesis improves. EBV may lower the mutational threshold for malignant transformation, create potential vulnerabilities related to viral alteration of cell metabolism and allow for improved immune targeting. However, these tumours may escape immune surveillance by affecting their immune microenvironment, limiting viral gene expression or potential loss of the viral episome. Methods to manipulate the latency state of the virus to enhance immunogenicity are emerging as well as the potential to detect so-called 'hit and run' cases where EBV has been lost. Finally, measurement of EBV DNA remains an important biomarker for screening and monitoring of LPD. Methods to distinguish EBV DNA derived from virions during lytic activation from latent, methylated EBV DNA present in EBV-associated neoplasms may broaden the utility of this testing, particularly in patients with compromised immune function. We highlight some of these emerging areas relevant to the diagnosis and treatment of EBV-associated LPD with potential applicability to other EBV-associated neoplasms.

Integrated Pan-Cancer Map of EBV-Associated Neoplasms Reveals Functional Host-Virus Interactions.

Epstein-Barr virus (EBV) is a complex oncogenic symbiont. The molecular mechanisms governing EBV carcinogenesis remain elusive and the functional interactions between virus and host cells are incompletely defined. Here we present a comprehensive map of the host cell-pathogen interactome in EBV-associated cancers. We systematically analyzed RNA sequencing from >1,000 patients with 15 different cancer types, comparing virus and host factors of EBV+ to EBV- tissues. EBV preferentially integrated at highly accessible regions of the cancer genome, with significant enrichment in super-enhancer architecture. Twelve EBV transcripts, including LMP1 and LMP2, correlated inversely with EBV reactivation signature. Overexpression of these genes significantly suppressed viral reactivation, consistent with a "virostatic" function. In cancer samples, hundreds of novel frequent missense and nonsense variations in virostatic genes were identified, and variant genes failed to regulate their viral and cellular targets in cancer. For example, one-third of patients with EBV+ NK/T-cell lymphoma carried two novel nonsense variants (Q322X, G342X) of LMP1 and both variant proteins failed to restrict viral reactivation, confirming loss of virostatic function. Host cell transcriptional changes in response to EBV infection classified tumors into two molecular subtypes based on patterns of IFN signature genes and immune checkpoint markers, such as PD-L1 and IDO1. Overall, these findings uncover novel points of interaction between a common oncovirus and the human genome and identify novel regulatory nodes and druggable targets for individualized EBV and cancer-specific therapies. SIGNIFICANCE: This study provides a comprehensive map of the host cell-pathogen interactome in EBV+ malignancies.See related commentary by Mbulaiteye and Prokunina-Olsson, p. 5917.

Interferon beta increases NK cell cytotoxicity against tumor cells in patients with nasopharyngeal carcinoma via tumor necrosis factor apoptosis-inducing ligand.

Nasopharyngeal carcinoma (NPC) is an EBV-associated neoplasm occurring endemically in Southeast Asia and sporadically all over the world. In children and adolescents, high cure rates have been obtained using chemotherapy, radiochemotherapy and maintenance therapy with interferon beta (IFNβ). The mechanism by which IFNβ contributes to a low systemic relapse rate has not yet been fully revealed. NK cells and serum samples from two patients with NPC were analyzed before and at different time points during IFNβ therapy, for assessment of TRAIL expression and NK cell cytotoxicity. Cytotoxicity was measured using the calcein release assay and the contribution of different death effector pathways was analyzed using specific inhibitors. Treatment with IFNβ induced TRAIL expression on patients' NK cells and increased their cytotoxicity against NPC targets in vitro. NK cell-mediated cytotoxicity was predominately mediated via TRAIL. IFNβ also induced the production of soluble TRAIL (sTRAIL) by NK cells and its release upon contact with NPC cells. IFNβ treatment increased serum levels of sTRAIL in patients. Moreover, sTRAIL concentrated from patients' serum samples induced apoptosis ex vivo in NPC cells from a patient-derived xenograft. Increased cytotoxicity of NK cells against NPC cells and increased serum levels of biologically active TRAIL in patients treated with IFNβ could be a means to eliminate micrometastatic disease and explain the low systemic relapse rate in this patient group.

Targeted Therapy for EBV-Associated B-cell Neoplasms.

Epstein-Barr virus (EBV) is directly implicated in several B-cell lymphoid malignancies. EBV-associated lymphomas are characterized by prominent activation of the NF-κB pathway and targeting this pathway establishes a rationale for a therapeutic approach. The ubiquitin/proteasome signaling plays an essential role in the regulation of the NF-κB pathway. Ixazomib is an FDA-approved, orally bioavailable proteasome inhibitor. Here we report the first preclinical evaluation of ixazomib-mediated growth-inhibitory effects on EBV-infected B-lymphoblastoid cell lines Raji and Daudi. Ixazomib induced apoptosis in these cell lines in a dose-dependent manner. Cell-cycle analysis demonstrated ixazomib treatment induced cell-cycle arrest at the G2-M phase with a concomitant decrease in G0-G1 and S phases. The results further revealed an increase in p53, p21, and p27 levels and a decrease in survivin and c-Myc protein levels. Mechanistically, ixazomib treatment resulted in the accumulation of polyubiquitinated proteins, including phosphorylated IκBα with a significant reduction of p65 subunit nuclear translocation. Altogether, our preclinical data support the rationale for in vivo testing of ixazomib in EBV-associated B-cell neoplasms. IMPLICATIONS: This preclinical study supports the use of oral proteasome inhibitor ixazomib for targeting NF-κB signaling in the treatment of EBV-associated B-cell neoplasms.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/4/839/F1.large.jpg.

The Presence of Defective Epstein-Barr Virus (EBV) Infection in Patients with EBV-Associated Hematological Malignancy

IntroductionEpstein-Barr virus (EBV) is a double-stranded DNA virus that infects >95% of the human population and is associated with a substantial risk of cancer development. Most infections in children and adolescents are asymptomatic or result in infectious mononucleosis; however, in some patients, EBV is associated with various hematological malignancies including Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), and extranodal NK/T-cell lymphoma. EBV infection is also present in a portion of epithelial cell neoplasms such as gastric cancer and nasopharyngeal carcinoma. Despite the large population risk of cancer associated with EBV, it is poorly understood why only a small subset of EBV-infected individuals develop neoplasms, while others do not.Patients and MethodsWe designed a target enrichment system to capture several EBV strains including the Akata strain, which is responsible for the majority of EBV infections in Japan. We analyzed the genomes of EBV strains in 139 patients with various EBV-associated diseases and 17 EBV-positive cell lines. Next-generation sequencing reads were aligned to the Akata reference genome to analyze nucleotide variations, copy number alterations, and structural variations including sequence insertions in the human genome. The institutional review board of Nagoya University Graduate School of Medicine approved this study.ResultsWe identified a median of 645 single nucleotide variants (SNVs) in the EBV genomes, 78% of which affected coding sequences. SNVs in coding sequences were significantly biased toward synonymous variants, suggesting negative selection pressure. The SNVs detected in noncoding sequences were enriched in two evolutionarily conserved viral noncoding RNAs (EBER1 and EBER2), particularly in the PAX5-binding domain of EBER2. However, most SNVs identified in the EBV genome do not seem to affect the development of neoplasms, as hierarchical clustering of EBV genomes from neoplastic and non-neoplastic diseases based on SNVs revealed no significant association between the EBV strain and disease type.In addition to SNVs, we identified frequent intragenic deletions in the EBV genomes of patients with EBV-positive DLBCL (10/14, 71%), extranodal NK/T-cell lymphoma (10/23, 43%), chronic active EBV infection (27/77, 35%), and other EBV-associated neoplasms (2/7). Such deletions were also identified in several EBV-associated cell lines (6/17), but not in non-neoplastic diseases such as infectious mononucleosis (0/4) and post-transplant lymphoproliferative disorders (0/14), suggesting a unique role of these mutations in the neoplastic proliferation of EBV-infected cells. Frequent deletions were detected in BamHI A rightward transcripts microRNA clusters (31/156), which suppress viral transcription factors (BZLF1 and BRLF1) required for the lytic reactivation of EBV. Deletions also were associated with several genes essential for virus production (20/156). These observed deletions are thought to upregulate lytic cycle-associated genes, some of which benefit neoplasms by inducing genomic instability and immune escape and mitigate cell damage caused by the production of viral particles. In fact, deletion of one essential gene, BALF5, resulted in upregulation of the lytic cycle and promotion of lymphomagenesis in a xenograft model.DiscussionAlthough the essential roles of several latency-associated genes, such as LMP-1 and EBNA-2, in EBV-mediated immortalization and transformation of human lymphocytes have long been discussed, our finding raises the possibility that lytic cycle-associated genes also contribute to lymphomagenesis. This agrees with reports that lytic cycle-associated genes are expressed in Burkitt lymphoma, DLBCL, and chronic active EBV infection, and that BZLF1-deficient lymphoblastoid cells exhibit significantly impaired tumorigenicity in mice. In addition, essential gene deletions lead to the protection of EBV-infected cells from lysis. Further studies are warranted to exploit these findings for the design of novel therapeutics for EBV-associated neoplasms. DisclosuresKiyoi:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria. Nakamura:Roche/Chugai,: Research Funding; Kyowa-Kirin: Research Funding.

Current Trends and Alternative Scenarios in EBV Research.

Epstein-Barr virus (EBV) infection is associated with several distinct hematological and epithelial malignancies, e.g., Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and others. The association with several malignant tumors of local and worldwide distribution makes EBV one of the most important tumor viruses. Furthermore, because EBV can cause posttransplant lymphoproliferative disease, transplant medicine has to deal with EBV as a major pathogenic virus second only to cytomegalovirus. In this review, we summarize briefly the natural history of EBV infection and outline some of the recent advances in the pathogenesis of the major EBV-associated neoplasms. We present alternative scenarios and discuss them in the light of most recent experimental data. Emerging research areas including EBV-induced patho-epigenetic alterations in host cells and the putative role of exosome-mediated information transfer in disease development are also within the scope of this review. This book contains an in-depth description of a series of modern methodologies used in EBV research. In this introductory chapter, we thoroughly refer to the applications of these methods and demonstrate how they contributed to the understanding of EBV-host cell interactions. The data gathered using recent technological advancements in molecular biology and immunology as well as the application of sophisticated in vitro and in vivo experimental models certainly provided deep and novel insights into the pathogenetic mechanisms of EBV infection and EBV-associated tumorigenesis. Furthermore, the development of adoptive T cell immunotherapy has provided a novel approach to the therapy of viral disease in transplant medicine and hematology.

Abstract 3079: Targeted therapy for EBV-associated B-cell neoplasms

Abstract Epstein-Barr virus (EBV) is directly implicated in several B-cell lymphoid malignancies. EBV associated B-cell neoplasms occur in both organ and allogeneic hematopoietic stem cell transplantations due to immunosuppression in these patients to maintain the function of transplant. Treatment of these B-cell neoplasms is limited to reduction in immunosuppression and antineoplastic chemotherapy. EBV associated lymphomas are characterized by prominent activation of Nuclear Factor kappa (NF-kB) pathway and targeting this pathway establishes a rationale for therapeutic approach. The ubiquitin/proteasome signaling pathway plays an important role in regulation of NF-kB pathway. Bortezomib (BZ) is the first FDA approved proteasome inhibitor for treating both newly diagnosed and relapsed/refractory multiple myelomas, and mantle cell lymphomas. BZ acts through inhibition of the 26S proteasome, a large protease complex that degrade ubiquitinated proteins. BZ stabilizes various cellular proteins involved in cell cycle arrest and apoptosis including p21, p27, p53, and IkBα by inhibiting proteasome function. Stabilization of IkBα results in inhibition of NF-kB signaling pathway which promotes tumor cell survival, growth, and angiogenesis. The major limiting factor for long-term administration of BZ through intravenous or subcutaneous is risk of peripheral neuropathy. There is a need to develop bioavailable proteasome inhibitor with low toxicity profile to overcome this conundrum. Ixazomib is an investigational orally bioavailable proteasome inhibitor which inhibits the activity of the 20S catalytic core subunit of the proteasome and currently in clinical trials for patients with relapsed/refractory multiple myeloma. Here we report the first preclinical evaluation of oral proteasome inhibitor ixazomib growth inhibitory effects on EBV-infected B-lymphoblastoid cell lines Raji and Daudi. In these cell lines, treatment with Ixazomib significantly induced apoptosis dose dependently (10-50 nM), in association with poly (ADP-ribose) polymerase cleavage. Cell cycle analysis by flow cytometry showed that Ixazomib treatment induced G2/M arrest dose dependently with concomitant decreases in the percentage of cells in G0/G1 and S phases. Immunoblotting analysis revealed increase in p53 and p27 levels without significant change in the relative levels of p21. Mechanistically, ixazomib treatment resulted in accumulation of polyubiquitinated proteins including accumulation of phosphorylated IkBα as demonstrated by western blot analysis concomitant with significant reduction in NF-kB activity and subunit translocation to the nucleus. Altogether, our pre-clinical data indicate that ixazomib induces apoptosis in EBV-associated lymphoma cells by blocking NF-kB signaling cascade and support the rationale for testing ixazomib in patients with EBV-associated B-cell neoplasms. Citation Format: Siddhartha Ganuguly, Satyanarayana Alleboina, Sudhakiranmayi Kuravi, Joseph McGuirk, Ramesh Balusu. Targeted therapy for EBV-associated B-cell neoplasms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3079.

Epstein-Barr virus reactivation is induced, but abortive, in cutaneous lesions of systemic hydroa vacciniforme and hypersensitivity to mosquito bites

BackgroundEpstein-Barr virus (EBV)-associated T/natural killer (NK)-lymphoproliferative disorders (LPDs) include hydroa vacciniforme (HV) and hypersensitivity to mosquito bites (HMB). The pathomechanisms of these diseases are still unclear. ObjectiveTo understand the inflammatory process, we examined EBV reactivation markers, BZLF1 and BDRF1 mRNA in the tissue and blood from patients with EBV-associated T/NK-LPDs. MethodsSixty-four patients with EBV-associated LPDs and epithelial neoplasms, and EBV+ cell line cells were studied. DNase-treated and resistant EBV DNA load in blood and cell culture supernatants were calculated. An EBV reactivation signal was analyzed in the tissue, blood and cell line cells. ResultsIn the tissue, BZLF1 mRNA was detected in 5 of 6 (83%) samples of EBV+ epithelial neoplasms, 16 of 21 (76%) of EBV+ lymphomas, and 5 of 15 (33%) of systemic HV and/or HMB, but negative in all 15 patients with classical HV. In the blood, BZLF1 mRNA was detected in only one of 19 (5.3%) samples of EBV-associated T/NK-LPDs. A down-stream reactivation signal, BDRF1 mRNA was expressed in all 6 samples of EBV+ epithelial neoplasms, but it was positive in only one of 15 (6.7%) samples from systemic HV and HMB in the tissue. EBV+ T/NK-cell line cells treated with phorbol 12-myristate 13-acetate produced BZLF1 and BDRF1 mRNA, and encapsidated EBV DNA was detected in the culture supernatants of cell line cells. ConclusionStimulation-induced EBV reactivation occurred both in vivo and in vitro, but it was almost abortive in vivo. Reactivation-related EBV antigens might be responsible for induction of systemic HV and HMB.

Similarities between the Epstein-Barr Virus (EBV) Nuclear Protein EBNA1 and the Pioneer Transcription Factor FoxA: Is EBNA1 a "Bookmarking" Oncoprotein that Alters the Host Cell Epigenotype?

EBNA1, a nuclear protein expressed in all EBV-associated neoplasms is indispensable for the maintenance of the viral episomes in latently infected cells. EBNA1 may induce genetic alterations by upregulating cellular recombinases, production of reactive oxygen species (ROS) and affecting p53 levels and function. All these changes may contribute to tumorigenesis. In this overview we focus, however, on the epigenetic alterations elicited by EBNA1 by drawing a parallel between EBNA1 and the FoxA family of pioneer transcription factors. Both EBNA1 and FoxA induce local DNA demethylation, nucleosome destabilization and bind to mitotic chromosomes. Local DNA demethylation and nucleosome rearrangement mark active promoters and enhancers. In addition, EBNA1 and FoxA, when associated with mitotic chromatin may “bookmark” active genes and ensure their reactivation in postmitotic cells (epigenetic memory). We speculate that DNA looping induced by EBNA1-EBNA1 interactions may reorganize the cellular genome. Such chromatin loops, sustained in mitotic chromatin similarly to the long-distance interactions mediated by the insulator protein CTCF, may also mediate the epigenetic inheritance of gene expression patterns. We suggest that EBNA1 has the potential to induce patho-epigenetic alterations contributing to tumorigenesis.

Risk factors for death and temporal trends in overall survival in patients with AIDS-associated primary central nervous system lymphoma (AIDS-PCNSL)

Background AIDS-PCNSL is a rare EBV-associated B-cell neoplasm that continues to carry a poor prognosis, even in the highly active antiretroviral therapy (HAART) era. We hypothesized that overall survival (OS) is affected by prior diagnosis of central nervous system (CNS) infections as well as treatment approaches to both HIV and AIDS-PCNSL. We evaluated risk factors and temporal trends for OS in patients with AIDS-PCNSL.

Lymphoepithelioma-like carcinoma of lung – a case report

We report a case in 49-year-old Asian female who presented with a radio-dense mass in the posterobasal segment of right lower lobe, located in a subpleural location. Sectioning showed a well defined, firm, circumscribed, nodular tumour 30 × 30 × 25 mm in a subpleural location leading to puckering of the overlying pleura. Three subcarinal lymph nodes were also submitted. Histopathology showed the nodule was a poorly differentiated carcinoma, forming a syncytium of tumour cells with vesicular nuclei. Accompanying the tumour was heavy lymphoid infiltrate, infiltrating within and surrounding the tumour mass. Tumour cells were postive for AE1/AE3, p63 and EBV ISH. Keratin 7 and 20 were negative. One of three lymph nodes contained metastatic carcinoma. Lymphoepithelioma-like carcinoma of the lung occurs as an EBV-associated neoplasm. The virus may be demonstrated by expression of latent membrane protein-1 and EBV-deoxyribonucleic acid in tumour cells by immunohistochemistry, and in situ hybridisation for EBV-encoded small nuclear ribonucleic acid. It needs to be distinguished histologically from large cell lymphoma, germ cell tumour, poorly differentiated squamous cell carcinoma and mucoepidermoid carcinoma. A thorough evaluation of other primary sites such as the nasopharynx should be done to exclude a metastatic tumour. The behaviour of lymhoepithelioma-like carcinoma is highly variable, ranging from apparently curable (by excision) to highly aggressive with extensive disease at presentation.

Patogenesi ed epidemiologia dei tumori associati all’infezione da virus di Epstein-Barr

The Epstein-Barr virus (EBV) is one of the most successful human viruses: it is ubiquitous worldwide, and more than 80% of people over the age of 30 show serological evidence of infection. In addition, EBV infection tends to be persistent, so that, once it has occurred, it remains for the lifetime, a feature that is common to many other herpesviral infections. EBV is strongly associated with the development of several cancers, in particular with Burkitt’s lymphoma, Hodgkin’s disease, and lymphoproliferative disorders which complicate immune suppression conditions. These EBV-associated neoplasms are characterized by peculiar geographic distributions and distinctive epidemiologic features.This review refers to recent advances in the mechanisms of EBV-induced cell transformation, and is focused on the main epidemiological aspects of virus-associated malignancies.

A spectrum of clinical manifestations caused by host immune responses against Epstein-Barr virus infections.

Epstein-Barr virus (EBV), or human herpesvirus 4 (HHV-4), infects the vast majority of adults worldwide, and establishes both nonproductive (latent) and productive (lytic) infections. Host immune responses directed against both the lytic and latent cycle-associated EBV antigens induce a diversity of clinical symptoms in patients with chronic active EBV infections who usually contain an oligoclonal pool of EBV-infected lymphocyte subsets in their blood. Episomal EBV genes in the latent infection utilize an array of evasion strategies from host immune responses: the minimized expression of EBV antigens targeted by host cytotoxic T lymphocytes (CTLs), the down-regulation of cell adhesion molecule expression, and the release of virokines to inhibit the host CTLs. The oncogenic role of latent EBV infection is not yet fully understood, but latent membrane proteins (LMPs) expressed during the latency cycle have essential biological properties leading to cellular gene expression and immortalization, and EBV-encoded gene products such as viral interleukin-10 (vIL-10) and bcl-2 homologue function to survive the EBV-infected cells. The subsequent oncogenic DNA damage may lead to the development of neoplasms. EBV-associated NK/T cell lymphoproliferative disorders are prevalent in Asia, but quite rare in Western countries. The genetic immunological background, therefore, is closely linked to the development of EBV-associated neoplasms.

EBV-associated neoplasms: alternative pathogenetic pathways

We propose that there are two main classes of Epstein-Barr virus (EBV) associated lymphomas: primarily malignant Burkitt’s Lymphoma (BL) and Hodgkin’s Disease (HD), on one hand, and primarily benign lymphoproliferations, e.g., post-transplant lymphoproliferative disease (PTLD) on the other hand. PTLD may start as a benign lymphoproliferation which becomes malignant if out of T cell control for too long. Our discovery of a binding site for the oncoprotein c-Myc at a central position of the EBV genome favours a distinction of pathogenetic pathways or scenarios for the proposed lymphoma classes. In the first scenario nuclear maintenance of the EBV genome and activation of viral anti-apoptotic functions with the help of c-Myc are indispensable for the origin of malignant tumours (BL, HD) from the germinal centre B-cell. In the second scenario expression of the main viral transforming protein EBNA2 is essential for immortalisation and non-malignant morphological transformation of any (germinal centre derived or non-germinal centre) B-cell in the absence of T cell control. Although EBNA2 expression is permissible, under specific circumstances, in malignant B-cells, it is not required for oncogenesis.

Epstein-Barr virus-associated B-cell lymphoproliferative disorders in angloimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified.

Various patterns of Epstein-Barr virus (EBV)-associated B-cell lymphoproliferation occur in patients with immunodeficiency. We studied 17 cases of T-cell lymphoma displaying extensive EBV-driven B-cell lymphoproliferation or simultaneous/subsequent EBV-associated B-cell lymphoma. In 10 cases of angioimmunoblastic T-cell lymphoma, an uncommonly prominent population of EBV+ atypical, activated, focally confluent large transformed B cells was found in the background of T-cell lymphoma. In 4 cases, an EBV-associated B-cell neoplasm (3 diffuse large B-cell lymphomas, 1 plasmacytoma) occurred in patients with T-cell lymphoma. Three cases were composite lymphomas of a peripheral T-cell lymphoma, unspecified, combined with EBV-associated diffuse large B-cell lymphoma. The transformed B-cell population displayed EBV latency types 2 and 3. Monoclonal and oligoclonal B-cell populations were detected in 5 and 6 cases, respectively. Similar to other states of immunodeficiency, disease-related and therapy-induced immunosuppression in T-cell lymphoma may lead to a prominent EBV-associated B-cell lymphoproliferation and to EBV+ B-cell neoplasms.