EBV Copy Number Research Articles

211 Plasma EBV estimation in Nasopharyngeal carcinoma from a nonendemic region: Impact of probe assay type, EBV copy numbers as a predictive and prognostic marker.

211 Plasma EBV estimation in Nasopharyngeal carcinoma from a nonendemic region: Impact of probe assay type, EBV copy numbers as a predictive and prognostic marker.

Could Active TB be a Major Contributor to the Disparity in Presentation and Mortality for cHL in HIV + and HIV - Individuals in South Africa?

CONCLUSIONS Classical Hodgkin lymphoma (cHL) outcomes in people living with HIV (PLWH) in South Africa are substantially worse than those in HIV-negative patients. Prior and current tuberculosis (TB) infection are common amongst PLWH and cHL in South Africa. We set out to evaluate the effect of active TB disease on presenting characteristics and outcomes in newly diagnosed cHL patients receiving care in Johannesburg, South Africa. With approvals from the Human Research Ethics Committee of the University of the Witwatersrand and the Johns Hopkins Institutional Review Boards, we conducted a prospective, observational study of newly diagnosed patients with cHL ≥18 years at Chris Hani Baragwanath Academic and Netcare Olivedale Hospitals in Johannesburg, South Africa between March 2021 and April 2023. Participants' data include history, abstracted HIV status, CD4 count, HIV viral load at the time of cHL diagnosis, baseline laboratory data, staging work-up, and review of diagnostic pathology. TB diagnosis was based on routine clinical procedures. Pre-treatment whole blood was collected in cell stabilizing tubes and processed locally using a plasma cell-free DNA (cfDNA) protocol. cfDNA was then isolated in the USA with subsequent analysis of fragment length, cfDNA and gDNA concentrations, and EBV copy number. Among 43 patients, 28 (65%) were PLWH. Fifty-one percent were female, mean age was 41 years and 98% of patients were diagnosed with advanced stage disease and B-symptoms. Forty-seven percent had marrow involvement including 18/28 (64%) PLWH. Among PLWH, mean CD4 count was 180 and 61% of patients had viral load <50 copies at time of diagnosis. There was no difference in age, sex, stage, performance status or International Prognostic Index (IPI) by HIV serostatus. PLWH had higher plasma EBV copy number at presentation (3.6 vs. -0.2 median Log 10copies/mL, p=0.0123) and higher cfDNA (41.8 vs. 19.8 ng/mL), and lower overall survival at 1-year (57.1% vs. 86.7%, p=0.0489) compared to HIV-seronegative. Overall, 10 cHL patients were receiving treatment for active TB (23%); Most 9/10, were PLWH. Patients with TB and cHL were more likely to have bone marrow involvement (90% vs 33%; p=0.002) and significantly lower mean CD4 count (70.6 vs 232.2;p=0.009), hemoglobin (6.9 vs 9.6;p=0.01), platelet count (77.7 vs 363.5;p=0.0002) and albumin (22.7 vs 33.1; p=0.0004) at time of diagnosis compared to those without active TB. In univariate analysis, HIV serostatus, active TB infection, bone marrow involvement, lower mean platelet count, absolute lymphocyte count and albumin, IPI, baseline EBV and plasma cfDNA were all associated with increased mortality. Active TB infection was common amongst PLWH with newly diagnosed cHL in South Africa. Bone marrow involvement and traditional laboratory markers of poor prognosis including anemia and hypoalbuminemia were more common among patients with TB. The role that active TB and its treatment plays in the increased mortality for HIV(+) cHL patients warrants further investigation.

Epstein-Barr virus copy number in peripheral blood mononuclear cells predicts prognosis in diffuse large B cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with variable outcomes. In this study, data of 84 DLBCL patients, who were tested EBV DNA in peripheral blood, were retrospectively analyzed. Patients were divided into three subgroups according to EBV copy number (EBV-CN) values: the negative (<500 copies/ml), low (500–104 copies/ml), and high EBV-CN group (≥104 copies/ml). The higher EBV-CN was associated with male and elderly patients. No significant difference was found between the three subgroups regarding immunophenotype, cytogenetic features, and molecular features. Patients of the high EBV-CN group had significantly worse progression-free and overall survival (OS) compared to other groups. After adjusting conventional risk factors, high EBV-CN was an independent prognostic factor for OS in multivariate analysis. Taken together, peripheral blood EBV-CN can predict outcomes of patients with DLBCL and 104 copies/ml is a more suitable boundary value than the traditional normal value in predicting prognosis.

Detection of Epstein-Barr virus encoded small RNA genes in oral squamous cell carcinoma and non-cancerous oral cavity samples

BackgroundEpstein-Barr Virus (EBV) is a human oncogenic virus that can lead to cancer in lymphoid and epithelial cells and is one of the hypothesized causes of oral cavity lesions including oral squamous cell carcinoma (OSCC), but the etiological association remains undetermined. The present investigation aimed to explore the EBV presence, viral load, and EBV-encoded small RNA (EBER) sequence variation in tissue samples of patients with OSCC and other oral cavity lesions including oral lichen planus (OLP), and oral irritation fibroma (OIF).MethodsIn total, 88 oral cavity samples (23 with OSCC, 29 with OLP, and 36 with OIF diagnosis) were examined by Real-Time PCR technique and some of them were sequenced.ResultsViral EBER sequence was detected in 6 out of the 23 OSCC (31.4%), 6 out of the 29 OLP (20.7%), and 3 out of the 36 OIF cases (8.3%). The mean EBV copy number was higher in OSCC samples (1.2 × 10−2 ± 1.3 × 10−2 copies/cell) compared to OLP (2.2 × 10−3 ± 2.6 × 10−3 copies/cell) and OIF (2.4 × 10−4 ± 2.0 × 10−4 copies/cell) samples, although this difference was not statistically significant (P = 0.318). The EBER gene was amplified and sequenced in 5 OSCC, 3 OLP, and 2 OIF samples with high EBV viral load. One OSCC, two OLP, and two OIF isolates showed different nucleotide variations compared with EBV-WT and AG876 prototype sequences: C6834T, C6870T, C6981T, C7085T, C7085G, and C7094T.ConclusionIn our study the presence of more than one genome copies per tumor cell indicates the possible role of EBV infection in oral cancers. However, more studies should be conducted to clarify the role of EBV in OSCC carcinogenesis.

Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

BackgroundGerminal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections.Case presentationWe present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001.ConclusionsTo the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

Correction: Genetic factors affecting EBV copy number in lymphoblastoid cell lines derived from the 1000 Genome Project samples.

[This corrects the article DOI: 10.1371/journal.pone.0179446.].

A study of Epstein-Barr virus infection in the Chinese tree shrew(Tupaia belangeri chinensis)

BackgroundEpstein–Barr virus (EBV) is closely associated with many human diseases, including a variety of deadly human malignant tumours. However, due to the lack of ideal animal models,the biological characteristics of EBV, particularly its function in tumourigenesis, have not been determined. Chinese tree shrews (Tupaia belangeri chinensis), which are similar to primates, have been used to establish a variety of animal models and have recently received much attention. Here, we established tree shrews as a model for EBV infection by intravenous injection.MethodsTen tree shrews were inoculated with EBV by intravenous injection,and blood was collected at regular intervals thereafter from the femoral artery or vein to detect EBV markers.ResultsEight of 10 tree shrews showed evidence of EBV infection. In the 8 EBV-infected tree shrews, EBV copy number increased intermittently or transiently, EBV-related gene expression was detected, and anti-EBV antibodies increased to varying degrees. Macroscopic hepatomegaly was observed in 1 tree shrew, splenomegaly was observed in 4 tree shrews, and enlarged mesenteric lymph nodes were observed in 3 tree shrews. Haematoxylin and eosin (HE) staining showed splenic corpuscle hyperplasia in the spleens of 4 tree shrews and inflammatory cell infiltration of the liver of 1 tree shrew and of the mesenteric lymph nodes of 3 tree shrews. EBER in situ hybridization(ISH) and immunohistochemical (IHC) staining showed that EBER-, LMP1- and EBNA2- positive cells were present in the spleens and mesenteric lymph nodes of some tree shrews. Western blotting (WB) revealed EBNA1-positive cells in the spleens of 4 tree shrews. EBV markers were not detected by HE, EBER-ISH or IHC in the lung or nasopharynx.ConclusionsThese findings suggest that EBV can infect tree shrews via intravenous injection. The presented model offers some advantages for exploring the pathophysiology of EBV infection in humans.

Investigation on the association between thyroid tumorigeneses and herpesviruses.

Herpesviruses have been associated with various human malignancies and with thyroid autoimmunity. Aiming to investigate the presence of these viruses in thyroid nodules, we analyzed serum and thyroid tissue from 183 patients (83 benign and 100 malignant thyroid nodules). We also obtained 104 normal thyroid tissues extracted from the contralateral lobe of these patients. We used ELISA to screen the serology of all patients and a real-time quantitative PCR to analyze thyroid tissue viral load in antibody-positive patients. In addition, the presence of herpesviruses was tested by histological analysis in 20 EBV-positive tissues using the expression of LMP-1 by immunohistochemistry (IHC) and EBER by in situ hybridization (ISH). There was no evidence of HSV-2 or CMV DNA, but we found EBV DNA sequences in 29 (16%) thyroid tissue samples. We also found 7 positive EBV cases out of 104 normal tissues. Viral load was higher in tumors than in their respective normal tissues (p = 0.0002). ISH analysis revealed EBER expression in 11 out of 20 (52%) EBV-positive tissues, mostly in malignant cases (8/11, 73%). The presence of high EBV copy numbers in thyroid tumors and the expression of EBER only in malignant cases suggest an association between EBV and thyroid malignancies. However, we did not find any association between the presence of EBV and/or its viral load and any clinical or pathological tumor feature. Further studies aiming to clarify the mechanisms of EBV infection in thyroid cells are necessary to support a possible role in the development of thyroid cancer.

The clinical significance of EBV DNA in the plasma and peripheral blood mononuclear cells of patients with or without EBV diseases

AbstractEpstein-Barr virus (EBV) is a ubiquitous virus that establishes a latent infection within the host and in some cases can lead to the development of EBV-associated lymphomas, lymphoproliferative disorders, hemophagocytic lymphohistiocytosis, solid tumors, and other diseases. We studied the clinical significance of detecting EBV DNA in the plasma and peripheral blood mononuclear cells (PBMCs) of 2146 patients who had blood specimens sent to the Johns Hopkins Hospital clinical laboratory for viral quantitative real-time polymerase chain reaction assay over a 5-year period. Within this largely immunocompromised and hospitalized cohort, 535 patients (25%) had EBV detected in plasma or PBMCs. When EBV was detected in the absence of an EBV+ disease (n = 402), it was present only in PBMCs in 69% of cases. Immunocompromised patients were less likely to have EBV in plasma than in PBMCs in the absence of EBV+ disease. In patients with active, systemic EBV+ diseases (n = 105), EBV was detected in plasma in 99% of cases but detected in PBMCs in only 54%. Across a range of copy number cutoffs, EBV in plasma had higher specificity and sensitivity for EBV+ disease as compared with EBV in PBMCs. EBV copy number in plasma distinguished untreated, EBV+ lymphoma from EBV+ lymphoma in remission and EBV− lymphoma, and also distinguished untreated, EBV+ posttransplantation lymphoproliferative disorder (PTLD) from EBV+ PTLD in remission and EBV− PTLD. EBV copy number quantification is a useful diagnostic marker across the spectrum of EBV+ diseases, even among immunocompromised patients, with plasma specimens more indicative of EBV+ disease than PBMCs.

Abstract 4772: Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which helps explain such observation. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5′Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptional repression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 was correlated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Our results reveal a novel mechanism wherein HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 may use as biomarkers for monitoring NPC treatment. Citation Format: CHEN-CHING PENG. Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2015-4772

Abstract 2300: Silencing of the transcription repressor HOXA2 by aberrant hypermethylation enhances invasion via MMP-9 activation in NPC

Abstract Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5′Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptionalrepression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 wascorrelated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment. Note: This abstract was not presented at the meeting. Citation Format: Chen-Ching Peng. Silencing of the transcription repressor HOXA2 by aberrant hypermethylation enhances invasion via MMP-9 activation in NPC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2300. doi:10.1158/1538-7445.AM2014-2300

Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in Nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5'Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptional repression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 was correlated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment.

Role of plasma EBV DNA levels in predicting recurrence of nasopharyngeal carcinoma in a western population

BackgroundLoco-regionally advanced nasopharyngeal carcinomas can be cured by the combination of chemotherapy and radiotherapy. In Eastern countries, plasma levels of viral Epstein-Barr deoxyribonucleic acid (DNA) are accurate in predicting recurrence, but few data are available in Western populations. The aim of this prospective study was to evaluate the relationship between viral Epstein-Barr DNA copy numbers in plasma and the response rate, progression-free survival and overall survival in a cohort of Western patients with stage IIb-IVb nasopharyngeal cancer.MethodsWe evaluated plasma samples from 36 consecutive patients treated with induction chemotherapy followed by chemoradiation. EBV copy numbers were determined after DNA extraction using real-time quantitative polymerase chain reaction. Survival curves were estimated using the Kaplan–Meier method.ResultsCirculating Epstein-Barr virus DNA levels were measured before treatment, at the end of concomitant chemo- and radiotherapy, and during the follow-up period. Pre-treatment levels significantly correlated with the initial stage and probability of relapse. Their increase was 100% specific and 71.3% sensitive in detecting loco-regional or metastatic recurrence (an overall accuracy of 94.4%). Three-year progression-free and overall survival were respectively 78.2% and 97.1%.ConclusionsThe results of this study confirm that patients from a Western country affected by loco-regionally advanced nasopharyngeal carcinoma have high plasma Epstein-Barr virus DNA levels at diagnosis. The monitoring of plasma levels is sensitive and highly specific in detecting disease recurrence and metastases.

Phase 2 study of rituximab-ABVD in classical Hodgkin lymphoma

AbstractIn classical Hodgkin lymphoma, circulating clonotypic malignant cells express CD20, which potentially explains the observed activity of rituximab. This multicenter phase 2 study investigated the combination of rituximab-ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) for stage II-IV untreated classical Hodgkin lymphoma. A goal was to assess the behavior of circulating clonotypic B cells clinically. Of 49 evaluable patients, 69% had stage IIB-IV disease; 8% had CD20+ Hodgkin and Reed-Sternberg cells. Rituximab-ABVD was generally well tolerated. Delivered relative dose intensity was 94% for AVD and 79% for bleomycin. After 6 cycles, 81% of patients were in complete remission. Only 8% received radiation therapy. The actuarial 3-year event-free and overall survival rates were 83% and 98%, respectively. EBV copy number in plasma fell dramatically during cycle 1 in patients with EBV+ tumors. Persistence of detectable circulating clonotypic B cells was associated with a greater relapse frequency (P < .05). Rituximab-ABVD and clonotypic B cells warrant additional study in classical Hodgkin lymphoma. This trial was registered at www.clinicaltrials.gov as NCT00369681.

Quantitation of EBV-DNA In Peripheral Blood In Hodgkin Lymphoma: Associations with Other Biomarkers and Patient Characteristics

AbstractAbstract 2678The Epstein-Barr virus (EBV) is present in the malignant Hodgkin/Reed-Sternberg (HRS) cells of 20–40% cases of Hodgkin lymphoma (HL) in western countries. Detection and quantitation of cell-free plasma EBV-DNA has been proposed as a biomarker in EBV-associated malignancies, including HL. We analyzed the presence of EBV in HRS cells, peripheral blood compartments, and correlated these data to EBV serology, other circulating biomarkers and clinical characteristics. In a single institution cohort of 93 HL patients, EBV-DNA was quantitated using real-time PCR for the EBNA region in whole blood (WB, n=69), plasma (PL, n=75) and peripheral blood mononuclear cells (MNC, n=74). Cell-free DNA levels were determined using a real-time PCR for the globin gene (n=84), cytokine concentrations were measured in 85 patients. EBV status of HRS cells was studied using in-situ hybridization with EBER. EBV-DNA was positive in 32% WB, 21% PL and 20% MNC at HL diagnosis, with concordant WB and PL results in 86% of cases. EBER was present in HRS cells in 32% of cases. EBV-DNA copy number over 1500/ml was predictive for the presence of EBER in HRS cells in 86% and 88% WB and PL, respectively, while EBV copy numbers <1500 had only 55% and 57% PPV for WB and PL. Older patients (>50 years) were frequently EBER-positive (66%), with higher EBV copy numbers (median 12800/ml), while younger patients (<50 years) were less frequent EBER-positive (23%), and copy numbers were lower (median 1200/ml) (p=0.01). EBER-positive HL cases frequently had EBNA-1 antibody titers < 100 U/ml when compared to EBER-negative cases (44% vs. 13%, p=0.01), and there was a trend for a negative correlation between EBV copy number and EBNA1 titers (p=0.07). In patients with EBER+ HL the plasma EBV-DNA copy number correlated to the level of circulating cell-free DNA (rho=0.65), IL-10 levels (rho=0.87), IL-6 levels (rho=0.74) and inflammation parameters, including CRP (rho=0.79) and ESR (rho=0.57) (all p<0.02), and were higher in stage IV disease (p=0.02). In conclusion, a high EBV-copy number in whole blood and/or plasma at diagnosis is a good indicator for EBV-association of HL, and EBV copy numbers strongly correlate to other biological parameters of disease activity. Moreover, the inverse correlation between EBV copy number and EBNA1 antibody titers may indicate a reduction in immune surveillance favoring the expansion of EBV. Disclosures:No relevant conflicts of interest to declare.

Cellular factors associated with latency and spontaneous Epstein–Barr virus reactivation in B-lymphoblastoid cell lines

EBV-immortalized B-lymphoblastoid cell lines are used as models for cellular transformation and as antigen-presenting cells in immunological assays. LCLs vary in surface markers and other phenotypic properties, but it is not known how this heterogeneity relates to the EBV life cycle. To explore correlations, we examined 62 LCLs for cellular and viral phenotypes. LCLs generated from pediatric and adult donors could similarly be categorized as either low in EBV copy number or fluctuating within a high range. High-copy status accompanied higher lytic viral gene expression and lower latent gene expression. Inhibiting lytic EBV replication did not affect cellular phenotype or lytic switch protein expression, indicating that an LCL's lytic permissivity was a stable property. Among the cellular genes overexpressed in permissive LCLs were unfolded protein response genes and plasma cell markers. Among genes overexpressed in non-permissive LCLs were transcription factors involved in maintaining B cell lineage, in particular EBF1. This study suggests previously undetected mechanisms by which cellular pathways influence the lytic reactivation of EBV.

Dysregulated Epstein-Barr virus infection in patients with CIDP

Ubiquitous viruses have frequently been proposed as a cause or trigger of chronic immune-mediated diseases. Infections are reported to be temporally associated with clinical exacerbations in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). We examined immunological parameters of herpesvirus infections in untreated patients with CIDP compared to demographically matched controls. Patients with CIDP were uniformly seropositive for EBV-specific IgG and the disease was associated with a moderately enhanced IgG reactivity to EBV-encoded antigens expressed during both B cell transformation and productive viral replication. Moreover, cellular EBV copy numbers were 3-fold increased in patients with CIDP. In contrast, humoral immune responses to other herpesviruses (HCMV, HSV) as well as virus-specific IgM responses were unchanged in CIDP. These data indicate that host–pathogen interactions during chronic EBV infection are dysregulated in treatment-naïve patients with CIDP.

Epstein-Barr Virus-Associated Haemophagocytic Lymphohistiocytosis in Adults Characterised by High Viral Genome Load within Circulating Natural Killer Cells.

Abstract 3672Poster Board III-608 IntroductionEpstein Barr virus (EBV) is predominantly B lymphotrophic both in-vitro and in vivo, where in immunocompetent individuals the virus persists asymptomatically in the B lymphoid compartment under host T cell control. EBV's association with B cell malignancies, such as Hodgkin and Burkitt lymphoma, can be viewed as rare accidents of the virus' lifelong interaction with the B cell system. By contrast, EBV infection of NK and T cells is considered a rare event but is nonetheless strongly associated with a spectrum of rare lymphoproliferations: EBV-associated haemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV), aggressive NK leukaemia and NK/T lymphoma all characterised pathogenically by the presence of monoclonal EBV in the T and/or NK cells. The mechanism of viral entry and its contribution to lymphoproliferation in these cell lineages remains to be clearly defined. The majority of reported cases of EBV-HLH occur in the context of primary EBV infection in children or adolescents, some of whom have a defined inherited immune defect. Adult cases of EBV-HLH occur extremely rarely and appear to be more frequent in individuals of East Asian origin. Furthermore, the vast majority of analysed cases of EBV-HLH have identified CD8+ lymphocytes as the predominant virus-bearing cell. To-date, EBV infection of (CD3-CD56+) circulating NK cells has not been reported and the pattern of viral gene expression remains unclear. Methods/PatientsWe analysed peripheral blood from three consecutive cases of EBV-HLH, referred to our laboratory between 2007-2009, to identify the predominant virus-harbouring cell. All three cases occurred in adults (mean age 44yrs), with no history of inherited immunodeficiency, who presented with clinical and laboratory features consistent with a diagnosis of HLH; fever, hepatosplenomegaly, pancytopenia, markedly elevated serum ferritin and lactate dehydrogenase and EBV copy number of 105-106 per millilitre of whole blood. Haemophagocytosis was unequivocally present on tissue biopsy from two patients. Mononuclear cells were separated using the MoFlo™ cell sorter into pure populations. Patient 1 and 2: CD19+CD3 −CD56−, CD3+CD19−CD56−, and CD56+CD3−CD19−. Patient 3: CD19+CD3 –CD16−, CD3+CD19−CD16−, CD16+CD3−CD19− and CD3−CD19−CD16−. DNA was subsequently extracted from each population and assayed by quantitative PCR, expressed as genome copies per million cells. ResultsIn all three cases we found the predominant EBV load within the non-B, non-T lymphocyte populations; definitively shown to be the CD56+CD3− cell fraction in 2 cases and for case 3 within CD3−CD19−CD16− lymphocytes likely to represent CD56+CD16- NK cells (a minority population in normal peripheral blood). A representative figure is shown: [Display omitted] We then quantitatively examined latent and lytic viral gene transcripts by real-time PCR and, in contrast to previously published data, we found a tightly restricted pattern of EBV gene expression with extremely high levels of EBER (EBV-encoded RNA) transcripts present. Lymphocytes derived from tonsillar tissue and peripheral blood, from both healthy and immunosuppressed individuals, served as control samples and demonstrated the predominant EBV genome load in the CD19+ B-cells but not the T or NK fractions. ConclusionThis novel finding of high EBV genome copy numbers and a restricted pattern of viral gene expression, within circulating natural killer cells in the context of adult EBV-HLH, is both pathogenically intriguing and importantly, has relevance for the investigation of targeted therapies for this aggressive disease. Disclosures:No relevant conflicts of interest to declare.

Increased Frequency of EBV-Specific Effector Memory CD8+ T Cells Correlates with Higher Viral Load in Rheumatoid Arthritis

EBV is a candidate trigger of rheumatoid arthritis (RA). We determined both EBV-specific T cell and B cell responses and cell-associated EBV DNA copies in patients with RA and demographically matched healthy virus carriers. Patients with RA showed increased and broadened IgG responses to lytic and latent EBV-encoded Ags and 7-fold higher levels of EBV copy numbers in circulating blood cells. Additionally, patients with RA exhibited substantial expansions of CD8(+) T cells specific for pooled EBV Ags expressed during both B cell transformation and productive viral replication and the frequency of CD8(+) T cells specific for these Ags correlated with cellular EBV copy numbers. In contrast, CD4(+) T cell responses to EBV and T cell responses to human CMV Ags were unchanged, altogether arguing against a defective control of latent EBV infection in RA. Our data show that the regulation of EBV infection is perturbed in RA and suggest that increased EBV-specific effector T cell and Ab responses are driven by an elevated EBV load in RA.

Epstein-Barr Virus-Associated Smooth Muscle Tumors Are Distinctive Mesenchymal Tumors Reflecting Multiple Infection Events

Epstein-Barr virus-associated smooth muscle tumors (EBV-SMT) are rare lesions that occur in immunocompromised patients. Because they have not been fully characterized pathologically or at the molecular level, we have studied 29 tumors from 19 patients, the largest series to date. Cases coded as EBV-SMT were identified in 19 patients from consultation files and from the renal transplant database at Singapore General Hospital. EBV-SMT occurred in adults (mean age 39 years; range, 21-57 years) and predominantly affected males (12 male, 7 female). Causes of immunocompromise were renal transplantation (10), AIDS (8), and steroid therapy (1). Tumors were located in soft tissue (5), lung (5), liver (4), and miscellaneous sites (15). In 13 patients (68%), the tumors were multiple. Infection with EBV was confirmed in all cases by in situ hybridization for EBV early RNAs (EBER). EBV-SMT were typically well-differentiated smooth muscle tumors with little atypia and usually a low level of mitotic activity. Unlike classic leiomyosarcomas, they lacked significant pleomorphism but frequently displayed primitive round cell areas and prominent intratumoral T lymphocytes. No consistent relationship between histologic features and clinical outcome was noted. All expressed actin (29 of 29) and less frequently desmin (14 of 26). Multiple tumors in a given patient were clonally distinct as assessed by the long terminal repeat region of the virus, supporting the view that multifocal tumors arise from multiple infection events rather than from metastasis. Strain typing by analysis of the EBNA-3C gene confirmed the presence of EBV type 2. Two of four tumors assessed were positive for a 30-bp deletion in the LMP1 gene. EBV copy number per cell ranged greatly between patients and between tumors from the same patient. Follow-up information was available in 18 of 19 patients (mean, 25 months; range, 1-105 months). Fifteen patients were alive: 11 with disease and 4 without. Three patients died, 1 due to disease. We conclude that EBV-SMT are histologically distinct from classic soft tissue smooth muscle tumors, are not readily evaluated by means of conventional histologic criteria, and in the case of multifocal tumors are the result of multiple infection events rather than metastasis. EBV-2 can transform smooth muscle cells, independent of the presence of the LMP1 deletion associated with greater virulence.