Epstein-Barr Virus Viral Load Research Articles

Analysis of antibody binding specificities in twin and SNP-genotyped cohorts reveals that antiviral antibody epitope selection is a heritable trait

Human immune responses to viral infections are highly variable, but the genetic factors that contribute to this variability are not well characterized. We used VirScan, a high-throughput epitope scanning technology, to analyze pan-viral antibody reactivity profiles of twins and SNP-genotyped individuals. Using these data, we determined the heritability and genomic loci associated with antibody epitope selection, response breadth, and control of Epstein-Barr virus (EBV) viral load. 107 EBV peptide reactivities were heritable and at least two Epstein-Barr nuclear antigen 2 (EBNA-2) reactivities were associated with variants in the MHC class II locus. We identified an EBV serosignature that predicted viral load in peripheral blood mononuclear cells and was associated with variants in the MHC class I locus. Our study illustrates the utility of epitope profiling to investigate the genetics of pathogen immunity, reports heritable features of the antibody response to viruses, and identifies specific HLA loci important for EBV epitope selection.

934. Epidemiology and Outcomes of Epstein-Barr Virus DNAemia in Adult Solid Organ Transplant Recipients

BackgroundImmunosuppression in solid organ transplantation (SOT) increases the risk of Epstein-Barr virus (EBV) DNAemia, which may herald development of post-transplant lymphoproliferative disease (PTLD). The objective of this study was to characterize the epidemiology and risk factors for EBV DNAemia and to describe the impact of these factors on development of PTLD in a cohort of SOT recipients.MethodsWe retrospectively examined adult (≥18 years) SOT recipients between 1/1/2015 to 12/31/2019. Demographics, immunosuppression, and clinical outcomes were examined. Subjects were stratified as having a positive (EBV viral load > 200 copies/mL) or negative viral load. Sustained EBV DNAemia was defined as EBV DNA detection ( > 200 copies/mL) on at least 3 consecutive samples. Categorical variables were analyzed using chi-square or Fishers exact test. Mann-U Whitney test was used for continuous variables. Results442 SOT recipients (258 kidney, 141 liver, 22 kidney-pancreas (KP), 15 kidney-liver, 6 pancreas) were examined. Most subjects (430, 97%) were EBV intermediate risk (recipient (R)+). 8 subjects (2%) were EBV high risk (donor (D)+, R-) and 4 (1%) were EBV low risk (D-/R-). EBV viral loads were obtained in 177/442 (40.0%) recipients. DNAemia was detected in 18/442 subjects (4.1%). It was most common in pancreas recipients(1/6; 16.7%) compared with kidney (10/258; 3.9%), liver (5/141; 3.5%), and KP recipients (1/22; 4.5%). DNAemia was most frequently observed in the D+/R- (3/8; 37.5%) group compared to intermediate risk (R+) (15/430; 3.5%) and D-/R- (0%) groups. Median time to EBV viral load detection was 14 months (range 3-60). In univariate analysis, EBV high-risk serostatus was the factor most strongly associated with development of EBV DNAemia (p< 0.001). Sustained DNAemia (median viral load 1829 copies/mL; peak viral load 1.9 million copies/mL) was observed in 8 subjects (1.8%). PTLD developed in 6 subjects (1.4%); 50% had sustained DNAemia prior to diagnosis.ConclusionWhile uncommon, development of sustained EBV DNAemia was associated with subsequent development of PTLD in our cohort of adult SOT recipients. These data provide guidance for identifying subjects at risk for PTLD above and beyond baseline EBV high-risk serostatus. Disclosures All Authors: No reported disclosures

EBV Positive DLBCL Is Effectively Treated with Commercial CAR T Cell Therapy but Is Associated with Higher Rates of Severe Immune Effector Cell Associated Neurologic Syndrome (ICANS): A Single Institution Experience

Introduction:CAR T-cell therapy (CART) has revolutionized the treatment landscape of aggressive B cell lymphoma (aBCL) in the relapsed/refractory (r/r) setting, including high-risk groups such as high grade B cell lymphoma (HGBCL), primary refractory disease, and chemo-resistant relapses. To date, however, there has been no data reported, on the outcomes of patients with Epstein-Barr virus positive (EBV+) DLBCL, NOS. EBV+ DLBCL, NOS is a high-risk group of aBCL characterized by the incorporation of EBV into the malignant cells, with the chronic inflammation associated with EBV infection being thought to contribute to DLBCL pathogenesis. It has been shown to be less responsive to standard chemotherapy with high relapse rates and overall poor prognosis. In the present study, we analyze outcomes and toxicities of patients with EBV+ DLBCL, NOS treated with CD19CART.Methods:We retrospectively analyzed charts of patients treated with commercially available CD19CAR T-cell products for the treatment of aBCL at City of Hope (COH) between April 2018 and March 2021. EBV status was determined by immunohistochemical stains from EBER in situ hybridization on paraffin embedded tissues. Patients were included in the analysis if they had positive or negative EBER staining, referred as EBV+ or EBV-, respectively. The investigation was approved by the COH IRB. Cytokine release syndrome (CRS) and immune effector cell associated neurologic syndrome (ICANS) were graded using ASTCT criteria (Lee et al. BBMT 2019). Demographics were analyzed with descriptive statistics. Univariate analysis was performed by Chi Square test.Results:Seventy-three patients had tumor samples that were evaluable for analysis, 92% (n=67) were negative for EBER and 8% (n=6) were positive for EBER. In the entire cohort, the median age was 62 years (range, 21-90), and included 18% (n=13) transformed follicular lymphoma, 73% (n=53) DLBCL, NOS, and 10%(n=7) DH. The median number of prior lines of therapy was 2 (range, 2-6) and 15% (n=11) patients had prior autologous stem cell transplant.Comparing the EBV+ patients to the EBV-, the CR rate was 67% (n=4) vs 58% (n=34), with 16% (n=1) PR vs 27% (n=15) (ORR 83% vs 74%). With a median follow up of 17.0 months (range, 3.6-38.3), 33% (n=2) of EBV+ patients had progressed vs 42% (n=28). In the EBV+ DLBCL, NOS group, 3 CR patients had absence of detectable EBV PCR in the peripheral blood at the time of CART; 1 CR patient did not have clinical indication for EBV viral load (VL) evaluation (EBV PCR not measured); 1 PR and 1 PD patients had detectable viremia. At the time of analysis, 31% (n=21) of EBV- patients died vs 33% (n=2) of EBV+ patients, both with non-CR response to CAR T and persistent EBV VL during therapy. In terms of toxicity, overall CRS rates were similar with 83% (n=5) in the EBV+ patients and 86% (n=58) in EBV- patients. Although overall incidences of ICANS were similar between the two groups (50% [n=3] vs 45% [n=30]), grade 3 ICANS was higher in EBV+ patients (50% [n=3]) than in EBV- patients( 7.5% [n=5]; p = 0.02). EBV VL did not correlate with CRS or neurotoxicity.Conclusion:We present a retrospective analysis of EBV+ DLBCL, NOS patients treated with CART at our institution. Based on our analysis, despite the limited number of cases, the CART is an effective therapy for EBV+ DLBCL,NOS with response rates similar to what has been reported in the literature for other aBCL groups. Responses appear to be improved if EBV VL is undetectable at the time of CART. Interestingly, we demonstrate a statistically significant higher rates of grade 3 ICANS in patients with EBV+ DLBCL,NOS. Our data suggests that close monitoring for ICANS during CART should be considered in EBV+ DLBCL patients with early therapeutic intervention to prevent severe toxicity. Further investigation of a larger cohort of EBV+ DLBCL patients, as well as a deeper analysis of inflammatory markers and EBV viremia in these patients undergoing CART may provide further insight to response and toxicity profile. DisclosuresNikolaenko: Rafael Pharmaceuticals: Research Funding; Pfizer: Research Funding. Herrera: Seagen: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Takeda: Consultancy; ADC Therapeutics: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Tubulis: Consultancy; Kite, a Gilead Company: Research Funding; Karyopharm: Consultancy; Gilead Sciences: Research Funding. Budde: Mustang Bio, Inc: Research Funding; Gilead: Consultancy; Merck, Inc: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; IGM Biosciences: Research Funding; Roche: Consultancy; BeiGene: Consultancy; Novartis: Consultancy. Shouse: Kite Pharma: Speakers Bureau; Beigene: Honoraria.

The Association of EBV and HHV-6 Viral Load with Different NK and CD8+ T Cell Subsets in The Acute Phase of Relapsing-Remitting Multiple Sclerosis.

Objective Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) are believed to involve in multiple sclerosis (MS) pathogenesis. Natural killer (NK) and CD8+ T cells have essential roles in handling viral infections and their phenotypic and functional properties may be influenced following exposure to viral infections. Here, we investigated the association of NK and CD8+ T cells subpopulations frequency with EBV and HHV-6 viral load in MS patients. Materials and Methods In this case-control study, EBV and HHV-6 viral load were evaluated in plasma of newly diagnosed relapsing-remitting MS (RRMS) patients at relapse phase (n=23), who were not on disease-modifying therapy (DMT), and sex- and age-matched healthy controls (n=19) using real-time polymerase chain reaction (PCR). The frequency of NK and CD8+ T cells subsets were assessed by CD27, CD28, CD45RO, CD56, and CD57 markers using flow cytometry. Results Despite the increased level of EBV viral load in RRMS patients compared to the control group, there was no statistically significant difference in EBV and HHV-6 copy numbers between the studied groups. In addition, a significant decrease was observed in the percentages of CD56brightCD57- and CD56dimCD57+CD8lowCD45RO- NK cells in RRMS patients in comparison to healthy controls. Analysis of CD8+ T cell subsets showed a substantially high proportion of CD27+CD28+CD45RO+CD57-CD8hiT cells in patients at relapse phase compared to controls. The frequency of NK and T cells subtypes was not associated with EBV and HHV6 plasma viral loads. ConclusionThese findings further highlight the variation of NK and CD8+ T cells subsets frequency in clinically active RRMS patients. Since the composition of cells was not associated with EBV and HHV-6 viral load, perhaps other viral infections may be involved in altered NK and CD8+ T cells subpopulation. Larger cohort studies are needed to confirm these results.

Management of Cytomegalovirus, Epstein-Barr Virus, and HIV Viral Load Quality Control Data Using Unity Real Time.

ABSTRACTQuality control (QC) rules (Westgard rules) are applied to viral load testing to identify runs that should be reviewed or repeated, but this requires balancing the patient safety benefits of error detection with the cost and inefficiency of false rejection. In this study, we identified the total allowable errors (TEa) from the literature and utilized a commercially available software program (Unity Real Time; Bio-Rad Laboratories) to manage QC data, assess assay performance, and provide QC decision support for both FDA-approved/cleared (Abbott cytomegalovirus [CMV] and HIV viral load) as well as laboratory-developed (Epstein-Barr virus [EBV] viral load) assays. Unity Real Time was used to calculate means, standard deviations (SDs), and coefficient of variation (CV; in percent) of negative, low-positive, and high-positive control data from 73 to 83 days of testing. Sigma values were calculated to measure the test performance relative to a TEa of 0.5 log10. The sigma value of 5.06 for EBV predicts ∼230 erroneous results per million individual patient tests (0.02% frequency), whereas sigma values of >6 for CMV (11.32) and HIV (7.66) indicate <4 erroneous results per million individual patient tests. The Unity Real Time QC Design module utilized these sigma values to recommend QC rules and provided objective evidence for loosening the laboratory’s existing QC rules for run acceptability, potentially reducing false rejection rates by 10-fold for the assay with the most variation (EBV viral load). This study provides a framework for laboratories, with Unity Real Time as a tool, to evaluate assay performance relative to clinical decision points and establish optimal rules for routine monitoring of molecular viral load assay performance.

O01 Post-transplant lymphoproliferative disorder (PTLD) in a patient with rheumatoid arthritis

O01 Post-transplant lymphoproliferative disorder (PTLD) in a patient with rheumatoid arthritis

Non-Hodgkin lymphoma after pediatric kidney transplantation

Non-Hodgkin lymphoma (NHL) that develops after kidney transplantation belongs to post-transplant lymphoproliferative disorders (PTLD) occurring with an incidence of 2–3%. Most pediatric cases are related to primary infection with Epstein-Barr virus (EBV), able to transform and immortalize B cells and widely proliferate due to the lack of relevant control of cytotoxic T cells in patients receiving post-transplant immunosuppression. NHL may develop as a systemic disease or as a localized lesion. The clinical pattern is variable, from non-symptomatic to fulminating disease. Young age of transplant recipient, seronegative EBV status at transplantation, and EBV mismatch between donor and recipient (D+/R-) are regarded as risk factors. Immunosuppression impacts the development of both early and late NHLs. Specific surveillance protocols, including monitoring of EBV viral load, are used in patients at risk; however, detailed histopathology diagnosis and evaluation of malignancy staging is crucial for therapeutic decisions. Minimizing of immunosuppression is a primary management, followed by the use of rituximab in B-cell NHLs. Specific chemotherapeutic protocols, adjusted to lymphoma classification and staging, are used in advanced NHLs. Radiotherapy and/or surgical removal of malignant lesions is limited to the most severe cases. Outcome is variable, depending on risk factors and timing of diagnosis, however is positive in pediatric patients in terms of graft function and patient survival. Kidney re-transplantation is possible in survivors who lost the primary graft due to chronic rejection, however may be performed after at least 2–3 years of waiting time, careful verification of malignancy-free status, and gaining immunity against EBV.

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.

S2703 A Rare Case of EBV Reactivation in an Immunocompetent Patient

Introduction: Hepatic involvement during an active primary Epstein-Barr virus (EBV) infection often results in mild, self-resolving elevation of transaminases and is associated with infectious mononucleosis (IM). EBV rarely reactivates, and most cases occur in immunocompromised individuals. We present a rare case of EBV reactivation in an immunocompetent patient only presenting with cholestatic liver injury. Case Description/Methods: A 19-year-old female with a history of resolved EBV IM one year prior presented with dark urine and jaundice. She had no fever, abdominal pain, nausea, vomiting, weight changes or night sweats. She had no history of illicit drug, tobacco or alcohol use, recent travel, or sick contacts. Her medications were loratadine and an OCP;she denied use of acetaminophen or herbal supplements. Family history was unrevealing. On exam she was jaundiced but otherwise normal. Initial laboratory tests showed AST 97 U/L, ALT 75 U/L, ALP 165 U/L, T-BILI 8.1 mg/dL, D-BILI 5.7 mg/dL. CT abdomen revealed splenomegaly. Work up revealed ASMA titer (1:40), but otherwise negative/ normal ANA, IgG, AMA, HCV Ab, HBV serologies, HAV IgM, CMV PCR, iron studies, tick borne serologies, SARS CoV-2 PCR, A1AT and ceruloplasmin. On the second day, the T-BILI peaked to 12.4 mg/dL, AST 151 U/L, ALT 131 U/L, ALP 237 U/L. (Fig.1) EBV serologies were positive (EBV VCA IgG >750.0 U/ml, EBV VCA IgM >160.0 U/ml), EBV viral load of 23,500 copy/ ml and negative monospot test. On day four, the LFTs improved and she was discharged. One month later LFTs had normalized, and she was asymptomatic. Discussion: EBV reactivation causing cholestatic liver injury is extremely rare in immunocompetent individuals. Furthermore, EBV-induced hepatitis is usually associated with IM but our patient's sole complaint was jaundice and dark urine. Interestingly, she had mononucleosis due to EBV one year prior with unremarkable LFTs. The fact that the hepatitis was caused by the reactivation of the virus instead of a primary infection may be the reason for lack of other symptoms. It is unclear whether EBV reactivation increases the risk of hepatitis. The diagnosis can be made without a liver biopsy if thorough work up for hepatitis is negative and the EBV serologies are positive. The disease is often self-limited and the use of antivirals is controversial and thus there is no clear indication. Our case demonstrates that EBV reactivation presenting without the 'classic' IM symptoms may be under-recognized as a cause of cholestatic liver injury. (Figure Presented).

Epidemiological risk factors associated with primary infection by Epstein–Barr virus in HIV-1-positive subjects in the Brazilian Amazon region

To identify the prevalence and risk factors for primary Epstein–Barr virus (EBV) infection in human immunodeficiency virus (HIV)-1-positive adult treatment-naïve patients between January 2018 and December 2019 in a state of the Brazilian Amazon region. A total of 268 HIV-1 positive patients and 65 blood donors participated in the study. Epidemiological data were obtained from medical records and through a designed questionnaire. EBV infection was screened by the semiquantitative detection of anti-viral capsid antigen (VCA) EBV IgM and IgG, followed by molecular detection of the EBNA-3C gene. The plasma viral loads of HIV-1 and EBV were quantified using a commercial kit. The prevalence of primary coinfection was 7.12%. The associated risk factors were education level, family income, history of illicit drug use and sexually transmitted infections, homosexual contact and condom nonuse. Approximately 58.5% had late initiation of highly active antiretroviral therapy, which influenced the risk of HIV-EBV 1/2 multiple infection (odds ratio (OR): 4.76; 95% CI 1.51–15.04) and symptom development (p = 0.004). HIV viral load was associated with patient age (OR: 2.04; 95% CI 2.01–2.07; p = 0.026) and duration of illicit drug use (OR: 1.57; 95% CI 1.12–2.22; p = 0.0548). EBV viral load was associated with younger age (OR: 0.82; 95% CI 0.79–1.03; p = 0.0579). The replication of both viruses was associated with symptom development (HIV = OR: 2.06; 95% CI 1.22–3.50; p = 0.0073; EBV = OR: 8.81; 95% CI 1–10; p = 0.0447). The prevalence of HIV/EBV coinfection was lower than that observed in other studies, and social vulnerability and promiscuous sexual behavior were associated risk factors. A long time of HIV-1 infection, without therapy, influenced the risk of coinfection and disease progression. The viral loads of both viruses may be associated with some epidemiological aspects of the population.

Acute Epstein‐Barr virus associated haemophagocytosis in an Asian female: What is the diagnosis?

Acute <scp>Epstein‐Barr</scp> virus associated haemophagocytosis in an Asian female: What is the diagnosis?

Novel Mutations in RASGRP1 Are Associated with Immune Dysregulation and Predispose to EBV-Induced Lymphoma

& I. Somekh and B. Marquardt contributed equally§ C. Klein and R. Somech contributed equallyBackgroundRAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary immunodeficiency disorder in one patient (Salzer et al, Nat Immunol 2016). Clinically, the disease was characterized by recurrent episodes of pneumonia, bronchiectasis, severe failure to thrive, CD4 T-cell lymphopenia, elevated TCRγδ cell proportions as well as low-grade Epstein-Barr virus (EBV)-associated B cell lymphoma. Immunologically, RASGRP1 deficiency is associated with defective proliferation and activation of T and B cells, respectively. Here, we report 3 novel patients with RASGRP1 deficiency from 2 unrelated consanguineous kindred.MethodsOne patient of Turkish origin (P1) and 2 Palestinian 1st degree cousins (P2, P3) were evaluated. Genetic analysis using whole exome sequencing was conducted. Immunological and biochemical assays were performed on primary patient material and genetically engineered T-cell lines using CRISPR/Cas9.ResultsClinical findings for all three patients included failure to thrive, hepatosplenomegaly, lymphadenopathy, and recurrent pulmonary infections resulting in bronchiectasia. Two patients had overt auto-immunity (AIHA and autoimmune thrombocytopenia, respectively). One patient had increased anti-nuclear antibody titers. One patient (P1) developed EBV-associated diffuse large B cell lymphoma. He underwent autologous hematopoietic stem cell transplant due to relapse after combined rituximab and NHL-BFM-90 treatment protocol and is currently in remission at the age of 14 years. P2 had deceased at the age of 3 years due to disseminated intravascular coagulation. P3 developed diffuse large B cell lymphoma at the age of 3 years, treated with R-CHOP protocol and is currently in remission.Immune workup of P2 and P3 revealed low levels of T-cell receptor excision circles (TREC), an abnormal T-cell receptor (TCR) Vß repertoire and a markedly increased proportion of TCRγδ cells. Phytohemagglutinin (PHA)-induced lymphocyte proliferation and anti-CD3 mitogen proliferation responses were decreased. EBV-viral load in peripheral blood was elevated for both patients, as measured by PCR.Genetic analysis by whole exome sequencing identified an autosomal recessive homozygous mutations in RASGRP1. P1 had an inversion (c.649_650inv; p.Glu217Ser) and P2/P3 had a deleterious frameshift mutation (c.1111_1114del; p.Asp371Ilefs*7). The variants were confirmed by Sanger sequencing and segregated with the disease phenotype. To confirm the functional relevance of the RASGRP1 mutations, we engineered Jurkat cells via CRISPR/Cas9 editing. RASGRP1 -/- Jurkat cells showed decreased phosphorylation of ERK1/2 and decreased CD69 expression in comparison to wildtype cells. Jurkat cells with RASGRP1 knockin mutants are currently under analysis.ConclusionsRASGRP1 deficiency is a novel primary immunodeficiency disorder associated with immune dysregulation and susceptibility to EBV-induced B-cell malignancies. Early diagnosis of RASGRP1 deficiency is critical. Even though clinical remission of EBV-lymphoma may be reached using high dose chemotherapy regimens, long term cures may only be reached by allogeneic hematopoietic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Incidence and Risk Factors of Epstein-Barr Virus Viremia in Patients Undergoing Haploidential Transplantation with Post Translant Cyclophosphamide

INTRODUCTIONHaploidentical stem cell transplants are increasingly performed for a wide variety of malignant and non-malignant hematological conditions. Epstein - Barr virus (EBV) viremia and subsequent development of Post-Transplant Lymphoproliferative Disorder (PTLD) are potential adverse sequelae of the transplantation process and associated immunosuppression. Traditionally, haploidentical transplants have been associated with increased risk of EBV reactivation and subsequent PTLD. This was largely due to graft versus host disease (GVHD) prophylaxis protocols using intense immunosuppression with either Ex-vivo T-cell depletion or in-vivo T- cell depletion with anti-thymocyte globulin (ATG) and Alemtuzumab. However, most haploidentical transplants in recent years have utilized post-transplant cyclophosphamide (PTCy) for GVHD prophylaxis. Rituximab has been used for preemptive treatment of EBV viremia/PTLD, but there is scarce data on its use in this setting, as well as the risk factors and outcomes of EBV viremia associated with haploidentical transplants with PTCy. RESULTSA total of 63 patients with hematological malignancies received haploidentical transplant at our institution from 2013 from 2016. Patient characteristics are summarized in Table 1.All patients received EBV monitoring using plasma viral copies once weekly starting 20 days post-transplant. EBV monitoring continued for 6 months after immunosuppression ended or when CD4 count >200. Among these 63 patients, 18 (28.5%) became EBV DNA positive upon follow-up. Per institutional protocol, EBV titer was confirmed by repeat testing when viral load was 1000 copies/mL and treatment with Rituximab was initiated if repeat titer was >1000 copies/ml.The median time to detection of EBV viremia from the time of transplant was 99 days (range: 20-277). The median peak level for the EBV reactivation cohort was 7650 viral copies /ml. The median time from detection of viremia to peak viremia was 1.5 days (range 0-253).8 of the 18 (44.4%) patients that developed EBV viremia required Rituximab due to rising or persistent EBV viremia. The median EBV level at initiation of therapy was 31,000 copies/ml for the 8 patients that received Rituximab treatment. EBV seronegativity was achieved in 6 patients (75%) with treatment at a median of 13.5 days (range 3-26). The median number of Rituximab cycles administered was 1.5 (range: 1-16). None of the patients in either cohort developed PTLD or other EBV associated diseases.A univariate analysis was performed comparing EBV reactivation cohort and no EBV reactivation cohort to look for risk factors predisposing to EBV reactivation (Table 1) There were no statistically significant differences seen in the variables studied other than patients not in complete remission at the time of transplant were statistically more likely to develop EBV viremia. 9 out of 18 patients (50%) that developed EBV viremia were not in complete remission at the time of transplant, whereas 11 out of the 45 patients (24.44 %) that did not develop EBV viremia were in an incomplete remission at the time of transplant (p value: 0.05) (Table 1).With a median follow up of469 days, there were a total of 22 deaths among the 45 (48.9 %) patients that had no viremia, and 8 deaths amongst the 18 (44.4 %) patients that had viremia (p value: 0.75). There was no statistically significant difference in graft versus host disease (GVHD) incidence (p value: 0.79) or overall survival (555 days for patients with EBV reactivation versus not reached for patients without EBV reactivation, p value: 0.45) between these two groups (p value: 0.79) CONCLUSIONSIn this retrospective study assessing risk factors and outcomes of EBV viremia in patients following haploidentical transplants, we observed a 28.5 percent incidence of EBV viremia. Rituximab was effective in preemptive treatment of EBV viremia. However, no cases of PTLD were observed, even in those patients with sustained elevation in EBV titers. It is unclear how to manage asymptomatic elevations in viral titers after allogeneic transplantation in patients who are otherwise at low risk for development of PTLD. Previous data suggests that even high levels of EBV viremia lacks specificity for prediction of PTLD in post-transplant setting. Given these results, the utility of routine monitoring of EBV viral load after haploidentical transplant should be questioned in those who have received PTCy. [Display omitted] DisclosuresLin:Jazz Pharmaceuticals: Consultancy. Ganguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau.

Genetic Background of Chronic Active Epstein-Barr Virus Disease

IntroductionEpstein-Barr virus (EBV) is associated with various benign and malignant diseases. Chronic active EBV disease (CAEBV) is a rare EBV-associated disease characterized by persistent EBV infection in natural killer (NK) and/or T cells, which is mainly prevalent in East Asia. While patients with CAEBV initially present with persistent or recurrent symptoms similar to ones present in infectious mononucleosis (IM), they often develop peripheral NK/T-cell lymphoma or multiple organ failure in the long run, and prognosis is dismal.Numerous aspects of the molecular pathogenesis of CAEBV are unresolved; it is unclear whether this disease is a clonal disorder. Although several case reports indicate the presence of primary immunodeficiency (PID) in patients with persistent EBV disease, the prevalence of PID or other inherited diseases in patients with CAEBV remains unclear. To reveal the genetic background of CAEBV, we performed whole-exome and targeted sequencing.Patients and MethodsWe studied 83 patients with CAEBV. The diagnosis of CAEBV was based on the fulfillment of the following criteria: (1) persistent or recurrent symptoms similar to those present in IM, (2) increased quantities of EBV viral load in either affected tissues or peripheral blood, and (3) exclusion of other possible diagnoses. The Institutional Review Board of Nagoya University Graduate School of Medicine approved this study.We performed whole-exome sequencing (WES) of T-, B-, and NK-cell subsets. Subsequently, we performed targeted gene sequencing of 605 genes associated with PID, inherited blood diseases, and hematological malignancies.ResultsRegarding germline mutations, we identified two patients who carried nonsense mutations in EVC2, which led to the diagnosis of Weyers acrofacial dysostosis (WAD). We also identified a patient who carried POLH mutations, which led to the diagnosis of the variant type of xeroderma pigmentosum, and a patient who carried an APC mutation, which led to the diagnosis of familial adenomatous polyposis. Mutations leading to the diagnosis of PID were not identified in any patient.WES led to the identification of at least one somatic mutation in 58% of patients. We identified DDX3X (n= 14; 16%), KMT2D (n= 4; 4.8%), BCOR / BCORL1 (n= 3; 3.6%), KDM6A (n= 3; 3.6%), and TET2 (n= 2; 2.4%) as undergoing recurrent mutations.We analyzed multiple EBV-infected cell subsets within a patient. One patient (UPN125) developed CAEBV in which both T and NK cells were positive for EBV DNA. The patient's CD4+ and CD56+ cells carried 21 and 27 mutations, respectively, of which 8 were shared including a DDX3X p.Arg534Cys driver mutation. Similar to UPN125, CD3+ and CD56+ cells of UPN404 carried a shared DDX3X p.Thr198Pro mutation and private mutations. CD19+ cells of UPN404 also carried the identical DDX3X mutation.In the survival analysis, patients carrying DDX3X mutations at diagnosis demonstrated a significantly worse OS compared with others (5-year OS: 40.6% vs. 78.6%; P= 0.007). Patients carrying KMT2D mutations also demonstrated poor prognosis (5-year OS: 50.0% vs. 73.9%; P= 0.02).DiscussionIn the present study, we found that CAEBV was a malignant disease carrying somatic driver mutations, the spectrum of which showed similarity with that of extranodal NK/T-cell lymphoma (ENKTL) and Burkitt lymphoma. DDX3X mutations and other shared mutations, which were present in multiple cell subsets in a patient, strongly suggest the presence of common ancestry, possibly lymphoid progenitor cells, which acquired driver mutations.We identified the presence of somatic DDX3X mutations as a marker of poor prognosis. A prospective study can be designed using genetic testing for DDX3X mutations and subsequent interventions, possibly chemotherapies used for ENKTL and early hematopoietic stem cell transplantation, to improve patient outcome.Furthermore, we identified several germline mutations. WAD caused by EVC / EVC2 mutations is characterized by the malformation of mesoderm and ectoderm caused by decreased hedgehog signaling mediated via EVC/EVC2 . Together with POLH and APC mutations, EVC / EVC2 mutations may be a genetic predisposition to CAEBV.In conclusion, we revealed the genetic background of CAEBV, which will lead to further clarification of its molecular pathogenesis and the development of therapeutic strategy. Clinical trials utilizing genetic diagnosis and stratified therapy are warranted. DisclosuresNo relevant conflicts of interest to declare.

Combination of Gemcitabine and Thymosin alpha 1 exhibit a better anti-tumor effect on nasal natural killer/T-cell lymphoma

BackgroundNasal natural killer/T-cell lymphoma (NNKTL) is an aggressive and poor prognostic malignant tumor along with high-level infection of Epstein-Barr virus (EBV). Gemcitabine (Gem) and Thymosin alpha 1 (Tα1) exert an anti-tumor effect in various cancers. However, the effect of the combination of Gem and Tα1 in NNKTL remains unknown. MethodsSNK6 cells were treated with Gem, Tα1 and Gem plus Tα1 for 48 h. The expression levels of EBV and inflammatory factors were measured by qRT-PCR assay. The effect of Gem and Tα1 on cell viability, proliferation, apoptosis, autophagy was detected by CCK-8, colony formation, flow cytometry, autophagic flux measurement, respectively. Western blot was used to evaluate the expression of proteins related to epithelial-mesenchymal transition (EMT), apoptosis and autophagy. In vivo xenograft models were used to further verify the roles of Gem and Tα1. Tumors were removed for weight measurement, H&E and IHC staining. ResultsWe identified that the half maximal inhibitory concentration (IC50) of Gem and Tα1 was 116.5 μmol/ml and 1.334 μmol/ml. Alone or combined administration of Gem and Tα1 dramatically attenuated the EBV viral load and promoted inflammatory factors expression in SNK6 cells, among which the combination of Gem and Tα1 treatment showed the most significant effect. Besides, combination treatment with Gem and Tα1 markedly inhibited cell growth and EMT progress, and enhanced apoptosis and autophagy. Similarly, Gem combined with Tα1 suppressed tumor growth, promoted apoptosis and autophagy in vivo. Additionally, combination treatment with Gem and Tα1 inhibited PI3K/AKT/mTOR pathway. ConclusionIn summary, combination administration of Gem and Tα1 suppressed the progression of NNKTL in vivo and in vitro. Our study provided an effective therapeutic strategy potentially for the clinical treatment of NNKTL.

Risk factors for post-transplant Epstein-Barr virus events in pediatric recipients of hematopoietic stem cell transplants.

Epstein-Barr virus (EBV) can cause severe disease following hematopoietic stem cell transplant (HSCT), including post-transplant lymphoproliferative disorder (PTLD). The objective was to analyze risk factors associated with post-transplant EBV outcomes among pediatric allogeneic HSCT recipients. We used data from 156 pediatric allogeneic HSCT recipients enrolled in the Canadian multicenter TREASuRE study. Cox and Prentice-Williams-Petersen models were used to analyze risk factors for post-transplant EBV events including occurrence and recurrence of EBV DNAemia, increase in EBV viral load (EBV-VL), and preemptive use of rituximab, an effective therapy against PTLD. Females were at higher risk for increasing EBV-VL (adjusted hazard ratio (HR)=2.83 [95% confidence intervals (CI): 1.33-6.03]) and rituximab use (HR=3.08 [1.14-8.30]), but had the same EBV DNAemia occurrence (HR=1.21 [0.74-1.99]) and recurrence risks (HR=1.05 [0.70-1.58]) compared to males. EBV DNAemia was associated with recipient pre-transplant EBV seropositivity (HR=2.47 [1.17-5.21]) and with graft from an EBV-positive donor (HR=3.53 [1.95-6.38]). Anti-thymocyte globulin (ATG) was strongly associated with all EBV outcomes, including the use of rituximab (HR=5.33 [1.47-19.40]). Mycophenolate mofetil (MMF) significantly decreased the risk of all EBV events including the rituximab use (HR=0.13 [0.03-0.63]). This study in pediatric allogeneic HSCT patients reveals a reduced risk of all EBV outcomes with the use of MMF. Risk factors for EBV events such as EBV-VL occurrence and recurrence include EBV positivity in the donor and recipient, and use of ATG, whereas risk factors for the most severe forms of EBV outcome (EBV-VL and the use of rituximab) include female sex and ATG use.

MO971LYMPHOPROLIFERATIVE DISEASE AFTER KIDNEY TRANSPLANTATION: DESCRIBING OUR EXPERIENCE

Abstract Background and Aims Post-transplant lymphoproliferative disorders (PTLD) are one of the most common malignancies in kidney transplant (KT) recipients. Immunosuppressive therapy and Epstein Barr Virus (EBV) play a main role in their pathogenesis. Method In this study we retrospectively analyze the characteristics, clinical evolution and treatments of a group of KT recipients performed between 1986 and 2020 in a single center. Results We included 31 patients (64.5% males). Polycystic kidney disease was the most frequent cause of renal failure. Before KT a 6.5% of the patients presented another malignancy, 10% were EBV seronegative and one received immunosuppressive therapy secondary to his primary disease. Mean age at KT was 43±12 years. 68% of the KT came from brain-dead donors. The most frequent immunosuppressive regime consisted in tacrolimus, mycophenolic acid and prednisone (61.3%). Basiliximab and Timoglobulin were used in the same proportion for the induction therapy (22.6%). Before PTLD appearance the immunosuppressive therapy was reduced in the 54.8% of the recipients. 13% of them presented acute allograft rejection. The majority of PTLD were diagnosticated between 2016 and 2020. Median time to develop PTLD was 13 years. 54.3% of the patients presented with extranodal involvement. Although all the patients positivized EBV serology, 60% of them had undetectable EBV viral load. The main therapeutical strategy after PTLD consisted in a reduction of the immunosuppressive therapy. In this way, 28.6% of the recipients was treated with monotherapy with a calcineurin inhibitors and 21.5% with monotherapy with a mTOR inhibitor. In other hand 16.7% received a combination of tacrolimus with a mTOR inhibitor. Rejections were not observed in our group and all the patients presented a preserved kidney function at the end of follow up. Four recipients died because of PTLD. The remaining 27 presented a complete response or stabilization of the disease. Conclusion Most of the PTLD were detected between 2016-2020. The time from transplantation to PTLD appearance was long, being EBV viral load negative in the majority of the cases. Graft survival after chemotherapy and reduction of immunosuppressive therapy was excellent, with a low risk of rejection and a good prognosis for hematologic disease. It is possible that a reduction in immunosuppression in selected patients could prevent the development of PTLD.

Natural History of Epstein-Barr Virus Replication and Viral Load Dynamics after Alemtuzumab-Based Allogeneic Stem Cell Transplantation

Epstein-Barr virus (EBV) load monitoring after allogeneic hematopoietic stem cell transplantation (HSCT) enables earlier detection of EBV replication and often serves as a trigger for preemptive therapies aimed at reducing EBV-related diseases. Our institutional strategy is to treat patients with clinical signs of EBV-related disease accompanied by a rising viral load, rather than to intervene based solely on viral load. This affords an opportunity to study the natural history of EBV replication and to assess whether our strategy reduces overtreatment without compromising outcomes. The objectives of the present study were to assess the natural history of untreated EBV replication in patients who underwent an alemtuzumab-based allogeneic HSCT and to examine whether our clinical strategy reduced overtreatment without compromising patient outcomes. In this retrospective single-center observational study of 515 consecutive patients (age ≥18 years) undergoing T cell-depleted allogeneic HSCT incorporating alemtuzumab, patients underwent surveillance monitoring for EBV by quantitative PCR in the peripheral blood at least weekly up to 100 days post-transplantation and longer if they remained on immunosuppressive therapy. The cumulative incidence of EBV detection and EBV-related disease were assessed. Among the 515 patients, 192 had EBV DNA detectable on ≥1 occasion, with a cumulative incidence of 35.8% (31.8% to 40.4%), although this remained below the limit of quantification in 93 patients. The median time to first detection was 89.5 days (range, 0 to 2254 days). The incidence was higher in recipients of sibling donor transplants (45.4% versus 30%; P=.00021) compared with recipients of unrelated donor transplants. Twenty patients developed EBV-related disease (cumulative incidence, 3.9%). Two patients had immunosuppression reduction alone, 18 received rituximab, and 5 required additional therapies. Five patients died from post-transplantation lymphoproliferative disorder, all of whom had received rituximab. The positive predictive value of EBV load for disease was higher in the unrelated donor cohort but remained <75% regardless of EBV threshold (57.1% to 72.7%). The cumulative incidence of EBV-related disease in our study (3.9%) is comparable to that reported in other studies incorporating alemtuzumab, and our clinical strategy reduced overtreatment in this patient population. PCR-based surveillance strategies have limitations, as reflected in the relatively low sensitivity of the assay coupled with the low positive predictive value, which may influence the potential choice of a threshold for preemptive intervention. We conclude that it remains unclear whether treatment based on a rising EBV viral load alone provides superior overall results to treatment based on the development of clinical signs of EBV-related disease in the context of a rising viral load.

Identification of Epstein-Barr Virus Related Risk Factors for the Occurrence of Post-Transplant Lymphoproliferative Disorder in Lung Transplant Recipients

<h3>Purpose</h3> Post-transplant lymphoproliferative disorder (PTLD) is the second most common malignancy in solid organ transplantation. Based on recent registry data, ≈ 1.1% of lung transplants (LTx) develop PTLD within the first year. PTLD confers significant morbidity and mortality and has been associated with Epstein-Barr virus (EBV) infection. The threshold for EBV viremia predictive of PTLD is unknown. In the current study we examined risk factors for PTLD development and the relationship between EBV viremia and PTLD in LTx was assessed. <h3>Methods</h3> A single-center, retrospective review of all LTx recipients from 1/1/2009 - 7/31/2019 with EBV viremia was conducted. All patients (pts) with at least one positive EBV PCR test were reviewed for EBV viremia progression and PTLD occurrence. PTLD was confirmed by review of oncology notes, biopsy results, computed tomography and positron emission tomography scans. <h3>Results</h3> One hundred and forty-seven pts were included. Thirty-four (24.8%) underwent work-up for PTLD, of whom ten were diagnosed with PTLD. Median time from transplant to EBV viremia was 368 days (IQR 111-1201) with PTLD diagnosis occurring at a median of 521 days (IQR 150-741) after LTx. The PTLD(+) group had a higher baseline median EBV PCR of 2613 copies/mL (IQR 680-6099) vs 528 copies/mL (IQR 369-930) in PTLD(-) pts and an increased number of positive PCRs; 3.5 (IQR 2-10) vs 1 (IQR 1-2). In patients with EBV IgG data available, (<i>n</i>=66), Donor(+)/Recipient(-) pts were more likely to develop PTLD than other groups, RR = 18.3 (95% CI 3.93, 85.32); p<0.001. A cut-point analysis of EBV viral load determined that pts with a baseline EBV viral load >1930 copies/mL had a significantly higher risk of PTLD development, RR = 31.03 (95 %CI 9.44, 102.02); p<0.001. <h3>Conclusion</h3> EBV mismatch, higher viral loads, and persistence of viremia may be indicative of PTLD development. Patients with an EBV viral load >1930 copies/ml may benefit from closer surveillance.

Epstein–Barr Virus (EBV) Viral Load in Tumor Cells Did Not Predict Tumor Extensiveness in Nasopharyngeal Cancer

Background: Nasopharyngeal cancer is commonly associated with Epstein–Barr virus (EBV) infection, especially undifferentiated non-keratinized histology. EBV DNA quantification through nasopharyngeal brushing was previously reported to be not related to disease stage. This study aimed to reinvestigate the relationship of EBV viral load in tumor tissue with tumor extensiveness by more accurate EBV DNA quantification through microscopically confirmed tumor cells from nasopharyngeal biopsy. Method: The specimens for EBV DNA quantification were derived from histopathology slides which were pre-treated following the QIAsymphony® SP protocol for tissue DNA extraction. Then, the extracted DNA underwent real-time polymerase chain reaction (RT-PCR) using the artus® EBV RG PCR Kit for EBV DNA quantification. The tumor volume was determined by delineating the gross tumor based on 3D imaging of the patient’s nasopharynx. Result: Twenty-four subjects were included in this study. All subjects were stage III and above, with more males (75%) than females. EBV viral load in tumor cells was found to have no correlation to tumor volume both in local and nodal regions. The median local tumor volume was 81.3 cm3 ± 80 cm3. The median EBV viral load in tumor cells was 95,644.8 ± 224,758.4 copies/100 ng of DNA. The median nodal or regional tumor volume was 35.7 ± 73.63 cm3. Conclusion: EBV viral load from tumor cells from nasopharyngeal biopsy has no relationship with tumor extensiveness in nasopharyngeal cancer. The presence and amount of EBV in tumor cells did not translate into larger or smaller tumors. The EBV viral proteins and RNAs were perhaps more likely to confer some prognostic information due to the fact that those molecules were related to carcinogenesis.