Epstein-Barr Virus Viral Load Monitoring Research Articles

Diagnostic Value of Whole-Blood and Plasma Samples in Epstein-Barr Virus Infections.

Epstein-Barr virus (EBV) is an oncogenic virus classified by the World Health Organization as a class 1 carcinogen. Post-transplant lymphoproliferative disorders are believed to be strongly related to an EBV infection. Monitoring of EBV DNAemia is recommended to assess the risk of reactivation of latent infection and to assess the effectiveness of therapy. Currently, various types of clinical specimens are used for this purpose. The aim of the study was to assess a reliable method of EBV viral load investigation depending on the clinical material used: whole blood or plasma samples. We found that of 134 EBV-DNA-positive whole-blood samples derived from 51 patients (mostly hemato-oncology or post-transplantation), only 43 (32.1%) were plasma-positive. Of these, 37 (86.0%) had lower plasma DNAemia compared to the corresponding whole-blood samples. We conclude that whole-blood samples have a higher sensitivity than plasma samples in EBV DNA detection. The clinical utility of the tests is unclear, but our results suggest that either whole blood or plasma should be used consistently for EBV viral load monitoring.

EBV-seronegative Status In Recipients Worsens Post Heart Transplant Survival

<h3>Background</h3> Although the incidence of Post-transplant lymphoproliferative disease which is associated with Epstein-Barr virus (EBV) is relatively high and its presence is associated with severe complications, impact of EBV serostatus on post heart transplantation (HT) survival is not clear. We sought to determine the impact of donor and recipient EBV serostatus on post HT survival. <h3>Methods</h3> We identified adult patients who underwent HT between 2000 and 2018 in the Scientific Registry of Transplant Recipients. These patients were categorized as per recipient and donor EBV serological status. We compared survival with the Kaplan-Meier method. We constructed Cox proportional hazards regression models to determine the risk-adjusted influence of EBV serostatus on post HT survival. <h3>Results</h3> Out of 20, 533 HT identified, 16,222 were +/+, 1081 were +/-, 2544 were -/+ and 186 were -/- EBV serological statuses. Compared to positive donor to negative recipient EBV serostatus, other donor and recipient EBV serostatuses were associated with decreased mortality (Figure and Table). <h3>Conclusion</h3> A primary EBV infection in EBV-seronegative patients receiving an EBV-seropositive donor organ or a reactivation in those who are already latently infected pre-transplantation can lead to uninhibited growth of EBV-infected B cells which can impact post HT survival. In the absence of effective preventive strategies, such as vaccines and antiviral agents the role of EBV viral load monitoring and immunosuppression regimen modulation needs investigation.

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.

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.

Roles of Epstein–Barr virus viral load monitoring in the prediction of posttransplant lymphoproliferative disorder in pediatric liver transplantation

This study is aimed to investigate the risk factors and clinical characteristics of posttransplant lymphoproliferative disorder (PTLD) after conducting Epstein-Barr virus (EBV) viral load monitoring in pediatric liver transplant (LT) patients in Taiwan, where EBV infection is endemic. From 2007 to 2013, pediatric LT recipients who underwent EBV viral load monitoring within 3 months after LT were recruited in this study. The impact of clinical parameters-including age at LT, sex, peak EBV viral load and immunosuppressant levels after LT-on the risk of PTLD were assessed. A total 39 patients underwent LT at a median age of 1.3 years (range: 0.6-14.0 years), and 5 patients developed PTLD during follow-up. Cox's proportional-hazards model identified two predictors of PTLD: peak EBV viral load within 3 months of LT >4100copies/μg peripheral blood mononuclear cells (PBMC) DNA and peak tacrolimus level within 3 months of LT>14.8ng/mL (Hazard ratio=17.14 and 11.54, P=0.02 and 0.03, respectively). Kaplan-Meier survival analysis revealed significant higher cumulative incidence rates of PTLD (27.3% and 41.8% at 0.3 and 1.2 years after LT) in subjects with peak EBV viral load >4100copies/μg PBMC DNA within 3 months after LT. (P=0.001, log-rank test). Close monitoring of EBV viral load within 3 months after LT is helpful to predict a high risk of PTLD. Tapering of immunosuppressants is suggested if the EBV viral load is >4100copies/μg PBMC DNA in LT children.

The Changing Epidemiology of Posttransplant Lymphoproliferative Disorder in Adult Solid Organ Transplant Recipients Over 30 Years: A Single-center Experience.

Posttransplant lymphoproliferative disorders (PTLD) are a complication of solid organ transplantation (SOT) associated with Epstein-Barr virus (EBV). We analyzed the incidence of and risk factors for PTLD among adult SOT recipients at our center over 30 years (1984-2013). We also compared PTLD incidence before and after a prevention strategy of EBV viral load monitoring in EBV serology mismatched patients was adapted in 2001 (ie, transplant era 1 [1983-2001] vs era 2 [2002-2013]). Among 4171 SOT patients, 109 developed PTLD. Cumulative incidence at 1, 10, and 20 years posttransplant was 0.95, 2.3, and 3.5 per 100 person-years, respectively. Beyond the first year peak of almost exclusively EBV-positive PTLD, a lower incidence of PTLD, predominantly EBV negative, persisted for 20 years. Thoracic transplant (hazard ratio [HR], 2.1; P = 0.007) and negative EBV serology (HR, 7.7; P < 0.001) were independent risk factors for PTLD on multivariate Cox regression analysis. EBV seronegativity significantly increased risk of early (HR, 18.5) and EBV-positive PTLD (HR, 14.2), as well as late (HR, 4.9) and EBV-negative PTLD (HR, 3.6) on univariate analyses. Risk of early PTLD was significantly reduced in the recent transplant era (0.8% era 2 vs 1.9% era 1 at 5 years, P = 0.002); this reduction was seen in recent era EBV seropositive (P = 0.035 at 5 years) but not seronegative recipients (P = 0.90 year 5), suggesting lack of impact of viral load monitoring. Adult SOT recipients face a prolonged risk of late PTLD, whereas risk of early PTLD may have declined in recent years.

Clinical Utility of Epstein-Barr Virus Viral Load Monitoring and Risk Factors for Posttransplant Lymphoproliferative Disorders After Kidney Transplantation: A Single-Center, 10-Year Observational Cohort Study.

BackgroundPosttransplant lymphoproliferative disease (PTLD) is an important cause of morbidity and mortality in solid organ transplants. Epstein Barr virus (EBV) plays a major role in PTLD development. Guidelines recommend EBV viral load (VL) monitoring in high-risk populations in the first year.MethodsRetrospective observational study in all adult patients who had at least 1 EBV-VL performed in the postkidney transplant (KT) period from January 2005 to December 2014 at the Policlinico Modena Hospital. We compared patients with negative EBV-DNA to patients with positive EBV-DNA and we described PTLD developed in the study period.ResultsOne hundred ninety (36.3%) KT patients of 523 were screened for EBV-DNA with 796 samples. One hundred twenty-eight (67.4%) of 190 tested patients presented at least 1 positive sample for EBV. Older age, the use of sirolimus, everolimus, and steroids were associated with EBV-DNA positivity in the univariate analysis. Nine (1.7%) of 523 patients had PTLD. Incidence rate of PTLD in the KT cohort was 0.19/100 person year follow-up (95% confidence interval, 0.09-0.37). One of 9 patients developed early PTLD and was a high-risk patient. Only this PTLD case was positive for EBV. No PTLD case had an EBV-VL superior to 4000 copies/mL.ConclusionsOur results suggest that the keystone of PTLD diagnosis is the clinical suspicion. Our study suggests that, in line with guidelines, EBV-VL assays may be avoided in low-risk patients in the absence of a strong clinical PTLD suspicion without increasing patients' risk of developing PTLD. This represents a safe and cost-saving clinical strategy for our center.

Quantitative PCR for Plasma Epstein-Barr Virus Loads in Cancer Diagnostics.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with posttransplant lymphoproliferative disease (PTLD), Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma, and HIV-related lymphomas. It infects nearly all humans and then persists for the life of the host in a small proportion of benign B lymphocytes. EBV reactivation, usually in the setting of immunosuppression, is the main risk factor for development of EBV-associated malignancies. EBV reactivation can be detected in tissue specimens using EBV-encoded RNA (EBER) in situ hybridization (ISH), which is routinely used for diagnosis of PTLD and nasopharyngeal carcinoma. However, EBER ISH cannot be routinely used for screening asymptomatic or monitoring posttreatment outcome due to difficulty in obtaining tissue specimens for testing and the nonquantitative nature of the assay. Recent studies have shown that EBV genomic DNA can be detected in blood of patients with EBV-associated diseases, and that monitoring of EBV viral load in blood could be an effective method of distinguishing disease-associated EBV reactivation from incidental EBV present in benign B lymphocytes, and could be used for diagnostic screening and monitoring of EBV-associated diseases. In this chapter we discuss a protocol for quantitative plasma EBV DNA analysis.

Persistent Epstein-Barr viral load in Epstein-Barr viral naïve pediatric heart transplant recipients: Risk of late-onset post-transplant lymphoproliferative disease.

AIMTo examine the risk of late-onset post-transplant lymphoproliferative disorder (PTLD) in the presence of persisting high Epstein-Barr virus (EBV) in EBV naïve pediatric heart transplant (HT) recipients.METHODSA retrospective review of the medical records of the 145 pediatric HT recipients who had serial EBV viral load monitoring at our center was performed. We defined EBV naive patients whose EBV serology either IgM or IgG in the blood were negative at the time of HT and excluded passive transmission from mother to child in subjects less than 6 mo of age.RESULTSPTLD was diagnosed in 8 out of 145 patients (5.5%); 6/91 (6.5%) in those who were EBV seropositive and 2/54 (3.7%) in the EBV naïve group at the time of HT (P = 0.71). We found 32/145 (22%) patients with persistently high EBV load during continuing follow-up; 20/91 (22%) in EBV seropositive group vs 12/54 (22%) in EBV naïve group (P = 0.97). There was no significant association between pre-HT serostatus and EBV load after transplant (P > 0.05). In the EBV seropositive group, PTLD was diagnosed in 15% (3/20) of patients with high EBV vs 4.2% (3/71) of patients with low or undetectable EBV load (P = 0.14) whereas in EBV naïve patients 8.3% (1/12) of those with high EBV load and 2.3% (1/42) with low or undetectable EBV load (P = 0.41). There was a highly significant association between occurrence of PTLD in those with high EBV load and duration of follow up (4.3 ± 3.9 years) after HT by Cochran-Armitage test for the entire cohort (P = 0.005). At least one episode of acute rejection occurred in 72% (23/32) of patients with high EBV vs 36% (41/113) patients with low or undetectable EBV after HT (P < 0.05).CONCLUSIONThere is an association between persistently high EBV load during post-HT follow up and the occurrence of late-onset PTLD in pediatric HT recipients irrespective of serostatus at the time of transplant. The occurrence of allograft rejection increased in patients with high EBV load presumably due to reduction in immunosuppression.

An Uncommon Presentation of Non-Hodgkin’s Lymphoma in a Renal Transplant Recipient

Dear Sirs, Primary cutaneous posttransplantation B-cell lymphoproliferative disorder is rare. Posttransplantation lymphoproliferative disorders (PTLDs) are usually of non-Hodgkin’s lymphoma (NHL), accounting for 93% of lymphomas in the transplant population versus 65% in the general population. We present a rare case of cutaneous manifestation of a NHL in a renal transplant recipient. A 44-year-old woman was referred to our department due to skin lesion recognized on the right arm (level of deltoid muscle) (Fig. 1). She had undergone renal transplantation from a living donor 7 years ago due to end-stage renal disease attributed to immunoglobulin A nephropathy. Her immunosuppressive regimen included azathioprine, corticosteroids, and cyclosporine. Clinically, skin lesion resembled a soft-tissue sarcoma or a squamous cell carcinoma. Skin biopsy demonstrated diffuse large non-Hodgkin’s B-cell lymphoma (DLBCL; centroblastic cells with appearance of medium- to large-sized lymphocytes with scanty cytoplasm). On immunohistochemistry, cells of DLBCL expressed B-cell–associated antigens (CD19, CD20, and CD22). Serologic analysis for Epstein-Barr Virus (EBV) was positive (seroconversion of a previously seronegative patient) and in situ hybridization techniques identified EBV sequences in the nuclei of lymphoid cells within the lesion. Computed tomography scan applied for staging the disease showed large lung, mesothoracic, and inguinal lymph-node blocks. She was diagnosed with EBV-related PTLD. Immunosuppression reduction, chemotherapy with CHOP-R regimen, was administrated, which induced complete remission of the tumor. No surgical intervention was applied. However, the patient succumbed to recurrent disease 18 months later.FIGURE 1: Skin lesion on the right arm (level of deltoid muscle).DISCUSSION Solid organ transplant recipients have elevated cancer risk due to immunosuppression and oncogenic viral infections. Among transplant recipients, NHL represents one extreme of EBV-driven proliferative disease acknowledged as PTLD, which ranges from benign hyperplasia and infectious mononucleosis to lymphoid malignancy (1 ). The most common NHL subtype among transplant recipients is DLBCL and most cases are EBV positive (2 ). In situ hybridization techniques using the EBV-encoded RNA probe for EBV messenger RNA are used to identify EBV sequences in the nuclei of lymphoid cells within the lesion (1 ). A diagnosis of presumed PTLD was based on documentation of EBV seroconversion of a previously seronegative patient or reactivation of immunoglobulin M against EBV viral capsid antigen, along with symptoms such as fever, fatigue, and anorexia and the detection of pulmonary nodules or extrapulmonary masses or both on radiologic evaluation (1 ). PTLD presenting with skin involvement rarely manifests as lesions and such lesions develop slowly, when they occur. Intensity of immunosuppressive therapies also influences the risk for PTLD. The use of EBV viral load monitoring facilitates the diagnosis and management of EBV/PTLD as well as being used to inform preemptive therapy with reduction of immunosuppression, the most effective intervention for prevention of and treatment for PTLD (3, 4 ). Other therapies, including rituximab (anti-CD20 monoclonal antibody) and traditional chemotherapy, are also useful in the treatment of established PTLD (3). These lesions regress after dose reduction or cessation of treatment (4 ). Future perspectives on treatment of PTLD may include adoptive immunotherapy, intravenous immunoglobulin, mammalian target of rapamycin inhibitors, monoclonal antibodies to interleukin-6 and galectin-1, and even EBV vaccination (5). Demetrios Moris 1,3 Spiridon Vernadakis2,3 Vasileios Zavvos1 Georgios Zavos3 1 1st Department of Surgery Athens University School of Medicine “Laikon” General Hospital Athens, Greece 2 Department of General, Visceral and Transplantation Surgery University Hospital Essen Essen, Germany 3 Transplantation Unit “Laikon” General Hospital Athens, Greece

Epstein–Barr Virus Infection and Posttransplant Lymphoproliferative Disorder

Epstein-Barr virus (EBV) is an important pathogen in recipients of solid organ transplants (SOT). Infection with EBV manifests as a spectrum of diseases/malignancies ranging from asymptomatic viremia through infectious mononucleosis to posttransplant lymphoproliferative disorder (PTLD). EBV disease and its associated PTLD is more frequently seen when primary EBV infection occurs after transplant, a common scenario in pediatric SOT recipients. Intensity of immunosuppressive therapies also influences the risk for PTLD. The use of EBV viral load monitoring facilitates the diagnosis and management of EBV/PTLD as well as being used to inform preemptive therapy with reduction of immunosuppression, the most effective intervention for prevention of and treatment for PTLD. Other therapies, including the rituximab (anti-CD20 monoclonal antibody) and traditional chemotherapy, are also useful in the treatment of established PTLD. The future development of standards for management based on EBV viral load and routine monitoring of EBV-specific CTL responses promise further improvement in outcomes with EBV and PTLD.

Immunosuppression-Related Lymphomas and Cancers in IBD: How Can They Be Prevented?

Most of the immunosuppressive therapy-associated excess lymphomas in IBD are due to a loss of control of Epstein-Barr virus (EBV) infection. Systemic EBV viral-load monitoring and preemptive treatments are extensively used in the posttransplant setting, but these methods have not yet been evaluated in IBD patients and cannot therefore be recommended in this context. However, the systemic EBV viral load should be measured in cases of unexplained fever, lymphadenopathy or hemophagocytic syndrome, in order to optimize the diagnostics of early EBV-related lymphoproliferations. The risk of hepatosplenic T cell lymphoma can, theoretically, be limited by avoiding prolonged combination therapy with thiopurines and anti-tumor necrosis factor (anti-TNF) beyond 2 years in young males. Young males seronegative for EBV are at risk for fatal forms of primary EBV infection, with postmononucleosis lymphoproliferation. This incidence could be limited by considering avoiding treatment with thiopurines in this subgroup of patients. There is a marked excess risk of nonmelanoma skin cancer in IBD patients currently or previously treated with thiopurines, which justifies lifelong sun protection and dermatological screening in these patients. The level of risk is still unclear for monotherapies with anti-TNF. An excess of human papilloma virus (HPV)-related uterine cervix dysplasia and cancer has been reported in various populations of women with IBD, but the proper role of immunosuppressive therapy remains to be quantified. However, yearly screening for uterine cervix abnormalities is recommended for all female IBD patients, along with HPV vaccination in young girls.

Epstein - Barr virus - associated Diseases in Allogeneic Hematopoietic Stem Cell Transplantation

Epstein - Barr virus - associated Diseases in Allogeneic Hematopoietic Stem Cell Transplantation

Quantitative Monitoring of EBV Viral Load in 222 Hematopoietic Stem Cell Transplant Patients: Risk Analysis and Development of EBV-Associated Post-Transplant Lymphoproliferative Diseases (PTLD)

Abstract 3018Background and Purposes Epstein-Barr virus (EBV)-associated PTLD is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Independent risk factors include use of ATG, acute GVHD, CMV antigenemia, T-depleted graft, and unrelated donor in our previous study. Quantitative real-time polymerase chain reaction (Q-PCR) was developed for early detection and intervention of EBV reactivation to prevent PTLD-related mortality.Patients and Methods Between Apr 2004 and Oct 2010, EBV viral load in plasma was monitored by Q-PCR in 222 HSCT patients (total 2945 samples) in NTUH. EBV reactivation was defined as > 500 copies/mL in two consecutive assays or > 10-fold elevation than baseline level.Results EBV reactivation occurred in 50 (22%) patients. The cumulated incidence of EBV reactivation was 28% at 1-year and 32% at 2-year. Median time to EBV reactivation was 40 days (ranges, 26–406) after SCT and median peak EBV-viral load, 10888 copies/ml (ranges, 948–3×107). The risk of EBV reactivation was significantly higher in patients receiving ATG (52% vs. 13%, p< 0.001), use of TBI (58% vs. 26%, p<0.001), mismatched donor (69% vs. 27%, p<0.001), CMV reactivation (44% vs. 19%, p<0.001) in univariate analysis. In multivariate analysis, independent risk factors include: ATG use (HR 5.44, p<0.001), TBI (HR 3.52, p<0.001), and Fludara-based conditioning (HR 3.16, P=0.01). PTLD developed in 8 patients (5%) who didn’t monitor EBV viral load regularly, and two were dead.Conclusions Monitoring of EBV viral load is a sensitive and useful tool in the surveillance of EBV-reactivation. Frequent monitoring in high-risk patients is important to prevent occurrence of EBV-PTLD. Disclosures:No relevant conflicts of interest to declare.

Features of Epstein-Barr Virus (EBV) reactivation after reduced intensity conditioning allogeneic hematopoietic stem cell transplantation

This single centre study assessed the incidence, kinetics and predictive factors of Epstein-Barr Virus (EBV) reactivation and EBV-related lymphoproliferative diseases (LPDs) in 175 consecutive patients who received a reduced-intensity conditioning (RIC) before allogeneic hematopoietic stem cell transplantation (allo-HSCT). The cumulative incidence of EBV reactivation at 6 months after allo-HSCT defined as an EBV PCR load above 1000 copies of EBV DNA/10(5) cells was 15%, and none of these patients experienced any sign or symptom of LPD. A total of 17 patients, who had EBV DNA levels exceeding 1000 copies/10(5) cells on two or more occasions, were pre-emptively treated with rituximab. With a median follow-up of 655 (range, 92-1542) days post allo-HSCT, there was no statistically significant difference in term of outcome between those patients who experienced an EBV reactivation and those who did not. In multivariate analysis, the use of antithymocyte globulin as part of the RIC regimen was the only independent risk factor associated with EBV reactivation (relative risk=4.9; 95% confidence interval, 1.1-21.0; P=0.03). We conclude that patients undergoing RIC allo-HSCT using anti-thymocyte globulin as part of the preparative regimen are at higher risk for EBV reactivation. However, this did not impact on outcome, as quantitative monitoring of EBV viral load by PCR and preemptive rituximab therapy allowed for significantly reducing the risk of EBV-related LPD.

Epstein Barr Virus (EBV) Reactivation After Reduced Intensity Conditioning (RIC) Allogeneic Hematopoietic Stem Cell Transplantation

AbstractAbstract 1259Following allogeneic hematopoietic stem cell transplantation (allo-HSCT), EBV reactivation and EBV lymphoprolipherative disease (LPD) are well recognized complications. To date, few data has been reported regarding the features of EBV LPD following RIC allo-HSCT. The aim of this study was to define the incidence and risk factors of EBV reactivation in 175 consecutive adult patients undergoing RIC allo-HSCT between January 2005 and June 2009 in our institution and to assess its impact on clinical outcome.In this series, the median age of recipients was 56 (range 18–71) years. In all, 85 patients (49%) had a myeloid malignancy, whereas 86 (49%) patients were diagnosed with lymphoid malignancies. The remaining 4 patients (2%) were treated for severe aplastic anemia. In total, 165 patients (94%) received peripheral blood stem cells (PBSC) whereas 10 patients (6%) received a bone marrow (BM) graft. 84 grafts (48%) were obtained from HLA identical sibling donors, 80 (46%) from HLA matched unrelated donors and 11 (6%) from 1 Ag HLA mismatched unrelated donors. The conditioning regimen associated fludarabine, busulfan, and ATG in 107 cases (61%), while 37 patients (21%) received fludarabine and low-dose TBI. The remaining 31 patient (18%) received different chemotherapy-based RIC regimens. EBV reactivation was defined as any EBV PCR load above 1000 copies of EBV DNA /105 cells. EBV LPD was defined as biopsy or autopsy proven post-transplantation lymphoma, or reactivation along with computerized tomography nodal or soft tissue abnormalities consistent with LPD. Patients with EBV viral load >1000 copies/105 cells on at least two consecutive occasions were treated with rituximab at a dose of 375 mg/m2 weekly until clearance of viremia.In our series, the median time to neutrophil recovery (absolute neutrophil count >0.5×109/L) was 17 (range, 6–48) days. Clinically significant grade II to IV acute GVHD occurred in 61 of cases (35%) and severe grade III to IV acute GVHD occurred in 37 of cases (21%). The cumulative incidence of EBV reactivation at 6 months after allo-HSCT was 15% (95% CI, 10–21%). EBV reactivation was observed at a median of 58 (range 0–930) days after allo-HSCT, with 27 (79%) of reactivations occurring during the first 6 months. In 141 patients (81%), the EBV load remained less than 1000 EBV copies/105 cells at all time. The remaining 34 patients (19%) experienced at least one EBV reactivation episode. Among these 34 patients, 17 patients had an EBV load superior to 1000/105 cells at a single time point after allo-HSCT. In these 17 cases, there were no concomitant clinical symptoms and the EBV load normalized spontaneously. The 17 patients who had EBV DNA levels exceeding 1000 copies/105 cells on 2 or more occasions were pre-emptively treated with a median number of 3 (range, 1–4) rituximab infusions which resulted in complete clearance of EBV viremia in all, but one patient (97%). This patient was severely immunosupressed, receiving three different immunosupressive agents and experienced both EBV and adenovirus (ADV) infection. This patient had symptoms mainly related to ADV infection, and died of multiorgan failure. Most importantly, none of the patients from this series developed EBV induced LPD. With a median follow-up of 655 (range, 92–1542) days post allo-HSCT among surviving patients, 104 patients (59%) were still alive and the overall survival (OS) was 47% at 4 years. There was no statistically significant difference in terms of OS or transplant related mortality (TRM) between patients who experienced an EBV reactivation and patients who did not (OS: log rank test, p=0.62; TRM: Gray test, p=0.99). In univariate analysis for risk factors associated with EBV reactivation only the use of ATG as part of the RIC regimen prior to allo-HSCT was significantly different between subgroups with and without EBV reactivation (Fisher's exact test, p=0.006). In the multivariate analysis, the use of ATG remained the only independent risk factor associated with EBV reactivation (Fine and Gray test; RR=4.9; 95%CI, 1.1–21.0; p=0.03).In all, we conclude that patients undergoing RIC allo-HSCT using ATG as part of the preparative regimen are at higher risk for EBV reactivation. However, this did not translate into a significant impact on outcome since monitoring of EBV viral load with quantitative PCR and early systematic pre-emptive rituximab therapy allowed for significantly reducing the risk of EBV-related LPD. Disclosures:No relevant conflicts of interest to declare.

Post-transplant lymphoproliferative disease

Post-transplant lymphoproliferative disease (PTLD) emerged in the mid-1990s as a major graft- and life-threatening complication of pediatric kidney transplantation. This condition, usually involving uncontrolled B lymphocyte proliferation, straddles the border between infection and malignancy, since Epstein–Barr virus (EBV) is intimately associated with the pathogenesis. PTLD is seen more in younger children (more likely to be EBV seronegative), Caucasian race, and in association with the more potent immunosuppression drugs. The clinical presentation typically involves multiple enlarged lymph nodes but varies based on localization of the lymphadenopathy. The diagnosis is based primarily on histopathological features. Treatment strategies include reduction of immunosuppression, use of anti-B cell antibodies, infusion of EBV-specific cytotoxic T lymphocytes, and chemotherapy. Many different strategies have been tried to prevent PTLD, ranging from serial EBV viral load monitoring and pre-emptive immunosuppression reduction to anti-viral prophylaxis. None of the major treatment or prevention strategies has been subject to randomized clinical trials, so their relative efficacy is still unknown. PTLD remains a risk factor for graft loss, though re-transplants have not, to date, been associated with repeat PTLD.

Quantitative EBV Viral Loads and Immunosuppression Alterations can Decrease PTLD Incidence in Pediatric Liver Transplant Recipients

Epstein-Barr virus (EBV) is a common viral infection in pediatric liver transplant patients and can lead to development of post-transplant lymphoproliferative disorder (PTLD). Differing studies have used immunosuppression reduction, antiviral medications or i.v. CMV-immunogloublin for EBV prevention and treatment. The purpose of this study was to determine whether implementation of a protocol for frequent EBV monitoring and EBV viral load-driven immunosuppression reduction could decrease the incidence of PTLD in our patient population. All data were prospectively collected between 2001 and 2004 at a single institution. Seventy-three patients were entered into the study. Patients were divided into a historical control group (pre-2001, 30 patients) and a treatment group (post-2001, 43 patients). Approximately 1271 blood samples of 73 patients were collected between 2001 and 2004. Eleven out of 43 patients received immunosuppression tapering due to high EBV viral loads (>4000 copies/microg DNA). One patient developed allograft rejection after immunosuppression modulation. Prior to 2001, the incidence of PTLD at our institution was 16%. After instituting a protocol for EBV monitoring, the incidence of PTLD decreased to 2% (p-value<0.05). These findings illustrate that frequent EBV viral load monitoring and preemptive immunosuppression modulation have an integral role in preventing PTLD in the pediatric liver transplant population.

Low-dose immunosuppression reduces the incidence of post-transplant lymphoproliferative disease in pediatric liver graft recipients.

In pediatric solid organ transplantation, the Epstein-Barr virus (EBV)-related lymphoproliferative disorders (PTLD) still play a major role in post-transplant morbidity and mortality. The aim of the study was to determine the incidence of PTLD in pediatric patients with liver transplant who receive low-dose immunosuppression protocols. All pediatric patients (n = 269) received a dual immunosuppression therapy consisting of cyclosporine A (initial trough levels, 170-200 microg/L; trough levels for maintenance immunosuppression after 1 year, 80-100 microg/L) and prednisolone (starting dose, 60 mg/m2). Steroids were reduced to 30 mg/m2 after 1 week, followed by a weekly tapering to 5 mg/m2. Seventy-seven of 269 patients were switched to tacrolimus therapy. The authors evaluated the significance of EBV-DNA monitoring by quantitative polymerase chain reaction in identifying patients at risk for PTLD. Patient survival was 90.3%; graft survival was 85.9%. Eight patients lost their grafts because of chronic rejection. The incidence of PTLD was low (0.7%), although a significant EBV viral load was found in 42.4% of the patients. One third of the patients with a viral load of 3,000 genomes/10(5) cells or greater had clinical signs of EBV infection. The authors conclude that low-dose immunosuppressive protocols significantly reduce the incidence of PTLD. In patients treated with that regimen, the monitoring of EBV viral load seems not to be helpful. It can be assumed that low-dose immunosuppression does not suppress EBV-specific cytotoxic CD8+ T cells, thus allowing the host to control EBV infection without the risk of PTLD. Our low-dose immunosuppression protocol did not increase the risk of chronic rejection.

Low‐Dose Immunosuppression Reduces the Incidence of Post‐transplant Lymphoproliferative Disease in Pediatric Liver Graft Recipients

ABSTRACTObjectivesIn pediatric solid organ transplantation, the Epstein‐Barr virus (EBV)‐related lymphoproliferative disorders (PTLD) still play a major role in post‐transplant morbidity and mortality. The aim of the study was to determine the incidence of PTLD in pediatric patients with liver transplant who receive low‐dose immunosuppression protocols.MethodsAll pediatric patients (n = 269) received a dual immunosuppression therapy consisting of cyclosporine A (initial trough levels, 170–200 μg/L; trough levels for maintenance immunosuppression after 1 year, 80–100 μg/L) and prednisolone (starting dose, 60 mg/m2). Steroids were reduced to 30 mg/m2 after 1 week, followed by a weekly tapering to 5 mg/m2. Seventy‐seven of 269 patients were switched to tacrolimus therapy. The authors evaluated the significance of EBV‐DNA monitoring by quantitative polymerase chain reaction in identifying patients at risk for PTLD.ResultsPatient survival was 90.3%; graft survival was 85.9%. Eight patients lost their grafts because of chronic rejection. The incidence of PTLD was low (0.7%), although a significant EBV viral load was found in 42.4% of the patients. One third of the patients with a viral load of 3,000 genomes/105 cells or greater had clinical signs of EBV infection.ConclusionsThe authors conclude that low‐dose immunosuppressive protocols significantly reduce the incidence of PTLD. In patients treated with that regimen, the monitoring of EBV viral load seems not to be helpful. It can be assumed that low‐dose immunosuppression does not suppress EBV‐specific cytotoxic CD8+ T cells, thus allowing the host to control EBV infection without the risk of PTLD. Our low‐dose immunosuppression protocol did not increase the risk of chronic rejection.