High Epstein-Barr Virus Viral Load Research Articles

Unusual Indolent Course of a Chronic Active Epstein-Barr Virus-Associated Natural Killer Cell Lymphoproliferative Disorder.

Natural killer (NK) cell lymphoproliferative disorders are uncommon and the Epstein-Barr virus (EBV) plays an important aetiological role in their pathogenesis. We report a 20-year-old male with a chronic active EBV infection associated with a NK cell lymphoproliferative disorder which had an unusual indolent course. He presented to the Sultan Qaboos University Hospital in Muscat, Oman, in December 2011 with a history of intermittent fever and coughing. Examinations revealed generalised lymphadenopathy, hepatosplenomegaly, leukocytosis, transaminitis, diffuse bilateral lung infiltrates and bone marrow lymphocyte involvement. A polymerase chain reaction (PCR) test revealed a high EBV viral load in the peripheral blood cells. The patient received a course of piperacillin-tazobactam for Klebsiella pneumoniae, but no active treatment for the lymphoproliferative disorder. However, his lymphocyte count, serum lactate dehydrogenase and liver enzymes dropped spontaneously. In addition, EBV PCR copies fluctuated and then decreased significantly. He remained clinically asymptomatic over the following four years.

Epstein Barr virus induces HERV-K and HERV-W expression in pediatrics liver transplant recipients?

Human endogenous retrovirus (HER Vs) constitute approximately 8% of the human genome. Induction of HER V transcription is possible under certain circumstances, and may have a possible role in some pathological conditions. Aim of the present study was to verify whether HER V-W and K activation by Epstein Barr Virus (EBV) might occur also in vivo, during EBV infection, in pediatric liver transplant recipients. A total of 35 pediatric liver transplant (LT) patients who received LT at the University Hospital City of Science and Health of Turin, Regina Margherita Children's Hospital were included. The samples were grouped in EBV negative and positive. We found that HER V-K, and HER V-W expression levels showed no differences between the two groups (P=0.533 HERV-W and P=0.6017 HERV-K). There was not was a significant difference P=0.1894 and 0.1705 for HERV-W and -K respectively when we compared transplant recipients' group with high EBV viral load vs. others transplant recipients. Our data suggest that EBV does not facilitate in-vivo HERV activation.

Epstein–Barr virus serology and PCR: conflicting results in an immunocompetent host. A case report and review of literature

We present the case of a 27-year-old immunocompetent man who progressively developed a generalized lymphadenopathy and B symptoms. Results of Epstein–Barr virus (EBV) serology were suggestive for a past infection, but the EBV viral load in whole blood was high. Also, core needle biopsy of the largest lymph node showed an image which could fit an EBV-driven reactive lymphoproliferation. Despite the absence of an immune disorder, all medical evidence points to an EBV-driven lymphoproliferative proces. In immunocompetent patients, it seems extremely uncommon to detect a high EBV viral load in the absence of serological evidence of an acute EBV infection or reactivation. We reviewed literature on this topic and on the selection of the appropriate sample type for EBV PCR, as this is known to be a critical point. Serological testing for the diagnosis of EBV infection is the gold standard in immunocompetent patients. Measuring EBV viral load is only recommended when dealing with immunocompromised patients. Although extremely rare, this case report shows that there is a place for EBV PCR in certain situations in immunocompetent patients. Besides, there is still no consensus regarding the specimen of choice for the determination of the EBV viral load. The preferred specimen type seems to depend on the patient’s underlying condition.

Concurrent Epstein Barr Virus Viremia and Hodgkin Lymphoma In Two Patients With Chronic Lymphocytic Leukemia

IntroductionChronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder (LPD) derived from mature B-cells with heterogeneous outcomes. High-grade lymphoma can arise from CLL in 3-10% of cases, a process known as Richter's transformation (RT). The majority of RT results in high grade B-cell lymphomas, though rarely transformation to Hodgkin lymphoma (HL) has been reported. Epstein Barr virus (EBV) viremia can arise in patients with CLL secondary to immunosuppression and can lead to an EBV driven LPD. In patients with CLL, this has been seen with fludarabine based treatments but has been reported with alemtuzumab. HL is also been associated with EBV virus in tumor cells but cases of HL and EBV viremia have not been reported. We report the first two cases in the English literature of CLL with EBV viremia and HL. CasesCase 1 A 66 year old male was diagnosed with a RAI stage I, del13q14, Zap-70 (-), IgvH mutated, CD38 (+) CLL in 2004. Worsening thrombocytopenia resulted in the initiation of treatment in April 2012 using Ofatumumab and high dose methylprednisolone followed by lenalidomide maintenance therapy (HiLOG clinical trial). The patient responded to induction therapy and was continued on lenalidomide maintenance for 12 cycles. In May of 2013 the patient developed progressive fevers, chills, cough, weakness, weight loss, and hypercalcemia. Evaluation revealed an EBV viremia with whole blood PCR titers of 484,550 copies/mL. Bone marrow biopsy was consistent with CLL and full body PET CT scan showed diffuse lymphadenopathy with max SUV of 30.7 and splenomegaly. Biopsy of a left external iliac node showed large atypical lymphoid cells that were surrounded by small lymphocytes. The large atypical lymphoid cells were CD30 (+), EBV (+), Pax-5 (+), CD 20 (-), CD15 (-), CD5 (-), and CD3 (-) confirming HL. The patient has currently received 4 weekly treatments with rituximab for EBV driven LPD and one cycle of Adriamycin, bleomycin, vinblastine, and dexamethasone (ABVD) for HL with resolution of symptoms and a whole blood EBV PCR to <500 copies/mL. Case 2A 68 year old male was diagnosed in England with an unknown stage del13q and IgVH unmutated CLL in 2002. He was started on treatment with 5 cycles of fludarabine, cyclophosphamide, rituximab in 2011 and achieved a complete response. His post therapy course was complicated with infection resulting in septic shock with renal failure resulting in dialysis. He stabilized but continued to have hypogammaglobulenemia and required intravenous immunoglobulin (IVIG) replacement. In April of 2013 he developed fevers to 104F, night sweats, weight loss and, fatigue. PET CT scan showed diffuse adenopathy with max SUV of 22.5, and bone marrow biopsy showed 60% CLL and a component of large cells that were CD 30 (+), CD15 (+), Mum1 (+), and EBER-ISH (+). The patient's whole blood EBV PCR was 276,600 copies/mL. Based on the clinical picture the patient was diagnosed with an EBV positive HL, EBV viremia, and chronic CLL. He has completed 4 weekly treatments with rituximab for EBV driven LPD and cycle 1 of ABVD for HL with resolution of symptoms and a whole blood EBV PCR of <500 copies/mL. DiscussionAlthough EBV viremia has been reported in CLL and EBV staining can be positive in HL, these two cases are the first to report CLL with EBV viremia and EBV positive HL. The high EBV viral load likely provided a base for an EBV driven LPD, in these cases HL. In patients with constitutional symptoms who have stable CLL disease burden, the clinician should screen for RT and EBV viremia. When a concurrent process is found, as in our cases, treatment against both the EBV driven LPD and HL should be initiated. Further research is needed to establish a biological relationship between EBV viremia and HL in patients with CLL. Disclosures:No relevant conflicts of interest to declare.

Impact of Epstein–Barr virus load, virus genotype, and frequency of the 30 bp deletion in the viral BNLF‐1 gene in patients harboring the human immunodeficiency virus

Patients infected with the human immunodeficiency virus (HIV) are at higher risk of developing Epstein-Barr Virus (EBV)-associated lymphomas. The usefulness of monitoring EBV in peripheral blood mononuclear cells (PBMCs) of patients infected with HIV has not been established. The aim of this study was to evaluate the EBV viral load in PBMCs, the frequency of viral genotypes, and the presence of the 30-bp deletion in the BNLF-1 gene. DNA samples from 156 patients attending the HIV/AIDS Day Clinic at Botucatu School of Medicine, Sao Paulo State University were evaluated. The EBV viral load was detectable by real time PCR in 123/156 (78.8%) cases and was higher in patients not receiving antiretroviral treatment or under therapeutic failure than in patients under successful highly active antiretroviral therapy (HAART) (P = 0.0076). Overall, the profile of patients with high EBV viral load included elevated HIV viremia (P = 0.0005), longer time of HIV diagnosis (P = 0.0026), and increased levels of T CD8 (+) lymphocytes (P = 0.0159). The successful amplification of the EBNA-2 gene by nested-PCR was achieved in 95 of 123 (77.2%) cases, of which 75.8% were EBV-1, 9.5% EBV-2, and 14.7% were co-infected with both EBV-1 and -2. The analysis of the BNLF-1 gene was possible in 99 of 123 (80.5%) cases, of which 50.5% had the 30-bp deletion. EBV-1 was more common than EBV-2, which may reflect the fact that the cohort was predominantly Caucasian and heterosexual.

Significance of Persistent EBV Viral Load in Pediatric Heart Transplant Recipients

PurposeAs most cases of post-transplant lymphoproliferative disorder (PTLD) are driven by EBV, most centers monitor EBV viral load (VL) by quantitative PCR. Optimal treatment for patients (pts) with persistent EBV VL is unknown. Aim: To determine the outcome of reduced immunosuppression (IS) and anti-viral therapy on the frequency of PLTD and acute rejection (AR) in pediatric heart transplant (Tx) recipients.Methods and MaterialsWe reviewed medical records of 113 pts whose EBV serostatus was known at Tx. We ascertained their EBV PCR VL, IS regimen, anti-viral therapy, CT/PET scan and biopsy results, and AR episodes. Whole blood PCR was used for EBV VL monitoring. Pts were divided into 3 groups based on EBV VL: not detected (ND) (<1000), low (1000-10,000) and high (>10,000) copies/ml. Pts who had persistently high VL twice within 2 months underwent whole body CT/PET imaging to exclude PTLD. Diagnosis of PTLD was based on histopathology findings of abnormal lymphoid proliferation and positive in situ hybridization using EBER probe for EBV.ResultsThe findings of this study are shown in Figure. Abnormal CT/PET scan concerning for PTLD was observed in 66% in EBV naïve heart Tx recipients who had high VL. Of 10 pts who had abnormal PET/CT scan, 3 had PTLD on biopsy. Antiviral therapy was initiated in 4 pts who showed only temporary decrease in VL. Reduction in IS in pts with high VL resulted in AR in 3 cases. [figure 1]ConclusionsPediatric heart Tx recipients with high EBV VL are at risk for PTLD despite reduction in IS. We recommend frequent surveillance of the allograft for AR with IS reduction. Multicenter study is needed to focus on strategies not only to prevent PTLD, but also to protect the allograft. As most cases of post-transplant lymphoproliferative disorder (PTLD) are driven by EBV, most centers monitor EBV viral load (VL) by quantitative PCR. Optimal treatment for patients (pts) with persistent EBV VL is unknown. Aim: To determine the outcome of reduced immunosuppression (IS) and anti-viral therapy on the frequency of PLTD and acute rejection (AR) in pediatric heart transplant (Tx) recipients. We reviewed medical records of 113 pts whose EBV serostatus was known at Tx. We ascertained their EBV PCR VL, IS regimen, anti-viral therapy, CT/PET scan and biopsy results, and AR episodes. Whole blood PCR was used for EBV VL monitoring. Pts were divided into 3 groups based on EBV VL: not detected (ND) (<1000), low (1000-10,000) and high (>10,000) copies/ml. Pts who had persistently high VL twice within 2 months underwent whole body CT/PET imaging to exclude PTLD. Diagnosis of PTLD was based on histopathology findings of abnormal lymphoid proliferation and positive in situ hybridization using EBER probe for EBV. The findings of this study are shown in Figure. Abnormal CT/PET scan concerning for PTLD was observed in 66% in EBV naïve heart Tx recipients who had high VL. Of 10 pts who had abnormal PET/CT scan, 3 had PTLD on biopsy. Antiviral therapy was initiated in 4 pts who showed only temporary decrease in VL. Reduction in IS in pts with high VL resulted in AR in 3 cases. [figure 1] Pediatric heart Tx recipients with high EBV VL are at risk for PTLD despite reduction in IS. We recommend frequent surveillance of the allograft for AR with IS reduction. Multicenter study is needed to focus on strategies not only to prevent PTLD, but also to protect the allograft.

Association of HLA-DR7 with the Development of a Symptomatic Epstein-Barr Virus (EBV) Infection after Pediatric Renal Transplantation

Introduction: So far, it is unclear why some patients exhibit pronounced acute clinical EBV-related symptoms during post-transplant EBV primary infection, whilst others remain asymptomatic despite a high, persistent EBV viral load, over months or years. A genetic predisposition is likely. Methods: In the framework of a prospective, multicenter trial among 106 pediatric kidney allograft recipients (aged 11.1 ± 5.9 years), we therefore analyzed the prevalence of HLA class I and II alleles and a potential association of HLA alleles with the development of a symptomatic EBV infection (flu-like symptoms or infectious mononucleosis (IM)) in the first year post-transplant. Results: Patients expressing HLA-DR7 bore a significantly increased risk of developing a symptomatic EBV infection (univariate regression analysis: OR 4.77; 95%-CI 1.60-14.2; p=0.005; multivariate analysis: Fig. 1). The prevalence of the HLA-DR7 allele amounted to 25%. Having a comparable EBV risk constellation, HLA-DR7-positive patients suffered significantly more often (9/26 (35%)) a symptomatic EBV infection than HLA-DR7-negative recipients (8/80 (10%); p=0.008). Asymptomatic patients with a high EBV viral load tended to express HLA-DR7 less frequently than symptomatic patients with a high EBV viral load (3/17 (18%) vs. 6/11 (55%), p=0.095).Figure: [EBV HLA]Conclusions: Pediatric renal transplant recipients expressing HLA-DR7 develop significantly more often a symptomatic EBV infection than HLA-DR7-negative patients, possibly as a consequence of a pronounced immune response which, in the long run, may protect against PTLD. Indeed, HLA-DR7 induces a T helper cell reaction through presentation of EBNA1 (1) and is known to be a protective factor against the development of PTLD (2).

Association of HLA-DR7 with the Development of a Symptomatic Epstein-Barr Virus (EBV) Infection after Pediatric Renal Transplantation

Introduction: So far, it is unclear why some patients exhibit pronounced acute clinical EBV-related symptoms during post-transplant EBV primary infection, whilst others remain asymptomatic despite a high, persistent EBV viral load, over months or years. A genetic predisposition is likely. Methods: In the framework of a prospective, multicenter trial among 106 pediatric kidney allograft recipients (aged 11.1 ± 5.9 years), we therefore analyzed the prevalence of HLA class I and II alleles and a potential association of HLA alleles with the development of a symptomatic EBV infection (flu-like symptoms or infectious mononucleosis (IM)) in the first year post-transplant. Results: Patients expressing HLA-DR7 bore a significantly increased risk of developing a symptomatic EBV infection (univariate regression analysis: OR 4.77; 95%-CI 1.60-14.2; p=0.005; multivariate analysis: Fig. 1). The prevalence of the HLA-DR7 allele amounted to 25%. Having a comparable EBV risk constellation, HLA-DR7-positive patients suffered significantly more often (9/26 (35%)) a symptomatic EBV infection than HLA-DR7-negative recipients (8/80 (10%); p=0.008). Asymptomatic patients with a high EBV viral load tended to express HLA-DR7 less frequently than symptomatic patients with a high EBV viral load (3/17 (18%) vs. 6/11 (55%), p=0.095).Figure: [EBV HLA]Conclusions: Pediatric renal transplant recipients expressing HLA-DR7 develop significantly more often a symptomatic EBV infection than HLA-DR7-negative patients, possibly as a consequence of a pronounced immune response which, in the long run, may protect against PTLD. Indeed, HLA-DR7 induces a T helper cell reaction through presentation of EBNA1(1) and is known to be a protective factor against the development of PTLD(2).

Editorial Commentary: Understanding Risk and Enhancing Safety in Immunotherapy Trials

these patients for herpesvirus infections has provided novel insights into the effects of common viral infections with the use of potent exogenous immunosuppression in the nontransplant setting. In the current study, the authors performed careful laboratory and clinical monitoring for Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex virus (HSV), and varicella zoster virus (VZV). Both primary infections and reactivations were observed but significantly increased morbidity was not seen in patients receiving either one or both immunosuppressive drugs compared with placebo. There was a trend toward greater EBV viral burden (P= .06) in patients receiving both daclizumab and MMF compared to those on no immunosuppression. However, most patients were either asymptomatic or had a self-limited mononucleosis-like syndrome. EBV is an important herpesvirus in transplantation; pediatric transplant patients especially are at greater risk of posttransplant lymphoproliferative disease (PTLD), primarily because they are often EBV-seronegative prior to transplantation and may receive a seropositive organ. In this study, EBV viral loads were reportedly not significantly different from viral loads measured in transplant patients at the same lab. In the transplant setting, high EBV viral loads have been correlated with an increased risk of PTLD [3]; however, other

Microgeographic variations in Burkitt's lymphoma incidence correlate with differences in malnutrition, malaria and Epstein-Barr virus.

Background:Endemic Burkitt's lymphoma (eBL) has been associated with Epstein–Barr virus (EBV) and holoendemic Plasmodium falciparum malaria. But recent evidence suggests that other risk factors are involved.Methods:We hypothesised that selenoprotein glutathione peroxidase (GPx), a surrogate of nutritional status, is an important biomarker for eBL risk. We measured plasma GPx, anthropometric markers of malnutrition, EBV viral loads and malaria parasitaemia in children aged 1–9 years (n=258) from two locations in Nyanza Province, Kenya, with higher-than-expected and lower-than-expected incidence of eBL. The study participants were malaria asymptomatic children from the community.Results:Children from eBL high-incidence areas had significantly lower GPx levels, high EBV viral load and more evidence of chronic malnutrition than children from eBL low-incidence areas (all P<0.001). Additionally, GPx levels were significantly lower in children with the highest EBV viral load and for those with P. falciparum infections (P=0.035 and P=0.004, respectively).Conclusions:These results suggest that selenium deficiency may be a risk factor for eBL.

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

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

P.020 Presence of high T cell counts and EBV-lytic antigen specific cells in paediatric renal transplant patients with high EBV viral loads: Predictive markers for risk PTLD

Epstein–Barr virus (EBV) is frequently associated with post-transplant lymphoproliferative disorders (EBV+ PTLD). In these cases, impaired Epstein–Barr virus (EBV)-specific CD8+ T-cell immunity is strongly implicated and antigen presentation within the malignant B-cell is intact. Interestingly, several studies have reported HLA class I alleles with protective or susceptibility associations. However, results are conflicting, likely influenced by methodology including inconsistent use of multiple hypothesis testing. By contrast, HLA class I associations have been repeatedly reported for classical Hodgkin Lymphoma (cHL), in which EBV is also implicated in a proportion of cases. In contrast to EBV+ PTLD which expresses the immunodominant EBV latency III EBNA3A/B/C proteins, EBV+ cHL is restricted to the subdominant EBNA1/LMP1/LMP2 proteins. Herein, we report a study of HLA class I associations in EBV+ PTLD, with 263 patients with lymphoma (cHL or PTLD) evaluated. Two Australian population cohorts, n = 23,736 and n = 891 were used for comparison. Contrary to previous reports, no HLA class I associations with EBV+ PTLD were found, whereas for cHL known HLA class I associations were confirmed, with HLA-A*02 homozygous individuals having the lowest odds of developing EBV+ cHL. Our results suggest that HLA class I does not influence susceptibility to the viral latency III expressing lymphoma, EBV+ PTLD. Further studies are required for definitive confirmation.

Prospecitve Quantitative PCR Monitoring of Allogeneic Stem Cell Transplant (SCT) Patients at High Risk for EBV Associated PTLD.

Background: EBV associated PTLD is a severe complication after allogeneic SCT with high mortality. Several studies have reported identified risk factors for development of PTLD such as an EBV serological mismatch between donor and recipient, and the use of T-cell depleted or mis-matched stem cell grafts including cord blood grafts. Detection of high EBV viral load in blood specimens has also been associated to the development of PTLD. Treatment with rituximab at the time of EBV viremia has been suggested as a way of reducing the risk for PTLD.Methods: A strategy for prospective monitoring was introduced 050701 in “high risk” patients while the “standard risk” patients were to be sampled only based on clinical suspicion. Patients were to be monitored at least weekly if they had a diagnosis of lymphoma, there was a serological mismatch for EBV between donor and recipient, a recent primary EBV infection before SCT; or were to be transplanted with a cord blood graft. 124 patients transplanted between 050701 and 070515 were included; 41 patients were classified as “high risk” while 83 were “standard risk”. High risk features were: EBV serological mismatch n= 21, cord blood grafts n=15, lymphoma n=10, primary EBV infection n=1 (patients could have more than one high risk feature). The EBV viral load was determined by a TaqMan PCR technique using the EBV BNRF gene. EBV DNA was extracted from serum with MagNa Pure LC. Rituximab were to be given if a patient developed a viral load of 10000 copies/ml or had symptoms suggestive of EBV associated disease.Results: A total of 746 blood samples were analyzed for EBV DNAemia; a median of 6 samples in the “high risk” and 2 samples in the “standard risk” group (p=0.03). 14/41 (34%) patients in the “high risk” group compared to 11/83 (13%; p=.009) in the “standard risk” group had EBV detected at least once. In patients experiencing EBV infection, the median viral was 5500 copies/ml (225-1.4 x106) load in the “high risk” group compared to 225 (225-2.3 x106 in the “standard risk” group (p=.10). Eight patients (19%) in the “high risk” group and 3 (3.6%) in the “standard risk” group received rituximab (p=.006). Three patients (7.3%) in the “high risk” and one (1.2%) in the “standard risk” group (p=.10) developed PTLD giving a total frequency of 3.2%. One patient died from PTLD in each group (ns). Thus, the mortality in EBV associated PTLD was 2/124 (1.6%) in the entire group, 2.4% in the “high risk”, and 1.2% in the “standard risk” group.Conclusions: EBV viral load monitoring directed to SCT patients at “high risk” for EBV associated PTLD resulted in a higher number of detected EBV DNAemias and more patients receiving preemptive rituximab therapy while the risks for PTLD and PTLD associated mortality were no higher than in “standard risk” patients.

EBV Viral Load in Tumor Tissue Is an Important Prognostic Indicator for Nasal NK/T-Cell Lymphoma

We retrospectively studied 19 cases of nasal NK/T-cell lymphoma for various potential prognostic factors and performed real-time quantitative polymerase chain reaction for Epstein-Barr virus (EBV) viral load in tumor tissue. Patients with a low EBV viral load (<1 copy per cell) more frequently survived for more than 2 years compared with patients with a high EBV viral load (>/=1 copies/cell) (7/7 vs 3/9; P = .014; Fisher exact test). Furthermore, the patients with low EBV viral loads had a better overall survival than patients with high viral loads (50% accumulative survival: not reached vs 4-5 months; Kaplan-Meier survival analysis; P = .049). In contrast, the overall survival of the patients did not correlate with the extent of lesion, age, stage, necrosis, histologic subtypes, CD56 expression, or angiocentric or angiodestructive growth pattern. Our findings suggest that the EBV viral load in tumor tissues is a useful indicator for predicting outcome of nasal NK/T-cell lymphoma.

Factors involved in the generation of memory CD8+ T cells in patients with X-linked lymphoproliferative disease (XLP)

We have analysed the phenotype of T lymphocytes in two X-linked lymphoproliferative disease (XLP) patients with the same SH2D1A mutation differing in initial exposure to Epstein-Barr virus (EBV) and treatment. While memory T lymphocytes (with low CCR7 and CD62L expression) prevailed in both XLP patients, in patient 9, who developed acute infectious mononucleosis (AIM) and received B cell ablative treatment, the predominant phenotype was that of late effector CD8 T cells (CD27-, CD28-, CCR7-, CD62L-, CD45 RA+, perforin+), while in patient 4 (who did not suffer AIM) the prevalent phenotype of CD8 T lymphocytes was similar to that of normal controls (N) or to that of adult individuals who recovered from CD27+ , CD28+, CCR7-, CD62L-, CD45 RO+ and perforin-. CD57 expression (related to senescence) was also higher in CD8 T cells from patient 9 than in patient 4, AIM or N. Persistently high EBV viral load was observed in patient 9. The results obtained from this limited number of XLP patients suggest that events related to the initial EBV encounter (antigen load, treatment, cytokine environment) may have more weight than lack of SH2D1A in determining the long-term differentiation pattern of CD8 memory T cells.

Analysis of EBV-Specific T Cell Responses in Transplant Recipients with PTLD.

EBV causes a chronic infection in >95 % of the population and despite its strong growth transforming capacity the majority of EBV infected individuals remain asymptomatic. In contrary, in immunosuppressed patients (pts) the risk of EBV reactivation and development of posttransplant lymphoproliferative disease (PTLD) is high. This is assumed to be due to a defective T cell response. Here we analyzed the EBV-specific CD8 and CD4 T cell response to different EBV latent and lytic antigens in pts with newly diagnosed PTLD. A prospective study of 10 pts after solid organ transplantation at time of diagnosis of PTLD was performed. EBV-specific CD8 T cells were examined by flow cytometric analysis using HLA-A2, HLA-B7 and HLA-B8 restricted tetramers incorporating BMLF1 (lytic), EBNA3 and LMP2 (both latent)-derived peptides. Staining was done in conjunction with mAbs against CD8 and CCR7. The ability of CD8 T cells to produce IFN-γ in response to the same EBV-derived peptides was measured by cytokine secretion assay. In healthy, EBV+ donors, we previously have found a consistent CD4 T cell response to the latent EBV antigen EBNA1. Therefore, EBNA1-specific CD4 T cell responses were monitored for IFN-g / IL-4 secretion after protein stimulation. T cell analysis was combined with EBV-DNA quantiation by real time PCR. We found EBV-specific CD8 T cell responses at low frequency in most pts with PTLD (8/10). Half of the pts showed low frequency EBNA1 specific CD4+ T cell responses. All pts with an EBNA1 specific CD4 T cell response showed an EBV-specific CD8 T cell response. In 2/10 pts we found no EBV-specific CD4 and CD8 T cell responses and both pts died under initial therapy. EBV-viral load was found to inversely correlate to absolute CD4 T cell counts. In comparison to healthy normal donors, no significant differences in EBV-specific T cell response could be observed. However, pts EBV-specific T cells were decreased in comparison to pts with high EBV viral load after TX and no PTLD as well as in comparison to pts with infectious mononucleosis. These results indicate that impairment of EBV-specific T cells is not due to clonal depletion, but rather seems to be due to impaired functional activation and expansion. We therefore conclude that pts with PTLD have an inadequatly low EBV-specific T cell responses which correlates to a low absolute CD4 T cell count. We propose a combined immunomonitoring of EBV viral load, absolute CD4 T cell count and EBV-specific T cell enumeration in pts at risk for development of PTLD. Further studies are needed to evaluate the role of EBV-specific T cell monitoring in immunosuppressed pts for prediction of PTLD and the potential usefulness of T cell monitoring as a prognostic marker in 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.

Risk factors for pediatric human immunodeficiency virus-related malignancy.

Although cancers occur with increased frequency in children with human immunodeficiency virus (HIV) infection, the specific clinical, immunological, and viral risk factors for malignancy have not been identified. To identify risk factors for malignancy among HIV-infected children. A multicenter case-control study of children with HIV at 26 institutions participating in the Pediatric Oncology Group. Forty-three case patients with a new malignancy and 74 control patients without a malignancy were matched based on the duration of their infection. Patients were enrolled between January 1992 and July 1998. Clinical and laboratory factors assessed as putative risk factors included demographic characteristics, HIV characteristics, prior antiretroviral treatment, and CD4 cell count. Coviral infections with Epstein-Barr virus (EBV), cytomegalovirus, and human herpesvirus 6 were assessed by semiquantitative polymerase chain reaction assays and serological testing. Case malignancy diagnoses included 28 non-Hodgkin lymphoma, 4 B-cell acute lymphoblastic leukemia, 1 Hodgkin disease, 8 leiomyosarcoma, 1 hepatoblastoma, and 1 schwannoma. Epstein-Barr virus viral load of more than 50 viral genome copies per 105 peripheral blood mononuclear cells was strongly associated with cancer risk but only for children with CD4 cell counts of at least 200/ microL (odds ratio [OR], 11.33; 95% confidence interval [CI], 2.09-65.66, P<.001). High EBV viral load was not associated with cancer for children with CD4 cell counts of less than 200/ microL (OR, 1.12; 95% CI, 0.13-9.62; P =.99). Zidovudine antiretroviral therapy did not confer a significant protective effect for either the high (OR, 0.81; 95% CI, 0.22-3.09; P =.77) or the low CD4 cell count groups (OR, 0.27; 95% CI, 0.04-1.46; P =.16). The route of HIV infection was not associated with increased cancer risk. Route of infection, demographic characteristics, and zidovudine use were not associated with the development of malignancy in HIV-infected children. High viral burden with EBV was associated with the development of malignancy in HIV-infected children although the effect was modified by CD4 cell count. The pathogenesis of HIV-related pediatric malignancies remains unclear and other contributing risk factors can be elucidated only through further study.