Clinical significance of peripheral blood Epstein-Barr viral load monitoring using polymerase chain reaction in renal transplant recipients

PTLD is a complication of EBV infection. We examined the efficacy of EBV-PCR monitoring to detect early replication in an attempt to prevent EBV-associated PTLD. Blood EBV levels in 156 renal transplant recipients (58 children) from three institutions over nine yr were retrospectively analyzed. Patients who were asymptomatic and at high risk for PTLD were monitored for EBV infection by PCR or serology followed by PCR at the time of EBV seropositivity. More children than adults had positive EBV-PCR (12/58 vs. 2/98, p < 0.001). Adults remained asymptomatic and viremia resolved post-therapy. 3/12 EBV-PCR positive children developed PTLD (3/12 children vs. 0/2 adults, p = NS). Two out of three with PTLD were initially monitored by serology, and later by PCR. PTLD resolved post-therapy in all three patients. The remaining 9/12 EBV-PCR positive children stayed asymptomatic. None of the children and adults with negative EBV-PCR developed PTLD. EBV-PCR monitoring in high-risk renal transplant recipients, especially in children, may allow early diagnosis and intervention, and therefore may help in preventing EBV-associated PTLD.     

Chronic high Epstein–Barr viral load carriage in pediatric small bowel transplant recipients

Lau AH, Soltys K, Sindhi RK, Bond G, Mazariegos GV, Green M. Chronic high Epstein–Barr viral load carriage in pediatric small bowel transplant recipients.
Pediatr Transplantation 2010: 14:549–553. © 2010 John Wiley & Sons A/S. Abstract: The development of EBV infection and PTLD is normally associated with a high EBV load in peripheral blood. Often, children undergoing primary or reactivation of EBV infection subsequent to ITx will have chronically elevated EBV loads. To better understand this phenomenon and its consequences, we retrospectively reviewed the records of children who underwent ITx (either isolated or part of multivisceral transplantation) at our center from 1992 to 2007, to identify chronic high EBV load carriers in this population. CHL state was defined as the presence of high load for >50% of samples for greater than or equal to six months following either asymptomatic infection or complete clinical resolution of EBV disease/PTLD. Thirty‐five CHL carriers were identified from our patient population. Pretransplant serologies were available on 34 of these patients: 17 were EBV negative and 17 seropositive; one had unknown EBV serostatus prior to transplant. Seven of the 17 seronegative patients developed their CHL carrier state at the time of their primary EBV infection. Thirteen of the 35 (37%) HLC patients developed EBV disease after meeting the definition of high‐load carrier states. EBV‐related diseases developing in CHL carriers included EBV adenitis (n = 1), EBV enteritis (n = 7), PTLD (n = 4), and EBV+ spindle cell tumor (n = 1). Disease was seen in 7/17 of the seronegative (one PTLD) and 6/17 of the seropositive patients (three PTLD). Thirteen of 35 patients (37%) resolved their CHL state without apparent sequelae while nine remain asymptomatic CHL carriers. Three children have had more than one episode of CHL. These data provide important information about the outcome of chronic EBV high‐load carriage in pediatric intestinal transplant recipients.     

Post-transplant multiple myeloma in a pediatric renal transplant patient

Post-transplant lymphoproliferative disease (PTLD) is a well-recognized complication of the intense immunosuppression required in solid organ and bone marrow transplant recipients. The clinical presentation is varied and can range from a benign infectious mononucleosis-like syndrome to malignant lymphoma. PTLD manifesting as multiple myeloma occurs rarely. We report the unique occurrence of Epstein-Barr virus (EBV)-associated post-transplant multiple myeloma in a 16-year-old male. In contrast to previously described cases of PTLD-myeloma type, this patient was very young, had a clear association with EBV, and an indolent clinical course.     

Chronic high Epstein-Barr viral load carriage in pediatric liver transplant recipients

Abstract:  Development of EBV disease and PTLD is usually accompanied by the detection of a high EBV load in peripheral blood. However, many children undergoing primary EBV infection following LTx will maintain chronically elevated EBV loads in the absence of clinical symptoms. To better understand this phenomenon, we retrospectively reviewed the records of children undergoing LTx at our center from 1997 to 2007 to identify chronic high EBV load carriers in this population. A CHL state was defined by the presence of a high load for >50% of samples for greater than or equal to six months following either asymptomatic or complete clinical resolution of EBV disease/PTLD. A total of 35 CHL carriers were identified. Pretransplant serologies were available for 29 of the 35; 22/29 (76%) were EBV negative prior to LTx; eight of these 22 developed their CHL state at the time of their primary EBV infection. Fourteen of the 35 had EBV disease (n = 7) or PTLD (n = 7) prior to development of the CHL state. Only one of 35 CHL carriers developed PTLD or lymphoma while they were a high load carrier. In all, 23/35 resolved their CHL state without apparent sequelae while 11 children continue to be asymptomatic high load carriers. These data provide important information about the outcome of chronic EBV high load carriage in pediatric liver transplant recipients.     

Epidemiology and outcome of chronic high Epstein‐Barr viral load carriage in pediatric kidney transplant recipients

The development of EBV infection and PTLD is normally associated with a high EBV viral load in peripheral blood. Observations have previously identified existence of a CHL carrier state that demonstrated variable outcomes based upon the organ which was transplanted. Data defining the incidence and outcome of CHL in pediatric KTx are not well described. The charts of children undergoing isolated KTx at Children's Hospital of Pittsburgh between January 2000 and December 2014 were retrospectively reviewed. EBV loads in the peripheral blood were routinely measured as part of surveillance protocols at our center. CHL was defined as the presence of high load for >50% of samples for ≥6 months. PTLD was defined histologically using WHO definitions. Of 188 isolated KTx recipients, we identified a total of 16 (8%) children who developed CHL carrier state. No patient developed EBV‐driven late‐onset PTLD. Age at the time of KTx was significantly lower in the CHL group (median 3.9 years, interquartile range: IQR 2.9‐6.6, P = .0004). Children in the CHL group were more likely to be EBV‐seronegative prior to KTx (94%, 15/16), compared to the UVL and LVL groups (55% and 50%, respectively, P < .002). The median duration of CHL carrier state was 20 months (IQR 10.7‐35.8). Fifteen of the 16 CHL carriers experienced spontaneous resolution of CHL carrier state. Children in the CHL group were younger at the time of primary EBV infection (P = .023). Finally, antiviral medication was not effective in either preventing or decreasing the EBV viral load in blood (P = .84). Overall incidence of late‐onset PTLD is very low compared to heart and intestinal transplant, even though KTx recipients can develop CHL carrier state. The CHL carriers in KTx recipients were EBV‐seronegative prior to transplant and were younger both at the time of KTx and at the time of primary EBV infection compared to those in the UVL and HVL groups. Antivirals did not prevent EBV infection or decrease EBV viral loads.     

Epstein-Barr virus associated disease following blood or marrow transplant

EBV associated disease continues to be a problem in the post-BHT population. The pathogenesis, risk factors, and diagnosis of PTLD, as well as the rationale, efficacy and advantages/disadvantages of various prophylactic, pre-emptive and treatment strategies are discussed.     

Emerging therapeutic strategies for Epstein-Barr virus+ post-transplant lymphoproliferative disorder

De novo malignancies represent an increasing concern in the transplant population, particularly as long-term graft and patient survival improves. EBV-associated B-cell lymphoma in the setting of PTLD is the leading malignancy in children following solid organ transplantation. Therapeutic strategies can be categorized as pharmacologic, biologic, and cell-based but the variable efficacy of these approaches and the complexity of PTLD suggest that new treatment options are warranted. Here, we review current therapeutic strategies for treatment of PTLD. We also describe the life cycle of EBV, addressing the viral mechanisms that contribute to the genesis and persistence of EBV+ B-cell lymphomas. Specifically, we focus on the oncogenic signaling pathways activated by the EBV LMP1 and LMP2a to understand the underlying mechanisms and mediators of lymphomagenesis with the goal of identifying novel, rational therapeutic targets for the treatment of EBV-associated malignancies.     

Post‐transplant lymphoproliferative disorder in pediatric intestinal transplant recipients: A literature review

Intestinal transplantation is a successful treatment for children with intestinal failure, but has many potential complications. PTLD, a clinically and histologically diverse malignancy, occurs frequently after intestinal transplantation and can be fatal. The management of this disease is particularly challenging. The rejection‐prone intestinal allograft requires high levels of immunosuppression, a precondition for PTLD. While EBV infection clearly plays a role in disease pathogenesis, the relatively naïve immune system of children is another likely contributor. As a result, pediatric intestine recipients have a higher risk of developing PTLD than other solid organ recipients. Other risk factors for disease development such as molecular and genomic changes that precipitate malignant transformation are not fully understood, especially among children. Studies on adults have started to describe the molecular pathogenesis of PTLD, but the genomic landscape of the malignancy remains largely undefined in pediatric intestinal transplant patients. In this review, we describe what is known about PTLD in pediatric patients after intestinal transplant and highlight current knowledge gaps to better direct future investigations in the pediatric population.     

Effective control of Epstein-Barr virus infection following pediatric liver transplantation by monitoring of viral DNA load and lymphocyte surface markers

Imadome K-I, Fukuda A, Kawano F, Imai Y, Ichikawa S, Mochizuki M, Shigeta T, Kakiuchi T, Sakamoto S, Kasahara M, Fujiwara S. Effective control of Epstein-Barr virus infection following pediatric liver transplantation by monitoring of viral DNA load and lymphocyte surface markers. Abstract: EBV-associated PTLD is a serious complication of liver transplantation. We performed periodical molecular EBV monitoring in 140 consecutive pediatric patients who had living-related liver transplantation in the National Center for Child Health and Development, Tokyo. Sixty-three of the 140 patients showed elevation of EBV DNA level to >10(2) copies/μg DNA and were further examined immunologically by flow cytometry, and the dose of tacrolimus and/or cyclosporine A was adjusted according to the results. The decrease in CD4/CD8 ratio and the increase in the number of HLA-DR(+) CD8(+) cells were observed in parallel with the decrease in EBV DNA load and in the number of CD19(+) CD23(+) cells following the reduction in immunosuppressive drugs. Analysis with HLA tetramers in a patient demonstrated a dramatic increase in the number of CD8(+) T cells specific to the EBV latent protein LMP2 accompanying the decline of EBV DNA load, suggesting that T cells of this specificity were actually involved in the control of EBV infection. No clinically apparent PTLD has developed in the 140 recipients, suggesting that our program of EBV control by molecular EBV monitoring coupled with lymphocyte phenotype analyses is effective in controlling EBV infection in pediatric liver transplant recipients.     

Intraventricular rituximab and systemic chemotherapy for treatment of central nervous system post-transplant lymphoproliferative disorder after kidney transplantation

Twombley K, Pokala H, Ardura MI, Harker-Murray P, Johnson-Welch SF, Weinberg A, Seikaly M. Intraventricular rituximab and systemic chemotherapy for treatment of central nervous system post-transplant lymphoproliferative disorder after kidney transplantation. Abstract: PTLD of the CNS is a rare complication of solid organ transplantation, and there are only case reports/series available in the literature. Current literature suggests that CNS PTLD carries a worse prognosis than PTLD outside the CNS, and most are of B-cell lineage, predominately monomorphic, and are associated with EBV infection. Because this disorder is so rare, there is no standard chemotherapy for pediatric patients with CNS PTLD and reported therapies for EBV-associated CNS PTLD are heterogeneous with mixed results. Since outcomes of CNS PTLD are historically poor, we attempted to develop a novel therapeutic treatment regimen. Based on a review of the literature and with the help of a multidisciplinary team, we created a regimen of chemotherapy that included dexamethasone and high-dose methotrexate in addition to intravenous and intraventricular Rituximab in two pediatric patients. The intraventricular chemotherapy succeeded in shrinking the tumor in both of our patients; however, as shown in the second case, the clinical outcome depends on the location of the tumor. Systemic and intraventricular therapies hold promise in the management of EBV-associated CNS PTLD; however the rarity of this entity prevents the development of well-designed studies necessary for the establishment of an evidence-based treatment standard.     

Monitoring of Epstein–Barr virus load and antibody in pediatric renal transplant patients

Background: Epstein–Barr virus (EBV) infection can lead to life-threatening post-transplant lymphoproliferative disorder (PTLD). The aim of the present study was to establish EBV monitoring methods to prevent PTLD.
Methods: EBV-DNA load was investigated, using real-time polymerase chain reaction (PCR) and anti-EBV antibody titers, in peripheral blood mononuclear cells of 21 renal transplant patients (seven recipients who were EBV-seronegative, R[−]; 14 who were EBV-seropositive, R[+]) before grafting. The mean age at entry and the mean follow-up period was 7.8 years of age (range, 3.3–12.0 years) and 1.8 years (range, 0.4–4.0 years), respectively, in the R(−) group, and 12.5 years of age (range, 3.9–17.7 years) and 3.8 years (range, 0.8–8.2 years) in the R(+) group, respectively.
Results: The mean maximum load of the EBV genome was 1071 copies/μg DNA (range, 106–20700 copies/μg DNA) in the R(−) group, and 61 copies/μg DNA (range, <50–552 copies/μg DNA) in the R(+) group. During follow up no patient in the R(+) group had any noticeable symptoms that could be related to EBV, but three recipients in the R(−) group developed EBV-related symptoms including adenoid hypertrophy, cervical lymphadenopathy, and PTLD (B cell lymphoma), in one patient each. In the R(−) group the first leukocyte-associated viremia was detected at 30–180 days, and seroconversion at 43–266 days after transplantation.
Conclusions: Viral DNA detection using PCR is a useful tool for EBV surveillance, but the maximum EBV load was not markedly elevated (2474 copies/μg DNA) in a patient with PTLD. Therefore, EBV surveillance using only monitoring of EBV load in peripheral leukocyte may be insufficient. Histology may therefore be necessary to accurately diagnose PTLD.     

Epstein-Barr virus DNA load and seroconversion in pediatric renal transplantation with tacrolimus immunosuppression

EBV infection is one of major complications arising in pediatric patients who have undergone renal transplantation. A strong correlation between the grade of immunosuppression and the development of PTLD, one of the most severe EBV-associated diseases, has been recognized. In this study, we monitored the serologic profile in conjunction with peripheral blood EBV-DNA load of 32 children who underwent renal transplantation with tacrolimus as an immunosuppressant. Six patients were EBV-seronegative (EBV-) before the transplantation, and the mean DNA load in the EBV- group was significantly higher than that in the EBV-seropositive (EBV+) group. Seroconversion occurred in five of these patients in a mean period of 22 weeks after the transplantation. The EBV-DNA load in the EBV+ group was maintained at a low level for a year, whereas it increased rapidly to over 1 x 10(5) copies/mL in two patients in the EBV- group three to seven months after the transplantation, which corresponds to the timing of seroconversion, and one of them developed PTLD. These observations suggest that the close monitoring of the EBV-DNA load, along with longitudinal observation of seroconversion, is essential in pediatric renal transplantation, particularly for younger children who are more likely to be EVB-.     

Diagnosis and treatment of post-transplantation lymphoproliferative disorder in pediatric heart transplant patients

PTLD is a severe complication in transplant recipients. Detection of increased EBV load in the peripheral blood acts as a surrogate marker for increased risk of PTLD development. We analyzed the time course of the disease, its severity, the organs involved, and mortality rates in our institutional experience of pediatric heart transplantation. This paper identifies risk factors for PTLD and describes the different ways of diagnosing and treating the disease. PTLD was screened for in 146 pediatric heart transplant patients using a retrospective analysis in patients who received transplantation before 1998. Prospective determination was performed in 72/146 patients transplanted after 1998 within the post-transplant follow-up. The occurrence of PTLD with all interventions, including tapering of immunosuppression, surgery, viral monitoring, and antiviral interventions, was recorded. PTLD was diagnosed in 12/147 (8.2%) children at a mean age of 7.2 +/- 3.3 yr after a mean post-transplant period of 3.2 +/- 2.2 yr. PTLD manifested in: lymph nodes (n = 4), intestine (n = 3), tonsils and adenoids (n = 2), eye (n = 2), and lung (n = 1). It was diagnosed in 7/12 as a monomorphic B-cell lymphoma and in four patients as a monomorphic Burkitt lymphoma, a polymorphic B-cell lymphoma, a T-cell rich or angiocentric lymphoma (Liebow) and as reactive plasmacytic hyperplasia (early lesion), respectively. Histology was not possible in one patient with ocular manifestation. EBV association was 83%. Risk factors in the comparison with patients without PTLD were age at time of Tx, primary EBV infection after Tx, use of Azathioprine and >or=3 doses of ATG. CMV mismatch and CMV infection, rejection episodes and steroids were not risk factors. Despite reduction of immunosuppression, treatment consisted of surgical procedures to remove tumor masses (n = 6), Rituximab (n = 5), polychemotherapy (n = 3), antiviral (n = 1) and autologous T-cell therapy (n = 1). All patients demonstrated full remission without death related to PTLD or treatment at 3.9 (1.3-6.2) yr median follow-up time. The manifestation of PTLD in pediatric heart transplant recipients is associated with EBV infection and is predominantly in the form of a B-cell lymphoma. A tight and specific follow-up including early assessment of immunity status and specific therapeutic intervention to improve cellular immunity is warranted and may contribute to a significant reduction of PTLD-related morbidity and mortality.     

Epstein-Barr virus-associated peripheral T-cell lymphoma and hemophagocytic syndrome arising after liver transplantation: case report and review of the literature

Post-transplantation lymphoproliferative disorders (PTLD) are a well-recognized complication of solid organ transplantation. The vast majority of PTLD are Epstein-Barr virus (EBV)-related infections that manifest as B-cell malignancies. We report an unusual case of an EBV-associated T-cell lymphoma in a 10-year-old boy who had previously undergone liver transplantation at age 4 years. He presented with hemophagocytic syndrome (HPS) and active EBV infection, with positive serum titers and polymerase chain reaction (PCR) for EBV in blood, colon, and antral samples.     

Epstein-Barr virus-associated post-transplant lymphoproliferative disease after bone marrow transplantation mimicking graft-versus-host disease

In contrast to solid organ transplantation (Tx), the incidence of post-transplant lymphoproliferative disease (PTLD) after hematopoietic stem cell Tx (HSCT) is generally low. This risk, however, is significantly elevated in patients receiving human leukocyte antigen (HLA) mis-matched or T-cell-depleted grafts, or after treatment for severe graft-versus-host disease (GvHD). An 18-yr-old patient with positive Epstein-Barr virus (EBV) serology received a fully matched, unmanipulated bone marrow graft from an unrelated EBV-positive donor for treatment of acute myeloid leukemia (AML) in second complete remission. GvHD prophylaxis was performed with cyclosporin A (CsA) and a short course of methotrexate. Four months after Tx, the patient developed ulcerative tonsillitis that was unresponsive to antibiotic treatment. Diarrhea appearing simultaneously was interpreted as gastrointestinal GvHD and steroids were added to CsA. A few days later the patient was admitted to hospital because of generalized seizure and pneumonia. Despite reduction of immunosuppression, intensification of anti-viral treatment, and subsequent mechanical ventilation, the patient died of acute respiratory distress 6 days later. Autopsy demonstrated disseminated EBV-induced, multi-nodular lymphoma infiltration of the entire colon but no signs of GvHD. Moreover, both lungs, paratracheal lymph nodes, kidneys, thyroid gland, and liver were infiltrated with large B-cell non-Hodgkin's lymphomas. This case underlines the rapid and aggressive course of EBV-induced disseminated PTLD after HSCT, initially mimicking intestinal GvHD because of massive colonic lymphoma infiltration. Tissue biopsies should be performed early for establishing correct diagnosis, thus enabling specific therapy, e.g. infusion of donor leukocytes with cytotoxic T-lymphocytes.     

The impact of monitoring Epstein–Barr virus PCR in paediatric bone marrow transplant patients: Can it successfully predict outcome and guide intervention?

BACKGROUND: Epstein-Barr virus (EBV) associated lymphoproliferative disease is a complication of haemopoietic stem cell transplantation (HSCT). In certain groups (unrelated and mismatched donor transplants, T-cell depleted) the risk may be as high as 25% with significant morbidity and mortality. Strategies to predict the impending development of this disorder and allow early intervention have therefore assumed importance. We routinely screen the peripheral blood of all recipients of allogeneic HSCT to detect EBV DNA by quantitative polymerase chain reaction (PCR) technology and report here how this correlates with clinical disease and management. PROCEDURE: Data on 28 successive patients who underwent HSCT at our institution were reviewed. The relationship between EBV reactivation demonstrated by quantitative PCR and development of post transplant lymphoproliferative disease (PTLD) was determined. RESULTS: EBV reactivation occurred in 68% of patients, however only 7% developed clinical PTLD. Patients with high level reactivation (n = 9) had more frequent episodes of reactivation and all patients who progressed to overt PTLD were found in this group. In contrast none of those patients with low level reactivation (n = 10) or persistently negative results (n = 9) showed any signs of clinical disease. Anti-CD20 monoclonal antibody (Rituximab) therapy was instigated in both cases of proven PTLD and three cases of high level reactivation with successful outcomes. Response to treatment was associated with a prompt decline in viral copy number. CONCLUSIONS: Our results indicate that EBV reactivation is a common occurrence in the paediatric allogeneic transplant setting and that only a proportion of patients will progress to PTLD. Frequent monitoring may help to predict those at highest risk and guide intervention.     

Increased Ig-null B lymphocytes in the peripheral blood of pediatric solid organ transplant recipients with elevated Epstein-Barr viral loads

Abstract:  In this study, the characteristics of Ig-null B cells in high viral load carriers were examined by four-color flow cytometry. The frequency of Ig-null B cells in patients with high, low or undetectable virus loads was found that while patients with a high load had more Ig-null cells, these cells were also present in the low and undetectable load groups. As Ig-null cells from patients with no viral load were EBV-negative, EBV infection was not absolutely required for the generation or survival of Ig-null cells. Ig-null cells were CD19⁺, sIg^-, CD5⁻, CD10⁻, CD27⁻, CD23⁻, CD38⁻, and CD69⁻ with variable surface expression of CD20 and CD40. Ig-null cells did not have a proliferating cell phenotype (Ki67^-) and a high proportion were HLA class I^- and class II^-. Virus copy number in CD19⁺ Ig-null cell populations may be much higher than in CD19⁺ Ig⁺ cell populations. EBV infected Ig-null cells were common in blood specimens from pediatric solid organ transplant recipients and infected Ig-null cells may pose potential problems for immunotherapies that target infected B cells directly.     

Epstein-Barr virus associated post-transplantation lymphoproliferative disorder with hemophagocytosis in a child with Wiskott-Aldrich syndrome

A 23-month-old boy with Wiskott-Aldrich syndrome (WAS) received human leukocyte antigen (HLA)-one locus mismatched, unmanipulated allogeneic bone marrow graft from his mother. An Epstein-Barr virus (EBV)-associated post-transplantation lymphoproliferative disorder (PTLD) of donor cell origin and hemophagocytosis syndrome with fever, lymphadenopathy, hepatosplenomegaly, seizures, involuntary movements and pancytopenia developed 52 days after transplantation. It was difficult to decide on the treatment strategy because the patient presented with B-cell hyperplasia that morphologically appeared malignant but was oligoclonal by heavy-chain analyses. Despite of donor leukocyte transfusion, low dose chemotherapy, and anti-B cell monoclonal antibody immunotherapy, the patient died 107 days after transplantation.