Posttransplant lymphoproliferative disease (PTLD) is a major cause of morbidity and mortality after pediatric liver transplantation. Primary Epstein-Barr virus (EBV) infection and intensity of immunosuppression have been identified as important risk factors for the development of PTLD (1–3).
Epstein-Barr virus is a Herpes virus that typically infects the epithelial cells of the oropharynx; active replication of the virus takes place during this phase (lytic infection). The virus subsequently infects and transforms B lymphocytes, maintaining a constant number of genome copies (latent infection) (1). The EBV-naïve solid organ transplant recipient may acquire EBV via latently infected lymphocytes in the donor organ. These lymphocytes may undergo a lytic phase and the recipient's lymphocytes may be infected. In the immunocompetent host, the proliferation of infected B lymphocytes is controlled by cytotoxic T lymphocytes and natural killer cells. Immunosuppression causes an iatrogenic defect in the T cells, allowing uncontrolled proliferation of EBV-infected B lymphocytes.
Seronegative children who acquire primary EBV infection after liver transplantation are at risk for development of symptomatic EBV disease, including PTLD. By 6 months after transplant, up to 60% of the seronegative patients become infected with EBV, as demonstrated by quantitative competitive polymerase chain reaction or by serologic analysis (2,3). Reduction in immunosuppressive therapy allows the host to restore the immune system, especially cytotoxic function of T cells, and therefore ameliorate EBV-related PTLD (4). Therefore, it may be useful to minimize immunosuppression in transplant recipients with active EBV infection.
We prospectively observed EBV viral load using a quantitative competitive polymerase chain reaction (QCPCR) assay in pediatric patients after liver transplantation to identify patients at increased risk for the development of PTLD (5). In addition, we assessed the safety and usefulness of preemptive reduction of immunosuppressive therapy in those patients.
PATIENTS AND METHODS
We prospectively measured EBV viral load in 23 consecutive patients (mean age, 6.2 years; range, 17 days–20.3 years) who underwent liver transplantation at The Mount Sinai Medical Center between July 1997 and November 1998 (Table 1). The patients were divided into two groups for analysis. Group 1 consisted of EBV-naïve patients, including patients who were either seronegative for EBV or who were less than 12 months of age at the time of transplantation. Primary EBV infection in infants less than 12 months of age is uncommon, and the presence of antibodies at this age is most likely related to the transplacental transfer of maternal antibodies (6).
Group 2 included patients who were seropositive for EBV before transplantation. The donor's EBV serologic status was not available in all the cases. Serial QCPCR assays to measure EBV viral load in peripheral blood lymphocytes (PBL) were performed on a monthly basis for the first year and every 2 months during the second year after liver transplant.
All patients with either negative serologic results for EBV or who were less than 12 months of age before transplantation received intravenous cytomegalovirus immune globulin (MedImmune, Gaithersburg, MD) and intravenous ganciclovir (Roche Pharmaceuticals, Nutley, NJ). The following regimen was used: cytomegalovirus immune globulin was given at a dose of 150 mg/kg within 72 hours of liver transplantation and then every 2 weeks for a total of five doses; subsequently, 100 mg/kg was given every 4 weeks for two doses (7). Ganciclovir was administered intravenously at 5 mg/kg every 12 hours for 14 days (8).
Primary immunosuppressive therapy consisted of Tacrolimus (Fujisawa, Deerfield, IL) and prednisone in all patients. Target trough Tacrolimus levels (IMX Tacrolimus II; Abbott Laboratories, Abbott Park, IL) were 15 ± 2 ng/mL during the first month after transplantation, 10 ± 2 ng/mL during the second and third month, and 5 ± 2 ng/mL after 3 months. The prednisone dosing was 0.6 mg/kg for the first 6 weeks, 0.3 mg/kg for weeks 7 to 12, 0.2 mg/kg for weeks 8 to 52. One year after transplantation, they received 0.2 mg/kg thrice weekly.
Tacrolimus dose was reduced by at least 50% to obtain a trough Tacrolimus level of 2 to 3 ng/mL if QCPCR demonstrated a viral load of more than 200 genome copies per 10 5 PBL in two consecutive samples. Dosage reduction took place at the time of the second positive QCPCR assay. Tacrolimus dosing was not reduced or maintained at low levels if medically contraindicated (see patients 7 and 11). Liver biochemistries and Tacrolimus levels were monitored monthly for at least 6 months after immunosuppression reduction.
Quantitative competitive polymerase chain reaction
Epstein-Barr virus viral load was measured in the PBL using QCPCR (5). This test was performed on a fee-for-service basis (University of Pittsburgh, laboratory of David Rowe).
The patients were followed up prospectively for a mean time of 73.1 weeks after transplantation. Group 1 consisted of 13 EBV-naïve patients with a mean age of 2.2 years (range, 17 days–14 years). Group 2 included 10 patients with seropositive EBV titers before transplantation with a mean age of 11.4 years (range, 5–20 years). Three of these patients were recipients of a second graft at the time of the study (patients 17, 22, and 23). Demographic characteristics and polymerase chain reaction findings are shown in Table 1. Nine of the 13 patients (69%) in group 1 had positive QCPCR results (>200 genome copies/10 5 PBL) at a mean time of 22.4 weeks (range, 4.4–60.9 weeks) after liver transplantation. All but one of these patients (patient 11; see case report below) were asymptomatic at the time of positivity (e.g., no fever, sore throat, snoring, weight loss, or occult blood in the stools). In seven of these nine patients, the donor's EBV serologic status was known, and all of them were positive.
Seven of the nine patients in group 1 subsequently had their immunosuppression (Tacrolimus) dose immediately decreased by at least 50% to reach Tacrolimus target levels of 2 ng/mL. None experienced acute cellular rejection or PTLD. All these patients were maintained with low Tacrolimus levels. There were two patients in whom the immunosuppression was not permanently reduced; one because of chronic rejection, and the second one because of a kidney transplant, which required higher levels of immunosuppression. Both of these patients experienced PTLD (see cases below).
Patient and graft survival in this cohort was 91%, with one death in each of groups 1 and 2, both of which were the result of causes unrelated to EBV disease. Seven of the nine patients in whom primary infection with EBV developed had received a living related graft (left lateral segment). The other two patients received cadaveric livers, one being a split graft and the other a whole liver. None of the patients who were seropositive for EBV at the time of transplantation (group 2) had a positive EBV viral load or PTLD during the follow-up period.
This 4-year-old male with primary hyperoxaluria and renal failure underwent living related liver transplantation as part of planned sequential living related liver and kidney transplantation. His postoperative course was complicated by hepatic artery thrombosis, biliary leak, chylothorax, fungal infection, and persistent fever. Primary EBV infection developed 21 weeks after orthotopic liver transplantation, and his immunosuppression was decreased by 50%. His EBV viral load decreased to 40 genomes/10 5 PBL without evidence of PTLD or acute cellular rejection. Forty-seven weeks after orthotopic liver transplantation, he underwent kidney transplantation. At the time of transplantation, enlarged mesenteric lymph nodes were noted; a biopsy showed nonspecific plasmacytic hyperplasia but no evidence of PTLD. Five days after his renal transplantation, he experienced an episode of acute cellular rejection of the kidney requiring OKT3 (murine monoclonal anti-CD3 antibody; Ortho-McNeil, Raritan, NJ). His EBV viral load increased to 1,000 genomes/10 5 PBL and repeat mesenteric lymph node biopsy showed polyclonal PTLD 52 weeks after orthotopic liver transplantation. Tacrolimus was discontinued, and the rejected transplanted kidney eventually was removed. This patient subsequently died of complications of oxalosis before kidney retransplantation could be performed. An autopsy was performed that did not reveal evidence of PTLD.
An 8-month-old male (previously reported in part (9)) with subfulminant hepatic failure of unknown cause underwent split liver transplantation. After surgery, he had multiple episodes of acute cellular rejection requiring pulses of steroids and OKT3. Six weeks after transplantation, he was persistently febrile and his EBV polymerase chain reaction results were found to be 5,000 genomes/10 5 PBL. Computerized tomographic scanning of the chest showed mediastinal adenopathy, but a biopsy was not performed because of the risks of the procedure. Upper endoscopy with random biopsies, liver biopsy, bone marrow biopsy, and upper gastrointestinal series were negative for PTLD. Tacrolimus dosing was decreased by 75%, and trough Tacrolimus levels dropped from 11.0 to 2.7 ng/mL. The patient experienced chronic rejection (total bilirubin, 20 mg/dL) and was treated with increased dosing of Tacrolimus and supplemental immunosuppression with Mycophenolate Mofetil (Roche Pharmaceuticals, Nutley, NJ). His bilirubin level fell to less than 1 mg/dL. In an effort to treat both his chronic rejection and EBV disease, ganciclovir was reinstituted at a dose of 5 mg/kg every 12 hours for 14 days and intravenous cytomegalovirus immune globulin (100 mg/kg per month) was continued for 6 months. He continued to have persistently high EBV viral loads, and Tacrolimus levels were maintained between 7 to 10 ng/mL. Twenty months after orthotopic liver transplantation, he sought treatment for recurrent pneumonia. A computed tomography scan of the chest was significant for persistent mediastinal lymphadenopathy. Mediastinal lymph node biopsy was significant for low-grade polyclonal PTLD. Lung biopsy, liver biopsy, and bone marrow biopsy results showed no evidence of PTLD. Cytomegalovirus immune globulin infusions were restarted, and immunosuppression remained unchanged (Tacrolimus target level, 8 ng/mL) in an effort to manage his chronic rejection. Thirty months after transplantation, his bilirubin is 0.9 mg/dL and his chest lesions have not recurred. He had a persistently high viral load of EBV (500–2,000 genomes/10 5 PBL) up until 28 months after liver transplantation, as has been described in children who have had PTLD (10–12). Thirty-two months after transplantation, his viral load has reduced to 100 genomes/10 5 PBL.
Posttransplant lymphoproliferative disease after liver transplantation in children remains a significant clinical problem. Different factors have been associated with the development of PTLD, including pretransplant EBV status (2,3), the recipient's age (13), and the level of immunosuppression (3). It has been shown that pediatric liver transplant recipients with primary EBV infection are at greater risk for the development of PTLD (3). One of the major concerns in pediatric liver transplantation is the EBV mismatch that typically exists between the seronegative recipient and an EBV-infected donor. With the increased use of living donors and split grafts, this problem has become more evident, because there is greater age disparity between donor and recipient. We considered infants less than 1 year of age as being seronegative patients, because the presence of antibodies is most likely related to the transplacental transfer of maternal antibodies (6). Our data revealed that 69% of the seronegative patients acquired primary EBV infection, and the only two patients in whom PTLD developed in our series had been EBV seronegative before transplant.
McDiarmid et al. (14) advocated the use of a minimum of a 100-day course of intravenous ganciclovir after orthotopic liver transplantation followed by oral acyclovir up to 2 years after transplant in seronegative patients who received a graft from a seropositive donor. We believe that EBV seronegative patients who receive an EBV-positive graft are likely to be infected near the time of transplantation. Therefore, to inhibit the lytic phase of the virus, all seronegative patients received ganciclovir in the immediate postoperative period for a 14-day course, as described by Green et al. (8). The approach advocated in this report avoids the need for continuous intravenous access for 3 months.
The use of immunosuppressive agents in organ transplant recipients interferes with host immunity, affecting cytotoxic T lymphocytes and natural killer cells, the major cell lines responsible for the natural immune surveillance of EBV infection. The deficient immune system together with the high risk of primary EBV infection in a seronegative patient increases the incidence of PTLD (2,3). Primary immunosuppression in pediatric liver transplant recipients in our center consists of a combination of steroids and Tacrolimus. It has been previously shown that the use of antithymocyte agents such as OKT3 for steroid-resistant rejection increases the risk of development of PTLD (15,16). Both of the patients in whom PTLD developed in our series were treated with enhanced levels of immunosuppression, one for ongoing chronic rejection and the other patient for a subsequent kidney transplant. Both patients also received treatment with OKT3.
Starzl et al. (4) demonstrated the reversibility of malignancies in transplant recipients by stopping or drastically reducing immunosuppression. Similarly, Cacciarelli et al. (17) were successful in treating pediatric liver transplant recipients with PTLD by decreasing or discontinuing the immunosuppression. More recently, McDiarmid et al. (14) showed a decreased incidence of PTLD in pediatric liver recipients by lowering immunosuppression before clinical EBV disease is evident. In our practice, serial monitoring with QCPCR as an indication of viral replication allowed us to detect patients in early asymptomatic stages of EBV infection. Monitoring of EBV serologic status in the setting of the administration of cytomegalovirus hyperimmunoglobulin is of little value. Preemptive immunosuppression reduction may have prevented progression to PTLD (18). Rowe et al. (5) determined that viral loads of more than 200 genome copies per 10 5 PBL were considered consistent with an increased risk of PTLD. In the present report, when the EBV viral load increased to more than 200 copies per 10 5 PBL, immunosuppression was lowered immediately by at least 50%. None of the seven asymptomatic patients who were treated with this approach experienced PTLD or allograft rejection. The absence of rejection suggests that lower target trough levels of Tacrolimus may be sufficient in pediatric liver transplant recipients. At a minimum, this experience shows that the approach of preemptive immunosuppression reduction is safe in the short term. The EBV viral load was quite variable after immunosuppression reduction. Peak loads were observed from the time of onset to 27 weeks later. Resolution of viral load, empirically defined as viral load less than 200 genomes/10 5 PBL, occurred in six of nine patients over a period of as long as 58 weeks.
Based on this experience, we suggest that the early detection of EBV replication with serial monitoring by QCPCR may be helpful in the management of pediatric liver transplant recipients. The preemptive reduction of immunosuppression in such patients may prevent the development of PTLD. Adjunctive antiviral therapy including cytomegalovirus immune globulin and ganciclovir may also be necessary to achieve these results. An ongoing study of cytomegalovirus immunoglobulin will address the issue of its role in prevention of development of PTLD. Future randomized controlled studies of preemptive immunosuppression reduction may also be warranted.
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