*Abbreviations: EBV, Epstein-Barr virus; OLTx, orthotopic liver transplant; PBL, peripheral blood lymphocytes; PCR, polymerase chain reaction; PTLD, posttransplant lymphoproliferative disease; QC-PCR, quantitative competitive PCR.
Epstein-Barr virus-associated posttransplant lymphoproliferative disease (EBV/PTLD*) is an important cause of morbidity and mortality after solid organ transplantation. Recent observations have identified an association between a high EBV viral load in the blood (as measured by semiquantitative or quantitative polymerase chain reaction [PCR] assays) and the presence of EBV/PTLD in recipients of solid organ and bone marrow transplantation (1-4). However, only limited data are available describing the natural history of the EBV viral load after EBV/PTLD. The purpose of this study was to prospectively follow the EBV viral load from the time of diagnosis of EBV/PTLD in pediatric orthotopic liver transplant (OLTx) recipients.
Beginning in November 1995, the EBV viral load in the peripheral blood was followed in all patients diagnosed with EBV/PTLD after pediatric OLTx at the Children's Hospital of Pittsburgh. The diagnosis of EBV/PTLD was initially suspected clinically in all patients. Routine evaluation for EBV/PTLD included a computed tomography scan of the chest and abdomen in all patients. Lymph node biopsies were performed on all patients with clinically significant peripheral adenopathy. Endoscopy was performed in any patient with symptoms suggestive of PTLD involvement of the gastrointestinal tract (e.g., diarrhea, microscopic or gross blood in stool). The diagnosis of PTLD was subsequently confirmed histologically and graded using previously published criteria (5,6). The presence of EBV within PTLD lesions was confirmed using the EBER probe (5). Determinations of clonality of PTLD lesions were not routinely performed during the study period.
EBV viral load was measured in the peripheral blood by quantitative competitive (QC) PCR using our previously described methods (4). Briefly, peripheral blood lymphocytes (PBL) were separated from whole blood by centrifugation onto a Histopaque (Sigma Chemical Co., St. Louis, MO) cushion. After washing and counting, aliquots of PBL were lysed and chilled before performance of PCR. For each QC-PCR assay, five tubes containing 5000, 1000, 200, 40 or 8 copies of a competitor plasmid which carries the EBV latent membrane protein 2a (LMP2a) target sequence with a 46-base pair deletion cloned into it. Reaction tubes were inoculated with lysates of 105 PBL from the patients and then subjected to PCR using the TP1Q5′ and TP1Q3′ primers (4). After amplification, quantification of the viral load was initially determined by ethidium bromide staining and was subsequently confirmed by phosphorimaging of Southern blots.
Blood specimens for performance of QC-PCR to determine EBV viral load in the peripheral blood were obtained at the time of diagnosis of PTLD. Follow-up specimens were initially obtained every 2 to 4 weeks from the time of diagnosis of PTLD; the frequency of measurement of EBV viral load was decreased to every 1 to 3 months once the patients were clinically stable.
EBV viral loads >200 genome copies/105 PBL measured on our assay were considered to be consistent with an increased risk of PTLD (4). Viral loads ≤200 genome copies/105 PBL obtained during the treatment of PTLD were considered virologic evidence of clearance of EBV/PTLD. Any subsequent viral loads >200 genome copies/105 PBL were considered evidence of a virologic "rebound." Persistent rebound was defined as continuous measurement of EBV viral load >200 on all assays after the initial clearance of EBV from the peripheral blood. Intermittent rebound was defined as a fluctuating EBV viral load that went above and below the rebound value of 200 genome copies/105 PBL.
The diagnosis of rejection in patients with a recent history of EBV/PTLD was also confirmed histologically using standard definitions (7). In general, EBER staining was routinely performed on all liver biopsies obtained within the first few months of the diagnosis of EBV/PTLD.
Management of the patients as well as their clinical and histologic response to treatment and long-term follow-up were recorded from a complete review of their medical records.
Seven pediatric OLTx recipients who received their transplants at the Children's Hospital of Pittsburgh between August 1990 and June 1997 who were diagnosed with EBV/PTLD between November 1995 and December 1997 were followed during this study. Standard immunosuppression treatment in all seven patients had consisted of tacrolimus and corticosteroids. The patients have been followed for a median of 6 months after the diagnosis of PTLD (range 3.5-28 months).
The age, primary diagnosis leading to liver transplantation, pretransplant donor/recipient EBV serologic status, time to the diagnosis of EBV/PTLD, sites of involvement, and histology of the PTLD lesions for the seven children with diagnosed EBV/PTLD are shown in Table 1. The mean age at the time of OLTx was 2.0 years (range 0.8-6.0 years). Six of the seven children were EBV-seronegative before transplant. Three of their donors were EBV-seropositive, three were seronegative, and the donor status was not determined in the seventh case. The median time to the development of EBV/PTLD after transplant was 6 months (range 0.2-5.3 years). The gastrointestinal tract was the only site of involvement of PTLD in five of the seven patients. The remaining sites of involvement included the liver and tonsils in one patient and a cervical lymph node in another patient. Six of the seven patients whose PTLD lesions were considered to have a polymorphic histology were managed by reduction (one patient) or discontinuation (five patients) of tacrolimus and steroids in combination with the use of intravenous ganciclovir (10 mg/kg per day) and high-titered anti-cytomegalovirus intravenous immunoglobulin (CytoGam, MedImmune, Inc., Gaithersburg, MD) at a dose of 100 mg/kg given three times after the initial diagnosis and then weekly. This therapeutic strategy was generally continued until there was clinical resolution of the PTLD. A single patient who presented with PTLD 5.3 years after OLTx was diagnosed as having a Burkitt's lymphoma and was treated with withdrawal of immunosuppression and traditional chemotherapy.
The results of EBV viral load testing and clinical course of the patients after the diagnosis of EBV/PTLD are shown in Table 2. A total of 69 EBV viral loads were measured for the seven patients (range 4-13) after the diagnosis of PTLD. The median EBV viral load in the peripheral blood at the time of diagnosis of PTLD was 500 genome copies/105 PBL (range 300-4000). Clearance of EBV from the peripheral blood (EBV QC-PCR <200 genome copies/105 PBL) was documented a median of 2 weeks (range 1.5 weeks to 8 months) after the diagnosis of EBV/PTLD and initiation of appropriate management. The fall in EBV viral load in the peripheral blood seemed to correlate with clinical and histologic evidence of regression of PTLD.
Five patients developed rejection within the first month after the diagnosis of PTLD. Of interest, the time to clearance of EBV from the peripheral blood was similar to the time of onset of first episode of rejection after the diagnosis of PTLD in these five children: mean onset of early rejection=13.8 days (range 5 days to 4 weeks) compared with mean time to clearance of EBV virus from peripheral blood=18.8 days (range 10 days to 5 weeks). When available, the EBV viral load at the time of diagnosis of rejection was always <100 (range 8-80 genome copies/105 PBL). The child treated with chemotherapy for Burkitt's lymphoma developed rejection 10 months after her initial diagnosis; her EBV viral load at the time of diagnosis of rejection was 40 genome copies/105 PBL. The remaining child has not developed rejection and remains off of immunosuppression now, 6 months after the diagnosis of PTLD.
A rebound in EBV viral load in the peripheral blood to a value of >200 genome copies/105 PBL was noted in five of the seven patients a median of 3.5 months (range 2.3-13 months) after the diagnosis of EBV/PTLD. An intermittent rebound pattern was noted in four of the five children, and a persistent rebound was noted in the child with a history of Burkitt's lymphoma. A short course of intravenous ganciclovir and CytoGam was used on two occasions in one child (patient 3) during an intermittent rebound and on two occasions for the child with the history of Burkitt's lymphoma with the persistent rebound (patient 1). The remaining children were not treated during rebounds in EBV viral load. Of interest, no child has persistent nondetectable values (<8 genome copies/105 PBL) of EBV viral load, and values as high as 200 EBV genome copies/105 PBL were intermittently seen but not treated in the two remaining children. In none of the seven patients has there been any clinical or histologic evidence of recurrent EBV/PTLD. All of the children are currently alive, and their initial hepatic allografts are functioning well.
Results of several recent studies have correlated the EBV viral load in the peripheral blood with the presence of EBV/PTLD in recipients of solid organ and bone marrow transplantation using semiquantitative (1-3,8) or quantitative PCR (4). However, these studies have not followed serial measurements of the EBV viral load after the diagnosis of PTLD in organ transplant recipients. Accordingly, we prospectively followed seven pediatric OLTx recipients with serial measurements of the EBV viral load after the diagnosis and treatment of PTLD. Results of this study suggest that the serial measurement of the EBV viral load after a diagnosis of PTLD provides an insight into the initial clinical response of patients to the management of PTLD. However, the meaning of the EBV viral load during long-term surveillance after the diagnosis of PTLD seems less clear.
Results of this study demonstrated that an initial drop in EBV viral load in the peripheral blood was correlated with clinical and histologic evidence of regression of PTLD. This would be consistent with current theories regarding the role of specific anti-EBV cytotoxic T cells in recovery from immunoresponsive EBV/PTLD once immunosuppression has been reduced or withdrawn (8,9). A fall in the EBV viral load seems to be a surrogate marker of the presence of circulating anti-EBV cytotoxic T cells and hence a predictor of the subsequent regression of PTLD lesions in affected patients. An alternate explanation for the fall in viral loads might be the antiviral effect of ganciclovir therapy. Hanto et al. (10,11) reported that the use of acyclovir in the treatment of EBV-induced PTLD resulted in clearance of salivary EBV and clinical improvement in a small group of renal transplant recipients. Although both acyclovir and ganciclovir are active against the lytic form of EBV (12), PTLD lesions consist primarily of EBV-immortalized B cells in which the EBV episome is maintained by human DNA polymerase (13). There is the possibility that a component of the viral load in the circulation is supported by the introduction of newly infected B cells. To the extent that this is the case, antiviral therapy with acyclovir or ganciclovir could reduce or eliminate the contribution of newly infected cells to the peripheral blood. However, the principal contributers to EBV viral load in the peripheral blood are considered to be immortalized or latently infected B cells rather than cells in the lytic state (1). Since neither acyclovir nor ganciclovir is able to affect this form of viral DNA replication, the use of either of these agents seems unlikely to account for the fall in EBV viral loads noted in this study. Further evidence against the role of nucleoside analogs like acyclovir or ganciclovir in the falling viral load seen in our patients is provided by Kenagy et al. (3), who noted that EBV viral loads increased and PTLD developed in some patients receiving acyclovir or ganciclovir. We and others have also observed the rise in viral load and development of PTLD on these therapies (9,14). Finally, it is possible that the viral load may have been decreased through the use of immunoglobulin therapy via antibody-dependent cell-mediated cytotoxicity.
Not all patients seemed to experience a drop in viral load in this evaluation before the onset of regression of PTLD, although such an occurrence was anticipated. A possible explanation for this is that we had only obtained viral load assays every 2 to 4 weeks after the diagnosis of PTLD. More frequent monitoring of our patients might have detected the fall in viral load in the period preceding the clinical and histologic evidence of regression. On the other hand, we were able to demonstrate a temporal relationship between the fall in the EBV viral load in the peripheral blood and the onset of rejection. The mean time of development of rejection and the time to a decline in the EBV viral load <200 genome copies/105 PBL were nearly identical, and all patients with diagnosed rejection had a viral load <100 genomes/105 cells. We believe that more rigorous follow-up in the first several months after the diagnosis of PTLD not only should identify patients who are responding to their therapeutic regimen, but also may identify the timing of increasing risk of rejection, permit an intervention, and thereby minimize the likelihood of graft loss to severe or chronic rejection. Accordingly, we are now monitoring our patients every 1 to 2 weeks after the diagnosis of PTLD until the time of viral clearance, clinical improvement, and/or onset of rejection.
An interesting feature of our PTLD monitoring was the development of intermittent or persistent rebounding of the EBV viral load in five of our seven patients. In general, this was correlated with the reinstitution of immunosuppression. However, an intermittent rebound also occurred in the one patient who never developed rejection after PTLD and for whom immunosuppression was not restarted. Viral loads approaching our treatment threshold of >200 genome copies/105 PBL were also seen in our two remaining patients. Since the normal EBV viral load in latently infected immunocompetent individuals is <0.1 genome copies/105 PBL, these convalescent EBV viral loads are >2000 times the expected value in the immunocompetent host. However, to date rebounding of the EBV viral load to these levels has not been associated with clinical symptoms or histologic evidence suggestive of EBV/PTLD. A brief course of ganciclovir and CytoGam was administered during rebound episodes in two patients. However, subsequent rebound episodes in these two children as well as in the remaining patients have been observed without any therapeutic intervention. Since this rebound was seen in five of seven of our patients, and since recurrent PTLD occurs in only about 5% of children who develop PTLD after OLTx (15), it suggests that these children maybe developing a weak but nonetheless adequate immunity against EBV and recurrent PTLD. Although more long-term observations are necessary to confirm this hypothesis, we currently do not recommend institution of antiviral therapy or dramatic modifications in immunosuppression in patients developing rebounds in their EBV viral loads in the absence of other clinical or histologic evidence of recurrent PTLD.
The increasing awareness of the problem of EBV/PTLD has led to the emergence of new diagnostic tools that may enhance the diagnosis and management of this problem. We believe that the EBV viral load in the peripheral blood as measured by the QC-PCR assay provides useful information into the clinical status and response of patients with PTLD. Additional and ongoing observations are necessary to confirm the observations of our study.
1. Riddler SA, Breinig MC, McKnight JLC. Increased levels of circulating Epstein-Barr virus-infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of posttransplant lymphoproliferative disease in solid-organ transplant recipients. Blood 1994; 84: 972.
2. Savoie A, Perpete C, Carpentier L, Joncas K, Alfieri C. Direct correlation between the load of Epstein-Barr virus-infected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood 1994; 83: 2715.
3. Kenagy DN, Schlesinger Y, Weck K, Ritter JH, Gaudreault-Keener MM, Storch GA. Epstein-Barr virus DNA in peripheral blood leukocytes of patients with posttransplant lymphoproliferative disease. Transplantation 1995; 60: 547.
4. Rowe DT, Qu L, Reyes J, et al. Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in peripheral blood of pediatric transplant recipients with lymphoproliferative disorders. J Clin Microbiol 1997; 35: 1612.
5. Randhawa PS, Jaffe R, Demetris AJ, et al. Expression of Epstein-Barr virus-encoded small RNA (by the EBER-1 gene) in liver specimens from transplant recipients with post-transplantation lymphoproliferative disease. N Engl J Med 1992; 327: 1710.
6. Nalesnik MA, Jaffe R, Starzl TE, et al. The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine A-prednisone immunosuppression. Am J Pathol 1988; 133: 173.
7. Fung JJ, Todo S, Jain A, Demetris AJ, McMichael JP, Starzl TE. The Pittsburgh randomized trial of tacrolimus versus cyclosporine for liver transplantation. J Am Coll Surg 1996; 183: 117.
8. Rooney CM, Loftin SK, Holladay MS, Brenner MK, Krance RA, Heslop HB. Early identification of Epstein-Barr virus-associated post-transplant lymphoproliferative disease. Br J Haematol 1995; 89: 98.
9. Green M, Reyes J, Rowe D. New strategies in the prevention and management of Epstein-Barr virus infections and posttransplant lymphoproliferative disease following solid organ transplantation. Curr Opin Solid Organ Transplant 1998; 3: 143.
10. Hanto DW, Frizzera G, Gajl-Peczalska KJ, et al. Epstein-Barr virus induced B-cell lymphoma after renal transplantation: acyclovir therapy and transition from polyclonal to monoclonal B-cell proliferation. N Engl J Med 306; 913: 1982.
11. Hanto DW, Frizzera G, Gajl-Peczalska KJ, Balfour HH, Simmons RL, Najarian JS. Acyclovir therapy of Epstein-Barr virus-induced posttransplant lymphoproliferative diseases. Transplant Proc 17; 89: 1985.
12. Lin JC, Smith MC, Pagano JS. Prolonged inhibitory effect of 9-(1,3-dihyroxy-2-propoxymethyl)guanine against replication of Epstein-Barr virus. J Virol 1984; 50: 50.
13. McKnight JLC, Cen H, Riddler S, et al. EBV gene expression, EBNA antibody responses and EBV + peripheral blood lymphocytes in post-transplant lymphoproliferative disease. Leuk Lymphoma 1994; 15: 9.
14. Yao Qy, Ogan P, Rowe M, Wood M, Rickinson AB. Epstein-Barr virus-infected B cells persist in the circulation of acyclovir treated carriers. Int J Cancer 1989; 43: 67.
15. Wu TT, Swerdlow SH, Locker J, et al. Recurrent Epstein-Barr virus-associated lesion in organ transplant recipients. Hum Pathol 1996; 27: 157.
The 17th Annual Meeting of the American Society of Transplant Physicians, May 9-13, 1998, Chicago, Illinois