Cellular Immunity to Epstein-Barr Virus in Liver Transplant Recipients Treated with Rituximab for Post-Transplant Lymphoproliferative Disease

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1 American Journal of Transplantation 5; 5: Blackwell Munksgaard Copyright C Blackwell Munksgaard 4 doi:./j x Cellular Immunity to Epstein-Barr Virus in Liver Transplant Recipients Treated with Rituximab for Post-Transplant Lymphoproliferative Disease Barbara Savoldo a,b,, Cliona M. Rooney a,b, Ruben E. Quiros-Tejeira b,yvette Caldwell a, Hans-Joachim Wagner a,timothy Lee c, Milton J. Finegold d, Gianpietro Dotti a,e, Helen E. Heslop a,b,e and John A. Goss c a Center for Cell and Gene Therapy, b Departments of Pediatrics, c Surgery, d Pathology and e Medicine, Baylor College of Medicine, Texas Children s Hospital and The Methodist Hospital, Houston, Texas, USA Corresponding author: Barbara Savoldo, bsavoldo@bcm.tmc.edu The evaluation of long-term cellular immunity to EBV in pediatric orthotopic liver transplant (OLT) recipients after treatment with the humanized anti-cd2 monoclonal antibody (Rituximab) has not yet been explored. At our institution, one child with EBV-related mononucleosis-like syndrome and five children with polymorphic-ebv-ptld occurring 6 88 months after OLT were treated with Rituximab. Treatment was well tolerated. All children achieved complete remission. After Rituximab, B-lymphocytes were undetectable in the peripheral blood and EBV-load, monitored with real-time PCR, decreased to undetectable levels in all children from >4 copies/lg DNA at diagnosis. Four to eight months after Rituximab, EBV-load increased (>4 copies/lg DNA) in four children, and PTLD recurred in three. Their frequency of EBV-specific T-cell precursors, measured by Elispot analysis, remained lower than in healthy controls. Rituximab effectively induced regression of PTLD in OLT recipients. However, EBV-specific T-cell immunocompetence, which may be crucial for the long-term control of EBV-mediated proliferation, did not improve. Key words: EBV, liver transplant, PTLD, rituximab Received 6 July 4 revised 8 September 4 and accepted for publication 2 September, 4 Introduction The use of intensive immunosuppressive regimens to prevent allograft rejection has resulted in the increased occurrence of a heterogeneous group of lymphomas known as PTLD. EBV plays an essential role in the development of these lymphomas, and impaired T-cell function allows the uncontrolled proliferation of B-lymphocytes infected and transformed by the virus (). The incidence of PTLD ranges from 2 to 2% depending in part on the type of transplanted organ, immunosuppressive regimen and EBV serology before transplant (reviewed in 2). PTLD can present as localized disease, sometime involving the transplanted organ, mononucleosis-like syndrome or disseminated lymphoma. Similarly, the histological pattern is widely heterogeneous, ranging from polymorphic lympho-proliferation to monomorphic lymphoma; treatment remains controversial (reviewed in 2,3). Reduction or withdrawal of immunosuppression induces regression of PTLD in 2 8% of cases in different studies and is usually recommended as first-line therapy. However, graft rejection occurs in more than 4% of patients as a consequence and represents the major limitation of this intervention. Alternatively, anti-viral agents, interferon-alpha (IFN-a) and chemotherapy have been adopted with variable results. In a few small series adoptive transfer of autologous or closely HLA-matched EBV-specific cytotoxic T-lymphocytes (CTL) has shown promise (4 6). Recently, anti-b-lymphocyte antibodies and in particular the humanized anti-cd2 monoclonal antibody (Rituximab), first introduced for treatment of non-hodgkin s follicular lymphomas, have been explored as an alternative treatment for PTLD. Several small studies have demonstrated the safety and efficacy of Rituximab for treatment of PTLD affecting both hematopoietic stem cell and SOT recipients (7 3). Overall, Rituximab allows the achievement of complete regression of PTLD in 5 6% of patients. Rituximab eliminates normal B cells as well as EBVinfected and/or transformed lymphocytes, predominately by in vivo complement activation and antibody-dependent cellular cytotoxicity (ADCC). However, since the restoration of EBV-specific cellular immunity is a crucial mechanism to sustain the long-term control of PTLD in patients in whom immunosuppression has been reduced or suspended, we evaluated EBV-specific cellular immunity in pediatric liver transplant recipients with PTLD who were treated with Rituximab. 566

2 EBV-Immunity in Pediatric Liver Transplant Recipients Patients and Methods Patients Between January 999 and June 3, 5 children and young adults (median age 5.8 years, range 2 years) who received orthotopic liver transplant (OLT) were referred to our department for monitoring of EBV-DNA load in the peripheral blood. All but patient who was treated with cyclosporin A (CSA) received tacrolimus (FK56) as immunosuppressive treatment. As a standard approach, FK56 trough levels were generally maintained around 2 ng/ml in the first 3 months after transplant and were then reduced to 8 ng/ml up to year. After the first year, the trough level is progressively reduced to reach 5 8 ng/ml. Prednisone is progressively reduced and withdrawn at a median of 6 months post-transplant. In patients with a high virus load or diagnosis of PTLD, trough levels of FK56 may be reduced to 5 8 ng/ml earlier. Since a high EBV load in peripheral blood is considered predictive of PTLD, we regularly measure EBV-DNA in transplanted patients (4,5). Our previous analysis in hematopoietic stem cell transplant recipients showed that EBV-DNA loads greater than 4 copies/lg DNA strongly correlate with occurrence of PTLD (6). This value was also used in our series of pediatric liver transplant recipients as an indicator to adjust the degree of immunosuppression. Thirty-two pediatric liver recipients (63%) followed for a median of 2.5 years (range.2 months 6 years) maintained an EBV load below 4 copies/lg DNA and developed no signs or symptoms of PTLD. Nineteen patients (37%) had EBV loads over 4 copies/lg DNA in at least two consecutive determinations at a median follow-up of months after transplant (range 2 96 months). In these patients the FK56 dose was adjusted to maintain trough levels around 5 8 ng/ml, regardless of the time from transplant. In patient receiving CsA, the dose was reduced to maintain a trough level around ng/ml. No patients developed graft rejection, but despite this immunosuppression adjustment, EBV-DNA remained persistently elevated (>4 copies/lg DNA) in 4 patients. Eight of these patients with persistent high EBV-DNA load developed PTLD, with a median of 9 months post-transplant (range months). Biopsies of suspected sites were analyzed by the Pathology Department of the Texas Children s Hospital, using conventional histologic examination and immunophenotyping. PTLDs were classified according to the criteria recommended by the Society of Hematopathology. Computerized tomography (CT) was performed for staging purposes. Treatment and response evaluation Two of 8 patients who developed PTLD were treated before Rituximab was available. PTLD regressed after chemotherapy treatment in patient and surgical resection of the localized mass in other. The remaining 6 patients were treated with Rituximab after consent was obtained from the legal guardian. Each patient received weekly intravenous doses of Rituximab (375 mg/m 2 ) for a total of four doses except the first one who received a single dose. All patients received acetaminophen as pre-medication. To minimize the risk of graft rejection, FK56 or CsA doses, already reduced when high EBV load was noted, were maintained at the trough level of 5 8 ng/ml and ng/ml, respectively. Peripheral blood B cells and T cells were monitored monthly after treatment using CD9 and CD3 antibodies (Becton Dickinson, San Jose, CA), respectively, and analyzed with a FACSscan flow cytometer (Becton Dickinson). Serum immunoglobulins were quantified and immuno-electrophoresis was performed every 3 months after Rituximab therapy. The response to treatment was evaluated within 4 weeks of the last dose of Rituximab. Complete response was defined as complete regression of all clinical (physical and radiographic) evidences of disease and decrease of the peripheral blood EBV-DNA load to <4 copies/lg DNA for at least 4 weeks. Relapse was defined as reappearance of signs or symptoms of PTLD in a patient who had previously achieved a complete remission. Physical and instrumental examinations and biopsy were repeated in case of suspected relapse. For these analyses the median follow-up was 23 months (range years) post-rituximab administration. EBV-DNA and Elispot analysis EBV load was measured on peripheral blood blood mononuclear cells (PBMC) using a real-time PCR assay specific for the highly conserved EBER region of the EBV genome, as previously reported by Savoldo et al. (7). The sensitivity of the real-time-pcr is 4 copies/lg of DNA. Values detected in the peripheral blood of 38 normal EBV-seropositive donors ranged from <4 to (median <4) copies/lg of DNA. To assess EBV-immunity, we measured the frequency of circulating interferon-gamma (IFN-c )producing cells in response to autologous EBVtransformed cells (LCL) by Elispot analysis as previously described (7,8). Elispot analysis was performed retrospectively on batches of frozen PBMC. In our experience no significant differences are observed when fresh or frozen samples are compared (data not shown). To minimize inter- and intraassay variability, at least four samples from the same donors were analyzed in the same assay. In developing the EBV-specific Elispot assay, we determined that inter- and intra-assay variability is acceptable when use of the same lot of antibody and plates and viability of the antigen-presenting cells is assured. In addition, the same operator performed all the assays of this study. PBMC were available pre- and post-rituximab for 4 patients. For patient only PBMC collected at different time-points >6 months after Rituximab therapy were available for analysis. Spots were counted using a high-resolution automated Elispot Reader System (KS Elispot 4.3 software; Zellnet Consulting Inc., NY). The number of IFN-c spot-forming cells (SFC)/ 5 PBMC was normalized for the percentage of CD3+ cells. A negligible number of spots (<4) were produced by medium, EBV-LCL or PBMC alone. As a positive control, PBMC were incubated with 25 ng/ml of PMA and lg/ml of Ionomycin and IL-2 ( U/mL). In all cases, > spots/5 4 CD3+ PBMC were produced. For comparison, cryopreserved PBMC from 9 EBV seropositive healthy donors and samples from 3 other EBV seropositive solid organ transplant recipients (age at transplant: mean.8 years, range.5 6 years; age at testing: 3. years, range 4 years; 7 patients were EBV seronegative and CMV seronegative pre-olt) were also analyzed for frequency of EBV-responding T cells by IFN-c Elispot assay. The frequency of IFN-c SFC/ 5 CD3+ PBMC from these donors is summarized as mean ± one standard deviation (SD). Results Patient details Clinical details of the 6 patients who received Rituximab are summarized in Table. In 4 patients, the diagnosis of PTLD was made based on histology, while patient was treated for a mononucleosis-like syndrome and patient 6 did not have easily accessible disease for biopsy but had persistent high viral load and mediastinal adenopathy detected by CT scan. Treatment was well tolerated. All patients had resolution of clinical signs and symptoms, disappearance of circulating peripheral B cells and a fall in EBV-DNA to undetectable or low normal levels (< copies/lg DNA). Longterm follow-up data are shown in Table 2. Two patients are still in complete remission (no PTLD and EBV load <4 copies/lg DNA). All patients had complete reconstitution of peripheral blood B cell 6 months post-rituximab therapy (mean 3.7% ± 2.6%, pre-rituximab vs. 6% ± 4.2%, American Journal of Transplantation 5; 5:

3 Savoldo et al. Table : Characteristics of patients treated with Rituximab Time EBV load Gender a / EBV/CMV OLT- high Time (copies/ Patients Age b status c EBV DNA d OLT-PTLD e lg DNA) PTLD: sign/symptoms PTLD histology M/3 Neg/Pos ,3 Fever, diarrhea Not available 2 F/3 Pos/Neg ,56 Fever, anemia, cervical Reactive polyclonal adenopathy hyperplasia, CD2+ EBER+ 3 F/9 Neg/Neg Adenotonsillar Plasmacytic, polyclonal hypertrophy, reduction hyperplasia, CD2+ EBER+; of the vision in the white cell infiltrates in the left eye anterior chamber 4 M/5 Neg/NA ,52 Adenotonsillar hypertrophy, Polymorphic, pulmonary nodules, polyclonal hyperplasia, mediastinal adenopathy CD2+ EBER+ 5 F/4 Neg/Neg Fever, diarrhea Lymphohistiocytic infiltrate and plasma cells, CD2+ EBER+; polyclonal 6 M/2 Neg/Neg ,247 Fever, adenopathy Not available (site not accessible) a M = male; F = female; b age in years at time of diagnosis of PTLD; c Serological status at transplant; d Time from orthotopic liver transplant (OLT) to peripheral blood EBV load >4 copies/lg DNA, expressed in months; e Time from orthotopic liver transplant (OLT) to PTLD, expressed in months. Table 2: Evaluation of patients after Rituximab therapy Time to EBV load at relapse Last EBV load Patients PTLD: localization PTLD: histology PTLD a (copies/lg DNA) Outcome (copies/lg DNA) N/A b N/A N/A N/A Alive (3.3 years) 2 Cervical lymph node Early PTLD 2,697 Deceased due to 4,459 (polyclonal, CD2+) graft failure (.8 years) 3 Left eye Not accessible Alive (2.3 years) N/A N/A N/A N/A Alive (2.2 years) 22 5 Gut Early PTLD Alive (.5 years) 798 (polyclonal, CD2+) 6 N/A N/A N/A N/A Alive (.6 years),763 a Time to relapse after last cycle of Rituximab, expressed in months; b N/A: not applicable. 6 months post-rituximab) except in patient 4 in whom B cells were recovered 7 months post-therapy. The percentage of gamma globulin fraction at immunoelectrophoresis showed no significant differences pre- versus post- Rituximab (mean 2.3 ± % and 7.8 ± 8%, respectively). Although IgG modestly decreased during the first 6 months post-treatment (from 49 ± 4 mg/dl to 952 ± 395 mg/dl), they recovered by month 2 after Rituximab therapy ( ± 46 mg/dl). In 4 patients, EBV load increased above 4 copies/lg of DNA, between 4 and 2 months after completion of Rituximab therapy and 3 developed evidence of recurrent disease, with histology showing early PTLD in 2 patients and recurrence of left eye involvement in the third. Two patients were treated with further reduction of immunosuppression. While viral load stabilized around 4 copies/lg DNA for patient 3, EBV- DNA remained elevated for patient 2, who suffered from frequent episodes of graft rejection and died of graft failure at a follow-up of.5 years post-rituximab treatment. Patient 5 and 6 were enrolled in our phase I study of adoptive therapy with EBV-specific CTLs and remained well 3 and >6 months after therapy. Evaluation of cellular immunity to EBV in solid organ transplant recipients To assess cellular immunity to EBV, we measured the release of IFN-c by PBMC following stimulation with EBVantigens in Elispot assay. Autologous EBV-LCL were used as the source of EBV antigens. The frequency of EBVspecific T cells was investigated in 3 of the solid organ transplant recipients referred to our laboratory for the determination of EBV-DNA load and for whom autologous EBV-LCL were available. For comparison purpose, the frequency of EBV-responding T cells was also evaluated in PBMC obtained from healthy EBV seropositive donors using the IFN-c Elispot assay. As expected the frequency of EBV-responding T cells was reduced in the solid organ transplant recipient group (mean 59, range IFN-c SFC/ 5 CD3+ PBMC) compared to our series of healthy EBV-seropositive donors (mean 588, range 568 American Journal of Transplantation 5; 5:

4 EBV-Immunity in Pediatric Liver Transplant Recipients Figure : Monitoring of EBV-DNA and EBV-specific T-cell precursor frequency in circulating peripheral blood of 2 patients with complete and durable response after Rituximab. The graph shows the EBV-DNA load (line) and frequency of EBV-specific T cells (black bars) in patients (panel A) and 4 (panel B) followed over time after Rituximab therapy. The arrow indicates when Rituximab was administered. The x-axis on the left indicates the number of IFN-c SFC/ 5 CD3+ PBMC, while the x-axis on the right indicates the number of EBV copies/lg of DNA in a log scale. The dotted line indicates the value of 4 copies/lg of DNA. Their EBV load remained below 4 copies/lg of DNA after Rituximab therapy. IFN-γ SFC/ 5 CD3+ PBMC Rituximab B cells > % Rituximab B cells > % Days post-transplant EBV copies/µg DNA A B 3 46 SFC/ 5 CD3+ PBMC), confirming that the immunosuppressive therapy taken by the solid organ transplant patients to prevent graft rejection impairs the function of EBV-responding T cells. In the 6 treated patients, we evaluated EBV-specific immune responses during and after Rituximab therapy (Figures and 2). In the 2 patients whose EBV load remained below 4 copies/lg of DNA after B cell recovery, the frequency of EBV-responding T cells at -year follow-up (25 ± 7 and 345 ± 8 IFN-c SFC/ 5 CD3+ PBMC for patient and 4, respectively) was comparable to that observed in solid organ transplant recipients with EBV load <4 copies/lg DNA (mean of 226 ± 5 SFC/ 5 CD3+ PBMC in our series). For the other 4 patients who had recurrence of high EBV-DNA (>4 copies/lg of DNA) or PTLD after recovery of circulating B cells, the frequency of EBV-responding T cells assessed by IFN-c Elispot did not significantly change after Rituximab. Surprisingly, patient 3 had a high frequency of EBV-responding cells of 96 ± 25 SFC/ 5 PBMC at the time of PTLD diagnosis. Such frequency is in the range seen in healthy controls. We can only speculate whether this is related to the occurrence of the PTLD 7 years after transplant and whether this implies that EBV immunity may play a less crucial role for the development of late PTLD or whether this reflected the immunoprivileged site of her disease (the eye). Nevertheless, the frequency of EBV-specific T cells in this patient did not significantly change after Rituximab therapy. Discussion Treatment of EBV-driven PTLD remains controversial, although it is widely accepted that reduction or suspension of immunosuppression can induce sustained regression (3). However, acute and chronic graft rejection secondary to modulation of immunosuppression remain the major drawback of this therapeutic approach, particularly for heart, liver and lung transplant recipients in whom graft rejection can be a life-threatening complication. A promising approach is represented by the use of anti-b-lymphocyte monoclonal antibodies and in particular by the humanized anti-cd2 antibody, Rituximab (7 3). Here we present our experience with the use of Rituximab in 6 pediatric solid organ transplant recipients presenting with PTLD (4 with biopsy-proven polyclonal disease, 2 with no accessible site for biopsy). Despite the small sample size, our study provides further evidence that Rituximab is well tolerated and induces significant regression of EBV+, CD2+ PTLD. As expected, B-lymphocytes rapidly disappeared from the peripheral blood and, in parallel, EBV-DNA load declined to low/undetectable level in all cases. No significant impairment of the humoral response was observed after Rituximab therapy. However, in our experience the remission was not durable. Four to 8 months after the last dose of Rituximab, at the time of B-lymphocyte recovery, EBV load increased in 4 of 6 patients all of whom had persistently low levels of T-cell immunity to EBV and early PTLD recurred in 3 of them. The role of EBV-specific cellular immunity in long-term control of PTLD has been demonstrated in hematopoietic stem cell recipients (9). Moreover, Porcu et al. recently emphasized the correlation between increased frequency of EBVspecific precursors observed after reduction/suspension of immunosuppression and durable regression of EBV-driven PTLD in renal transplant patients (2). We observed that Rituximab directly induced regression of PTLD by eliminating B-lymphocytes but that there was no recovery of circulating EBV-responding T cells. The continued impaired cellular immunity to EBV observed in these patients after treatment with Rituximab American Journal of Transplantation 5; 5:

5 Savoldo et al. IFN-γ SFC/ 5 CD3+ PBMC A B Rituximab B cells > % Rituximab B cells > % EBV copies/µg DNA IFN-γ SFC/ 5 CD3+ PBMC C Days post-transplant Rituximab B cells > % D B Rituximab B cells > % Days post-transplant EBV copies/µg DNA Figure 2: The frequency of EBVspecific T cells did not change after treatment with Rituximab in patients who had relapse of EBV load and/or disease after Rituximab. The graph shows the EBV-DNA load (line) and frequency of EBV-specific T cells (black bars) in patients 2 (panel A), 3 (panel B), 5 (panel C) and 6 (panel D) followed over time before and after Rituximab therapy. The arrow indicates when Rituximab was administered. The x-axis on the left indicates the number of IFN-c SFC/ 5 CD3+ PBMC, while the x-axis on the right indicates the number of EBV copies/lg of DNA in a log scale. The dotted line indicates the value of 4 copies/lg of DNA. In all 4 patients, the frequency of EBV-responding T cells did not significantly change after Rituximab therapy. High EBV load recurred in these patients at B cell recovery. was likely determined by the iatrogenic immunosuppression maintained to prevent graft rejection. FK56 trough levels between 5 and 8 ng/ml, which are significantly higher than the levels generally maintained in allograft recipients with PTLD, allowed an efficient graft protection in our series, but likely inhibited the function of circulating EBV-responding T cells, resulting in relapse in 4 of them. Since Rituximab induces early regression of PTLD, the choice not to discontinue or modify immunosuppression regimen, thus reducing the risk for acute or chronic graft rejection might be taken (2). Although limited, our experience however suggests that if immunosuppression is not significantly reduced, the patients maintain their risk of developing PTLD when B cells recover. Thus, Rituximab should be complemented with restoration of the T-cell function, for example, by reducing immunosuppression or by adoptive transfer of EBV-specific T cells. For transplant recipients with PTLD, the ideal treatment should allow an efficient reconstitution of EBV-specific cellular immunity to control virus reactivation while maintaining an adequate immunosuppression to protect the graft. EBV-specific T-cell precursors can be determined accurately by the use of immunological assays, such as tetramer and Elispot analysis, and future prospective studies could identify a threshold of CTL-specific precursor frequency that allows EBV protection in patients receiving immunosuppression (2,22). However, this balance may be difficult to achieve in practice. 57 American Journal of Transplantation 5; 5:

6 EBV-Immunity in Pediatric Liver Transplant Recipients The adoptive transfer of EBV-specific CTL might represent an alternative strategy to increase the frequency of EBV-specific T-cell precursors in immunosuppressed patients. Our group and others have shown that it is possible to expand autologous EBV-specific CTL from SOT recipients (4 6,7). Their infusion can induce PTLD regression, reduce EBV-DNA load in the peripheral blood and, most importantly, increase EBV-specific CTL frequency (4 6). However, EBV-specific CTL line generation requires 8 2 weeks, while PTLD can present as aggressive disease requiring immediate treatment. Prophylactic administration of EBV-specific CTL to patients at high risk of developing PTLD would be ideal, but the clinical and biological criteria to identify these patients are still not well defined. At our institution we use Rituximab to induce rapid tumor regression and provide a clinical respite while EBV-specific CTL are established. Detection of increased peripheral blood viral load has been proposed as a predictive marker (4,5). However, other groups have observed that in the solid organ transplant setting EBV load, while sensitive, lacks specificity for the development of PTLD (5). We also have observed that pediatric solid organ transplant recipients can continue to carry high viral load without developing PTLD. While additional tests that might improve the specificity of EBV load are under investigation (tetramer, elispot and viral gene expression), instead of withdrawing the immunosuppression that puts the graft at risk, we are adoptively transferring EBV-specific CTL to restore virus surveillance. Multicenter trials are obviously required to test the efficacy of this approach. In conclusion, Rituximab was safely used to induce regression of PTLD in 6 liver transplant recipients. B cells and humoral immunity were restored 6 8 months after treatment. However, although reduced immunosuppression was not discontinued, and therefore virus-specific cellular immunity did not improve, disease/high virus load recurred in 4 of them. Although more extensive analysis, including adult patients and cases of monoclonal PTLD, are required to support this observation derived from a small single-center experience, our data suggest that Rituximab therapy should be complemented with treatment aimed at improving the cellular immune surveillance. Acknowledgments We are grateful to Teresita Lopez and Lan Zhang, from the Center for Cell and Gene Therapy at Baylor College of Medicine, for excellent technical assistance. We thank Jaymee Scott and Lisa Bristow, from the Department of Surgery at Baylor College of Medicine, for taking care of the patients and for proving samples for this study. We also thank Dr. Malcolm Brenner for critical review of the manuscript. This work was supported by NIH grants PO CA94237, DK62329, the GCRC at Baylor College of Medicine (RR88), a Specialized Center of Research Award from the Leukemia Lymphoma Society, a Doris Duke Distinguished Clinical Scientist Award (HEH), a Methodist Foundation grant (BS) and a Glaser Foundation fellowship (BS). References. Rickinson AB, Kieff E. Epstein-Barr virus. Fields Virology. Philadelphia: Lipincott-Raven, 996: Straathof KC, Savoldo B, Heslop HE, Rooney CM. Immunotherapy for post-transplant lymphoproliferative disease. Br J Haematol 2; 8: Green M. Management of Epstein-Barr virus-induced posttransplant lymphoproliferative disease in recipients of solid organ transplantation. Am J Transplant ; : Khanna R, Bell S, Sherritt M et al. Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease. Proc Natl Acad Sci U S A 999; 96: Comoli P, Labirio M, Basso S et al. 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