Long-Term Maintenance Therapy for Post–Cardiac Transplant Monoclonal Lymphoproliferative Disorder: Caveat Mammalian Target of Rapamycin

Long-Term Maintenance Therapy for Post–Cardiac Transplant Monoclonal Lymphoproliferative Disorder: Caveat Mammalian Target of Rapamycin Z. Khalpey, D.V. Miller, J.D. Schmitto, and S.S. Kushwaha ABSTRACT A 53-year-old Caucasian male suffering from idiopathic dilated cardiomyopathy underwent cardiac transplantation. Fifty-seven days following transplant, he developed posttransplant lymphoproliferative disorder (PTLD), which was Epstein-Barr virus positive. The initial episode of PTLD was treated with a dose reduction in cyclosporine (CsA) and a 4-week course of rituximab. Subsequent biopsies showed resolution of PTLD. One year posttreatment, his evaluation revealed severe cardiac allograft vasculopathy (CAV). The patient was switched to sirolimus-based immunosuppression regimen with gradual uptitration of sirolimus in combination with complete withdrawal of previously administered Calcineurin-based immunosuppression approach. The switchover was carried out over a 6-week period. In the following 3 years, there was CAV regression as well as PTLD remission, without any significant episode of rejection. Despite frequent relapses with this form of PTLD, the patient remains in remission, 8 years posttransplantation. In summary, sirolimus has been demonstrated to attenuate the progression of CAV, and this case report illustrates that regression of CAV is possible. In addition to preventing rejection, mammalian target of rapamycin inhibitors directly suppress signaling pathways leading to PTLD and may be effective monotherapy for preventing rejection and suppressing PTLD. INCE THE FIRST cardiac transplantations by Barnard, Kantrowitz, and Shumway,1 there has been substantial improvement in terms of survival rate after cardiac transplantation, which has gone up to 1 year. Survival beyond 1 year, however, still remains elusive. Posttransplant lymphoproliferative disorder (PTLD) has been labeled as the main culprit and in combination with other posttransplant malignancies, it comprises the second most common cause of death in cardiac transplant recipients. Reduction in immunosuppressive drugs is the first line of treatment against PTLD with more aggressive therapies reserved for nonresponders. Monoclonal forms of PTLD particularly respond less often to the reduction of immunosuppression, with chances of recurrence. There is a definite need for identification of optimal agents for maintenance therapy in patients with this form of PTLD.

S

CASE REPORT Cardiac transplantation was performed on a 53-year-old Caucasian male with end-stage idiopathic dilated cardiomyopathy in November 2002. The donor was a 54-year-old male smoker with a history of hypertension who had died from trauma to the brain. Donor coronary angiography showed no significant vascular obstruction in

the heart and a normal left ventricular function. Ischemic time was 175 minutes. From the second day postoperation, the patient underwent induction therapy with OKT3 (2.5 mg/d) for 3 consecutive days. This was switched to antithymocyte globulin due to the development of persistent fever. Cyclosporine (CsA) was started day 10 postoperation and its dose was progressively increased (target: 350 – 400 ng/mL). Prior to this, the patient was on a dosage regimen of 2 mg/kg/d of azathioprine, started day 1 postoperation. A tapering course of prednisone was initiated upon discontinuation of the induction therapy. The patient was put on long-term aspirin (325 mg/d) and pravastatin (20 mg/d) prior to discharge.

From the Division of Cardiovascular Diseases (Z.K., S.S.K.), Mayo Clinic, Rochester, Minnesota, USA; Division of Anatomic Pathology (D.V.M.), Mayo Clinic, Rochester, Minnesota, USA; and Division of Cardiac Surgery (J.D.S.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusett. Z.K. and S.S.K. authors contributed equally to this work. Address reprint requests to Sudhir S. Kushwaha, MD; William J. von Liebig Transplant Center, Division of Cardiovascular Diseases, Gonda 6, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail: [email protected]

© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/–see front matter doi:10.1016/j.transproceed.2011.03.033

Transplantation Proceedings, 43, 1893–1899 (2011)

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1894 On day 46 postoperation, the patient was readmitted due to development of cytomegalovirus (CMV) viremia, with a CMV copy count of 46,000/mL. Upon readmission, he was administered ganciclovir intravenously for 3 days, then switched to oral valganciclovir following discharge. Subsequent testing revealed clearance of viremia. On day 57, a routine surveillance endomyocardial biopsy revealed no rejection but showed Epstein-Barr virus (EBV) associated PTLD, plasma cell myeloma type with monoclonal kappa light chain restriction (Fig 1 and 2). Cardiac magnetic resonance imaging showed normal allograft function and no discrete mass lesions were observed. Computed tomography scan of the chest, abdomen, and pelvis was negative for lymphadenopathy or lesions. Bone marrow biopsy revealed no evidence of PTLD. As the PTLD was confined to the cardiac region, the patient was initially treated with a reduction in the standard dose of CsA, followed a week later with rituximab, 375 mg/m2 weekly for 4 weeks. Subsequent biopsies over the next several weeks showed gradual dissolution of the PTLD infiltrate. However, on day 92, routine biopsy showed grade 2R (formerly 3A) rejection. CsA was increased as a cautionary measure, and subsequent biopsies were negative for PTLD or significant rejection. Coronary angiography at 12 months posttransplantation revealed severe diffuse coronary artery disease with an occluded proximal left anterior descending artery (LAD) and severe vasculopathy affecting all vessels (Table 1, Fig 3A). The immunosuppressive regimen was altered by reducing CsA in a tapered dose over 6 weeks and replacing it with sirolimus to achieve a level of 10 to 14 ng/mL. A repeat coronary angiography 6 months later showed interval regression of cardirac allograft vasculopathy (CAV) (Table 1, Fig 3B) with proximal and mid-LAD diameter stenosis decreasing from 40% and 100% to 30% and 40%, respectively. A left coronary artery intravascular ultrasound (IVUS) study correlated well with the angiogram, showing only a 52% plaque volume (Fig

Fig 1. Photomicrographs from the first endomyocardial biopsy showing posttransplant lymphoproliferative disease (day 57 posttransplant). (A) High magnification view showing an unusual interstitial infiltrate that is rich in plasma cells; lower right (hematoxylin and eosin, ⫻400). (B) Intermediate magnification view showing positive in situ hybridization for Epstein-Barr virus (blue staining nuclei) (EBV-ISH, ⫻200). (C) Intermediate magnification view showing positive immunoperoxidase staining for kappa immunoglobulin light chain (brown stain) (Kappa-IP, ⫻200). (D) Intermediate magnification view showing immunoperoxidase staining for lambda immunoglobulin light chain (brown stain); only rare positive cells are seen (Lambda-IP, ⫻200).

KHALPEY, MILLER, SCHMITTO ET AL 3C). Serial IVUS studies (up to 36 months) and coronary angiograms (up to 48 months) after the switchover to sirolimus indicated no worsening of the CAV. During this time, the patient has been symptom-free, and left ventricular dimensions and function remains preserved (Table 1). The patient’s glomerular filtration rate (GFR) and serum creatinine level also improved.

DISCUSSION

In the case presented, the patient’s PTLD is unique in several respects. Plasma cell myeloma PTLD mostly involves the bone marrow or sometimes presents as a mass lesion. Diffuse infiltrative involvement limited to heart is distinctly unusual. It is possible that the incidental diagnosis by endomyocardial biopsy was particularly fortuitous for this patient and allowed detection of the process at an early stage, before its dissemination to other areas. Whether this may have contributed to the uncharacteristically dramatic response to conservative therapy is open to speculation, as there is no basis for comparison in the available literature. Still, the potential for recurrence was real and considerable. Sirolimus Attenuates Progression of Allograft Vasculopathy

CAV remains the major limiting factor to long-term survival following cardiac transplantation and accounts for 30% of deaths 5 years following cardiac transplantation.2 CAV develops insidiously, characterized by persistent perivascular inflammation with the development of intimal hyperplasia, and affects large epicardial vessels and the microcirculation, resulting in progressive luminal narrowing and decreased myocardial blood flow.3 It is likely that CAV is primarily an immune-mediated disease. Following car-

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Fig 2. Photomicrographs from an endomyocardial biopsy shortly after initiating therapy with rituximab (day 76 posttransplant). (A) High magnification view showing an unusual interstitial infiltrate that is rich in plasma cells but also with scattered eosinophils (hematoxylin and eosin, ⫻400). (B) High magnification view showing positive in situ hybridization for Epstein-Barr virus (blue staining nuclei) (EBV-ISH, ⫻400). (C) Intermediate magnification view showing less dominant immunoperoxidase staining for kappa immunoglobulin light chain (brown stain) (Kappa-IP, ⫻200). (D) Intermediate magnification view showing immunoperoxidase staining for lambda immunoglobulin light chain (brown stain); approximately equal numbers of positive cells are seen when compared to the Kappa-IP stain (Lambda-IP, ⫻200).

diac transplantation, humoral or cellular responses to human leukocyte antigen (HLA) antigens and vascular endothelial cell antigens may result in endothelial cell damage.3 Alloreactive B cells and anti-HLA antibodies are seminal components of the rejection process.4 Importantly, memory alloreactive B cells can persist for years after the initial immunizing event and therefore have the potential to initiate chronic allograft rejection at any time, even years after the initial inciting event. In heart grafts, the development of anti-HLA antibodies is known to correlate with CAV development.4,5 Following major histocompatability complex (MHC) class I antigen detection by CD8 lymphocytes, CD4 lymphocytes induce up-regulation of MHC class II antigens on endothelial cells resulting in a cellular immune response. A variety of stimulatory cytokines (interleukin [IL]-1, IL-2, IL-4, IL-6, and tumor necrosis factor [TNF]-alpha) and growth factors (including PDGF and transforming growth factor-beta) are also released, which are thought to promote the development of CAV.2 The chronic inflammation, which results from the initial endothelial cell injury, ultimately results in migration and proliferation of VSMC.2,5 Although the primary cellular effect of calcineurin inhibiTable 1. Posttransplant Lymphoproliferative Disorder Malignant transformation of lymphocytes, resembling lymphomas that develop in immunocompetent patients, but driven by different cellular mechanisms that require: 1. Profound suppression of cell-mediated immunity 2. Epstein-Barr virus infection

tors is to suppress transcription of proinflammatory cytokine genes,6 the clinical course of CAV is not arrested by CsA, and there are data to suggest that cytotoxic and pro-oxidative effects of other molecules, such as homocysteine may accelerate the development of CAV.7 Sirolimus and derivatives (Everolimus, RAD) are macrolide antibiotics with antiproliferative and potent antirejection properties. Sirolimus does not inhibit cytokineinduced lymphocyte proliferation and the genes associated with cell cycle progression, which results in vascular intimal and smooth muscle proliferation—the signature of CAV. Sirolimus also blocks proliferative responses by inhibiting T- and B-cell responses to growth factors—a different pathway than calcineurin inhibitors.8 In cardiac transplant recipients, sirolimus, or its derivative everolimus, have generally been used as secondary immunosuppressive agents in place of azathioprine or mycophenolate retarding the progression of allograft vasculopathy. However, sirolimus has been increasingly used as a primary immunosuppressant in cardiac transplant recipients, often limited to combination with mycophenolate and steroids.9 Based on the evidence that sirolimus is a powerful immunosuppressive agent, with the potential to preserve renal function, it is currently the practice of the Mayo Clinic Cardiac Transplant Program to try to convert all fresh cardiac transplant recipients to sirolimus-based immunosuppression at or around 6 months following transplant.10 IVUS is performed as part of the annual evaluation with routine coronary angiography in all cardiac transplant recipients, and the structural effects on the coronary circulation of conversion to sirolimus have been examined.

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Fig 3. Photomicrographs from a subsequent endomyocardial biopsy showing acute cellular rejection, ISHLT grade 2R (day 92 posttransplant). (A) Intermediate magnification view showing an interstitial infiltrate containing mostly lymphocytes (hematoxylin and eosin, ⫻200). (B) Intermediate magnification view showing negative in situ hybridization for Epstein-Bar virus (no blue staining nuclei) (EBV-ISH, ⫻200). (C, D) Intermediate magnification views showing polytypic immunoperoxidase staining for kappa and lambda immunoglobulin light chains (brown stain) (respectively Kappa-IP and Lambda-IP, ⫻200).

Sirolimus-based immunosuppression has been shown to result in attenuation of CAV.11 Sirolimus as a Novel Treatment Regimen for Cardiac PTLD

PTLD is a collective term for several distinct entities classified by the World Health Organisation as: reactive lymphoid/plasmacytic hyperplasia, polymorphic, monomorphic, and Hodgkin lymphoma-like types.10 Hyperplastic and polymorphous forms tend to be more indolent and responsive to therapy, whereas monomorphic and Hodgkin-like PTLD are more resistant and more likely to recur.10 PTLD is known as the third leading cause of death beyond the immediate perioperative period2–5,10 and affects 6% to 10% of heart transplant recipients.2,5 It is a significant hindrance to improved long-term survival in heart transplant recipients collectively.5 Interestingly, the PTLD rate among heart recipients is somewhat higher than for renal transplants, but lower than what is seen in lung transplants.6 It is more common in younger patients and most often occurs during the first year posttransplant.11 The risk of developing PTLD increases with increasing intensity of immunosuppression.8 EBV-seronegative recipients are more likely to develop PTLD, especially with organs from EBV-positive donors.9,11 Although the presence of EBV genome in lymphoid cells is not required for the diagnosis of PTLD, this is a recognized and frequent (⬎80%) association.10 EBV is more likely to be detected when PTLD is diagnosed in the first year posttransplant.12 In heart recipients, PTLD most often arises in the oropharynx, colon, lymph nodes, brain, and lung.13 Unlike renal, lung, and small bowel transplants, involvement of

transplanted heart itself is uncommon (⬎5% of all patients with PTLD).3 Localization to the transplanted organ suggests that a local immune reaction may play a role in the proliferative stimulus.14 In the allograft heart, it can present as intramural ventricular and mitral valve masses as well as infiltrative and insidious involvement of the myocardium and epicardium.7,14 When present in the myocardium, PTLD may be amenable to diagnosis by endomyocardial biopsy.7,14 Additionally, there is a ninefold-increase in risk of PTLD following heart transplantation with the use of anti-CD3 lymphocytolytic therapy (OKT3) used for induction of immunosuppression or treatment of rejection14 (used in the current patient). Eighty-eight percent of PTLD tumors are B cell in origin, and the role of EBV in the pathogenesis of PTLD has been well described.15 A recent study showed ubiquitous activation of the mammalian target of rapamycin (mTOR) signaling pathway in cells in PTLD, regardless of their EBV genome expression status.16 The inhibition of mTOR kinase activity by sirolimus and its analogs impairs cell growth and proliferation and may be effective in PTLD treatment. Our case study revealed that conversion from a calcineurin inhibitor base of immunosuppressive treatment to low-dose sirolimus was beneficial. Conversion to sirolimus has also been shown to be a successful strategy in treating cardiac transplant patients who have a recurrence of PTLD while on conventional immunosuppressive therapy.17 There is limited published data on the significance of conversion to sirolimus in PTLD patients.18,19 Cullis et al presented a case of disseminated PTLD with graft involvement in a renal transplant patient, which completely re-

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solved with sirolimus administration.18 Zaltzman et al also showed PTLD remission very early after transplantation.19 In another report, pediatric patients converted to sirolimus after a positive PTLD diagnosis achieved remission in 23 months.20 In a recent report by Pascual from eight European transplant centers, 3 of 16 PTLD patients were successfully converted to sirolimus as the only therapeutic agent. Resolution of PTLD occurred in three patients. However, sirolimus was withdrawn in a patient because of interstitial pneumonitis.21 Our patient was treated with low-dose sirolimus upon diagnosis of CAV a year after the transplant. Levels were maintained to achieve a trough blood level between 10 and 14 ng/mL. These doses were sufficient to attenuate CAV progression and maintenance of PTLD remission and cardiac allograft function. According to recent trials, conversion of calcineurin inhibitors to mTOR inhibitors is safe and also reduces the risk of chronic allograft nephropathy.22 It is difficult to determine whether PTLD suppression is because of the antineoplastic effect of sirolimus, diminished immunosuppression, or a combination of both. A study from Mayo Clinic showed that only 20% to 25% of patients with PTLD achieved a long-term remission after reduction or withdrawal of calcineurm inhibitor– based immunosuppressive treatment.23 The outcome of PTLD remains poor and sirolimus treatment should be considered, regardless whether the regression of PTLD is caused by immune system reconstitution or direct anticancer effect as it has been shown to reduce the incidence of de novo malignancies. Multicenter trials are required to establish a definitive role and appropriate dosage for long-term use of sirolimus to maintain PTLD remission in cardiac transplant recipients. Treatment and Management

Switching cardiac transplant patients from calcineurin inhibitors to sirolimus is a safe and effective primary immunosuppression strategy and prevents the progression of CAV. Sirolimus-based regimens may circumvent known complications of calcineurin inhibitor regimens such as nephrotoxicity and preserve posttransplant GFR. Cardiac PTLD is rare, and in our case study was treated initially by curtailing CsA for a week followed by a 4-week course of rituximab. Sirolimus had the added antineoplastic or antiproliferative effect of maintaining suppression of the PTLD as well as suppressing de novo malignancies. Four years after transplantation, our patient remains asymptomatic with no worsening of CAV or recurrence of the cardiac PTLD; his donor heart maintains and preserves its normal cardiac function. This suggests that sirolimus can be an effective long-term immunosuppressant without the necessary complications of calcineurin inhibitors. Treatment for PTLD presents a particular challenging paradox in modulating the immune system: a balancing act between inadequate suppression resulting in rejection on one side and oversuppression leading to PTLD progression or recurrence on the other. This is especially precarious

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when PTLD affects the transplanted heart itself, as erring on either side directly results in myocardial loss. Factors associated with poor outcomes include monomorphic phenotype, more than one extra nodal site, and poor performance status.4 The mainstay of PTLD treatment is reduction in immunosuppression, allowing the patient’s intrinsic cell-mediated immunity to attack the neoplastic cells.24,25 Yet, overall, response occurs in less than half of patients and durable remission is uncommon.24,26 For B-cell malignancies, anti-CD20 antibody (rituximab) therapy has also been examined, with response rates of 42% and 73%.27 In a recent multicenter study of 80 patients, the progressionfree survival for 59 patients treated with both reduced immunosuppression and rituximab was 70% (compared to 21% for the remaining patients treated with reduced immunosuppression alone).28 Long-term follow-up data for rituximab use are few, but there does still seem to be a real risk of recurrence. Bexarotene use has also been reported for T-cell forms of PTLD.29 Patients not responding to these therapies typically go on to conventional chemotherapy regimens used to treat lymphomas in nonimmunosuppressed patients (eg, CHOP ProMACE-CytaBOM, etc). As expected, these are associated with more infectious complications and toxicities.24 More recently, the mTOR inhibitor sirolimus has been proposed as a potential therapy for PTLD. Sirolimus acts by preventing cell cycle progression from G1- to S-phase. Originally developed as chemotherapy agent, it has found application as an antibacterial agent, immunosuppressant, and antineoplastic drug.30 The potential dual benefit in transplant recipients with PTLD is obvious. Other demonstrated effects of this agent, relevant to PTLD, include antiangiogenesis, inhibition of IL-10 production, and apoptosis induction.31 The mTOR signaling pathway, with ultimate nuclear factor-kappa B activation, is important in the development of several conventional lymphomas32 and is constitutively activated in Kaposi sarcoma.32 Sirolimus has been shown to reduce PTLD (and other malignancy) development in renal transplants33 and lead to regression and sustained remission in renal transplant patients as well.33 The use of mTOR inhibitors as the primary immunosuppressive agent following cardiac transplant is much less common than other agents in this class, particularly tacrolimus and CsA (both calcineurin inhibitors). While the combined antineoplastic and immunosuppressive effects of

Table 2. Cell-Mediated Rejection in Cardiac Transplant Biopsies 2004 ISHLT Grading system 1R. Perivascular inflammation with up to 1 focus of myocyte damage. 2R. Perivascular inflammation with 2 or more foci of myocyte damage, but not widespread inflammation and myocyte damage. 3R. Diffuse, widespread inflammation and myocyte damage.

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Table 3. Critical Immunologic Factors in the Neoplastic Transformation of Posttransplant Lymphoproliferative Disease Presence of a transplanted (nonself organ) Profound suppression of cell-mediated immunity Epstein-Barr virus infection

ACKNOWLEDGMENT We would like to thank Tammy Burns in assisting in the preparation of this manuscript.

REFERENCES

mTOR inhibitors appear to reduce the risk of PTLD, the optimal method for conversion from calcineurin inhibitor varies from center to center. Because the implications associated with the risk of PTLD progression due to oversuppression are more serious than those of rejection caused by undersuppression, fairly rapid withdrawal of calcineurin inhibitor seems the most sensible.32 Because sirolimus was introduced at 1 year posttransplant in this patient, rather than at the time of initial PTLD treatment, the sustained remission cannot be causally linked to the use of an mTOR inhibitor. Successful remission has been achieved with rituximab and reduced immunosuppression alone. Still, this case raises the stimulating possibility of a multipronged approach to posttransplant management with a single agent. In conclusion, we report a case of a heart transplant patient who developed a PTLD, CAV, and renal impairment in the first year following his transplant. He was weaned off CsA and bridged onto sirolimus as a primary immunosuppressant. Four years posttransplant, the PTLD has been suppressed, progression of CAV was attenuated, donor heart function was preserved, and there was complete resolution of renal impairment, which was likely due to calcineurin inhibitor toxicity. This case highlights the multiple advantages that can be achieved with the use of sirolimus and possibly other mTOR inhibitors, reflecting on what might be a novel paradigm shift in the postoperative immunosuppressive treatment of cardiac transplant patients. While fortunately uncommon in cardiac transplant recipients, PTLD remains one of the leading causes of death in such patients due to high mortality from both direct and indirect effects on the transplanted heart. Involvement of cardiac allograft itself is unusual, but has important implications: biologically (as a localized process within an immunologically foreign microenvironment); diagnostically (as it may be amenable to endomyocardial biopsy); and clinically (as it is associated with a poor prognosis and increased risk of CAV). The successful long-term mTOR inhibitor maintenance management of this patient’s monomorphic PTLD involving the heart (typically an aggressive PTLD with a propensity for recurrence) and avoidance of cytotoxic chemotherapy is encouraging. It suggests a clinical basis for the bench top evidence supporting the potential of a single mTOR inhibitor providing manifold benefits including adequate immunosuppression, PTLD prevention and/or suppression, and reduced risk of CAV. Tables 2 and 3.

1. Schmitto JD, Grossmann M, Kubbe E, et al: Norman Edward Shumway—pioneer of cardiac surgery (February 9, 1923 to February 10, 2006). Herz 33:386, 2008 2. Kass M, Haddad H: Cardiac allograft vasculopathy: pathology, prevention and treatment. Curr Opin Cardiol 21:132, 2006 3. Kushwaha SS, Narula J, Narula N, et al: Pattern of changes over time in myocardial blood flow and microvascular dilator capacity in patients with normally functioning cardiac allografts. Am J Cardiol 82:1377, 1998 4. Weiss MJ, Madsen JC, Rosengard BR, et al: Mechanisms of chronic rejection in cardiothoracic transplantation. Front Biosci 13:2980, 2008 5. Takemoto SK, Zeevi A, Feng S, et al: National conference to assess antibody-mediated rejection in solid organ transplantation. Am J Transplant 4:1033, 2004 6. Ho S, Clipstone N, Timmermann L, et al: The mechanism of action of cyclosporin A and FK506. Clin Immunol Immunopathol 80(3 Pt2):S40, 1996 7. Kutschka I, Pethig K, Harringer W, et al: Increased plasma homocysteine concentrations accelerate cardiac allograft vasculopathy. J Heart Lung Transplant 23:1260, 2004 8. Gustafsson F, Ross HJ: Proliferation signal inhibitors in cardiac transplantation. Curr Opin Cardiol 22:111, 2007 9. Raichlin E, Khalpey Z, Kremers W, et al: Replacement of calcineurin-inhibitors with sirolimus as primary immunosuppression in stable cardiac transplant recipients. Transplantation 84:467, 2007 10. Raichlin E, Bae JH, Khalpey Z, et al: Conversion to sirolimus as primary immunosuppression attenuates the progression of allograft vasculopathy after cardiac transplantation. Circulation 116:2726, 2007 11. Harris NL, et al: Lymphoma classification—from controversy to consensus: the R.E.A.L. and WHO Classification of lymphoid neoplasms. Ann Oncol 11(suppl 1):3, 2000 12. Nelson BP, et al: Epstein-Barr virus-negative post-transplant lymphoproliferative disorders: a distinct entity? Am J Surg Pathol 24:375, 2000 13. Opelz G, Henderson R: Incidence of non-Hodgkin lymphoma in kidney and heart transplant recipients. Lancet 342:1514, 1993 14. Swinnen LJ, Costanzo-Nordin MR, Fisher SG, et al: Increased incidence of lymphoproliferative disorder after immunosuppression with the monoclonal antibody OKT3 in cardiac-transplant recipients. N Engl J Med 323:1723, 1990 15. Tsai DE, Douglas L, Andreadis C, et al: EBV PCR in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: results of a two-arm prospective trial. Am J Transplant 2008 16. El-Salem M, Raghunath PN, Marzec M, et al: Constitutive activation of mTOR signaling pathway in post-transplant lymphoproliferative disorders. Lab Invest 87:29, 2007 17. Bonatti H, Hoefer D, Rogatsch H, et al: Successful management of recurrent Epstein-Barr virus-associated multilocular leiomyosarcoma after cardiac transplantation. Transplant Proc 37: 1839, 2005 18. Cullis B, D’Souza R, McCullagh P, et al: Sirolimus-induced remission of posttransplantation lymphoproliferative disorder. Am J Kidney Dis 47:e67, 2006 19. Zaltzman JS, Prasad R, Chun K, et al: Resolution of renal allograft-associated post-transplant lymphoproliferative disorder with the introduction of sirolimus. Nephrol Dial Transplant 20: 1748, 2005

LONG-TERM MAINTENANCE THERAPY 20. Jimenez-Rivera C, Avitzur Y, Fecteau AH, et al: Sirolimus for pediatric liver transplant recipients with post-transplant lymphoproliferative disease and hepatoblastoma. Pediatr Transplant 8:243, 2004 21. Pascual J: Post-transplant lymphoproliferative disorder—the potential of proliferation signal inhibitors. Nephrol Dial Transplant 22(suppl 1):i27, 2007 22. Ponticelli C: Can mTOR inhibitors reduce the risk of late kidney allograft failure? Transpl Int 21:2, 2008 23. Ghobrial IM, Habermann TM, Maurer MJ, et al: Prognostic analysis for survival in adult solid organ transplant recipients with post-transplantation lymphoproliferative disorders. J Clin Oncol 23:7574, 2005 24. Swinnen LJ, Mullen GM, Carr TJ, et al: Aggressive treatment for postcardiac transplant lymphoproliferation. Blood 86: 3333, 1995 25. Starzl TE, et al: Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet 1:583, 1984 26. Knight JS, et al: Lymphoma after solid organ transplantation: risk, response to therapy, and survival at a transplantation center. J Clin Oncol 27:3354, 2009

1899 27. Choquet S, et al: Efficacy and safety of rituximab in B-cell post-transplantation lymphoproliferative disorders: results of a prospective multicenter phase 2 study. Blood 107:3053, 2006 28. Evens AM, et al. Multicenter analysis of 80 solid organ transplantation recipients with post-transplantation lymphoproliferative disease: outcomes and prognostic factors in the modern era. J Clin Oncol 28:1038, 2010 29. Tsai DE, et al: Successful treatment of T-cell post-transplant lymphoproliferative disorder with the retinoid analog bexarotene. Am J Transplant 5:2070, 2005 30. Andrassy J, Graeb C, Rentsch M, et al: mTOR inhibition and its effect on cancer in transplantation. Transplantation 80: S171, 2005 31. Guba M, et al: Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 8:128, 2002 32. Monaco AP: The role of mTOR inhibitors in the management of posttransplant malignancy. Transplantation 87:157, 2009 33. Kahan BD, et al: Low incidence of malignancy among sirolimus/cyclosporine-treated renal transplant recipients. Transplantation 80:749, 2005