Posttransplant Lymphoproliferative Disorder in Solid Organ and Hematopoietic Stem Cell Transplantation

Posttransplant Ly mphoprolif erat ive D i s o rde r i n S o l i d Or g a n and Hematopoietic Stem C ell Tr ansplantat ion Sarah J. Nagle, MDa, Ran Reshef, MDb, Donald E. Tsai, MD, PhDa,* KEYWORDS  Posttransplant lymphoproliferative disorder  Epstein–Barr virus  Epstein–Barr virus cytotoxic T cells  Allogeneic hematopoietic stem cell transplantation  Solid organ transplantation

KEY POINTS  Posttransplant lymphoproliferative disorders (PTLD) represent immunosuppression-related lymphoid or plasmacytic proliferation that occur in the setting of solid organ or allogeneic hematopoietic cell transplantation.  PTLD is most commonly related to Epstein–Barr virus (EBV) infection.  Incidence depends on type of transplant, age of the patient and degree of immunosuppression. Clinical presentation is nonspecific and there have been 5 identified histologies.  Initial treatment involves the withdrawal of immunosuppression, and refractory disease can be treated with antibody therapy or cytotoxic chemotherapy.  More recently, there is evidence for the use of EBV-specific cytotoxic T cells from either the donor or a third party and a phase II trial is ongoing.

Posttransplant lymphoproliferative disorders (PTLD) represent immunosuppression-related lymphoid or plasmacytic proliferation that occur in the setting of solid organ or allogeneic hematopoietic cell transplantation (HSCT). PTLD after solid organ transplant almost always originates from recipient lymphoid cells, whereas PTLD after HSCT is usually of donor origin. PTLD is a relatively

common malignancy after transplantation and incidence varies by type of transplant, age of the patient, and degree of immunosuppression. Although most cases of PTLD are due to Epstein– Barr virus (EBV) infection, EBV-negative disease does occur and is becoming increasingly common. Treatments aim to restore the host immune system to combat EBV. Novel therapeutic approaches including EBV-specific cytotoxic T lymphocytes are currently being studied.

a Department of Medicine, Division of Hematology and Oncology, Hospital of the University of Pennsylvania, Perelman Center for Advanced Medicine, West Pavilion, 4th Floor, 3400 Civic Center Boulevard, Philadelphia, PA 19104, USA; b Blood and Marrow Transplantation Program and Columbia Center for Translational Immunology, Columbia University College of Physicians and Surgeons, New York, NY, USA * Corresponding author. E-mail address: [email protected]

Clin Chest Med - (2017) -–http://dx.doi.org/10.1016/j.ccm.2017.08.001 0272-5231/17/Ó 2017 Elsevier Inc. All rights reserved.

chestmed.theclinics.com

INTRODUCTION

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Nagle et al PATHOPHYSIOLOGY The majority of PTLD are related to EBV infection. EBV is a member of the herpesvirus family that infects more than 90% of humans and persists for the lifetime of the person. The virus is typically transmitted via oral secretions, but can also be transmitted via blood, transplanted allogeneic hematopoietic cells, or solid donor organs.1 EBV infections of infants and children are generally asymptomatic or have nonspecific symptoms. However, infections of adolescents and adults frequently result in infectious mononucleosis, characterized by the classic triad of fever, posterior cervical lymphadenopathy, and pharyngitis.2 Acute infection with EBV leads to a polyclonal expansion of B lymphocytes that harbor EBV. Viral antigens expressed by these B lymphocytes elicit both a humoral and cellular immune response. Antibodies are produced to viral capsid and nuclear proteins. Natural killer cells and CD41 and CD81 T cells control proliferating EBV-infected B lymphocytes during primary infection.3,4 However, a small population of B lymphocytes escape the immune response and are transformed into a state of B-cell activation and continuous proliferation that persist throughout life. In an immunocompetent host, these latently infected B lymphocytes are controlled by cytotoxic T cells (CTLs). However, if T-cell immunity wanes, these cells can proliferate and may lead to PTLD.2 EBV-encoded proteins drive signaling events that directly contribute to B-cell growth and survival. The EBV genome is a linear DNA molecule that encodes approximately 100 viral proteins. During latency, the viral genome expresses only 10 proteins. This makes T-cell recognition more challenging, aiding in persistence of EBV infection. The proteins expressed during latency are EBV latent membrane proteins and EBV nuclear antigens (EBNAs). Latent membrane protein (LMP)-1 and LMP-2A transmit signals that mimic certain aspects of antigen-mediated B-cell activation. LMP-1 increases expression of CD23 (a B-cell activation antigen) and induces expression of bcl-2 (an inhibitor of apoptosis) in infected cells. LMP-2 prevents EBV reactivation in latently infected cells. EBNA-2 upregulates the expression of LMP-1 and LMP-2 that are necessary for transformation of B cells. Additionally, EBNA-2 in concert with EBV nuclear antigen-leader protein (EBNA-LP) forms a transcriptional regulatory complex that upregulates pro-growth factors that can transform B cells into immortal lymphoblastoid B cells.2

The EBV-infected cells that give rise to PTLD can originate in the host or the donor. After solid organ transplantation, PTLD is typically of host origin, manifests as a multisystem disease and most likely represents reactivation of a latent EBV infection. Primary EBV infection may also cause PTLD, but this more commonly occurs in children who have not had prior EBV infection.5 In solid organ transplant recipients, if the PTLD is donor derived, it is typically limited to the allograft tissue.6 Conversely, most cases of PTLD after allogeneic stem cell transplantation are donor derived and may present with systemic involvement. In this situation, the host immune system has been destroyed by the conditioning regimen enabling EBV-infected donor cells to proliferate.7 PTLD that is not related to EBV infection is less well-understood. A recent study of 176 solid organ transplant recipients with PTLD demonstrated that the proportion of EBV-negative PTLD cases is increasing, and that this subtype has distinct characteristics from the EBVpositive counterpart. EBV-negative PTLD is typically characterized by a later onset of disease, monomorphic histology, and a more aggressive course.8 Interestingly, EBV-negative PTLD may still be responsive to immunotherapy/immune reconstitution approaches, such as withdrawal of immunosuppression.9 Further studies have shown that EBV-positive and EBV-negative PTLD have distinct gene expression profiles. The gene expression profile of EBV-negative PTLD is similar to that of diffuse large B-cell lymphoma in immunocompetent individuals.10

EPIDEMIOLOGY AND RISK FACTORS Incidence PTLD is the most common malignancy complicating solid organ transplantation (excluding nonmelanoma skin cancer and in situ cervical cancer), comprising approximately 20% of all cancers after transplantation.11,12 Conversely, PTLD accounts for a minority of second cancers after allogeneic hematopoietic stem cell transplantation. It is more common in pediatric patients owing to high rates of EBV seronegativity at the time of transplantation. The incidence of PTLD varies based on patient population, allograft type, and immunosuppressive regimen. In adults, the incidence rates in kidney and liver transplant recipients are approximately 1% to 3%. The rate in small intestine transplant recipients is up to 20%, most likely related to the intensity of immunosuppression, young age of the patients, and high lymphocyte content of the graft.13 The incidence of PTLD in thoracic transplant recipients ranges

Posttransplant Lymphoproliferative Disorder from 1.2% to 6.5% in heart transplant recipients, 1.8% to 7.9% in lung transplant recipients, and 5.26% to 7.59% in heart–lung transplant recipients (Table 1).14–23 PTLD is most likely to develop during the first year after transplantation. The incidence in the first year is 224 per 100,000, but decreases to 54 per 100,000 in the second year, and continues to decrease the further from transplant the recipient is.16

Risk Factors The main risk factors for the development of PTLD are the EBV serostatus of the recipient and the degree of T-cell immunosuppression. The most important risk factor for the development of PTLD is the EBV serostatus of the recipient. The incidence of PTLD in EBV-seronegative recipients is higher than for EBV-seropositive recipients because patients who were not exposed to EBV before transplantation usually acquire the infection from the donor.24 This likely accounts for the observation that PTLD is more common in children than in adults.25 For example, among 1316 kidney transplant recipients, the incidence of PTLD was 10.1% in children and 1.2% in adults.26 Interestingly, in adults, the risk for PTLD continues to decrease with age. In 1 study that looked at more than 164,000 solid organ transplant recipients, the 5-year risk of PTLD was 1.74% to 3.28% in patients younger than 34 years, and 0.36% to 2.22% in those older than 50 years.27

The degree of immunosuppression is also an important factor in the development of PTLD, with the degree of T-cell immunosuppression being of greater importance than the degree of B cell immunosuppression. EBV-infected B cells are normally held in check by CTLs. This defense mechanism is lost when T-cell function is impaired, potentially leading to uncontrolled B-cell proliferation and the development of PTLD. Attempts to identify the risk associated with individual immunosuppressive drugs have been hampered by frequent use of multidrug regimens, leading to conflicting results.11 Among induction agents, T-cell–depleting agents such as the anti-CD3 antibody muromonab-CD3 and antithymocyte globulin are associated with an increased risk for PTLD.19,28 More recently, the use of the CTLA-4 modulator belatacept as maintenance for adult kidney transplant recipients was found to have a high rate of PTLD in EBV seronegative recipients.29–31

CLINICAL PRESENTATION PTLD can develop at any time after transplantation. Approximately 60% of patients present with early PTLD, within 1 year after transplantation. The median onset is 6 months after transplantation.32–34 The clinical presentation of PTLD is highly variable and depends on the type of PTLD, rate of progression, and involved organs. Some patients are asymptomatic; others present with nonspecific systemic symptoms such as fever, malaise, and

Table 1 Incidence of posttransplant lymphoproliferative disorder in thoracic transplant recipients Paper 14

Kumarasinghe et al,

2015

Opelz et al,15 1993 Oplez et al,16 2004

Walker et al,24 1995 Armitage et al,18 1991 Swinnen et al,19 1990 Aris et al,20 1996 Levine et al,21 1999 Paranjothi et al,22 2001 Gao et al,23 2003

Population

Incidence (%)

Relative Risk

Incidence Rate

Heart/lung Heart Lung Heart Heart/lung Heart Lung Heart Lung Heart Lung Heart Lung Lung Lung Heart Heart/lung

7.59 5.37 3.1 1.2 —

—

—

— 239.5 27.6 58.6 —

— —

— 3.4 7.9 6.5 6.4 1.8 6.1 6.52 5.26

—

2.0/100 person-years 6.2/100 person-years —

— — — — —

— — — — —

3

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Nagle et al weight loss. In more than one-half of cases, PTLD involves sites outside lymphoid tissue (ie, extranodal). Involved organs include the gastrointestinal tract, lungs, skin, liver, central nervous system (CNS), and the allograft itself. Involvement of the allograft can lead to allograft dysfunction (renal failure, heart failure, or respiratory dysfunction). The CNS is involved in 3.4% to 11.7% of patients.35

DIAGNOSIS AND STAGING In the appropriate clinical setting, the diagnosis and staging of PTLD relies on the following:  Laboratory evaluation,  Radiographic imaging, and  Biopsy.

Laboratory Tests Initial tests in patients with suspected PTLD are similar to nontransplant patients with lymphoproliferative disorders and include a complete blood count to evaluate for anemia, thrombocytopenia, or leukopenia; a comprehensive metabolic panel, because there may be serum chemistry abnormalities depending on the location of the PTLD lesion; serum uric acid and lactate dehydrogenase levels, which may be elevated; and serum and urine protein electrophoresis, which may reveal a monoclonal protein in the urine or serum.35

Imaging There are no large studies comparing different imaging techniques for PTLD, and guidelines developed for other lymphoid malignancies are generally used for diagnosis, staging, and response assessment.36 Initial imaging is typically accomplished with a contrast-enhanced computed tomography scan of the neck, chest, abdomen and pelvis that can help to identify a site to biopsy, stage the disease, and be used in monitoring treatment response. PET/computed tomography scanning has the added benefit of identifying metabolically active sites of disease that have not yet increased in size. It is also better in detecting bone marrow involvement. However, a PET/computed tomography scans cannot distinguish between malignancy and the other causes of increased uptake, such as inflammation or infection. Additionally, certain slow-growing and indolent PTLD are not fluorodeoxyglucose avid. Further imaging depends on the location of suspected lesions. Often, gadolinium-enhanced MRI of the brain and/or spinal cord is used to assess for disease involving the CNS if relevant symptoms are present.

Biopsy As is the case with all lymphomas, a diagnosis of PTLD requires histologic examination of the tumor tissue. An excisional biopsy is preferred over a core biopsy and a core needle biopsy should only be done when it is impossible to obtain an excisional biopsy.35,37–39 The different forms of PTLD are distinguished by a number of different histopathologic features. Special studies to confirm the diagnosis of PTLD include immunophenotyping by flow cytometry or immunohistochemistry, and molecular studies such as fluorescent in situ hybridization for EBV early RNA and polymerase chain reaction for the EBV genome.40,41 In unusual cases where a biopsy is not feasible owing to tumor location or the patient’s general condition, a presumptive diagnosis can be made based on radiologic appearance in addition to detection of EBV viremia. This step is reserved for unusual situations and should be discouraged owing to the broad differential diagnosis of mass lesions in immunosuppressed individuals, which includes infections and other malignancies.

Staging and Prognosis There is no specific staging system for PTLD. The Ann Arbor staging system has been used to describe sites of involvement and the presence of symptoms. Additionally, involvement of the transplanted organ should be evaluated in all patients by imaging, assessment of graft function and biopsy, if indicated, to differentiate between PTLD involvement, graft rejection, and other causes for graft dysfunction such as infection. Additional studies such as bone marrow biopsy and lumbar puncture are useful in diagnosing bone marrow and CNS involvement. EBV viral load in the peripheral blood should be checked and monitored during treatment.35,39 There is no universally accepted scoring system for the prognosis of PTLD. The International Prognostic Index used for non-Hodgkin’s lymphoma in the nontransplant setting does not include some important factors associated with poor prognosis in PTLD, including tumor monoclonality, EBVnegative tumor, and graft involvement.34,42,43 Therefore, the International Prognostic Index score used alone to predict prognosis in PTLD is probably inappropriate, but could be used in concert with additional information.9,34,39

CLASSIFICATION The World Health Organization uses morphologic, immunophenotypic, genetic, and clinical features to classify PTLD into 5 main categories (Fig. 1):

Posttransplant Lymphoproliferative Disorder

Fig. 1. Histologic subtypes of posttransplant lymphoproliferative disorder (PTLD). (A) Monomorphic PTLD. (B) Polymorphic PTLD (H&E stain, original magnification 40).

 Plasmacytic hyperplasia and infectious mononucleosis-like PTLD,  Polymorphic PTLD,  Monomorphic PTLD,  Florid follicular hyperplasia, and  Classical Hodgkin lymphoma-like PTLD.

Plasmacytic Hyperplasia and Infectious Mononucleosis-like Posttransplant Lymphoproliferative Disorder Early lesions tend to present in children and adult solid organ recipients who have not had a prior EBV infection. They more often involve lymph nodes or tonsils and adenoids as compared with extranodal sites. The lesions are composed of polyclonal B cells, plasma cells, and T cells, which form polyclonal masses with preservation of the normal architecture of the underlying tissue. EBV is present in most cases. These lesions often regress without intervention or with a reduction in immunosuppression. The early lesions of PTLD can be differentiated from reactive lymphoid hyperplasia based on the extent the proliferation, the clinical situation, and the presence or absence of EBV.44

Polymorphic Posttransplant Lymphoproliferative Disorder Polymorphic PTLD is the most common form of PTLD in children. Lesions are composed of all types of B cells and form a polytypic lymphoid infiltrate that destroys the normal architecture of surrounding tissue. These lesions do not fulfill the criteria for any of the recognized types of lymphoma in immunocompetent hosts.44

origin that fulfill the criteria for one of the B-cell or T-/natural killer cell lymphomas recognized in immunocompetent patients. Notably, small B-cell lymphoid neoplasms (follicular lymphoma, small lymphocytic lymphoma) and marginal zone lymphomas arising in the posttransplant setting are not considered PTLD. The histology of the monomorphic lesions shows near obliteration of the normal tissue architecture by a lymphoid infiltrate. The lesions are further classified based on the subtype of lymphoma. Most monomorphic B-cell PTLD tumors fulfill criteria for diffuse large B-cell lymphoma. Less commonly, patients present with Burkitt lymphoma or a plasma cell neoplasm. Lymphomas of natural killer and T-cell origin are less common, but when they occur are typically classified as peripheral T-cell lymphoma not otherwise specified. In each case, patients present in a similar way to the lymphoma or plasma cell neoplasm that they resemble.44

Florid Follicular Hyperplasia Florid follicular hyperplasia is a new classification of early lesions. The lesions are reactive, but may have chromosomal abnormalities and may lead to uncontrolled lymphoid proliferation in solid organ transplant recipients.45

Classical Hodgkin Lymphoma-like Posttransplant Lymphoproliferative Disorder Classical Hodgkin lymphoma-like PTLD is the least common major form of PTLD. It most commonly occurs in renal transplant recipients. Lesions are almost always EBV positive and should fulfill criteria for classical Hodgkin lymphoma.44

Monomorphic Posttransplant Lymphoproliferative Disorder

MONITORING AND PREVENTION

Monomorphic PTLD comprises a heterogeneous group of tumors of monoclonal B-cell or T-cell

The best strategy for the management of PTLD is currently focused on prevention; early detection

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Nagle et al of PTLD may allow for prompt therapy and improved outcomes. Most commonly, this is achieved through measurement of EBV viral load in the peripheral blood by polymerase chain reaction amplification. High risk patients should be identified before transplantation and monitored for the development of EBV infection. Patients who are considered high risk include those who are EBV or cytomegalovirus seronegative before transplantation, those receiving lung or small bowel transplants, and patients who receive high doses of immunosuppression for the prevention of allograft rejection.46 Several studies have demonstrated that high viral loads precede clinical manifestations of PTLD.47,48 However, the optimal frequency of monitoring is unknown. Given the doubling time of the EBV viral load is between 1 and 8 days, weekly to twice weekly monitoring during the first year has been recommended.35,39,49–51 Furthermore, little is known about the natural course of an increase in EBV viral load and there is no clear cutoff point, so it is unclear when an intervention should take place.35,52 The decision on when to initiate preemptive therapy should be based not only on an arbitrary cutoff, but also on each patient’s individual risk factors and the rate of increase in viral load. The typical approach to an increasing EBV viral load is reduction in immunosuppression, which has been shown to decrease the incidence of PTLD.53 Alternative approaches are sometimes necessary, because rapid tapering of immunosuppression is not always feasible. Alternative approaches include the introduction of antiviral agents or the use of rituximab. Historical comparisons between patients receiving and not receiving acyclovir or ganciclovir prophylaxis either immediately after transplantation or during antilymphocyte antibody therapy for acute rejection suggest that the use of either prophylactic antiviral drug may be of some benefit.54–56 However, although EBV DNA levels decrease with anti-EBV therapy, this may not always correlate with PTLD tumor response. The preemptive use of rituximab has been studied in solid organ transplant recipients and allogeneic stem cell transplant recipients with an increasing EBV viral load. In 1 study, allogeneic HSCT patients with EBV reactivation received 1 dose of rituximab. This measure resulted in a complete response in 14 of 15 patients with complete clearance of EBV DNA from the peripheral blood and prevention of PTLD.57 In heart transplant recipients, rituximab was given to patients with EBV reactivation and viral loads of greater than 105 copies/mL or to patients with primary infection if they were not responding after 1 month of withdrawal of immunosuppression.

This approach was found to be safe and effective.57,58

THERAPY The 2 main goals of therapy are eradication of the PTLD while preserving function of the allograft. The main options for treatment of PTLD are reduction of immunosuppression, rituximab, cytotoxic chemotherapy, radiation therapy, or surgery for localized disease, and adoptive immunotherapy with EBV-specific CTLs. There are no randomized trials comparing these treatment options and data largely come from expert opinion, retrospective analyses, and uncontrolled prospective trials (Table 2).

Reduction of Immunosuppression Reduction of immunosuppression is the first step in therapy, unless concern regarding graft rejection makes this approach impossible. Recovery of the host’s immune system will allow for EBVspecific CTLs to proliferate and control the disease. However, reduction of immunosuppression increases the risk of allograft failure and the optimal reduction of immunosuppression to control the PTLD while preserving the allograft is unknown. General consensus is that immunosuppression should be reduced as much as possible while carefully monitoring allograft function. Therefore, the reduction regimen used is based on the severity of the PTLD and the risk associated with possible loss of the allograft. Often, a reduction of 25% to 50% from baseline is used if alternative organ support is available. For heart and lung transplant recipients, a maximum reduction of 50% to 75% is recommended.28,39 The majority of early lesions either resolve completely or improve significantly to the reduction of immunosuppression.9,18,34 However, complete responses are less common in other types of PTLD.59 Factors that are associated with poor response include increased lactate dehydrogenase levels, organ dysfunction, multiorgan involvement, and older age.9,34,39

Rituximab For those who fail to achieve a complete response with reduction of immunosuppression or in patients who are not candidates for this approach, rituximab is standard therapy for CD201 PTLD. Rituximab may be administered in combination with cytotoxic chemotherapy (either sequentially or concurrently) or as a single agent. Rituximab monotherapy is well-tolerated. It is typically given at 375 mg/m2 either in 4 weekly doses or until best response.

Posttransplant Lymphoproliferative Disorder

Table 2 Treatment response rates for PTLD Study 9

Reshef et al, 2011 Oertel et al,60 2005 Blaes et al,61 2005 Choquet et al,62 2006 Gonzalez-Barca et al,64 2007 Mamzer-Bruneel et al,71 2000 Norin et al,70 2004 Choquet et al,94 2007 Trappe et al,65 2012

Treatment

Patients (n) ORR (%) CR (%) OS

Reduction of immunosuppression 4 weekly doses rituximab 4 4 weekly doses rituximab every 6 mo  2 y 4 weekly doses rituximab 4 weekly doses rituximab

67

45

37

44 mo

17

59

53

37 mo

11

64

55

14 mo

43 38

44.2 79

28 34

67% at 1 y 47% at 27.5 mo

10

90

60

27 mo

12 26 59

42 65 90

42 50 68

— 13.9 mo 6.6 y

54

72

54

83% at 2 y

126

88

70

6.6 y

13 19 6 33

85 64 50 64

85 68 50 14/33

69% at 10.5 y — — 79% at 6 mo

11

36

27

—

CHOP  surgical resection CHOP CHOP 4 weekly doses rituximab followed by CHOP 73 Gross et al, 2012 Low-dose cyclophosphamide d1, Prednisone or methylprednisolone days 1–5, rituximab d 1, 8, 15 on cycles 1 and 2, every 3 wk  6 cycles 4 weekly doses of Trappe et al,74 2016 rituximab CR / rituximab every 3 wk  4 PR/SD/PD / R-CHOP every 3 wk  4 EBV-CTLs Heslop et al,79 2010 Doubrovina et al,80 2012 EBV-CTLs Moosmann et al,81 2010 EBV-CTLs EBV-CTLs Haque et al,90 2007; Haque et al,95 2010 Gallot et al,96 2014 EBV-CTLs

Abbreviations: CHOP, cyclophosphamide, doxorubicin, vincristine and prednisone; CR, complete response; CTL, cytotoxic T-cell; EBV, Epstein–Barr virus; ORR, overall response rate; OS, overall survival; PD, progressive disease; PR, partial response; PTLD, posttransplant lymphoproliferative disorder; SD, stable disease.

Several phase II trials showed response rates of up to 55%.60–64 However, up to 57% of patients with an initial partial or complete response to rituximab monotherapy will have disease progression 1 year after treatment and the median overall survival has been reported between 14.9 and 42.0 months in different studies.63,65–68

Chemotherapy Increasing the intensity of treatment for PTLD results in higher response rates, but with increased toxicity. Cytotoxic chemotherapy should be

considered for patients who do not achieve a complete response to rituximab monotherapy or progress after rituximab monotherapy, those with clinically aggressive lymphoma, or patients with critical organ compromise who require a rapid response to therapy. Additionally, cytotoxic chemotherapy is the initial treatment after reduction of immunosuppression in patients with CD20-negative PTLD. There are no randomized trials comparing different chemotherapy regimens and patients are generally treated with chemotherapy regimens used for non-Hodgkin lymphoma. Chemotherapy

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Nagle et al is usually administered in conjunction with rituximab for patients with CD201 PTLD. Patients with PTLD involving the CNS should be treated with protocols used for primary CNS lymphoma.69 The remainder of patients are often treated with the combination of cyclophosphamide, doxorubicin, vincristine and prednisone. A number of small, retrospective studies have evaluated the utility of combination chemotherapy. Overall response rates for anthracycline-based regimens are higher than for rituximab monotherapy and range from 42% to 100%. However, this response is at the expense of high treatment-related mortality of 25% to 50%.63,66,70–72 More recently, an international phase II trial of rituximab followed by a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy demonstrated overall response rates of 90%, but with an 11% treatment-related mortality rate.65 Given the toxicity of combination chemotherapy, it may not be necessary in all circumstances. In a phase II study of 55 pediatric patients with EBV-positive PTLD after solid organ transplant, patients received a total of 6 cycles of chemotherapy every 3 weeks. The first 2 cycles included cyclophosphamide 600 mg/m2 on day 1, prednisone 1 mg/kg twice a day on days 1 to 5, and rituximab 375 mg/m2 on days 1, 8, and 15. The remaining 4 cycles were administered without rituximab. The complete response rate was 69%. The 2-year event-free survival was 71% and overall survival was 83%. There were 10 deaths, 3 from infections and 7 from progressive PTLD.73 Another reasonable approach to treatment of this population is response-adjusted therapy. One recently published phase II study used response to rituximab to determine the need for cytotoxic chemotherapy. One hundred fifty-two patients with CD201 PTLD were treated with 4 weekly doses of rituximab. Those who achieved a complete response continued with rituximab monotherapy every 21 days for 4 doses. Patients who did not achieve a complete response received 4 doses of rituximab plus a combination of cyclophosphamide, doxorubicin, vincristine and prednisone chemotherapy every 21 days. Despite withholding chemotherapy from the 88 patients who achieved a complete response to rituximab induction, the overall response rate of the entire cohort was 88% and the median overall survival was 6.6 years.74

Antiviral Agents Antiviral treatment for EBV infection uses nucleoside analogs which target the virus-specific

enzyme, thymidine kinase, which is expressed in typically infected cells. This viral enzyme target is not expressed in EBV-positive tumors during viral latency, making nucleoside analogs ineffective as a sole antineoplastic agent. However, butyrate has been shown to sensitize EBV-positive lymphoma cells to in vitro apoptosis induced by ganciclovir. A trial of arginine butyrate in combination with ganciclovir included 6 patients with PTLD and demonstrated complete responses in 2 patients and partial response in 3 patients.75

Adoptive Immunotherapy Adoptive immunotherapy involves the transfer of T cells into a patient for therapeutic purposes. EBVrelated PTLD tumors typically express all latent EBV viral proteins and are, therefore, responsive to adoptive immunotherapy. This strategy uses several different approaches with the goal of restoring the underlying immune defect while avoiding the organ toxicity or immune suppression caused by other treatment modalities.76 The T cells may be donor derived (in the case of an HSCT recipient), patient derived (autologous), or from a third-party donor (“off the shelf”). The T cells may also be EBV specific or multiviral. Although EBV-specific CTLs target all the latent EBV viral proteins, multiviral CTLs are able to target at least one antigen from 2 or more viruses (Fig. 2).77 Unmanipulated donor lymphocyte infusions can reconstitute EBV-specific immunity and have been reported to achieve clinical response rates of more than 70% in patients with PTLD after HSCT. However, this measure is at the risk of severe and life-threatening graft-versus-host disease.76,78 To avoid this toxicity, donor EBV-specific CTLs have been used in this population as both prophylaxis and treatment for PTLD. In this case, donor T cells directed against EBV antigens are expanded selectively. These cells recognize multiple viral antigens, and when infused into patients can reconstitute an immune response to EBV and eradicate PTLD.67 Multiple centers have reported the use of donor EBV-specific CTLs to prevent and treat PTLD resistant to conventional therapy.48,79–86 The largest study evaluated more than 100 HSCT recipients who received donor EBV CTLs prophylactically. Among all patients, none developed PTLD and no patients developed de novo graft-versus-host disease after CTL infusion. In this same study, when used as a treatment for PTLD, EBV CTLs induced a sustained complete response in 11 of 13 patients.79 Autologous EBV CTLs for solid organ transplant recipients have also been used. In this setting,

Posttransplant Lymphoproliferative Disorder

Fig. 2. A cytotoxic T-cell (CTL) library of inventory is made for each development program. (1) The process starts with healthy white blood cells (peripheral blood mononuclear cells [PBMCs]) collected from a third-party donor. (2) The B cells are separated and exposed to the target antigen (protein) of interest so they can present the antigen to T cells. (3) T cells from the same third-party donor are then exposed to the antigen-presenting B cells. Only those T cells with a receptor that recognizes the antigen of interest become activated to eliminate target diseased cells, and their numbers significantly expand with activation. Those T cells unable to recognize the antigen do not become activated and do not expand. (4) The result is a CTL line enriched with cytotoxic T cells specifically recognizing the target antigen. This CTL line is characterized by its HLA and restriction profile and categorized in a library of cryopreserved inventory. (5) The library is composed of a relatively small number of ready-to-use CTL lines that provide broad coverage to match to each patient’s individual immune profile. APC, antigen-presenting cell. (Courtesy of Atara Biotherapeutics, San Francisco, CA; with permission.)

PTLD most commonly arises from recipient B cells. In this situation, cells are collected from the patient and exposed to EBV antigens. The resulting activated T cells are expanded ex vivo and reinfused into the patient. Several small studies have evaluated the use of autologous EBV CTLs in this population both in patients at high risk for PTLD or with active disease. These studies have demonstrated that patients who receive EBV CTLs do not suffer from organ rejection or other adverse events. In vivo T-cell expansion seems to be lower than observed in HSCT patients who received similar doses; however, response rates are high.87–89 More recently, in an attempt to improve processing times and make this therapy accessible to an increasing number of patients, HLA-typed EBV-specific T-cell lines have been generated. With this technology, T cells are collected from the blood of third-party donors and exposed to EBV antigens. T cells are expanded and stored for future therapeutic use in an appropriate human leukocyte antigen matched patient. Using this “offthe-shelf” approach, several studies have shown high response rates in otherwise refractory disease. The best results are observed in patients

who received donor T cells that are best HLA matched.80,82,90–93 An HLA-specific EBV T-cell band not only improves processing time, but is less expensive than using autologous T cells with the same benefit of limited toxicity (as compared with cytotoxic chemotherapy). For this reason, a phase II trial of third-party EBV CTLs is currently ongoing.

SUMMARY PTLD is a devastating consequence of HSCT and solid organ transplantation with a high morbidity and mortality. Although most PTLD is due to EBV, an increasing number of cases are EBV negative. EBV-negative PTLD has a longer latency as compared with EBV-positive disease. Therefore, the increase in EBV-negative PTLD may be explained by the improvement in survival in transplant recipients and the longer time from transplant to the development of PTLD. Additionally, changes in immunosuppression may be associated with the increase in risk for EBV-negative PTLD. Incidence depends on the type of transplant and degree of immunosuppression. Clinical presentation is heterogeneous and nonspecific. Initial

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Nagle et al treatment involves the withdrawal of immunosuppression, whereas refractory disease can be treated with antibody therapy or cytotoxic chemotherapy. More recently, there is evidence for the use of EBV-specific CTLs from either the donor or a third party and a phase II trial is ongoing.

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