Post-transplant lymphoproliferative disease (PTLD): Pharmacological, virological and other determinants

Pharmacological Research 63 (2011) 1–7

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Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Review

Post-transplant lymphoproliferative disease (PTLD): Pharmacological, virological and other determinants Jana Stojanova a,b , Sophie Caillard c , Annick Rousseau a,b,d , Pierre Marquet a,b,e,∗ a

INSERM U850, Limoges, France Univ Limoges, Faculty of Medicine, Laboratory of Medical Pharmacology, Limoges, France Service de Néphrologie Transplantation, Hôpitaux Universitaires de Strasbourg, France d Univ Limoges, Faculty of Pharmacy, Laboratory of Biophysics, Limoges, France e CHU Limoges, Department of Pharmacology, Toxicology and Pharmacovigilance, Limoges, France b c

a r t i c l e

i n f o

Article history: Received 21 September 2010 Received in revised form 27 October 2010 Accepted 28 October 2010 Keywords: Immunosuppressants Transplantation Lymphoproliferative disorders Risk factors Pharmacogenetics

a b s t r a c t Post-transplant lymphoproliferative disorders (PTLDs) represent a serious complication in solid organ transplantation and are the first cause of cancer related mortality in this population. Pre-transplant Epstein Barr Virus seronegativity and receipt of T cell depleting agents for induction or severe/refractory rejection are known risk factors, but they primarily impact early occurring disease. On the other hand, late occurring disease, which has typically not correlated with the above or other specific risk factors, has recently been shown to be associated with older recipient age and prolonged receipt of calcineurin inhibitors. Furthermore, recent data has contributed to and, in some instances shed light on, previous debate concerning the role of viruses other than EBV and the level of HLA mismatches as risk factors for PTLD. Gene association studies focusing on key cytokines and their receptors have identified several polymorphisms that may prove useful to identify patients at risk, with distinction for early and late occurring disease. Determining the influence of individual maintenance immunosuppressive agents on lymphomagenesis has been limited by the complexity of the multi-drug regimens used and absence of measures of drug exposure and time-dependent covariates in multivariable analyses. Biomarkers that measure the extent of immunosupression may have a role in avoiding PTLD, and other post-transplant complications. © 2010 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PTLD natural history: the role of EBV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relationship to the transplanted organ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of cytokines: cytokine gene polymorphisms and association with PTLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of other viruses: CMV, HVB and HVC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pre-transplant HLA matching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk factors: immunosuppressive drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The post transplant lymphoproliferative disorders (PTLDs) follow skin cancer as the second most common group of malignancies

∗ Corresponding author at: INSERM U850, Department of Pharmacology, Toxicology and Pharmacovigilance, CHU, 2 Avenue Martin-Luther King, 87042 Limoges cedex, France. Tel.: +33 555 05 64 18; fax: +33 555 05 61 62. E-mail address: [email protected] (P. Marquet). 1043-6618/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2010.10.016

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occurring after solid organ transplantation, however they represent the leading cause of cancer related death and graft loss. Compared to lymphoma in the general population, the occurrence can be 10–1000-fold higher, depending on the recipient age and the type of organ received [1]. In addition, lymphomatous PTLD in this setting is more frequently extranodal, can behave more aggressively and tends to have a worse prognosis [2]. PTLD encompasses a spectrum of diseases, primarily of B cell origin, that include early virally driven polymorphic lymphoproliferations that may regress with reduced immunosuppression, and monomorphic forms that are

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J. Stojanova et al. / Pharmacological Research 63 (2011) 1–7

rarely regressive [3]. A notable feature is the frequent involvement of the Epstein Barr virus (EBV). EBV establishes a life-long latent infection in approximately 90% of adults but this rarely leads to cancer in immune competent individuals. Reduction of immunosuppression is often the first treatment approach, with the aim to restore functionality in EBV specific cytotoxic T-lymphocytes (CTLs). However, for most cases additional treatment modalities are necessary and typically include (alone or in combination) antivirals, radiotherapy, chemotherapy, surgery and the anti-B cell agent rituximab for CD20 positive tumours. High mortalities have been reported for cohorts representing heterogeneously treated cases [1,4,5]. Reconstitution of EBV-specific immunity by infusion of autologous CTLs expanded in vivo is another option for EBV positive disease of recipient origin but is limited by the continued use of immunosuppressants. Very recently, two independent groups generated CTLs resistant to calcineurin inhibitors (CNI) which may improve the success of adoptive T-cell therapy [6,7]. Identifying the patients at risk of PTLD is important as there is a clear benefit from early diagnosis and treatment, and several strategies (e.g. reducing immunosuppression, antiviral therapy, rituximab and adoptive CTL therapy) may be useful as pre-emptive interventions. To this end, serial quantitative viral load monitoring targeted to those at risk has been a reasonable strategy to identify impending EBV-positive disease: a sudden rise is a sensitive indicator, although is not highly specific [8]. This approach is limited in detecting EBV negative and localised PTLD and may not be practical to perform ongoing testing for detection of late occurring disease. Most of the work in this area has been conducted in paediatric cohorts and more data are required for other risk strata. Coupling EBV viral load monitoring with assays for EBV-specific CTL activity appears to enhance specificity in individuals with a high viral load but this approach is not currently in widespread clinical use [8]. The aim of the following review is to revisit the pathophysiology and the known risk factors for PTLD in solid organ transplant recipients and present recent findings on the topic.

B cells are highly immunogenic and outgrowth is normally controlled by specific CTLs. When naïve B cells undergo the germinal centre (GC) reaction, the latency program switches to Latency II where only a few of the latency genes are expressed. Viral driven cell differentiation results in a pool of long-lived memory B cells, harboring the virus. Memory B cells in peripheral circulation exist in Latency 0 or Latency I, where there is either no expression or expression of a single, poorly antigenic protein. In this manner the virus can persist for the life time of the host, existing in an equilibrium that is permissive for survival but not outgrowth. The majority of EBV positive PTLD exhibit gene expression consistent with type III latency, although a mixed picture that includes the other latency states, and occasionally lytic gene expression, has been described. These cells can be at various stages of their development, but are most frequently of GC and post-GC origin. LMP1 expression influences growth and survival of infected cells by various distinct mechanisms. One is engaging intracellular processes that ultimately result in expression of cytokines that can serve as growth factors or inhibitors of immune recognition/effector functions. Moreover, it has a role in the upregulation of intra-cellular anti-apoptotic proteins (for example, bcl-2 and A20). Finally, LMP1 and other latency proteins can interfere with specific extra-cellular apoptosis stimuli by activating internal survival signaling. This is of particular importance in the rescue of post-GC infected B cells lacking a functional B cell receptor (approximately 50% of PTLD) that are otherwise committed to die [13,14]. Genetic and epigenetic alterations of host DNA can occur, and in particular are an important feature of monoclonal disease [14]. Some examples include alterations in BCL-6, c-MYC and p53. The mutator phenotype, characterised by microsatellite instability and faulty mismatch repair, was found amongst immunodeficiencyassociated lymphomas (8.1% of PTLD and 2.3% of HIV-associated lymphomas) but not amongst tumours derived from immunocompetent individuals [15]. Importantly, it was found amongst both EBV positive and negative tumours.

2. PTLD natural history: the role of EBV 3. Relationship to the transplanted organ EBV seronegative recipients receiving an organ from an EBV seropositive donor are at the highest risk of PTLD and non-Hodgkin lymphoma (NHL) [5,9]. The period of highest risk is early posttransplant (typically the first year), likely coinciding with primary infection. Seronegative recipients were not at increased risk for NHL occurring beyond the 2nd post-transplant year [10]. This is consistent with the finding that EBV is detected in the majority of early B-cell PTLD, while more than 50% of late PTLD can be EBV negative [11]. The high incidence of PTLD in paediatric recipients is largely due to the higher proportion of EBV seronegative recipients, although it is noteworthy that NHL occurs at a significantly higher rate in EBV seropositive children (age <19 years) compared to seropositive adults [9]. Despite the higher proportion of EBV seronegativity amongst the paediatric transplant population, it has been highlighted that adult seronegative recipients far outnumber their paediatric counterparts [9]. The role of EBV in the pathogenesis of PTLD has been the focus of several reviews [12,13]: a brief summary will be presented here. The first association between EBV and the human host occurs in the epithelial cells of the nasopharynx and salivary glands, the site of active lytic replication during primary infection. Via interaction with the CD21 receptor, linear EBV genome enters naïve B cells where it forms the circular episome, initiating the expression of latency genes. The first of a series of latency programs is Latency III (also known as the unrestricted growth program) where the entire repertoire of latency genes is expressed: EBV nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C and LP, and latent membrane proteins (LMPs) 1, 2A and 2B. At this stage, the infected

The risk of PTLD varies according to the organ transplanted. The relative risk of lymphoma compared to the background population (matched for age, sex and geographical location) is 239.5 for heart–lung, 58.6 for lung, 34.9 for pancreas, 29.9 for liver, 27.6 for heart and 12.6 for cadaver kidney recipients at five years post transplant [1]. For all organs, the risk is highest during the first post-transplant year and it is this period that is largely responsible for between-organ differences. In the subsequent years, the less pronounced but steady rise in cumulative lymphoma incidence is similar between the various organs, with the exception of heart recipients for whom a consistently steep rise remains [1]. The difference in risk between the different organs, and the temporal relationship observed, is consistent with a contribution by the intensity of immunosuppression (higher immunosuppressant dosing and a greater total exposure to antibody preparations for both induction and treatment of rejection). The degree of antigenicity afforded by each organ, and the amount of lymphoid tissue contained in the graft, both of which would vary in degree according to the size and type of organ transplanted, have also been discussed to play a role [16]. Furthermore, PTLD localised in the allograft occurs more frequently during the first post-transplant year, and represents the majority of PTLD that occur in this period [1,17]. Direct antigenic stimulation of host B lymphocytes by the allograft is unlikely given the proportion of PTLD with non-functioning B cell receptors and the purported lack of evidence for antigenic stimulation in the remainder of lesions [14]. This is in contrast with AIDS associated lymphomas where antigenic stimulation is an important

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aspect of the pathophysiology [14]. On the other hand, the amount of lymphoid tissue transplanted with the graft may contribute significantly to the total EBV viral load. For solid organ transplant recipients, allograft-localised, early PTLD may derive from donor lymphocytes [18–20], but the majority of tumours are thought to be of host origin [21–23]. One hypothesis for the preferential localisation within the graft is that allogeneic stimulation of the suppressed immune system results in a local microenvironment favourable for EBV driven lymphoproliferation [17]. A minority of PTLD (approximately 1%) occur at the site of previous surgical intervention [24]. 4. The role of cytokines: cytokine gene polymorphisms and association with PTLD Cytokines have an important role in the immortalisation, growth and survival of EBV infected B cells. Some are expressed under the influence of EBV latency genes and can serve as autocrine growth factors or inhibitors of host immune attack. In addition, immunosuppressants influence the normal picture of cytokine expression, and this has been shown to influence tumorigenesis. For example, calcineurin mediated expression of TGF-␤ causes phenotypic changes that lead to malignant transformation [25–27]. Elevated blood levels of IL4, IL6, IL10 and TNF-␣ have coincided with PTLD [28,29], however it is difficult to know if this is a primary cause or a secondary outcome of the disease. The precise details of the cytokine networks that may favour lymphomagenesis is yet to be revealed in full, however it is a reasonable hypothesis that gene polymorphisms that influence the expression of cytokines and their receptors may

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associate with PTLD and prove useful in identifying patients at risk. With the hypothesis that high anti-inflammatory or low inflammatory responses may cause low cytotoxic T cell frequency or efficacy, VanBuskirk et al. [30] genotyped for SNPs in the genes expressing TNF-␣, TGF-␤, IL-6, IL-10 and IFN-␥ (Table 1). They found the IFNG +874 A/A genotype, low secretor phenotype, present in a greater proportion of PTLD cases. Using a severe combined immunodeficiency (SCID) mouse model, the same group demonstrated that peripheral blood mononuclear cells (PBMCs) causing rapid, high-penetrance lymphoproliferative disease (LPD) more often originated from individuals carrying the IFNG +874 A/A genotype [31]. They propose that an alteration in the INF-␥–TGF-␤ relationship might be the cause, given that each has a counter regulatory effect on the other. Stimulating PBMCs representing each of the genotypes with exogenous TGF-␤, they showed INF␥ production was inhibited in carriers of the A allele but not those homozygous for the T allele. Antibody directed against TGF-␤ attenuated the aggressive lymphomagenesis observed in carriers of the A/A genotype. Babel et al. [32] focused their study on late-onset, EBV associated PTLD and chose SNPs in IL-10, TGF-␤, TNF-␣ and INF-␥ reasoning that the former two are involved in B cell activation and/or proliferation, while the latter two in the anti-viral immune response (Table 1). The TGFB1 codon 25 +915 C/C genotype, low secretor phenotype, was more frequent in PTLD cases and the IL10 −1082 G/G genotype, high secretor phenotype, was more frequent in controls. That the low secretor TGF-␤ phenotype is associated with PTLD is, according to the authors, consistent with its in vivo action as a tumour suppressor and inhibitor of B cell acti-

Table 1 Location of single nucleotide polymorphisms in cytokine genes investigated in association with post-transplant lymphoproliferative disease. Study

No association found

Associated with PTLD

VanBuskirk et al. [30] 9 adult renal transplant recipients, early PTLD 143 renal transplant controls

TNF TGFB1 IL6 IL10 (unspecified SNP locations)

IFNG promoter +874 A/T*

Lee et al. [34]

59 pediatric liver transplant recipients (6 developed PTLD)

TNF promoter −308 G/A TGFB1 codon 25 +915 G/C, codon 10 +869 T/C IL2 promoter −330 T/G IL2 +166 G/T (silent)

IFNG promoter +874 A/T*

Babel et al. [32]

38 adult solid organ transplant recipients (mixed organ type), late EBV-PTLD 408 organ-matched controls

TNF promoter −308 G/A

IL10 promoter-1082 A/G*

TGFB1 codon 10 +869 T/C IFNG promoter +874 A/T*

TGFB1 codon 25 +915 G/C

McAulay et al. [29]

Study population

45 adult solid organ transplant recipients (mixed organ type), EBV positive PTLD 65 heart transplant controls 183 EBV seropositive healthy controls

TNF promoter

LTA exon 3 +720 C/A

−857 C/T −308 G/A −238 G/A TNFRSF1A promoter −230 A/G −845 A/G −839 G/A TNFRSF1B exon 10: −1663 A/G −1668 T/G −1690 C/T TNFRSF1B exon 6 +676 T/G

LTA intron 1 −1031 T/C +365 C/G −863 C/A +249 A/G TNFRSF1A IL1A −889 C/T −201 G/T IL1R1 −1339 C/T −1135 T/C IL6 promoter −174 C/G IL6 intron 4 3331 A/G IL10 promoter: −1082 A/G* −819 C/T −592 C/A IL10RA −241 G/A

Thomas et al. [35]

37 adult solid organ transplant recipients (mixed organ type), EBV positive PTLD [13 early, 24 late] 109 transplant controls (organ type unspecified) 181 healthy controls

IFNG promoter +874 A/T*

Stern et al. [33]

Discovery cohort: 100 renal transplant recipients Validation cohort: 100 kidney, 24 heart, 12 liver recipients Control cohort: 100 renal transplant recipients (age not reported)

IFNG promoter +874 A/T* IL10 promoter −1082 A/G* TGFB1 codon 25 +915 G/C

TNF promoter

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vation/proliferation. However, some in vitro models have shown that TGF-␤ fails to inhibit B cells once immortalisation has occurred [28]. Also, TGF-␤ is implicated in calcineurin inhibitor mediated tumorigenesis in a variety of experimental models [25,26]. The authors discussed the apparent inconsistency between high-level IL-10 secretion and lack of PTLD given that high blood levels have coincided with PTLD diagnoses. They offer as a possible explanation IL-10 mediated increase in humoral activity or IL-10 mediated control over IL-6. There was no apparent linkage between the two gene loci. McAulay et al. [29] found variant alleles in the TNF-␣ promoter and TNF-␣ receptor 1 promoter regions significantly associated with PTLD. Variants containing TNF-863A had previously been shown in experimental models to result in increased TNF-␣ production as a consequence of decreased NF␬B binding to the promoter region, NF␬B being a transcription down regulator. The functional significance of the other polymorphisms is as yet unknown, although there exists a degree of linkage between the −863 and the −1031 loci in the TNF promoter. Furthermore, the significantly elevated TNF-␣ levels found in PTLD cases could not be fully explained by the genetic variant types [29]. A recent study with a large number of cases and controls found no influence of polymorphisms in IFNG, IL10, TGFB on neither lymphoma incidence nor survival [33] (Table 1). In addition to including the largest number of cases amongst similar studies to date, cases were split into two cohorts, one serving as the validation cohort. Cases and controls differed in several noteworthy parameters, including transplant center of origin, period of transplantation and immunosuppressant regimen; only the latter was included in the multivariable model for lymphoma incidence. Although cytokine genes were investigated, the primary aim of this study was to explore polymorphisms in and expression of specific natural killercell receptors: none were found to influence lymphoma incidence that could be confirmed in the validation cohort. There were significant findings for lymphoma survival, however these are outside of the scope of the present review. Table 1 summarises all SNP locations investigated to date, with controversial findings marked by an asterisk. The INFG +874 A/A genotype, significantly associated with PTLD in two studies with small numbers of homogenous cases and controls [30,34], but not in other studies [32,33,35]. Similarly, the IL10 −1082 G/G was higher in PTLD-free organ recipient controls compared to cases [32], while others did not achieve this finding [29,33]. 5. The role of other viruses: CMV, HVB and HVC Viruses other than EBV have been investigated for their role in PTLD. Kidney recipients that were CMV seronegative prior to transplantation were not found to be at increased risk for PTLD or NHL [10,36] in univariate analyses. In contrast, in two multivariate models, both using data from the OPTN/UNOS database but covering different periods (1997–2001, 2000–2004), CMV seronegative kidney recipients had an elevated risk for PTLD, regardless of donor status [37,38]. In an analysis of kidney recipients stratified for both EBV and CMV pre-transplant serostatus, CMV status did not make a significant contribution to risk. It was shown, however, that pre-transplant CMV and EBV serostatus were correlated (r = 0.23, P = 0.001) [9]. Hospitalisation for CMV disease in the first posttransplant year increased NHL risk in years 2–6 post-transplant in one study [9], while others found CMV disease at any stage prior to diagnosis was not associated with increased PTLD risk [4]. CMV prophylaxis with acyclovir or ganciclovir did not influence the incidence of post-transplant lymphoma in kidney transplant recipients: a multivariate Cox model confirmed the finding for the data at one-year post transplant [36]. Although the dosing strategy used was outlined in general terms in the study methods, the

outcome as a function of duration of treatment was not specifically evaluated. Despite a much smaller number of study participants (n = 100 cases, n = 375 controls, compared to a cohort of 44,282 subjects in the former study), one case–control study found that kidney transplant patients who had received ganciclovir as CMV prophylaxis were at a reduced risk for early PTLD [39]. A longer duration of treatment was noted to have a positive bearing on the outcome. Pre-transplant positivity for HBV or HCV did not associate with PTLD in large cohorts of renal transplant recipients [4,40]. Although some smaller, single centre analyses have noted a significant association between HCV infection and PTLD amongst heart [41] and liver recipients [42], others did not encounter the same finding for liver recipients transplanted at a different centre [43] (n < 700 for all studies). Pre-transplant HCV positivity had no effect on PTLD in a very large retrospective cohort of solid organ transplant recipients, regardless of the type of organ transplanted and transplant indication (n = 210,763). Sub-analyses of subjects with confirmed HCV infection, persistent infection and acute infection did not reveal an impact on risk [44]. 6. Pre-transplant HLA matching A greater number of HLA mismatches has not been typically associated with PTLD risk in large cohorts of renal transplant recipients [5,37] although it was found to be a risk factor in one multivariate model [4]. Emerging evidence suggests that the type of mismatching is important [45,46]. More mismatches in HLA B were found to be an independent risk factor for PTLD in one analysis that also accounted for immunosuppressants [45], however, the authors noted the small number of subjects as a limitation (n = 1013). Although mismatching in HLA-DR resulted in significantly increased overall lymphoma risk, particularly for those that received T cell depleting antibody induction, stratification for site of disease revealed a significant relationship where there had been two mismatches in HLA-DR for CNS disease, and any combination of two mismatches in HLA-B and/or HLA-DR for graft localised disease [46] (n = 152,728). 7. Risk factors: immunosuppressive drugs Of all strategies used to achieve immunosuppression following solid organ transplantation, use of antibody preparations that deplete T cells, but not both T and B cells, consistently emerges as a risk factor for lymphoma in both univariate and multivariate analyses [1,4,5,9,10,38,47]. The increased risk is evident in the first post-transplant year [1,47], and is not associated with late occurring disease [10]. This is generally accepted as evidence that the intensity of immunosuppression has the highest bearing on lymphoma risk, however differences in risk between the various antibody preparations suggest that their mechanism of action is an important consideration. The IL-2 receptor antagonists, which specifically target activated T cells and are non-depleting, do not increase lymphoma risk [1,4,38,48]. Induction with alemtuzumab, which depletes both T and B lymphocytes, was not found to be associated with PTLD in kidney recipients [38], however more data are needed to confirm this finding for solid organ transplant recipients. Despite being grouped together in the majority of analyses, individual preparations of the polyclonal T cell depleting agents may differ in their contribution to risk. Induction with Thymoglobulin® and Atgam® similarly increased NHL risk at the third post-transplant year, while ATG Fresenius® (marketed outside of the United States) carried a risk similar to no induction [48], which was suggested to be due to its lack of activity against CD3. Of note is that only Thymoglobulin® and the IL-2 receptor antagonists were associated with significantly improved graft survival at the third

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post-transplant year compared to no induction, whereas monoclonal Muromonab-CD3, Atgam® and ATG Fresenius® were not [48]. The cumulative dose received is likely to be a relevant consideration for some of these agents, given that some have several possible dose administration regimens, and others could be used to treat steroid resistant rejection, with varying treatment periods. Amongst heart transplant recipients, the PTLD risk associated with Muromonab-CD3 further increased for cumulative doses above 75 mg: this cut-off distinguished patients receiving induction only, and those who received induction and subsequently anti-rejection treatment [49]. Treatment of refractory acute rejection increased the NHL risk for kidney recipients who had not received induction, and increased the risk further when induction had been used [1]. Quantifying the cumulative dosage of T cell depleting antibodies in risk analyses may be useful to identify a safety threshold for these agents which remain in widespread clinical use. At this point in time, the calcineurin inhibitors, cyclosporin and tacrolimus, continue as the pillars of maintenance immunosuppressive regimens and are taken by the majority of recipients. Looking at calcineurin inhibitor use as a time-dependant variable (reflecting data collected during the post-transplant follow-up period), it was found that kidney transplant recipients taking either of these drugs were at a higher risk of late occurring NHL, but not early occurring NHL, compared to individuals that were not receiving them [10]. Several groups have found tacrolimus to be associated with an increased risk of NHL or PTLD compared to cyclosporin, amongst kidney transplant recipients who had not received antibody induction [1,4,40], a difference that did not hold amongst those that had received induction [40]. The same was not found for liver transplant recipients [1]. Different methods were used to come to these discordant findings, notably some considered drugs received at discharge post-transplant while others included vaguely defined data on maintenance treatment collected during follow-up. In one of these studies, stratification for concomitantly taken mycophenolate or azathioprine did not significantly alter the higher incidence of NHL found for the tacrolimus treated patients (drug regimen received at discharge, analysis unadjusted for nondrug factors) [1]. A different group found there was no statistical difference in the PTLD rate between two periods of tacrolimus use (1996–1998 and 1998–2000), suggesting that the higher dosing employed when the drug first came into clinical use was not the reason for the difference between the two drugs found in this study [4]. Whether a difference in risk between tacrolimus and cyclosporin exists when IL-2 receptor antagonists or alemtuzumab are used for induction is unknown. To date, data from experimental models have shown similar effects by both drugs on tumourigenisis, though through a promoting, rather than a causal effect on tumour growth [27]. Furthermore, both calcineurin inhibitors inhibited apoptosis in a lymphoblastoid cell line [50]. It has been suggested that any difference in risk might be due to a higher level of immunosuppression provided by tacrolimus compared to cyclosporin [51]. The place of the antimetabolites, mycophenolate and azathioprine, is for the most part alongside the calcineurin inhibitors as adjunct therapy for preventing allograft rejection. Though not specifically in lymphomagenesis, azathioprine may promote tumour formation by interfering with DNA mismatch repair mechanisms, associated with an increased rate of spontaneous mutations [51]. Mycophenolate, on the other hand, has been shown to inhibit growth and induce apoptosis in a variety of human non Hodgkin lymphoma cells, both EBV positive and negative [52]. With both antimetabolites considered together as time dependent variables, no association was found with the receipt of these drugs and neither early nor late occurring NHL in kidney recipients [10]. In head-to-head comparisons, some have found a reduced risk with mycophenolate [37] while others did not find a signifi-

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cant difference [40] (heterogeneous methods). At the present time, mycophenolate is predominantly used due to improved graft outcomes when combined with the calcineurin inhibitors. There was initial concern that the additional immunosuppressive potency provided by mycophenolate might increase the PTLD rate, and two case–control studies were conducted specifically to address the issue. Looking at PTLD cases (n = 108) and PTLDfree controls (n = 404), no difference in risk was found between triple therapy maintenance regimens containing mycophenolate and those that did not (however, these latter triple regimens were not further defined) [53]. Comparing kidney recipients receiving mycophenolate at discharge to those that did not, even a decrease in risk was found amongst cases derived from the CTS registry (representing European and Canadian recipients), but not from the UNOS-OPTN registry (recipients from the United States) [54]. A post hoc sub-analysis of the UNOS-OPTN data examined all recipients exposed to mycophenolate, including those that had been switched from azathioprine following initial designation, and found a lower PTLD rate in this group. However, this study was limited in that neither use of antibody preparations nor concomitant immunosuppressants were considered. All the data put together appear to show that, despite being a potent immunosuppressant, mycophenolate does not increase the risk of PTLD. A number of studies have shown the use of mTOR inhibitors, sirolimus and everolimus, to be associated with a lower incidence of any de novo, skin and non-skin malignancies in comparison to, or when used together with, the calcineurin inhibitors [55–57]. Both everolimus and sirolimus have been found to inhibit the growth of EBV positive lymphoblastoid B cells in pre-clinical experimental models [58,59]. There have been a few case reports of complete remission of PTLD following conversion from a calcineurin inhibitor based regimen to sirolimus without further treatment (e.g. rituximab, chemotherapy, radiation) [60,61], but for the most part the data reflect therapy with multiple strategies, thus it is difficult to draw firm conclusions about any potential curative effect of mTOR inhibitors. A protective effect by these agents has not been found in retrospective, registry based studies examining lymphoma risk in kidney recipients [4,9,10]. Indeed, one group reported a significant increase in PTLD following kidney transplantation amongst individuals receiving sirolimus based discharge regimens compared to those that did not contain the drug [38]. This study contained a significant number of mTOR inhibitor treated subjects (n = 8755), however is limited by a lack of distinction for how these drugs were used, especially given that their place in therapy is not firmly established and can vary (e.g. use as adjunct therapy with or without dose adjustment of the primary drug, or as primary immunosuppression with or without dose adjustment of the associated antimetabolite). The low dose sirolimus arm of a prospective randomised controlled trial (ELITE-Symphony) conducted in adult renal transplant recipients was the only treatment group in which lymphoma and PTLD occurred (one case each of B-cell NHL, T-cell NHL and PTLD, follow-up 12 months, n = 411) [62]. The other treatment arms were low dose tacrolimus, low dose cyclosporin and standard-dose cyclosporine. All low dose arms received daclizumab induction while for the standard-dose cyclosporine arm there was no induction, and all arms received concomitant mycophenolate and corticosteroids. The sirolimus arm was also associated with the highest rate of acute rejections that may have possibly resulted in an increased use of T cell depleting agents as a consequence. The novel agent belatacept, a fusion protein that selectively blocks T cell co-stimulation, is a recent addition to the drugs available for maintenance immunosuppression and does not appear to cause the nephropathy associated with the calcineurin inhibitors. In phase III studies comparing two different belatacept dosing strategies against a standard cyclosporine regimen (without induction, and similar adjunctive therapy) there were more PTLD

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cases amongst belatacept treated patients. Seven of the 11 PTLD cases across these two studies occurred in individuals that had been exposed to known PTLD risk factors (pre-transplant EBV seronegative, were treated with a T cell depleting agent, or both) [63,64]. 8. Conclusion and future directions PTLD remains a serious complication following solid organ transplantation. The move towards more careful use of immunosuppression and lowest possible dosing [65] in the modern era of transplantation may serve to reduce the rate of these and other malignancies, provided it does not favor a higher rate of rejection episodes whose aggressive treatment may ultimately impact the rate (as may have been the case in the Symphony study). Converse approaches based on early robust immunosuppression with the intention to ‘spare’ the adverse effects of particular agents (such as corticosteroids in the pediatric setting) have not gone without an associated increase in PTLD rate [66]. Despite more careful dosing in general, the number of transplants performed annually is increasing globally, and recipients are surviving longer with transplant organs in situ, which will likely result in a greater absolute number of cases. EBV serostatus is now routinely performed and recorded by transplant registries, and whereas large proportions of recipients with unknown serostatus may have muddied multivariate analyses in the past, this may serve to clarify future analyses. It has been an important and revealing step in risk factor analyses to distinguish early from late occurring disease [10,32] and localization site [46], as well as incorporating time dependent variables, in particular for measures of maintenance immunosuppressive drugs [10]. Recipients of non-kidney organs, the young and the elderly, those who receive T cell depleting agents and EBV seronegative recipients remain important at-risk patient groups. Qualitative analysis of HLA mismatches may also prove useful for identifying those at risk, particularly for early and graft-localized disease. Viruses other than EBV do not appear to have a clear association with PTLD, hence are probably not useful for prospective risk stratification. More data are needed from large cohorts of non-renal solid organ recipients. Gene association studies have revealed potentially useful polymorphisms in cytokine genes but further studies are required to confirm these preliminary results, which may be serve to enhance predictive tests such as viral monitoring [34]. Concerning maintenance immunosuppressive medication, the findings to date have been limited in the absence of detailed data, including measures of drug exposure. Although it has been shown that higher drug exposure resulted in an increased rate of overall malignancy and PTLD for cyclosporine treated patients [67], this may not be the best indicator of degree of immunosuppression. Pharmacodynamic monitoring promises to narrow the gap that exists between blood drug concentration measurements and drug effect. Measures of NFAT gene expression (a pharmacodynamic marker), but not cyclosporine plasma levels, significantly predicted non-melanoma skin cancer (NMSC) amongst a small group of elderly transplant recipients [68]. The same group later used this technique to guide dose adjustment of immunosuppression and successfully prevented recurrent NMSC in an elderly gentleman [69]. This may not be of practical relevance for PTLD, which has a comparatively lower incidence, but if used globally may lead to improved individualized immunosuppression and serve to reduce the malignancy rate overall. At the present time, there is vast heterogeneity in methods of pharmacodynamic monitoring and none have attained wide-spread clinical use. In the same vein, gene polymorphisms associated with drug response may be useful to predict those at risk of over-immunosuppression and consequently PTLD.

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