Post Transplant Lymphoproliferative Disorder

High incidence of PTLD after non-T-cell-depleted allogeneic haematopoietic stem cell transplantation as a consequence of intensive immunosuppressive treatment

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Summary:

The occurrence of post-transplant lymphoproliferative disorder (PTLD) in relation to immunosuppressive treatment was determined in 257 patients treated with non-T-cell-depleted allogeneic stem cell transplantation from an HLA-matched sibling (173 patients) or unrelated donor (84 patients). The conditioning consisted of total body irradiation and cyclophosphamide (myeloablative conditioning, 250 patients), or fludarabine combined with cyclophosphamide or a single 2 Gy dose of TBI (nonmyeloablative conditioning, seven patients). In transplantations from an unrelated donor, the patients also received antithymocyte globulin (ATG). The prophylaxis against graft-versus-host disease (GVHD) consisted of cyclosporine A, methotrexate, and methylprednisolone. The autopsy reports of deceased patients were systematically reviewed, and the autopsy materials of cases suggestive of PTLD were re-examined histologically for Epstein–Barr virus (EBV). Nineteen patients with EBV-positive PTLD were identified, of whom six had been transplanted from a sibling donor and 13 from an unrelated donor. All the patients who developed PTLD had been given ATG either for the treatment of steroid-resistant acute GVHD (all PTLD patients with a sibling donor and one with an unrelated donor), or as part of the conditioning (all patients with an unrelated donor). In conclusion, in transplantations from an HLA-identical donor with a non-T-cell-depleted graft, the risk of PTLD correlated strongly with the intensity of the immunosuppressive treatment.

Main

In post-transplant lymphoproliferative disorder (PTLD), T-lymphocyte dysfunction caused by immunosuppressive treatment allows uncontrolled proliferation of B cells hosting Epstein–Barr virus (EBV).1,2,3 After allogeneic stem cell transplantation, PTLD has been considered to be rare (<1%), but intensive immunosuppression, such as T-cell depletion of the graft, may markedly increase the risk.4,5,6,7,8 With effective immunosuppressive treatment, the risk of graft-versus-host disease (GVHD) can be maintained at a low level, but, at the same time, the risk of infections, including PTLD, may increase. Solid organ transplantation-associated PTLD can arise either early or late after the transplantation,2,9 while in association with allogeneic stem cell transplantation PTLD tends to be mostly of the early-onset rapidly progressive form.4,7,10 The treatment possibilities of PTLD have been very limited and the prognosis dismal. Recently, however, donor lymphocyte infusions (DLI),11 anti-B-cell antibodies,12,13,14,15,16 or infusions of EBV-specific cytotoxic T cells17,18 have been shown to be useful in the treatment of EBV-induced lymphoproliferation. In the present study, the relation between the occurrence of PTLD and the intensity of the immunosuppressive treatment was studied in transplantations from an HLA-matched donor with a non-T-cell-depleted graft in order to identify high-risk patient groups for prophylactic and pre-emptive treatment.

Patients and methods

In the years 1994–1999, a total of 257 adult patients underwent allogeneic stem cell transplantation at Helsinki University Central Hospital. Of the donors, 173 were siblings and 84 unrelated. In 250 cases, the conditioning was myeloablative and in seven cases nonmyeloablative. In 252 patients the indication for transplantation was a haematological malignancy and in five patients, aplastic anaemia. All donors, except one sibling donor with a single HLA A locus difference, were matched for HLA A, B, and DR. Approximately 80% of the patients and donors were typed by standard serologic cytotoxicity tests and the other 20% by polymerase chain reaction (PCR)-based methods for class I alleles both in transplantations from a sibling and in those from an unrelated donor. When serology was used, uncertain findings were confirmed with PCR-based techniques. Class II alleles were typed by serologic tests in 10% and by PCR methods in 90% of the cases, in approximately one half of those with low-resolution and one half with high-resolution techniques. In the sibling transplantations, the graft was bone marrow in 143 and blood stem cells in 30 cases. In the unrelated transplantations, the graft was bone marrow with two exceptions. The grafts were nonmanipulated apart from the red cell and plasma removal from the bone marrow grafts in the case of ABO incompatibility.

The myeloablative conditioning consisted of cyclophosphamide 60 mg/kg on two successive days and six doses of 2 Gy total body irradiation given during the following 5 days. In the transplantations from an unrelated donor, the conditioning also included antithymocyte globulin (ATG). In the first 50 cases ATG was Atgam® (Upjohn, Kalamazoo, USA) 10 mg/kg/day on days −5 to −3, and in the subsequent 33 cases Thymoglobuline® (Merieux/Sangstat, Lyon, France), 10 mg/kg/day on days −3 to −1 (four patients) or 4 mg/kg/day on days −3 to −1 (the last 29 patients). The prophylaxis against GVHD consisted of cyclosporine A (CyA), a short course of methotrexate (MTX, 15 mg/m2 on day +1 and 10 mg/m2 on days +3, +6, and in the 138 first cases, also on day +11), and methylprednisolone (MP) as described previously.19 The CyA treatment was initiated with an i.v. dose of 3 mg/kg/day and continued after approximately 2 weeks by mouth. The dose was modified according to the side effects and the whole blood cyclosporine concentrations (target level of 300–400 μg/l on days 0–14 in all patients; after that 100–200 μg/l in sibling transplantations and 200–300 μg/l in transplantations from an unrelated donor). MP was initiated either on day +14 (the first 138 patients) or on day +8 (112 patients) with a dose of 0.5 mg/kg/day. After 1 week, the dose was increased to 1 mg/kg/day for 2 weeks, whereafter MP was slowly tapered and discontinued on day +110.

The nonmyeloablative conditioning consisted of fludarabine 30 mg/m2 daily on 3 days combined with cyclophosphamide 1000 mg/m2 on 2 days (five patients) or one dose of 2 Gy TBI (two patients). The only patient trans-planted from an unrelated donor received Thymoglobuline® 4 mg/kg/day on days −3 to −1. For GVHD prophylaxis, the patients given conditioning containing cyclophosphamide received CyA and MTX (3 days), as described above. The patients conditioned with TBI were given mycophenolate mofetil 30 mg/kg/day for 30 days and CyA orally.

Acute GVHD was assessed and graded according to previously published criteria.20,21 Acute GVHD was treated independently of the grade with intravenous MP 10 mg/kg/day divided into four doses. The dose was halved thrice every third day and thereafter according to the clinical situation. In the corticosteroid-resistant cases, ATG was used as the second-line treatment.

DLIs were given to 36 patients, 25 with a sibling donor and 11 with an unrelated donor, for post-transplant relapse to enhance graft-versus-malignancy effect. The dose of donor T lymphocytes was 0.4–3.2 × 108/kg with a median of 1.5 × 108/kg in 30 patients. Six patients, three with a sibling donor and another three with an unrelated donor, received escalating doses of lymphocytes starting with a dose of 1 × 107 T cells /kg from a sibling donor and 1 × 106 T cells/kg from an unrelated donor.

The post-mortem reports of the deceased patients were reviewed for findings suggestive of PTLD. In suspect cases, archived post-mortem paraffin blocks were re-examined immunohistologically for EBV antigens (LMP1, EBNA) and by in situ hybridization for EBV-RNA (EBER 1 and 2) (Table 1). In addition, EBV-DNA, in sera collected before and after the transplantation, was measured by real-time PCR, as described separately22 (Table 1).

Table 1 Clinical, laboratory, and autopsy data of 19 patients with PTLD

Statistical analysis

The χ2-test with continuity correction or Fisher's exact test, when appropriate, was used to compare GVHD and the use of ATG in relation to the risk of PTLD. The role of GVHD was examined by crosstabulating PTLD and ATG, also separately, in the groups with or without GVHD.

Results

Of the 257 patients, 133 (52%) were alive by June 2001 with a median follow-up time of 1490 days (range 556–2711 days), and 124 (48%) had died with a median follow-up time of 274 days (range 4–1661 days). The cause of death was transplant-related in 76 patients (61%), relapse in 42 patients (34%), and other in seven cases (5%).

In all, 77 (62%) of the 124 deceased patients underwent autopsy. The analysis of the post-mortem reports and the re-evaluation of the autopsy materials revealed or confirmed 19 cases of histopathologically verified PTLD, each presenting with disseminated, multiorgan infiltration of lymphocytes (Tables 1 and 2). According to the WHO classification, PTLD was monomorphic in 12 patients, polymorphic in six patients, and Hodgkin-like in one case. Of all patients, 10 showed monoclonality by immunoglobulin heavy-chain rearrangement as detected by PCR. In 14 cases, PTLD had been diagnosed before the present analysis. In seven of them PTLD had been diagnosed while the patients were alive based on the histology and positive EBV staining of lymph node and kidney biopsies (LMP) (Patient No. 9) or liver biopsy (LMP) (No. 15), the appearance of EBV positive (EBER and EBNA) and CD20 positive atypical lymphocytes in circulation (Nos. 5 and 6), or the presence of high copy numbers of EBV-DNA in plasma by PCR (Nos. 3, 16, and 19). After death, autopsy confirmed the diagnosis of PTLD in all of these patients (Table 1). In addition, PTLD was diagnosed in seven further patients at autopsy (Table 1). In five cases, PTLD was diagnosed as a result of the present study. PTLD was not diagnosed in any of the survivors.

Table 2 Characteristics of the patients with post-transplant lymphoproliferative disorder (PTLD)

Six of the 19 patients with PTLD had been transplanted from a sibling donor and the remaining 13 patients from an unrelated donor. One PTLD patient with an unrelated donor had received nonmyeloablative conditioning (fludarabine+TBI), and she was also the only PTLD patient having received a blood stem cell graft. All other PTLD patients had been transplanted with a bone marrow graft after myeloablative conditioning. In three patients with an unrelated donor, PTLD occurred after GVHD induced by DLI given for post-transplant relapse. Confirmed PTLD occurred in 19 of the total of 257 patients (7.4%), in 19 of the 124 deceased patients (15.3%), in 13 of the 84 patients with an unrelated donor (15.5%), and in six of the 173 patients with a sibling donor (3.5%).

Table 1 shows the clinical and autopsy findings of the patients with PTLD. In 16 patients the onset of PTLD was early, and the patients died in a median of 96 days (range 67–221 days) from the transplantation. In the remaining three patients, DLI with subsequent GVHD and its treatment was apparently the triggering event for PTLD. The clinical picture of PTLD lacked distinctive features. With one exception, all patients had fever which appeared in a median of 72 days (range 29–162 days) after the transplantation (or DLI), and in a median of 16 days (range 3–155 days) before death. During life only four patients had palpable lymphadenopathy, and in two other patients lymphadenopathy was shown by computerized tomographic scan. At autopsy, only six patients had enlarged lymph nodes, although all patients had multiorgan lymphocyte infiltrations. In all, 11 patients showed reactive lymphocytes in the circulation. The lactate dehydrogenase concentration was elevated with a rapid increase towards death in 18 patients. Sera were available for EBV-PCR analysis from 13 patients and high copy numbers of EBV-DNA were detectable in all of them at the onset of the symptoms and signs of PTLD. Before transplantation, none of the 13 patients had EBV-IgM and all patients except one (No. 7) had EBV-IgG in serum, indicating prior latent infection.

As a treatment for PTLD, three patients (Nos. 5, 6, and 16) received DLI, two patients (Nos. 3 and 19) were treated with one infusion of rituximab, and one patient (No. 15) with local irradiation and one course of cytotoxic treatment (CHOP). The response to the treatment was poor in all patients.

Table 3 shows the occurrence of PTLD according to the immunosuppressive treatments given. All patients with PTLD had been treated with ATG either before the transplantation as part of the conditioning (unrelated donor), or after the transplantation for GVHD, or both. Of the 257 patients, 64 (35 patients with a sibling donor and 29 patients with an unrelated donor) had grade II–IV acute GVHD and were treated with high-dose MP. In 30 of these 64 patients (15 with a sibling donor and 15 with an unrelated donor), the response to MP was poor, and the patients were given ATG as the second-line treatment. Of the 36 patients given DLI, 11 (seven with a sibling donor and four with an unrelated donor) had grade II-IV acute GVHD after DLI. They were treated with varying doses of MP and only two patients with an unrelated donor received ATG as the second-line treatment.

Table 3 Correlation of post-transplant lymphoproliferative disorder with immunosuppressive treatment in allogeneic transplantation patients

Among 173 transplantations from a sibling donor, PTLD was diagnosed in six patients (Table 3). They all had experienced grade II–IV steroid-resistant acute GVHD and had been treated with ATG, whereas no PTLD was found among the 158 patients who had a sibling donor, had not been given ATG, and of whom 20 patients with acute GVHD of grade II–IV had been treated with high-dose MP only. Of the 84 patients transplanted from an unrelated donor with ATG as part of the conditioning, 13 developed PTLD (Table 3). In nine PTLD patients, ATG had been given only before the transplantation. Three of the four patients given Thymoglobuline® 10 mg/kg/day and five of the 26 patients given Thymoglobuline® 4 mg/kg/day on 3 days pretransplantation developed PTLD. Eight of the 13 PTLD patients had not been treated for acute GVHD, one patient had had acute GVHD grade II treated only with MP, and one PTLD patient had had steroid-resistant acute GVHD treated with ATG. In addition, PTLD occurred in three of the 11 patients transplanted from an unrelated donor who had post-transplant relapse and were treated with DLI. All the three patients experienced grade II–IV acute GVHD induced by DLI and two of them were treated with ATG (Table 2). The use of ATG was a statistically significant risk factor for PTLD (P<0.0001), and the PTLD/ATG correlation also remained significant when analysed separately in the groups with or without GVHD.

Discussion

PTLD is generally a rare complication of allogeneic stem cell transplantation, but intensive immunosuppressive measures increase its incidence.4,5,6,7,8 The present findings are in agreement with this previous experience.

In the present study, 19 cases of PTLD were found among 257 consecutive allogeneic transplantations of non-T-cell-depleted grafts from HLA-identical siblings or unrelated donors. In all cases, PTLD was an early event with the first signs appearing at 29–162 days after the transplantation or donor lymphocyte infusion, the median being 72 days. The incidence of 7.4% (19/257 patients) may be an underestimate. The present analysis was retrospective and mainly based on the autopsy material, and only about two-thirds of the succumbed patients were autopsied. No cases of PTLD were diagnosed among the survivors. The present findings may, however, reflect relatively well the actual situation, as the early type of PTLD is usually fatal.6,7,10

The occurrence of PTLD was strongly associated with the intensity of the immunosuppressive treatment. No PTLD developed among the patients receiving a graft from a sibling donor in the absence of steroid-resistant acute GVHD treated with ATG. Of the 55 patients transplanted from an unrelated donor with ATG as part of the conditioning, eight (15 %) developed PTLD despite the absence of GVHD. The larger immunological differences in the unrelated donor situation as compared to the sibling transplantation – despite A, B, DR antigen match – may have contributed to the immunosuppression. In the transplantations from an unrelated donor, the addition of low-dose corticosteroid to the GVHD prophylactic regimen consisting of cyclosporine and methotrexate may have been a further contributing factor, although in our earlier study in the sibling transplantation setting there were fewer infections in the group of patients given the triple prophylaxis compared to patients given the combination of cyclosporine and methotrexate.19

The present study also suggests a difference in the immunosuppressive effect between the different ATG products in the doses used, which is in accordance with the previous study by Gaber et al.23 They showed that after renal transplantation, the degree of T-cell depletion caused by Thymoglobuline® was significantly greater and longer-lasting when compared to the effects of Atgam®. Thymoglobuline® was also more effective in the treatment of acute rejection. In line with these observations, the incidence of acute GVHD was clearly higher and the incidence of PTLD lower among the present patients who had been transplanted from an unrelated donor and given pretransplant Atgam® 10 mg/kg/day for 3 days, compared to those who had been given pre-transplant Thymoglobuline® 4 or 10 mg/kg/day for 3 days. In the immunosuppressive treatment after stem cell transplantation, the balance between effective GVHD prophylaxis and the complications of immunosuppression is delicate. With the intense immunosuppressive treatment used, the incidence of GVHD was maintained at a low level, but at the cost of an increased incidence of infections, especially EBV infections and stubborn CMV infections. Since then we have reduced immunosuppression in transplantations from an unrelated donor by omitting corticosteroid from the GVHD prophylaxis as well as by reducing the dose of Thymoglobuline® to 2 mg/kg/day for 3 days in the conditioning. The incidence of GVHD has somewhat increased, but that of viral infections including PTLD significantly decreased.24

EBV-induced PTLD is a serious and often fatal complication of allogeneic stem cell transplantation, and it is of vital importance to identify high-risk groups for early diagnosis and treatment. Intensive immunosuppression is a central factor in the development of this complication, and, in the present material, the relation between the PTLD and ATG treatment was found to be very close. None of the present patients who did not receive ATG developed PTLD. Even in those who were given ATG as part of the conditioning and who did not show any acute GVHD, the risk seems to be increased, but the effect of the dose and the type of ATG still need further evaluation. The clinical diagnosis of PTLD is difficult, as the symptoms and signs are nonspecific. However, the quantitative measurement of EBV DNA in the serum has recently been shown to be very useful in the prediction and early diagnosis of PTLD.16,22 The demonstration of increasing EBV-copy numbers allows early intervention with antibodies (e.g. rituximab) or donor lymphocyte infusion.

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Acknowledgements

This study was financially supported by the Finnish Cancer Research Foundation, the HUCH (EVO) Fund, and the Finnish Technology Advancement Fund.

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Correspondence to E Juvonen.

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Juvonen, E., Aalto, S., Tarkkanen, J. et al. High incidence of PTLD after non-T-cell-depleted allogeneic haematopoietic stem cell transplantation as a consequence of intensive immunosuppressive treatment. Bone Marrow Transplant 32, 97–102 (2003) doi:10.1038/sj.bmt.1704089

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Keywords

  • PTLD
  • allogeneic stem cell transplantation
  • non-T-cell-depleted graft
  • immunosuppressive treatment
  • ATG

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