Peripheral Blood Progenitor Cells

Failure of sustained engraftment after non-myeloablative conditioning with low-dose TBI and T cell-reduced allogeneic peripheral stem cell transplantation

Abstract

We investigated whether a T cell-reduced allogeneic stem cell transplant (SCT) with minimal conditioning and subsequent donor lymphocyte infusions (DLI) could reduce the incidence and severity of GVHD while retaining stable engraftment. Five patients with hematological malignancies (three MM, one CLL, one Chediak-Higashi syndrome) were conditioned with TBI (200 cGy). One patient additionally received fludarabine (120 mg/m2). CsA and mofetyl-mycophenolate (MMF) were administered to prevent GVHD. All patients were grafted with >3 × 106/kg highly purified CD34+ cells together with 2 × 106/kg CD3+ cells (three patients) or 1 × 105/kg CD3+ cells (two patients). Quick hematopoietic recovery and initial mixed donor chimerism was observed. Treatment-related toxicity was minimal in all but one patient who died of treatment-refractory GVHD on day 112. The four other patients only achieved partial donor T cell chimerism. BM and PBMC donor chimerism was lost between day 40 and 209 despite DLI. Three patients are alive with disease and one is in CR. We conclude that T cell-reduced SCT using 200 cGy as the conditioning regimen does not result in stable hematopoietic engraftment. Predominant donor T cell chimerism is not a prerequisite for initial allogeneic hematopoietic proliferation. However for sustained long-term engraftment it is of major importance. Bone Marrow Transplantation (2001) 28, 157–161.

Main

In conventional allogeneic SCT, myeloablative conditioning therapy serves the purpose of eradicating the underlying disease and ensuring engraftment of donor hematopoiesis. Treatment-related toxicity and GVHD remain the major post-transplantation complications resulting in high morbidity and mortality. Allogeneic SCT with reduced intensity conditioning regimens are increasingly used in order to decrease the procedure-related complications in patients not eligible for standard SCT because of age, comorbidities or prior excessive therapies.1,2 Storb and colleagues3,4,5 showed in a canine model that stable engraftment can be reached with minimal myelosuppressive conditioning regimens and minimal treatment-related toxicities. In addition, they provided evidence that a state of mixed donor–recipient chimerism is connected with a reduced incidence of GVHD.5,6 However, an ongoing clinical trial revealed a 36% acute GVHD rate in this setting in spite of intensive prophylactic immunosuppression.1 One way to circumvent the problems of GVHD is T cell depletion of the donor stem cell product. However, these allografts are more likely to be rejected and the graft-versus-leukemia potential is reduced.7,8,9 DLI subsequent to establishment of donor chimerism represents one possibility to immunologically eradicate the underlying disease. We report on the results of a pilot trial which combines the 200 cGy conditioning with transplantation of T cell-reduced grafts followed by prophylactic DLI.

Materials and methods

Patients

Patient data are summarized in Table 1. All patients and related donors provided written informed consent to the pilot-protocol No. 40 which was approved by the local Ethics Committee of the Landesärztekammer Rheinland-Pfalz. Included were patients not eligible for conventional allogeneic SCT because of age, significant comorbidities or prior excessive therapies. Between June and December 1999, five patients underwent nonmyeloablative allogeneic peripheral stem cell transplantation (PBSCT) from their molecularly typed HLA-identical sibling or unrelated donors (details shown in Table 1). Because of the graft failures the study was terminated early for ethical reasons.

Table 1 Transplant characteristics

Conditioning and transplant procedure

Donors received filgrastim (Amgen, Thousand Oaks, CA, USA) 10 μg/kg BW s.c. every 12 h. On day 4, large volume leukaphereses were performed until at least 6 × 106 CD34-positive cells per kg of recipient body weight were collected. A CD34-positive cell selection was performed using the Clini-MACS-System (Miltenyi, Bergisch-Gladbach, Germany). The CD34-positive fraction was cryopreserved in 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. In the CD34-positive and negative fractions, the CD3-positive cells were quantified by flow cytometry (FACScan, Coulter, Krefeld, Germany). The CD34-positive fraction had a purity between 97.1 and 98.2% and contained residual CD3+ cells between 0.2 and 0.6 × 105 cells/kg. The CD34-negative fraction was cryopreserved in DMSO in aliquots of defined numbers of CD3-positive cells. All patients received total body irradiation of 200 cGy on day −1 as the preparative regimen. They were immunosuppressed using CsA (3 mg/kg/day i.v.) and MMF (3 mg/kg p.o. in divided doses) starting on day −6. Patients were switched from the i.v. to the oral formula as soon as tolerated. One patient (UPN 5), in addition received fludarabine i.v. from day −5 to −2 with a total dose of 120 mg/m2. CsA and MMF were tapered off between day 15 and day 50 in patients free of signs of GVHD.

Patients received either 2 × 106/kg (UPN 1 to 3) or 1 × 105/kg CD3+ cells (UPN 4 to 5) on day 0 to facilitate engraftment. Patient-specific transplant characteristics are shown in Table 1. In the case of mixed chimerism, additional DLI were not considered before day +70. Patients with decreasing donor-chimerism after day +70 were scheduled to receive DLI every 3 to 4 weeks.

Chimerism assay

The degree of donor recipient chimerism was monitored by a semi-quantitative analysis of the chimeric genotypes based on nine autosomal short tandem repeat (STR) polymorphisms (D3S1358, VWA, FGA, THO1, TPOX, CSF1PO, D5S818, D13S317, D7S820), as well as the genomic amelogenin locus.10 Briefly, DNA was extracted from bone marrow samples, from FACS-separated mononuclear cells as well as from CD3+ and CD3 lymphocytes. Furthermore, buccal swabs were collected from the donors and recipients to establish their genotypes prior to transplantation. The DNA was subjected to PCR typing using the ‘AmpFlSTR Profiler Kit’ in a multiplex PCR assay following the manufacturer's instructions (Applied Biosystems, Weiterstadt, Germany). The resulting STR fragments were separated by capillary electrophoresis with an ABI Prism 310 automated sequencing system. Results were analysed using the GeneScan software, and the genotypes were identified by comparison to allelic ladders for all loci. To assess the relative proportion of donor and recipient cells from these STR profiles, five loci were selected in each case exhibiting allelic differences, eg homozygosity vs heterozygosity for the same two alleles, or the presence of three or four alleles. The peak areas for each allele based on relative fluorescence units (rfu) were recorded. The total number of cells was defined as the sum of peak areas for each STR system. The relative proportion of donor vs recipient alleles was then calculated in percent, and the mean value and standard deviation was determined across the five selected systems. It could be demonstrated that this approach provides a reliable estimate of relative proportion of donor recipient mixtures in mock experiments (data not shown), as well as in transplantation cases.

Results

Patient UPN 1

A 50-year-old male patient diagnosed with CLL in 1995 received six cycles of fludarabine and an autologous PBSCT in 1996. He relapsed with CLL shortly thereafter. Because of rapid and symptomatic disease progression he was considered to undergo allogeneic transplantation. He was not eligible for standard allogeneic SCT because of prior autologous SCT, his poor performance status, the lack of a fully matched family or unrelated donor and his extensive prior treatment. Following experimental SCT he recovered neutrophil counts (>500/μl) on day 32. On day 71 he developed CMV pneumonia which was successfully treated with foscarnet and CMV hyperimmunoglobulin. Cytologically and immunophenotypically the malignant population could no longer be detected in BM and PBMC. His chimerism on day 26 was >70% donor in the bone marrow and on day 60 he reached a complete chimerism in all subsets (Figure 1). Initially, he experienced grade I GVHD of the skin and later developed a chronic extensive high risk GVHD with progressive and steroid refractory involvement of the liver. The patient died on day +112 from liver failure.

Figure 1
figure1

Percent of molecular donor chimerism ± s.d. (as described in Materials and methods) in bone marrow (..▪..), CD3 positive (T) cells (–♦–), CD3 negative cells (–•–) and whole blood mononuclear cells (----) from patient Nos 1 (a), 2 (b), 3 (c), and 4 (d). Dosage of donor lymphocyte infusions (DLI) are shown in Table 1.

Patient UPN 2

This patient was a 29-year-old women with Chediak-Higashi syndrome and no prior therapy.11 She was not eligible for standard allogeneic SCT because of severe neurological deficits and multiple infectious complications. Following experimental SCT her neutrophil counts never dropped below 500/μl or her thrombocyte counts below 20 000/μl. On day 20 she displayed >70% donor chimerism in a bone marrow sample which rapidly declined until day 68 (Figure 1). Currently, she is alive and well with disease on G-CSF treatment.

Patient UPN 3

This patient was a 46-year-old male patient with an 18-month history of lambda light chain multiple myeloma with Bence–Jones proteinuria. In the past he had received multiple chemotherapies including autologous PBSCT in March 1999. He only achieved a PR following high-dose therapy (HDT) and autotransplantation. Because of prior autologous SCT the patient was considered ineligible for standard allogeneic SCT. Following experimental SCT his neutrophil counts never dropped below 500/μl or thrombocyte counts below 20 000/μl. The patient showed a myeloid donor chimerism of 72% on day +26, which decreased to 9% until day +271 despite multiple DLIs (0.2 × 106/kg CD3+ cells on day 112; 1 × 106/kg on day 229; 10 × 106/kg on day 277; see Figure 1). Cytologically and immunophenotypically he maintained a PR and is currently well without further therapy.

Patient UPN 4

This was a 56-year-old female patient with Ig-A multiple myeloma with Bence–Jones proteinuria diagnosed 3 years before transplantation with a history of multiple chemotherapies. In 1997, she received HDT plus autologous PBSCT. She experienced a relapse 19 months later. Because of this therapy she was ineligible for standard allogeneic SCT. Three months before experimental SCT she had a second autologous SCT and was in CR thereafter. Post allogeneic SCT she recovered her neutrophil counts at day 11 and her thrombocyte counts never dropped below 20 000/μl. On day 59, her donor chimerism was complete but on day 115 it had decreased to 60% in peripheral blood leukocytes, which could not be improved by DLI (0.1 × 106/kg CD3+ cells) given on day 118. At day +152 her chimerism was 90% recipient. Her further clinical course was complicated by a neutropenic colitis. Because of loss of chimerism, she was retransplanted on day 163 with unmanipulated PBSC from her original donor (3.6 × 106 CD34+ cells/kg) without conditioning therapy (Figure 1). Again she did not engraft and received her autologous ‘back-up’ on day 182. Then she recovered completely with autologous hematopoiesis. Currently, she is in CR, alive and well.

Patient UPN 5

This a 57-year-old female diagnosed with Ig-G multiple myeloma 15 months before allogeneic transplantation. She has a history of multiple chemotherapies including HDT plus autologous PBSCT 6 months before allo-transplantation. Thereafter she relapsed and at the time of experimental SCT was in progressive disease. Following alloSCT she recovered her neutrophil counts on day 3 and her thrombocyte counts never dropped below 20 000/μl. On day 16, her donor chimerism was 81% which was completely lost on day 37. She developed prolonged neutropenia starting from day +29 with reactivation of CMV. The patient also had neutropenic fever which resolved under antibiotic therapy. On day +48 she received her autologous back-up and recovered her hematopoiesis promptly. She is alive and well with progressive disease.

Discussion

In a canine animal model it has been shown by Storb et al5 that sustained mixed chimerism can be achieved with low-dose TBI and strong post-transplant immunosuppression with CsA and MMF. In this study four of five dogs had stable mixed chimerism for longer than 1 year and no signs of GVHD were recognised. In spite of this promising preclinical data, 36% of the patients enrolled in a clinical trial developed clinical significant GVHD.1 Stable hematopoietic engraftment was reached in 33 of 46 patients (72%). The aim of the present study was the establishment of stable hematopoietic chimerism in the absence of GVHD using a T cell-reduced PBSCT. A bidirectional graft–host tolerance should then provide a safe platform from which GVL reactivity can be established by DLI. Only one out of five patients in our series showed sustained donor chimerism. After initial mixed myeloid chimerism with predominance of donor cells, starting from day 37 all but one patients lost detectable donor chimerism. The additional immunosuppression provided by earlier HDT plus autologous SCT in patient UPN Nos 3 and 5 was not relevant. Interestingly, predominantly donor T cell chimerism was never established in the patients who had eventual graft failure, suggesting a key role of donor T cells in long-term graft protection. As has been shown earlier in the setting of myeloablative conditioning, T cell depletion is not always connected with delayed graft failure.12,13 In addition, we have previously shown that with a slightly stronger but still nonmyeloablative conditioning regimen engraftment was possible in spite of a T cell-depleted PBSCT.14 In our study two out of four patients with decreasing myeloid donor chimerism received DLI. However, the DLI had no significant impact on the already progressing graft rejection. This might be related to the dose and time point of administration. Georges et al15 showed recently in an canine model that DLI were unable to convert mixed to complete chimeras after nonmyeloablative conditioning and transplant, confirming the not yet elucidated role of DLI in graft protection.

The first three patients in our series received an initial dose of 20 × 105/kg CD3-positive cells on day 0. One of them developed lethal GVHD. Therefore the initial T cell dose was reduced to 1 × 105/kg and no GVHD was observed in the further patients. The development of GVHD correlated with complete or almost complete donor chimerism of the T cell compartment, confirming recently published data on patients receiving a matched sibling transplant after nonmyeloablative conditioning.16 In this study, five of nine patients had a mixed myeloid chimerism, but a 100% donor T cell chimerism. These patients developed GVHD despite their mixed myeloid donor chimerism.

In conclusion, we confirm the importance of donor T cells for maintenance of long-term myeloid engraftment following a minimal conditioning regimen. However, transient engraftment of donor myeloid cells was not disturbed by T cell depletion. Future efforts should be directed to defining safe dosage and time points of DLI to sustain hematopoietic engraftment without the development of GVHD.

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Acknowledgements

We would like to thank Beate Stradmann-Bellinghausen and Jutta Lummer for their excellent technical assistance. This work was supported by a grant from the Deutsche Krebshilfe (No. 70–2428-Schm 2). Martin Schuler is a fellow of the Dr Mildred Scheel Stiftung für Krebsforschung.

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Correspondence to S Kreiter.

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Kreiter, S., Winkelmann, N., Schneider, P. et al. Failure of sustained engraftment after non-myeloablative conditioning with low-dose TBI and T cell-reduced allogeneic peripheral stem cell transplantation. Bone Marrow Transplant 28, 157–161 (2001). https://doi.org/10.1038/sj.bmt.1703107

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Keywords

  • non-myeloablative
  • fludarabine
  • T cell depletion
  • multiple myeloma
  • allogeneic stem cell transplantation
  • chimerism analysis

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