Unrelated peripheral blood stem cell transplantation with - Nature

Bone Marrow Transplantation (2000) 25, 513–517  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt

Unrelated peripheral blood stem cell transplantation with ‘megadoses’ of purified CD34+ cells in three children with refractory severe aplastic anemia W Schwinger1, Ch Urban1, H Lackner1, R Kerbl1, M Benesch1, HJ Dornbusch1, P Sovinz1, K Schauenstein3, M Schumm2 and R Handgretinger2 1

Division of Pediatric Hematology/Oncology, Department of Pediatrics, and 3Institute of Experimental Pathology, University of Graz, Austria; and 2Department of Pediatrics, University of Tu¨bingen, Germany

Summary: Three children with refractory severe aplastic anemia were transfused with high numbers of unrelated matched (n = 2) or C-locus haploidentical mismatched (n = 1) CD34-selected peripheral blood stem cells in the absence of an HLA-identical family donor. Two leukaphereses of the donors yielded a median number of 10.1 ⴛ 1010 nucleated cells (range 9.7–15.4) with a median number of 9.89 ⴛ 108 CD34+ cells (range 7.46– 26.1) and a median percentage of CD34+ cells of 0.98% (range 0.77–1.7). After positive selection by magnetic cell sorting the patients received a median of 14.3 ⴛ 106 CD34+ cells/kg (range 11.7–24.3) and of 1.3 ⴛ 104 CD3+ cells/kg (range 0.57–5.8). Median time to ANC ⭓0.5 ⴛ 109/l was 7 days (range 7–12) and to platelets ⭓20 ⴛ 109/l 13 days (range 13–27). Chimerism analysis of peripheral blood after transplantation revealed permanent 100% donor hematopoiesis in all patients. The patient with the C-locus haploidentical mismatch presented with acute GVHD (grade III–IV) of the skin, liver and lower gastrointestinal tract (onset day +40) and died despite intensive immunosuppressive treatment on day +238. The two survivors developed lymphopoietic recovery of B and T lymphocytes within 3 months after transplantation. To our knowledge this experience represents the first report of transplantation with unrelated CD34+ enriched peripheral blood stem cell in children with refractory severe aplastic anemia. Bone Marrow Transplantation (2000) 25, 513–517. Keywords: MACS; CD-34 selection; unrelated donor transplantation; severe aplastic anemia

Allogeneic stem cell transplantation is the only curative treatment in children with severe aplastic anemia (SAA). Lacking a suitable HLA-matched family donor, some patients with SAA may benefit from immunosuppressive therapy (IS). In patients who fail conventional IS, transCorrespondence: Dr W Schwinger, Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Graz, Auenbruggerplatz 30, A-8036 Graz, Austria Received 19 April 1999; accepted 18 October 1999

plantation of unrelated stem cells may be a further alternative. However, it is limited by a high risk of graft rejection and graft-versus-host disease (GVHD).1–5 To overcome these problems, megadoses of unrelated highly purified peripheral CD34+ cells were used in this study. Magnetic cell sorting (MACS) was performed to purify the peripheral blood mononuclear cells with adequate recovery of CD34+ cells.6 We present the clinical, hematopoietic and immunologic data of three children who underwent transplantation with ‘megadoses’ of unrelated purified CD34+ cells for the treatment of refractory SAA. Patients and methods Patients Patients enrolled in this study previously failed immunosuppressive treatment and did not have an appropriate HLA-matched sibling or related donor. In all patients, Fanconi anemia was excluded by diepoxybutane testing of peripheral blood lymphocytes for increased chromosome breakage. Patient 1 presented with constitutional SAA at the age of 3 months, and did not respond to various immunosuppressive agents within 3 years, including repeated courses of antilymphocyte globulin (ALG), cyclosporin A (CsA) and methylprednisone (MP) as well as to stimulation of hematopoiesis with filgrastim, erythropoietin and huR-IL-3. He underwent splenectomy but remained erythrocyte and platelet transfusion-dependent. Patient 2 also suffered from constitutional SAA. He did not respond to similar immunosuppressive treatments as patient 1. After conditioning with cyclophosphamide (200 mg/kg) and ALG, the patient received a 5/6 HLA-matched (C-locusmismatched) umbilical cord blood (UCB) graft. After initial engraftment he showed graft rejection and autologous reconstitution by day +45 and remained erythrocyte and platelet transfusion-dependent. Patient 3 suffered from acquired SAA and did not respond to ALG, CSA and MP. Patients characteristics are summarized in Table 1. Unrelated donor selection, mobilization and collection of stem cells International search for potential unrelated donors was performed by the ‘Austrian Bone Marrow Donor Registry’ and

Unrelated PBSCT with CD34+ cells for SAA W Schwinger et al

514

Table 1

Patient and donor characteristics

Patient

1 2 3

Recipient

Donor

CMV status

Age (years)

Sex

Weight (kg)

Disease

Sex

Relationship

HLA analysis

11.2 7.5 14.0

M M F

50 30 59

SAA SAA SAA

M M M

unrelated unrelated unrelated

matched C-locus mismatched matched

donors were found in the ‘Deutsche Knochenmarksspenderdatei’ (DKMS) (Tu¨bingen, Germany). Informed consent for stem cell apheresis and mobilization with filgrastim (GCSF) was obtained from all donors. Confirmatory HLA typing of donors and patients was performed using standard serological techniques identifying common specifities for HLA-A, -B and -C antigens and PCR techniques for identification of HLA-DRB1 and DRB3. Host-specific cytotoxic T lymphocyte precursor (CTLP) frequencies were measured according to standard techniques. All donors tested negative for cytomegalovirus (CMV), hepatitis-A/-B/-C virus, parvo-B19 virus and Epstein–Barr virus (EBV) infection (Table 1). Stem cells were mobilized by subcutaneous injection of G-CSF (10 ␮g/kg body weight/day) once daily for 5 consecutive days. Leukaphereses were performed with a Cobe Spectra blood cell separator (Cobe, Vienna, Austria) on days 5 and 6 using ACD-A as the anticoagulant. Apheresis products were resuspended with autologous plasma to a final volume of 200 ml.

negative negative negative

position of cells within the negative fraction (unselected cells = total NC minus CD34+ cells) were assessed by flow cytometry. Conditioning regimen Informed consent was obtained from the parents. Patient 1 was conditioned with ALG (Sero Merieux) 0.75 ml/kg/day (× 4), thiotepa 250 mg/m2/day (× 2), cyclophosphamide 60 mg/kg/day (× 2), and total lymphoid irradiation (TLI, 5 Gy). Patient 2 who had a relapse after a previous umbilical cord blood transplantation, was treated with ALG (Sero Merieux) 0.75 ml/kg/day (× 4), busulphan 4 mg/kg/day (× 4), fludarabine 40 mg/m2/day (× 5) and cyclophosphamide 60 mg/kg/day (× 1). Patient 3 was conditioned with thiotepa 250 mg/m2/day (× 2), cyclophosphamide 50 mg/kg/day (× 4), total lymphoid irradiation (TLI, 5 Gy) and instead of ALG OKT3 (day −4 to day +14) 2.5 mg together with MP. All patients were transplanted in laminar air flow (LAF) units.

Magnetic positive selection of CD34+ cells In patient 1 positive selection of CD34+ cells was performed using the CD34+ Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) applying a magnetic cell separation (MACS) technique using the Super-MACS sorting device (Miltenyi Biotec) according to the manufacturer’s instructions.6–8 In the other two cases CD34+ cells were selected using the CliniMACS-system (Miltenyi Biotec). Apheresis products were diluted (1:1) in buffer solution consisting of phosphate-buffered saline (PBS) w/o Ca/Mg (Gibco, Paisley, UK), 5 mm EDTA and 5% human albumin at a final concentration of 0.8% (Immuno, Vienna, Austria) and centrifuged for 15 min at 350 g for removal of platelets. Then, sedimented cells were resuspended in the same buffer solution at 2 ⫻ 108 nucleated cells (NC)/ml and incubated with 5 mg 7-Simmunoglobulin/1011 NC for 5 min at room temperature to block nonspecific Fc-receptors. Thereafter 7.5 ml of CliniMACS CD34 Reagent (Miltenyi Biotec) was added to 5 ⫻ 1010 NC and incubated for 30 min at room temperature. Then, antibody-labeled cells were resuspended in buffer solution to a final concentration of ⭐2 ⫻ 108 NC/ml and transferred into a 350 ml blood collection bag (Baxter, Deerfield, IL, USA). Connection to the CliniMACS tubingset as well as the fully automated selection procedure was performed according to the instructions of the manufacturer. The purity and recovery of the isolated CD34+ cells, the contamination with T lymphocytes as well as the comBone Marrow Transplantation

GVHD prophylaxis and cytokine stimulation after transplantation None of the patients received prophylactic immunosuppression. Patients were evaluated daily for acute GVHD during hospitalization and at least once weekly as outpatients until day +100. Diagnosis of GVHD was based on WHO approved clinical signs. All patients received filgrastim (GCSF) 5 ␮g/kg/day subcutaneously starting at day +4 until white blood counts stabilized ⭓5 ⫻ 109/l and erythropoietin (EPO) at a dosage of 150 IU/day by subcutaneous injection until reaching hemoglobin levels ⭓10 g/dl without erythrocyte transfusions for 10 consecutive days. Proliferation assays A whole blood stimulation assay was used.9 Briefly, various volumes (5, 10 and 20 ␮l) of fresh, heparinized blood were diluted in microtiter plates to a final volume of 200 ␮l with medium (RPMI-1640 with antibiotics and 10−5 m 2mercaptoethanol) containing the mitogens (all from Sigma, St Louis, MO, USA) phytohemagglutinin (PHA 20, 10, 5, 2.5 0.2 ␮g/ml), concanavalin A (ConA 40, 20, 10, 5 ␮g/ml), or pokeweed mitogen (PWM 160, 80, 40, 20 ␮g/ml). Each culture was incubated for 72 h at 37°C in 5% CO2 humidified air. The proliferative response was monitored by 3HTdR uptake during the last 6–8 h of culture. The results were expressed as maximal counts per minute


Table 2

Selection of CD34+ cells

Patient

Number of aphereses

1 2 3

Table 3 Patient

2 2 2

515 Before selection

After selection

Recovery %

NC ×1010

CD34+ %

CD34+ cells/kg ×106

NC ×108

CD34+ cells %

CD34+ cells/kg ×106

CD3+ cells/kg ×105

10.1 9.7 15.4

0.98 0.77 1.70

20.0 24.9 45.6

7.02 3.6 14.4

99.2 97.7 99.2

14.3 11.7 24.3

0.057 0.13 0.58

70.1 46.9 53.2

Engraftment and clinical outcome Transplanted cells

ANC ⭓5 × 108/l day

Platelets ⭓20 × 1010/l day

Acute GVHD grade

CD34+ cells ×106/kg

CD3+ cells ×105/kg

1

14.3

0.057

7

13

—

2

11.7

0.13

12

27

Skin, gut (III–IV)

3

24.3

0.58

7

11

—

of a sample (c.p.m.max) divided by the calculated number of lymphocytes present in culture (c.p.m.max/lymphocyte). Results Collection and manipulation of CD34+ cells The median time between initial donor search and HLA confirmation of a suitable donor was 7 months (range 4– 13). Two donor–recipient pairs (patients 1 and 2) were sexmatched. Patients 1 and 3 received HLA-identical grafts, whereas patient 2 received stem cells which were disparate at one HLA-locus (C-locus). Two donor leukaphereses yielded a median number of 10.1 ⫻ 1010 NC (range 9.7– 15.45) with a median of 9.89 ⫻ 108 CD34+ cells (range 7.46–26.1) and a median percentage of 0.98% (range 0.77– 1.7). After positive selection of CD34+ cells the median number of NC was 7 ⫻ 108 (range 3.6–14.4) with a median number of CD34+ cells of 6.96 ⫻ 108 (range 3.51–14.28), reaching a median purity of 99.2% (range 97.7–99.2%). Recovery of CD34+ cells compared to the initial apheresis product was a median of 53.2% (range 46.9–71.1). Contamination with CD3+ cells was a median of 3.9 ⫻ 105 (range 2.85–34.2). Viability of thawed CD34+ cells was ⬎95% in all units. Results are summarized in Table 2.

Outcome

Follow-up months

de novo SAA → reversed → well/alive death

28

well/alive

10

8

eral blood at a median of 11 days (range 11–14). Repeated chimerism analysis of peripheral blood revealed permanently 100% donor hematopoiesis. Two patients (patients 1 and 3) are well and alive, whereas patient 2 who had a Clocus mismatched peripheral blood stem cell transplantation (PSCT) died at day +238 due to GVHD (grade III–IV) of the liver, lower gastrointestinal tract and skin despite intensive immunosuppression with CsA, MP, mycophenolate mofetil (MMF) and tacrolimus (FK506). Patient 1 developed pancytopenia with persistence of donor cells at day +591, after a trivial virus infection, which was successfully reversed by antithymocyte globulin (ATG) and cyclophosphamide followed by CD34+-selected stem cells of the same donor. GVHD Acute GVHD (grade III–IV) of the liver, lower gastrointestinal tract and skin beginning on day +40, was diagnosed in patient 2, who had been previously transplanted with a 5/6 HLA-matched (C-locus-mismatch) UCB-graft, followed by an unrelated C-locus mismatched PSCT, which failed to respond high doses of prednisone, CsA, MMF and FK506 (Table 3). Immunologic reconstitution

Engraftment and outcome (Table 3) Patients received a median of 14.3 ⫻ 106 CD34+ cells/kg body weight (range 11.7–24.3) with a median of 1.3 ⫻ 104 CD3+ cells/kg/body weight (range 0.57–5.8). All patients showed complete hematological reconstitution of donor origin. Median time to ANC ⭓0.5 ⫻ 109/l was 7 days (range 7–12) and median time to platelets ⭓20 ⫻ 109/l was 13 days (range 13–27). Reticulocytes appeared in the periph-

A measurable number of circulating lymphocytes was detected in all patients 1 month after transplantation. The majority of early detectable lymphocytes was analyzed as CD56+/CD16+ natural killer (NK) cells.10 There was a correlation between the number of CD3+/CD19+ lymphocytes occurring within 1 month after transplantation and the number of CD34+ cells transfused (correlation coefficient for CD3+ cells: 0.95 and for CD19+ cells: 0.87) (Figure 1). The Bone Marrow Transplantation


516

Lymphocytes (×106/l)

250 200 150 100 50 0 0

5

10

15

20

25

30

Number of CD34+ cells transfused (×106/kg) CD3+ T lymphocytes (correlation coefficient 0.95) CD19+ B lymphocytes (correlation coefficient 0.87)

Figure 1 Correlation between number of CD34+ cells infused and number of T/B lymphocytes occurring within 1 month after transplantation.

occurrence of CD8+ cells was earlier than that of CD4+ cells, resulting in an inverted CD4+/CD8+ ratio within the first 6 months after transplantation. In proliferative assays T lymphocytes showed an increasing response to polyclonal activators (PHA, ConA, PWM). The distribution of lymphocyte subtypes and mitogen-induced lymphocyte proliferation is depicted in Table 4. Discussion Allogeneic stem cell transplantation is the only curative approach in children with SAA. Patients lacking an appropriate family donor may also benefit from immunosuppressive therapy. The results of mismatched family donor or matched unrelated bone marrow transplantations in patients failing such therapies, however, are not promising even though attempts have been made to overcome the risk of graft rejection as well as of GVHD by increasing the dose of the conditioning regimens and by reducing the number of transplanted T lymphocytes, respectively.1,11 Recently an Table 4

encouraging approach to prevent graft rejection has been reported by the use of ‘megadose’ stem cell transplants, assuming that the use of megadoses of CD34+ cells might abrogate resistance to engraftment and overcome even major HLA barriers in the unrelated donor setting.12–16 High purification of CD34+ cells provides sufficient T cell depletion and therefore combines the advantage of high numbers of unrelated peripheral blood stem cells with a tolerable incidence of GVHD. Presently magnetic cell sorting of peripheral blood CD34+ cells has been shown to be the most effective selection procedure in recovering high numbers of stem cells with high purity and low T cell numbers and therefore was used in our patients.6–8 Donors were provided by the ‘Deutsche Knochenmarsspenderdatei’ (DKMS) (Tu¨bingen, Germany). Two cell separations after mobilization with G-CSF yielded sufficient numbers of progenitors. Magnetic positive selection of CD34+ cells resulted in excellent purity (median 99.2%, range 97.7–99.2%) with a very low contamination of residual T lymphocytes (⭐0.25 × 105/kg). All patients received more than 10 × 106 CD34+ cells/kg body weight. Conditioning chemotherapy was well tolerated except for allergic reactions after ALG administration in two children. Patients showed rapid hematological reconstitution with sufficient lymphopoietic recovery of B and T lymphocytes within 3 months after transplantation (Figure 1). Patient 2 previously transplanted with a 5/6 matched UCB, followed by a C-locus mismatched PSC graft developed acute GVHD and died at day +238 due to GVHD (grade III–IV) of the liver, lower gastrointestinal tract and skin despite intensive immunosuppression with CsA, MP, MMF and FK506. The other two children are alive with a median follow-up of 15 months although patient 1 developed pancytopenia with persistence of 100% donor hematopoiesis 1 month after an unclassified viral infection at day +591. This aplastic episode, however, could be reversed successfully by a further course of ATG and cyclophosphamide followed by the retransfusion of highly purified positively selected CD34+ cells from the original donor. To our knowledge, this experience represents the first

Immunological reconstitution Lymphocytes (×107/l)

Patient

Stimulation (c.p.m./lymphocyte)

CD3

CD4

CD8

CD19

CD56/CD16

Ratio CD4/8

Con A

PHA

PWM

55 134 496 694 1120

28 38 117 256 500

28 153 306 361 648

14 484 292 301 420

1171 859 496 407 356

1.0 0.24 0.38 0.70 0.77

3.5 3.4 14.6 31.8 40.0

0.9 4.6 17.5 52.2 56.2

0.09 0.5 2.2 4.4 8.1

2 1 Month 3 Months

33 30

19 24

14 3

6 65

237 177

1.35 8.0

3 1 Month 3 Months 4 Months

147 1025 841

47 209 172

110 759 652

80 361 480

933 455 309

0.42 0.27 0.26

1 1 Month 3 Months 6 Months 12 Months 18 Months

Bone Marrow Transplantation

0.98 0.4 4.74

ND 0.64 3.5 ND 14.9

0.29 0.23 0.98


report of transplantation of unrelated megadoses of highly purified CD34+ cells in children with refractory SAA. Rapid engraftment was observed in all children with immunologic reconstitution demonstrated by quantitative and qualitative functional tests of the emerging lymphocytes. We conclude that this procedure could be a promising alternative for children with SAA who lack a suitable related donor and have failed previous immunosuppressive therapies. Note added in proof: Meanwhile a fourth patient, a 7-year-old girl with refractory SAA was also given an unrelated peripheral stem cell transplant containing 7.85 × 106 highly purified CD34+ cells/kg (HLA-C mismatched at one locus) with 0.11 × 105 CD3+ cells/kg following conditioning with thiotepa, cyclophosphamide, TLI and OKT-3. She also has uncomplicated engraftment up to now (day +60 post transplant).

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