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Transplantation of CD34-enriched peripheral stem cells from an HLA-haplotype mismatched donor to a patient with severe aplastic anemia

Abstract

A 14-year-old girl developed very severe aplastic anemia unresponsive to steroids, cyclosporine, ATG and filgrastim. She experienced repeated bacterial infections, hypermenorrhagia and epistaxis and received numerous transfusions. Lacking a matched family or unrelated donor, she was transplanted 6 months after diagnosis with CD34+cell-enriched peripheral stem cells from her HLA-haploidentical uncle. Conditioning included fludarabine, cyclophosphamide, 800 cGy TLI and OKT3. Prompt and sustained trilineage engraftment occurred. Acute GVHD grade 1 and herpes esophagitis were successfully treated. Eight months after grafting she was well with stable hematopoiesis. She then succumbed to fulminant hepatic failure due to adenovirus infection. Bone Marrow Transplantation (2001) 27, 111–113.

Main

Transplantation of allogeneic hematopoietic stem cells is a curative treatment option for patients with severe aplastic anemia (SAA). It represents the first choice of treatment for most patients who have an HLA-identical family donor and yields a failure-free survival rate of 66% after 5 years (1699 patients, EBMT data).1 For pediatric patients, survival rates of 66% to 84% are reported.23 If the alternative treatment, ie immunosuppression, fails, a bone marrow transplant (BMT) with cells from an alternative donor is considered. Graft failure and graft-versus-host disease (GVHD) which have been frequent problems of BMT for SAA must be expected even more often when using alternative donors. In 168 patients transplanted between 1981 and 1994, graft failure occurred in 20%, acute GVHD grades II–IV in 75% and chronic GVHD in 50%; 35% of patients survived disease-free.4 To enforce engraftment, attempts were made to intensify the immunosuppressive conditioning, and T cell depletion (TCD) was used to prevent GVHD. A retrospective analysis of 141 patients with SAA transplanted from unrelated donors after failure of immunosuppressive therapy revealed engraftment failure in 11%, acute GVHD grades II–IV in 52% but an outcome of still only 36% of patients surviving after a median of 3 years. Besides duration of disease before BMT, the HLA mismatch was the only significant predictor for death in a multivariate analysis.5 However, grafting of unmanipulated marrow from haploidentical family donors has been reported for six patients with SAA, two of whom had a three-locus HLA disparity. All six engrafted. One of the three-antigen mismatched patients died of GVHD.6 To our knowledge, we report here the first SAA patient to engraft after transplantation of peripheral stem cells from an HLA-haploidentical family donor which had been substantially T cell-reduced by CD34+ cell enrichment for prevention of GVHD.

Case report

A 14-year-old girl presented with pharyngitis, multiple bruises and hypermenorrhagia. Her previous history was uneventful. Her blood count revealed a hemoglobin of 7.5 g/dl, 0.7 leukocytes/nl with less than 0.1 PMN/nl and 41 platelets/nl. Bone marrow aspirate and biopsy showed trilineage hypoplasia of the hematopoiesis and no signs of myelodysplasia or myelofibrosis. Fanconi's anemia and PNH were ruled out by appropriate methods and a diagnosis of very severe aplastic anemia of unknown origin was made. Since an HLA-identical family donor was not available, she was treated with steroids, cyclosporine, ATG and filgrastim, 5 μg/kg first and 10 μg/kg after 4 weeks of no response. During the 6 months until hematopoietic stem cell transplantation, the patient suffered from repeated febrile episodes requiring antibiotic treatment which was supplemented with intravenous amphotericin B and acyclovir. The hypermenorrhagia never stopped completely despite the use of estrogen and of the LH-RH antagonist leuprorelin. Her maximum leukocyte count was 1.8/nl, with PMNs less than 0.1/nl at any time. She required 48 bags of RBCs and 71 concentrated platelet transfusions during this period.

Due to the rare HLA phenotype of the patient, a completely matched unrelated donor was not found despite an extensive search. The best unrelated donor identified had an HLA-A mismatch. In view of her relentlessly unresponsive disease, an HLA-haploidentical donor was considered. As both parents were not suitable, one because of psychosocial reasons, the other due to long-term treatment with methotrexate for psoriasis arthritis, a paternal uncle was chosen being HLA-identical to the father (Table 1).

Table 1  Donor–recipient characteristics

Conditioning included irradiation of all major lymphoid areas (whole abdomen ap-pa, thoracic lymphatic regions via mantle field exposure and pharyngeal lymphoid organs via opposed Waldeyer fields) with 10 MeV photons in four fractions up to a total dose of 8 Gy, fludarabin 180 mg/m2 for 6 days, cyclophosphamide 200 mg/kg for 4 days and anti-CD3 monoclonal antibody (OKT3) from day −4, with doses increasing up to 0.1 mg/kg/day. OKT3 was stopped on day 0 because of two episodes of seizures the cause of which could not be elucidated. Methylprednisolone, 2 mg/kg was administered from day −4 for 14 days and then tapered down. Filgrastim (5 μg/kg) was given from day 4 onwards until neutrophil recovery.

After informed consent, the donor underwent two filgrastim-stimulated peripheral stem cell collections. Both were T cell-reduced by magnetic positive selection of CD34+ cells using the CliniMACS sorting device7 (Miltenyi Biotec, Gladbach, Germany) and cryopreserved. On day 0 the patient, whose weight was 72 kg, received a transplant containing 11.9 × 106/kg CD34+ cells and 2.8 × 104/kg CD3+ cells. The marrow engrafted promptly: 0.5 PMN/nl were reached on day 8, 1.0 leukocytes/nl on day 9 and >20 platelets/nl for 3 consecutive days on day 17. Besides the seizures, side-effects included diabetes mellitus while on steroid treatment, fever during which Staphylococcus epidermidis was isolated from one of several blood cultures, mucositis grade 2, a herpes labialis infection and subsequently an esophagitis where herpes simplex virus resistant to acyclovir was cultured from a mucosal biopsy during gastroscopy. Antiviral treatment was changed from acyclovir to cidofovir and the clinical and endoscopic signs of the infection disappeared. Acute GVHD of the skin, stage II (no liver, no gut; thus grade 1), developed 3 weeks after transplant and was controlled with steroids and cyclosporine. The hypermenorragia had ceased since the platelets had recovered. The patient was well and free of any symptoms, with steroids being tapered, on cyclosporine, cidofovir treatment and prophylactic intravenous immunoglobulins 8 months after transplant, when diarrhea and subsequent fulminant hepatic failure developed from which she died. Adenovirus was grown from stools and from a liver specimen. Blood counts up to this time had been stable without transfusions. Immunologic studies had shown a lack of circulating B-lymphocytes, CD3 and CD4 cell counts of about 50% of lower normal limits and an increasing in vitro reactivity to mitogens and to recall antigens such as herpes simplex virus, varizella zoster virus, Candida albicans and other infectious agents.

Discussion

Hematopoietic stem cell transplantation in patients with SAA who lack an HLA-identical donor and who have failed immunosuppressive treatment must be carried out with alternative donors. Engraftment failure and GVHD with subsequent infections are the two major problems which result in survival rates of 20–50% (reviewed in Ref. 8). Removing T cells from grafts significantly decreases the incidence of severe GVHD, but raises the risk of graft failure.9 In haploidentical transplantation with HLA-A, B and DRB1 mismatches for patients with leukemia, GVHD could be successfully prevented by highly effective T cell depletion while engraftment was supported by giving a high dose of stem cells.10

In the case of our patient, BMT was mandatory since severe neutropenia and bleeding problems were not influenced at all by immunosuppressive treatment plus G-CSF. The patient's rare HLA phenotype and her familial situation left us with two alternatives: an unrelated donor with HLA-A mismatch vs an HLA-haploidentical relative. We considered GVHD to be the most imminent problem in this setting and T cell reduction by selection of CD34+ cells to be the most effective means to prevent it. Of course, the low T cell dose added another risk factor for non-engraftment to the pre-existing ones, that is, HLA mismatches, a high number of previous transfusions and the underlying SAA. To counterbalance this risk, an intensive immunosuppressive conditioning was chosen, adding fludarabine and irradiation of the complete lymphoid system to cyclophosphamide and OKT3. In addition, a large stem cell dose was aimed for. Since no data exist to guide a choice between a one antigen-mismatched unrelated donor and a three antigen-mismatched family donor with T cell reduction, we chose the family donor who was more easily available for two subsequent leukaphereses in order to achieve a high stem cell dose for transplantation. The highly selective method of magnetic enrichment of CD34+ cells allowed us to graft this adult size adolescent girl with 11.9 × 106 CD34+ cells/kg, but with only 2.8 × 104 CD3+ cells/kg. Engraftment was prompt, although the treatment with anti-CD3 antibody had to be discontinued on the day of the transplant. GVHD did not exceed grade I, but still required treatment which may have contributed to a slow immunologic recovery and to the fatal outcome in our patient. Schwinger et al11 have just reported four patients with SAA with T cell-depleted, CD34+ cell-enriched transplants from unrelated donors, two of them matched, two with a HLA-C mismatch. These patients received a mean of 14.3 × 106 CD34+ cells/kg; three patients survived, one child with a mismatched transplantation died of severe GVHD.

These results and the prompt and stable engraftment we observed in our patient lead us to suggest that patients with refractory SAA lacking an HLA-matched donor should be considered for an alternative donor transplant even if no matched unrelated donor can be identified. In HLA-mismatched settings, up to a complete haplotype mismatch, the strategy we propose is to prevent GVHD by highly quantitative TCD and to overcome the resulting risk of graft failure by very immunosuppressive conditioning and by large numbers of stem cells. The immunologic reconstitution after such transplants should be carefully studied in order to learn how to cope with viral and fungal infections during this period.

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Correspondence to B Kremens.

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Kremens, B., Basu, O., Grosse-Wilde, H. et al. Transplantation of CD34-enriched peripheral stem cells from an HLA-haplotype mismatched donor to a patient with severe aplastic anemia. Bone Marrow Transplant 27, 111–113 (2001). https://doi.org/10.1038/sj.bmt.1702748

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Keywords

  • bone marrow transplantation
  • haploidentical
  • T cell depletion
  • severe aplastic anemia

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