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CD34-enriched peripheral stem cells infusion for graft insufficiency treatment in a pediatric patient with severe aplastic anemia after allogeneic bone marrow transplantation

Allogeneic transplantation of hematopoietic stem cells is a treatment of choice for patients with severe acquired aplastic anemia, who have an HLA-identical family donor. This treatment is now able to cure 80–90% of young SAA patients.1 Meanwhile, early and late graft failure still remains a major problem, compromising the survival of these patients, and often leads to second allogeneic stem cell transplantation from the same or an alternative donor.1, 2 The intensification of the preparative regimen and the use of high doses of stem cells have led to further reduction of the graft failure rate, but the problem is still unpredictable. Moreover, deep immunosuppression associated with second-transplant procedure and graft-versus-host disease (GVHD) onset may further decrease the survival rate of this category of patients. We report about a pediatric SAA patient with transient engraftment after allogeneic bone marrow transplantation (BMT), compromised with acute GVHD, who received a CD34-selected stem cells boost from the same donor and achieved full donor chimerism and stable hematopoietic reconstitution.

A 14 year-old girl presented with very severe acquired aplastic anemia in September 2002. Fanconi Anemia and PNH were ruled out by appropriate diagnostic methods. In November 2002, the patient underwent allogeneic BMT from a HLA-matched sibling donor. Patient and donor characteristics are shown in Table 1. The conditioning regimen consisted of cyclophosphamide (50 mg/kg/day on days –5 to –2) and antithymocyte globulin (ATGAM, 30 mg/kg/day on day –3 to day −1). On day 0, the patient received unmanipulated bone marrow (NC – 3.2 × 108/kg, MNC – 1 × 108/kg). GVHD prophylaxis included cyclosporin A and a short course of methotrexate. Engraftment was delayed till day +27; the patient remained dependent on blood component transfusions till day +34. Acute GVHD of the skin (grade 2) and liver (grade 1) developed on day +45 post BMT and was successfully controlled with methylprednisolone and CsA. On day +50, the patient's blood count revealed hemoglobin 84 g/l, leukocytes 1.8 × 106/l with PMN less than 0.6 × 106/l, and 16 × 106 platelets/l. The patient started stimulation with G-CSF, but there was no evidence of hematological improvement after 4 weeks of treatment with the growth factor. Chimerism analysis based on sex chromosome polymorphism was carried out on day +80, and 95% of donor cells were discovered despite hypocellular bone marrow aspirate and high transfusion dependency. Blood count performed on day +80 revealed hemoglobin 89 g/l, leukocytes 1.5 × 106/l, and 19 × 106 platelets/l. There were no confirmations of CMV, HHV-6 or Parvovirus infection by qualitative PCR technique. On day +100, the patient underwent a boost of CD34-enriched stem cells from the same donor. After a 5-day mobilization with G-CSF, a single apheresis product was T-cell-reduced using the CliniMACS sorting device (Miltenyi Biotech, Gladbach, Germany). The transplant contained 9 × 106/kg CD 34+ cells, and 1 × 108/kg CD3+ cells were added to enhance engraftment. GVHD prophylaxis consisted of basiliximab and mofetil mycophenolate. Pancytopenia gradually improved over the next 3 weeks after a second infusion of stem cells, the patient became transfusion independent. Acute GVHD grade 2 (skin/grade 1/and gut/grade 1) reappeared on the day +151/+54 and was treated successfully with steroids. A chimerism study performed 3 months post second CD34-enriched PBSC infusion showed all bone marrow cells to be of donor origin. Now, 16 months after the first transplantation, the patient remains alive and well, with full hematological reconstitution and without evidence of GVHD.

Table 1 Patient and donor characteristics

Despite the increased survival after bone marrow transplantation for SAA, early and late graft failure occurs in a significant number of patients and has been related to transfusion-induced sensitization, low stem cell dose and persistence of host-derived alloreactive lymphocytes.3 The strategy for further treatment of SAA patient with mixed chimerism and graft insufficiency after bone marrow transplantation is still unclear. Management of this life-threatening condition may consist of long-lasting treatment with growth factors, infusions of hematopoietic stem cells without additional conditioning or performing of a second transplant from the same or alternative donor.4 Execution of a second transplant is usually associated with high morbidity and mortality linked to prolonged neutropenia and GVHD, which may not be sustainable especially for the patients with mixed chimerism or at risk for acute GVHD. The fact that donor leukocyte infusions (DLI) may restore hematopoietic function in the SAA patients with late graft failure and mixed chimerism means that DLI bears a potential for the eradication of host-derived alloreactive T cells.5 In the meantime, the advantage of DLI for the treatment of graft failure is limited especially by the high incidence of acute and chronic GVHD, and thus restricted only for those patients without symptoms of this often fatal complication of BMT. The possibility that DLI contains a sufficient number of stem cells capable of repopulating bone marrow also cannot be ruled out. The use of T-depleted or CD34-positively selected stem cells collected from peripheral blood as a graft source for the SAA patients significantly decreases the GVHD rate, but raises the risk of graft failure.6

The attempt of graft dysfunction treatment with growth factors in our patient was unsuccessful. The patient remained transfusion-dependent for more than 3 months after BMT, in spite of 95% of donor cells in the bone marrow. In these circumstances, we considered an immune mechanism of the graft dysfunction and decided to perform a second infusion of stem cells from the same donor, without additional cytotoxic conditioning. Based on the data that a larger stem cell dose is accompanied with higher rate of engraftment,7 we decided to use PBSC as a source of stem cells for the boosting. Taking into account the high risk of acute and chronic GVHD reactivation in our patient associated with PBSC infusion,8 we performed CD34-positive selection of the apheresis product and added 1 × 108 CD3 cells to the CD34-cell fraction (1-log reduction compare to native PBSC product) as a powerful suppressive mechanism of the host-mediated immunity. For further amelioration of the risk of GVHD, we enhanced immunosuppression with addition of mofetil mycophenolate and basiliximab. In spite of that, acute GVHD reappeared, but was mild and successfully treated with first-line therapy. Full reconstitution of hematopoietic function appeared shortly after the second infusion of CD34-enriched PBSC, with reversion to full donor-type chimerism.

The use of large numbers of CD34-positively selected stem cells with the addition of CD3 cells may be an additional alternative for the patients after stem cell transplantation with mixed chimerism and graft insufficiency at risk for GVHD reactivation. Further studies are required to evaluate all the risks and benefits of CD34-enriched PBSC boosting (especially time interval before boosting, the cell doses and the risk of acute and chronic GVHD) for SAA patients with transient engraftment after BMT.

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Acknowledgements

We thank Dr Chiara De Luka for her assistance in preparation of the manuscript.

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Trakhtman, P., Shipicina, I., Balashov, D. et al. CD34-enriched peripheral stem cells infusion for graft insufficiency treatment in a pediatric patient with severe aplastic anemia after allogeneic bone marrow transplantation. Bone Marrow Transplant 34, 465–466 (2004). https://doi.org/10.1038/sj.bmt.1704599

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