The cure for thalassemia involves correcting the genetic defect in a hematopoietic stem cell that results in reduced or absent β-globin synthesis and an excess of α-globin dimers. Intracellular precipitation and accumulation of α-dimers results in ineffective erythropoiesis and hemolytic anemia. Replacing the abnormal thalassemic marrow with allogeneic normal or heterozygous stem cells carrying the functional gene restores appropriate β-globin chain synthesis. Eighty to ninety per cent of patients transplanted from an HLA-identical sibling or parent become ex-thalassemic after transplant.1,2 Haploidentical hemopoietic stem cell transplantation has been explored as an option for treating patients with leukemia who lack an HLA-identical sibling or parent donor. However, severe graft versus host disease and high graft failure/rejection rates have limited the application of this transplant modality for patients with thalassemia. Advances using high doses of T cell-depleted peripheral blood stem cells (PBSCs) and intensive pre-transplant conditioning regimens have helped to overcome these limitations. Grafts containing mega doses of enriched CD34+ progenitor cells can be achieved by combining bone marrow with G-CSF-mobilized PBSCs. Thereafter, T-cells can be removed by positive selection for CD34. Limiting the numbers of CD3+ cells in the graft might allow retention of rapid engraftment kinetics provided by the mobilized PBSCs while reducing the risk of extensive GVHD. In this pilot study, we used a similar approach involving mega dose haploidentical positively selected CD34+ marrow and peripheral hematopoietic stem cell transplantation to treat patients with thalassemia who lack an HLA-identical familial or unrelated marrow donor. Positive selection of CD34+ stem cells results in an approximately 3–4 log reduction of CD3+ cells, which reduces the risk of GVHD but increases the risk of graft failure.3–5 Adding a defined dose of CD3+ marrow cells to the cellular suspension at the time of transplant can help to reduce the graft rejection rate. In contrast to positive selection of stem cells, marrow graft depleted of CD3+ and CD19+ cells contains significant amounts of monocytes, NK cells, dendritic cells, precursor T-cells, and other cell types that may play important roles in engraftment while accelerating the post-transplant immune reconstitution. Therefore, in a second prospective phase of this pilot study, we evaluated the use of haploidentical CD3+/CD19+-depleted marrow graft combined with CD34-selected mobilized PBSCs and CD3+ marrow cells that were added back at the time of infusion.6,7
Here, we report the outcomes of 31 children with thalassemia who were transplanted from haploidentical donors, 27 was mothers, two brothers and two fathers.8,9
Graft processing and transplant procedures
All donors received recombinant human G-CSF 15 µg/kg/day in two daily subcutaneous boluses to mobilize PBSCs. CD34+ cells from leukapheresis and bone marrow harvests were select using the CliniMACS one-step procedure (Milteny Biotech, Germany) for 14 donors. Two-step selection (CD34 positive selection leukapheresis followed by negative selection using anti-CD3 and anti-CD19 monoclonal antibodies) of bone marrow cells was employed for eight donors. We attempted to suppress erythropoiesis by intensive hypertransfusion and chelation. Between day -59 and day -11 before the transplantation, 40 mg/kg deferoxamine was continuously infused through a central venous catheter each 24 hours. Red cells were transfused every 3 days to maintain the hemoglobin level between 140 and 150 g/L (14 and 15 g/dL). During this time interval hydroxyurea 60 mg/kg daily and azathioprine 3 mg/kg daily were administered to eradicate marrow, and growth factors, granulocyte colony-stimulating factor and erythropoietin, were given twice weekly to maintain stem cell proliferation in the face of hypertransfusion, thereby facilitating the effect of the hydroxyurea. Fludarabine was administered at a dosage of 30 mg/m2/d from day -17 through day -13. Starting on day -10, 14 doses of busulfan (BU) 1 mg/kg were administered orally 3 times daily over 4 days (total dose 14 mg/kg over 4 days) in the first 17 patients, and corresponding dose of busulfan give intravenous in the following 14 patients, followed by intravenous cyclophosphamide (CY) 50 mg/kg daily on each of the next 4 days (total dose 200 mg/kg), and 10 mg/kg thiotepa, and 12.5 mg/kg anti-thymocyte globulin daily from days -5 to -2, (ATG-Fresenius S).
All patients received cyclosporine for GVHD prophylaxis for the first two months post transplantation.
Eight patients received T cell-depleted peripheral blood progenitor cells (CD34+ immunoselection) and CD3+– and CD19+-depleted bone marrow stem cells. Median infused cell doses per kilogram of recipient body weight were CD34+: 15.2×106 (range, 8.2–26×106); CD3± T cells: 1.8×105; and 0.27×106/kg CD19. Twenty-three patients received CD34+ mobilized peripheral and bone marrow progenitor cells. Positive selection was performed using the CliniMACS procedure. The CD34+ grafts contained a median of 14.2×106/kg CD34+ cells (range, 5.4–39×106/kg), 2×105/kg CD3+ cells, and 0.19×106/kg CD19+. No side effects were associated with graft infusion.