Haploidentical haematopoietic stem cell transplantation using CD3 or CD3/CD19 depletion and conditioning with fludarabine, cyclophosphamide and antithymocyte globulin for acquired severe aplastic anaemia (2011)

The treatment of patients with severe aplastic anaemia (SAA) who lack a suitable donor remains a significant challenge. Although haematopoietic stem cell transplantation (HSCT) using a haploidentical related donors is a possible alternative for patients without a suitable donor, limited reports are available on the use of haploidentical HSCT for patients with acquired SAA (Tzeng et al, 1996; Kremens et al, 2001; Tsutsumi et al, 2004; Woodard et al, 2004; Lacerda et al, 2005; Wang et al, 2010). The majority of reports to date have involved a T-cell depleted strategy using purified CD34+ cells to prevent fatal graft-versus-host disease (GVHD). However, haploidentical HSCT using CD34+-selected stem cells was complicated by a high rate of graft rejection and opportunistic infection due to delayed immune reconstitution. In this prospective trial, CD3- or CD3/CD19-depleted grafts instead of purified CD34+ cells were employed to reduce the risk of severe GVHD and facilitate engraftment by selectively removing T cells and repleting non-CD34+ cells. For conditioning, a fludarabine-based regimen with dose-reduced cyclophosphamide and rabbit-antithymocyte globulin (rATG) without total body irradiation (TBI) was used to enhance engraftment and reduce regimen-related toxicity. Here, we report the successful preliminary results of haploidentical HSCT using CD3- or CD3/CD19-depleted grafts and a non-TBI, dose-reduced regimen for children and adolescents with acquired SAA. Between 2009 and 2010, four patients with acquired SAA (including two with very severe aplastic anaemia) who lacked a suitable sibling or an unrelated donor received HSCT from a haploidentical related donor at the Asan Medical Centre, Seoul, Korea. Clinical characteristics of the patients are shown in Table I. This study was registered at http://www.clinicaltrials.gov (NCT01105273). The conditioning regimen consisted of fludarabine (30 mg/m2 once daily on days −6 to −2), cyclophosphamide (60 mg/kg once daily on days −3 to −2) and rATG (Thymoglobulin; Genzyme, Cambridge, MA, USA) (2·5 mg/kg once daily on days −3 to −1). For prophylaxis of GVHD, a combination of mycophenolate mofetil and ciclosporin was used in cases where the CD3+ cell count in the graft exceeded 5 × 104/kg. Peripheral blood stem cells (PBSCs) were processed for CD3 or CD3/CD19 depletion by negative selection using the Milteny CliniMACS® system (Miltenyi-BioTec, Bergisch-Gladbach, Germany). The depletion procedure was described previously in detail (Bethge et al, 2008). If infused CD34+ cells were less than 3·0 × 106/kg of recipient body weight, a second PBSC collection was performed and the pooled product was processed for CD34 enrichment by positive selection of CD34+ cells. Total infused CD34+ cells ranged from 3·0–5·5 × 106/kg of recipient weight. T-cell depletion was variably effective, with 1·3–3·1 log reduction. In terms of CD3+ cells, two grafts contained 15·6 and 19·0 × 104/kg, while the remaining 3 grafts contained unexpectedly higher doses of 125, 155, and 795 × 104/kg, respectively (Table I). All patients achieved rapid neutrophil and platelet engraftment (Table I). Patients 1 and 2 achieved sustained engraftment with full donor chimerism and remained transfusion-independent. After initial engraftment, Patient 3 developed secondary graft failure at day 31. Following reconditioning, the patient received a second CD3/CD19-depleted graft from an alternative haploidentical donor at 78 days after the first transplant. The patient remained transfusion-independent for 9 months with complete donor chimerism after the second transplant. Patient 4 showed 90–95% donor chimerism, but had poor graft function requiring infrequent transfusion. The patient received a boost of CD34+-selected cells without further conditioning from the same donor as the first transplant at 11 months after transplantation. The patient was alive and transfusion-independent for 4 months. T-cell and Natural Killer (NK) cell reconstitution data are depicted in Fig 1. Apart from patient 4 who showed a prolonged delay in immune reconstitution, the other patients achieved CD3+ cell counts of >0·15 × 109/l on day 90, >0·4 × 109/l on day 180, and >0·7 × 109/l on day 365, and CD4+ cell counts of > 0·15 × 109/l on day 180 and >0·4 × 109/l on day 365 (Fig 1). Reconstitution of T and NK cells after haploidentical HSCT. (A) CD3+ cells (B) CD4+ cells, and (C) CD56+ cells at 1, 3, 6, 12 months after HSCT. Data from Patient 3 were acquired after the second HSCT. HSCT, haematopoietic stem cell transplantation. Three patients experienced cytomegalovirus (CMV) reactivation, which was managed with preemptive ganciclovir or foscarnet. Two displayed complications of CMV retinitis, which were resolved with intravenous and intraocular ganciclovir, leaving no visual impairment. Patient 1 developed Epstein-Barr virus-related post-transplant lymphoproliferative disorder, which was treated successfully with rituximab, cyclophosphamide, and corticosteroids (Table I). No patients developed clinically significant acute or chronic GVHD. The regimen was well tolerated without significant toxicity or transplant-related mortality. All four patients survived for a median period of 19 (range, 16–23) months. All patients were transfusion-independent with sustained complete donor chimerism for a median period of 14 (range, 4–21) months (Table I). While this preliminary study only involved four patients with refractory SAA, all subjects were long-term survivors without transfusion requirement. Our findings suggest that rapid engraftment without significant GVHD can be achieved even in heavily transfused patients using CD3- or CD3/CD19-depleted grafts and fludarabine-based, dose-reduced conditioning, and haploidentical HSCT using negative depletion of T cells can be a curative treatment option for SAA patients with no suitable donors. T-cell reconstitution in our study was comparable with previous reports of haploidentical HSCT using CD3 or CD3/CD19 depletion and reduced-intensity conditioning (Chen et al, 2006; Bethge et al, 2008; Federmann et al, 2011), and appeared more rapid than that of haploidentical HSCT using CD34 selection and myeloablative conditioning (Eyrich et al, 2001). Notably, Patient 4 experienced prolonged CMV reactivation and CMV retinitis due to delayed immune reconstitution. Accordingly, we will adopt early CMV prophylaxis with foscarnet or ganciclovir instead of preemptive strategy or transfer of antigen-specific donor T cells in the next trial. Despite successful initial engraftment, graft rejection remained a problem for one patient who had a long history of disease and had been heavily transfused. In our next trial, strategies to facilitate successful engraftment will include increasing the dose of CD34+ cells to more than 5·0 × 106/kg and introduction of low-dose TBI to enhance recipient immunosuppression for heavily transfused patients before HSCT. In addition, the decision to transplant in cases of highly transfusion-dependent patients without a suitable donor needs to be made without prolonged delay. KN Koh analysed the data and wrote the article. HJ Im conceived and designed the prospective study, including the conditioning regimen. BE Kim collected clinical data. ES Choi was responsible for collection of mononuclear cells and acquisition of the related data. SW Kwon took part in graft manipulation. S Jang and CJ Park performed flow cytometric analysis of the products. HJ Im and JJ Seo revised the article critically for improvement of its content. The authors have no relevant financial relationships to disclose or conflicts of interest to report.