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Partial T Cell-Depleted Peripheral Blood Stem Cell Transplantation from HLA-Identical Sibling Donors for Patients with Severe Aplastic Anemia
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Partial T Cell-Depleted Peripheral Blood Stem Cell Transplantation from HLA-Identical Sibling Donors for Patients with Severe Aplastic Anemia 1, Juan Montoro1, Isabel Cano1, Manuel Guerreiro1, Jaime Sanz1,3,4,*, Federico Moscardo 2 1,3 s Go mez-Segui1,3, Pau Montesinos1,3, María A. Dasí , Pilar Solves , Ignacio Lorenzo1,3, Ine 1 1 1,3 Elvira Mora , Mario Arnao , Amparo Sempere , Isidro Jarque1,3, Carlos Carretero1, Leonor Senent1,3, 1, Nelly Carpio1, Ana Vicente1, Rafael Andreu1, Irene Luna1, Aitana Balaguer-Rosello 1,3,4 1,3,4 1 L. Pin ~ ana , Miguel A. Sanz , Jose Guillermo F. Sanz Hematology Department, Hospital Universitari i Politecnic La Fe, Valencia, Spain Hematology Unit, Department of Pediatrics, Hospital Universitari i Politecnic La Fe, Valencia, Spain ncer, Instituto Carlos III, Madrid, Spain Centro de Investigacion Biomedica en Red de Ca 4 Faculty of Medicine, University of Valencia, Valencia, Spain 1 2 3
Article history: Received 7 June 2019 Accepted 28 August 2019 Keywords: Severe aplastic anemia Allogeneic stem cell transplantation Ex vivo T cell depletion Matched sibling donor
A B S T R A C T We analyzed the outcomes of 26 consecutive patients with acquired severe aplastic anemia (SAA) undergoing peripheral blood stem cell transplantation (PBSCT) with partial ex vivo T cell depletion with a targeted T cell dose from HLA-identical sibling donors. The median patient age was 37 years (range, 3 to 63 years). Four patients with uncontrolled pneumonia at the time of transplantation died, on days +1, +2, +21, and +26. All evaluable patients engrafted, with a median time to neutrophil recovery of 11 days (range, 10 to 14 days) and a median time to platelet recovery of 19 days (range, 8 to 53 days). Two patients had transient grade I acute graft-versus-host disease (GVHD) with skin involvement, but no patients developed grade II-IV acute GVHD. Two patients had mild skin chronic GVHD, and 1 patient had moderate chronic GVHD with ocular involvement. No relapse was observed after a median follow-up of 114 months (range, 4 to 233 months). The overall cumulative incidence of TRM at 10 years was 19%, whereas it was 5% for those with a Karnofsky Performance Status (KPS) score >60 at the time of transplantation. Disease-free survival, overall survival, and GVHD and relapse-free survival at 10 years were 81%, 81%, and 80%, respectively, for all patients and 95%, 95%, and 90%, respectively, for patients with a KPS score >60 at transplantation. Our data indicate that PBSCT with partial ex vivo T cell-depleted targeted cell dose grafts from an HLA-identical sibling donor is a feasible, safe, and effective approach to reduce GVHD and cure patients with SAA. © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
INTRODUCTION Allogeneic hematopoietic cell transplantation (alloHCT) from HLA-identical siblings is the treatment of choice for young adults with severe aplastic anemia (SAA), as well as for patients age >40 years who do not respond to first-line immunosuppressive therapy [1-3]. There is a general consensus that bone marrow (BM) is the preferred stem cell source over peripheral blood (PB) for alloHCT from HLA-identical sibling donors in patients with SAA [4]. The most consistent evidence leading to this consensus comes from 3 large retrospective studies conducted by the Center for International Blood and Marrow Transplant Research (CIBMTR) and European Society for Blood and Marrow Transplantation (EBMT) [5-7] that
Financial disclosure: See Acknowledgments on page 4. * Correspondence and reprint requests: Jaime Sanz, Hospital Universitari i cnic La Fe, Av. Fernando Abril Martorell, 106, 46026 Valencia, Spain. Polite E-mail address: [email protected] (J. Sanz).
compared outcomes of patients with SAA who underwent alloHCT from HLA-identical sibling donors using either BM or PB as the hematopoietic stem cell source. Overall, these studies showed a lack of benefit of PB for alloHCT to reduce graft failure, along with worse outcomes in terms of an increased incidence of graft-versus-host disease (GVHD) and poorer survival. It should be noted, however, that these studies were based on patients undergoing alloHCT with nonmanipulated grafts. Thus, the question that arises is whether the use of T cell-depleted PB grafts to decrease GVHD would improve outcomes in patients with SAA compared not only with unmanipulated PB stem cell transplantation (PBSCT), but also with alloHCT using BM grafts. The use of such efficient graft manipulation to reduce GVHD would seem to be particularly appealing in patients needing alloHCT for nonmalignant hematologic disorders, such as SAA, in whom the closely linked graft-versus-tumor effect is not required. Additional potential advantages with the
https://doi.org/10.1016/j.bbmt.2019.08.020 1083-8791/© 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
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use of PB are faster engraftment and lower risk of fatal infection in the early post-transplantation period. The number of reports on the use of ex vivo T cell depletion in patients with SAA undergoing PBSCT from sibling donors is very limited, however [8-11]. In a previous pilot study [8], we reported the feasibility, safety, and GVHD prevention efficacy of the use of partial ex vivo T cell-depleted grafts with targeted PBSC doses in alloHCT from HLA-identical sibling donors in patients with SAA. In the present study, we aimed to confirm the safety and efficacy of such strategy in a larger cohort of patients with a longer follow-up. METHODS Patients Patients were included in the study who met the following criteria: (1) diagnosis of SAA, defined as BM cellularity <25% and severe PB cytopenia in at least 2 lineages (neutrophil count .5 £ 109/L, platelet count 20 £ 109/L, or reticulocyte count 20 £ 109/L; (2) age <40 years, or >40 years for patients who had failed at least 1 line of immunosuppressive treatment with antithymocyte globulin (ATG) with or without cyclosporine (CSA); (3) an available HLA-identical sibling donor; and (4) written informed consent from the donor and patient. The clinical protocol was Institutional Review Board-approved. Conditioning Regimen All patients received a combination of cyclophosphamide (200 mg/kg) rieux, Lyon, France, 60 mg/kg total dose in and ATG (Lymphoglobuline; Me the first 7 patients or Thymoglobulin; Genzyme Transplant, Cambridge, MA at a total dose of 6 mg/kg). Total lymphoid irradiation (5 Gy) was added to the preparative regimen in 2 pediatric patients age 3 and 6 years. Rituximab (200 mg on day -1 before transplantation) was added in the last 2 patients). GVHD Management GVHD prophylaxis consisted of CSA and prednisone. Prednisone was empirically chosen to avoid myelotoxicity of methotrexate in a context of T cell- depleted grafts. CSA 1.5 mg/kg i.v. every 12 hours was started on day -1 and was followed by 3 to 5 mg/kg orally every 12 hours when oral intake was possible until day +180, with slow tapering until months +9 to +12. Prednisone was given at a dose of .5 mg/kg/day on days +7 to +14 and 1 mg/kg/ day on days +14 to +28, and then slowly tapered until day +180. Chronic GVHD was treated with prednisone 1 mg/kg/day. Supportive Care Patients were maintained in HEPA-filtered rooms. Granulocyte colonystimulating factor was administered subcutaneously at 5 mg/kg/day from day +7 until neutrophil engraftment. Antibacterial and antifungal prophylaxis consisted of oral ciprofloxacin during neutropenia or until the initiation of broad-spectrum antibiotics and a mold-active azole (itraconazole or voriconazole) until day +100 or while the patient was on steroids (prednisone dose equivalent to 15 mg/day) to treat GVHD. Prophylaxis against Pneumocystis jiroveci included cotrimoxazole from day 7 to day 2, which was then resumed after engraftment with a dosing scheme of 2 days a week and maintained for a minimum of 1 year or until immunosuppressive therapy was stopped. All blood products were irradiated and leukocyte-depleted. Cytomegalovirus (CMV)-seropositive patients received high-dose acyclovir 500 mg/m2 i.v. every 8 hours until neutrophil recovery. Thereafter, patients received prophylaxis with either i.v. ganciclovir (5 mg/kg 3 days per week until 2004) or oral valganciclovir (900 mg once daily from 2004 to 2017) until day +100, as reported previously [12]. From 2017 to date, patients did not receive preemptive strategies without prophylaxis. Nonspecific immunoglobulin was administered i.v. at a dose of 500 mg/kg weekly until day +100 and then monthly during the first year after transplantation. Since 2005, stringent weekly surveillance for the presence of Epstein-Barr virus (EBV) by quantitative RT-PCR and a preemptive strategy with i.v. rituximab for patients with EBV reactivation was maintained, as described previously [13]. Donor Mobilization and Graft Manipulation For mobilization, all donors received granulocyte colony-stimulating factor for at least 5 days at a dose of 10 mg/kg every 12 hours. PBSC collection was started at day 5. CD34+ cells were positively selected using a biotin avidin immunoadsorption column (Ceprate SC System; CellPro, Bothell, WA) in the first 3 donors and the CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) in the subsequent 23 donors. To obtain a pre-fixed targeted T cell dose of .3 £ 106 CD3+ cells/kg in the final hematopoietic cell graft product, add-backs were performed to the CD34+ cell harvest. T cells were not administered after transplantation in any instance. CD34+ and CD3+ cells were quantified as described previously [14]. The fresh CD34-selected donor stem cell product was infused to the patient without further manipulation.
Definitions Myeloid engraftment was defined as an absolute neutrophil count of .5 £ 109/L on 3 consecutive days. Platelet engraftment was defined as the first of 7 consecutive days with a platelet count >20 £ 109/L without receipt of platelet transfusion support. Patients who survived for >28 days after transplantation and who failed to achieve myeloid engraftment were considered graft failures. The time to myeloid or platelet engraftment was defined as the time required to reach the first day of engraftment. Secondary graft failure was defined as the loss of engraftment. Mixed donor chimerism was defined as the presence of <90% donor hematopoiesis. Acute and chronic GVHD were defined and graded according to standard criteria [15-17]. Performance status was graded according to the Karnofsky Performance Status (KPS) scale for patients age >16 years and according to the Lansky scale for those age 16 years. Statistical Analysis The probabilities of engraftment and transplantation-related mortality (TRM) were estimated using the cumulative incidence method (marginal probability). For cumulative incidence analyses of engraftment, death before engraftment was considered a competing cause of failure, and relapse was the competing event for TRM. Unadjusted time-to-event analyses were performed using the Kaplan-Meier method. Disease-free survival (DFS) was calculated from the date of transplantation. In the analysis of DFS, relapse or death in complete remission, whichever occurred first, was considered an uncensored event. For the analysis of the composite endpoint GVHD-free/ relapse-free survival (GRFS), grade III-IV acute GVHD, chronic GVHD necessitating systemic therapy, relapse, and death were considered uncensored events. Statistical analysis was conducted using R version 3.0.1 (R Project for Statistical Computing, Vienna, Austria) [18].
RESULTS Patient and Graft Characteristics Between July 1996 and October 2018, 26 consecutive patients with SAA underwent PBSCT at our institution (Hospital Universicnic La Fe) using partial ex vivo T cell-depleted grafts tari i polite with targeted cell doses from PBSCs from HLA-identical sibling donors. The main characteristics of patients are summarized in Table 1. The median patient age was 37 years (range, 3 to 63 years), and 15 (58%) were female. Seven patients (27%) were younger than 20 years, and 10 (38%) were older than 40 years. Nine patients (35%) had received previous immunosuppressive therapy with at least 1 course of ATG plus CSA without response (1 course in 8 patients and 2 courses in 1 patient). The median infused CD34+ cell dose was 5.1 £ 106/kg (range, 1.7 to 13.0 £ 106/ kg). A fixed CD3+ cell dose of .3 £ 106/kg was infused. A female donor to male recipient match was present in 4 cases (15%). Eighteen donor-recipient pairs (69%) were CMV-seropositive. The median time from diagnosis to transplantation was 56 days (range, 15 to 1783 days). In a desperate salvage attempt, 4 patients underwent transplantation despite of a very poor clinical condition (KPS score 60) and active uncontrolled pneumonia. Engraftment Except for 2 patients who proceeded to transplantation with uncontrolled pneumonia and who died on days +1 and +2, all remaining patients experienced neutrophil and platelet engraftment at a median time of 11 days (range, 10 to 14 days) and 19 days (range, 8 to 53 days), respectively. There were no primary or secondary graft failures. Sequential samples for analyzing long-term chimerism in myeloid and T cell lineages were available in 19 patients (79%). The last chimerism study was performed at a median of 21 months after transplantation (range, 4 to 185 months). At this time point, the median donor T cell chimerism was 84% (range, 2% to 100%), with 14 of 19 patients (74%) showing mixed chimerism. The median donor myeloid chimerism was 94% (range, 48% to 100%), with 11 of 19 patients (58%) showing mixed chimerism with donor predominance. All patients had normal PBSC counts in all cell lineages irrespective of the degree of myeloid or T cell chimerism.
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Table 1 Patient and Transplantation Characteristics Characteristic
Value
Number of patients
26
Age, yr, median (range)
37 (3-63)
Age 20 yr, n (%) Age 20-40 yr, n (%)
Successful pregnancy was achieved by 1 female recipient and 2 female partners of male recipients. All pregnancies were conceived without any medical intervention, all births were via vaginal delivery, and all 3 newborns were healthy without any malformations.
7 (27) 9 (35)
Age 40 yr, n (%)
10 (38)
Female sex, n (%)
15 (58)
Weight, kg, median (range)
73 (15-100)
KPS or Lansky score at transplantation, n (%) 60
4 (15)
>60
22 (85)
Previous treatment, n (%) None
3
16 (62)
Immunosuppression (ATG + CSA)*
9 (35)
Oxymetholone
1 (4)
TRM and Causes of Death Four patients died from pulmonary infections. All 4 patients were age >30 years and at the initiation of or during conditioning therapy were in poor clinical condition and required mechanical ventilation. Three of these patients started respiratory support during conditioning and died of uncontrolled active infection, on days +1, +2, and +26, and the fourth patient developed severe respiratory insufficiency at day 0 and died on day +21. One other patient died due to chemorefractory EBV-PTLD at 8 months after transplantation. The cumulative incidence of TRM at 10 years was 19% overall but only 5% for those with a KPS score >60 at the time of transplantation.
Donor/recipient sex match, n (%) Male/male
7 (27)
Male/female
10 (38)
Female/male
4 (15)
Female/female
5 (19)
Donor/recipient pair CMV serologic status, n (%) Positive/positive
18 (69)
Negative/positive
5 (19)
Negative/negative
3 (12)
Infused CD34+ cell dose (£ 106/kg)
5.1 (1.7-13.0)
Days from diagnosis to transplantation
56 (15-1783)
Percentages may not sum to 100 because of rounding. * One patient received 2 courses of immunosuppression.
GVHD Two patients developed skin grade I acute GVHD that rapidly responded to topical steroids, and no patient had grade II-IV acute GVHD. Two patients had mild skin chronic GVHD. One patient developed moderate chronic GVHD with ocular involvement (ie, dry eye syndrome) that was effectively treated with the insertion of a tear duct plug. No patient had severe chronic GVHD. All patients were off local or systemic immunosuppressive therapy at last follow-up.
Post-Transplantation Events Nine patients had CMV reactivation and were treated with preemptive valganciclovir 900 mg every 12 hours. All 9 patients achieved CMV seronegativity without end organ damage. Three patients had neutrophil and platelet counts <.5 and 50 £ 109/L during CMV reactivation, respectively. In all cases, blood counts recovered after withdrawal of antiviral therapy. One patient developed probable invasive pulmonary aspergillosis during the conditioning regimen that was treated with antifungal therapy, followed by successful transplantation. Five patients had detectable EBV reactivation at a median time of 36 days (range, 25 to 90 days). Three of these patients subsequently cleared EBV DNAemia without medical intervention; the other 2 developed biopsy-proven EBV-associated post-transplantation lymphoproliferative disorder (PTLD), at days +71 and +91. One of these patients achieved complete response with immunosuppression tapering and rituximab treatment (4 weekly doses as induction, followed by 4 monthly doses as consolidation) and remained disease-free 59 months later. The other patient was refractory to R-CHOP chemoimmunotherapy and died of progressive PTLD at day +262.
Outcomes The median duration of follow-up of the 21 surviving patients was 114 months (range, 4 to 233 months). No patient experienced relapse, and all patients maintained normal blood counts. DFS, OS, and GRFS at 2, 5, and 10 years were 81%, 81%, and 80%, respectively, overall (Figure 1) but 95%, 95%, and 90%, respectively, for patients with a KPS score >60 at transplantation (Figure 2). DISCUSSION This study shows that allogeneic PBSCT from HLA-identical siblings using partial T cell depletion with a targeted T cell dose of .3 £ 106 CD3+ cells/kg is an easy, safe, and effective strategy for curing patients with SAA while avoiding GVHD. Rapid and sustained engraftment was achieved in all evaluable patients. The procedure was associated with low TRM in patients with a good KPS score at the time of transplantation, an extremely low incidence of acute and chronic GVHD, and a high cure rate. Despite the relatively small sample size, this study provides evidence that this partial T cell depletion technique could be an attractive strategy not only for patients with SAA, but also for patients with other nonmalignant hematologic or immunologic disorders in whom a graft-versus-tumor effect is not required. Although alloHCT with unmanipulated BM from HLA-identical sibling donors remains the standard of care for patients with SAA, due mainly to the lower risk of developing acute and chronic GVHD compared with allogeneic PBSCT, this latter
Figure 1. Probability of DFS overall.
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Figure 2. Probability of DFS in patients with a KPS score >60 at the time of transplantation.
procedure is still widely used, not only in developing countries, but also in high- income countries [19]. Furthermore, although BM is by far the most frequently used graft source for patients with SAA, severe GVHD still occurs in a relatively high proportion of patients in this setting [4-6,18]. Several large studies have reported an incidence of grade II-IV acute GVHD ranging from 10% to 13% in patients transplanted with BM [4-6,18], increasing to 20% in those age >20 years [5]. The reported incidence of chronic GVHD in these studies is up to 11% to 16% at 3 years and 31% at 5 years, respectively. The incidence of acute and chronic GVHD observed in our study, using ex vivo T cell depletion of PBSC grafts, was clearly lower than that reported not only with nonmanipulated PBSCs, but also with BM, in the previously mentioned EBMT and CIBMTR studies [4-6,18]. In fact, none of our patients developed grade II-IV acute GVHD or severe chronic GVHD. Apart from our previous pilot study [8], only 2 other studies have reported outcomes in patients with SAA after transplantation from HLA-identical siblings using more intensive ex vivo T cell-immunodepleted PBSC grafts [10,11]. These latter studies used alemtuzumab (anti-CD52 monoclonal antibody) “in the bag” as a T cell depletion method. One of these studies included 30 patients [10]; the median time to engraftment was 12 days, and 2 patients developed graft failure that was successfully overcome with retransplantation and donor lymphocyte infusion. Unfortunately, the infused CD3+ cell dose was not reported in that study, but it presumably was much lower than in the present study. No acute or chronic GVHD was observed, and all patients were alive and transfusion-free after a median follow up of 65 months. The other study included only 12 patients with SAA [11], who underwent alloHCT using partially T cell-depleted PBSC grafts with alemtuzumab “in part of the bag,” whereas the rest of the bag, adjusted to contain 100 £ 106 CD3+ cells/kg, was given as a T cell add-back the following day. All patients engrafted (at a median of 15 days) and achieved durable complete remission at a median follow-up of 91 months, and only 1 of them developed moderate chronic GVHD. As in our study, the arbitrarily predefined infused CD3+ cell dose was able to maintain an excellent rate of engraftment and prevent GVHD without significantly increasing the risk of viral or fungal infections. The 2 patients with EBV-PTLD in our series merit separate consideration. An increased incidence of this complication has been previously recognized in patients with SAA undergoing alloHCT with unmanipulated grafts, attributed to the use of previous immunosuppressive therapy with ATG [20]. In our patients, we believe that, apart from the use of previous
immunosuppressive therapy with ATG, the partial ex vivo T cell depletion and the conditioning regimen that included ATG also might have played a role. In fact, a recent study by our group analyzing 21 patients with malignant diseases who developed PTLD after alloHCT reported a significantly higher incidence of this complication in patients who received a conditioning regimen with ATG compared with those who did not (5% versus 0%; P < .0001) [21]. Based on our previous experience, we are currently using prophylaxis with rituximab, 200 mg i.v. on day -1 before transplantation, to prevent EBV DNAemia and PTLD in patients undergoing alloHCT [22,23]. Regarding mortality and morbidity, we emphasize the low morbidity and mortality related to our procedure. Apart from the 4 patients who died early (on days +1, +2, +21, and +26) because of uncontrolled pulmonary infection and severe neutropenia when they started the conditioning regimen, who underwent transplantation in a desperate attempt to attain rapid improvement in neutrophil count, only 1 death can be attributed to transplantation-related complications. Consequently, longterm results can be considered very satisfactory and compare favorably with other series of patients with SAA undergoing alloHSCT with unmanipulated BM grafts [4-6,18], particularly in patients in good clinical condition at transplantation. In conclusion, our study shows that PBSCT with partially T cell-depleted grafts with a targeted T cell dose of .3 £ 106 CD3+ cells/kg from HLA-identical sibling donors is associated with rapid and durable engraftment, low morbidity and mortality, and encouraging long-term DFS, OS and GRFS. The additional advantages of using mobilized PBSCs instead of BM as the graft source for both donors and transplant teams are also evident. We believe that this strategy is a very useful alternative to BM for alloHCT in patients with SAA and merits further investigation in future randomized clinical trials in this setting. ACKNOWLEDGMENTS Financial disclosure: This work was partially financed with FEDER funds (CIBERONC; CB16/12/00284). Conflict of interest statement: There are no conflicts of interest to report. Authorship statement: M.A.S., G.F.S., F.M., J.L.P., and J.S. conceived the study, interpreted the data, and drafted the manuscript. J.S. performed the statistical analyses. J.M., I.C., M.G., M. A.D., P.S., I. Lorenzo, I.G.-S., P.M., E.M., M.A., A.S., I.J., C.C., L.S., A. V., R.A., I. Luna, A.B-R., and N.C. participated in data collection, reviewed the manuscript, contributed to the final draft, and gave final approval for manuscript submission. REFERENCES 1. Gupta V, Eapen M, Brazauskas R, et al. Impact of age on outcomes after bone marrow transplantation for acquired aplastic anemia using HLAmatched sibling donors. Haematologica. 2010;95:2119–2125. 2. Scheinberg P. Aplastic anemia: therapeutic updates in immunosuppression and transplantation. Hematology Am Soc Hematol Educ Program. 2012;2012:292–300. 3. Bacigalupo A. Bone marrow transplantation for acquired severe aplastic anemia. Hematol Oncol Clin North Am. 2014;28:1145–1155. 4. Bhella S, Majhail NS, Betcher J, et al. Choosing wisely BMT: American Society for Blood and Marrow Transplantation and Canadian Blood and Marrow Transplant Group’s list of 5 tests and treatments to question in blood and marrow transplantation. Biol Blood Marrow Transplant. 2018;24:909–913. 5. Schrezenmeier H, Passweg JR, Marsh JC, et al. Worse outcome and more chronic GVHD with peripheral blood progenitor cells than bone marrow in HLA-matched sibling donor transplants for young patients with severe acquired aplastic anemia. Blood. 2007;110:1397–1400. 6. Chu R, Brazauskas R, Kan F, et al. Comparison of outcomes after transplantation of G-CSF-stimulated bone marrow grafts versus bone marrow or peripheral blood grafts from HLA-matched sibling donors for patients with severe aplastic anemia. Biol Blood Marrow Transplant. 2011;17:1018–1024. G, Schrezenmeier H, et al. Bone marrow versus periph7. Bacigalupo A, Socie eral blood as the stem cell source for sibling transplants in acquired
ARTICLE IN PRESS J. Sanz et al. / Biol Blood Marrow Transplant 00 (2019) 1 5
8.
9.
10.
11.
12.
13.
14.
aplastic anemia: survival advantage for bone marrow in all age groups. Haematologica. 2012;97:1142–1148. de la Rubia J, Cantero Peral S, Sanz GF, et al. Transplantation of CD34+selected peripheral blood to HLA-identical sibling patients with aplastic anaemia: results from a single institution. Bone Marrow Transplant. 2005;36:325–329. Novitzky N, Thomas V, du Toit C, McDonald A. Reduced-intensity conditioning for severe aplasia using fludarabine and CY followed by infusion of ex vivo T-cell-depleted grafts leads to excellent engraftment and absence of GVHD. Bone Marrow Transplant. 2009;43:779–785. Novitzky N, Thomas V, du Toit C. Conditioning with purine analogs leads to good engraftment rates of immunodepleted grafts for aplastic anemia. Biol Blood Marrow Transplant. 2013;19:584–588. Masouridi-Levrat S, Simonetta F, Beauverd Y, et al. Excellent outcome with a high proportion of mixed chimerism in patients with severe aplastic anemia treated with partially T-cell-depleted peripheral hematopoietic stem cell transplants. Bone Marrow Transplant. 2016;51:860–862. Montesinos P, Sanz J, Cantero S, et al. Incidence, risk factors, and outcome of cytomegalovirus infection and disease in patients receiving prophylaxis with oral valganciclovir or intravenous ganciclovir after umbilical cord blood transplantation. Biol Blood Marrow Transplant. 2009;15:730–740. Sanz J, Arango M, Senent L, et al. EBV-associated post-transplant lymphoproliferative disorder after umbilical cord blood transplantation in adults with hematological diseases. Bone Marrow Transplant. 2014;49:397–402. F, Sanz J, Carbonell F, et al. Effect of CD8⁺ cell content on umbiliMoscardo cal cord blood transplantation in adults with hematological malignancies. Biol Blood Marrow Transplant. 2014;20:1744–1750.
5
15. Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation. 1974;18:295–304. 16. Shulman HM, Sullivan KM, Weiden PL, et al. Chronic graft-versus-host syndrome in man. A long-term clinicopathologic study of 20 Seattle patients. Am J Med. 1980;69:204–217. 17. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant. 1995;15:825–828. 18. R Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2014. Available at: http://www.R-project.org/. Accessed 1 may 2019. 19. Kumar R, Kimura F, Ahn KW, et al. Comparing outcomes with bone marrow or peripheral blood stem cells as graft source for matched sibling transplants in severe aplastic anemia across different economic regions. Biol Blood Marrow Transplant. 2016;22:932–940. 20. Buyck HC, Ball S, Junagade P, Marsh J, Chakrabarti S. Prior immunosuppressive therapy with antithymocyte globulin increases the risk of EBVrelated lymphoproliferative disorder following allo-SCT for acquired aplastic anaemia. Bone Marrow Transplant. 2009;43:813–816. 21. Romero S, Montoro J, Guinot M, et al. Post-transplant lymphoproliferative disorders after solid organ and hematopoietic stem cell transplantation. Leuk Lymphoma. 2019;60:142–150. 22. Corti P, Peters C, Balduzzi A, et al. Reconstitution of lymphocyte subpopulations in children with inherited metabolic storage diseases after haematopoietic cell transplantation. Br J Haematol. 2005;130:249–255. 23. Dominietto A, Tedone E, Soracco M, et al. In vivo B-cell depletion with rituximab for alternative donor hemopoietic SCT. Bone Marrow Transplant. 2012;47:101–106.
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Partial T Cell-Depleted Peripheral Blood Stem Cell Transplantation from HLA-Identical Sibling Donors for Patients with Severe Aplastic Anemia 1, Juan Montoro1, Isabel Cano1, Manuel Guerreiro1, Jaime Sanz1,3,4,*, Federico Moscardo 2 1,3 s Go mez-Segui1,3, Pau Montesinos1,3, María A. Dasí , Pilar Solves , Ignacio Lorenzo1,3, Ine 1 1 1,3 Elvira Mora , Mario Arnao , Amparo Sempere , Isidro Jarque1,3, Carlos Carretero1, Leonor Senent1,3, 1, Nelly Carpio1, Ana Vicente1, Rafael Andreu1, Irene Luna1, Aitana Balaguer-Rosello 1,3,4 1,3,4 1 L. Pin ~ ana , Miguel A. Sanz , Jose Guillermo F. Sanz Hematology Department, Hospital Universitari i Politecnic La Fe, Valencia, Spain Hematology Unit, Department of Pediatrics, Hospital Universitari i Politecnic La Fe, Valencia, Spain ncer, Instituto Carlos III, Madrid, Spain Centro de Investigacion Biomedica en Red de Ca 4 Faculty of Medicine, University of Valencia, Valencia, Spain 1 2 3
Article history: Received 7 June 2019 Accepted 28 August 2019 Keywords: Severe aplastic anemia Allogeneic stem cell transplantation Ex vivo T cell depletion Matched sibling donor
A B S T R A C T We analyzed the outcomes of 26 consecutive patients with acquired severe aplastic anemia (SAA) undergoing peripheral blood stem cell transplantation (PBSCT) with partial ex vivo T cell depletion with a targeted T cell dose from HLA-identical sibling donors. The median patient age was 37 years (range, 3 to 63 years). Four patients with uncontrolled pneumonia at the time of transplantation died, on days +1, +2, +21, and +26. All evaluable patients engrafted, with a median time to neutrophil recovery of 11 days (range, 10 to 14 days) and a median time to platelet recovery of 19 days (range, 8 to 53 days). Two patients had transient grade I acute graft-versus-host disease (GVHD) with skin involvement, but no patients developed grade II-IV acute GVHD. Two patients had mild skin chronic GVHD, and 1 patient had moderate chronic GVHD with ocular involvement. No relapse was observed after a median follow-up of 114 months (range, 4 to 233 months). The overall cumulative incidence of TRM at 10 years was 19%, whereas it was 5% for those with a Karnofsky Performance Status (KPS) score >60 at the time of transplantation. Disease-free survival, overall survival, and GVHD and relapse-free survival at 10 years were 81%, 81%, and 80%, respectively, for all patients and 95%, 95%, and 90%, respectively, for patients with a KPS score >60 at transplantation. Our data indicate that PBSCT with partial ex vivo T cell-depleted targeted cell dose grafts from an HLA-identical sibling donor is a feasible, safe, and effective approach to reduce GVHD and cure patients with SAA. © 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
INTRODUCTION Allogeneic hematopoietic cell transplantation (alloHCT) from HLA-identical siblings is the treatment of choice for young adults with severe aplastic anemia (SAA), as well as for patients age >40 years who do not respond to first-line immunosuppressive therapy [1-3]. There is a general consensus that bone marrow (BM) is the preferred stem cell source over peripheral blood (PB) for alloHCT from HLA-identical sibling donors in patients with SAA [4]. The most consistent evidence leading to this consensus comes from 3 large retrospective studies conducted by the Center for International Blood and Marrow Transplant Research (CIBMTR) and European Society for Blood and Marrow Transplantation (EBMT) [5-7] that
Financial disclosure: See Acknowledgments on page 4. * Correspondence and reprint requests: Jaime Sanz, Hospital Universitari i cnic La Fe, Av. Fernando Abril Martorell, 106, 46026 Valencia, Spain. Polite E-mail address: [email protected] (J. Sanz).
compared outcomes of patients with SAA who underwent alloHCT from HLA-identical sibling donors using either BM or PB as the hematopoietic stem cell source. Overall, these studies showed a lack of benefit of PB for alloHCT to reduce graft failure, along with worse outcomes in terms of an increased incidence of graft-versus-host disease (GVHD) and poorer survival. It should be noted, however, that these studies were based on patients undergoing alloHCT with nonmanipulated grafts. Thus, the question that arises is whether the use of T cell-depleted PB grafts to decrease GVHD would improve outcomes in patients with SAA compared not only with unmanipulated PB stem cell transplantation (PBSCT), but also with alloHCT using BM grafts. The use of such efficient graft manipulation to reduce GVHD would seem to be particularly appealing in patients needing alloHCT for nonmalignant hematologic disorders, such as SAA, in whom the closely linked graft-versus-tumor effect is not required. Additional potential advantages with the
https://doi.org/10.1016/j.bbmt.2019.08.020 1083-8791/© 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
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use of PB are faster engraftment and lower risk of fatal infection in the early post-transplantation period. The number of reports on the use of ex vivo T cell depletion in patients with SAA undergoing PBSCT from sibling donors is very limited, however [8-11]. In a previous pilot study [8], we reported the feasibility, safety, and GVHD prevention efficacy of the use of partial ex vivo T cell-depleted grafts with targeted PBSC doses in alloHCT from HLA-identical sibling donors in patients with SAA. In the present study, we aimed to confirm the safety and efficacy of such strategy in a larger cohort of patients with a longer follow-up. METHODS Patients Patients were included in the study who met the following criteria: (1) diagnosis of SAA, defined as BM cellularity <25% and severe PB cytopenia in at least 2 lineages (neutrophil count .5 £ 109/L, platelet count 20 £ 109/L, or reticulocyte count 20 £ 109/L; (2) age <40 years, or >40 years for patients who had failed at least 1 line of immunosuppressive treatment with antithymocyte globulin (ATG) with or without cyclosporine (CSA); (3) an available HLA-identical sibling donor; and (4) written informed consent from the donor and patient. The clinical protocol was Institutional Review Board-approved. Conditioning Regimen All patients received a combination of cyclophosphamide (200 mg/kg) rieux, Lyon, France, 60 mg/kg total dose in and ATG (Lymphoglobuline; Me the first 7 patients or Thymoglobulin; Genzyme Transplant, Cambridge, MA at a total dose of 6 mg/kg). Total lymphoid irradiation (5 Gy) was added to the preparative regimen in 2 pediatric patients age 3 and 6 years. Rituximab (200 mg on day -1 before transplantation) was added in the last 2 patients). GVHD Management GVHD prophylaxis consisted of CSA and prednisone. Prednisone was empirically chosen to avoid myelotoxicity of methotrexate in a context of T cell- depleted grafts. CSA 1.5 mg/kg i.v. every 12 hours was started on day -1 and was followed by 3 to 5 mg/kg orally every 12 hours when oral intake was possible until day +180, with slow tapering until months +9 to +12. Prednisone was given at a dose of .5 mg/kg/day on days +7 to +14 and 1 mg/kg/ day on days +14 to +28, and then slowly tapered until day +180. Chronic GVHD was treated with prednisone 1 mg/kg/day. Supportive Care Patients were maintained in HEPA-filtered rooms. Granulocyte colonystimulating factor was administered subcutaneously at 5 mg/kg/day from day +7 until neutrophil engraftment. Antibacterial and antifungal prophylaxis consisted of oral ciprofloxacin during neutropenia or until the initiation of broad-spectrum antibiotics and a mold-active azole (itraconazole or voriconazole) until day +100 or while the patient was on steroids (prednisone dose equivalent to 15 mg/day) to treat GVHD. Prophylaxis against Pneumocystis jiroveci included cotrimoxazole from day 7 to day 2, which was then resumed after engraftment with a dosing scheme of 2 days a week and maintained for a minimum of 1 year or until immunosuppressive therapy was stopped. All blood products were irradiated and leukocyte-depleted. Cytomegalovirus (CMV)-seropositive patients received high-dose acyclovir 500 mg/m2 i.v. every 8 hours until neutrophil recovery. Thereafter, patients received prophylaxis with either i.v. ganciclovir (5 mg/kg 3 days per week until 2004) or oral valganciclovir (900 mg once daily from 2004 to 2017) until day +100, as reported previously [12]. From 2017 to date, patients did not receive preemptive strategies without prophylaxis. Nonspecific immunoglobulin was administered i.v. at a dose of 500 mg/kg weekly until day +100 and then monthly during the first year after transplantation. Since 2005, stringent weekly surveillance for the presence of Epstein-Barr virus (EBV) by quantitative RT-PCR and a preemptive strategy with i.v. rituximab for patients with EBV reactivation was maintained, as described previously [13]. Donor Mobilization and Graft Manipulation For mobilization, all donors received granulocyte colony-stimulating factor for at least 5 days at a dose of 10 mg/kg every 12 hours. PBSC collection was started at day 5. CD34+ cells were positively selected using a biotin avidin immunoadsorption column (Ceprate SC System; CellPro, Bothell, WA) in the first 3 donors and the CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) in the subsequent 23 donors. To obtain a pre-fixed targeted T cell dose of .3 £ 106 CD3+ cells/kg in the final hematopoietic cell graft product, add-backs were performed to the CD34+ cell harvest. T cells were not administered after transplantation in any instance. CD34+ and CD3+ cells were quantified as described previously [14]. The fresh CD34-selected donor stem cell product was infused to the patient without further manipulation.
Definitions Myeloid engraftment was defined as an absolute neutrophil count of .5 £ 109/L on 3 consecutive days. Platelet engraftment was defined as the first of 7 consecutive days with a platelet count >20 £ 109/L without receipt of platelet transfusion support. Patients who survived for >28 days after transplantation and who failed to achieve myeloid engraftment were considered graft failures. The time to myeloid or platelet engraftment was defined as the time required to reach the first day of engraftment. Secondary graft failure was defined as the loss of engraftment. Mixed donor chimerism was defined as the presence of <90% donor hematopoiesis. Acute and chronic GVHD were defined and graded according to standard criteria [15-17]. Performance status was graded according to the Karnofsky Performance Status (KPS) scale for patients age >16 years and according to the Lansky scale for those age 16 years. Statistical Analysis The probabilities of engraftment and transplantation-related mortality (TRM) were estimated using the cumulative incidence method (marginal probability). For cumulative incidence analyses of engraftment, death before engraftment was considered a competing cause of failure, and relapse was the competing event for TRM. Unadjusted time-to-event analyses were performed using the Kaplan-Meier method. Disease-free survival (DFS) was calculated from the date of transplantation. In the analysis of DFS, relapse or death in complete remission, whichever occurred first, was considered an uncensored event. For the analysis of the composite endpoint GVHD-free/ relapse-free survival (GRFS), grade III-IV acute GVHD, chronic GVHD necessitating systemic therapy, relapse, and death were considered uncensored events. Statistical analysis was conducted using R version 3.0.1 (R Project for Statistical Computing, Vienna, Austria) [18].
RESULTS Patient and Graft Characteristics Between July 1996 and October 2018, 26 consecutive patients with SAA underwent PBSCT at our institution (Hospital Universicnic La Fe) using partial ex vivo T cell-depleted grafts tari i polite with targeted cell doses from PBSCs from HLA-identical sibling donors. The main characteristics of patients are summarized in Table 1. The median patient age was 37 years (range, 3 to 63 years), and 15 (58%) were female. Seven patients (27%) were younger than 20 years, and 10 (38%) were older than 40 years. Nine patients (35%) had received previous immunosuppressive therapy with at least 1 course of ATG plus CSA without response (1 course in 8 patients and 2 courses in 1 patient). The median infused CD34+ cell dose was 5.1 £ 106/kg (range, 1.7 to 13.0 £ 106/ kg). A fixed CD3+ cell dose of .3 £ 106/kg was infused. A female donor to male recipient match was present in 4 cases (15%). Eighteen donor-recipient pairs (69%) were CMV-seropositive. The median time from diagnosis to transplantation was 56 days (range, 15 to 1783 days). In a desperate salvage attempt, 4 patients underwent transplantation despite of a very poor clinical condition (KPS score 60) and active uncontrolled pneumonia. Engraftment Except for 2 patients who proceeded to transplantation with uncontrolled pneumonia and who died on days +1 and +2, all remaining patients experienced neutrophil and platelet engraftment at a median time of 11 days (range, 10 to 14 days) and 19 days (range, 8 to 53 days), respectively. There were no primary or secondary graft failures. Sequential samples for analyzing long-term chimerism in myeloid and T cell lineages were available in 19 patients (79%). The last chimerism study was performed at a median of 21 months after transplantation (range, 4 to 185 months). At this time point, the median donor T cell chimerism was 84% (range, 2% to 100%), with 14 of 19 patients (74%) showing mixed chimerism. The median donor myeloid chimerism was 94% (range, 48% to 100%), with 11 of 19 patients (58%) showing mixed chimerism with donor predominance. All patients had normal PBSC counts in all cell lineages irrespective of the degree of myeloid or T cell chimerism.
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Table 1 Patient and Transplantation Characteristics Characteristic
Value
Number of patients
26
Age, yr, median (range)
37 (3-63)
Age 20 yr, n (%) Age 20-40 yr, n (%)
Successful pregnancy was achieved by 1 female recipient and 2 female partners of male recipients. All pregnancies were conceived without any medical intervention, all births were via vaginal delivery, and all 3 newborns were healthy without any malformations.
7 (27) 9 (35)
Age 40 yr, n (%)
10 (38)
Female sex, n (%)
15 (58)
Weight, kg, median (range)
73 (15-100)
KPS or Lansky score at transplantation, n (%) 60
4 (15)
>60
22 (85)
Previous treatment, n (%) None
3
16 (62)
Immunosuppression (ATG + CSA)*
9 (35)
Oxymetholone
1 (4)
TRM and Causes of Death Four patients died from pulmonary infections. All 4 patients were age >30 years and at the initiation of or during conditioning therapy were in poor clinical condition and required mechanical ventilation. Three of these patients started respiratory support during conditioning and died of uncontrolled active infection, on days +1, +2, and +26, and the fourth patient developed severe respiratory insufficiency at day 0 and died on day +21. One other patient died due to chemorefractory EBV-PTLD at 8 months after transplantation. The cumulative incidence of TRM at 10 years was 19% overall but only 5% for those with a KPS score >60 at the time of transplantation.
Donor/recipient sex match, n (%) Male/male
7 (27)
Male/female
10 (38)
Female/male
4 (15)
Female/female
5 (19)
Donor/recipient pair CMV serologic status, n (%) Positive/positive
18 (69)
Negative/positive
5 (19)
Negative/negative
3 (12)
Infused CD34+ cell dose (£ 106/kg)
5.1 (1.7-13.0)
Days from diagnosis to transplantation
56 (15-1783)
Percentages may not sum to 100 because of rounding. * One patient received 2 courses of immunosuppression.
GVHD Two patients developed skin grade I acute GVHD that rapidly responded to topical steroids, and no patient had grade II-IV acute GVHD. Two patients had mild skin chronic GVHD. One patient developed moderate chronic GVHD with ocular involvement (ie, dry eye syndrome) that was effectively treated with the insertion of a tear duct plug. No patient had severe chronic GVHD. All patients were off local or systemic immunosuppressive therapy at last follow-up.
Post-Transplantation Events Nine patients had CMV reactivation and were treated with preemptive valganciclovir 900 mg every 12 hours. All 9 patients achieved CMV seronegativity without end organ damage. Three patients had neutrophil and platelet counts <.5 and 50 £ 109/L during CMV reactivation, respectively. In all cases, blood counts recovered after withdrawal of antiviral therapy. One patient developed probable invasive pulmonary aspergillosis during the conditioning regimen that was treated with antifungal therapy, followed by successful transplantation. Five patients had detectable EBV reactivation at a median time of 36 days (range, 25 to 90 days). Three of these patients subsequently cleared EBV DNAemia without medical intervention; the other 2 developed biopsy-proven EBV-associated post-transplantation lymphoproliferative disorder (PTLD), at days +71 and +91. One of these patients achieved complete response with immunosuppression tapering and rituximab treatment (4 weekly doses as induction, followed by 4 monthly doses as consolidation) and remained disease-free 59 months later. The other patient was refractory to R-CHOP chemoimmunotherapy and died of progressive PTLD at day +262.
Outcomes The median duration of follow-up of the 21 surviving patients was 114 months (range, 4 to 233 months). No patient experienced relapse, and all patients maintained normal blood counts. DFS, OS, and GRFS at 2, 5, and 10 years were 81%, 81%, and 80%, respectively, overall (Figure 1) but 95%, 95%, and 90%, respectively, for patients with a KPS score >60 at transplantation (Figure 2). DISCUSSION This study shows that allogeneic PBSCT from HLA-identical siblings using partial T cell depletion with a targeted T cell dose of .3 £ 106 CD3+ cells/kg is an easy, safe, and effective strategy for curing patients with SAA while avoiding GVHD. Rapid and sustained engraftment was achieved in all evaluable patients. The procedure was associated with low TRM in patients with a good KPS score at the time of transplantation, an extremely low incidence of acute and chronic GVHD, and a high cure rate. Despite the relatively small sample size, this study provides evidence that this partial T cell depletion technique could be an attractive strategy not only for patients with SAA, but also for patients with other nonmalignant hematologic or immunologic disorders in whom a graft-versus-tumor effect is not required. Although alloHCT with unmanipulated BM from HLA-identical sibling donors remains the standard of care for patients with SAA, due mainly to the lower risk of developing acute and chronic GVHD compared with allogeneic PBSCT, this latter
Figure 1. Probability of DFS overall.
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Figure 2. Probability of DFS in patients with a KPS score >60 at the time of transplantation.
procedure is still widely used, not only in developing countries, but also in high- income countries [19]. Furthermore, although BM is by far the most frequently used graft source for patients with SAA, severe GVHD still occurs in a relatively high proportion of patients in this setting [4-6,18]. Several large studies have reported an incidence of grade II-IV acute GVHD ranging from 10% to 13% in patients transplanted with BM [4-6,18], increasing to 20% in those age >20 years [5]. The reported incidence of chronic GVHD in these studies is up to 11% to 16% at 3 years and 31% at 5 years, respectively. The incidence of acute and chronic GVHD observed in our study, using ex vivo T cell depletion of PBSC grafts, was clearly lower than that reported not only with nonmanipulated PBSCs, but also with BM, in the previously mentioned EBMT and CIBMTR studies [4-6,18]. In fact, none of our patients developed grade II-IV acute GVHD or severe chronic GVHD. Apart from our previous pilot study [8], only 2 other studies have reported outcomes in patients with SAA after transplantation from HLA-identical siblings using more intensive ex vivo T cell-immunodepleted PBSC grafts [10,11]. These latter studies used alemtuzumab (anti-CD52 monoclonal antibody) “in the bag” as a T cell depletion method. One of these studies included 30 patients [10]; the median time to engraftment was 12 days, and 2 patients developed graft failure that was successfully overcome with retransplantation and donor lymphocyte infusion. Unfortunately, the infused CD3+ cell dose was not reported in that study, but it presumably was much lower than in the present study. No acute or chronic GVHD was observed, and all patients were alive and transfusion-free after a median follow up of 65 months. The other study included only 12 patients with SAA [11], who underwent alloHCT using partially T cell-depleted PBSC grafts with alemtuzumab “in part of the bag,” whereas the rest of the bag, adjusted to contain 100 £ 106 CD3+ cells/kg, was given as a T cell add-back the following day. All patients engrafted (at a median of 15 days) and achieved durable complete remission at a median follow-up of 91 months, and only 1 of them developed moderate chronic GVHD. As in our study, the arbitrarily predefined infused CD3+ cell dose was able to maintain an excellent rate of engraftment and prevent GVHD without significantly increasing the risk of viral or fungal infections. The 2 patients with EBV-PTLD in our series merit separate consideration. An increased incidence of this complication has been previously recognized in patients with SAA undergoing alloHCT with unmanipulated grafts, attributed to the use of previous immunosuppressive therapy with ATG [20]. In our patients, we believe that, apart from the use of previous
immunosuppressive therapy with ATG, the partial ex vivo T cell depletion and the conditioning regimen that included ATG also might have played a role. In fact, a recent study by our group analyzing 21 patients with malignant diseases who developed PTLD after alloHCT reported a significantly higher incidence of this complication in patients who received a conditioning regimen with ATG compared with those who did not (5% versus 0%; P < .0001) [21]. Based on our previous experience, we are currently using prophylaxis with rituximab, 200 mg i.v. on day -1 before transplantation, to prevent EBV DNAemia and PTLD in patients undergoing alloHCT [22,23]. Regarding mortality and morbidity, we emphasize the low morbidity and mortality related to our procedure. Apart from the 4 patients who died early (on days +1, +2, +21, and +26) because of uncontrolled pulmonary infection and severe neutropenia when they started the conditioning regimen, who underwent transplantation in a desperate attempt to attain rapid improvement in neutrophil count, only 1 death can be attributed to transplantation-related complications. Consequently, longterm results can be considered very satisfactory and compare favorably with other series of patients with SAA undergoing alloHSCT with unmanipulated BM grafts [4-6,18], particularly in patients in good clinical condition at transplantation. In conclusion, our study shows that PBSCT with partially T cell-depleted grafts with a targeted T cell dose of .3 £ 106 CD3+ cells/kg from HLA-identical sibling donors is associated with rapid and durable engraftment, low morbidity and mortality, and encouraging long-term DFS, OS and GRFS. The additional advantages of using mobilized PBSCs instead of BM as the graft source for both donors and transplant teams are also evident. We believe that this strategy is a very useful alternative to BM for alloHCT in patients with SAA and merits further investigation in future randomized clinical trials in this setting. ACKNOWLEDGMENTS Financial disclosure: This work was partially financed with FEDER funds (CIBERONC; CB16/12/00284). Conflict of interest statement: There are no conflicts of interest to report. Authorship statement: M.A.S., G.F.S., F.M., J.L.P., and J.S. conceived the study, interpreted the data, and drafted the manuscript. J.S. performed the statistical analyses. J.M., I.C., M.G., M. A.D., P.S., I. Lorenzo, I.G.-S., P.M., E.M., M.A., A.S., I.J., C.C., L.S., A. V., R.A., I. Luna, A.B-R., and N.C. participated in data collection, reviewed the manuscript, contributed to the final draft, and gave final approval for manuscript submission. REFERENCES 1. Gupta V, Eapen M, Brazauskas R, et al. Impact of age on outcomes after bone marrow transplantation for acquired aplastic anemia using HLAmatched sibling donors. Haematologica. 2010;95:2119–2125. 2. Scheinberg P. Aplastic anemia: therapeutic updates in immunosuppression and transplantation. Hematology Am Soc Hematol Educ Program. 2012;2012:292–300. 3. Bacigalupo A. Bone marrow transplantation for acquired severe aplastic anemia. Hematol Oncol Clin North Am. 2014;28:1145–1155. 4. Bhella S, Majhail NS, Betcher J, et al. Choosing wisely BMT: American Society for Blood and Marrow Transplantation and Canadian Blood and Marrow Transplant Group’s list of 5 tests and treatments to question in blood and marrow transplantation. Biol Blood Marrow Transplant. 2018;24:909–913. 5. Schrezenmeier H, Passweg JR, Marsh JC, et al. Worse outcome and more chronic GVHD with peripheral blood progenitor cells than bone marrow in HLA-matched sibling donor transplants for young patients with severe acquired aplastic anemia. Blood. 2007;110:1397–1400. 6. Chu R, Brazauskas R, Kan F, et al. Comparison of outcomes after transplantation of G-CSF-stimulated bone marrow grafts versus bone marrow or peripheral blood grafts from HLA-matched sibling donors for patients with severe aplastic anemia. Biol Blood Marrow Transplant. 2011;17:1018–1024. G, Schrezenmeier H, et al. Bone marrow versus periph7. Bacigalupo A, Socie eral blood as the stem cell source for sibling transplants in acquired
ARTICLE IN PRESS J. Sanz et al. / Biol Blood Marrow Transplant 00 (2019) 1 5
8.
9.
10.
11.
12.
13.
14.
aplastic anemia: survival advantage for bone marrow in all age groups. Haematologica. 2012;97:1142–1148. de la Rubia J, Cantero Peral S, Sanz GF, et al. Transplantation of CD34+selected peripheral blood to HLA-identical sibling patients with aplastic anaemia: results from a single institution. Bone Marrow Transplant. 2005;36:325–329. Novitzky N, Thomas V, du Toit C, McDonald A. Reduced-intensity conditioning for severe aplasia using fludarabine and CY followed by infusion of ex vivo T-cell-depleted grafts leads to excellent engraftment and absence of GVHD. Bone Marrow Transplant. 2009;43:779–785. Novitzky N, Thomas V, du Toit C. Conditioning with purine analogs leads to good engraftment rates of immunodepleted grafts for aplastic anemia. Biol Blood Marrow Transplant. 2013;19:584–588. Masouridi-Levrat S, Simonetta F, Beauverd Y, et al. Excellent outcome with a high proportion of mixed chimerism in patients with severe aplastic anemia treated with partially T-cell-depleted peripheral hematopoietic stem cell transplants. Bone Marrow Transplant. 2016;51:860–862. Montesinos P, Sanz J, Cantero S, et al. Incidence, risk factors, and outcome of cytomegalovirus infection and disease in patients receiving prophylaxis with oral valganciclovir or intravenous ganciclovir after umbilical cord blood transplantation. Biol Blood Marrow Transplant. 2009;15:730–740. Sanz J, Arango M, Senent L, et al. EBV-associated post-transplant lymphoproliferative disorder after umbilical cord blood transplantation in adults with hematological diseases. Bone Marrow Transplant. 2014;49:397–402. F, Sanz J, Carbonell F, et al. Effect of CD8⁺ cell content on umbiliMoscardo cal cord blood transplantation in adults with hematological malignancies. Biol Blood Marrow Transplant. 2014;20:1744–1750.
5
15. Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation. 1974;18:295–304. 16. Shulman HM, Sullivan KM, Weiden PL, et al. Chronic graft-versus-host syndrome in man. A long-term clinicopathologic study of 20 Seattle patients. Am J Med. 1980;69:204–217. 17. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant. 1995;15:825–828. 18. R Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2014. Available at: http://www.R-project.org/. Accessed 1 may 2019. 19. Kumar R, Kimura F, Ahn KW, et al. Comparing outcomes with bone marrow or peripheral blood stem cells as graft source for matched sibling transplants in severe aplastic anemia across different economic regions. Biol Blood Marrow Transplant. 2016;22:932–940. 20. Buyck HC, Ball S, Junagade P, Marsh J, Chakrabarti S. Prior immunosuppressive therapy with antithymocyte globulin increases the risk of EBVrelated lymphoproliferative disorder following allo-SCT for acquired aplastic anaemia. Bone Marrow Transplant. 2009;43:813–816. 21. Romero S, Montoro J, Guinot M, et al. Post-transplant lymphoproliferative disorders after solid organ and hematopoietic stem cell transplantation. Leuk Lymphoma. 2019;60:142–150. 22. Corti P, Peters C, Balduzzi A, et al. Reconstitution of lymphocyte subpopulations in children with inherited metabolic storage diseases after haematopoietic cell transplantation. Br J Haematol. 2005;130:249–255. 23. Dominietto A, Tedone E, Soracco M, et al. In vivo B-cell depletion with rituximab for alternative donor hemopoietic SCT. Bone Marrow Transplant. 2012;47:101–106.