Sustained Donor Cell Engraftment Research Articles

Sustained Engraftment in Fanconi Anemia (FA) Mice Using in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction FA is the most common inherited bone marrow failure syndrome caused by a mutation in one of 23 genes involved in DNA repair; the disease manifests as hematologic abnormalities by age seven with near uniform bone marrow failure in adult patients. The gold standard therapy is bone marrow transplantation (BMT), which portends best outcomes when performed at a young age from Human Leukocyte Antigen (HLA)-matched sibling donors. However, due to the underlying pathophysiology of the disease, FA patients are especially sensitive to the chemoradiation conditioning regimens used with BMT, leading to high short-term morbidity and an increased long-term cancer risk. Due to these challenges of conventional BMT in FA, IUHCT is an attractive non-myeloablative and non-immunosuppressive option capable of inducing donor-specific tolerance when in utero testing has identified pathogenic FA mutations. Here we analyzed whether non-conditioned IUHCT confers fetal engraftment as a potential therapy in the FA mouse disease model. Methods Fanconi anemia type C homozygous mice (Fancc-/-) underwent IUHCT on gestation day 14 (E14) via vitelline vein injections of 1x107 T-cell depleted bone marrow cells from green fluorescent protein (GFP)-expressing congenic donor mice. Littermate wild-type mice served as controls. Donor cell engraftment in peripheral blood and bone marrow was assessed by flow cytometry. To confirm long-term donor HSC engraftment, we performed primary and secondary transplantation of an equivalent number of BM mononuclear cells from either Fancc -/- or wild-type mice that had undergone IUHCT. Statistical analysis was performed via Welch's t-test. P values <0.05 were considered statistically significant; for graphical purposes ***p<0.001; **p<0.002; *p<0.033; nonsignificant, ns. Results Fancc-/- homozygous mice had significantly higher donor GFP+ cell engraftment than wild-type controls at one, three, and six months post-transplantation (Figure 1). This higher engraftment was maintained across cell types in peripheral blood including CD3+ T cells (0.7% vs 3.4%), B220+ B cells (1.2% vs 6.9%), GR1+ granulocytes (1.7% vs 10.6%), and CD11B+ myeloid cells (1.9% vs 11.5%). Analysis of the bone marrow of these mice at 6 months post-transplant similarly showed a trend toward increased donor cell engraftment of progenitor cell populations including LSK, HSPC, LTHSC, STHSC, MPP2, MPP3, MPP4, CLP, CMP, GMP, and MEP cells in Fancc -/- recipients compared to wild-type mice. At all timepoints after primary and secondary transplants, recipient mice engrafted with BM mononuclear cells from prenatally transplanted Fancc -/- mice had higher peripheral blood GFP+ chimerism than those engrafted with BM mononuclear cells from prenatally transplanted wild-type mice (Figure 2). Conclusion Fancc-/- homozygous mice have higher sustained donor cell engraftment compared with wild-type littermates, indicating successful engraftment and possible early selection for wild-type donor cells in this preclinical model. These initial studies support the potential of IUHCT as a therapy for FA and highlight the need for additional studies to identify approaches to enhance donor cell engraftment to more clinically relevant levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Role of interferon-γ in immune-mediated graft failure after allogeneic hematopoietic stem cell transplantation.

Pathophysiology of graft failure (GF) occurring after allogeneic hematopoietic stem cell transplantation (HSCT) still remains elusive. We measured serum levels of several different cytokines/chemokines in 15 children experiencing GF, comparing their values with those of 15 controls who had sustained donor cell engraftment. Already at day +3 after transplantation, patients developing GF had serum levels of interferon (IFN)-γ and CXCL9 (a chemokine specifically induced by IFNγ) significantly higher than those of controls (8859±7502 vs. 0 pg/mL, P=0.03, and 1514.0±773 vs. 233.6±50.1 pg/mlL, P=0.0006, respectively). The role played by IFNγ in HSCT-related GF was further supported by the observation that a rat anti-mouse IFNγ-neutralizing monoclonal antibody promotes donor cell engraftment in Ifngr1−/−mice receiving an allograft. In comparison to controls, analysis of bone marrow-infiltrating T lymphocytes in patients experiencing GF documented a predominance of effector memory CD8+ cells, which showed markers of activation (overexpression of CD95 and downregulation of CD127) and exhaustion (CD57, CD279, CD223 and CD366). Finally, we obtained successful donor engraftment in 2 out of 3 children with primary hemophagocytic lymphohistiocytosis who, after experiencing GF, were re-transplanted from the same HLA-haploidentical donor under the compassionate use coverage of emapalumab, an anti-IFNγ monoclonal antibody recently approved by the US Food and Drug Administration for treatment of patients with primary hemophagocytic lymphohistiocytosis. Altogether, these results suggest that the IFNγ pathway plays a major role in GF occurring after HSCT. Increased serum levels of IFNγ and CXCL9 represent potential biomarkers useful for early diagnosis of GF and provide the rationale for exploring the therapeutic/preventive role of targeted neutralization of IFNγ.

Administration of BPX-501 Following α-T and B-Cell Depleted Haplo-HSCT in Children with Transfusion-Dependent Thalassemia

▪BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-identical sibling has been shown to cure most children with transfusion-dependent b-thalassemia major. Results obtained using an HLA-partially matched (haploidentical, haplo) donor have been historically less satisfactory, the thalassemia-free-survival (TFS) probability being ~ 60%. αβ T and B-cell depleted haplo-HSCT has been proposed as a novel strategy able to minimize the risk of graft-versus-host disease (GvHD) and to protect patients from the risk of life-threatening viral/fungal infections. However, recovery of adaptive immunity after this type of allograft is still suboptimal.BPX-501 is an allogeneic product consisting of T cells modified to express the inducible caspase-9 (iC9) safety switch and truncated CD19 to allow monitoring and expansion of BPX-501 following transplant. The polyclonal nature of the BPX-501 provides broad virus and tumor-specific immunity, while the safety switch provides the unique ability to promptly and durably resolve graft-versus-host disease (GvHD) symptoms following the administration of rimiducid which induces dimerization and activation of iC9, inducing apoptosis of BPX-501.AimsTo evaluate the safety and efficacy (immune recovery and risk of acute and chronic GvHD) of BPX-501 administered after a αβ T and B-cell depleted haplo-HSCT in pediatric patients with b-thalassemia.MethodsThis is a multicenter US (NCT03301168) and EU (NCT020658), prospective trial utilizing αβ-T and B-cell-depleted haplo-HSCT followed by infusion of donor lymphocytes genetically modified with iC9 (BPX-501) in patients with malignant or non-malignant disorders. A subset of patients enrolled had b-thalassemia. BPX-501 was planned to be infused on day14±4 after the allograft. No post-transplant pharmacological GvHD prophylaxis was employed. Patients who develop GvHD resistant to conventional steroid therapy can receive ≥1 dose of dimerizing rimiducid activating iC9.The efficacy-evaluable population (EEP) was defined as any patient with b-thalassemia who received HSCT, BPX-501 infusion and had at least one follow-up assessment.ResultsAt the time of clinical cut-off (June 30, 2018) 24 patients (EU [n=22] US [n=2]; Male [n=14] Female [n= 10]; median age 9.16 yrs (2.15 - 14.34 yrs) met the EEP definition. The median follow-up was 11.3 mos (0.5-37.4 mos).The majority (83.3%) of patients received a busulfan-based conditioning regimen. The median HSCT CD34+ and ab TCR+ cell doses were 20.5 x 106 /kg and 0.15 x 105 /kg, respectively. The donor was a parent in all children (100%).The median time to BPX-501 infusion was 17 days (10- 36 days).Two children experienced primary graft failure (8.7% [95% CI: 0-20.3]). One patient underwent a second HSCT with successful engraftment and remains disease free with a median follow up of 31 months. In patients who obtained sustained engraftment of donor cells, the median time for neutrophil and platelet engraftment was 15 (15 - 18) and 12 (11 - 13) days, respectively. Of 22 evaluable patients 3 patients (Grade 1 [n=1] and Grade 2 [n= 2]) developed Grade 1-II aGvHD (13.6% [95% CI: 0 - 28). No patients developed Grade III-IV aGvHD or chronic GvHD. The dimerizing agent rimiducid was administered in one patient with Grade 2 Skin aGvHD who subsequently achieved a complete response.Two of the twenty-four patients died after transplantation from infections (9.3% [95% CI: 0 - 21.6%]). Disease-free survival (DFS) was 82.2% (95% CI: 66.3 - 98%). Overall Survival (OS) was 90.7% (95% CI: 78.4 - 100). The median time from last red blood cell transfusion was 11.1 mos (1 day - 37.2 mos).CD3+ and CD3+CD4+ T cells above 500 cells/ml were achieved by 180 and 270 days, respectively. IgA and IgM levels achieved normal values by 30 and 180 days, respectively. The percentage of circulating and median absolute BPX-501 at Day 100 were 2.53% ± 2.62% (0 - 7.6%) and 23.94 ± 28.58 cells/ml (0 - 76.5 cells/ml), respectively.ConclusionAn αβ-T and B-cell depleted haplo-HSCT followed by infusion of BPX-501 is a suitable and effective option for children with transfusion-dependent b-thalassemia major lacking an HLA-identical donor. The low cumulative incidence of TRM and the absence of severe aGvHD or chronic GvHD observed support further study in this patient population. DisclosuresLocatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Administration of BPX-501 Cells Following Αβ T and B-Cell-Depleted HLA-Haploidentical HSCT (haplo-HSCT) in Children with Malignant or Non-Malignant Disorders

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) is an established treatment for children with leukemia or life-threatening non-malignant disorders. Approximately 25% of patients have a HLA-matched sibling and ~50% have a suitable matched unrelated donor, leaving ~25% of patients who require an alternative donor. HLA-partially matched (haploidentical, haplo) donors represent a suitable alternative for these children; extensive T-cell depletion of the graft is largely employed to minimize the risk of graft-versus-host disease (GvHD).BPX-501 is an allogeneic product consisting of T cells modified to express the inducible caspase-9 (iC9) safety switch and truncated CD19 to allow monitoring and expansion of CD3+ CD19+ T-cells following transplant. The polyclonal nature of the BPX-501 T cells provides broad virus and tumor-specific immunity, while the safety switch provides the unique ability to promptly and durably resolve graft-versus-host disease (GvHD) symptoms following the administration of rimiducid.AimsTo evaluate the safety and efficacy of BPX-501 T cells administered after a T-cell receptor αβ and B-cell-depleted haplo-HSCT in pediatric patients with malignant or non-malignant disorders. The primary endpoint is event-free survival at 180 days (events include TRM (or NRM for malignant patients), severe GVHD (acute Grade 2-4 organ or extensive chronic GVHD) and life-threatening infections (Grade 4).MethodsThis multicenter EU (NCT02065869), prospective trial utilizes αβ-T and B-cell-depleted haplo-HSCT followed by infusion of donor lymphocytes genetically modified with iC9 (BPX-501). BPX-501 cells were planned to be infused on day14±4 after the allograft. No post-transplant pharmacological GvHD prophylaxis was employed. Patients who develop GvHD resistant to conventional steroid therapy could receive ≥1 dose of dimerizing rimiducid activating iC9.The efficacy evaluable population (EEP) was defined as any patient who received HSCT, BPX-501 infusion and at least one follow-up assessment.ResultsAt the time of clinical cut-off (June 30, 2018) 166 patients met the EEP definition. All patients were from EU sites. Key baseline and transplant characteristics are shown in Table 1.In patients who obtained sustained engraftment of donor cells, the median time for neutrophil and platelet engraftment was 16 (15-17) and 11 (11-12) days, respectively. No patients experienced graft failure. Thirty-one patients developed Grade I-IV aGvHD (18.7% [95% CI;12.8 - 24.7%]). Three patients developed Grade III-IV aGvHD (1.8% [95% CI; 5.2 - 14.1%]). Of 132 evaluable patients, 9 developed cGvHD (7.2% [95% CI; 2.6 - 11.7%]) with only 2 patients experiencing moderate - severe cGvHD (95% CI: 0.0 - 3.1). Rimiducid was administered to 11 patients. Ten patients had ≥ 1 response assessment following administration of rimiducid. The best overall response rate (CR/PR) was 100% with 9 patients (90%) achieving complete response.At the time of clinical cut off, EFS at 180 days was 92.7% (95% CI: 88.7 - 96.7%). An interim analysis with approximately 100 patients from a concurrent Matched Unrelated Donor (MUD) HSCT comparator trial and previously published data is planned for the time of the congress.At a median follow-up of 17.6 mos (1.5 - 43.7 mos) 5 patients experienced transplant related mortality (TRM) (3.3% [95% CI: 0.4 - 6.2%]). DFS was 89.4% (95% CI: 84.7 - 94.2%). Overall survival (OS) was 94.2% (95% CI: 90.5 - 97.9%).CD3+ and CD3+CD4+ T cells above 500 cells/ml were achieved by day 100. IgA and IgM levels achieved normal values by day 180. The percentage of circulating and median absolute BPX-501 T-cells at Day 100 were 9.06% ± 10.52% (0 - 54.9%) and 109.49 ± 213.99 cells/ml (0 - 1001 cells/ml), respectively.ConclusionPreliminary evaluation of the primary endpoint and additional time-dependent efficacy outcomes, shows that the adoptive transfer of BPX-501 T cells following αβ-T and B-cell depleted haplo-HSCT followed by infusion of BPX-501 represents a novel and highly effective transplantation strategy for pediatric patients with malignant or non-malignant disorders. Despite the addition of BPX-501, overall rates of GvHD were low with few cases of high-grade aGvHD or cGvHD. Rimiducid was shown to be an effective treatment for patients who developed steroid-refractory GvHD. [Display omitted] DisclosuresQasim:Bellicum: Research Funding; Autolus: Equity Ownership; Servier: Research Funding; Orchard: Equity Ownership. Slatter:Medac: Other: Travel assistance. Locatelli:bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Reduction of Graft Rejection By Fludarabine Combined Busulfan, Cyclophosphamide and Antithymocyte Globulin Conditioning in Cord Blood Transplantation for Children with Wiskott-Aldrich Syndrome

Background and Objectives: Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency with microthrombocytopenia. Hematopoietic stem cell transplantation is a curative treatment for WAS. Unrelated cord blood transplantation (UCBT) become a more successive alternative donor transplantation for WAS with the improvement of high resolution HLA-typing method. However, graft rejection is the primary cause for mortality of WAS after UCBT. We hypothesized that the addition of fludarabine to a myeloablative conditioning regimen with antithymocyte globulin (ATG) would support engraftment for these children.Methods: Seventeen Children with WAS underwent UBCT matched 6-10/10 at higher resolution (HLA-A, -B, -C, -DRB1and DQB1) in our center between 2013 and 2018. The conditioning regimen consisted of fludarabine 160 mg/m2 , busulfan (BU)12.8 mg/kg, cyclophosphamide (CY)120 mg/kg, ATG 7.5 mg/kg, and cyclosporine and mycophenolate for GVHD prophylaxis. Median follow-up time is 2.5 years (range, 0.2 to 3.5 years).Results: All children had sustained donor cell engraftment and are stable engraftment. Twelve (71%) patients had cytomegalovirus (CMV) antigenemia and no one developed into CMV disease. Six (35%) children underwent acute Graft-versus-host disease (GVHD) (grade II-IV) and four (24%) children developed into chronic GVHD (cGVHD). Among these cases, only one child with HLA matched 6/10 died of kidney failure because of cGVHD. He experienced aGVHD with skin bullosa. Sixteen (94%) children are survival.Conclusion: Conditioning regimen consisted of Fludarabine combined BUCY and ATG was able to reduce graft rejection in the WAS children after UCBT without increasing infection related mortality. Future efforts will focus on further reducing rates of acute GVHD and extensive cGVHD. DisclosuresNo relevant conflicts of interest to declare.

Post-Transplant Cyclophosphamide and Sirolimus in Matched Related and Unrelated Allogeneic Transplant with a Treosulfan-Based Conditioning

Abstract Introduction: Post transplant cyclophosphamide (PT-Cy) has recently emerged as a very promising pharmacological strategy to overcome human leukocyte antigen (HLA) barriers in the setting of haploidentical hematopoietic cell transplant (HCT). We recently reported a promising preliminary experience on the use of PT-Cy and sirolimus as graft-versus-host disease (GVHD) prophylaxis in matched allo-HSCT (Greco R et al, Blood 2016). Herein we describe long-term outcomes of matched allogeneic HSCT, using treosulfan-based conditioning, and GVHD prophylaxis with PT-Cy and sirolimus. Methods: In our center, we collected 104 adult patients (pts) receiving matched HSCT for high-risk hematological malignancies, mainly acute myeloid leukemia (n=43). Donor was matched related (MRD) for 45 pts, 10/10 matched unrelated (MUD) for 39 pts and 9/10 MUD for 20 pts. Median age was 48 years (range 19-78). At HSCT, 51% of patients were not in complete remission (CR), 39% were in CR1 and 11% in subsequent CR. Graft source was mainly PBSCs (95%). All pts received a conditioning regimen based on treosulfan and fludarabine; 89% received an intensified conditioning with the addition of melphalan. All pts received PT-Cy (50 mg/kg/day) on days 3 and 4. Sirolimus was given from day 5, and withdrawn 3 months after HSCT. Mycophenolate mofetil (MMF) was added from day 5 to day 30, only in MUD. All patients were treated according to current institutional programs upon written informed consent for transplant procedures. Results: Median follow up was over 16 months (range 3-51). Median CD34+ and CD3+ cell doses were 5.6x10^6/Kg (range, 1.5-10.9) and 2.0x10^8/Kg (range, 0.2-8.0), respectively. All the recipients of allo-HSCT experienced a sustained donor cell engraftment. The cumulative incidence of grades II-IV and III-IV acute GVHD at 100 days was 21% and 9%, respectively. The cumulative incidence of chronic GVHD was 25% at 2 years; we observed severe chronic GVHD only in 4% of pts. The cumulative incidences of relapse and non-relapse mortality (NRM) were 33% and 8% at 2 years, respectively. Two-year overall survival (OS) was 67% and progression free survival (PFS) 59%. The composite end point of GVHD-free/relapse-free survival (GRFS) was 52% at 2 years, in which events include grade 3-4 acute GVHD, systemic therapy-requiring chronic GVHD, relapse, or death. There was a longer OS, 2-year OS was 77% (p = 0.05), and a trend towards higher PFS and GRFS, 63% (p=0.07) and 58% (p=0.06) respectively, for pts with CR status at HSCT. We did not found a significant effect of HLA-matching (9/10 versus 10/10) or donor type (related versus unrelated) on major transplant outcomes (NRM, PFS, GRFS, relapse, acute and chronic GvHD). Conclusion: These outcomes confirmed that matched allogeneic HSCT using treosulfan-based chemotherapy, PT-Cy and sirolimus, is associated with low NRM and acceptable severe GVHD, providing relevant long-term survival in high-risk diseases. A randomized trial comparing this strategy with other kind of in-vivo T-cell depletion (i.e. ATG) is warranted. Disclosures Vago: Moderna TX: Research Funding; GENDX: Research Funding.

Allogenic peripheral stem cell transplantation from HLA-matched related donors for adult sickle cell disease: remarkable outcomes from a single-center trial.

Adult patients with sickle cell disease (SCD) are highly susceptible to stem cell transplant complications, including drug toxicity, graft versus host disease (GVHD), and graft rejection due to SCD-related tissue damage, endothelial activation, and inflammation. The scarcity of compatible stem cells for transplantation further limits treatment options, with only 43 cases of adult allogeneic peripheral blood stem cell transplantation (allo-PSCT) from human leukocyte antigen (HLA)-identical sibling donors reported in the international registry for the period 1986-2013. Herein we report remarkable outcomes in a cohort of adult SCD patients who underwent allo-PSCT using a fludarabine (Flu), busulfan (Bu), and anti-T-cell lymphocyte globulin (ATG)-based conditioning regimen in combination with very low dose total body irradiation (TBI), followed by post-transplant cyclophosphamide (Cy) and sirolimus as GVHD prophylaxis. We performed a single-center, retrospective study consisting of 20 consecutive patients (mean age 33.4 years) who underwent allo-PSCT from HLA-matched related donors with a conditioning regimen of Flu 150/Bu 3.2/Cy 29/ATG 30 (Fresenius)/TBI 200 between September 2013 and September 2017. Data were validated by an independent data audit group of the affiliated JACIE-accredited transplantation center. All patients experienced a sustained donor cell engraftment. Full donor chimerism (total cell) occurred within 180 days in all patients. Mean duration of follow-up was 13.8 months (range: 0.3-50 months), with 12 (60%) patients completing 12 months. No non-relapse mortality or graft rejection occurred. Successful treatment was achieved without the presence of graft loss, grade III-IV acute GVHD, extensive chronic GVHD, or other major complications. Allo-PSCT in combination with Flu 150/Bu 3.2/Cy 29/ATG 30(Fresenius)/TBI 200- Cy/Sirolimus therapy yielded encouraging outcomes with no mortality and low incidence of GVHD. Further controlled studies will be necessary to compare transplant protocols and long-term outcomes.

Outcomes of Children with Hemophagocytic Lymphohistiocytosis Given Allogeneic Hematopoietic Stem Cell Transplantation in Italy

We report on 109 patients with hemophagocytic lymphohistiocytosis (HLH) undergoing 126 procedures of allogeneic hematopoietic stem cell transplantation (HSCT) between 2000 and 2014 in centers associated with the Italian Pediatric Hematology Oncology Association. Genetic diagnosis was FHL2 (32%), FHL3 (33%), or other defined disorders known to cause HLH (15%); in the remaining patients no genetic abnormality was found. Donor for first transplant was an HLA-matched sibling for 25 patients (23%), an unrelated donor for 73 (67%), and an HLA-partially matched family donor for 11 children (10%). Conditioning regimen was busulfan-based for 61 patients (56%), treosulfan-based for 21 (20%), and fludarabine-based for 26 children (24%). The 5-year probabilities of overall survival (OS) and event-free survival (EFS) were 71% and 60%, respectively. Twenty-six patients (24%) died due to transplant-related causes, whereas 14 (13%) and 10 (9%) patients experienced graft rejection and/or relapse, respectively. Twelve of 14 children given a second HSCT after graft failure/relapse are alive and disease-free. Use of HLA-partially matched family donors was associated with higher risk of graft failure and thus with lower EFS (but not with lower OS) in multivariable analysis. Active disease at transplantation did not significantly affect prognosis. These data confirm that HSCT can cure most HLH patients, active disease not precluding successful transplantation. Because in HLH patients HLA-haploidentical HSCT performed through CD34+ cell positive selection was found to be associated with poor sustained engraftment of donor cells, innovative approaches able to guarantee a more robust engraftment are warranted in patients given this type of allograft.

Unrelated Umbilical Cord Blood Transplantation for Sickle Cell Disease Following Reduced-Intensity Conditioning: Results of a Phase I Trial

Hematopoietic stem cell transplantation from HLA-matched sibling donors results in disease-free survival of >90% in patients with sickle cell disease (SCD); however, only approximately 18% of these patients have suitable donors available. Unrelated cord blood transplantation (UCBT) is one way to expand donor options for patients with severe SCD, but historically has been associated with high graft rejection rates (50% to 62%). We hypothesized that the addition of thiotepa to a previously tested reduced-intensity conditioning (RIC) regimen would support engraftment after UCBT in patients with SCD. Nine children (age 3 to 10 years) with cerebrovascular complications of SCD underwent 5-6/6 HLA-matched (A, B, and DRB1 loci) UCBT after conditioning with hydroxyurea, alemtuzumab, fludarabine, thiotepa, and melphalan. A calcineurin inhibitor and mycophenolate mofetil were used for graft-versus-host-disease (GVHD) prophylaxis. With median follow up of 2.1 years (range, 1 to 4.2 years), 7 patients had sustained donor cell engraftment and are free of SCD, and 2 patients had autologous recovery. Acute GVHD (grade II-IV) and mild and moderate chronic GVHD developed in 3 patients, 2 patients, and 1 patient, respectively. At >2 years post-UCBT, 4 of 5 patients discontinued systemic immunosuppression. Seven patients had viral infections (cytomegalovirus, Epstein-Barr virus, respiratory syncytial virus, or adenovirus) and recovered. The 1-year overall survival and disease-free survival rates were 100% and 78%, respectively. Thus, this RIC regimen was able to achieve donor engraftment in the majority of patients. Future efforts will focus on further reducing rates of acute GVHD and viral infection.

Successful Engraftment of Donor Umbilical Cord Blood (UCB) Cells after Co-Transplantation of Nicord® (Ex Vivo Expanded UCB Progenitor Cells with Nicotinamide) and a Second Unmanipulated Cord Blood Unit after Myeloablative Chemotherapy in Pediatric Patients with Severe Sickle Cell Disease

Background: Patients with severe sickle cell disease (SCD) experience organ damage, poor quality of life, and are at high risk of premature mortality. To date, allogeneic hematopoietic stem cell transplant remains the only available curative therapy for SCD. However, patients with SCD have difficulties finding matched related or unrelated donors for transplantation. UCB could be an alternative graft option for most of the SCD patients but, to date, results with unrelated UCB have not been satisfactory for these hard to engraft patients. NiCord is an ex vivo expanded product manufactured from an entire UCB unit which has been shown to produce rapid and sustained engraftment in combination with a second unmanipulated CB unit or as a standalone graft in adult patients with high-risk hematologic malignancies. We hypothesized that the combination of NiCord with an unmanipulated UCB unit might overcome the engraftment barriers which have limited the success of UCBT in patients with SCD. This strategy is currently evaluated for safety and efficacy in a phase I/II multi-center study in pediatric patients with SCD undergoing myeloablative conditioning therapy. Here we report the results of the first 8 patients in the study.Methods: Patients with SCD and high risk features were eligible if they were between 2 and 45 years of age and if they had adequate performance status and organ function, 2 suitable unrelated UCB units that minimally matched the patient at 4/6 HLA alleles, and if they did not have a fully HLA matched related or unrelated adult donor. The NiCord graft, manufactured by the study sponsor, consisted of a CD133+ expanded cell fraction and an uncultured CD133- T-cell containing fraction. All patients received hydroxyurea beginning on day -35. Patients were subsequently prepared for transplantation with myeloablative chemotherapy: Busulfan/Cyclophosphamide and Anti Thymocyte Globulin (ATG). After the first 3 patients an adverse effect of ATG on the expanded graft was suspected and ATG was replaced by Fludarabine for the subsequent 5 patients. On day 0, the unmanipulated CB unit and NiCord graft were infused. Engraftment, GVHD incidence, treatment related mortality and survival were assessed by conventional parameters.Results: Eight patients, at a median age of 12 years (range 3-16 years) and a median weight of 41 kg (range 17-67 kg) were transplanted. HLA matching between patients and NiCord units was 4/6 (n=7) and 5/6 (n=1); and that between patients and unmanipulated units was 4/6 (n=5) and 5/6 (n=3). Median CD34+ cell dose infused in the NiCord graft was 107 x 105/kg. All 8 transplanted patients initially engrafted neutrophils at a median of 7 days (range 6-20 days) with the NiCord graft. One patient experienced secondary graft failure and died after a second transplant. Ultimately, long term engraftment was derived from NiCord in 2 patients, from the unmanipulated unit in 4 patients and mixed donor chimerism is still sustained in one patient. Patients were hospitalized for initial transplant for a median of 55 days (range 43-100 days). The 7 patients with sustained full donor cell engraftment are currently alive at a median follow-up of 33 months (range 3-45 months), transfusion free and without sickle cell related symptoms. Engraftment and clinical outcomes of the 8 patients are shown in Table 1.Conclusions: NiCord appears to overcome the engraftment barriers of UCB in patients with SCD. Remarkably, sustained donor cell engraftment was obtained in 7/8 patients, despite 4/6 matched UCB units and 5 patients without ATG. NiCord has the potential to increase access to transplantation for patients with SCD by enabling the successful use of unrelated UCB donors. Further optimization of this approach with strategies to further decrease GVHD is warranted. [Display omitted] DisclosuresMartin:Novartis: Other: Support of clinical trials; Jazz Pharmaceuticals: Other: One time discussion panel. Galamidi:Gamida Cell Ltd.: Employment. Peleg:Gamida Cell Ltd.: Employment. Goshen:Gamida Cell Ltd.: Employment. Schwarzbach:Gamida Cell Ltd.: Employment. Snyder:Gamida Cell Ltd.: Employment. Peled:Gamida Cell Ltd.: Employment.

Children with Acute Leukemia Given Hematopoietic Stem Cell Transplantation (HSCT) from an HLA-Compatible Sibling, or an Unrelated Donor (UD) or an HLA-Haploidentical Relative after Alpha/Beta T-Cell Depletion Have a Comparable Outcome

Background: Allogeneic HSCT is a widely used treatment for children with acute leukemia (AL) either relapsed or at high risk of treatment failure. However, an HLA-identical sibling is available for only 20-25% of patients and an UD can be located in a suitable time only for a portion of the remaining population. HSCT from an HLA-haploidentical relative (haplo-HSCT) is now considered an alternative option, especially in view of the recent insights in graft manipulation. We recently developed a novel method of more selective T-cell depletion based on physical elimination of α/β T cells (ClinicalTrial.gov identifier: NCT01810120), shown to be effective for both preventing graft-versus-host disease (GvHD) and for conferring improved protection against infections in comparison to haplo-HSCT performed through the infusion of positively selected CD34+ cells. The initial results on 40 patients with AL were reported at the ASH Meeting in 2013 (Bertaina et al). We now present the comparison of the outcome of 80 children with AL given haplo-HSCT after α/β T-cell depletion (group 1) with that of patients transplanted from an HLA-identical sibling (group 2) or an UD (group 3) in the same time period.Patients and methods: All patients with AL were transplanted at the Bambino Gesù Children's Hospital in Rome, Italy, between December 2010 and September 2014; 80 patients were included in group 1, 41 in group 2 and 51 in group 3. Patients were offered α/β T-cell-depleted haplo-HSCT in the absence of suitable conventional donor (HLA identical sibling or 10/10 UD evaluated using high resolution typing) or if affected by rapidly progressive disease not permitting time to identify an UD. Clinical characteristics of patients assigned to the 3 groups and those of their donor are shown in Table1. All children were given a fully myeloablative regimen. No group 1 patient was given any post-transplantation GvHD prophylaxis, while patients of group 2 and 3 were given Cyclosporine-A and short-term methotrexate. Group 1 and 3 patients received ATG Fresenius® (4 mg/Kg/day) from day -5 to -3 for preventing both graft rejection and GvHD.Results: All group 2 and 3 patients had sustained engraftment of donor cells, while 1 of the 80 patients included in group 1 experienced primary graft failure and was rescued by haplo-HSCT from the other parent. The cumulative incidence (CI) of acute GvHD was 30%, 41% and 42%, respectively. Remarkably, all children of the group 1 who developed acute GvHD had a skin-only involvement, while 17% and 16.3% of those of group 2 and 3 had either gut or liver involvement (p<0.001). The CI of chronic GvHD was significantly lower in group 1 children than in those of groups 2 and 3 (p=0.02, see also Figure 1-Panel A). None of the 4 group 1 patients experiencing chronic GvHD had the extensive form of the disease, while the CI of extensive chronic GvHD of group 2 and 3 was 8% and 14%, respectively (p=0.01). Four, 1 and 6 children of patients assigned in group 1, 2 and 3, respectively, died for transplant-related causes; the CI of transplantation-related mortality (TRM) in the 3 groups is shown in Figure 1-Panel B. Relapse was the main cause of treatment failure and occurred at a comparable CI in all the 3 groups (see also Panel C of Figure 1). The 3-year probability of Event-Free Survival (EFS) was comparable in the 3 groups (Figure 1 - Panel D). In multivariate analysis, a Total Body Irradiation (TBI)-containing regimen was the only variable favourably influencing EFS of group 1 children (hazard ratio 2.93, 95% Confidence Interval 1.24-6.95). No variable influenced EFS of group 2 and 3 patients.Conclusions: Overall, these data indicate that haplo-HSCT after α/β T-cell depletion is associated with a risk of TRM and leukemia recurrence comparable to that of transplantation from an HLA-identical sibling or an UD, this translating in a similar probability of EFS. In view of the low incidence of chronic GvHD, this transplant option has to be considered a competitive alternative for children with AL in need of an allograft.TableSibling (n=41)MUD (n=51)Haplo (n=80)Sexp=0.77M273255F141925Age at Transplantation (years)10.69.49.7p=0.20Diseasep=0.23ALL343556AML71624Disease status at Transplantationp=0.13CR1203030CR2212047≥CR3013CMV serology (Donor/Recipient)p=0.001neg/neg856neg/pos8217pos/neg1411pos/pos242156Source of Stem Cellsp<0.0001BM40400PBSC11180Conditioning regimensp=0.10TBI-based262960non TBI-based152220 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HLA-Identical Embryos Selected after in Vitro Fertilization and Pre-Implantation Genetic Diagnosis Combined with HLA Typing: A Promising Option for Successful Allogeneic Hematopoietic Stem Cell Transplantation for Children with Genetic or Malignant Disorders Who Lack an HLA-Matched Related or Unrelated Donor

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative option for many children with various genetic or malignant diseases. However not all children in need for HSCT have a suitable donor available. For such cases in vitro fertilization (IVF) with pre-implantation genetic diagnosis (PGD) combined with human leukocyte antigen (HLA) tissue typing has been used to select an IVF healthy and HLA-matched embryo in order to give birth to a child who may serve as a stem cell donor. We report our experience with five children transplanted from HLA-matched siblings selected after IVF and PGD in our center.Methods and Patients: The clinical protocol was approved by the center’s Institutional Research and Ethics Committee. Written informed consent was obtained from parents.Hormonal stimulation for IVF, culture techniques, PGD and HLA-typing were performed according to standard protocols. Detection of the genetic disease mutation was performed by using mini-sequencing. Embryos genotyping was performed by using multiplex polymerase chain reaction (PCR) analysis of informative polymorphic short tandem repeat (STR) markers.Healthy and HLA-identical embryos were transferred 6 days after fertilization to the uterus of the respective mothers. Confirmatory genetic testing and HLA-typing were performed during the first trimester of pregnancy by using chorionic villus sampling.Patient’s characteristics are shown in Table 1. None of our patients had an available matched sibling donor. All patients underwent myeloablative conditioning consisting of busulfan, cyclophosphamide and antithymocytic globulin. GVHD prophylaxis consisted of CyA plus Methotrexate. Stem cell graft consisted of bone marrow plus cord blood in 4 out of 5 patients, while one patient received bone marrow only.Results: All couples underwent a single cycle of IVF resulting into the generation of 78 embryos. Fourteen embryos were healthy and HLA-identical with their sibling and 10 of them were transferred to the uterus of the respective mother giving birth to a total of 6 healthy children. Results of IVF, PGD and HLA-typing are shown in more detail in Table 2.The median age of the donor at the time of HSCT was 16 months. The median total nucleated cell (TNC) concentration of cord blood grafts was 2.3x107/kg, while the median CD34+ cell was 0.47x105/kg. The median TNC and CD34 cell concentration of bone marrow grafts was 1,97x108/kg and 5,09x106/kg respectively. All patients achieved sustained engraftment of donor cells. Mild acute GVHD of the skin was observed in one out of 5 patients. As of today, with a median follow up period of 4 years, all patients are alive with complete donor chimerism, without chronic GVHD and free of disease.Conclusions: IVF and PGD/HLA typing methodology should be considered and discussed with parents in cases where no HLA-matched donor is available. The procedure can be applied only in cases requiring non-urgent HSCT, and in parents of reproductive age. Table 1:Patient’s characteristicsPatientSexAge (years)DiseasePrevious treatmentsDate of transplant1Male4,5Chronic granulomatous diseaseInterferon-gamma, antibiotics2/10/20072Male4Chronic granulomatous diseaseantibiotics1/7/20083Male11Chronic myeloid leukemiaHydroxyurea, imatinib21/7/20094Male4Thalassemia majorRBC transfusions4/5/20105Female4,5Diamond-Blackfan anemiaCorticosteroids, RBC transfusions30/4/2013Table 2:Results of IVF, PGD and HLA-typingPatientsIVF (cycles)Number of embryos after IVFNumber of healthy and HLA-identical embryosNumber of healthy and HLA-identical transferred embryosNumber of healthy and HLA-identical borned children11376212111221311022141101115110332 DisclosuresNo relevant conflicts of interest to declare.

Long-term functional benefits of human embryonic stem cell-derived cardiac progenitors embedded into a fibrin scaffold

Cardiac-committed cells and biomimetic scaffolds independently improve the therapeutic efficacy of stem cells. In this study we tested the long-term effects of their combination. Eighty immune-deficient rats underwent permanent coronary artery ligation. Five to 7 weeks later, those with an echocardiographically measured ejection fraction (EF) ≤55% were re-operated on and randomly allocated to receive a cell-free fibrin patch (n = 25), a fibrin patch loaded with 700,000 human embryonic stem cells (ESC) pre-treated to promote early cardiac differentiation (SSEA-1(+) progenitors [n = 30]), or to serve as sham-operated animals (n = 25). Left ventricular function was assessed by echocardiography at baseline and every month thereafter until 4 months. Hearts were then processed for assessment of fibrosis and angiogenesis and a 5-component heart failure score was constructed by integrating the absolute change in left ventricular end-systolic volume (LVESV) between 4 months and baseline, and the quantitative polymerase chain reaction (qPCR)-based expression of natriuretic peptides A and B, myosin heavy chain 7 and periostin. All data were recorded and analyzed in a blinded manner. The cell-treated group consistently yielded better functional outcomes than the sham-operated group (p = 0.002 for EF; p = 0.01 for LVESV). Angiogenesis in the border zone was also significantly greater in the cell-fibrin group (p = 0.006), which yielded the lowest heart failure score (p = 0.04 vs sham). Engrafted progenitors were only detected shortly after transplantation; no grafted cells were identified after 4 months. There was no teratoma identified. A fibrin scaffold loaded with ESC-derived cardiac progenitors resulted in sustained improvement in contractility and attenuation of remodeling without sustained donor cell engraftment. A paracrine effect, possibly on innate reparative responses, is a possible mechanism for this enduring effect.

Unmanipulated haploidentical BMT following non-myeloablative conditioning and post-transplantation CY for advanced Hodgkin’s lymphoma

Twenty-six patients with advanced Hodgkin's disease received a related HLA haploidentical unmanipulated BMT, following a non-myeloablative conditioning with low-dose TBI, proposed by the Baltimore group; GvHD prophylaxis consisted of high-dose post-transplantation CY (PT-CY), mycophenolate and a calcineurin inhibitor. All patients had received a previous autograft, and 65% had active disease at the time of BMT. Sustained engraftment of donor cells occurred in 25 patients (96%), with a median time to neutrophil recovery (>0.5 × 10(9)/L) and platelet recovery (>20 × 10(9)/L) of +18 and +23 days from BMT. The incidence of grade II-IV acute GVHD and of chronic GVHD was 24% and 8%, respectively. With a median follow-up of 24 months (range 18-44) 21 patients are alive, 20 disease free. The cumulative incidence of TRM and relapse was 4% and 31%, respectively. The actuarial 3-year survival is 77%, the actuarial 3-year PFS is 63%. In conclusion, we confirm that high-dose PT-CY is effective as prophylaxis of GVHD after HLA haploidentical BMT, can prevent rejection and does not appear to eliminate the allogeneic graft versus lymphoma effect.

Outcomes of Unrelated Umbilical Cord Blood Transplantation Using a Conditioning Regimen of TBI/Ara-c/CY without ATG for Adolescent and Adults Patients with High-Risk Hematologic Malignancies

AbstractAbstract 4525To retrospectively analyze the curative efficacy of umbilical cord blood transplantation (UCBT) using total body irradiation (TBI)-based myeloablative conditioning regimen without antithymocyte globulin (TBI/Ara-c/CY w/o ATG) in adolescent and adult patients with high-risk hematological malignancies.Outcomes of forty-five consecutive adolescent and adult patients with high-risk hematological malignancies treated with TBI-based myeloablative UCBT without ATG in a single center between September 2006 and February 2012 were retrospectively analyzed. The conditioning regimen included TBI/Ara-c/CY:TBI 12GY (four fractionated) + Ara-C 8g/m2 (two days fractionated) + CY 120mg/kg (two days fractionated), and rhG-CSF was administered for myeloid leukemia by continuous infusion at a dose of 5μg·kg−1·d−1. Infusion of G-CSF was started 24 hours before the first dose of Ara-C and stopped at the completion of the last dose. The patients included 31 males and 14 females, with a median age of 21 years (range: 14–40) and a median weight of 58 kg (range: 42–76). Of those, 17 patients (37.8%) had advanced disease. Double UCB grafts were used for 16 patients, while single UCB graft was used for 29 patients. The median number of nucleated cells infused was 3.57 (1.94∼6.76)×107/kg and the median CD34+cells infused was 2.11 (0.71∼4.95)×105/kg. All patients received a combination of cyclosporine (CSA) and mycophenolate mofetil (MMF) for graft-versus-host disease (GVHD) prophylaxis.All patients successfully engrafted. The median times to neutrophil (ANC≥0.5×109/L) and platelet (PLT PLT≥20×109/L) recovery were 19 days (range: 13–35 days) and 36 days (range: 24–90 days) respectively after transplantation in 40 evaluable patients. Acute GVHD occurred in 21 patients and the cumulative incidences of gradeII-‡W and grade III-‡W acute GVHD were 24.4% and 11.1%, respectively. Chronic GVHD occurred in five of 40 evaluable patients (12.5%). Of the 45 patients, 9 (20%) had relapse. After a median follow-up of 25.1 months (range: 6–65.1) among survivors, treatment-related mortality (TRM) within 100 days and within one year was 8.9% and 24.4%, respectively. The main causes of death were pneumonia and severe acute GVHD. The probability of three-year disease-free survival and overall survival (OS) was 53.3% and 57.8%, respectively.The TBI/Ara-c/CY myeloablative conditioning regimen has been well tolerated by patients at our institution and seems to be able to establish sustained donor cell engraftment and decrease the risk of transplant-related death. For high risk patients and patients with advanced disease, this conditioning regimen could reduce relapse and chronic GVHD, indicating the feasibility of TBI/Ara-c/CY as a conditioning regimen for CBT in adolescent and adult patients with hematologic malignancies. Disclosures:Sun:Key Scientific and Technological Project of Anhui province “Twelfth Five-Year Plan” (11010402164): Research Funding; the Fund of the Key Medical Project of Anhui Provincial Healthy Department (2010A005): Research Funding; Anhui Provincial “115” Industrial Innovation Program (2009): Research Funding.

Unrelated umbilical cord blood transplantation with TBI/Ara-c/CY non-ATG conditioning regimen for adults with hematologic malignancies

To retrospectively analyze the curative efficacy of umbilical cord blood transplantation (UCBT) with improved myeloablative conditioning regimen (total body irradiation (TBI)/cytosine arabinoside (Ara-c)/cyclophosphamide (CY) without antithymocyte globulin (ATG)) in adult patients with hematological malignancies. Forty consecutive adult patients with hematological malignancies received improved myeloablative unrelated CBT at a single center from September 2006 to May 2011. Their average age was (23 ± 6) years and the average weight (58 ± 9) kg. Thirty-five (87.5%) patients were high-risk and 15 (37.5%) at the advanced disease status at pre-transplantation. They received 1 (n = 23) or 2 (n = 17) cord blood units. Seventy-five percent of them were transplanted with 1/2-human leukocyte antigen mismatched unit. The conditioning regimen consisted of 12 Gy TBI, granulocyte colony-stimulating factor (G-CSF) plus Ara-c and CY without ATG. All patients received a combination of cyclosporine (CsA) and mycophenolate mofetil (MMF) for the prophylaxis of graft-versus-host disease (GVHD). For the entire group of patients, the average cell doses infused were (4.1 ± 1.1)×10⁷ total nucleated cells/kg and (2.4 ± 1.0)×10⁵ CD34(+) cells/kg. All patients acquired engraftment with an implantation rate of 100%. The average time of absolute neutrophil count (ANC) ≥ 0.5×10⁹/L was (20 ± 5) days and the average time of platelet ≥ 20×10⁹/L was(38 ± 12) days. Acute GVHD occurred in 23 patients (57.5%) and 4 (10.0%) were of grade III-IV. Chronic GVHD occurred in 22.9% (8/35) evaluable patients. Relapse occurred in 12.5% (5/40) patients. During a median follow-up period of 19.8 (range 4.6 - 55.0) months, the transplantation-related mortality was 15.0% (6/40) within 100 days and 35.0% (14/40) within 1 year. The main causes of mortality were pneumonia and severe acute GVHD. Two-year overall survival (OS) or disease-free survival was 58.8% and 58.8%, respectively. Two-year OS for patients with advanced or complete remission disease was 48.6% and 63.8%, respectively. The TBI/Ara-c/CY myeloablative conditioning regimen is well-tolerated and capable of establishing sustained donor cell engraftment and decreasing the risks of transplant-related death in adults with hematologic malignancies. For the high-risk and advanced patients, it may reduce the occurrences of relapse and chronic GVHD.

Favorable Immune Reconstitution After Nonmyeloablative, T-Cell Replete, HLA-Haploidentical BMT with Post-Transplant Cyclophosphamide

Abstract 1009Delayed immune reconstitution with increased risk of opportunistic infection is a major complication of HLA-haploidentical stem cell transplantation, especially in protocols employing extensive T cell depletion of the graft. Previous studies at our institution with high-dose, post-transplantation Cy (PT/Cy) have reported low rates of non-relapse mortality and serious opportunistic infections. Here we characterize immune reconstitution in fifty-three consecutive hematologic malignancies patients receiving nonmyeloablative conditioning, T cell-replete, HLA-haploidentical bone marrow transplantation (BMT), and graft versus host disease prophylaxis including PT/Cy. Patients with advanced hematologic malignancies (median age 51, range 14–71; 5 AML, 2 ALL, 4 MDS, 2 CML, 4 CLL, 1 CMML, 25 NHL, 7 Hodgkins, 3 mantle cell) received Cy 14.5 mg/kg/day IV on days −6 and −5, fludarabine 30 mg/m2/day IV on days −6 to −2, 200 cGy of TBI on day -1 and T cell replete bone marrow from donors with a median age of 44 (range 14–68). GVHD prophylaxis consisted of Cy (50 mg/kg/day) on days 3 and 4, mycophenolate mofetil for 30 days, and tacrolimus for 6 months. Grafts contained an infused median TNC/kg of 4.1 e8 (range 2.6–6.6 e8), CD3+/kg 3.6 e7 (range 1.7–6.7e7) and a CD34+/kg of 3.5e6 (range 1.4–7.0e6). Sustained engraftment of donor cells occurred in 86% of evaluable patients (44/51).The median times to neutrophil (>500/μL) and platelet recovery (>20,000/μL) were 17 days (range, 13–92 days) and 28 days (range, 13–580 days), respectively. Post-transplantation recovery of lymphocyte subsets is shown in Table 1 and Figure 1 and is notable for the following: 1) The median lymphocyte count at day 30 after transplantation is >180/ml and recovers to over 800/ml by day 60; 2) CD4+ T cell counts recover to a median >120/ml by day 60 and >220/ml by day 180 after transplantation; and 3) recovery of CD31+ recent thymic emigrants and CD45RA+ naïve T cells is delayed compared to recovery of memory T cells. T cell receptor spectratyping analysis on a subset of 10 patient/donor pairs chosen specifically for having no relapse/no GVHD (n=4), GVHD and no relapse (n=3), or late relapse (n=3) revealed that patients without relapse, GVHD, or recent viral infection had excellent reconstitution of the T cell repertoire to the level of the pre-transplant donor, as early as 6 months post-transplant (Figure 2). CMV specific T cell response using ELISPOT measured on a subset of 17 patients whose donors were reactive to CMV, revealed that donor-derived immunity to CMV returns by Day 60 in about 70% of patients (12/17) (Figure 3). In conclusion, immune reconstitution after non-myeloablative haploidentical T cell replete BMT with PT/Cy compares favorably with other reduced intensity conditioning alternative donor regimens and suggests that PT/Cy selectively preserves pathogen-specific memory T cells necessary to protect against infection. Further correlations of immune reconstitution with specific infectious and overall outcomes are being analyzed. [Display omitted] [Display omitted] [Display omitted] Table 1Post-transplantation Lymphocyte Subset RecoveryMedian (cells/μL) (N)Interquartile range (cells/μL)ALCDonor1765 (46)1480–2100Recipient pre-BMT840 (45)425–1295Day 30184 (49)54–402Day 60820 (38)470–1260Day 180915 (34)670–1560Day 3653060 (22)820–2030CD3+CD4+CD45RA+ (naïve)Donor119 (33)82–189Recipient pre-BMT22 (34)4–38Day 300.33 (35)0.07–1Day 603 (29)1–9Day 18011 (23)5–31Day 36523 (13)13–92CD3+CD4+CD45RA−CCR7+ (central memory)Donor135 (33)95–158Recipient pre-BMT54 (34)11–79Day 302 (35)0.5–11Day 6034 (29)10–79Day 18061 (23)35–117Day 36589 (13)60–122CD3+CD4+CD45RA−CCR7− (effector memory)Donor187 (33)130–245Recipient pre-BMT87 (34)14–134Day 303 (35)1–18Day 6059 (29)14–122Day 180102 (23)43–179Day 365142 (12)64–204CD3+CD4+CD45RA+CD31+ (recent thymic emigrants)Donor61 (33)30–97Recipient pre-BMT6 (34)1–16Day 300.9 (35)0.03–0.4Day 601 (29)0.5–2Day 1804 (23)1–11Day 3657 (13)2–12CD3+CD4+Foxp3+Donor28 (33)23–35Recipient pre-BMT13 (34)6–24Day 301 (35)0.1–5Day 608 (29)4–16Day 18013 (23)7–25Day 36514 (13)7–17ALC, absolute lymphocyte count; WBC, white blood cell count; Treg, regulatory T cell Disclosures:Jones:Aldagen: Patents & Royalties.

Nonmyeloablative, HLA-Haploidentical Bone Marrow Transplantation with High Dose, Post-Transplantation Cyclophosphamide

Allogeneic stem cell transplantation (SCT) from an HLA-haploidentical relative provides a potentially curative treatment option for hematologic malignancies patients who lack a suitably HLA-matched donor. The greatest challenge to performing HLA-haploidentical SCT has been high rates of graft failure and severe graft-versus-host disease (GVHD). Our group has been exploring high dose, post-transplantation cyclophosphamide (Cy) as prophylaxis of GVHD after nonmyeloablative, HLA-haploidentical bone marrow transplantation, or mini-haploBMT. Among 210 recipients of mini-haploBMT, 87% of patients have experienced sustained donor cell engraftment. The cumulative incidences of grades II-IV acute GVHD and chronic GVHD are 27% and 13%, respectively. Five-year cumulative incidence of non-relapse mortality is 18%, relapse is 55%, and actuarial overall survival and event-free survivals are 35% and 27%, respectively. These outcomes suggest that mini-haploBMT with post-transplantation Cy is associated with acceptably low toxicities and can provide longterm survival, if not cure, for many patients with advanced hematologic malignancies.

Unrelated Umbilical Cord Blood Transplantation Using a TBI/FLAG Conditioning Regimen for Adults with Hematologic Malignancies

A combined chemotherapy regimen comprising fludarabine, cytosine arabinoside, and granulocyte colony-stimulating factor (FLAG) has been used in the treatment of relapsed or refractory leukemias. We here report 38 patients with hematologic malignancies who underwent single-unit cord blood transplantation (CBT) with a conditioning regimen comprising 12-Gy total-body irradiation (TBI) and FLAG therapy (TBI/FLAG). Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus or cyclosporin A and/or methotrexate. The median nucleated cell dose was 2.43 × 10 7/kg (range: 1.96-3.55 × 10 7/kg). Of 34 evaluable recipients, the cumulative incidence of donor engraftment was 97%. The median time to reach an absolute neutrophil count of 500/μL was 23 days (range: 18-35 days). The median time to an untransfused platelet count of 50,000/μL was 45.5 days (range: 28-208 days). Sixteen patients developed grades II-IV of acute GVHD. Fourteen patients were alive at a median follow-up of 46 months (range: 4-77 months). The estimated event-free survival at 3 years for all patients was 33.5%, with 72.7% in the standard-risk group (n = 11) and 17.7% in the high-risk group (n = 27) ( P = .0075). These results showed that this novel regimen was well tolerated by patients and able to establish sustained donor cell engraftment, indicating the feasibility of TBI/FLAG as a conditioning regimen for CBT in adults with hematologic malignancies.

139 INVITED Non-myeloablative transplantation in solid tumors-results in other solid tumors

We previously described a nonmyeloablative hematopoietic stem cell transplantation regimen in dog leukocyte antigen (DLA)-identical littermate recipients consisting of low-dose total body irradiation (TBI) before and mycophenolate mofetil (MMF)/cyclosporine (CSP) given after transplant to control both graft-vs-host and residual host-vs-graft reactions. In this study, we sought to develop a reduced-intensity regimen to achieve engraftment across major histocompatibility complex barriers in DLA-haploidentical littermate recipients.We tested a regimen of 450-cGy TBI with or without postgrafting MMF/CSP for 28 and 35 days, respectively, and with the administration of monoclonal antibody (mAb) S5 (anti-CD44), at a dose of 0.2 mg/kg/day from days −7 through −2, prior to receiving TBI.One of six dogs conditioned with 450-cGy TBI alone achieved engraftment of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells. Three of six dogs achieved sustained donor cell engraftment using 450-cGy TBI and posttransplantation MMF/CSP. None of three dogs given mAb S5 followed by 450-cGy TBI showed signs of donor cell engraftment. However, when S5 mAb pretreatment was added to 450-cGy TBI and postgrafting MMF/CSP, 10 of 12 dogs achieved sustained engraftment (p = 0.008 or 0.007 vs 450-cGy alone or to S5 + 450-cGy TBI without MMF/CSP, respectively), with only three dogs developing severe graft-vs-host disease on this short regimen of immunosuppression.These results show that engraftment across a DLA haplotype-mismatched barrier can be achieved after reduced-intensity conditioning when mAb S5 directed at CD44 is added to this regimen.