Autologous Hematopoietic Recovery Research Articles

Neither Conditioning Chemotherapy Nor GvHD-Prophylaxis: CD7 CAR-T Treatment Bridging to Haplo-HSCT

BACKGROUND: Relapsed/refractory (R/R) acute leukemia patients ineligible for allo-HSCT have poor prognosis. CAR-T therapy has shown promising therapeutic outcomes for these patients, but simultaneously confers the risk of pancytopenia and potential tumor relapse. Bridging CAR-T therapy with allo-HSCT has the potential to restore hematopoiesis and consolidate long-term efficacy. However, GvHD remains a major impediment, and the myeloablative conditioning regimen eradicates residual CAR-T cells and compromises CAR-mediated antitumor function. Furthermore, the implementation of conditioning regimen and GvHD-prophylaxis could lead to severe toxicities. Novel strategies are therefore warranted to preserve CAR-T cells, enhance antitumor potency and minimize complications. METHODS: In this study, we presented 10 patients (enrolled between November 2021 and June 2023) with R/R CD7-positive acute leukemia who proceeded directly to haploidentical HSCT (haplo-HSCT) after CAR-T therapy. All patients received allogeneic CD7 CAR-T therapy, manufactured from either haploidentical donors (n=8) in a phase I clinical trial of donor-derived CAR-T cells (NCT04599556) or (n=1) universal CD7 CAR-T cells (RD13-02) therapy (NCT05716113) or as a compassionate use program after the end of a phase I clinical trial of universal CD7 CAR-T cells (RD13-01) (NCT04538599). CAR-T cell doses were at 2´10 6(haploidentical donor, n=8), 4´10 6 (RD13-02, n=1) or 5´10 6 (RD13-01, n=1) cells/kg. After achieving complete remission with incomplete hematological recovery (CRi), all patients underwent haplo-HSCT without conditioning regimen or GvHD prophylaxis. The primary end point was efficacy, safety, hematopoiesis recovery, and donor chimerism. RESULTS: All patients relapsed from multiple lines of chemotherapy or immunotherapy (median, 7; range, 4-13), were ineligible for allo-HSCT and received palliative care. After CAR-T infusion, 9 patients achieved MRD-negative CRi, and 1 achieved MRD-positive CRi, on day 15 after CD7 CAR-T therapy. 9 patients developed grade 1-2 cytokine release syndrome, and no neurological toxicity was reported. All patients exhibited grade 4 pancytopenia and bone marrow hypocellularity and were directly bridging to haplo-HSCT. The median time from CAR-T infusion to haplo-HSCT was 19 (range, 15-89) days. One patient died of HHV6 infection in central nervous system on day 13 after haplo-HSCT and was not evaluable for the following safety and efficacy. 3 patients experienced short-term grade II acute GvHD, which was resolved completely after low dose steroid, anti-TNF-α or ruxolitinib therapy. One patient developed PTLD at 2.1 months post-HSCT. In 9 evaluable patients, 8 showed full donor chimerism at 1-month post-HSCT, and 1 showed autologous hematopoiesis recovery. At a median follow-up of 10.2 (range, 2.2-20.7) months after CAR-T therapy, 6 patients remained in MRD-negative CR with normal hematopoiesis (including the patient with autologous hematopoietic recovery), 1 had CD7-negative relapse at 5.6 months, 1 developed CD7-negative relapse at 4.3 months and died at 4.8 months, and 1 died from septic shocks at 3.7 months. OS, PFS, NRM, and CIR at 1 year were 64.3% (95% CI, 38.5%-100%), 51.4% (95% CI, 26.2%-100%), 22.9% and 25.7%, respectively. Peripheral normal T cells in 8/10 of patients remained CD7-negative at the last follow-up, illustrating a well-maintained CAR-T function post-HSCT. CONCLUSIONS: Our findings demonstrate a novel strategy to simultaneously achieve CAR-T persistence and complete HSC engraftment, while reducing the risks of GvHD, providing valuable insights into combining cellular therapy with allo-HSCT for the treatment of R/R acute leukemia patients ineligible for conventional allo-HSCT.

High-Exposure, Targeted Daily Busulfan and Fludarabine-Based Conditioning for Children Undergoing Hematopoietic Stem Cell Transplantation for Inherited Metabolic Disorders: Outcomes at a Single Center

Allogeneic hematopoietic stem cell transplantation (HSCT) has emerged as standard therapy for children suffering from various life-threatening inherited metabolic disorders (IMD). Historically, successful outcomes have been limited by high rates of both graft failure and regimen-related toxicity. Real-time busulfan pharmacokinetic monitoring, combined with once daily dosing, has allowed for more precise delivery of targeted exposures during transplant conditioning. Recently, the Utrecht group reported highly favorable outcomes following a high-dose busulfan and fludarabine-based preparative regimen in patients with a variety of diagnoses, particularly in comparison to historical regimens that employ two or more alkylating agents. We report our experience using the same regimen specifically in patients with IMD. Since July 2014, 24 children undergoing first HSCT for IMD were conditioned with once daily IV busulfan (4 days; total regimen exposure of 90 mg x h/L) and once daily IV fludarabine (40 mg/m2/day x 4 days). Busulfan pharmacokinetic monitoring was performed on each of the first 3 doses (days); subsequent doses were altered to achieve the desired total regimen exposure. Serotherapy use was donor-dependent: HLA-matched related donor (MRD, n = 5), none; unrelated UCB (n = 16), thymoglobulin (2.5 mg/kg/day, days - 8 to -5); HLA-matched unrelated donor (MUD, n = 1), thymoglobulin (2.5 mg/kg/day, days - 5 -to -2); and, related HLA haploidentical marrow (n = 2), alemtuzumab (0.5 mg/kg divided days -11 and -10), +/- rituximab (375 mg/m2, day -12). All patients received CSA for GvHD prophylaxis. Additional GvH prophylaxis depended upon the allograft source: UCB, prednisolone; MRD/MUD, MMF; related haploidentical, MMF and post-transplant cyclophosphamide. Growth factor support was administered to recipients of UCB or haploidentical grafts. IMD diagnoses were as follows: Hurler syndrome (n = 10); cerebral adrenoleukodystrophy (n = 9); metachromatic leukodystrophy (n = 1); osteopetrosis (n = 1); fucosidosis (n = 1); alpha-mannosidosis (n = 1); and, beta-mannosidosis (n = 1). At a median follow-up of 229 days (range, 15 - 743), 22 of 24 patients survive, with a Kaplan Meier estimate of survival at one year of 86%. Causes of death were pulmonary bronchiolitis obliterans (BOS, n = 1) and graft failure (n = 1). Twenty-two patients had initial donor neutrophil recovery at a median day +14 (range, +11 to +21); for these patients, the median duration of severe neutropenia (ANC < 500/μL) was 7.5 days (range, 4 to 16 days). Of 20 patients who at the time of analysis are ≥ 30 days from HSCT, 18 had platelet engraftment (≥20,000/μL, transfusion unsupported) at a median day +22.5 (range, +10 to +94). One patient developed Grade II aGvHD (haploidentical marrow graft) which was successfully treated; no patients developed Grade III-IV aGvHD. One patient developed chronic GvHD with fatal BOS (UCB). Regimen-related complications were as follows: veno-occlusive disease, n = 0; hemorrhagic cystitis, n = 1; diffuse alveolar hemorrhage n = 1; idiopathic pneumonia syndrome, n = 1; immune-mediated cytopenias, n = 2; CMV viremia, n = 3; and, adenovirus infection, n = 1. Twenty patients (83%) have demonstrated durable, complete donor myeloid engraftment following the regimen. Of these, all have demonstrated mixed donor-recipient T-cell chimerism. Four patients, (all UCB grafts), have experienced graft failure: aplastic graft failure, n = 3, and autologous hematopoietic recovery, n = 1. Three of these patients underwent second HSCT and 2 are alive and engrafted while the other died of complications following second transplant. One patient awaits second transplant. In summary, this regimen of high-exposure, targeted daily busulfan and fludarabine in children with IMD provides favorable engrafted survival with minimal toxicities. DisclosuresNo relevant conflicts of interest to declare.

Optimal Outcomes after Second Hematopoietic Stem Cell Transplantation in Patients with Thalassemia Recurrence Following Graft Failure/Rejection of the First Graft

Introduction. Graft failure/rejection (GF/R) is a significant complication following HCT in non- malignant diseases. The risk of GF/R in patients with thalassemia is particularly high as a consequence of alloimmunization related to transfusion exposures and hyperproliferative marrow. Although the GF/R rate after myeloablative HLA-matched sibling transplants for thalassemia has been reduced, its incidence still remains high following alternative donor transplantation. Typically, GF/R after HCT for thalassemia is accompanied by autologous hematopoietic recovery, but occasionally patients develop GF with prolonged marrow aplasia. Given great variability in conditioning regimens and outcomes after second HCT for GF/R, it is important to develop a uniform treatment approach to patients receiving second transplantation for GF/R with autologous recovery.Methods. We report on 21 consecutive patients with median age of 9 years (range, 4-24 years) with thalassemia who underwent a second HCT for GF/R with autologous recovery. Ten patients had primary and 11 patients secondary GF/R following the first BMT. Median time to second transplant was 41 months (range, 8- 204). Treatment protocol consisted of preconditioning cytoreduction/immunosuppression with hydroxyurea (30 mg/kg/day) and azathioprine (3 mg/kg/day) from day −45 pre-transplant, and fludarabine (30 mg/m2/day) from day −16 through day −12. Conditioning regimen included oral or intravenous (i.v.) busulfan, thiotepa 10 mg/kg/day, cyclophosphamide 200 mg/kg total dose, and thymoglobulin 12.5-10 mg/kg total dose. Cyclosporine, methylprednisolone and methotrexate were given for GVHD prophylaxis. All but 4 patients received second HCT from the same donor. Thirteen patients received bone marrow (BM), and 8 patients PBSC. One patient was in class 1, 7 patients in class 2, and 13 patients in class 3 of risk. Most patients had moderately severe iron overload with median serum ferritin of 2692 ng/ml (range, 500-10126 ng/ml). The median liver fibrosis score 1 (range 0-5).Results. All but 1 patient achieved sustained engraftment with a 5 year overall and disease-free survival (DFS) of 85% (95% CI, 59-95%). DFS was 92% (95% CI, 54-99%) in patients receiving BM and 75% (95% CI, 32-93%) in those given PBSC, although the difference was not statistically significant (p=0.27) (Figure 1).One patient had primary GF with the cumulative incidence of 5% (95% CI, 0-13%). The incidence of grade 2-4 acute GVHD was 50% (95% CI, 0-75%) in the PBSC and 8 % (95% CI, 0-24%) in the BM group (p=0.025). Respective incidences of moderate to severe chronic GVHD were 43% (95% CI, 0-70%) and 0% (p=0.012). The high incidence of both acute and chronic GVHD after PBSC transplantation prompted us to abandon its use for second transplantation since 2006. At the time of survival analysis, 18 patients were alive, with median follow-up duration of 10 years (range, 1.2-12.8 years). At present all patients are off immunosuppressive medication. There were 3 deaths due to pneumonia, postsplenectomy sepsis and rejection with cerebral bleeding. The most frequently observed toxicities were grade 2 elevations in AST and ALT, followed by grade 2 oral mucositis. Few patients experienced grade 3 liver and gut toxicities. None of the patients developed liver VOD. Three patients had pneumonia, and 4 patients developed grade 2-3 late onset BK virus -related hemorrhagic cystitis. None of the patients developed lymphoproliferative disorder.Conclusions. We demonstrate the excellent DFS in patients with thalassemia treated with second transplantation for GF/R following the first graft. Importantly, the intensified treatment protocol was not associated with increased nonhematological toxicity, even though most patients suffer from pre-existing organ damage due to iron overload and/or hepatitis. This study clearly showed that the same donor could be successfully used for second transplantation in these patients. Due to significantly high incidence of both acute and chronic GVHD after PBSC, bone marrow graft is preferred source for second transplantation in patients with thalassemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Multicenter Phase II Trial of Unrelated Donor Reduced Intensity Bone Marrow Transplantation for Children with Severe Sickle Cell Disease (SCURT): Results of the Blood and Marrow Transplant Clinical Trials Network (BMT CTN 0601) Study

▪Background:Children with severe sickle cell disease (SCD) experience organ damage, poor quality of life, and a risk of premature mortality. Allogeneic bone marrow transplantation (BMT) halts disease progression when stable donor erythropoiesis is established. Myeloablative conditioning (MAC) followed by HLA matched sibling donor BMT is effective, but most patients with SCD lack a matched sibling donor and long term side effects from MAC (e.g., infertility) can be barriers to patient acceptance of BMT. The SCURT trial was designed as a prospective, multicenter phase II trial to evaluate outcomes after HLA matched unrelated donor (MUD) BMT with reduced intensity conditioning (RIC). We hypothesized that an intensely immunosuppressive RIC regimen would be sufficient for donor engraftment and could limit toxicity. The primary endpoint was one year event free survival (EFS) (alive and disease free) ≥75%. All patients were evaluable for the primary endpoint for the purpose of this report.Methods:Children between 3 and 19.75 years of age who had ≥1of the following complications - stroke, high transcranial doppler velocity, recurrent acute chest syndrome, or significant vaso-occlusive pain crises were eligible. An independent review committee confirmed eligibility. The preparative regimen consisted of alemtuzumab (48 mg) (day-22 to -19), fludarabine (150 mg/m2) (day-8 to -4), and melphalan (140mg/m2) (day-3). Donors and recipients were matched at the HLA allele-level at HLA-A, -B, -C and -DRB1 loci. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus or cyclosporine, methotrexate (7.5 mg/m2)on days +1, +3 and +6, and methylprednisolone (1 mg/kg/day) from day +7, tapered after day +28.Results:Thirty patients were enrolled between 2008 and 2014. One patient was excluded from analysis for a major eligibility violation at the DSMB's recommendation. Twenty-nine patients (16 M, 13F), 5.9-19.3 years old (median 14.0 years) from 19 centers were evaluable with median follow up of 25.2 months (range, 12-61.5 months). All patients recovered neutrophils (median 12 days) and platelets >50x109/L (median 24 days). Graft rejection (GR) in 3 patients (10%) occurred before day +100, and was followed by autologous hematopoietic recovery. No late GR has been observed. The primary endpoint was met with1-year EFS of 76% (Figure 1A). The 1-year probability of overall survival (OS) was 86% (Figure 1B). The cumulative incidence (CINC) of grade II-IV and III-IV acute GVHD at day 180 were 31% and 17% respectively (Figure 2A). The CINC of chronic GVHD at 1-year was 62%. Chronic GVHD was extensive in 38% (Figure 2B). GVHD when present was noted by 12 months post-BMT. There were 7 deaths, all in patients >14 years at time of BMT. One death occurred after a second transplant for graft rejection. The other 6 deaths occurred between 143 and 960 days post BMT, from GVHD. Non-fatal toxicities included posterior reversible encephalopathy syndrome (PRES) associated with hypertension and seizures in 35%. All cases were observed in the first 6 months post-BMT; all patients recovered with no obvious sequelae. No patient developed veno-occlusive disease, idiopathic pneumonia syndrome or cerebral hemorrhage. Infections were common, and occurred in 76% of patients. CMV and EBV reactivation each occurred in 23% of recipients. Surviving patients report performance scores of 100% (n=14), 90% (n=5), and 80% (n=3) at 2 years or at the time of last assessment. At last follow up, 13 patients had successfully discontinued immunosuppressive therapy.Conclusion:In summary, the RIC regimen employed in this trial of MUD BMT in children with severe SCD resulted in low rates of regimen-related organ toxicity (other than a high rate of PRES) and 1-year EFS of 76%, associated with regression of SCD manifestations. . The risk of graft rejection/disease recurrence was similar to that observed after conventional HLA-identical sibling donor BMT. Chronic GVHD occurred in 62% of patients and was the leading cause of toxicity and mortality. Future trials should focus on effective GVHD prophylaxis after URD BMT. Secondary outcome analyses including health-related quality of life and neurocognitive performance are ongoing. [Display omitted] [Display omitted] DisclosuresWalters:ViaCord and AllCells, Inc: Other: Medical director. Levine:Novartis: Consultancy.

Graft Failure after Hematopoietic Cell Transplantation for Hemoglobin Disorders: Successful Application of a Rescue Regimen

BackgroundAlthough patients with clinically significant hemoglobinopathies can be supported with blood transfusions and iron chelation therapy, the only curative option is allogeneic hematopoietic stem cell transplantation (HCT). Unfortunately, there is a risk of graft rejection as a consequence of donor sensitization and alloimmunization related to transfusion exposures and a hyperproliferative marrow niche. Typically, graft rejection after HCT for hemoglobinopathies is accompanied by autologous hematopoietic recovery, but there is also a risk of graft failure and marrow aplasia which can cause a fatal outcome. To accommodate individuals who lack a suitable sibling donor, transplant options are being expanded to include alternative sources of hematopoietic donors which carry higher risks of graft rejection, graft-versus-host disease and transplant-related mortality. Given the significant risk of graft failure after alternative donor HCT, it is important to develop a plan for stem cell rescue when these complications occur.Methods and ResultsWe reviewed 3 patients with hemoglobin disorders who were successfully rescued by alternative transplantation regimens following primary or secondary graft failures. Two patients with sickle cell disease and one with β-thalassemia major were treated at our institution with a reduced-intensity regimen. All 3 patients had graft rejection accompanied by marrow aplasia within 2 months after HCT. Patient 1 was successfully rescued with mobilized peripheral blood stem cells from the same HLA mismatched sibling donor after a minimal toxicity preparative regimen. Patients 2 and 3 were successfully rescued from HLA-haploidentical donors after a minimal toxicity preparative regimen that included in vivo T-cell depletion by post-grafting high dose cyclophosphamide. Refer to Table 1 for patient and transplant characteristics.ConclusionThere are 3 methods to rescue patients from graft rejection: donor lymphocyte infusions, the intensification of immunosuppression or a rescue allogeneic transplant. Here we report our local experience with graft failure and its successful management in 3 patients with hemoglobin disorders. Although graft rejection remains a major obstacle to successful transplantation for hemoglobinopathies, better donor selection and screening by lymphocyte cross matching, optimized conditioning regimens and improved supportive care should improve outcomes after alternate donor HCT. We speculate that it is also possible now to rescue from graft failure in most cases and that an allogeneic HCT rescue might be utilized for a curative outcome.Table 1Patient characteristics, rescue transplant regimen, engraftment, graft-versus-host-disease (GVHD), and duration of immune suppression post-rescue HCT. BM, Bone Marrow; UCB, Umbilical Cord Blood; ATG, Anti-thymocyte Globulin; HLA, Human Leukocyte Antigen; MMF, Mycophenolate Mofetil; TBI, Total Body IrradiationPatient 1Patient 2Patient 3Age, Gender14 yo Male3 ½ yo Male14 yo MaleDiagnosisHemoglobin SSβ-Thalassemia majorHemoglobin SSIndication for TransplantSubarachnoid Hemorrhage Veno-occlusive crisesAlternative to life-long transfusions and iron chelation therapyMultiple Cerebral infarcts Acute Chest Syndrome Veno-occlusive crisesTransplant #1 Conditioning RegimenBusulfan Thiotepa Cyclophosphamide Rabbit ATGBusulfan Thiotepa Fludarabine Rabbit ATGBusulfan Fludarabine Rabbit ATGTransplant #1 Cell SourceBM, 9/10 HLA mismatch at A locusSingle 6/6 HLA matched UCBBM, 10/10 HLA matched siblingGVHD Prophylaxis for Transplant #1Cyclosporine MMFCyclosporine MethotrexateType of Graft FailureSecondaryPrimarySecondaryRescue Transplant Conditioning RegimenFludarabine 30mg/m2 on days -4 through -2; TBI 400cGy on day 0Fludarabine 30 mg/m2 on days -6 through -2; TBI 400cGy on day 0; Cyclophosphamide 14.5 mg/kg/day on days -6, -5; and 50 mg/kg/day on days +3,+4Recue Transplant Cell SourceSame donor, Mobilized PBSCHaploidentical motherHaploidentical fatherRescue GVHD ProphylaxisTacrolimus MMFTime to Neutrophil Engraftment (days)141415Time to Platelet Engraftment (days)182522Day 28 Percent Donor Cells98%100%99%Acute GVHDNoneSkinNoneChronic GVHDNoneOral and Integument (resolved)Pericardial effusion (resolved)Duration of Post-Rescue Transplant Immune Suppression17 months37 months16 months DisclosuresNo relevant conflicts of interest to declare.

Successful matched sibling donor marrow transplantation following reduced intensity conditioning in children with hemoglobinopathies.

Fifty-two children with symptomatic sickle cell disease sickle cell disease (SCD) (N = 43) or transfusion-dependent thalassemia (N = 9) received matched sibling donor marrow (46), marrow and cord product (5), or cord blood (1) allografts following reduced intensity conditioning (RIC) with alemtuzumab, fludarabine, and melphalan between March 2003 and May 2014*. The Kaplan-Meier probabilities of overall and event-free survival at a median of 3.42 (range, 0.75-11.83) years were 94.2% and 92.3% for the group, 93% and 90.7% for SCD, and 100% and 100% for thalassemia, respectively. Treatment-related mortality (all related to graft versus host disease, GVHD) was noted in three (5.7%) recipients, all 17-18 years of age. Acute and chronic GVHD was noted in 23% and 13%, respectively, with 81% of recipients off immunosuppression by 1 year. Graft rejection was limited to the single umbilical cord blood recipient who had prompt autologous hematopoietic recovery. Fourteen (27%) had mixed chimerism at 1 year and beyond; all had discontinued immunosuppression between 4 and 12 months from transplant with no subsequent consequence on GVHD or rejection. Infectious complications included predominantly bacteremia (48% were staphylococcus) and CMV reactivation (43%) necessitating preemptive therapy. Lymphocyte recovery beyond 6 months was associated with subsidence of infectious complications. All patients who engrafted were transfusion independent; no strokes or pulmonary complications of SCD were noted, and pain symptoms subsided within 6 months posttransplant. These findings support using RIC for patients with hemoglobinopathy undergoing matched sibling marrow transplantation (*www.Clinical Trials.gov: NCT00920972, NCT01050855, NCT02435901).

Clinical study of unrelated umbilical cord blood transplantation for 25 patients with hematological diseases

This study was purposed to investigate the engraftment, graft-versus-host disease (GVHD), transplantation related mortality (TRM), relapse and survival in hematologic patients received unrelated umbilical cord blood transplantation (UCBT). A total of 25 patients with hematological disease underwent UCBT, including 8 pediatric and 17 young adult patients. Among them 3 cases received single unit of UCBT and 22 cases received double units of UCBT. For donor/recipients human leukocyte antigen (HLA) matching: HLA 6/6 loci matched in 9 cases, HLA 4-5/6 loci matched in 16 cases. There were 19 patients with hematologic malignancies, including 3 cases in the period of disease progression and 6 cases of non-hematologic malignancies. Conditioning regimens were TBI/Cy ± Flu ± ATG or BuCy ± Flu ± ATG for 21 patients and Cy+Flu+ATG for 4 patients. For prophylaxis of acute graft-versus-host disease (aGVHD) the regimen of cyclosporine (CsA) as dominant drug was used. The results showed that among 16 patients (80.0%) achieved engraftment, 20 patients survived for more than 42 d after transplantation. The cumulative neutrophil recovery rate on day 42 after transplant was 64.0%, with a median time of 17.0 d;the cumulative platelet recovery rate on day 100 after transplant was 60.0 %, with a median time of 35.0 d. The cumulative rate of grade II-IV and III-IV aGVHD after transplantation 100 d was 44.0% and 30.7%, respectively. Until the end of the follow-up, the cumulative rate of TRM was 54.3%. For all the patients, overall survival rate was 42.7%. Out of 17 evaluable patients with hematologic malignancies 7 cases (41.2%) survived to date, and only 1 case relapsed, so event-free survival rate was 35.3%. Out of 5 evaluable patients with non-hematologic malignancies, 4 patients survived and 2 patients were in stable engraftment state, 2 cases with autologous hematopoietic recovery. Among 3 cases of hematologic malignancies at advanced stage, only 1 case survived to date. It is concluded that HLA-4-6/6 loci matched UCBT is an effective option to treat hematological diseases. Double cord blood transplantation (dUCBT) can overcome the disadvantage of insufficient cells of single cord blood UCBT to treat overweight children and adult.

Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies Using a Reduced-Intensity Conditioning Regimen and Third-Party Mesenchymal Stromal Cells

Allogeneic hematopoietic stem cell transplantation for patients with a hemoglobinopathy can be curative but is limited by donor availability. Although positive results are frequently observed in those with an HLA-matched sibling donor, use of unrelated donors has been complicated by poor engraftment, excessive regimen-related toxicity, and graft-versus-host disease (GVHD). As a potential strategy to address these obstacles, a pilot study was designed that incorporated both a reduced-intensity conditioning and mesenchymal stromal cells (MSCs). Six patients were enrolled, including 4 with high-risk sickle cell disease (SCD) and 2 with transfusion-dependent thalassemia major. Conditioning consisted of fludarabine (150 mg/m2), melphalan (140 mg/m2), and alemtuzumab (60 mg for patients weighing > 30 kg and .9 mg/kg for patients weighing <30 kg). Two patients received HLA 7/8 allele matched bone marrow and 4 received 4-5/6 HLA matched umbilical cord blood as the source of HSCs. MSCs were of bone marrow origin and derived from a parent in 1 patient and from an unrelated third-party donor in the remaining 5 patients. GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. One patient had neutropenic graft failure, 2 had autologous hematopoietic recovery, and 3 had hematopoietic recovery with complete chimerism. The 2 SCD patients with autologous hematopoietic recovery are alive. The remaining 4 died either from opportunistic infection, GVHD, or intracranial hemorrhage. Although no infusion-related toxicity was seen, the cotransplantation of MSCs was not sufficient for reliable engraftment in patients with advanced hemoglobinopathy. Although poor engraftment has been observed in nearly all such trials to date in this patient population, there was no evidence to suggest that MSCs had any positive impact on engraftment. Because of the lack of improved engraftment and unacceptably high transplant-related mortality, the study was prematurely terminated. Further investigations into understanding the mechanisms of graft resistance and development of strategies to overcome this barrier are needed to move this field forward.

Second Umbilical Cord Blood Transplant (UCBT2) In Adult Patients With Engraftment Failure Or Autologous Hematopoiesis After UCBT

IntroductionUmbilical cord blood transplantation (UCBT) is often complicated by delayed engraftment and higher rates of engraftment failure. We sought to investigate the outcomes of patients undergoing UCBT2 after engraftment failure compared to those who had autologous hematopoietic recovery and did not receive UCBT2. MethodsWe reviewed the medical records of 186 patients who received UCBT between 2001 and 2011 at the Case Comprehensive Cancer Center [Seidman Cancer Center, University Hospitals Case Medical Center and Taussig Cancer Institute, Cleveland Clinic]. Standard definitions of engraftment were used. Patients who died or had progressive disease before day 30 were excluded (n=9), as they were considered inevaluable for engraftment. ResultsTwenty-seven patients (15%) had primary engraftment failure; twelve had autologous hematopoietic recovery and 15 presented no recovery (Table). The median total nucleated cell dose for the first UCBT was 2.56x107 cells/kg (range 1.23 x107 - 5.69x107), not statistically different from patients achieving engraftment (p = 0.29). The cause of engraftment failure could not be identified in 22 patients.Table 1Demographic characteristics and outcomes of patients with primary engraftment failure or autologous hematopoietic recovery after UCBT.Median Age (Range) (years)49 (20-65)Female / Male10 / 17DiagnosisAML13ALL3CML4NHL3PCM1MDS2Aplastic Anemia1UCBT #1UCBT #2Conditioning RegimenReduced Intensity2315Myeloablative40Number of UnitsUCBT #1UCBT #2110421411>230Autologous HematopoiesisSecond UCBTp valueOverall Survival (days)301273ns40 month cumulative incidence of relapse92%50%p = 0.017Among patients with engraftment failure without autologous recovery, 2 patients did not receive further salvage and died at 40 and 48 days after first UCBT; 2 others received backup autologous hematopoietic cell infusions and 11 received UCBT2. In patients with autologous hematopoietic recovery, 8 did not receive a UCBT2 because disease remission had been attained (n=4), active infection (n=1), relapsed disease beyond 40 days after UCBT (n=1) or absence of compatible donor (n=1).Fifteen patients (engraftment failure [n=11]); autologous recovery, [n=4]) underwent UCBT2, at a median of 43 days (range 33-244) from first to second UCBT. All patients received non–myeloablative second conditioning regimens. Four patients received one UCB unit and 11 received two units. Neutrophil engraftment after UCBT2 was observed in 10 of 15 patients at a median of 30 days (range, 13-35).The median survival for the entire cohort was 272 days (range 40–2130); one year overall survival was 36%. Engraftment and overall survival were not statistically different between those receiving 1 or 2 UCB units for UCBT2. Overall survival was not statistically different between patients who underwent UCBT2 and those who had autologous recovery or backup autologous infusions without further salvage.The proportion of patients in remission was similar in the 2 subgroups (UCBT2, 53%; autologous recovery, 50%). Relapse occurred in 2/15 patients who underwent UCBT2 and 8/10 patients who had autologous recovery or backup infusions. Forty months after transplantation, the cumulative incidence of relapse was 92% after autologous recovery vs. 50% after UCBT2 (Figure). Non–relapse mortality after UCBT2 was 73%. [Display omitted] ConclusionEngraftment failure after UCBT frequently has no identifiable cause, and is associated with high morbidity and mortality. UCBT2 is feasible and can result in engraftment in the majority of patients, but is limited by patient condition after transplant as well as the availability of compatible grafts. UCBT2 is associated with significantly better disease control than continued autologous hematopoiesis, but additional efforts are necessary to reduce the high risk of non-relapse mortality in this group of patients. Disclosures:Hill:Celgene: Honoraria, Research Funding.

Multicenter Investigation Of Unrelated Donor Hematopoietic Cell Transplantation (HCT) For Thalassemia Major After a Reduced Intensity Conditioning Regimen (URTH Trial)

BackgroundHCT cures thalassemia major (TM). In the absence of a family donor, marrow or less frequently umbilical cord blood (UCB) from unrelated donors (URD) has been used. Due to risks of graft rejection, myeloablative preparative regimens are primarily utilized. URD marrow and UCB HCT have 65-90% and 21-74% event free survival (EFS) and rejection rates of 9-17% and 17-57% respectively.The URTH trial was developed in collaboration with the Thalassemia Clinical Research Network (TCRN), National Heart, Lung and Blood Institute (NHLBI), Pediatric Blood and Marrow Transplant Consortium (PBMTC) and New England Research Institutes (NERI) to explore URD HCT for TM as a strategy to expand availability of HCT. It employed reduced intensity conditioning (RIC) as a means to decrease early and late toxicities. The study tested our hypothesis that an immunosuppressive RIC regimen was sufficient for engraftment in children with TM (age > 1 year to < 17 years) after URD HCT. The primary objective was to determine EFS at 1 year after URD marrow or UCB HCT. MethodsPatients with transfusion dependent beta thalassemia and a suitable URD (matched at 8/8 HLA-alleles in marrow donors or 5 to 6/6 HLA antigens in UCB donors) were conditioned with hydroxyurea (30mg/kg x 30 days) (day -50 to -21), alemtuzumab (48 mg) (-22 to -19), fludarabine (150 mg/m2) (-8 to -4), thiotepa (8mg/kg) (-4), and melphalan (140mg/m2) (-3). Patients received tacrolimus or cyclosporine with methotrexate and methylprednisone (marrow) or mycophenolate mofetil (UCB) after HCT to prevent graft-versus-host disease (GVHD). Suitable UCB units were defined as having a pre-thaw total nucleated cell content >4.0x10E7/Kg recipient weight. Patients were eligible irrespective of Pesaro classification but the presence of liver fibrosis by histology was an exclusion criterion. ResultsTwenty-three patients from 11 US centers (11M: 12F) with a median age of 10 years (2–17 years) received unrelated donor allografts: marrow (14) or UCB matched at 6/6 (1) or 5/6 HLA antigens (8). The median follow up time was 12 months (range 120 days-2 years). The median time to neutrophil engraftment was 13 days (range 10-25) and 34 days (range 12-46) after marrow and UCB HCT respectively. The median time to platelet engraftment after marrow and UCB HCT was 24 days (range 18-34) and 54.5 days (range 32-234) respectively. Primary graft rejection occurred in 1 patient (4% of all patients) following UCB HCT and was accompanied by autologous hematopoietic recovery 35 days after HCT. All others had >90% donor chimerism and achieved transfusion independence. There were no late graft rejections. The overall and EFS probabilities were 82% and 78% respectively at the most recent encounter.One patient developed mild VOD which resolved uneventfully. Of 15 patients who had CMV reactivation, 13 responded to pre-emptive therapy and had no progression to CMV disease. The probabilities of grade II-IV and grade III-IV acute GVHD were 30% and 9% respectively. Limited chronic GVHD was noted in 35% of the cohort; 9% developed extensive cGVHD. Four patients died on days 25, 86, 106 and 366. The causes of death included 1) pulmonary hemorrhage associated with CMV, adenovirus, and Pneumocystis jiroveci infections, 2) diffuse alveolar pulmonary hemorrhage, 3) cGVHD with pneumonia associated with CMV and adenovirus infections, and 4) cGVHD with pulmonary failure associated with CMV and EBV infections and presumed central nervous system post-transplantation lymphoproliferative disease. ConclusionHCT after RIC for thalassemia is feasible and sufficient for engraftment after URD marrow and UCB transplantation with survival exceeding 80%. The principal transplant- related complications we observed were early opportunistic viral reactivations; otherwise the preparative regimen was tolerated well with very little early toxicity. Fatal and late viral infections were noted only in the setting of severe GVHD. Patients should be monitored carefully and treated promptly for infectious complications after HCT until there is adequate immune reconstitution. The risk of severe GVHD was low despite unrelated and mismatched (UCB) donor sources. Longer follow up will determine if this regimen can reduce late toxicities. An extension of this trial is ongoing and currently recruiting patients to evaluate additional HCT related and quality of life measures. Disclosures:Neufeld:Shire: Consultancy. Kwiatkowski:Resonance Health: Research Funding; Shire: Consultancy. Thompson:Novartis: Consultancy, Research Funding; ApoPharma: Consultancy, Honoraria; Glaxo Smith Kline: Research Funding; Eli Lilly: Research Funding; Amgen: Research Funding; bluebird bio: Research Funding.

A Pilot Study Of Continuous Infusion Mycophenolate Mofetil For Graft Versus Host Disease Prophylaxis

Mycophenolate mofetil (MMF), an ester prodrug of mycophenolic acid (MPA) has been used successfully for graft-versus-host disease (GVHD) prophylaxis. Most MMF dosing regimens and pharmacokinetic (PK) parameters have been established in solid organ transplant recipients. Empiric fixed-dose-escalation strategies have failed to achieve target MPA exposure, especially in the early period following conditioning therapy. We hypothesized that a PK-based dosing approach using a novel continuous infusion (CI) method of MMF will be able to achieve and maintain target MPA exposure. The objective of this study was to evaluate the safety and feasibility of this MMF dosing approach in pediatric BMT recipients. Fifteen patients were enrolled in the study. The median age at transplant was 8.4 years (0.9 – 21.5); eight were female and seven were male. Thirteen patients were transplanted for hematologic malignancies and two patients for nonmalignant diseases. Stem cell sources included matched-sibling bone marrow in seven, 10/10 allele matched donor bone marrow in four, and unrelated cord blood in four cases. All patients received a myeloablative conditioning regimen with either cyclophosphamide-TBI (n=8) or busulfan-cyclophosphamide (n=7). In addition, of eight unrelated donor transplants, six patients received equine ATG and two received fludarabine. All patients received cyclosporine A and MMF for GVHD prophylaxis. Intravenous (IV) cyclosporine was initiated on day -2 as a continuous infusion (target blood level: 160 to 250 ng/mL). IV MMF was started on day “0” in a dose of 15 mg/kg every 8 hours. After a minimum of five doses, serial blood samples were collected within one dosing interval for the 1st PK analysis. Using these results, MMF was converted from intermittent IV to CI administration to target total MPA AUC0-24 of 40-80 mcg/mL/hr. MMF dose was adjusted to maintain a total MPA steady-state concentration (Css) of 1.7-3.3 mcg/mL. CI MMF was converted to every-8-hour oral dosing when patients were tolerating oral medications. A 2nd PK study was performed after a minimum of five oral doses, and dose adjustments were made to maintain a total MPA trough concentration (Ctrough) of 1-3.5 mcg/mL. MMF was continued until day 42, and in the absence of acute GVHD, tapered off over 8 weeks. On the initial 15 mg/kg q8 IV MMF dose schedule, MPA half-life was 1.3±1.3 hrs and AUC0-24 was 44.3±60.3 mg* hr/L. During the CI schedule, MMF dose was 44±16 mg/kg/day and MPA AUC0-24 38.4±20.7 mg* hr/L, and thirteen of fifteen (87%) patients achieved MPA Css within target of 1.7–3.3 mcg/mL. On conversion to oral route, median MMF dose was 45±19mg/kg/day, MPA half-life 1.95±2.3 hrs and AUC0-24 38.3±16.0 mg* hr/L. MPA clearance was 17±14 ml/min/kg. Higher steady state MPA concentrations were achieved with CI method (Css 1.6 ±0.9 mcg/mL) compared with intermittent IV or oral dosing (Ctrough 0.7±0.9 mcg/mL). All patients achieved neutrophil engraftment at 13 ± 11 days post-transplant, and platelet engraftment at 45 ± 72 days post-transplant. Seven out of 15 patients (47%) developed stage I-IV acute GVHD. Three patients had stage III-IV aGVHD (20%); they were steroid-refractory but went into remission with additional therapy. For 11 patients with a minimum of one year follow-up post-transplant, one (9%) developed “overlap” chronic GVHD. MMF was discontinued without weaning in four patients: in three of them due to concern of delay in blood count recovery and all of them achieved hematopoietic engraftment, and in one patient with severe aGVHD due to potential concern for MMF gut toxicity. One patient with severe congenital neutropenia developed autologous hematopoietic recovery by day 52. One patient with ALL died of relapse 150 days after transplant. With a median follow-up of 23 months (range 100 days to 3.6 y), 14 out of 15 patients are alive, and one-year event-free survival and overall survival are 83% and 92% respectively. In conclusion, we show that continuous infusion of MMF is feasible and well-tolerated. In this pilot PK study, we observed no toxic deaths, excellent engraftment, and low rates of grade III-IV aGVHD and cGVHD. The PK studies showed significantly lower half-life and higher drug clearance in pediatric BMT recipients compared to stable pediatric renal transplant patients. This regimen deserves further validation in a larger cohort of pediatric patients undergoing myeloablative transplantation. Disclosures: Off Label Use: Use of Mycophenolate Mofetil for Graft versus Host Disease Prophylaxis.

Rapid and complete reconstitution of autologous haemopoiesis after cord blood infusion in treatment‐naive patients with severe aplastic anemia receiving high‐dose cyclophosphamide/ATG therapy

Although high-dose cyclophosphamide seems to achieve durable complete remission, there are still concerns about its too much early toxicity. So, we designed a clinical study to investigate the effects of high-dose cyclophosphamide/ATG combined with cord blood infusion as first-line therapy for patients with severe aplastic anemia. Between January 2003 and September 2007, we treated 16 treatment-naive patients with severe aplastic anemia with cord blood infusion after high-dose cyclophosphamide (50mg/kg/d×2) and rabbit antithymocyte globulin (3mg/kg/d×5) therapy. Although only one patient had durable full donor engraftment, 14 of the enrolled 16 patients had rapid autologous hematopoietic recovery. The median recovery time for neutrophils and platelets was only 23 and 37d after infusion of cord blood. Of the 15 responding patients, all patients achieved treatment-free remission: nine patients met the criteria for a complete remission; six patients achieved a partial remission. Infusion of cord blood after high-dose cyclophosphamide/ATG resulted in a rapid autologous hematologic recovery and a high response rate in patients with treatment-naive patients with severe aplastic anemia. These promising results merit further investigation and confirmation on a larger number of patients.

Long-Term Remission in a Juvenile Myelomonocytic Leukemia Patient After Graft Rejection of Unrelated Bone Marrow Transplantation

Abstract 4962Juvenile myelomonocytic leukemia (JMML) is an aggressive clonal malignancy and mixed myeloproliferative and myelodysplastic disorder. Although cure in most cases requires hematopoietic stem cell transplantation (HSCT), the major cause of treatment failure is relapse. However, in some cases, symptoms improve without treatment. We report a case of a patient with JMML who sustained remission after graft rejection of an unrelated bone marrow transplantation (UBMT). CaseAn 18-month-old girl presented with marked splenomegaly and hemorrhagic diathesis. Laboratory blood tests revealed the following: white blood cell (WBC) count 12. 2 × 109/L, monocytes 22. 0%, hemoglobin 7. 6 g/dL, platelets 10. 0 × 109/L, and fetal hemoglobin 12. 8%. A bone marrow aspirate revealed a hypercellular marrow with mild dysplastic changes and 4. 4% blast cells. The BCR–ABL fusion gene was not detected. Following a diagnosis of JMML, she subsequently developed respiratory failure due to leukemic infiltration of the lungs, and was referred to our hospital. On admission, she developed severe thrombocytopenia due to splenic sequestration of platelets, and she needed frequent transfusions. She received chemotherapy with cytarabine and 6-mercaptopurine. Pulmonary leukemic infiltration improved, but transfusion frequency could not be reduced. After she had undergone splenectomy, platelet transfusion was not needed. When her clinical condition had improved, KRAS mutation was investigated by bone marrow aspiration, and the KRAS 13G>D mutation was detected. Five months after diagnosis, she was transplanted with major mismatch blood type, HLA-A 1-allele mismatch, from an unrelated female donor. The conditioning regimen consisted of busulfan (BU; 16 mg/kg), fludarabine (Flu; 120 mg/m2), and cyclophosphamide (CY; 120 mg/kg). Short-term methotrexate and tacrolimus (FK506) were administered for the prevention of graft-versus-host disease. The level of infused donor marrow cells was 1. 18 × 108/kg. Recovery of peripheral blood count was rapid, and no regimen-related toxicity was observed. Chimerism by short tandem repeat analysis of bone marrow mononuclear cells on day 28 after UBMT was 100% recipient type, indicating graft rejection with autologous hematopoietic cell recovery. FK506 was then discontinued. From day 48 after UBMT until the current day, WBC count has been almost 10. 0 × 109/L. Despite graft rejection, the KRAS 13G>D mutation was not detected by bone marrow aspiration on day 219, and her peripheral blood counts were normalized. Four years after diagnosis, the KRAS 13G>D mutation in the peripheral blood, nails, buccal mucosa, and hair was not detected, but the KRAS13G>D mutation was not. She has been managed without treatment and remained in complete remission for over 5 years since receiving UBMT. DiscussionIn JMML patients with specific RAS mutations, spontaneous improvement in hematologic abnormalities has been reported. HSCT was needed in this case because the patient developed respiratory failure due to pulmonary infiltration of JMML cells. In JMML patients with gene mutation, JMML-specific gene mutations could not be detected after engraftment of HSCT. In contrast, most JMML cases relapse and need a second HSCT after rejection of the first. However, this patient's condition normalized after rejection of UBMT. Nowadays, minimal residual disease in JMML is analyzed by detection of JMML cell-specific gene mutations. The KRAS mutation can be detected in spontaneously regressed JMML following hematological improvement. We suggest that a myeloablative conditioning regimen including BU, Flu, and CY could eradicate JMML clones, and in some JMML cases, this could prevent the need for a second HSCT after rejection of the first. Disclosures:No relevant conflicts of interest to declare.

Unrelated Donor Cord Blood Transplantation for Children with Severe Sickle Cell Disease: Results of One Cohort from the Phase II Study from the Blood and Marrow Transplant Clinical Trials Network (BMT CTN)

The Sickle Cell Unrelated Donor Transplant Trial (SCURT trial) of the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) is a phase II study of the toxicity and efficacy of unrelated donor hematopoietic cell transplantation in children with severe sickle cell disease (SCD) using a reduced-intensity conditioning regimen. Here we report the results for the cord blood cohort of this trial. Eight children with severe SCD underwent unrelated donor cord blood transplantation (CBT) following alemtuzumab, fludarabine, and melphalan. Cyclosporine or tacrolimus and mycophenolate mofetil were administered for graft-versus-host disease (GVHD) prophylaxis. Donor/recipient HLA match status was 6 of 6 (n = 1) or 5 of 6 (n = 7), based on low/intermediate-resolution molecular typing at HLA -A, -B, and high-resolution typing at -DRB1. Median recipient age was 13.7 years (range: 7.4-16.2 years), and median weight was 35.0 kg (range: 25.2-90.2 kg). The median pre-cryopreservation total nucleated cell dose was 6.4 × 10(7) /kg (range: 3.1-7.6), and the median postthaw infused CD34 cell dose was 1.5 × 10(5) /kg (range: 0.2-2.3). All patients achieved neutrophil recovery (absolute neutrophil count >500/mm(3)) by day 33 (median: 22 days). Three patients who engrafted had 100% donor cells by day 100, which was sustained, and 5 patients had autologous hematopoietic recovery. Six of 8 patients had a platelet recovery to >50,000/mm(3) by day 100. Two patients developed grade II acute GVHD. Of these, 1 developed extensive chronic GVHD and died of respiratory failure 14 months posttransplantation. With a median follow-up of 1.8 years (range: 1-2.6), 7 patients are alive with a 1-year survival of 100%, and 3 of 8 are alive without graft failure or disease recurrence. Based upon the high incidence of graft rejection after unrelated donor CBT, enrollment onto the cord blood arm of the SCURT trial was suspended. However, because this reduced-intensity regimen has demonstrated a favorable safety profile, this trial remains open to enrollment for unrelated marrow donor transplants. Novel approaches aimed at improving engraftment will be needed before unrelated CBT can be widely adopted for transplanting patients with severe SCD.

Autologous Hematopoietic Recovery with Aberrant Antigen Expression after Allogeneic Bone Marrow Transplantation

A 50-year-old woman was admitted in December 2006 with progressive petechiae. Bone marrow (BM) aspiration showed massive proliferation of leukemic myeloblasts with myeloperoxidase staining. Three-color flow cytometry (FCM) with a CD45 gate for the BM cells was performed in our laboratory ; the blasts were positive for CD7, CD11c, CD13, CD15, CD33, myeloperoxidase and HLA-DR and negative for CD34 and CD117. Karyotypes of the BM cells were normal. A diagnosis of acute myeloblastic leukemia (AML)M2 was made on the basis of French-American-British classification. The patient needed standard induction chemotherapy twice to achieve complete remission. Refractoriness to platelet transfusions due to anti-HLA antibody developed. She received an allogeneic BM transplant from an ABOmatched and HLA-DR-mismatched unrelated female donor in September 2007. The conditioning regimen consisted of total body irradiation (2 Gy twice daily for 3 days) followed by cyclophosphamide (60 mg/kg/day for 2 days). On day 0, 4.0 × 10 BM cells per recipient body weight were infused. Tacrolimus and short-course methotrexate were used as prophylaxis for graft-versus-host disease. After BM transplantation, severe pancytopenia persisted. BM aspiration on day 22 revealed marked hypocellularity, suggesting graft rejection. Peripheral blood neutrophils gradually increased from day 50. BM aspiration smears on day 75 showed recovery of the BM cells, especially myeloid cells, without significant morphologic abnormalities and proliferation of myeloblasts, indicating that complete remission was maintained. Chromosomal analysis of the BM cells showed various cytogenetic abnormalities (Table 1). A chimerism-based analysis of the BM cells using short tandem repeat-polymerase chain reaction showed that 100% of the cells originated from the recipient. A diagnosis of autologous (recipient) hematopoietic recovery after graft rejection was made. Multiparametric FCM based on a four-color method (ReproCELL, Yokohama, Japan), which had been approved by the Jichi Medical University Institutional Review Board (no. 06-70), did not show the abnormal expression of antigens in the blasts in the BM on days 75 and 183. However, it showed small populations of CD34CD7 cells and CD34CD15 cells in the blasts on day 253 (Fig. 1). The latter cells were characterized by a high intensity of CD34, which indicates abnormal expression of the antigen. BM aspiration was performed every three to six months ; various chromosomal abnormalities of BM cells were found in each sample (Table 1). After day 253, routine three-color FCM instead of the multiparametric FCM was conducted to analyze phenotypes of the blasts in the BM. This FCM can detect CD34CD7 cells but not CD34CD15 cells because of antibody combinations. The proportions of CD34CD7 cells among the blasts of the BM were as follows : on day 323, 37.2% ; on day 421, 30.9% ; on day 603, 35.5% ; on day 785, 21.5% ; on day 975, 49.2% ; on day 1,149, 36.0% ; and on day 1,232, 24.5%. Although chromosomal abnormalities were detected on and after day 75, obvious dysplastic features associated with myelodysplastic syndrome (MDS) were not found. The patient is clinically well with normal peripheral blood cell counts. There are several reports on autologous (recipient) hematopoietic recovery after allogeneic hematopoietic stem cell transplantation. All patients received total body irradiation

T-Cell Replete Haploidentical Transplantation Using Post-Transplant Cyclophosphamide Results in Equivalent Non-Relapse Mortality and Disease-Free Survival Compared to Transplantation From HLA-Identical Sibling and Matched Unrelated Donors: A Stratified Cox Model Analysis of Two Hundred and Sixty Contemporaneous Allogeneic Transplants From a Single Center

Abstract 833HLA-identical siblings (MRD) or 10 of 10 HLA - A, B, C, DRB1 and DQB1 matched unrelated volunteers (MUD) are considered optimal donors for patients who may benefit from allogeneic hematopoietic cell transplantation (allo-HCT). However, many patients, particularly those from ethnic minorities and mixed race backgrounds, will lack such donors.HLA-haploidentical (Haplo) relatives are available for virtually all patients. Traditional approaches to allo-HCT using Haplo donors have entailed stringent ex-vivo T-cell depletion and intense preparative regimens. Such transplants have been associated with slow immune reconstitution and a high-risk of opportunistic infections and non-relapse mortality (NRM). Recently, another approach to haploidentical transplantation using T-replete grafts and post-transplant cyclophosphamide (Cy) to prevent graft versus host disease (GVHD) and rejection has demonstrated promising results (Brunstein et al Blood 2011, vol118, p 282).Forty-Five patients underwent first allografts with a Haplo donor at our center between 02/05 and 08/10 (median age=46, range 20–73; diagnoses were AML 14, ALL 6, CML 4, MDS 4, CLL 7, NHL 4, HD 6; median KPS 90, range 70–90; CIBMTR disease risk score low 14, intermediate 7, high 24; median hematopoietic cell transplant co-morbidity index (HCTCI) score = 1, range 0–5; prior autotransplant = 7). Twenty-seven patients received a reduced-intensity conditioning regimen (RIC): fludarabine 30 mg/m2/d d −6 to −2; TBI 200cGy d-1, Cy 14.5 mg/kg/d on d-6,-5 and 50mg/kg/d on d+3,+4 with a bone marrow graft. Eighteen patients received myeloablative conditioning where the above regimen was modified by replacing low-dose TBI with iv busulfan 110–130 mg/m2/d × 4 days and bone marrow was replaced with G-CSF mobilized PBSC as the graft. Two patients receiving RIC failed to engraft and developed autologous hematopoietic recovery. All other patients engrafted with median 100% donor T-cell chimerism from initial testing at d+30. Outcomes of the Haplo transplants were compared to contemporaneous first allografts from MRD (n=115) and MUD (n=99) at our center. Median follow-up for surviving patients was 36 months (range 9–74.5 months). Cumulative probability of NRM at 36 months was 16%, 19% and 8% for MRD, MUD and Haplo respectively (p=NS) (see Fig). For both overall survival (OS) and disease-free survival (DFS), the effects of patient and disease characteristics were evaluated in Cox models with three transplant groups as strata. HCTCI score, CIBMTR risk category, type of diagnosis were identified as significant covariates on both OS and DFS, while age was significant on DFS only. The adjusted probabilities of OS and DFS were estimated from the stratified Cox models, weighted by the proportions of the significant covariates in the pooled sample. MRD had statistically superior OS to Haplo at 18 months (79% vs. 61%, p=0.033 in point wise comparison). However, adjusted OS was not significantly different between the three types of transplant at 36 months (71%, 58% and 58% for MRD, MUD and Haplo respectively (p=NS)). Adjusted DFS at 36 months was 47%, 46% and 55% respectively (p=NS) (see Fig). Ninety-three patients (35.7%) suffered relapse or progression of their malignancy at a median of 154 days post transplant (range 12–1445 days). Estimated cumulative incidence of relapse at 36 months was 35%, 38% and 36% respectively for MRD, MUD and Haplo (p=NS) (see Fig). The inverse probability of censoring weighted (IPCW) method was used to determine survival following relapse/progression in each group. Estimated survival at 1 year following relapse was 71%, 64% and 15% (p<0.0001 pointwise comparison for MRD vs. Haplo and MUD vs. Haplo).This study demonstrates that excellent NRM and DFS can be achieved using haploidentical donor transplantation with the above regimens. These outcomes are equivalent to those obtained in contemporaneously transplanted MRD and MUD at our center. T-cell replete haploidentical transplant is therefore a valid alternative for patients who lack a conventional donor. Survival following relapse/progression is inferior following haploidentical than conventional donor transplants. This may at least partly be related to the difficulty in using DLI following relapse in haploidentical transplants. Future strategies to improve outcomes following haploidentical transplants should focus on treatment approaches following relapse. [Display omitted] [Display omitted] Disclosures:Bashey:Otsuka America Pharmaceuticals, Inc: Research Funding. Sizemore:Otsuka America Pharmaceuticals, Inc: Research Funding. Manion:Otsuka America Pharmaceuticals, Inc: Research Funding. Brown:Otsuka America Pharmaceuticals, Inc: Research Funding. Holland:Otsuka America Pharmaceuticals, Inc.: Research Funding. Solomon:Otsuka America Pharmaceuticals, Inc: Research Funding. Morris:Otsuka America Pharmaceuticals, Inc: Research Funding.

Infusion of “off-the-shelf” Third Party Ex Vivo Expanded Cord Blood Progenitor Cells As Supportive Care Following Clofarabine with High Dose Cytarabine and Granulocyte Colony-Stimulating Factor Priming for the Treatment of AML,

Abstract 3640Regardless of patient age, prolonged neutropenia and infectious complications are common side effects of AML induction and salvage chemotherapy, requiring intensive supportive care and contributing to treatment failure. Thus, novel therapies that can abrogate prolonged pancytopenia and facilitate more rapid hematopoietic recovery are needed. Previously, we have demonstrated that the absolute number of repopulating cord blood (CB) hematopoietic stem/progenitor cells (HSPC) can be increased by culture with Notch ligand; recent data from a Phase I double CB transplant trial utilizing ex vivo expanded CB HSPC has suggested more rapid neutrophil recovery (Delaney et al, Nat med). We hypothesize that this expanded cell product, which is devoid of T cells, could be cryopreserved and then infused as an off-the-shelf non-HLA matched product to provide rapid but temporary donor myeloid engraftment and might also facilitate autologous hematopoietic recovery following AML therapy, thus reducing the infectious complications associated with therapy.In a cohort of 50 patients, Becker et al at the Fred Hutchinson Cancer Research Center have reported on the use of clofarabine and high dose ara-c, in combination with G-CSF (GCLAC) in a phase I/II trial for the treatment of relapsed/refractory AML (Becker et al, Br. J. Haematol, in press). GCLAC is profoundly immunosuppressive and myelosuppressive, with periods of prolonged neutropenia post-GCLAC ranging from 17 to 35 days (median time to ANC >500 is 21 days) in this cohort. Infection was the most frequent adverse event, with ≥ grade 3 bacterial and fungal infections seen in 40% of patients (including 1st and ≥ 2nd salvage patients). We now herein report the preliminary results of a phase I safety trial investigating the use of GCLAC plus infusion of off-the-shelf expanded and cryopreserved CB HSPC for patients with AML.To date, 12 adult patients with relapsed (n=8), primary refractory (n=2) or de novo high risk (n=2) AML have been enrolled and received GCLAC as their first salvage or initial induction therapy. The median age and weight was 55 years (range, 38 to 59 years) and 79.5 kg (range, 63.8 to 126) respectively. Patients received cycle 1 of GCLAC followed by infusion of off-the-shelf expanded CB HSPC 24 hours after completion of chemotherapy. Patients who achieved a complete morphologic remission and did not experience any toxicities associated with the expanded cell product were eligible to receive a 2nd cycle of GCLAC with cells if not proceeding directly to stem cell transplant. A total of 15 products have been infused in 12 patients. Five of the 12 patients were refractory to GCLAC and therefore non-evaluable for neutrophil recovery. The median time to achieve an ANC > 500 in the evaluable patients was 19 days (range 18 to 27) after cycle 1, as compared to 21 days (range 17 to 35) in the historical cohort of patients receiving GCLAC only without expanded cells. Importantly, in this phase I study, there have been no infusional toxicities from the expanded cells or adverse events attributed to the expanded cell product. Alloimmunization has not been observed as monitored by pre- and post-infusion panel reactive antibody assays to determine the presence of donor directed antibodies.Four of the 12 enrolled patients have experienced clinically significant infections (e.g., bacteremia, fungal infections) compared to 40% in the comparison cohort, and no infectious deaths have been observed. Finally, in contrast to the first cycle of chemotherapy where donor cells were detected in only one patient, 4 out of 4 patients who received a 2nd cycle of GCLAC with expanded cells were found to have transient donor contribution to myeloid recovery (by peripheral blood cell sorted DNA chimerism assay) one week after infusion of the cells ranging from 85 to 100% donor in the CD33/CD14 cell lineages. Expanded cells were also able to home to the marrow as evidenced by transient donor engraftment (range, 3 to 15%) in recipient marrows one week after expanded cell infusion. It is unclear whether this is related to increased marrow “space” or increased host immunosuppression following cycle 2. With this encouraging data, we are continuing accrual of newly-diagnosed patients. This approach may decrease complications of pancytopenia and allow delivery of more dose-intense therapy, particularly in patients with intermediate/favorable cytogenetics who are already relatively sensitive to GCLAC. Disclosures:Delaney:Sanofi-Oncology (formerly Genzyme): Clofarabine for the study supplied by Sanofi-Oncology (formerly Genzyme). Becker:Sanofi-Oncology (formerly Genzyme): Research Funding.

Therapeutic granulocyte transfusions for the treatment of febrile neutropenia in patients with hematologic diseases: a 10-year experience at a single institute

This single-center 10-year retrospective study assessed clinical efficacies and adverse events and determined prognostic factors in patients with hematologic disease and febrile neutropenia treated with granulocyte transfusions (GT) from unrelated healthy donors stimulated with recombinant human granulocyte-colony-stimulating factor (rhG-CSF) and dexamethasone. Between September 1999 and June 2009, 1027 therapeutic GT were performed for the treatment of 170 episodes of febrile neutropenia in 157 patients. Efficacy analysis included 979 GT for 138 episodes in 128 patients who received at least three GT per episode. Adverse event analysis included all patients who received at least one GT. The median granulocyte dose was 0.96 × 10(9)/kg/transfusion (range 0.47-1.80 × 10(9)/kg/transfusion). Infection was controlled in 73 episodes (52.9%). The 28-day infection-related survival rate was 64.7 ± 4.1%. The dose of granulocytes transfused did not correlate with clinical outcome. Multivariate analysis revealed that septic shock and pneumonia/multiple primary infection sites were related to infection control failure. Furthermore, refractory underlying disease and septic shock were associated with shorter infection-related survival. Massive hemoptysis (3.5%) and respiratory failure (5.9%) occurred in a few patients. Prior pneumonic infiltration, azotemia and a larger volume of daily GT were associated with serious respiratory complications. GT therapy is a viable adjunctive treatment option for febrile neutropenia as a bridge to autologous hematopoietic recovery in patients with hematologic disease with tolerable toxicity. GT therapy requires close monitoring in patients with prior pneumonic infiltration and azotemia. It is recommended that transfusion with higher volumes is avoided.

Disease-specific hematopoietic cell transplantation: nonmyeloablative conditioning regimen for dyskeratosis congenita

Dyskeratosis congenita (DC) is characterized by reticular skin pigmentation, oral leukoplakia and abnormal nails. Patients with DC have very short telomeres and approximately one-half have mutations in telomere biology genes. A majority of patients with DC develop BM failure (BMF). Hematopoietic cell transplantation (HCT) represents the only known cure for BMF in DC, but poses significant toxicities. We report six patients who underwent allogeneic HCT with a novel nonmyeloablative conditioning regimen specifically designed for DC patients. Graft sources included related PBSCs (1), unrelated BM (2) and unrelated double umbilical cord blood (3). Complete donor engraftment was achieved in five of six patients. One patient had initial autologous hematopoietic recovery, which was followed by a second transplant that resulted in 88% donor chimerism. With a median follow-up of 26.5 months, four patients are alive, three of whom were recipients of unrelated grafts. We conclude with this small study that encouraging short-term survival can be achieved with HCT in patients with DC using a preparative regimen designed to promote donor engraftment and minimize life-threatening disease-specific complications such as pulmonary fibrosis. Long-term follow-up will be crucial with respect to individualized patient care with each of the transplanted individuals.

Pentostatin and Alemtuzumab: An Immunosuppressive Nonmyeloablative Preparative Regimen for Allogeneic Peripheral Blood Stem Cell Transplantation (PBSCT).

Abstract 4312 IntroductionCurrent nonmyeloablative and reduced-intensity preparative regimens for allogeneic (allo) PBSCT are associated with significant myelosuppression and neutropenia. We evaluated a novel, purely immunosuppressive preparative regimen of continuous-infusion pentostatin (PEN) plus alemtuzumab (ALEM) in patients (pts) undergoing related and unrelated allo PBSCT for high-risk hematological malignancies. Patients and Methods12 pts (median age, 58.5 yr; range, 23-69 yr; 7 male, 5 female) with high-risk, relapsed or refractory acute myelogenous leukemia (AML; 5 pts), Hodgkin's disease (HD; 2 pts), multiple myeloma (MM; 2 pts), chronic lymphocytic leukemia (CLL; 1 pt), low-grade non-Hodgkin's lymphoma (NHL; 1 pt) or myelodysplastic syndrome (1 pt) were enrolled on this study; 6 pts had failed previous autologous PBSCT. The median interval between most recent conventional chemotherapy and allo PBSCT was 3.5 months (range, 1.5-19.5 months). Pts received continuous-infusion PEN (12 mg/m2 over 72 hr, days -8 through -6) and ALEM (20 mg/day, days -5 through -1) followed by allo PBSCT from 8/8 HLA-matched related (3 pts) or unrelated (9 pts) donors. All pts received single-agent cyclosporine (CSP) for prophylaxis of acute graft-versus-host disease (GVHD). ResultsMedian cell doses were 5.45 (range, 1.86-7.90) x 106 CD34+ cells/kg and 2.71 (range, 1.40-4.46) x 108 CD3+ cells/kg. Ten pts had asymptomatic transient elevations of AST and ALT (CTCAE grade 2-3) between days -6 and +21. No pts developed mucositis. Median duration of post-PBSCT hospitalization was 7 days (range, 3-62 days). The median nadir of the absolute neutrophil count (ANC) was 1.24 (range, 0.55–5.11) x 109/L at day +15, and the median nadir of the platelet count was 70 (range, 16-244) x 109/L at day -1. In contrast, the median nadir of the absolute lymphocyte count (ALC) was zero (range, 0-0.72) x 109/L at day -4. ALC remained low at day +28 (median, 0.09 × 109/L; range, 0.05-0.91 × 109/L) and increased by day +100 to a median of 0.34 × 109/L (range, 0.23-2.63 × 109/L). One pt developed grade 2 acute GVHD at day +21. No pts developed chronic GVHD. Evaluation of chimerism in bone marrow at day +28 showed that 3 pts had zero donor DNA and 9 pts had a median of 17% (range, 3-68%) donor DNA. At day +100, 2 of 8 evaluable pts (one with zero and one with 13% donor DNA at day +28) had zero donor DNA in bone marrow, and 6 pts had a median of 64% (range, 21.3-100%) donor DNA. Cumulative actuarial probability of sustained engraftment (defined as at least 50% donor DNA in bone marrow) is 53.4%. Doses of infused CD34+ cells or CD3+ cells were not statistically significantly different in pts with or without allogeneic engraftment (P=0.34, Mann-Whitney test). Three pts had asymptomatic reactivation of cytomegalovirus at 5, 11, and 35 days, respectively, after allo PBSCT. Two pts developed disseminated toxoplasmosis, which was fatal in 1 pt. One pt developed Mucor sinusitis and died with Enterococcus sepsis. One pt developed nonfatal Nocardia pneumonitis and sepsis at 175 days after allo PBSCT. Relapses occurred in 5 pts (1 AML, 1 CLL, 1 HD, 2 MM) at a median of 106 days (range, 28-360 days) after allo PBSCT; 1 of these pts had had engraftment failure and autologous hematopoietic recovery. The actuarial probability of relapse is 56.4%. Seven pts died at a median of 60 days (range, 44-347 days) after allo PBSCT: 1 relapse of HD, 2 infection (1 toxoplasmosis, 1 Enterococcus sepsis), 1 pulmonary hemorrhage with multiorgan failure, and 3 refractory acute GVHD that occurred following treatment of relapse with donor lymphocyte infusion (1 pt), withdrawal of CSP (1 pt) or second allo PBSCT (1 pt). Four subjects (2 AML, 1 HD, 1 NHL) are alive in remission at a median of 513 days (range, 267-787 days) after allo PBSCT. Actuarial probabilities of overall survival and event-free survival are 40.0% and 31.3%, respectively. ConclusionsA preparative regimen of continuous-infusion PEN plus ALEM can allow engraftment of HLA-matched allogeneic PBSCs without significant myelosuppression. However, engraftment failure is greater after this purely immunosuppressive regimen than with standard nonmyeloablative or reduced-intensity regimens. Relapsed or refractory disease at time of allo PBSCT, the absence of cytoreduction with this preparative regimen, and potentially a decrease in graft-versus-tumor effect as a result of profound and prolonged immunosuppression are factors that contribute to the relapse rate observed in this study. Disclosures:Off Label Use: Pentostatin administered as a continuous infusion according to FDA IND #67,067.