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Hematopoietic cell transplantation (HCT) is the only readily available cure for many life-threatening pediatric nonmalignant diseases (NMD), but most patients lack a matched related donor and are at higher risk for graft-versus-host disease (GVHD). Use of abatacept (Aba) to target donor T-cell activation has been safe and effective in preventing GVHD after unrelated donor (URD) HCT for malignant diseases (Aba2 trial). Our primary objective was to evaluate the tolerability of Aba added to standard GVHD prophylaxis (cyclosporine and mycophenolate mofetil) in pediatric patients with NMD undergoing URD HCT. In this single-arm, single-center phase 1 trial, 10 patients receiving reduced intensity or nonmyeloablative conditioning underwent URD HCT. Immune reconstitution was assessed longitudinally via flow cytometry and compared to pediatric patients on Aba2. Nine patients successfully engrafted, with 1 primary graft rejection in the setting of inadequate cell dose; secondary graft rejection occurred in 1 patient with concurrent cytomegalovirus viremia. Two deaths occurred, both unrelated to Aba. One patient developed probable posttransplant lymphoproliferative disease, responsive to rituximab and immune suppression withdrawal. No patients developed severe acute or chronic GVHD, and 8 patients were off systemic immunosuppression at 1 year. Immune reconstitution did not appear to be impacted by Aba, and preservation of naïve relative to effector memory CD4+ T cells was seen akin to Aba2. Thus, 4 doses of Aba were deemed tolerable in pediatric patients with NMD following URD HCT, with encouraging preliminary efficacy and supportive immune correlatives in this NMD cohort.

Background: Blood stream infections (BSI) are a major source of morbidity and mortality after allogeneic blood and marrow transplantation (BMT). In studies of risk for BSI various factors have been identified. The impact of cytomegalovirus (CMV) viremia, however, on risk has not been assessed. This is important since both CMV infection and ganciclovir (GCV), the mainstay of pre-emptive therapy, have myelosuppressive and immunosuppressive effects. We conducted a retrospective analysis to test the hypothesis that CMV viremia predisposes allogeneic BMT patients to BSI. Methods: We retrospectively analyzed 278 allogeneic BMTs performed at Children's Healthcare of Atlanta between January 1st 2005 and December 31st 2014 that met eligibility criteria. The primary outcome was the first episode of BSI occurring between day +30 and +100 seen in engrafted patients following allogeneic BMT. We compared clinical characteristics between patients with and without BSI. This analysis was based on a time dependent competing risk model. Risk events including acute GVHD and CMV viremia were counted only when they preceded the BSI. We performed a multivariate analysis to estimate the effect of CMV viremia on risk for BSI in the post-engraftment period (days +30-100). Results: The median age was 9 years (range 0-22 years). 44.6% of the patients were Caucasian, while 38.5% were African-American. 59.7% received an unrelated marrow, unrelated cord or the alternative donor transplant. 60.8% were transplanted for a hematologic malignancy. The cumulative incidence of BSI between day +30 and+100 was 21.9% (95% confidence interval (CI), 17.5-27.3%). The median day of BSI development was 54 (range 30-99). The leading cause of BSI was Staphylococcus epidermidis. Gram-positive cocci were responsible for 71.4% of all BSIs. In the multivariate analysis, three factors were significant: grade III/IV aGVHD (hazard ratio (HR)=3.490, 95% CI 1.530-7.980, P=0.003), CMV viremia (HR=3.410, 95% CI 1.410-8.250, P=0.007), and African-American ethnicity (HR=2.520, 95% CI 1.130-5.600, P=0.024). Form of insurance (Medicaid/no insurance vs. other), employed as a surrogate for income level, had no influence on risk. In the 57 patients with CMV viremia, the frequency of neutropenia ( Conclusion: Our analysis of risk for post-engraftment BSI in children yielded three factors. One, aGVHD, is well established. The other two, CMV viremia and African-American ethnicity, have not been previously recognized. That CMV viremia would predispose to subsequent BSI is highly plausible. Our results suggest that the risk of CMV viremia may be mediated in part by neutropenia stemming from CMV or its treatment with ganciclovir. That African-American ethnicity has not previously been recognized as a risk factor may simply reflect the ethnic make up of previous studies. Our findings are consistent with those of studies performed in other settings, showing patients of African descent to be at increased risk for bacteremia and sepsis. An increased risk for BSI among patients of African descent could partly explain the concerning excess risk for transplant related mortality previously observed among these patients. Disclosures No relevant conflicts of interest to declare.

Introduction: Major ABO mismatch occurs when the recipient has preformed isoagglutinins against the donor. Since unmanipulated bone marrow transplant (BMT) grafts contain donor red blood cells (RBCs), major ABO mismatch BMT infusions can cause hemolytic reactions that lead to renal dysfunction. To prevent this complication, RBC depletion of the graft can be performed by various techniques. Strong evidence-based recommendations regarding RBC depletion are lacking. In particular, it is unclear what residual volume of incompatible RBCs in the graft is safe, especially for pediatric patients. Methods: We conducted a retrospective cohort study of pediatric patients who had major ABO mismatch BMT at a single center. No patients had pre-BMT plasmapheresis to reduce isoagglutinin titers. RBC depletion was performed on the BMT grafts with either hydroxyethyl starch (HES) sedimentation or Ficoll density gradient separation. All patients received similar supportive care around the BM infusion that included hyperhydration and premedication with acetaminophen, diphenhydramine, and hydrocortisone. Serum creatinine the day after the BM infusion was compared to creatinine the day of the BM infusion. Since 2006 all patients had urine screened for blood post-BMT. Results: Sixty-three patients were identified who received a major ABO mismatch BMT between 9/2004 and 6/2015 (39 had RBC depletion with HES, 24 with Ficoll). Compared to Ficoll RBC depletion, patients who received BM grafts that had RBC depletion with HES received significantly more incompatible RBCs (Table 1). Hemoglobinuria was significantly more common in HES patients, but evidence of post-BM infusion renal impairment was not (Table 1). All patients had donor engraftment with a similar time to neutrophil engraftment for both groups (Table 1). Considering only the HES patients, 8/8 (100%) patients with >25% rise in creatinine had hemoglobinuria compared to 8/21(38%) patients with ≤25% rise in creatinine (p=0.003). Also among just the HES patients, the median amount of incompatible RBCs infused was not significantly different between patients with (0.74 ml/kg) and without (0.62 ml/kg) hemoglobinuria (p=0.42), or between patients with (0.56 ml/kg) and without (0.62 ml/kg) a >25% rise in creatinine (p=0.86). Table 1: Comparison of patients who had RBC depletion with HES vs. Ficoll Table 1.HES n=39Ficoll n=24p-valueMedian volume of RBCs/patient weight 0.62 ml/kg0.04 ml/kg25% rise in creatinine 8 (21%)5 (21%)1.0Number with >50% rise in creatinine 4 (10%)0 (0%)0.29Median day of neutrophil engraftment 20210.11 *considering only patients transplanted after 2006. Conclusion: Our study is the first to analyze pediatric major ABO mismatch BMT infusion hemolysis after two different RBC depletion techniques: HES and Ficoll. Our results suggest that RBC depletion with Ficoll achieves less residual RBCs in the BM graft and likely less resulting hemoglobinuria. However, after both techniques hemolysis-induced renal impairment is rare. The volume of residual incompatible RBCs in the infused BM graft does not appear to strongly determine if clinical hemolysis occurs. The volume that is safe likely depends on other variables that influence the risk of clinical hemolysis. Disclosures No relevant conflicts of interest to declare.

Insufficient expression of factor VIII (fVIII) is a major hurdle in the development of successful nucleic acid treatments for hemophilia. However, we recently showed that under myeloablative and reduced-intensity total body irradiation (TBI) conditioning, transplantation of hematopoietic stem cells (HSCs) transduced with recombinant retroviruses containing B domain–deleted porcine fVIII (BDDpfVIII) sequences provides curative fVIII levels in a hemophilia A mouse model. In the current study, we tested BDDpfVIII activity after nonmyeloablative conditioning with busulfan, cyclophosphamide, or fludarabine and immunosuppressive agents CTLA4-Ig + anti-CD40L or anti-(murine)thymocyte serum (ATS). ATS is similar in action to anti-(human)thymocyte globulin (ATG), which is used clinically with busulfan in bone marrow transplantations to increase donor cell engraftment. Mice conditioned with busulfan + ATS and that received a transplant of BDDpfVIII-transduced stem-cell antigen 1-positive cells exhibited moderate levels of donor cell chimerism (between 20% and 60%) and achieved sustained fVIII levels more than 1 U/mL. Similar results were observed in mice preimmunized with human fVIII and conditioned with 5 Gy TBI + ATS or busulfan + ATS. These data demonstrate that it is possible to achieve sufficient fVIII expression after transplantation of BDDpfVIII-transduced HSCs following low-toxicity pretransplantation conditioning with targeted immunosuppression, potentially even in the context of preexisting inhibitors.

Abstract 3487 Hematopoietic stem cell transplantation (HSCT) is associated with poor response to platelet (PLT) transfusions. It has been posited that vascular endothelial cell (EC) damage from the effects of high dose chemoradiotherapy is responsible for PLT consumption. To test this hypothesis, we assessed the relationship between serial levels of plasma markers of vascular damage and repair, and PLT transfusion requirement. Plasma samples were obtained on 40 adult and pediatric recipients of allogeneic HSCT with myeolablative conditioning for leukemia, lymphoma, or myelodysplastic syndrome at days -10, +5, +15, and +30. Single donor apheresis PLT transfusions were given prophylactically for PLT count 125% of ideal BW, adjusted BW was calculated as 0.25 × (actual – ideal BW) + ideal BW. The influence of response to PLT transfusions prior to transplant admission was considered. Poor response to PLT transfusion was defined as a PLT count rise of Disclosures: Ofori-Acquah: Emory University: Patents & Royalties.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) for sickle cell disease (SCD) provides curative therapy but carries significant risk of transplant-related morbidity and mortality, graft failure, and disease recurrence. Past trials of myeloablative HSCT with busulfan in SCD patients showed a 10–13% rate of rejection and 8–9% rate of partial chimerism with busulfan steady-state concentration (Css) targeted to 400–600 ng/ml (area under curve (AUC) 575–877 mmol•min/L). In thalassemia and other diseases, total systemic exposure to busulfan is a critical determinant of engraftment yet is limited by risk of toxicity. Therapeutic drug monitoring (TDM) is used to measure variability in individual patient pharmacokinetics and allows dose adjustments to achieve desired level of busulfan exposure. A retrospective analysis of busulfan AUC in SCD patients during HSCT was performed to determine if higher systemic busulfan levels resulted in more effective engraftment or increased toxicities. METHODS: A retrospective review of busulfan pharmacokinetics, engraftment status, and clinical toxicities in a cohort of SCD patients who received MSD bone marrow grafts at a single pediatric institution was performed. All patients received a myeloablative preparative regimen with busulfan (initially dosed for total 14 mg/kg), cyclophosphamide (200 mg/kg), antithymocyte globulin (90 mg/kg), and graft versus host disease prophylaxis with methotrexate and cyclosporine. Anticonvulsant prophylaxis with phenytoin or with levitiracetam plus lorazepam was given during busulfan administration. Busulfan was administered every 6 hours for 16 doses. Busulfan AUC was determined for the first dose and adjustments were made on subsequent doses if needed. Total AUC was calculated as a weighted average of total doses. Target AUC varied widely over the study period; however, for the last 3 years, the intended AUC has been targeted to 900–1100 mmol•min/L (Css 618–753 ng/ ml). Busulfan-associated toxicities recorded were: hepatic veno-occlusive disease (VOD), interstitial pneumonitis (IP), and seizure during or 1 day following busulfan administration. RESULTS: Twenty-seven consecutive hemoglobin SS SCD patients received HSCT with MSD between December 1993 and August 2007. All patients transplanted since May 1996 (n=25) had busulfan TDM performed. Median age was 8.8 years (range 3.3–17.4 years). Event-free survival was 96% with a median follow-up time of 3.9 years. Engraftment occurred in all cases with no subsequent graft rejection. Full donor chimerism (>95% donor leukocytes) was seen in 21 (84%) patients, and high partial donor chimerism (50–95% donor) in 4 (16%) at >12 months post-HSCT. Busulfan was administered orally in 18 (72%) and intravenously in 7 (28%). Dose adjustments were made in 17 patients (8 increased, 9 decreased). Median dose increase was 16.3% (range 9.7–76.5%), and median decrease was 14.3% (range 4.2–57.1%). Five (20%) patients had dose alterations >20% of initial dose (2 increased, 3 decreased). Median total dose received was 13.9 mg/kg (range 10.8–24.0). Median AUC for first dose was 968 mmol•min/L (range 595–1379) and median total AUC was 992 mmol•min/L (range 780–1305). Busulfan-associated toxicities were observed in 9 (36%) patients: 8 with mild-moderate VOD, 1 with IP. No seizures occurred during busulfan administration. Patients with busulfan-associated toxicity had mean total AUC of 1044.7 mmol•min/L (range 821–1305) versus 962.4 mmol•min/L (range 780–1184) in those without toxicity (p=0.185). Patients with partial donor chimerism had lower total AUC (mean 861.5 mmol•min/L, range 780–932) compared to those with full donor chimerism (mean 1017 mmol•min/L, range 885–1305; p=0.0379) (Figure). Since the implementation of a target busulfan AUC range of 900–1100 mmol•min/L, all patients (n=10) achieved full donor chimerism. CONCLUSIONS: We conclude that higher busulfan levels are associated with decreased incidence of partial donor chimerism in SCD patients during myeloablative HSCT with MSD bone marrow grafts. A target AUC range of 900–1100 mmol•min/L, attainable with TDM and dose adjustment, provides effective engraftment without increased risk of busulfan-related toxicity. Figure: Total busulfan AUC in patients with full donor chimerism (mean 1017 mmol•min/L) vs. partial chimerism (mean 861.5 mmol•min/L, p=0.0379). Figure:. Total busulfan AUC in patients with full donor chimerism (mean 1017 mmol•min/L) vs. partial chimerism (mean 861.5 mmol•min/L, p=0.0379).

A major barrier to successful gene therapy for hemophilia A using bone marrow cells is achieving engraftment of a sufficient number of gene-modified cells to result in therapeutic factor VIII (fVIII) levels under conditions of acceptable patient risk. Previously, we demonstrated sustained levels of a high-expression porcine (HEP)-fVIII construct, at greater than 1 unit/mL for over one year, in hemophilia A mice following myeloablative HSC transplantation (HSCT) thus overcoming the barrier of low fVIII expression (Gangadharan et al, Blood, 2006). In the current study, we tested HEP-fVIII expression after several non-myeloablative and reduced-intensity stem cell transplantation (RIST) protocols incorporating a clinically-relevant dose of genetically-modified hematopoietic stem cells transduced at low multiplicity of infections of ≤ 2. Recipient fVIII exon 16-knockout mice received 3 × 105 HEP-fVIII-MSCV transduced, congenic stem cell antigen-1+ cells. Mice were conditioned using one of the following regimens: 5.5 Gy total body irradiation (TBI), 2 Gy TBI, 2 Gy TBI + 200 mg/kg cyclophosphamide (Ctx), 200 mg/kg Ctx, 200 mg/kg Ctx + 90 mg/kg fludarabine (Flu), 35 mg/kg busulfan (Bu), 35 mg/kg Bu + 200 mg/kg Ctx, 35 mg/kg Bu + costimulation blockade (0.5mg/day CTLA4-Ig and α-CD40L on days 0, 2 post-transplantation). Resulting donor cell engraftment, HEP-fVIII activity, and inhibitory anti-fVIII antibody levels were followed. No fVIII activity was seen in the 2 Gy, 2 Gy + Ctx, Ctx only, or Ctx + Flu cohorts. The Bu and Bu/Ctx treated animals expressed therapeutic levels of HEP-fVIII (0.05 ± 0.07 U/mL and 0.81 ± 0.37 U/mL, respectively) at 1 wk post-HSCT, but returned to baseline in all mice by 2 wks with inhibitor formation in 3 of 7 mice in the Bu group and 0 of 4 mice in the Bu/Ctx cohort. In order to suppress inhibitor formation following conditioning with Bu, recipient mice were administered a low dose course of costimulation blockade post-HSCT. Four of 10 mice in this group demonstrated therapeutic levels of HEP-fVIII activity on day 34-post HSCT (0.11 ± .09 U/mL). All animals demonstrated donor cell engraftment in peripheral blood mononuclear cells at day 23 post-HSCT (18.4 ± 12.1%) and at 34 days post-HSCT, none of the animals had detectable levels of fVIII inhibitors in a modified Bethesda assay. Additionally, we were able to identify specific hematopoietic lineages expressing HEP-fVIII in recipient mice using a novel enzyme-linked immunospot (ELISPOT) assay developed in our laboratory. These data demonstrate that it is possible to achieve sufficient fVIII expression under conditions of i) low MOI transduction with an optimized high-expression fVIII transgene, ii) mild pretransplantation conditioning, iii) limited genetically-modified HSC engraftment, and iv) mild post-HSCT immunosuppression. Furthermore, our results show that clinically-significant fVIII activity levels can be maintained without inhibitor formation suggesting that the current protocol may be on the threshold, in terms of the level of immunosuppression, of promoting tolerance to HEP-fVIII.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to specifically stimulate proliferation and differentiation of CD34+ hematopoietic progenitor cells. Although STAT3 was thought to be essential for the transduction of GM-CSF-induced cell proliferation, the downstream signaling mediated by STAT3 to support cell proliferation and growth has not been completely understood. Because the inhibitor of apoptosis protein (IAP) survivin is believed to regulate cell proliferation and survival via its anti-apoptotic function, we chose to study the link between STAT3 signaling and survivin expression in CD34+ cells. We constructed plasmids containing the survivin promoter sequence and performed luciferase reporter assay in CD34+ KG-1 cells stimulated with GM-CSF. These experiments showed that GM-CSF stimulated survivin promoter activity. Chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) revealed that STAT3 binds to the core survivin promoter containing a STAT response element (SRE) TT(N)5AA at sites −264 to −256. Mutation or deletion of this SRE completely abolished the effect of GM-CSF on survivin promoter activity. Furthermore, specific JAK inhibitor and STAT3 siRNA inhibited GM-CSF-induced survivin promoter activity and survivin expression. Inhibition of survivin by STAT3 siRNA or by withdrawal of GM-CSF in a GM-CSF-dependent CD34+ line TF-1 resulted in decreased cell growth and induction of apoptosis. These results suggest that the anti-apoptotic protein survivin is a transcriptional target of STAT3, and that GM-CSF stimulated-CD34+ cell proliferation is regulated by the JAK/STAT3/survivin signaling pathway.

Although the cytotoxic effect of vitamin K3 (VK3) on human cancer cells has been repeatedly reported, no clear conclusions from either in vitro or in vivo tests have so far been made for VK3 as an anticancer agent due to marked inter-tumor variability of efficacy in response to VK3 treatment. Here, we report that sensitivity of neoplastic cells to VK3-induced killing depends on IKKα expression/NF-kB activation in the cells. We tested the sensitivity to VK3 of 14 leukemic cell lines established from children with acute lymphoblastic leukemia (ALL). The 14 lines were classified into three groups: IKKα +/NF-kB+, IKKα +/NF-kB−, IKKα−/NF-kB−. IKKα +/NFkB+ cell lines that are generally resistant to doxorubicin are more sensitive to VK3 induced cell death than are the IKKα +/NFkB− lines that are usually sensitive to doxorubicin. The median of IC 50 values of VK3 and doxorubicin as tested by WST analysis for IKKα +/NFkB+ cells were 3.92 mM and 1.58 mM, respectively, compared to IKKα +/NFkB− cells (7.3 mM of VK3 and 0.71 mM of doxorubicin, p

UCBT may be curative in patients (pts) with high-risk SCD. Unrelated cord blood units matched at >4/6 HLA are acceptable for UCBTs and available to most pts. We reviewed UCBTs in 7 children with SCD and cerebrovascular accidents performed in 4 centers (1998 to 2003): 4 pts were conditioned with conventional myeloablative regimens, and 3 pts with reduced-intensity regimens. The former received busulfan (BU), cyclophosphamide (CY) and anti-thymocyte globulin (ATG) (3 pts) or BU/CY/ATG plus fludarabine (FLU) (1 pt). Of 3 pts who received 4/6 HLA-matched UCBTs after BU/CY/ATG and GVHD prophylaxis with methylprednisone (MP) and cyclosporine (CSP) or tacrolimus (TAC), 2 have durable engraftment, 1 had autologous reconstitution (BMT2004; 34:405–11). A 5 yr old (yo) female with transfusion-induced alloimmunization received a 5/6 HLA-matched UCBT (6.2 X 10^7/kg nucleated [NC] cells) after BU/CY/ATG/FLU, followed by CSP and MP post-transplant. PMN engraftment and VNTR > 98% of donor origin were documented 24 and 37 days, respectively, after UCBT. Complications included elevated liver enzymes during conditioning, grade IV GVHD of skin, GI and liver, grade III mucositis, VOD, parainfluenza 3 infection, candidemia and adenovirus viremia. Pt died of severe acute GVHD and multi-organ failure 73 days after UCBT. Two different reduced-intensity regimens were used in the other 3 pts. An 8 yo male received a 4/6 HLA-matched UCBT (3.2 X 10^7/kg NC) after conditioning with alemtuzumab, rituximab, hydroxyurea, thiotepa and TBI (600cGy, with 300 cGy total dose to liver and kidneys) and received TAC and mycophenolate mofetil (MMF) post-transplant. This patient failed UCBT 8 months earlier (FLU/CY/ATG/TLI, 4/6 HLA). PMN engraftment and VNTR > 98% of donor origin was documented within 18 days after UCBT. Complications included grade I skin GVHD, grade 1 mucositis and parainfluenza 1 infection. This pt remains engrafted and fully active 1.6 ys after UCBT. A 4 yo female and a 16 yo male received 4/6 or 5/6 HLA-matched cord units, respectively, after BU or CY, ATG, FLU and total lymphoid irradiation (200 or 500 cGy) and received CSP and MMF post-transplant. Neither had evidence of donor engraftment, and autologous reconstitution was documented within 2 weeks after UCBT. Sustained donor engraftment occurred in 3/4 pts (75%) who underwent UCBT after myeloablative conditioning and 1/3 pts (33%) who underwent UCBT after reduced-intensity conditioning. Two of the 3 pts who engrafted after myeloablative preparative regimens developed acute grade III–IV GVHD, and 1 of these pts developed extensive chronic GVHD. The 1 pt who engrafted after a reduced-intensity preparative regimen developed grade I acute GVHD only. Significant viral infections (CMV 1 pt, adenovirus, parainfluenzavirus 2 pts each) occurred in 4 pts. In this limited experience, following various conditioning regimens, optimal GVHD and infection prevention remains a challenge. Both apparent lesser risk and documented durable engraftment may justify further development of non-myeloablative strategies for UCBT in children with high-risk SCD.