Search

We report graft-versus-host disease (GVHD)-free relapse-free survival (GRFS) (a composite end point of survival without grade III-IV acute GVHD [aGVHD], systemic therapy–requiring chronic GVHD [cGVHD], or relapse) and cGVHD-free relapse-free survival (CRFS) among pediatric patients with acute leukemia (n = 1613) who underwent transplantation with 1 antigen–mismatched (7/8) bone marrow (BM; n = 172) or umbilical cord blood (UCB; n = 1441). Multivariate analysis was performed using Cox proportional hazards models. To account for multiple testing, P < .01 for the donor/graft variable was considered statistically significant. Clinical characteristics were similar between UCB and 7/8 BM recipients, because most had acute lymphoblastic leukemia (62%), 64% received total body irradiation–based conditioning, and 60% received anti-thymocyte globulin or alemtuzumab. Methotrexate-based GVHD prophylaxis was more common with 7/8 BM (79%) than with UCB (15%), in which mycophenolate mofetil was commonly used. The univariate estimates of GRFS and CRFS were 22% (95% confidence interval [CI], 16-29) and 27% (95% CI, 20-34), respectively, with 7/8 BM and 33% (95% CI, 31-36) and 38% (95% CI, 35-40), respectively, with UCB (P < .001). In multivariate analysis, 7/8 BM vs UCB had similar GRFS (hazard ratio [HR], 1.12; 95% CI, 0.87-1.45; P = .39), CRFS (HR, 1.06; 95% CI, 0.82-1.38; P = .66), overall survival (HR, 1.07; 95% CI, 0.80-1.44; P = .66), and relapse (HR, 1.44; 95% CI, 1.03-2.02; P = .03). However, the 7/8 BM group had a significantly higher risk for grade III-IV aGVHD (HR, 1.70; 95% CI, 1.16-2.48; P = .006) compared with the UCB group. UCB and 7/8 BM groups had similar outcomes, as measured by GRFS and CRFS. However, given the higher risk for grade III-IV aGVHD, UCB might be preferred for patients lacking matched donors.

Background: Pyruvate kinase deficiency (PKD) is a rare inherited hemolytic anemia caused by mutations in the PKLR gene resulting in decreased red cell pyruvate kinase activity and impaired erythrocyte metabolism. Manifestations include anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current treatments are palliative and limited to blood transfusions, chelation therapy, and splenectomy which are associated with significant side effects. Preclinical studies in a clinically relevant PKD murine model have demonstrated that infusion of gene-modified Lin− bone marrow (BM) cells may ameliorate PKD phenotype. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adult and pediatric subjects with severe PKD. Methods: Six adult and pediatric patients with severe PKD (defined as severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled. Peripheral blood (PB) hematopoietic stem cells (HSCs) are collected on 2 consecutive days via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. HSCs are enriched, transduced with PGK-coRPK-WPRE lentiviral vector (LV), and cryopreserved. Following release testing of the investigational product (IP), RP-L301, myeloablative therapeutic drug monitoring (TDM) busulfan is administered over 4 days. RP-L301 is then thawed and infused. Patients are followed for safety assessments, including replication competent lentivirus (RCL) and insertion site analysis (ISA), and for efficacy parameters including PB and BM genetic correction, decrease in transfusion requirements, clinically significant improvement in anemia, and reduction of hemolysis for 2 years post-infusion. Results: As of May 2021, 2 adult patients with severe anemia have received RP-L301. Patient 1 (age 31 years) received 3.9x106 CD34+ cells/kg with mean vector copy number (VCN) of 2.73. Patient 2 (age 47 years) received 2.4x106 CD34+ cells/kg with mean VCN of 2.08. Despite baseline hemoglobin (Hb) levels in the 7.0-7.5 g/dL range, both patients displayed normal-range hemoglobin (Hb), improved hemolysis markers, and have required no red blood cell transfusions post-engraftment at 9- and 6- months follow-up. Both report improved quality of life. PB mononuclear cell VCNs for both patients were >2.0 at last evaluated timepoint (6- and 3-months post-treatment, respectively). No serious adverse events have been attributed to RP-L301. Updated safety and efficacy data will be presented. Conclusions: Hematopoietic stem cell mobilization using G-CSF and plerixafor is feasible and effective in adult PKD patients. RP-L301 was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications. Efficacy was demonstrated by normalized Hb associated with engraftment confirmed by PB and BM VCN. Disclosures Shah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: Dr. Navarro has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla: Miltenyi: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Sevilla is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, and may be entitled to receive financial benefits from the licensing of such patents.; SOBI: Consultancy. Glader: Agios: Consultancy. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company. Beard: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Rao: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Bueren is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, may be entitled to receive financial benefits from the licensing of such patents and receives funding for research., Patents & Royalties, Research Funding. Schwartz: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding.

Solid organ transplantation (SOT) treats over 1500 children/year in the US. While short-term outcomes have improved, long-term outcomes still remain poor. With the rare exception of monozygotic twins as donors, recipients require lifelong immunosuppression with its accompanying toxicities, the risk of nonadherence, and chronic rejection. Further, up to half of recipients require a second transplant. We have pioneered translational approaches that abrogate rejection and the need for post-transplant immunosuppression. Previous attempts in adult patients utilizing allogeneic hematopoietic stem cell transplantation (HSCT) to eliminate the need for post-SOT immunosuppression in non-histocompatible kidney transplant (KT) recipients have failed(Lowsky R, BMT 2019). The establishment of mixed lymphoid chimerism has permitted a reduction in the number of immunosuppressive drugs needed, but not their discontinuation, whereas complete donor engraftment after HSCT has resulted in fatal graft-versus-host disease in some patients (Leventhal JR, Eur J Pediatr 2000). The use of HSCT to eliminate the need for immunosuppression following pediatric KT has not been evaluated. We have pioneered sequential haploidentical HSCT followed by KT in three patients with Schimke immuno-osseous dysplasia (SIOD) to abrogate kidney rejection and the need for post-transplant immunosuppression. SIOD is a monogenic disorder with progressive nephropathy correctable by KT and a T-cell immunodeficiency curable by HSCT. All three patients received αβT-cell/CD19 B-cell depleted HSCT (αβhaplo-HSCT) from a parent prior to a KT from the same parental donor. The pre-HSCT reduced intensity preparative regimen consisted of fludarabine (a starting dose of 1 mg/kg x 4 days, which was adjusted based on AUC), total body irradiation (TBI) 200 cGy, cyclophosphamide 1200 mg/m 2, anti-thymocyte globulin (ATG) thymoglobulin® 7.5 mg/kg, and rituximab 200 mg/m 2. Immunosuppressive drugs were not prophylactically administered after HSCT. After confirmation of full donor lymphoid chimerism post-HSCT, the patients received a living donor KT from their parental HSCT donor. Post-KT immunosuppression included intraoperative methylprednisolone and post-operative low-dose oral prednisone (0.5 mg/kg/day with taper) and tacrolimus (target serum level of 3-5 ng/ml) to reduce potential reperfusion inflammation. All immunosuppressive drugs were tapered off by Day +30 post-KT. To demonstrate functional tolerance after KT, Mixed Lymphocyte Cultures (MLC) were performed between peripheral blood mononuclear cells (PBMC) and patient/parent-derived irradiated EBV transformed lymphoblastoid cells lines (EBV-LCL) as stimulators (Figure 1). PBMC were isolated from SIOD patients (n=3) by Ficoll Hypaque separation and were labelled with Invitrogen™ CellTrace Violet Proliferation Kit (CTV). EBV-LCL were established from SIOD patients, parents and healthy donors. The assay was performed in round bottom microtiter plates with 100,000 labelled PBMC as responder cells (R) and 50,000 irradiated (30gy) EBV-LCL in RPMI medium with 10% fetal calf serum. Cells were harvested on Day 6, and T-cell proliferation determined by CTV expression of CD3+ T cells using a Novocyte Penteon flow cytometer (Agilent). Donor-derived T cells isolated from the peripheral blood of all three recipients > 1-year post-HSCT/KT, did not respond to the donor cells, while they did proliferate to the non-donor parental and control cells (Fig. 1). These results demonstrate that post-HSCT/KT the circulating donor-derived T cells are functionally tolerant to the transplanted kidney and, therefore, are unable to mediate graft rejection even in the absence of immune suppression. We have achieved two goals: first, ablating the recipient immune system before HSCT we safely established a new donor-derived immune system - that could also, if required, cure a patient's underlying disease- and, second, functional tolerance to the transplanted kidney. At 12 to 24 months after KT, all patients have full donor lymphoid and myeloid chimerism, normal renal function without immunosuppression, MLC demonstrated tolerance towards donor cells, and normal proliferative responses to non-donor parental cells. These findings suggest that the combination of αβhaplo-HSCT and SOT could be extended to other solid transplantation like liver and small intestine. Figure 1 Figure 1. Disclosures Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees. Shah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Parkman: Jasper Biotech: Consultancy.

Successful hematopoietic stem cell transplantation (HSCT) requires vacating recipient hematopoietic stem cell (HSC) niches in the bone marrow to permit donor HSC engraftment that can provide life-long hematopoietic and immune function. Currently, HSCT in SCID relies on DNA damaging chemotherapy to eliminate recipient HSC and achieve niche clearance. We have pursued a non-toxic approach to target and deplete HSC using a humanized monoclonal antibody, JSP191, that binds human CD117 (c-Kit). We previously showed the safety and successful HSC engraftment in a Phase 1 trial of the first 6 patients with severe combined immunodeficiency (SCID), who underwent a second transplant because of HSC engraftment failure and poor immunity after their first transplantation. In these re-transplant patients even a low level of stringently measured myeloid chimerism resulted in significant and sustained generation of naive T cells and clinical improvement. Based on these results, the study of JSP191 (NCT#02963064)has opened a cohort of newly diagnosed infants with SCID. Here we report data from the first patient in this cohort, a SCIDX1 patient who received a primary HSCT with haploidentical CD34+ cells after conditioning with JSP 191. The patient had a c.270-15A>G variant in the IL2RG gene, which is predicted to cause a null phenotype. Besides a T- B+ NK- phenotype typical of SCIDX1 including dysfunctional B cells, the patient had anemia and intermittent neutropenia and thrombocytopenia. Despite evidence of maternal T cell engraftment, the patient had no clinical graft-versus-host disease (GVHD). The patient was initially enrolled in a trial of lentiviral gene therapy, but harvested bone marrow cells died in vitro during transduction and culture. The patient also mobilized poorly with G-CSF/Plerixafor. Further investigation revealed heterozygosity for loss-of-function mutations in two genes involved in DNA repair, BRCA1 and RAD51; Diepoxybutane (DEB) breakage study showed greater than normal pathologic chromosomal breaks, but less than that seen in Fanconi anemia. Because of concern for possible hypersensitivity to alkylating agent-based conditioning, the patient was referred for transplant with JSP191 conditioning. The patient received a CD34+ peripheral blood HSCT from his father after conditioning with 0.3 mg/kg of JSP 191 antibody intravenously over an hour on Day -8 and rATG (Thymoglobulin) on Day -5, -4, -3 and -2 (3.5 mg/kg total) to prevent rejection by the maternal T cells. The cryopreserved donor CD34+ cells were administered after sufficient clearance of the JSP191 serum level. The antibody infusion was well tolerated without toxicity, and the post-transplant course was uneventful without acute toxicities or GVHD. As a surrogate marker for HSC engraftment, CD15+ myeloid cells from peripheral blood were stringently sorted by flow cytometry and donor levels were quantified by short-tandem repeat (STR) analysis. Progressive levels of myeloid engraftment were observed beginning at Week 4. The level of donor chimerism at 12 weeks was 8% in the sorted CD15+ blood cells, and a marrow aspirate showed 25% donor CD34+ cells. By 3 months pre-existing abnormal CD19-CD20+ host B lymphocytes were significantly reduced, and CD19+ donor-derived B lymphocytes were emerging. At 2 months, CD4+ recent thymic emigrant and naïve T lymphocytes were observed, and by 3 months, overall T and NK lymphocyte numbers were 390/uL and 117/uL, respectively. Normal blastogenic responses to the T cell mitogen PHA were observed at 3 months. These first-in-class results provide proof of concept of the safety and efficacy of the use of JSP191 antibody to clear host marrow niche space to enable sufficient donor HSC engraftment and immune reconstitution as primary therapy of SCID. Non-genotoxic conditioning with JSP191 may replace conventional conditioning for newly diagnosed infants with SCID, thereby avoiding toxicities of chemotherapy. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. De Oliveira:Orchard Therapeutics: Research Funding; bluebird bio, Inc.: Research Funding. Czechowicz:Rocket Pharmaceuticals, Inc.: Research Funding. Brown:Merck: Membership on an entity's Board of Directors or advisory committees; Ansun: Membership on an entity's Board of Directors or advisory committees; Cidara: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Cellerant Therapeutics: Membership on an entity's Board of Directors or advisory committees. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

Introduction: Pyruvate Kinase Deficiency (PKD) is a rare inherited hemolytic anemia that is caused by mutations in the PKLR gene leading to decreased red cell pyruvate kinase (RPK) activity and impaired erythrocyte metabolism. The disorder is characterized by anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current therapies are palliative and limited to chronic blood transfusions, iron chelation therapy, and splenectomy. The side effects of these supportive treatments include iron overload, end-organ damage and increased infection risks. AG-348, an allosteric activator of RPK, is under evaluation in clinical trials, predominantly in less severely-afflicted transfusion-independent patients. Allogeneic hematopoietic stem cell transplantation (HSCT) has been performed in selected cases and resulted in transfusion independence, suggesting that the disorder may be reversed when an adequate level of hematopoietic stem and progenitor cells (HSPCs) harboring a corrected PKLR gene engraft in the bone marrow (BM). The therapeutic efficacy of allogeneic transplant is limited by the availability of a suitable donor and transplant-associated toxicities. Preclinical studies conducted in a clinically relevant PKD murine model have demonstrated the safety and efficacy of Lin- BM cells transduced with the therapeutic lentiviral vector, PGK-coRPK-WPRE, in ameliorating the PKD phenotype. More specifically, transplantation of transduced cells resulted in increased erythrocyte survival, decreased reticulocytosis, and improvement in the secondary manifestations of hemolytic anemia, including splenomegaly and hepatic iron overload. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (clinicaltrials.gov#NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adults and pediatric subjects with severe PKD. Methods: 6 subjects with severe PKD (defined as having a history of severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled in the Phase 1 study; the first 2 subjects will be adults (age ≥18- Results: An adult female PKD subject (age 31 years) with significant anemia and transfusion requirement has received treatment as of July 2020. Mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target area under the curve (AUC). IP consisted of 3.9×106 CD34+ cells/kg body weight, with a mean vector copy number (VCN) of 2.73. Safety and preliminary efficacy results will be available at the time of presentation. Conclusions: Efficacy in pre-clinical models indicates promising potential for clinical gene therapy in severe PKDHematopoietic stem cell mobilization using G-CSF and plerixafor appears feasible and effective in adult PKD patientsIP was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications Disclosures Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: SN has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company. Glader:Agios Pharmaceuticals, Inc.: Consultancy. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

Flt3 is a class III tyrosine kinase receptor expressed on primitive human and murine hematopoietic progenitor cells (HPC). In previous studies using stroma-free short term assays, Flt3 ligand (FL) has been shown to induce proliferation of HPC at proportions similar to or less than c-kit ligand (steel factor, SF). Using long term stromal cocultivation assays, we studied the effects of FL on proliferation and differentiation of a highly primitive and cytokine nonresponsive subpopulation of human HPC, CD34+cd38- cells. Cell Proliferation was significantly greater with FL than with SF, when used individually or in combinations with interleukin-3 (IL-3) and/or IL-6. The effect of FL was greater on bone marrow (BM) CD34+CD38- cells than the more cytokine responsive cord blood CD34+CD38- cells. Little or no effect was seen with FL on more mature CD34+CD38+ cells from either BM or cord blood. The frequency of colony-forming units (CFU) and high proliferative potential-colony forming cells (HPP-CFC) during early culture ( < or = 30 days) was increased by both SF and FL to similar levels. However, in the LTC-IC period (35 to 60 days) and extended long-term culture initiating cell (ELTC-IC) period ( > 60 days), the frequency of CFU and HPP-CFC was significantly greater in cultures containing FL than those without FL (P < .0025). Fluorescence-activated cell sorter analysis of cultures after 21 days showed a significantly higher percentage of cells remained CD34+ in the combination of FL, IL-3, IL-6, and SF (F/3/6/S) than in 3/6/S (0.78% +/- 0.52% v 0.21% +/- 0.29% respectively, mean +/- SD). Cloning efficiency of BM CD34+CD38- cells was significantly increased by the addition of FL to the combination of 3/6/S (mean 11.7% v 0.5%, P < .0001). These data show that FL is able to induce proliferation of CD34+CD38-cells that are nonresponsive to other early acting cytokines and to improve the maintenance of progenitors in vitro.

We present cell cycling and functional evidence that the CD34+CD38- immunophenotype can be used to define a rare and primitive subpopulation of progenitor cells in umbilical cord blood. CD34+CD38- cells comprise 0.05% +/- 0.08% of the mononuclear cells present in cord blood. Cell cycle analysis with the fluorescent DNA stain 7- aminoactinomycin D showed that the percentage of CD34+ cells in cycle directly correlated with increasing CD38 expression. CD34+CD38- cord blood cells were enriched for long-term culture-initiating cells (LTCIC; cells able to generate colony-forming unit-cells [CFU-C] after 35 to 60 days of coculture with bone marrow stroma) relative to CD34+CD38- cells. In an extended LTCIC assay, CD34+CD38- cells were able to generate CFU-C between days 60 and 100, clearly distinguishing them from CD34+CD38+ cells that did not generate CFU-C beyond day 40. When plated as single cells, onset of clonal proliferation was markedly delayed in a subpopulation of CD34+CD38- cells; clones (defined as = 100 cells) appeared after 60 days of culture in 2.9% of CD34+CD38- cells. In contrast, 100% of CD34+CD38+ cells formed clones by day 21. Although the CD34+CD38- immunophenotype defines highly primitive populations in both bone marrow and cord blood, important functional differences exist between the two sources. CD34+CD38- cord blood cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and generate approximately sevenfold more progeny than do their counterparts in bone marrow.

Pediatric patients with hematologic malignancies undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) comprise a patient cohort at highest risk of developing disseminated fungal infections. Invasive aspergillosis post-HSCT is associated with unacceptably high mortality in pediatric patients with overall survival of only 15-34%. Hospital construction and excavation further potentiate the risk of fungal related morbidity and mortality in the post-HSCT setting. The current standard of care for anti-fungal prophylaxis for children undergoing aHSCT is the use of triazoles. However, 40% of healthcare associated mold infections in pediatric leukemia patients exposed to hospital construction are historically resistant to voriconazole. Methods to optimize prevention of fungal infections in this high-risk population are critical to improving pediatric aHSCT survival outcomes. We hypothesized that pre-emptive ambisome prophylaxis would be tolerated in the post-HSCT setting and would decrease the incidence of voriconazole resistant healthcare-associated fungal infections and resultant morbidity. We explored the use of intermediate dose daily ambisome (3mg/kg/day) as fungal prophylaxis in 5 patients with hematologic malignancies (2=ALL, 2=AML, 1=Lymphoblastic lymphoma) undergoing allogeneic stem cell transplantation in proximity to construction and excavation. Patient demographics, disease, donor source, conditioning regimen, methotrexate course and transplant related morbidity are shown in table 1. We found that 5 of 5 patients experienced delayed clearance of low dose methotrexate (MTX). This delayed clearance resulted in delayed methotrexate dosing in 3 of 5 patients and a missed methotrexate dose in 1 patient who subsequently developed GVHD and complications from high dose steroids. 4 of 5 patients experienced acute kidney injury (AKI), demonstrated by doubling of the pre-HSCT creatinine value following MTX administration. This identified creatinine elevation is increased as compared to historical controls receiving standard anti-fungal prophylaxis with azole agents. It has been described that AKI delays the clearance of high dose methotrexate. Here, we demonstrate that intermediate doses of daily ambisome following allogeneic HSCT is associated with AKI and delayed clearance of low doses of methotrexate used for GVHD prophylaxis in pediatric patients. Delayed methotrexate clearance was associated with development of morbidity including inadequate GVHD prophylaxis and possible resultant development of GVHD. Combined ambisome and low dose MTX is associated with AKI and delayed MTX clearance and thus is a constrained strategy for prevention of invasive fungal disease in the post-HSCT pediatric setting. The implication of these finding can be extrapolated beyond the construction setting implying cautious use of intermediate dose ambisome prophylaxis in combination with low dose MTX GVHD prophylaxis in pediatric patients post aHSCT. Table 1 Table 1. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Equity Ownership.

Avascular necrosis (AVN) of the bone is a painful and debilitating complication of allogeneic hematopoietic cell transplantation (HCT) that is associated with significant morbidity and often requires surgery. Risk factors for its development in pediatric allogeneic HCT recipients are not well described. To assess risk factors for AVN in children and adolescents following allogeneic HCT, we conducted a nested case-control study with a matched cohort of 638 patients reported to the Center of International Blood and Marrow Transplant Research who were ≤ 21 years of age, received their first allogeneic transplant between 1990 to 2008 in the United States and had survived ≥ 6 months from HCT. Overall, 160 cases with AVN were identified. Each case was matched with up to 3 controls by same year of HCT, similar length of follow-up and by transplant center (478 controls). Cases and controls were confirmed via central review of radiology, pathology and/or surgical procedure reports. The median age for cases was 15 (range 2-21) years, 49% were male, 65% had acute leukemia, 65% had received high-dose total body irradiation (TBI) based conditioning regimen, and 65% had received unrelated donor HCT. Among cases, 18% had a history of acute graft-versus-host disease (GVHD) while 56% had a history of chronic GVHD prior to development of AVN. Median time from HCT to diagnosis of AVN was 14 (range Disclosures: No relevant conflicts of interest to declare.

Background Hematopoietic stem cell transplantation (HSCT) is the only available cure for chronic granulomatous disease (CGD). Preexisting infections tend to put CGD patients at high risk for myeloablative HSCT related complications, making reduced intensity conditioning (RIC) an attractive option for these patients. However, published studies using RIC HSCT in CGD have shown a high rate of graft rejection. Objectives To characterize the clinical features and outcomes of high-risk children with chronic granulomatous disease transplanted at Children's Hospital of Los Angeles with a RIC regimen. Methods Patients with CGD with pre-existing co-morbidities that precluded the use of myeloablative HSCT and who received RIC HSCT were selected for review. Conditioning regimen consisted of targeted dose Campath (with level monitoring), Fludarabine 30 mg/m2/dose x 3 doses and Total Body Irradiation 200 cGy x 2 doses. Tacrolimus (or cyclosporine) and Mycophenolate Mofetil (MMF) were used for graft-versus-host disease (GVHD) prophylaxis. Results Four children (median age 102 months, range 24-180 months) with severe CGD (serious bacterial/fungal infections pretransplantation) and significant comorbidities (Table 1) received RIC matched unrelated donor HSCT. Two patients received a 9/10 HLA allele matched graft, and the other two received a 10/10 HLA allele matched graft. Three patients received peripheral blood stem cells (PBSCs), and the one other patient received a bone marrow (BM) graft. For the PBSC grafts, the mean total nucleated cell (TNC) dose was 24.3 x 10e8/kg (range 11.9-24.5 x 10e8/kg), the mean CD34+ cell dose was 9.4x10e6/kg (range 3.7-11.8 x 10e6/kg), and the mean CD3+ cell dose was 60.3x10e7/kg (range 33.8-77.1 x 10e7/kg). The patient who received a bone marrow graft developed primary graft failure and autologous recovery 4 months after HSCT. Only one patient who was non-compliant with immune-suppressive medications developed grade 4 acute graft-versus-host disease (GVHD) with subsequent extensive chronic GVHD. None of the other patients have any evidence of active GVHD at the most recent follow up. Median follow up of all patients is 23.5 months (range 13-28 months) and three of the four patients are alive with a high donor chimerism (100%). Discussion Patients with CGD often have multiple co-morbid conditions that lead to an increased risk of transplantation related mortality with myeloablative conditioning regimens. RIC regimens are increasing being used to decrease the short and long term toxicity of transplantation. Previous studies have suggested a high rate of graft rejection with RIC in CGD. Most of the published data on RIC HSCT in CGD patients pertains to the use of bone marrow grafts from HLA-matched siblings. In our series, we demonstrate that RIC (campath, fludarabine, and TBI) matched unrelated donor PBSC transplantation in CGD patients with life threatening infections leads to excellent engraftment, durable donor chimerism, and correction of neutrophil dysfunction, excellent survival and a low incidence of severe graft-vs-host disease. Thus, RIC HSCT represents a possible curative strategy for CGD patients with co-morbidities who are not eligible for myeloablative HSCT. Disclosures: No relevant conflicts of interest to declare.

Background HSCT recipients have increased incidence of acquiring infections, particularly by encapsulated bacteria such as Streptococcal pneumoniae and Haemophilus influenzae. Delayed immune reconstitution has a pivotal role in these complications. Although T-cell immune reconstitution has been well studied, long-term B-cell reconstitution remains less characterized. Patients and Methods We studied 73 patients, who received allogeneic HSCT at Childrens Hospital Los Angeles. Patients were in complete remission of their underlying disorder and with median follow up 4.15 years [yrs] (range 6 month -18yrs, mean 5 yrs) post-HSCT. All subjects were< 18 years of age. B (naive [IgD+CD27-CD19+], memory [IgD+CD27+CD19+] and switched memory [IgD-CD27+CD19]); and T (CD3+, CD3+CD4+, CD3+CD8+, CD4+CD25+CD127dim (T Regulatory) [Tregs], RA+CD4+) cell subtypes, quantitative Immunoglobulins levels and antibodies to both polyribosyle polyphospate (PRP) and tetanus toxoid (TT) antigens were assessed longitudinally after HSCT. Results Naive B Cells were the first B cell subtype to return to normal value at 6 month post-HSCT, while memory B cells were persistently low up to two years post-HSCT. PRP levels continued to be low up to 10 years post -HSCT in unrelated donor HSCT recipients. TT antibodies level was normal at 6 month post-HSCT following immunization with TT in patients not receiving IVIG therapy. Switched memory B cell counts correlated positively with RA+CD4+ counts at 6 month post-HSCT (r=0.459, P=0.021) and with CD3+CD+4 counts at 6 months (r=0.530, P=0.006) and 2-years post-HSCT (r=0.398, P=0.016). However, switched memory B cells did not correlate with Tregs at any time post-HSCT. Switched memory B cells correlated positively with serum level of IgG at 1 (r=0.443, P=0.039), and 2 years post transplantation (r=0.617, P=0.001) and with serum level of IgA at 2 years post-HSCT(r=0.567. P=0.004). Memory B-cells counts correlated positively with serum levels of IgM at 1 year post-HSCT (r=0.478, P=0.021) and with serum levels of both IgG and IgA (r=0.431 P=0.035, and r=0.416, P=0.043, respectively) at 2 years post-HSCT. However, memory B-cell counts did not correlate with RA+CD4+, CD3+CD4+, CD3+CD8+ or Tregs cell counts. The use of Total body irradiation (TBI) was associated with lower switched memory B cells at 2 years (P=0.01) post-HSCT. TBI recipients also have lower PRP levels at 6-month post-HSCT compared to patients who did not receive TBI. Age of the recipient at time transplantation is another independent factor affecting all the B cell subsets recovery after transplantation; increase in age at transplantation is associated with lower B cell recovery. Decreased memory B cells post-HSCT was observed in patients with history of acute graft versus host disease (GVHD) (P=0.034) and chronic GVHD (P=0.01), even after resolution of clinical manifestations of active GVHD at 6 month and 2 years follow up, respectively. Compared to cord blood graft recipients, Bone marrow graft recipients have increased switched memory B-cells at 6 month and higher (P=0.0001) PRP antibodies titer at 3 years post-HSCT, respectively. Patients who did not receive ATG or Alemtuzumab have increased recovery of naive B-cells (P=0.024) at 2 years post-transplantation. Patients received ATG have higher both naive B cells in univariate analysis and PRP levels (in multivariate analysis) than those received Alemtuzumab at 6 months post-HSCT. Multivariate regression analysis showed that patients received Alemtuzumab have higher TT antibodies titer at 6 month post -HSCT compared to those received ATG. Conclusion We have found that memory and switched memory B-cells and serum PRP levels are deficient post-HSCT in children. Switched memory B cells correlated positively with serum level of IgG and IgA and memory B-cells correlated positively with serum levels of IgM, IgG and IgA. This confirms that HSCT recipients have impaired humoral immune reconstitution, hindering both B-cells development and generation of immunoglobulins. We also studied the different factors affecting humoral immune reconstitution using backwards multivariate regression analysis model and found that the use of TBI, age of the recipient at transplantation, GVHD status and source of stem cells can affect the kinetics of humoral immune reconstitution after HSCT in children. Disclosures: No relevant conflicts of interest to declare.

Abstract 3052 The initial analysis of the impact of cGVHD on thymic function used TREC analysis and demonstrated a sustained decrease in thympoiesis in hematopoietic stem cell transplant (HSCT) recipients with a history of cGVHD (Blood 97:1458, 2011). We have reported that HSCT recipients, who had the clinical resolution of their cGVHD, had increases in their resting regulatory T lymphocytes (rTreg) as compared to HSCT recipients with active cGVHD [ASH 2011]. The increase in rTreg lymphocytes suggested that clinical resolution of cGVHD might be due to improved thymic function, which resulted in the increased production of rTreg lymphocytes. To measure the thymic function of pediatric HSCT recipients with cGVHD, we determined the frequency of recent thymic emigrants (RTE; CD4+, CD45RA+, CD31+) in the whole CD4 T lymphocyte population and the CD4 regulatory T (Treg; CD127-, CD25+) and CD4 conventional T lymphocytes (Tcon; CD127+, CD25+) subpopulations of normal individuals, HSCT recipients without a history of cGVHD (n =16) and HSCT recipients with a history of cGVHD (n=20). The frequency of RTE in total, Treg, and Tcon lymphocytes of the HSCT recipients with cGVHD was decreased (P=0.001) compared to HSCT recipients without a history of cGVHD but was the same as normal individuals. Thus, pediatric HSCT recipients with cGVHD have thymic function that is similar to normal individuals, but have decreased thymic reserves compared to HSCT recipients without cGVHD. However, since rTreg lymphocytes (CD4+, CD127-, CD25+, CD45RA+, FoxP3+) represent only 20% of total Treg lymphocytes, we specifically determined the thymic contribution to rTreg lymphocytes. The RTE content of rTreg lymphocytes was determined in HSCT recipients with active or resolved cGVHD. The RTE content of the rTreg lymphocytes (as a percentage of total CD4 T lymphocytes) was similar in HSCT patients with both active and resolved cGVHD. Thus, the increase in rTreg lymphocytes present in HSCT recipients with the clinical resolution of their cGVHD is not due to an increase in the frequency of RTE in their rTreg lymphocyte population. Therefore, the increase in rTreg lymphocytes, that is associated with the clinical resolution of cGVHD, may be due to either their decreased conversion to Th17 lymphocytes or their decreased apoptosis. Disclosures: No relevant conflicts of interest to declare.

Abstract 4076 Due to their clinical similarities, controversy has existed as to whether the pathophysiology of chronic graft-versus-host disease (GVHD) and systemic sclerosis (SSc) is the same. The principal cellular effector mechanism in SSc is clonal autoreactive CD4 T lymphocytes that activate fibroblasts, resulting in increased collagen production. We have previously reported that the principal cellular effector mechanism in histocompatible murine chronic GVHD is autoreactive CD4 T lymphocytes, which also stimulated fibroblast collagen production. More recently defects in naturally occurring regulatory T lymphocytes (Treg) have been reported in adult chronic GVHD patients. Using the Miyana classification (Immunity 30:899, 2009), which discriminates between resting Treg lymphocytes, activated Treg lymphocytes, and conventional T lymphocytes (Tcon), we assessed whole blood from pediatric patients, who were more than 1 year following allogeneic hematopoietic stem cell transplantation (HSCT), or patients with active juvenile SSc (jSSc), for their total number of Treg lymphocytes and their relative proportion of resting and activated Treg lymphocytes (r/a ratio). Normal individuals had an average of 15,300 Treg lymphocytes/mL with 10,890 resting Treg/mL and 4,410 activated Tregs/mL or a r/a ratio of 2.5. The resting Treg lymphocytes were 17.1% of the CD4+, CD25+, CD127- T lymphocytes while the activated Treg lymphocytes were 6.9%. Allogeneic HSCT recipients without a history of either acute or chronic GVHD had elevated numbers of total Treg lymphocytes (25,979/mL) (P=.05) with 19,199 resting and 5,780 activated Treg lymphocytes or a r/a ratio of 3.3, secondary to the relative increase in the proportion of resting Treg lymphocytes. Recipients, who had had only acute GVHD (Grade 2–4) but no history of chronic GVHD, also had an increased number of total Treg lymphocytes with a normal r/a ratio. Recipients, who had active chronic GVHD, had reduced numbers of total Treg lymphocytes (10,040/mL) (P=.02) with reduced numbers of both resting (7,631/mL) and activated (2,408/mL) Treg lymphocytes. Half of the recipients with active chronic GVHD had more activated Treg lymphocytes than resting T lymphocytes, resulting in an inverted r/a ratio of Patients with jSSc had Treg lymphocyte patterns similar to HSCT recipients with active chronic GVHD; their total number of Treg lymphocytes was reduced (843/mL) (P=.01) with reduced resting (532/mL) and activated (661/mL) Treg lymphocytes and an inverted r/a ratio of 0.8 due to the relative excess of activated Treg lymphocytes. Thus, pediatric patients with either active chronic GVHD or jSSc have reduced total numbers of Treg lymphocytes with a relative increase in the proportion of activated Treg lymphocytes, suggesting that the abnormalities in Treg lymphocytes are central to the pathogenesis of both clinical syndromes. HSCT recipients with a history of resolved chronic GVHD had increased numbers of Treg lymphocytes, indicating that the resolution of chronic GVHD requires increased numbers of Treg lymphocytes with a normal ratio of resting to activated Treg lymphocytes. Disclosures: No relevant conflicts of interest to declare.

Graft Versus Host Disease (GVHD) is a cause of serious morbidity and mortality in > 50% of recipients of unrelated hematopoietic stem cell transplantation (HSCT). We performed a trial using Campath 1 H pre- and post-HSCT in an attempt to decrease the incidence of GVHD without increasing the risk of infection or relapse. Patients were retrospectively compared to a population of patients who received antithymocyte globulin (ATG) pre-and post-HSCT. Materials and Methods: 27 patients were evaluated for this study. Fourteen patients received Campath 1H and 13 patients received ATG. Demographics of patients who received Campath 1H consisted of 9 males and 5 females, with a median age of 13 years (3 years–17.8 years). Thirteen patients received unrelated BM and 1 patient received unrelated PBSC. Demographics of patients receiving ATG consisted of 9 males, 4 females with a median age of 7.4 years (21 months–19 years). Twelve patients received unrelated bone marrow (BM) and 1 patient received unrelated peripheral blood stem cells (PBSC). Diagnoses were similar between the two groups. Patients who received Campath1H received a total dose of 52 mg/m2 pre-HSCT and 20 mg/m2 post-HSCT. Patients who received ATG received a total dose of 60 mg/kg pre-HSCT and 100 mg/kg post-HSCT. GVHD prophylaxis and supportive care measures were similar in both groups. Results: There was a significant difference in the incidence of severe (grade III and grade IV) GVHD between the two arms [Campath (0/14) vs. ATG (6/13), p=0.006]. Among the patients who were transplanted for leukemia, there was no significant difference between the two arms in terms of relapse [Campath (2/14) vs. ATG (4/9), p=0.162]. The 1- year and 2-year overall survival between the two arms was not significantly different [Campath 1H (64%) vs. ATG (69%), p=0.98]. Patients receiving Campath 1H had the presence of CD3+ T cells (>30 cells/ml) in their peripheral blood later than in those who received ATG [65 days (Campath 1H) vs. 27 days (ATG), p=0.001]. The median time to the development of a response to PHA occurred later in the Campath 1H arm [240 days (Campath 1H) vs. 90 days (ATG), p= 0.0005]. The median time to an antigen specific response also occurred later in those receiving Campath 1H [365 days (Campath 1H) vs. 150 days (ATG), p= 0.008]. Patients who received ATG had a greater chance of developing a candida infection [Campath 1H (2/14) vs. ATG (8/13) p=0.02]. Among other specific viral infections, there was no significant difference between the two groups. Conclusions: Campath 1H is effective in decreasing the incidence of GVHD without increasing the risk of relapse. Although there is a significant delay in immune reconstitution, there was no increase in infectious complications in recipients of Campath 1H.

Protective levels of anti-bacterial carbohydrate antibodies are necessary to protect HSC transplantation recipients from severe infections with encapsulated bacteria. Recipients of unrelated HSC transplants are at increased risk of severe infections regardless of their history of graft versus host disease (GVHD). Antibodies to a naturally occurring, prototypic bacterial carbohydrate antigen, polyribose phosphate (PRP), present in Hemophilus influenza type b and K100 stains of E. coli, were measured in the recipients of unrelated bone marrow (UBMT) and unrelated cord blood transplants (UCBT). UBMT and UCBT recipients, who were not on immunosuppressive therapy, were analyzed with follow-ups of 12 and 7 years, respectively. Of 20 UBMT recipients, only 3 had protective anti-PRP antibody levels. All 3 recipients with protective antibody levels were younger than 1 year old at the time of transplantation and did not have a history of GVHD. No recipients, who were older than 1 year old at the time of transplantation, had protective levels of anti-carbohydrate antibody. Of 16 UCBT recipients, 7 had protective antibody levels, including recipients as old as 53 at the time of UCBT. All unrelated recipients, who did not have protective levels of anti-carbohydrate antibodies, were able to produce protective levels of antibodies to a protein antigen, tetanus toxoid, after immunization. Therefore, the inability of the recipients of unrelated HSC to produce protective levels of anti-bacterial carbohydrate antibody is not part of a generalized antibody deficiency state, but is a specific defect. Since bacterial carbohydrate antigens are T lymphocyte-dependent antigens in humans, the results indicate that thymic and T lymphocyte function are greater in UCBT recipients than in UBMT recipients (p=.008 for protective anti-PRP antibody levels in recipients older than age 1). Defects in protective anti-bacterial carbohydrate antibody production are common in the recipients of HSC unrelated transplants regardless of the source of HSC. The recipients of unrelated HSC transplants, especially UBMT, should be tested for their capacity to produce anti-carbohydrate antibody, and, if deficiencies are detected, appropriate prophylactic measures undertaken to reduce the likelihood of severe bacterial infections.