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Background: Pyruvate Kinase (PK) deficiency is caused by mutations in the PKLR gene leading to chronic hereditary non-spherocytic hemolytic anemia. Diagnostic evaluation can be challenging with falsely normal PK enzyme activity levels in chronically transfused patients, in those with profound reticulocytosis, or in patients with mutations with unusual biochemical properties. Genetic testing can also be complex, as up to 20% of affected patients have new PKLR variants and up to 10% of patients have variants not routinely detected through exon sequencing. The phenotypic spectrum is broad, and biomarkers of clinical severity would be helpful for both prognosis and monitoring. Aims: To describe the correlation of PK enzyme activity, red cell PK (PK-R) protein level, and red cell metabolites [adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG)] with clinical phenotype. To estimate the sensitivity of PK enzyme activity to diagnose PK deficiency in a cohort of patients with genetically confirmed PK deficiency. Methods: Blood samples were collected from a subset of 41 patients enrolled in the PK Deficiency Natural History Study, all with two confirmed PKLR mutations and no red cell transfusions for ≥3 months prior to the blood sample. PK and hexokinase (HK) enzyme activity testing were performed using standard biochemical assays (Beutler 1985) at the Stanford Red Blood Cell Special Studies Laboratory (normal ranges: PK activity: 3.2-6.5 EU/gm Hb, HK activity: 0.14-0.37 EU/gm Hb). Baseline PK-R protein was quantitated by antibody-based capture and detection using a Meso Scale assay and the signal normalized to a control sample without PK deficiency. ATP and 2,3-DPG concentrations (μg/ml) in blood were analyzed using qualified tandem mass spectrometry methods and then normalized for individual hemoglobin (Hb) values (ATP and 2,3-DPG concentrations were converted to g/dL and divided by Hb (g/dL)). Spearman correlation coefficients were calculated to estimate the correlation between continuous variables, and Wilcoxon rank-sum testing was used to assess the association of continuous and binary variables. Results: The median age was 25.3 years (range 1.4-60.4) and 80% of patients were splenectomized (Table). The mean PK enzyme activity level was 1.1 EU/gm Hb with 90% (37/41) of patients with low PK activity. Normal PK activity was observed in 4 patients, 3 of whom had high HK activity and a low PK/HK ratio (normal mean PK/HK ratio 15.6, range: 8.7-22.5), with a sensitivity of 98% (40/41) when both PK activity and PK/HK ratio were used together. There were no correlations between PK enzyme activity and clinical severity indicators, including transfusion status (p=0.3), splenectomy status (p=0.4), or post-splenectomy Hb (r=0.109). Normalized ATP levels were significantly correlated with normalized 2,3-DPG levels (r=0.93) and higher in ever transfused (median=0.0022, range=0.0011-0.0029) compared with never transfused patients (median=0.0012 , range=0.0008-0.0020), (p=0.004). PK-R protein level was inversely correlated with the total number of transfusions prior to enrollment (r= -0.53) and was higher in never transfused (median=40.1%, range=9.8-73.9%) compared with ever transfused patients (median=7.7%, range=0.4%-15.1%), (p=0.0014). Conclusions: In this cohort with a molecularly confirmed diagnosis, PK deficiency was detected by enzyme testing with 98% sensitivity by utilizing both the PK activity and the PK/HK ratio. Although these assays cannot distinguish between heterozygotes and affected patients, the combination of PK activity and the PK/HK ratio is useful as a screening test to determine which patients require genetic testing. There was no correlation between PK enzyme activity and clinical phenotype. PK-R protein and ATP levels were inversely correlated with clinical severity and may be a helpful marker of disease phenotype. Disclosures Al-Samkari: Agios: Consultancy, Research Funding; Moderna: Consultancy; Dova: Consultancy, Research Funding. Glader:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chonat:Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Thompson:bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Research Funding. Kuo:Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Pfizer: Consultancy. Ravindranath:Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, Research Funding. Rothman:Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Agios: Honoraria, Research Funding. Kwiatkowski:Celgene: Consultancy; Terumo: Research Funding; Imara: Consultancy; Apopharma: Research Funding; Novartis: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy. Kung:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. Kosinski:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. London:United Therapeutics: Consultancy; ArQule, Inc: Consultancy. Grace:Novartis: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary non-spherocytic hemolytic anemia. The spectrum of disease in PKD is broad, ranging from an incidentally discovered mild anemia to a severe transfusion-dependent anemia. Splenectomy partially ameliorates the anemia and reduces the transfusion burden in the majority of patients. Because hemoglobin poorly correlates with symptoms in PKD, transfusion requirements are typically used clinically to classify disease severity with those who are regularly transfused despite splenectomy recognized as the most severely affected subgroup. Aim: To compare demographics, complications, and laboratory results between the most severely-affected PKD patients (those that are splenectomized and regularly transfused) with non-regularly transfused splenectomized PKD patients. Methods: After ethics committee approval, patients were enrolled on the PKD Natural History Study, a prospective 30 site international study. All patients had molecularly confirmed PKD. Only splenectomized patients were included in the analysis. Transfusion frequency was observed over a 3-year period. Patients were divided into two groups based on transfusion frequency: the severe phenotype group was defined as those who receive regular transfusions (≥6 discrete red cell transfusion episodes per year) and the control group did not receive regular transfusions. Phenotype stability over the 3-year period was also assessed. Results: 154 splenectomized patients with PKD were included: 30 patients in the severe PKD phenotype group and 124 patients in the comparison PKD group. Results of the analysis comparing the two groups are described in the Table. Severely affected patients were more likely to be female (77% versus 51%, p=0.013), older at the time of splenectomy (median age: 5 versus 3.6, p=0.011), have iron overload (93% vs. 51%, p Conclusions: Patients with PKD who are regularly transfused despite splenectomy appeared to have similar rates of PKD-associated complications (except for iron overload) and similar relevant laboratory values and genotypes when compared to those who are not regularly transfused after splenectomy. The similarity observed between severe phenotype patients and comparison patients with PKD may result from a protective effect of transfusion (e.g. reduction of bone fractures and extramedullary hematopoiesis) or could suggest transfusion-dependence is an artificial signifier of disease severity, reflective of provider practices and patient symptoms rather than an actual distinct phenotype. Transfusion requirements in severe PKD appear to fluctuate significantly over time. Disclosures Al-Samkari: Dova: Consultancy, Research Funding; Agios: Consultancy, Research Funding; Moderna: Consultancy. van Beers:RR Mechatronics: Research Funding; Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding. Barcellini:Agios: Consultancy, Other: Advisory board; Apellis: Consultancy; Incyte: Consultancy, Other: Advisory board; Bioverativ: Consultancy, Other: Advisory board; Novartis: Research Funding, Speakers Bureau; Alexion: Consultancy, Research Funding, Speakers Bureau. Eber:Agios Pharmaceuticals, Inc.: Consultancy. Glader:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chonat:Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Kuo:Pfizer: Consultancy; Novartis: Consultancy, Honoraria; Celgene: Consultancy; Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy. Despotovic:Dova: Honoraria; Novartis: Research Funding; Amgen: Research Funding. Kwiatkowski:Celgene: Consultancy; Terumo: Research Funding; Apopharma: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Imara: Consultancy; Novartis: Research Funding. Thompson:Baxalta: Research Funding; Novartis: Consultancy, Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Ravindranath:Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, Research Funding. Rothman:Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Agios: Honoraria, Research Funding. Verhovsek:Sickle Cell Awareness Group of Ontario: Membership on an entity's Board of Directors or advisory committees; Sickle Cell Disease Association of Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding; Canadian Haemoglobinopathy Association: Membership on an entity's Board of Directors or advisory committees; Vertex: Consultancy. Kunz:Novartis: Membership on an entity's Board of Directors or advisory committees. Sheth:CRSPR/Vertex: Other: Clinical Trial Steering committee; Apopharma: Other: Clinical trial DSMB; Celgene: Consultancy. London:United Therapeutics: Consultancy; ArQule, Inc: Consultancy. Grace:Novartis: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and lymphoma. Gene therapy (GT) using autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplant for patients who lack well matched donors, avoiding graft-versus-host-disease. An initial experience with gene therapy using a γ-retroviral vector showed correction of hematological defects in 9/10 patients, but was aggravated by development of leukemia in 7 of them. We report the outcomes of a phase I/II clinical trial in which 5 WAS patients underwent GT using a self-inactivating lentiviral (SIN-LV) vector expressing the human WAS cDNA under the control of a 1.6kB fragment of the human WAS promoter. Subjects and Methods Five patients with severe WAS (clinical score 3-5) were enrolled at a median age of 1.8 years (1.4 - 8 years) at a single pediatric tertiary care center. WAS protein (WASP) was absent or markedly decreased in 2 and 3 subjects, respectively. Purified CD34+ cells from mobilized peripheral blood (n = 4) or both mobilized peripheral blood and bone marrow (n = 1) were transduced ex-vivo with the SIN-LV vector and re-infused after conditioning with busulfan (target AUC of 70-80 mg*h/L) and fludarabine (120mg/m2). The median dose of CD34+ cells infused was 9.8 x 106 cells/kg (6.3 - 24.9 x 106 cells/kg) with a mean vector copy number (VCN) of 1.7 copies/cell in CD34+ cells (0.54 - 3.37). In addition to eczema, thrombocytopenia and WAS-related infections in all patients, two subjects also had autoimmunity pre-GT, manifested as skin vasculitis and autoimmune cytopenias. Results All 5 subjects were alive and well at median follow-up of 4.8 years (2.5 - 5.9 years). Multi-lineage vector gene marking was sustained over time. All subjects had improvement or resolution of eczema and none have had any intercurrent severe infectious events. WASP expression measured by flow cytometry in T cells was increased over baseline in all patients, but remained below normal levels and correlated with VCN and cell dose received. Proliferation of T cells in response to anti-CD3, which was initially defective in 4/5 patients, improved post-GT. Humoral immune deficiency was also ameliorated, as evidenced by independence from Ig replacement and vaccine responses in those tested. All subjects remained platelet transfusion-free and none have had severe bleeding events. Platelet levels increased to >50 x 103 cells/uL in two patients with a VCN ≥2 in transduced stem cells and myeloid VCN ~1 copy/cell in neutrophils; the other 3 subjects sustained platelet counts No severe GT-related adverse events have occurred to date. Replication-competent lentivirus was not detected. Analysis of integration site distributions in five subjects showed reconstitution to be highly polyclonal, with no clones expanded to >20% of the transgene-marked cell population. To date, there have been no malignancies reported, either related to GT or WAS itself. Conclusion In summary, our data confirm and extend the safety and efficacy of GT in correcting disease manifestations associated with WAS, as seen in other studies using SIN-LV. Higher VCN in the drug product and in transduced stem cells correlated with better reconstitution of platelets and myeloid function. In contrast to other groups, we found in our study that patients with poor lymphocyte reconstitution post-GT may be at risk of ongoing autoimmunity despite high-level gene marking. Disclosures London: ArQule, Inc: Consultancy; United Therapeutics: Consultancy. Despotovic:Novartis: Research Funding; Amgen: Research Funding; Dova: Honoraria. Forbes:Takeda: Consultancy. Galy:Genethon: Employment. Williams:Novartis: Membership on an entity's Board of Directors or advisory committees; bluebird bio: Other: License of certain IP relevant to hemoglobinopathies. Potential for future royalty/milestone income. Received payment in past through BCH institutional licensing agreement., Research Funding; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, potential for future royalty/milestone income, Research Funding; Alerion Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. OffLabel Disclosure: CliniMACS technology for CD34+ cell selection

Background: Immune thrombocytopenia (ITP) is the most common autoimmune cytopenia diagnosed in children, typically causing a severely low platelet count, variable bleeding symptoms, and reductions in health-related quality of life (HRQoL). Eltrombopag is an established therapy for pediatric patients with chronic ITP. Favorable safety and efficacy were shown in the PETIT and PETIT2 trials leading to FDA-approval for children with chronic ITP in 2015. Off-label use of eltrombopag for adults with newly diagnosed ITP has been described in two small single-center trials. Gomez-Almaguer et al. reported 100% response (platelets >30 x 109/L) at completion of therapy and 66.7% relapse-free survival at 1 year in a single-arm study of dexamethasone in combination with 4 weeks of eltrombopag upfront in adult patients with newly diagnosed ITP, better outcomes than expected for comparable patients treated with steroids alone. In a second study, Tripathi et al. found 76% of steroid-nonresponsive patients had a durable response to eltrombopag after 3 months of therapy. Pediatric hematologists are already using thrombopoietin receptor agonists (TPO-RAs) in some cases of newly diagnosed ITP, according to a retrospective study by Neunert et al. TPO-RAs may be an efficacious first-line therapy for newly diagnosed ITP patients who require treatment. Study Design and Methods: The PINES (Pediatric ITP Newly diagnosed patients Eltrombopag vs Standard therapy) Study, NCT03939637, is an investigator-initiated prospective, open label, randomized, multi-center trial sponsored by the ITP Consortium of North America (ICON) and funded by Novartis. The primary objective is to determine if the proportion of patients with a platelet response, defined as ≥ 6 of 8 weeks with platelets >50 x109/L during weeks 5-12 of therapy without rescue treatment, is significantly greater in patients with newly diagnosed ITP treated with eltrombopag than in those treated with standard first-line treatments. This is a primary outcome used in a previous pediatric study of eltrombopag in chronic ITP (PETIT2) and is a clinically relevant outcome measuring a sustained, rather than transient, platelet response. Patients (n=156) from 20 ICON centers will be randomized 2:1 to receive the experimental treatment, eltrombopag, or investigator's choice of 3 standard front-line therapies: prednisone, intravenous immune globulin, or anti-D globulin at protocol-specified doses (Figure). Eligible patients are ages 1 to Patients will be followed for 1 year. Patients may receive prednisone, intravenous immune globulin, or anti-D globulin as rescue treatments beyond their study-assigned treatment in the first 12 weeks of the study. Patients randomized to the eltrombopag arm may continue this treatment throughout the 1-year duration of study participation if needed, with guidelines given for dose adjustments. Treatment after the first 12 weeks of study in the standard therapy arm or for patients originally assigned to eltrombopag who do not respond is at the discretion of the treating physician. Randomization is stratified by age and treatment status (upfront treatment vs. treatment failure). A one-sided z-test, at alpha=0.025, will be used to compare the proportion of patients who have a platelet response between the two arms. All randomized patients will be analyzed in the intention-to-treat analysis. Secondary objectives include comparison of bleeding scores (WHO Bleeding Scale and Modified Buchanan Score), changes in HRQoL (measured by the Kids ITP Tool, Hockenberry Fatigue Scale, PROMIS, and Global Change Scale), and changes in percentage of CD4+25+Foxp3+ regulatory T cells. Samples will be banked for optional future biology studies. Site activation and enrollment began in May 2019, and updated enrollment data will be presented at the meeting. Figure Disclosures Shimano: Novartis: Research Funding; Pfizer: Research Funding; Daiichi Sankyo: Research Funding. Grace:Novartis: Research Funding; Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bennett:Novartis: Research Funding. Neufeld:Octapharma, Shire Pharmaceuticals (Baxalta), Novo Nordisk, Celgene, NHLBI/NIH: Research Funding; Octapharma, Agios, Acceleron, Grifols, Pfizer, CSL Behring, Shire Pharmaceuticals (Baxalta), Novo Nordisk, ApoPharma, Genentech, Novartis, Bayer Healthcare: Consultancy; Octapharma: Other: study investigator, NuProtect study (Octapharma-sponsored). London:United Therapeutics: Consultancy; ArQule, Inc: Consultancy. Despotovic:Novartis: Research Funding; Amgen: Research Funding; Dova: Honoraria. OffLabel Disclosure: Eltrombopag is a thrombopoietin receptor agonist FDA-approved for use in chronic ITP.

Introduction: Early preclinical studies demonstrating that eradication of intestinal bacteria could prevent the development of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) led many adult and pediatric HSCT programs to practice "gut decontamination" (GD) with non-absorbable oral antibiotics for aGVHD prophylaxis. However, there is insufficient data supporting a clear benefit for GD in HSCT patients. Recent adult HSCT studies show that GVHD-related mortality is associated with decreased bacterial diversity in the gut microbiome, suggesting that certain bacterial species are protective against aGVHD. GD may decrease microbiome diversity and lead to worse post-HSCT outcomes. Methods: We conducted a randomized phase 2 study to examine the impact of GD on intestinal microbiome composition in pediatric allogeneic HSCT patients. 20 patients were enrolled and randomized to receive (GD) or not receive (no GD) oral vancomycin-polymyxin per our institutional standard from day -5 through engraftment. Stool samples were collected pre-transplant, weekly until neutrophil engraftment, monthly through day +100, then at 6 months and 1-year post-HSCT. Our primary endpoint is bacterial diversity, quantified by Shannon diversity index, at 2 weeks post-HSCT. Change in diversity is defined as the delta of diversity before transplantation compared to the minimum diversity seen in a single patient in a one-week window (around 14 days post-transplantation). This change in diversity was then averaged (mean) across each treatment arm. The stool microbiome was characterized using shotgun metagenomic sequencing (Nextera Tagmentation and Illumina HiSeq4000 Sequencing) for 10 patients to date, with median count of 1.4x107 reads per sample (range 1.3x106 to 3.3x107). Following quality control (FastQC, Trim Galore, Super Deduper), reads are assigned taxonomic designations using Kraken2 and GenBank (Oct. 2018) database. Secondary endpoints include the frequency of diarrhea (defined as >3 bowel movements per day in the first 7 days post-HSCT), incidence of aGVHD and bacteremia during the first 100 days post-transplant, immune reconstitution, survival, and malignant disease relapse at 2 years after study entry. Comparisons by treatment group were performed with Fisher's exact test (binary) or Wilcoxon rank-sum test (continuous). Results: Baseline patient characteristics were similar between the two arms (Table 1). At present,5 patients (25%) have completed 2 years of follow up, 4 of 15 patients with malignant disease (27%) have had relapse (3 GD, 1 No GD), and 2 patients (10%) in the No GD arm have died (1 malignant relapse, 1 refractory chronic GVHD). Microbiome analysis in the first 10 patients (4 GD, 6 No GD) demonstrates that the average change in Shannon diversity score at 2 weeks is 1.7 (SD 0.86) in GD and 0.65 (SD 0.61) in No GD (Table 2). Diarrhea occurred in 4/10 patients in GD vs 4/10 patients in no GD (p≥0.99).1/10 patients in GD and 3/10 in No GD developed aGVHD grade II or above(p=0.58). Bacteremia within the first 100 days post-HSCT occurred in 1/10 patient in GD, whereas 5/10 patients in No GD had 8 different bacteremia events (p=0.14) (Table 3). Conclusions: This is the first randomized study of gut decontamination in pediatric patients undergoing HSCT with long-term prospective post-transplant stool sample collection and immune monitoring. Though the small sample size is currently not powered to detect differences in clinical outcomes between the 2 study arms, the descriptive microbiome and immune reconstitution data will inform larger studies. There is no statistical difference in the diarrhea, aGVHD, or bacteremia incidence between the two groups but it is interesting that there was 1 vs 8 bacteremia events in GD vs No GD. Microbiota diversity results from our pilot study with 10 patients indicate a trend toward lower diversity at 2 weeks post-transplant in the GD arm. Microbiome analysis of the remaining samples from the remaining 10 patients is underway as well as longitudinal analysis of samples from all patients. Describing the changes to the microbiota composition over time in two groups will provide insight to practice of GD, potential targets for aGVHD, and will provide preliminary data to understand which microbes and microbial gene-pathways may influence immune reconstitution. Disclosures London: United Therapeutics: Consultancy; ArQule, Inc: Consultancy. Ritz:Draper Labs: Consultancy; Kite Pharma: Research Funding; Avrobio: Consultancy; LifeVault Bio: Consultancy; Merck: Research Funding; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Equillium: Research Funding. Bhatt:ArcBio: Other: Scientific Advisory Board; January.ai: Other: Scientific Advisory Board; Caribou Bioscience: Other: Scientific Advisory Board; Kaleido Biosciences: Consultancy, Other: Paid Consultant; Janssen Human Microbiome Institute: Consultancy, Other: Paid Consultant; Illumina: Honoraria; 10x Genomics: Other: Research collaboration without funding support; Illumina: Other: Research collaboration without funding support; Agilent: Research Funding; Global Oncology, Inc: Other: Nonprofit Board.

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs.

Introduction: Germline mutations in GATA2 lead to myelodysplastic syndromes (MDS) and bone marrow failure (BMF). Stem cell transplantation (SCT) remains the only curative therapy. In our previously described cohort of pediatric MDS/BMF patients with GATA2 mutation we noted a high transplant related mortality (TRM), a low relapse rate and frequent toxicities including thromboses. We hypothesized that analogous to other inherited syndromes the underlying GATA2 mutation may contribute to increased toxicity. Methods: We performed a case control study of patients treated at our center in 2000-20014 comparing pediatric MDS/BMF patients with GATA2 mutations to MDS/BMF patients without the mutation (control A) and to patients with acute leukemia (ALL or AML) (control B). Controls were randomly selected and matched for age (0-10, 11-20), stem cell source (matched related donor (MRD), matched unrelated donor (MUD), mismatched unrelated donor (mmURD) or umbilical cord blood (UCB) and conditioning regimen. We assessed if the proportion of patients who experienced a) high grade transplant related toxicity (TRT) including: infections, organ toxicities, secondary malignancies, thrombotic events; b) acute (grade II-IV) or extensive chronic GVHD (cGVHD); and, c) TRM within 100 and 365 days from transplant day, differed between the investigational and control cohorts. All pairwise comparisons of these factors were performed using a one-sided Fisher's Exact test. We reported overall survival (OS), disease free survival (DFS) and event free-survival (EFS) (events: TRM, relapse, acute GVHD, cGVHD, secondary malignancy, thrombotic events using point estimates at 5 years post-transplant (± standard error). Results: 80 patients were analyzed: 15 GATA2, 25 control A and 40 control B. The diagnoses in the GATA2 vs. the control A cohort were: RCC (n=8 vs 9), RAEB/ RAEB-T/AML-MRC (n=4 vs 11), AML (n=2 vs 0), BMF (n=0 vs 1), aplastic anemia (n=0 vs 4) and ALL (n=1 vs 0). Control B patients had AML (n=20) or ALL (n=20). Monosomy 7 was the most common cytogenetic abnormality in the GATA2 cohort [(n=9 (60%)] vs. 9 (36%) and 3 (8%) in Control A and B. The median follow-up for the study population was 3 years (range=0.14-12.1). GATA2 patients were treated with the following conditioning regimens: cyclophosphamide (CY)/total body irradiation (TBI) +/-ATG (n=11), fludarabine/CY/TBI (n=2) and busulfan/CY (n=2) and transplanted using bone marrow from a MRD (n=3), MUD (n=5) and mmURD (n=3), or UCB (n=4). GVHD prophylaxis consisted of cyclosporine A (CSA)/MTX+/- prednisone (n=9) or CSA/ mycophenolate mofetil (n=3). Thrombotic events were seen only in the GATA2 cohort (p< 0.001). For all other toxicities 10 (67%) occurred in the GATA2 group vs. 7 (28%) in control A (p=0.019), and 8 (20%) in control B (p=0.0018)]. Other comparisons were not statistically significant. Secondary malignancy was rare, but 1 (6.7%) was observed in the GATA2 cohort vs. 1 (4%) and 2 (5%) in control A or B. Severe lung disease requiring lung transplant was observed in 2 (13.3%) of the GATA2 patients and in 0 (0%) and 1 (2.5%) in control A and B, respectively. Acute and cGVHD were seen in 5 (33.3%) and 5 (33%) in the GATA2 group vs. 5 (20%) and 8 (32%) in control A and 10 (25%), 9 (22.5%) in control B. TRM within 100 and 365 days occurred in 2 (13%), 3 (20%) in the GATA2 cohort and 2 (8%), 4 (16%) in control A and 2 (5%), 7 (17.5%) in control B. TRM was the most common cause of death within 365 days in all groups. Relapse rate was not different between groups. The 5-year OS in the GATA2 cohort was 65±13% compared to 58%±10% and 45%±8% for control A and B, respectively. The 5-year EFS were 7%±6%, 31%±11% and 33%±8% for cases, A, and B, respectively. The 5-year DFS was 51%±13% in the GATA2 group, 51% ±11% in control A, and 44%±8% in control B. Conclusions: Pediatric MDS/BMF patients with GATA2 mutations had a significantly higher rate of TRT and thromboses compared to those without themutation or pre-treated leukemia patients. The 5-year EFS was lower in the GATA2 patients, but 5-year OS and DFS was comparable across the groups. Toxicities observed, especially frequent thromboses appear unique to the GATA2 cohort and warrant specific treatment considerations in the future. Disclosures No relevant conflicts of interest to declare.

Background: PKD causes a defect in glycolysis resulting in a hereditary non-spherocytic hemolytic anemia. The prevalence of iron overload is not well described for PKD. Aim: We aim to describe the demographic features and prevalence of iron overload in transfusion dependent and transfusion independent patients with PKD. Methods: Between March 2014 and April 2016, 203 patients enrolled on the PKD Natural History Study at 29 IRB approved sites. All patients were confirmed to have two compound heterozygous or homozygous mutations in the PKLR gene. Children < 1 year of age (n=9) were excluded from this analysis, because elevated ferritin levels are less reliably related to iron overload. Patients were designated with iron overload at the time of enrollment if the plasma ferritin was >1000 ng/mL or the patient was on chelation therapy at any time during the prior 12 months. Patients were designated with having had iron overload if a MRI ever showed liver iron content (LIC) >3 mg/g dry weight or if they had ever received chelation therapy. Tests of association were performed using Fisher's exact test (categorical) and Wilcoxon rank sum test (continuous). Linear associations between variables were measured by Pearson correlation coefficient. P-values Results: Of the 194 patients, 111 (57%) were adults ≥18 years and 83 (43%) were children. The median age of enrollment was 20.6 y (range: 1.3-69.9). Splenectomy had been performed in 65% (126/194). Screening ferritin levels were available for 72% (140/194) and LIC for 32% (62/194). At enrollment, 48% (70/147) had iron overload as defined by ferritin and/or current chelation. Using the LIC criterion, iron overload had been present at some point in 86% (95/110) of patients. Ferritin positively correlated with LIC (n=45); r=0.62, p3 mg/g DW. Baseline characteristics in patients with and without iron overload are shown in the Table. Notably, even in patients that were never regularly transfused and had a hemoglobin (Hb) >8.7 g/dl, the prevalence of iron overload was 26% (8/31). The frequency of iron overload was significantly higher in patients who had a prior splenectomy (p Data on cardiac iron status was available for 66 patients.Only 2 had cardiac iron overload (defined as T2* Of the 194 patients, 52 (27%) were from the Pennsylvania Amish community. These patients were managed differently than the non-Amish, in that only 2% of the Amish patients were on iron chelation therapy in the 12 months prior to enrollment compared with 43% among the non-Amish cohort. In addition, the Amish had a significant higher prevalence of splenectomy (96% vs 52%, p Conclusion: Iron overload is a common, serious complication in PKD, regardless of age, disease severity, or transfusion status. Although ferritin correlates with LIC for the PKD population overall, at the individual patient level, ferritin is not a good predictor of LIC and a ferritin Disclosures Barcellini: Agios: Consultancy. Neufeld:Novartis: Consultancy. Morton:Agios Pharmaceuticals: Research Funding. Yaish:Octapharma: Other: Study investigator. Kuo:Agios Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Apotex: Other: unrestricted educational grant; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Thompson:bluebird bio: Consultancy, Research Funding; Eli Lily: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mast: Research Funding; Baxalta (now part of Shire): Research Funding; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Amgen: Research Funding. Grace:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Current treatment strategies for Hodgkin lymphoma result in excellent survival but often confer significant long-term toxicity. We designed ABVE-PC (doxorubicin, bleomycin, vincristine, etoposide, prednisone, cyclophosphamide) to (1) enhance treatment efficacy by dose-dense drug delivery and (2) reduce risk of long-term sequelae by response-based reduction of cumulative chemotherapy. Efficient induction of early response by dose-dense drug delivery supported an early-response–adapted therapeutic paradigm. The 216 eligible patients were younger than 22 years with intermediate- or high-risk Hodgkin lymphoma. ABVE-PC was administered every 21 days. Rapid early responders (RERs) to 3 ABVE-PC cycles received 21 Gy radiation to involved regions; RER was documented in 63% of patients. Slow early responders received 2 additional ABVE-PC cycles before 21 Gy radiation. Five-year event-free-survival was 84%: 86% for the RER and 83% for the slow early responders (P = .85). Only 1% of patients had progressive disease. Five-year overall survival was 95%. With this regimen, cumulative doses of alkylators, anthracyclines, and epipodophyllotoxins are below thresholds usually associated with significant long-term toxicity. ABVE-PC is a dose-dense regimen that provides outstanding event-free survival/overall survival with short duration, early-response–adapted therapy. This trial was registered at www.clinicaltrials.gov as #NCT00005578.

The X-linked disorder Wiskott-Aldrich Syndrome (WAS), characterized by thrombocytopenia, eczema, recurrent infections, autoimmunity and cancer, is typically treated with allogeneic transplantation. Somatic gene therapy (GT) using autologous CD34+ cells is a promising treatment alternative for high-risk patients lacking matched allogeneic donors, as it avoids graft versus host disease. GT using a γ-retroviral vector with intact viral enhancers was efficacious but had a high rate of leukemia due to insertional oncogenesis. We report here preliminary results of 4 WAS patients who underwent GT using a self-inactivating lentiviral (SIN-LV) vector, in which viral enhancers have been deleted and the human WAS cDNA is controlled by a 1.6kB fragment of the human WAS promoter (w1.6_hWASP_WPRE SIN-LV). WASP expression per cell induced by this vector was lower than in normal cells when examined in murine models and human trials. We hypothesized that the SIN-LV would readily correct immune abnormalities due to selective advantage for WAS protein (WASP) expressing T cells whereas correction of myeloid and platelet abnormalities would require high vector copy number (VCN) in the infused cells. Patients with severe WAS (clinical scores 3-5) were enrolled at a median age of 32 months (17 months-8 years). Patients 1 and 3 had detectable but low WASP expression. Patients 2 and 4 carried mutations that abrogated WASP expression but had evidence of somatic reversion in T and/or NK cells. CliniMACS purified CD34+ mobilized peripheral blood or bone marrow cells were transduced with the vector and infused after busulfan (12-15mg/kg) and fludarabine (120mg/m2) conditioning. CD34+ cell doses ranged from 6.3-24.91 x 106 cells/kg. VCN of the infused cells was variable (3.37, 1.34, 0.54, 1.01 copies/cell). Busulfan exposure was myeloablative or near-myeloablative in patients 1, 3, and 4 (81.2, 77.2, 84.5 mg*h/L) and submyeloablative in patient 2 (48.8 mg*h/L). All patients are alive with median follow-up of 13.5 months (9-24 months). All patients had improvement in eczema, remain platelet transfusion independent and have had no severe bleeding events. WASP expression in T cells was increased post-GT over baseline. Selective advantage for WASP expressing T cells was apparent in patients 1, 2 and 4, who had higher VCN in T cells at 6 months post-GT (0.93-2.21) than in B (0.48-1.7) or myeloid (0.13-0.89) cells. The presence of revertants in patients 2 and 4 did not appear to interfere with T cell reconstitution. In contrast patient 3 who had the highest WASP expression at baseline and the lowest VCN of infused cells (0.5 copies/cell), had the lowest VCN in T cells at 8 months (0.1 copies/cell). Defective T cell proliferation in response to anti-CD3 stimulation, characteristic of WAS, was improved post-GT. Next generation sequencing of T cell receptors in sorted naïve, memory and regulatory T cells revealed profound abnormalities of diversity, decreased entropy and marked clonal expansions pre-GT; most of these improved at 6 months post-GT. Cytoskeletal function in myeloid cells was highly abnormal pre-GT, as shown by absence of podosome formation in monocyte-derived dendritic cells (0-1% vs. 61% in controls). Podosome formation at 6 months post GT was improved but subnormal (4-40%). Only patient 1, who received the highest cell dose, the highest VCN, and myeloablative busulfan exposure had robust platelet reconstitution (pre-GT 24 versus 110 x 109/L 6 months post-GT) and high level gene marking in myeloid cells 0.89 copies/cell. At the same timepoint, patients 2, 3 and 4 had platelet counts of 20-30 x 109/L and correspondingly lower VCN in myeloid cells (0.13-0.27 copies/cell). No severe adverse events related to GT have occurred to date, with relatively short follow-up. Integration site analysis of sorted cells showed highly polyclonal reconstitution, with distributions of integration acceptor sites as expected for the lentiviral vector backbone. In summary, GT using a SIN-LV that induces expression of WASP at levels below wild type improved the clinical and laboratory manifestations of WAS, with better reconstitution in patients receiving cells with high VCN. Disclosures Off Label Use: Off-label use of CliniMACS purified CD34+ cells.

Background: PKD is the most common enzyme defect of the glycolytic pathway causing hereditary non-spherocytic chronic hemolytic anemia. PKD is transmitted as an autosomal recessive trait, caused by both homozygous and compound heterozygote mutations in the PKLR gene, and is characterized by molecular heterogeneity with > 200 different mutations reported. Aim: To describe the PKLR genotypes in the PKD NHS with an in depth characterization of 20 newly reported mutations. Methods: Participants (pts) were enrolled in the PKD NHS, a prospective international study open at 23 sites in North America and Europe. Pts with prior PKLR gene sequencing were not resequenced. DNA from all other pts was extracted and the PKLR gene analyzed by Sanger sequencing at 1 of 2 central labs. All new missense mutations affected highly conserved residues in multiple domains of the PKLR gene, were not detected in 1000 genomes and LOVD database, and were considered pathogenic by NCBI and/or UniProtKB and by Polyphen analysis. Results: Genotype information was available on 140 enrolled pts. Of these, 66 (47%) were related to other subjects enrolled in the study. Molecular characterization confirmed the wide heterogeneity of PKD with 65 different mutations identified, including: 42 missense, 20 disruptive mutations (7 splicing, 6 frameshift, 3 stop codons, and 4 large deletions), 2 inframe insertion/deletions, and 1 promoter variant. Sixty-six pts were homozygous, of whom 55 were of Amish origin carrying the p.R479H mutation. Of the 55, 46 had been transfusion dependent prior to splenectomy and 9 had only received transfusions for acute stressors; 93% had been splenectomized, and all were transfusion independent post-splenectomy. Thirty-nine cases had 2 different missense mutations; 18 had one missense and one disruptive mutation, and 16 had 2 disruptive mutations; 1 patient with 17% residual PK activity displayed 3 different mutations (R510W, E241X and V276WXfs45). Besides R479H, the most common mutations were: R510W (16% of the mutated alleles), R486W (12%), and G241X (9%). Frequencies of R510W and R486W were less than those reported in Europe (41% and 30%, respectively). Twenty mutations, all affecting the PK structural domains, have not been previously described: 14 missense, 3 splicing (c.966(-9) a>g; c.1116(+2) t>c; c.375(+1) g>a), 1 frameshift (R40R fsX7), 1 inframe insertion of 2 amino acids, and 1 large deletion spanning intron 2 to intron 3 (Table). The 3 new splice site mutations were predicted to affect normal splicing when analyzed by HSF3.0, using both HSF and MaxEnt algorithms; in particular, homozygous c.966(-9) a>g was detected in a patient with moderate anemia, reticulocytosis, and mental retardation of unclear etiology. The two new missense mutations detected at a homozygous level (A137V and N156G) were associated with moderate or severe anemia and need for regular transfusions. The latter is located in the Aβ3 catalytic domain/K binding site and probably affects the catalytic efficiency of the enzyme. All the remaining new variants were detected in compound heterozygosity making it difficult to predict their effect on clinical phenotype. Intra-family clinical variability was observed; no correlation was found among the kinds of mutations and the residual PK activity. Conclusion: The molecular features of the largest international cohort of PKD pts are described, including a report of 20 new mutations, thus confirming the wide heterogeneity of the molecular genotype in PKD. Figure 1. Figure 1. Disclosures Morton: Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees. Eber:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yaish:Agios: Membership on an entity's Board of Directors or advisory committees. Nottage:Janssen Pharmaceuticals: Employment. Kuo:Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees. Grace:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: An international, multicenter registry was established to collect clinical data on patients with PKD, the most common glycolytic defect causing congenital non-spherocytic hemolytic anemia. Aim: To describe and categorize the phenotypic spectrum of PKD, including the range of lab parameters, management, and complications. Methods: 144 patients enrolled on the PKD NHS at 23 sites from 3/2014 to 6/2015. For this report, baseline and retrospective data were included. PKD patients were categorized into 4 severity groups (Gp1-Gp4), with increasing severity of the disease: Gp1. Never regularly transfused +/- prior acute transfusions; Gp2. Regular transfusions prior to splenectomy, post-splenectomy baseline hemoglobin (hb) >8.7 g/dl; Gp3. Regular transfusions prior to splenectomy, post-splenectomy baseline hb ≤8.7 g/dl; and Gp4. Post-splenectomy and currently regularly transfused. To reduce the risk of chance associations when dividing the cohort by phenotype, the cohort was divided into a test set (n=73) to define the categories and a subsequent validation set (n=71). Results: Median age at enrollment was 19.9 years (0.1-70.7) with 47% males. This cohort was 95% Caucasian and 38% Amish. Perinatal complications were frequently reported including preterm birth (33%), perinatal transfusions (35%), and hydrops (10%). Newborn jaundice was common, requiring phototherapy (91%) and/or exchange transfusion (46%). 68% (98/144) had undergone splenectomy at a median age of 3.1 years (0.6-28.1). Common indications for splenectomy included reducing transfusion burden, improving anemia, and enhancing quality of life. The median pre-splenectomy hb was 7.0 g/dl (4.5-12.5). Splenectomy reduced the transfusion burden in 91%. Triggers for hemolysis included: pregnancy (59%), infections (61%), stress (35%), and medications (6%). In the 37% (53/144) who required a cholecystectomy, the median age was 14 years (2.6-60.4) and 35 (66%) occurred post-splenectomy. In both the test and validation cohorts, increased severity was associated with a younger age at diagnosis (p Gp4 pts required regular transfusions even after splenectomy (11/98). In this group, the median age of splenectomy was 5.0 y (2.1-11.8). Ferritin was higher in those who had a splenectomy, even after adjusting for transfusion status (p4 mg/g dry weight and 9/11 (82%) had ferritin >500 ng/ml. Chelation was used in 35 patients at a median age of 11.8 years (1-53.7) with a median ferritin 815 ng/ml (182-5630) and median LIC 5.7 mg/g dry weight (1.7-46.0). Conclusions: This NHS cohort is the largest assembly of PKD patients to date. We defined 4 severity Groups, based on transfusion history, anemia, and splenectomy status. Complications (e.g. iron overload) correlate with disease severity but also occur in milder phenotypes. Based on these findings, regular monitoring of iron status and screening for gallstones should be considered. Transfusion dependence persisted despite splenectomy in 11% of patients. Prospective data from the NHS will provide guidance for monitoring and treatment in this rare anemia. Figure 1. Figure 1. Disclosures Grace: Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Morton:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees. Eber:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yaish:Agios: Membership on an entity's Board of Directors or advisory committees. Nottage:Janssen Pharmaceuticals: Employment. Kuo:Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees. Neufeld:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction Pediatric acquired aplastic anemia (AA) is a hypocellular bone marrow condition that is often difficult to distinguish from inherited bone marrow failure syndromes (IBMFS) and hypoplastic refractory cytopenia of childhood (RCC). RCC is a provisional entity for which the clinical implications are still under investigation. Historically, patients with RCC have been classified as AA. In this study, we sought to assess the intra-observer reproducibility and prognostic value of the histological criteria for RCC and AA as defined in the WHO classification. Specifically, we evaluated if RCC is an independent prognostic factor of overall survival (OS) and event-free survival (EFS), including treatment failure, disease progression and clonal evolution. Methods We performed a retrospective analysis of 149 AA patients seen at a single center between 1976-2010. Patients that met clinical or molecular diagnostic criteria for inherited bone marrow failure syndromes were excluded. We evaluated 5-year EFS, OS, and response rate to immunosuppressive therapy (IST); outcomes after matched HLA-identical related donor transplant (MRD) and matched unrelated donor (MUD) stem cell transplantation, as well as toxicities and clonal evolution. Of the 149 eligible individuals, 72 had diagnostic pathology material available for review. A double-blinded analysis of bone marrow aspirate and biopsy slides was undertaken by 3 pediatric hematopathologists. Each pathologist reviewed the slides of the entire study set and independently made a determination of the diagnosis. In cases where one or more differed in their assessment, the three conferred with an additional senior pediatric hematopathologist and all four reached agreement on a ‘consensus diagnosis’. To describe the degree of concordance between the assessments of the pathologists, a kappa coefficient was calculated. A logistic regression model was used to identify factors prognostic of treatment failure. Survival curves were generated using the methods of Kaplan and Meier. Results The 149 patients were classified as moderate aplastic anemia (MAA) (n=58), severe aplastic anemia (SAA) (n=50) and very severe aplastic anemia (vSAA) (n=41). Ninety-one patients received IST, 50 underwent a MRD HSCT, and 8 were observed without treatment. The concordance between pathologists in the assessment of the diagnosis of AA or RCC was modest, but ultimately a consensus between the pathologists was reached. The overall EFS and OS were 50.8% and 73.1% for all patients. The 5-year OS and 5-year EFS for all patients receiving IST were 87.8% and 51.5%, respectively, with a failure to respond to IST in 48%. Patients with vSAA had worse 5-year OS compared to SAA and MAA patients, respectively (p=0.02). 5-year OS was comparable at 83.6%±7.2 and 85.3%±5.2 for patients receiving a MUD or MRD respectively. Within AA patients receiving IST, none of the following factors had prognostic value to predict failure of treatment: consensus diagnosis of RCC, macrocytosis (MCV > 100), or HgF% > 4%. An MCV >100and HgF% >4% likewise were not associated with consensus diagnosis of RCC. However, a significantly higher hemoglobin (Hgb) and absolute reticulocyte count was found in patients with RCC compared to AA. Surprisingly the diagnosis of RCC was associated with a trend towards improved 5- year OS (84.9% versus 72.5%) and 5-year EFS (71.3% versus 52.5%) compared to AA. Five patients (~3%) of the analytical cohort all with the clinical diagnosis of MAA experienced clonal evolution or disease progression. Interestingly, the clinical diagnosis of vSAA significantly correlated with the histologic appearance of AA, whereas SAA and MAA corresponded with RCC, strongly suggesting a correlation between lower disease severity as determined by peripheral counts and/or marrow cellularity and the diagnosis of RCC. Conclusion The EFS and OS of the IST group in this single institution study is consistent with larger series. Our data indicate a low rate of clonal evolution in pediatric AA that in this series was associated exclusively with MAA. Most important, our findings suggest that the histologic diagnosis of RCC, reached by consensus among four pediatric hematopathologists does not predict disease outcome in this retrospective series of AA. These data indicate the need for larger prospective studies to determine the clinical significance of the classification. Disclosures Sartain: Hemostasis and Thrombosis Research Society: Research Funding.

Dyskeratosis congenita (DC) is an inherited bone marrow failure (BMF) syndrome caused by defects in telomere biology genes. BMF occurs in approximately 80% of patients by 30 years of age and, in addition to pulmonary failure and malignancies, remains a major contributor to mortality. Hematopoietic cell transplantation (HCT) cures BMF in DC patients but is associated with a high incidence of graft failure and transplant-related mortality. Radiation and/or alkylating agents (e.g. cyclophosphamide, busulfan, melphalan) used in conditioning may have a role in the poor outcomes in DC patients following HCT. Recently, reduced-intensity conditioning approaches have improved short-term outcomes, but concerns remain about the long-term effects of DNA damaging agents in these regimens, given the multisystem disease and lifelong predisposition to malignancy conferred by telomere dysfunction. Radiation and alkylating agents are mainstays of allogeneic HCT preparative regimens: their highly effective myeloablative and immunosuppressive properties provide a niche for donor hematopoietic progenitors and decrease graft rejection. Based on a rationale that presumes (1) niche availability in DC patients with BMF, and (2) an intrinsic replicative defect in DC hematopoietic cells, we hypothesized that engraftment would be feasible in DC patients using a regimen of immunosuppressive therapy alone. Here, we report that in a single-institution prospective study, 4 consecutive patients with DC have undergone successful HCT using a radiation- and alkylator-free preparative regimen. Patients received alemtuzumab and fludarabine, followed by bone marrow transplantation (BMT) from unrelated donors. Cyclosporine A and mycophenolate mofetil were used for graft versus host disease (GVHD) prophylaxis. The primary outcome measures were neutrophil engraftment at day +30 and overall survival at day +100. Secondary outcome measures included acute toxicities and rates of graft failure, infections, and GVHD. The first two patients were 18 month-old twins with TINF2 mutations and transfusion-dependent BMF who received a BMT from a fully HLA-matched unrelated donor. The third patient was an 18 year-old female with CTC1 mutations and transfusion-dependent BMF, who underwent a B-mismatched unrelated donor BMT. The fourth patient, a 22 year-old male with clinical DC and short telomeres, had significant pulmonary disease (diffusing capacity of lung for carbon monoxide 28% of predicted) and BMF, and underwent a fully HLA-matched unrelated donor BMT. All 4 patients engrafted neutrophils by day +30, and are alive and well with follow-up ranging from 6 months (1 patient) to 2 years (3 patients). Platelet transfusion-independence occured between day +4 to +18, and red cell transfusion-independence occured between day 0 to +90. No patients required transfusions thereafter. All patients showed full donor myeloid chimerism by day +60. 3 of 4 patients showed full donor lymphoid chimerism by year 2; the fourth patient has high and increasing mixed donor lymphoid chimerism (60%) at day +180. There were no significant, unexpected toxicities, bacterial or fungal bloodstream or tissue infections. All patients were monitored weekly by serum PCR for CMV, EBV, adenovirus and HHV6 until day +100; only one patient showed CMV reactivation which was controlled with pre-emptive ganciclovir therapy. There was no acute GVHD. Only one patient developed chronic GVHD with limited skin involvement controlled with topical steroids. These results provide promising early data for engraftment and a favorable short-term toxicity profile using a radiation- and alkylator-free conditioning regimen in HCT for BMF due to DC. This approach could spare DC patients the acceleration of non-hematologic complications and malignancies, which has been attributed to HCT, and improve long-term survival. The limited toxicity of the alemtuzumab/fludarabine regimen may also allow patients with severe disease-related co-morbidities such as pulmonary dysfunction to safely undergo HCT. To our knowledge, this is the first prospective HCT study to achieve full donor myeloid engraftment in a series of patients following conditioning without radiation or alkylating agents. Disclosures Off Label Use: alemtuzumab - conditioning for bone marrow transplantation fludarabine - conditioning for bone marrow transplantation cyclosporine A - graft versus host disease prophylaxis in bone marrow transplantation mycophenolate mofetil - graft versus host disease prophylaxis in bone marrow transplantation.

A COG Phase II pilot study was conducted to assess the efficacy of the novel re-induction regimen of ifosfamide/vinorelbine (IV) in pediatric patients with heavily pre-treated relapsed/refractory Hodgkin disease. The treatment regimen consisted of : Ifosfamide 3 gm/m2/d continuous 24-hr IV infusion on Days 1–4; MESNA 3 gm/m2/d continuous 24-hr IV infusion on Days 1–5; and vinorelbine 25 mg/m2/dose IV bolus on Days 1, 5. Filgrastim was administered on Day 6 of Cycle 1 at a dose of 5 μg/kg until ANC ≥ 1000/μl for 3 consecutive days or ≥ 10,000/μl for 1 day and on Day 6 of Cycle 2 at a dose of 10 μg/kg to continue daily until completion of peripheral stem cell mobilization. We report the results of the first phase of the study, that evaluated the re-induction regimen IV which was substantiated as an effective re-induction regimen based on acceptable toxicity profile, capacity to mobilize hematopoietic stem cells (CD34+), and response sufficient to allow subsequent stem-cell transplantation in a large fraction of patients. Twenty-nine patients were accrued on study; 12 male (41%), 17 female (59%); median age at time of enrollment was 16 (range 5–20 yrs). The protocol mandated two cycles of therapy, although some patients received an optional third cycle. This report is based on data from the first 2 cycles only. Twenty-five patients had complete toxicity data. The predominant toxicity was hematologic, specifically leukopenia/neutropenia. The frequencies of grade 3–4 hematologic toxicities included: anemia (62%), leukopenia (100%), neutropenia (95%), and thrombocytopenia (50%). Hypophosphatemia (3%), febrile neutropenia (34.9%) and infection with grade 3 or 4 neutropenia (21.6%), and neurotoxicity (3%), each occurred in a minority of patients. There were no toxic deaths. Harvest was attempted in 25 patients. At least 2.5 x 106 CD34+ cells/kg were collected in 19 (76%), and at least 5.0 x 106 were collected in 16 (64%). Complete response data were available for twenty-five patients. Seven patients attained a complete response (CR) and eleven a partial response (PR) (CR/PR rate 72%). IV was substantiated as an acceptable re-induction regimen for children with Hodgkin disease. It was effective, well tolerated, and allowed for collection of sufficient CD34+ cells to support peripheral stem cell transplantation. Based upon these findings, 35 additional patients will be accrued to more precisely establish the definitive response rate in pediatric patients. In addition, minimally pretreated patients will now be included, allowing assessment of response, toxicity and hematopoietic stem cells (CD34+) mobilization in minimally pre-treated patients as well as in the heavily pre-treated population.