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Background: Sickle cell disease (SCD) is a group of genetic blood disorders characterized by hemolytic anemia, multi-organ damage and acutely painful events (vaso-occlusive crises [VOCs]) that can cause life-threatening complications. Vaso-occlusion is mediated, in part, by P-selectin, an adhesion molecule expressed on endothelial cells and platelets. Adults and children with SCD have similar P-selectin expression levels; however, disease severity worsens with increasing age because of cumulative endothelial damage caused by repeated vaso-occlusion events. Crizanlizumab is a humanized anti-P-selectin monoclonal antibody that binds P-selectin and blocks its interaction with its ligands. SUSTAIN, a Phase 2 study in adults with SCD, has shown that crizanlizumab, compared with placebo, is well tolerated and significantly decreases the number of VOCs requiring a healthcare visit (Ataga et al. N Engl J Med 2017). Aim: To describe the design of the first crizanlizumab study in pediatric patients (pts) with SCD and report demographics and baseline characteristics of a subset of enrolled pts (aged 6 to Methods: Primary objectives of this Phase 2, multicenter, open-label study are to confirm crizanlizumab dosing and assess safety. Pts aged 6 mo to Secondary objectives include assessment of crizanlizumab efficacy, measured by annualized rate of VOCs leading to a healthcare visit (clinic, emergency room [ER] or hospital), and annualized rate of VOCs managed at home. VOC subcategories (uncomplicated pain crisis, acute chest syndrome, hepatic and splenic sequestration, and priapism), overall hospitalizations and ER visits and dactylitis events are also assessed. Safety measures include frequency and severity of adverse events. Long-term PK and PD will also be characterized by measuring pre-dose concentrations and percentage of P-selectin inhibition prior to each study drug dose. Part A in each age group will confirm PK dose, beginning with ≥8 pts in G1. If unconfirmed, dose will be adjusted based on population PK model, and ≥8 additional pts enrolled. Once dose is confirmed, recruitment will be expanded for long-term safety and efficacy evaluation of the PK confirmed dose (Part B). This process will then repeat for G2 then G3 (2 to Results: As of January 28, 2020, 59 pts were enrolled: 46 pts in G1 (11 pts in Part A and 35 pts Part B) and 13 pts in G2 Part A. Pt demographics are available for Part A in G1 and 2 (Table), based on 2 Data Monitoring Committee analyses. The median age of G1 was 17.0 yr (range 13-17), 6 (54.5%) were male and 11 (100%) were Black/African American. 9 (81.8%) had HbSS disease, 1 (9.1%) HbSC and 1 (9.1%) HbSβ0. The median age of G2 was 9.0 yr (range 6-11), 8 (61.5%) were male, 7 (53.8%) were Black/African American, 4 (30.8%) were White, and 2 were of multiple race, specifically 'White, Asian' (n=1, 7.7%) and 'White, Black/African American' (n=1, 7.7%). 12 (92.3%) pts had HbSS disease and 1 (7.7%) had HbSC. Conclusions: This study aims to address an unmet treatment need in pediatric pts, exploring appropriate dosing and the safety of crizanlizumab. The annualized rates of VOCs, hospitalizations and ER visits will be assessed as secondary objectives. The primary analyses for Parts A and B of G1 and 2, and then G3, will occur consecutively when all pts enrolled in each group have either completed 26 weeks of treatment or discontinued the study treatment. Disclosures Heeney: UpToDate: Patents & Royalties: Author royalties; Micelle: Consultancy, Other; Keros: Consultancy; Novartis: Consultancy, Other; AstraZeneca: Consultancy, Other; Cyclerion: Consultancy, Other; Forma Therapeutics: Consultancy; Dova: Consultancy; Global Blood Therapeutics: Consultancy; Emerging Therapy Solutions (ETS): Consultancy. Rees:Alnylam Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AstraZeneca: Other: Data monitoring committee membership; Emmanus Medical: Consultancy, Honoraria; TauRx: Other: Data And Safety Monitoring. de Montalembert:Vertex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Addmedica: Honoraria, Membership on an entity's Board of Directors or advisory committees. Odame:Novo Nordisk: Other: Study Advisory Board; Global Blood Therapeutics: Other: Study Data Safety Monitoring Board; Novartis: Other: Study Steering Committee. Wali:Novartis: Research Funding. Nguyen:Novartis: Current Employment. Lam:Novartis: Current Employment. Herranz:Novartis: Current Employment. Kanter:NHLBI Sickle Cell Advisory Board: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; bluebird bio, inc: Consultancy, Honoraria; SCDAA Medical and Research Advisory Board: Membership on an entity's Board of Directors or advisory committees; AGIOS: Membership on an entity's Board of Directors or advisory committees; BEAM: Membership on an entity's Board of Directors or advisory committees; Guidepoint Global: Honoraria; Cowen: Honoraria; Jeffries: Honoraria; Wells Fargo: Honoraria; Medscape: Honoraria; GLG: Honoraria; Sanofi: Consultancy. OffLabel Disclosure: Crizanlizumab is a monoclonal antibody to P-selectin indicated in the USA for the prevention of vaso-occlusive crises in patients aged 16 years and over with sickle cell disease. This abstract described the new pediatric trial of crizanlizumab, which is not indicated for patients aged less than 16 years of age

The phase 3 HESTIA3 study assessed the efficacy and safety of the reversible P2Y12 inhibitor ticagrelor vs placebo in preventing vaso-occlusive crises in pediatric patients with sickle cell disease (SCD). Patients aged 2 to 17 years were randomly assigned 1:1 to receive weight-based doses of ticagrelor or matching placebo. The primary end point was the rate of vaso-occlusive crises, a composite of painful crises and/or acute chest syndrome (ACS). Key secondary end points included number and duration of painful crises, number of ACS events, and number of vaso-occlusive crises requiring hospitalization or emergency department visits. Exploratory end points included the effect of ticagrelor on platelet activation. In total, 193 patients (ticagrelor, n = 101; placebo, n = 92) underwent randomization at 53 sites across 16 countries. The study was terminated 4 months before planned completion for lack of efficacy. Median ticagrelor exposure duration was 296.5 days. The primary end point was not met: estimated yearly incidence of vaso-occlusive crises was 2.74 in the ticagrelor group and 2.60 in the placebo group (rate ratio, 1.06; 95% confidence interval, 0.75-1.50; P = .7597). There was no evidence of efficacy for ticagrelor vs placebo across secondary end points. Median platelet inhibition with ticagrelor at 6 months was 34.9% predose and 55.7% at 2 hours’ postdose. Nine patients (9%) in the ticagrelor group and eight patients (9%) in the placebo group had at least one bleeding event. In conclusion, no reduction of vaso-occlusive crises was seen with ticagrelor vs placebo in these pediatric patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT03615924.

Background: Hospitalization due to vaso-occlusive crisis (H-VOC) is common in individuals with sickle cell disease (SCD), with an increasing occurrence of SCD-related complications, including organ damage, as the disease progresses. Evidence regarding the relationship between H-VOC and SCD-related organ damage, however, is lacking. Aim: To assess whether H-VOC is associated with hospitalization due to SCD-related organ damage, through retrospective analysis of data collected prospectively during a 3-year, multicenter, observational US study (NCT01220115) that aimed to better understand disease burden and management of SCD in individuals aged ≥2 years. Methods: Of the 498 individuals with SCD who were recruited into the US study, data were analyzed from 202 (100 men and 102 women) who were aged ≥16 years and had available hospital admission data. Organ damage was defined based on hospital discharge diagnosis. 1 Variables tested at baseline, in addition to H-VOC, included demographics, blood measures, and treatment history. Age and sex were included by default in all models based on literature suggesting they are relevant factors influencing organ damage. Hazard ratios (HRs) for the time from H-VOC to the first subsequent hospitalization due to SCD-related organ damage were estimated using multivariable Cox regression. Worsening of pre-existing organ damage was not considered as an event due to potential confounding (ie worsening of organ damage related to the pre-existing condition rather than as a consequence of the VOC). Results: During median 3-year follow-up, 55 (27%) individuals experienced at least one hospitalization due to SCD-related organ damage; 2 19 (9%) had multiple visits. Within the 12 months preceding baseline, 22 (11%) individuals had a history of organ damage, there was a median of two H-VOC in the 90 (45%) individuals with history of H-VOC, and 43 (21%) individuals had received chronic transfusion (≥6). History of H-VOC (HR 2.54, 95% confidence interval [CI] 1.46 to 4.43 in past 12 months), genotype (HR 2.69, 95% CI 1.34 to 5.41 for HbSS), and sex (HR 1.90, 95% CI 1.08 to 3.34 for women) were all significantly associated with subsequent hospitalization for SCD-related organ damage. Discussion and conclusion: This analysis demonstrates that history of H-VOC within the preceding 12 months is significantly associated with a higher rate of subsequent hospitalization due to SCD-related organ damage, independent of age, sex, and genotype, and may therefore help identify individuals at high risk of developing organ damage. Despite 21% of individuals receiving chronic transfusions at baseline, this factor did not remain significantly associated with the outcome when also considering genotype and H-VOC. Age and sex were unexpectedly insignificantly associated with the outcome; this is likely due to the relatively short follow-up time. Extending the historical timeframe of organ damage to 5 years did not change our findings, except that age also became significantly associated with subsequent hospitalization for organ damage. Acute chest syndrome and pneumonia were the most common types of historical (baseline) organ damage, whilst gallbladder disease was the most common organ damage observed during the follow-up period that was not observed at baseline. Our data have limited statistical power and generalizability; additional studies are required to confirm these findings. Nevertheless, our findings support the existing evidence of the impact that VOCs may have on individuals with SCD, and highlights the importance of preventing and reducing H-VOC. 1Acute chest syndrome or pneumonia; avascular bone necrosis of hip(s), shoulder(s) or spine; cardiac failure; central nervous system disease (ie abnormal transcranial Doppler, silent infarct, stroke and transient ischemic attack); gallbladder disease; leg ulcer; liver disease (ie hepatic fibrosis/ cirrhosis, hepatic sequestration/sickle-hepatopathy/intrahepatic sickling, pulmonary fibrosis, pulmonary hypertension); priapism; renal disease (ie acute renal failure, chronic renal failure-supportive, dialysis, microalbuminuria/ proteinuria, transplant); retinopathy; and splenic sequestration. 2The top 3 reasons for hospitalization due to SCD-related organ damage were acute chest syndrome or pneumonia (n=29; 53%), renal disease (n=7; 13%) and gallbladder disease (n=6; 11%). Figure 1 Figure 1. Disclosures Heeney: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; FORMA: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Vertex / Crispr Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: DSMB; bluebird bio: Consultancy; Keros: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cyclerion: Consultancy, Membership on an entity's Board of Directors or advisory committees. Pattipaka: Novartis: Current Employment, Current holder of individual stocks in a privately-held company. Fermont: Novartis Pharma AG, Basel, Switzerland: Current Employment, Current equity holder in publicly-traded company.

Background: Vaso-occlusive crises (VOCs) are the hallmark of SCD. The cell adhesion molecule P-selectin plays a key role in the multicellular interactions that can lead to VOCs. In the SUSTAIN trial in adults, crizanlizumab 5.0 mg/kg, a humanized monoclonal antibody that blocks P-selectin, significantly reduced the median annualized rate of VOCs vs placebo and had a favorable safety profile (Ataga et al. N Engl J Med 2017). Aim: To describe initial safety and efficacy results for patients (pts) with SCD aged 12- Methods: SOLACE-kids is a Phase II study to confirm and establish appropriate dosing and evaluate safety of crizanlizumab in pediatric pts with SCD (any genotype) and ≥1 VOC leading to a healthcare (HC) visit within 12 mo prior to screening. Pts (N≥100) are stratified by age: Group 1 (G1; 12- Results: As of 28 August 2020, 50 pts were enrolled in G1 of SOLACE-kids. Mean (SD) age of pts was 15.0 (1.92) yr, 29 (58%) were female, 44 (88%) had the HbSS genotype, 32 (64%) were Black/African American and 42 (84%) were receiving HU. Median (range) duration of exposure to crizanlizumab was 36.6 (6-98) wk; 44 (88%) pts received treatment for ≥26 wk. The most commonly reported AEs were headache (n=14 [28%]), vomiting (n=12 [24%]) and back pain (n=9 [18%]). Grade ≥3 AEs were reported in 13 (26%) pts; most common were anemia (n=3 [6%]) and back pain (n=2 [4%]). Serious AEs were reported in 11 (22%) pts; none were deemed related to treatment. Incidence of AEs of special interest (AESI) is shown in Table 1. No AESI led to treatment discontinuation except 1 pt who died of meningitis (not related to treatment). No infusion-related reactions were serious; all had resolved at data cut-off (except for 1 case of Grade 1 dizziness). No case of anaphylactic reaction to crizanlizumab was reported. Pain events on the day of crizanlizumab infusion suspected to be related to treatment were reported in 3 (6%) pts. All pain events, regardless of relationship to treatment, were Grade 1/2, except for two Grade 3 events reported in the same pt (back pain and pain in extremity), which resolved on day of onset. All hemorrhage events were mild and not considered related to treatment. Increase from baseline (BL) in total bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was reported in 29 (58%), 17 (34%) and 20 (40%) pts, respectively. 2 (4%) pts had Grade 4 total bilirubin significantly above normal (1 pt was Grade 4 and 1 pt was Grade 3 at BL); 21 (42%) pts had Grade 3 total bilirubin significantly above normal (5 pts were Grade 3 at BL). Grade 3 increase in ALT and AST was reported in 2 (4%) pts each. All reported liver function parameters did not meet study criteria for severe drug-induced liver injury. The median (range) number of VOCs leading to a HC visit was 3.0 (1.0-26.0) at BL and 1.6 (0.0-12.7) on treatment (median absolute reduction: 1.0 [range: -13.3 to 5.8]). 18 (36%) pts did not experience a VOC leading to a HC visit while on treatment. The median (range) annualized rate of hospitalizations/ER visits at BL was 4.0 (1.0-36.0) vs 1.54 (0.0-14.3) on treatment (median reduction: 2.35 [range: -21.7 to 5.3]) (Table 2). Conclusion: This initial analysis of SOLACE-kids shows crizanlizumab 5.0 mg/kg is safe and well tolerated in pts aged 12- Figure 1 Figure 1. Disclosures Heeney: FORMA: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Vertex / Crispr Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: DSMB; bluebird bio: Consultancy; Keros: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cyclerion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rees: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Addmedica: Honoraria; TauRx: Membership on an entity's Board of Directors or advisory committees. De Montalembert: Vertex: Membership on an entity's Board of Directors or advisory committees; bluebird bio: Membership on an entity's Board of Directors or advisory committees; Addmedica: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Odame: Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Steering Committee; Global Blood Therapeutics: Other: DSMB. Brown: Imara: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Novo Nordisk: Consultancy; Forma Therapeutics: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding. Wali: Novatis Oncology: Research Funding. Nguyen: Novartis: Current Employment. Lam: Novartis Pharmaceuticals Corporation: Current Employment, Current equity holder in publicly-traded company. Pfender: Novartis: Current Employment, Current equity holder in publicly-traded company. Kanter: Fulcrum Therapeutics, Inc.: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Forma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees; Beam: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Consultancy; GuidePoint Global: Honoraria; Fulcrum Tx: Consultancy.

Adenosine A2A receptor (A2AR) agonists have been shown to decrease tissue inflammation induced by hypoxia/reoxygenation in mice with sickle cell disease (SCD). The key mediator of the A2AR agonist's anti-inflammatory effects is a minor lymphocyte subset, invariant natural killer T (iNKT) cells. We tested the hypothesis that administration of an A2AR agonist in patients with SCD would decrease iNKT cell activation and dampen the severity of vaso-occlusive (VO) crises. In a phase 2, randomized, placebo-controlled trial, we administered a 48-hour infusion of the A2AR agonist regadenoson (1.44 μg/kg per hour) to patients with SCD during VO crises to produce a plasma concentration of ∼5 nM, a concentration known from prior studies to suppress iNKT cell activation in SCD. The primary outcome measure was a >30% reduction in the percentage of activated iNKT cells. Ninety-two patients with SCD were randomized to receive a 48-hour infusion of regadenoson or placebo, in addition to standard-of-care treatment, during hospital admission for a VO crisis and had analyzable iNKT cell samples. The proportion of subjects who demonstrated a reduction of >30% in activated iNKT cells was not significantly different between the regadenoson and placebo arms (43% vs 23%; P = .07). There were also no differences between regadenoson and placebo groups in length of hospital stay, mean total opioid use, or pain scores. These data demonstrate that a low-dose infusion of regadenoson intended to reduce the activity of iNKT cells is not sufficient to produce a statistically significant reduction in such activation or in measures of clinical efficacy. This trial was registered at www.clinicaltrials.gov as #NCT01788631.

Background: DOVE (Determining Effects of Platelet Inhibition on Vaso-Occlusive Events) was a Phase 3, randomized, double-blind, placebo-controlled study conducted in children with sickle cell anemia at 51 sites in 13 countries across four continents. Procedure: Data from DOVE were assessed for regional differences in subject phenotype and treatment. Demographics, baseline clinical and laboratory data, hydroxyurea (HU) use, vaso-occlusive crisis (VOCs, composite endpoint of painful crisis or acute chest syndrome (ACS, Beijing, China)), serious adverse events (SAEs, Florence, Italy), hospitalization, and treatments were compared across the Americas, Europe, North Africa/Middle East, and Sub-Saharan Africa (SSA). Results: Race, body mass index, and blood pressures differed by region. Pre-enrollment VOCs were highest in the Americas. For subjects not on HU, baseline hemoglobin was lowest in SSA, reticulocyte count was lowest in the Americas. Within SSA, Kenya subjects presented higher baseline hemolysis. Painful crisis was the most common SAE, followed by ACS in the Americas and infections in other regions. VOC rate and percentage of VOC hospitalizations were highest in Europe. Regardless of region, most VOCs were treated with analgesics, approximately half were treated with intravenous fluids. The proportion of VOC-related transfusions was greatest in Europe. Lengths of hospital stay were similar across regions. Conclusions: Overall differences in SAEs and hospitalization for VOCs may be due to cultural diversities, resource utilization, disease severity, or a combination of factors. These data are of importance for the planning of future trials in SCA in a multinational setting.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited blood disorders characterized by pathological mitochondrial iron deposition in erythroid precursors. Each known cause has been attributed to a mutation in a protein associated with heme biosynthesis, iron-sulfur cluster biogenesis, mitochondrial translation, or a component of the mitochondrial respiratory chain. Here, we describe a recurring mutation, c.276_278del, p.F93del, in NDUFB11, a mitochondrial respiratory complex I-associated protein encoded on the X chromosome, in 5 males with a variably syndromic, normocytic CSA. The p.F93del mutation results in respiratory insufficiency and loss of complex I stability and activity in patient-derived fibroblasts. Targeted introduction of this allele into K562 erythroleukemia cells results in a proliferation defect with minimal effect on erythroid differentiation potential, suggesting the mechanism of anemia in this disorder.

TO THE EDITOR: Iron-refractory iron deficiency anemia (IRIDA) is characterized by congenital iron deficiency (ID) that is poorly responsive to oral iron treatment. Biallelic mutations in TMPRSS6 are found in most patients with IRIDA.[1][1] TMPRSS6 negatively regulates synthesis of the iron

Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.

BCL11A regulates the fetal-adult hemoglobin switch by repressing expression at the gamma (γ)-globin locus (Sankaran et al., Science, 2008), and thus it represents an appealing therapeutic target for sickle cell disease (SCD). BCH-BB694 is a lentiviral vector (LVV) encoding a shRNA targeting BCL11A embedded in a microRNA scaffold (shmiR) allowing erythroid-specific knockdown to induce γ-globin expression and concomitantly and coordinately repress β-sickle globin expression (Brendel et al. JCI, 2016). In a pilot and feasibility gene therapy study we are evaluating the safety of infusion of BCH-BB694-transduced autologous CD34+ cells in patients with severe SCD. The study is an IND enabled and IRB approved open label, non-randomized, single center trial (NCT 03282656). We report here data from the full adult cohort which has completed enrollment with > 6 months of follow up in all patients. The adult cohort included three patients >/= 18 years old. Autologous CD34+ cells were collected by plerixafor mobilization and then transduced ex vivo with the BCH-BB694 shmiR lentiviral vector. Cell doses and vector copy number (VCN) are shown in the Table. After testing and release, gene modified cells were infused into subjects who had received busulfan conditioning. There were no Grade 3 or 4 AEs associated with mobilization, collection or infusion. All three adults (age 21-26 years old) demonstrated neutrophil engraftment on day +22 with adverse events consistent with busulfan conditioning. These patients are now 7, 9, and 17 months post infusion. One subject resumed red cell transfusions at 3 months due to pre-existing moyamoya using a pre-defined conservative trigger value of 40% sickle Hb in whole blood and will be detailed separately. There have been no adverse events related to the gene therapy product. VCN has been stable in bone marrow (BM) and peripheral blood (PB) in all cell lineages during the length of the study, with the latest time point studied at 15 months (BCL002) and ranged from 0.45-2.85 copies per cell in erythroid progenitor cells. BCL11A protein levels evaluated by immunoblot in subject BCL002 at 30 days (PB) and 6 months (BM) post-infusion showed highly effective and selective knockdown of BCL11A in erythroid progenitors with no reduction in BCL11A expression in B lymphoid cells. The number of HbF-containing cells (F cells) was assessed by flow cytometry and the kinetics of F cell production was remarkably similar in all subjects. The two untransfused subjects (BCL002 and BCL004) produced 70% F-cells in PB at 3 and 5 months, which has remained stable until the last point assayed (15 months and 7.5 months, respectively) (table). Calculated average HbF per F cell was >10pg in all subjects (table) and quantitative single cell HbF flow analysis showed the majority of F cells had >4pg F/cell, a level that is believed to prevent sickling under physiological oxygen saturation (Rakotoson et al., ASH 2017). In both untransfused subjects, total Hb remained stable with evidence of reduced hemolysis by reticulocyte count (slightly elevated) and LDH (normal in one subject, slightly elevated in the other). At the 3-month timepoint before re-starting transfusions, the subject with moyamoya (BCL003) had a pre-transfusion Hb of 11 g/dL with 76% of non-transfused cells containing on average 17pg F/F cell. For all subjects, we estimated the fraction of RBCs containing significant Hb sickle polymers and the amount of polymer in each sickled RBC at physiologic oxygen tension (where 50% of monomeric hemoglobin was oxygen saturated, or the P50) (Di Caprio et al. PNAS 2019, in press). The results for all 3 subjects in this adult cohort showed fewer RBCs with significant Hb polymer than two hydroxyurea-responsive treated comparators and significantly less Hb polymer per sickled RBC than a third highly responsive hydroxyurea-treated comparator. In conclusion, these data demonstrate successful and sustained engraftment in three adult patients treated with LVV-delivered shmiR technology targeting BCL11A. Early results suggest an acceptable safety profile, validation of BCL11A as effective target for HbF induction in humans with high numbers of F cells in circulation containing high levels of HbF per F cell, and mitigation of cellular pathology of SCD. Disclosures Achebe: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacosmos: Membership on an entity's Board of Directors or advisory committees; Fulcrum Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Heeney:AstraZeneca: Research Funding; Micelle Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Research Funding; Ironwood / Cyclerion: Research Funding; Vertex / Crisper Therapeutics: Other: Data Safety Monitoring Board. Higgins:Sanofi: Consultancy, Research Funding. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Wood:Sanofi: Consultancy, Research Funding. Williams:Alerion Biosciences: Other: Co-founder; Novartis: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, Patents & Royalties: Potential for future royalty/milestone income, X-SCID., Research Funding; bluebird bio: Patents & Royalties: Licensed certain IP relevant to hemoglobinopathies to bluebird bio. Received payment in the past bluebird bio through a BCH institutional licensing agreement and there is a potential for future royalty/milestone income from this agreement., Research Funding.

Congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders identified by pathological erythroid precursors with perinuclear mitochondrial iron deposition in bone marrow. An international collaborative group of physicians and laboratory scientists collated clinical information on cases of CSA lacking known causative mutations, identifying a clinical subgroup of CSA associated with B immunodeficiency, periodic fevers, and development delay. Twelve cases from 10 families were identified. Median age at presentation was 2 months. Anemia at diagnosis was sideroblastic, typically severe (median hemoglobin, 7.1 g/dL) and markedly microcytic (median mean corpuscular volume, 62.0 fL). Clinical course involved recurrent febrile illness and gastrointestinal disturbance, lacking an infective cause. Investigation revealed B-cell lymphopenia (CD19⁺ range, 0.016-0.22 × 10⁹/L) and panhypogammaglobulinemia in most cases. Children displayed developmental delay alongside variable neurodegeneration, seizures, cerebellar abnormalities, sensorineural deafness, and other multisystem features. Most required regular blood transfusion, iron chelation, and intravenous immunoglobulin replacement. Median survival was 48 months, with 7 deaths caused by cardiac or multiorgan failure. One child underwent bone marrow transplantation aged 9 months, with apparent cure of the hematologic and immunologic manifestations. We describe and define a novel CSA and B-cell immunodeficiency syndrome with additional features resembling a mitochondrial cytopathy. The molecular etiology is under investigation.

Abstract 5147 Platelet activation/aggregation in sickle cell disease (SCD) may promote tissue ischemia, suggesting antiplatelet therapy may be useful. However, assessing platelet function and the effect of antiplatelet therapy in blood from SCD patients may be confounded by hemolysis with release of ADP. Here we evaluate levels of platelet activation markers in SCD adolescents vs. normal controls and compare, by multiple methods, the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Platelet activation markers in blood from SCD adolescents (n=15) and healthy adults (n=10), and the effect of R-138727 (0. 1 – 10 μM) added in vitro, were evaluated with and without ADP stimulation. Circulating levels of platelet-monocyte and platelet-neutrophil aggregates were significantly higher (p Disclosures: Frelinger: Eli Lilly: Consultancy, Research Funding; Daiichi Sankyo: Research Funding; GLSynthesis: Research Funding. Jakubowski:Eli Lilly: Employment. Heeney:Novartis: Consultancy, Research Funding; Eli Lilly and Company: Research Funding; Pfizer: Consultancy. Michelson:Eli Lilly: Data monitoring committee and idependent external monitor of clinical trials, Research Funding; Takeda: Research Funding; Oxygen Biotherapeutics: Research Funding; Alexion: Research Funding; Omthera: Research Funding.

To date, there is a lack of long-term safety and efficacy data for iron chelation therapy in transfusion-dependent patients with sickle cell disease (SCD). To evaluate the long-term safety and efficacy of deferasirox (a once-daily oral iron chelator), patients with SCD completing a 1-year, Phase II, randomized, deferoxamine (DFO)-controlled study entered a 4-year extension, continuing to receive deferasirox, or switching from DFO to deferasirox. Average actual deferasirox dose was 19·4 ± 6·3 mg/kg per d. Of 185 patients who received at least one deferasirox dose, 33·5% completed the 5-year study. The most common reasons for discontinuation were withdrawal of consent (23·8%), lost to follow-up (9·2%) and adverse events (AEs) (7·6%). Investigator-assessed drug-related AEs were predominantly gastrointestinal [including nausea (14·6%), diarrhoea (10·8%)], mild-to-moderate and transient in nature. Creatinine clearance remained within the normal range throughout the study. Despite conservative initial dosing, serum ferritin levels in patients with ≥ 4 years deferasirox exposure significantly decreased by -591 μg/l (95% confidence intervals, -1411, -280 μg/l; P = 0·027; n = 67). Long-term deferasirox treatment for up to 5 years had a clinically acceptable safety profile, including maintenance of normal renal function, in patients with SCD. Iron burden was substantially reduced with appropriate dosing in patients treated for at least 4 years.

Autologous gene therapy (GT) for beta-hemoglobinopathies has demonstrated encouraging early safety and efficacy using addition of a sickling-resistant globin gene to stem cells. Another therapeutic strategy for sickle cell disease (SCD) is erythroid-specific inactivation of BCL11A, which is a validated repressor of gamma globin expression (Sankaran et al. Science 2011). This approach has the distinct advantage of simultaneously inducing fetal hemoglobin (HbF) while coordinately decreasing sickle hemoglobin (HbS). Since hemoglobin (Hb) polymerization in sickle red cells is highly dependent on the intracellular concentration of HbS and is strongly inhibited by HbF, effective BCL11A repression should prevent the sickling phenotype within red cells. We have shown that erythroid-specific expression of microRNA-adapted shRNAs (shRNAmiR) targeting BCL11A effectively induces HbF in human erythroid cells derived from transduced HSCs, largely attenuating the hematologic effects of SCD in a murine model while avoiding negative effects in HSCs and B lymphocytes (Brendel et al. JCI 2016). Here we report the initial results of a pilot clinical study utilizing a shRNAmiR lentiviral vector (LVV) targeting BCL11A for autologous GT in SCD patients. Transduction of hematopoietic cells with GMP lentiviral vector (BCH_BB-LCRshRNA(miR)) expressing the shRNAmiR for BCL11A in an erythroid-specific fashion showed no toxicity in engraftment and genotoxicity assays, efficient transduction rates of 80-95% of CD34+-derived erythroid colony forming cells from healthy donors and SCD patients, and >95% of transduced erythroid colonies demonstrating HbF levels of 50-95% of total Hb. Transduction at clinical scale with plerixafor mobilized CD34+ cells from three SCD donors yielded vector copies of 3.7 - 5.2/cell. Patients with severe SCD were screened for eligibility according to an IND enabled, IRB-approved investigator-initiated protocol. The first cohort included patients ≥ 18 years old. After at least 3 months of protocol-required transfusions, autologous CD34+ cells were collected by plerixafor mobilization and apheresis, and then transduced under GMP conditions with the BCH_BB-LCRshRNA(miR) vector. As of July 28, 2018, 3 patients representing the adult cohort had undergone a total of 3 (n=2) or 4 (n=1) days of mobilization. Mean single-day apheresis yields were 3.2 (range 1.5 - 6.8) x 106 CD34+ cells/kg. No Grade 3 or 4 AEs were attributed to mobilization and collection, although one subject developed an incidentally-discovered line-associated atrial clot and pulmonary embolism. Transduced cell products for these 3 patients have cell doses of 3.3 - 6.7 x 106 CD34+ cells/kg, VCN of 3.3 - 5.1 copies per cell and >95% vector-positive CD34+-derived colonies. One subject (BCL002), who had been regularly transfused for 17 years, has undergone infusion of gene-modified cells after myeloablative busulfan conditioning and achieved neutrophil engraftment after 22 days. Post-infusion follow-up is 78 days. At the time of the last analysis 76 days after GT and 64 days after last RBC transfusion (Table 1) subject BCL002 had a sustained Hb of >10 g/dL and, compared to pre-GT, there was a notable absence of irreversibly sickled cells on peripheral smear and a persistently low absolute reticulocyte count consistent with markedly reduced hemolysis. Hb electrophoresis showed 23.3% HbF, 51.8% HbS and 22.3% HbA (from residual transfused cells) with a HbF/(HbF+HbS) ratio of 29.7%. At day 76, the number of F cells had risen to 59.7% with 12pg HbF/F cell. In flow-sorted immature erythroid cells γ-globin mRNA was >80% of total β-like globins in the graft-derived population and BCL11A protein was reduced by ~90%. Adverse events observed from the start of conditioning until latest follow-up were consistent with myeloablative conditioning, and there have been no product-related adverse events and no SCD-related complications. These early results show: (1) feasibility of enrollment, cell procurement, and GMP manufacturing of gene modified CD34+ cells in 3 adult SCD patients; (2) the first proof of principle demonstrating shRNAmiR-based gene knockdown in humans, and (3) successful rapid induction of HbF in the first patient infused, with marked attenuation of hemolysis in the early phase of autologous reconstitution. Based on the trajectory of increasing HbF/(HbF+HbS), near full suppression of the SCD phenotype is expected. Disclosures Esrick: Bluebird Bio: Honoraria. Negre:bluebird bio: Other: Spouse employed by bluebird Bio. Dansereau:Bluebird Bio: Consultancy. Braunewell:Bluebird Bio: Employment, Equity Ownership. Christiansen:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Nikiforow:Kite Pharma: Consultancy. Achebe:Luitpold Pharmaceutical: Consultancy; AMAG Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Syros pharmaceuticals: Consultancy. Negre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Heeney:Sancilio Pharmaceuticals: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee; Pfizer: Research Funding; Astra Zeneca: Consultancy, Research Funding. Williams:Bluebird Bio: Research Funding.

Human induced pluripotent stem cells (hiPSCs) hold remarkable capacity for disease modeling and the development of novel therapeutic treatments for sickle cell disease (SCD). hiPSCs can theoretically produce all cell types including induced red blood cells (iRBCs). Sickle cell patients, in particular, could benefit from autologous, engineered red blood cells (RBCs). Many patients possess rare Rh phenotypes, are allo-sensitized to blood products and are at risk of iron overload from recurrent transfusions. Therefore, the generation of personalized iRBCs is attractive. Yet, in vitro iRBC production has been hampered by an inability of these cells to differentiate into terminally-mature, enucleated, beta globin-expressing RBCs. Here, we describe updated strategies to improve in vitro production of iRBCs. hiPSCs from sickle cell patients as well as those with normal hemoglobin were differentiated into hematopoietic stem progenitor cells (HSPCs) and immortalized via the overexpression of a previously characterized set of transcription factors promoting self-renewal and multipotency under the control of a doxycycline-regulated promoter. Utilizing an in vitro protocol incorporating increasing concentrations of human plasma, HSPCs differentiated from these lines proceed through terminal erythroid differentiation, including the formation of CD71-/GlyA+/α4 integrin-/Band 3+ cells. Plasma-stimulated iRBCs achieved robust enucleation (11-60.7%) and underwent fetal to adult globin-switching. Further, nearly 21% of the enucleated iRBCs were RNA negative erythrocytes 5-8 microns in diameter. RNA sequencing analysis reveals similar transcriptional profiles between iRBCs and peripheral blood CD34+- derived cultured RBCs (cRBCs) yet distinct differences between SCD and WT iRBCs. SCA iRBCs have increased extracellular matrix organization, cell-cell adhesive properties and up-regulation of hypoxia-response genes. Heme metabolism, DNA repair, fatty acid metabolism and oxidative phosphorylation are all impaired in SCD iRBCs. Assessment of cell physiology exposes membrane damage in SCD iRBCs with increased phalloidin permeability in comparison to wild type controls. Intriguingly, SCD iRBCs co-expressing gamma and beta-globin also demonstrate sickling under hypoxic conditions. With the development of expandable source of erythroid progenitors capable of producing mature red cells, we now aim to utilize this platform for robust disease modeling and autologous cell therapy. Figure. Figure. Disclosures Heeney: Pfizer: Research Funding; Sancilio Pharmaceuticals: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee.

Hydroxyurea therapy offers promise for ameliorating the clinical course of children with sickle cell disease (SCD). Hydroxyurea is a prototypic therapeutic option; it can be administered with minimal side effects, has a relatively wide therapeutic window, and has mechanisms of action that address pathophysiologic pathways of sickling, vaso-occlusion, hemolysis, and organ damage. There are limited data regarding hydroxyurea's ability to prevent or diminish organ dysfunction, and the long-term risks of hydroxyurea therapy remain incompletely defined. Although clinical trials are underway to address long-term issues, hydroxyurea remains an effective but underutilized therapy for SCD.

Hematologic abnormalities following solid organ transplantation are infrequently autoimmune in origin. We present a series of autoimmune cytopenias developing as a late complication of pediatric cardiac transplantation. Autoimmune cytopenias represented include autoimmune hemolytic anemia, acquired Glanzmann thrombasthenia, and idiopathic thrombocytopenic purpura. Standard therapies were used in each patient without sustainable results. Eventually, each patient was treated with and responded to rituximab. In this report, we review these cases, propose potential mechanisms for development of autoimmune cytopenias, and discuss our experience with rituximab in managing refractory autoimmune cytopenias. Pediatr Blood Cancer 2007;48:339–344. © 2006 Wiley-Liss, Inc.

Resistance to apoptosis has been described in neutrophils from patients with PNH and related hematologic disorders (aplastic anemia, myelodysplastic syndrome), but its molecular basis is not understood. Using gene expression analysis, PNH granulocytes had relative overexpression of four anti-apoptosis genes (human A1, hHR23B, Mcl-1, and RhoA) compared to normal controls. These findings were confirmed by RT-PCR analysis and observed in both peripheral blood granulocytes and mononuclear cells of patients with PNH. Anti-apoptosis gene upregulation may confer resistance to apoptosis in PNH and related disorders, and provide a common compensatory mechanism after bone marrow injury that allows survival and growth of remaining hematopoietic stem cells.

Hydroxyurea therapy reduces hemolysis and decreases serum bilirubin levels in children and adults with sickle cell anemia (SCA) and may therefore help prevent the development of cholelithiasis in this patient population. We recently reported that a promoter polymorphism in the uridine diphosphoglucuronate glucuronosyltransferase 1A (UGT1A) gene affects steady-state bilirubin levels and the incidence of gallstones in children with SCA. We have now analyzed the influence of the UGT1A genotype on the therapeutic response to hydroxyurea. A large cohort of children with SCA taking hydroxyurea therapy at the maximum tolerated dose demonstrated significant reductions in hemolysis independent of UGT1A promoter polymorphism genotype, but the hydroxyurea-related decreases in serum bilirubin levels were significantly different. Children with the wild-type 6/6 UGT1A genotype demonstrated normalized bilirubin levels with hydroxyurea therapy, but children with the heterozygous 6/7 or abnormal 7/7 genotypes did not. Children with the abnormal 7/7 genotype, which confers the phenotype of Gilbert syndrome, had bilirubin levels greater than 3 mg/dL despite full-dose hydroxyurea therapy. These data indicate the UGT1A promoter polymorphism is a powerful nonglobin genetic modifier in SCA that influences serum bilirubin both at baseline and on hydroxyurea therapy. UGT1A promoter polymorphisms may therefore influence the ability of hydroxyurea to prevent gallstone formation in patients with SCA. (J Lab Clin Med 2003;141:279-82)

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance.

Congenital sideroblastic anemias (CSAs) are uncommon inherited diseases resulting from defects in heme biosynthesis, mitochondrial translation or mitochondrial iron-sulfur cluster (ISC) assembly. CSAs are characterized by pathological mitochondrial iron deposits in bone marrow erythroblasts. Recently, mutations in mitochondrialheat shock protein 70 (HSPA9), a critical chaperone involved in mitochondrial ISC assembly, have been reported as a cause of non-syndromic CSA. Human heat shock cognate protein 20 (HSCB), a highly conserved mitochondrial co-chaperone, is the primary binding partner of HSPA9. HSCB allows the transfer of nascent ISC to HSPA9 and stimulates its ATPase activity, promoting ISC transfer to target proteins. To identify novel genes responsible for CSA, we performed whole exome sequencing on more than 75 CSA probands and their family members. In one patient, a young woman, with pancytopenia characterized by a normocytic anemia with numerous bone marrow ringed sideroblasts, we identified two variants in HSCB : a paternally-inherited promoter variant (c.-134C>A) predicted to disrupt a conserved ETS transcription factor binding site, and a maternally-inherited frameshift (c.259dup, p.T87fs*27). A fibroblast cell-line derived from the proband showed a decrease in HSCB expression, but normal HSPA9 expression compared to healthy, unrelated controls. Impairment of ETS1-dependent transcriptional activation of the promoter variant was demonstrated in K562 cells transfected with an HSCB-luciferase reporter construct. K562 cells were also employed to determine if reduced expression of HSCB could result in impaired erythroid metabolism, maturation, or proliferation. K562 cells infected with shRNA directed against HSCB were deficient in multiple mitochondrial respiratory complexes, had abnormal iron metabolism and a defect of protein lipoylation, all consistent with defective ISC metabolism. In addition, both IRP1 and IRP2 expression were decreased and cell surface transferrin receptor 1 (TFR1) expression was enhanced, suggesting disturbed cellular iron metabolism. Nevertheless, cells lacking HSCB partially retained an ability to respond to iron chelation and iron overload. Cells lacking HSCB lose their ability to hemoglobinize in response to sodium butyrate treatment (Figure 1A). This defect was confirmed in vivo using a morpholino strategy in zebrafish, as fish lacking HSCB are also unable to hemoglobize (Fig 1B). We generated an Hscb conditional mouse to better elucidate the underlying pathophysiology of the disease. Heterozygous (Hscb+/-) animals have no discernable phenotype; however, null animals die prior to embryonic day E7.5. Thus, to avoid this lethality, we employed Vav-cre animals (Tg(Vav1-cre)1Graf) to evaluate the loss of HSCB specifically in the hematopoietic compartment. Hscbc/- Vav-cre+ pups are pale and growth retarded compared to control littermates and die at approximately p10 with severe pancytopenia. To assess the loss of HSCB specifically in the erythroid lineage, we bred conditional animals to EpoR-cre (Eportm1(EGFP/cre)Uk) mice. Hscbc/- EpoR-cre+ mice die at approximately E12.5 due to a complete failure of erythropoiesis (Figure 1C). Finally, temporally inducible, hematopoietic-specific deletion animals were generated by transplantation of fetal livers from Mx-Cre (Tg(Mx1-cre)1Cgn) positive Hscbc/- animals. After polyinosinic:polycytidylic acid (pIpC) induction, global defects of hematopoiesis were observed in Mx-Cre+ animals, leading to their death 3-weeks post-induction from profound pancytopenia. A transient siderocytosis was seen in the peripheral blood between days 6-8 post-pIpC. Flow cytometry using FSC-TER119-CD44 gating strategy confirmed the defect in erythropoiesis. Taken together, these data demonstrate that HSCB is essential for hematopoiesis; both whole animal and in vitro cell culture models recapitulate the patient's phenotype, suggesting that the two patient mutations are likely disease-causing. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The Stroke With Transfusions Changing to Hydroxyurea (SWiTCH) trial compared standard (transfusions/chelation) to alternative (hydroxyurea/phlebotomy) treatment to prevent recurrent stroke and manage iron overload in children chronically transfused over 7 years before enrollment. Standardized brain magnetic resonance imaging/magnetic resonance angiography (MRA) and transcranial Doppler (TCD) exams were performed at entry and exit, with a central blinded review. A novel MRA vasculopathy grading scale demonstrated frequent severe baseline left/right vessel stenosis (53%/41% ≥Grade 4); 31% had no vessel stenosis on either side. Baseline parenchymal injury was prevalent (85%/79% subcortical, 53%/37% cortical, 50%/35% subcortical and cortical). Most children had low or uninterpretable baseline middle cerebral artery TCD velocities, which were associated with worse stenoses (incidence risk ratio [IRR] = 5.1, P ≤ .0001 and IRR = 4.1, P < .0001) than normal velocities; only 2% to 12% had any conditional/abnormal velocity. Patients with adjudicated stroke (7) and transient ischemic attacks (19 in 11 standard/8 alternative arm subjects) had substantial parenchymal injury/vessel stenosis. At exit, 1 child (alternative arm) had a new silent infarct, and another had worse stenosis. SWiTCH neuroimaging data document severe parenchymal and vascular abnormalities in children with SCA and stroke and support concerns about chronic transfusions lacking effectiveness for preventing progressive cerebrovascular injury. The novel SWiTCH vasculopathy grading scale warrants validation testing and consideration for use in future clinical trials. This trial was registered at www.clinicaltrials.gov as #NCT00122980.

Introduction: The TCD with Transfusions Changing to Hydroxyurea (TWiTCH) trial was an NHLBI-funded multicenter phase 3 randomized trial that demonstrated non-inferiority of hydroxyurea versus continued transfusion therapy in children with sickle cell anemia and abnormal TCD velocities but no severe vasculopathy. Children who were randomized to continue transfusions (Standard Arm) received conventional iron chelation therapy with deferasirox, while children receiving hydroxyurea (Alternative Arm) received serial phlebotomy (10 mL/kg, maximum 500 mL) every 4 weeks. Extrahepatic iron burden was monitored by R2* MRI in the kidney, pancreas, and spleen to monitor the changes in iron overload phenotype between the two groups. Methods: R2* MRI of the abdomen was performed at baseline, 1 year, and 2 years following enrollment. Liver, spleen, pancreas, and kidney R2* were measured from axial and coronal multiecho, gradient echo images. Images were analyzed centrally at an experienced core laboratory using an exponential plus constant model for signal decay. Iron burden in the different organs was assessed using repeated measures analysis of variance (ANOVA) using JMP 11.0. Results: 120 patients underwent baseline R2* assessment, 83 completed the midpoint, and 89 patients completed the endpoint examination. Figure 1 (left) summarizes the changes in kidney R2* across the three time points. Kidney R2* was elevated at baseline in both treatment arms and correlated with LDH (r2 =0.28, p 30 mg albumin per g creatinine) trended lower in patients on hydroxyurea, (5/45 at baseline (p=1), 1/44 at midpoint (p=0.03), and 4/52 at endpoint (p=0.33). Figure 1 (right) demonstrates the temporal evolution of ACR in 11 study participants with baseline albuminuria; 4/6 patients on standard arm continued to have significant proteinuria on their terminal examination compared with only 1/5 patients on hydroxyurea (p=0.24). There were no groupwise differences in mean pancreas R2* at any time point and clinically significant pancreas iron deposition (R2* > 100 Hz) was only observed in two patients at the mid timepoint. Spleen R2* was markedly elevated at baseline (503 ± 396 Hz), but did not change systematically over time in either group. The change in spleen R2* was correlated with the change in liver iron (r2=0.21, p=0.003), regardless of the treatment group, suggesting that the spleen iron stores are part of the dynamic iron storage pool. 37% of the patients had no MRI-detectable spleen (surgical + autosplenectomy). The change of liver iron was not different in patients with or without a spleen. Conclusion: Hydroxyurea therapy was associated with lower intravascular hemolysis and kidney iron deposition. Further studies are necessary to determine whether these changes are associated with decreased proteinuria or improved renal function. Splenic iron burden remained high but stable in all patients, and its role in iron unloading needs further investigation. Pancreas iron loading remained rare, consistent with the low prevalence of endocrinopathies in SCD. Figure 1 Figure 1. Disclosures Wood: Apopharma: Consultancy; Ionis Pharmaceuticals: Consultancy; Vifor: Consultancy; Vifor: Consultancy; Biomed Informatics: Consultancy; AMAG: Consultancy; Biomed Informatics: Consultancy; Ionis Pharmaceuticals: Consultancy; World Care Clinical: Consultancy; World Care Clinical: Consultancy; Celgene: Consultancy; Apopharma: Consultancy; Celgene: Consultancy; AMAG: Consultancy. Heeney:Sancilio and Company: Consultancy, Research Funding; Eli Lilly and Company: Research Funding; Pfizer: Research Funding. Ware:Global Blood Therapeutics: Consultancy; Nova Laboratories: Consultancy; Biomedomics: Research Funding; Bristol Myers Squibb: Research Funding; Addmedica: Research Funding; Bayer Pharmaceuticals: Consultancy.

Introduction: The multicenter phase 3 randomized clinical trial TCD With Transfusions Changing to Hydroxyurea (TWiTCH, NCT01425307) investigated whether hydroxyurea could maintain TCD velocities and prevent stroke in children with sickle cell anemia and abnormal TCD velocities. The trial was stopped early after reaching the primary endpoint, when the average TCD velocity in the alternative (hydroxyurea) arm was found to be non-inferior and even slightly better (138 versus 143 cm/sec) than the standard (transfusion) arm. A planned secondary analysis was to determine the variation in serial TCD velocity measurements for individual study participants, to document the normal TCD velocity fluctuations that occur and to inform the need for frequent TCD evaluations in this clinical setting. Methods: All TCD examinations collected in TWiTCH were analyzed for the maximum time-averaged mean velocity (TAMV) measured in the main intracranial arteries. TCD studies were performed by certified examiners using identical non-imaging instruments, no more than one week before a scheduled transfusion or phlebotomy procedure. Measurements on the index side, defined as the cerebral hemisphere with the higher mean arterial velocity at baseline assessment, were used for these statistical analyses. TCD values were analyzed according to four phases of the trial: (1) screening with three monthly TCD examinations performed at Weeks -8, -4, and 0 before randomization; (2) initial treatment period (hydroxyurea dose escalation with overlap transfusions or transfusions only) with three quarterly TCD measurements at Weeks 12, 24, and 36; (3) steady-state treatment period (hydroxyurea only or transfusions only) with four quarterly TCD measurements at Weeks 48, 60, 72, and 84; and (4) study exit with three monthly TCD examinations performed at Weeks 96, 100, and 104 or during the accelerated close-out period. The within-patient variance and standard deviation of the TAMV values were found using a linear mixed model, which was calculated using SAS Version 9.3. Results: TAMV measurements on the index side from 1458 TCD examinations on 121 randomized patients formed the overall dataset. TAMV values ≥170 cm/sec were identified in 140 measurements on 40 children (9.6% of TCD values) and exceeded 200 cm/sec in 1.8% of examinations (26 values, 8 children) during the study treatment phase. The within-patient TCD variation, representing fluctuation of repeated TCD measurements in the same study participant, was 12.0 cm/sec for the entire cohort during the initial screening phase (363 TCD measurements). In the initial treatment phase, TCD variation in the hydroxyurea arm (180 values, 60 children) was 10.5 cm/sec, similar to 10.2 cm/sec on the transfusion arm (183 values, 61 children). In the steady-state treatment phase, the TCD variation in the hydroxyurea arm was 10.2 cm/sec (207 values, 57 children), compared to 12.3 cm/sec on the transfusion arm (212 values, 58 children). In the final exit phase, TCD variation in the hydroxyurea arm was 9.8 cm/sec (155 values, 58 children), similar to 10.2 cm/sec on the standard arm (155 values, 57 children). TCD variation was greater with higher baseline TCD velocity and shorter transfusion duration, but was not affected by age or gender. For example, participants with baseline TCD velocity of Conclusions: The TWiTCH trial provides prospective data regarding the biological variation in serial TCD values that occurs in children with previous abnormal TCD examinations but no severe vasculopathy. The observed within-patient variation in TCD velocities was consistently 10-14 cm/sec, with higher variation observed in children with higher baseline TCD velocities. This fluctuation can be expected with serial TCD examinations and should be considered before making treatment decisions based on a single TCD measurement. Since comparison of entry and exit TCD values in the TWiTCH trial documented that hydroxyurea at maximum tolerated dose was non-inferior to transfusions, frequent TCD examinations may not be warranted. Disclosures Ware: Bayer Pharmaceuticals: Consultancy; Addmedica: Research Funding; Global Blood Therapeutics: Consultancy; Bristol Myers Squibb: Research Funding; Biomedomics: Research Funding; Nova Laboratories: Consultancy. Heeney:Eli Lilly and Company: Research Funding; Pfizer: Research Funding; Sancilio and Company: Consultancy, Research Funding. Thompson:Baxalta (now part of Shire): Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mast: Research Funding; Eli Lily: Research Funding; Celgene: Research Funding; Amgen: Research Funding; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy, Research Funding.

Sickle cell disease (SCD) is a pleiotropic genetic disorder of hemoglobin that has profound multiorgan effects. The low prevalence of SCD ( approximately 100,000/US) has limited progress in clinical, basic, and translational research. Lack of a large, readily accessible population for clinical studies has contributed to the absence of standard definitions and diagnostic criteria for the numerous complications of SCD and inadequate understanding of SCD pathophysiology. In 2005, the Comprehensive Sickle Cell Centers initiated a project to establish consensus definitions of the most frequently occurring complications. A group of clinicians and scientists with extensive expertise in research and treatment of SCD gathered to identify and categorize the most common complications. From this group, a formal writing team was formed that further reviewed the literature, sought specialist input, and produced definitions in a standard format. This article provides an overview of the process and describes 12 body system categories and the most prevalent or severe complications within these categories. A detailed Appendix provides standardized definitions for all complications identified within each system. This report proposes use of these definitions for studies of SCD complications, so future studies can be comparably robust and treatment efficacy measured. Use of these definitions will support greater accuracy in genotype-phenotype studies, thereby achieving a better understanding of SCD pathophysiology. This should nevertheless be viewed as a dynamic rather than final document; phenotype descriptions should be reevaluated and revised periodically to provide the most current standard definitions as etiologic factors are better understood, and new diagnostic options are developed.

Transcranial Doppler (TCD) screening in children with sickle cell anemia (SCA) identifies abnormally elevated cerebral artery flow velocities that confer an elevated risk for primary stroke. Chronic transfusions offer effective stroke prophylaxis in this setting, but must be continued indefinitely and lead to transfusional iron overload. An alternative treatment strategy that offers similar effective protection against primary stroke, and provides control of iron overload, is needed. TCD With Transfusions Changing to Hydroxyurea (TWiTCH, NCT01425307) was an NHLBI-funded Phase III multicenter randomized clinical trial comparing 24-months of standard treatment (transfusions) to alternative treatment (hydroxyurea) in children with SCA and abnormal TCD velocities. All eligible children had received at least 12 months of transfusions. TWiTCH had a non-inferiority trial design; the primary study endpoint was the 24-month TCD velocity obtained from a linear mixed model, controlling for baseline (enrollment) values, with a non-inferiority margin of 15 cm/sec. The transfusion arm maintained children at HbS 240 cm/sec. Exit brain MRI/MRA exams documented no new parenchymal abnormalities but one child (transfusion arm) developed new vasculopathy. Sickle cell related serious adverse events were more common in the hydroxyurea arm than the transfusion arm (23 to 15), but none was related to study treatment or study procedures. Iron overload improved more in the hydroxyurea arm than in the transfusion arm, with a greater average change in serum ferritin (-1085 compared to -38 ng/mL, p Disclosures Ware: Eli Lilly: Other: DSMB membership; Bayer Pharmaceuticals: Consultancy; Bristol Myers Squibb: Research Funding; Biomedomics: Research Funding. Off Label Use: Hydroxyurea for children with SCA. Owen:Novartis: Speakers Bureau. Rogers:BioRad Labs: Consultancy; Apopharma: Consultancy; Baxter: Consultancy; Glaxo Smith Kline: Consultancy. Kwiatkowski:Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy; Sideris Pharmaceuticals: Consultancy; Novartis: Research Funding; ISIS: Membership on an entity's Board of Directors or advisory committees. Heeney:Sancillio: Consultancy; Eli Lilly: Research Funding. Nottage:Janssen Pharmaceuticals: Employment. Cohen:Novartis: Consultancy; ApoPharma: Other: DSMB member.

Immune thrombocytopenic purpura and autoimmune hemolytic anemia are typically idiopathic processes without underlying systemic illness. Four children with autoimmune cytopenia had low immunoglobulin levels that led to the diagnosis of common variable immunodeficiency. Routine screening of immunoglobulins is suggested for children with chronic or recurrent immune thrombocytopenic purpura and autoimmune hemolytic anemia.

Although paroxysmal nocturnal hemoglobinuria (PNH) has been recognized as an intriguing clinical entity for over a century, the past 10–15 years have witnessed remarkable progress toward a better understanding of the pathogenesis and pathophysiology of this mysterious blood disorder. After multiple proteins were found to be absent from the surface of PNH cells, it was recognized that each utilized a glycosylphosphatidylinositol (GPI) anchor for cell surface attachment. Elucidation of the biochemical abnormalities in PNH cells identified a specific defective step in GPI anchor bioassembly, which eventually led to the cloning of the PIG-A gene. All patients with PNH reported to date have acquired (non-germline) PIG-A gene mutations.

Introduction: Although advances in care have allowed individuals with SCD to live further into adulthood, treatment of adult SCD pts is challenging due to increased rates of comorbidities and complications. Here we present clinical outcomes and treatment patterns from a prospective registry of pediatric and adult pts with SCD at 3 y to better characterize disease and treatment patterns in adult pts. Methods: Pts ≥2 y old with HbSS, HbSC or HbS/β-thalassemia were enrolled from 57 US centers and assessed every 6 mo until 3 y. Differences between pediatric ( Results: Among 498 pts (317 pediatric; 181 adults) completing baseline visit, 74.1% had HbSS, 15.3% HbSC and 10.4% HbS/β-thalassemia; 61 pts discontinued the study. At baseline, pediatric pts had more asthma/airway reactive disease, dactylitis, and splenic sequestration (Table 1). Adults had significantly poorer performance status (P Conclusions: Patterns of disease and complications differed between children and adults. Adults had more hospitalizations due to transfusions/chelation and more classic chronic conditions (eg, AVN, GBD and PAH). Absenteeism from school/work was common in both groups. Limitations included the observational study design, variation in time from diagnosis, and lack of mandatory data collection. TABLE 1. Baseline Characteristics of Pediatric vs Adult Pts with SCD at 3 Y *P † P CNS, central nervous system; TCD, transcranial Doppler TABLE 2. Clinical Complications and Treatment Patterns in Pediatric vs Adult Pts with SCD at 3 Y 20 d in last 12 mo) 28 (14.7) 5 (25.0) During study (>20 d since last visit) 4 (2.5) 0 Missed work, n (%) Prior to study (>20 d in last 12 mo) 0 12 (22.2) During study (>20 d since last visit) 0 4 (9.5) ACS, acute chest syndrome *P † P ‡ P µUp to 3 discharge diagnoses may be listed for any hospital admission Disclosures Heeney: Novartis: Research Funding. Adams-Graves:Novartis Pharmaceuticals: Consultancy, Honoraria, Speakers Bureau. Paley:Novartis Pharma: Employment. Esposito:Novartis Pharma: Employment. McNamara:Novartis Pharmaceuticals Corporation: Employment. Vichinsky:Novartis : Consultancy, Research Funding, Speakers Bureau.

Introduction Patients (pts) with sickle cell disease (SCD) experience a heterogeneous clinical course, with a range of symptoms and sequelae. We describe clinical outcomes and treatment patterns from a prospective registry of pediatric and adult pts with SCD. Methods Pts ≥2 years old with HbSS, HbSC, or HbS/β-thalassemia were enrolled from 57 US centers and assessed every 6 months (mos) for up to 3 years. Differences between pediatric and adult pts at 24 mos follow-up are reported. (ClinicalTrials.gov NCT01220115). Results A total of 498 pts completed the baseline visit (74.1% HbSS disease, 15.3% HbSC, and 10.4% HbS/β-thalassemia) (Table 1 ). At baseline, the following conditions were more frequent in adults: avascular necrosis, gallbladder disease, leg ulcers, and pulmonary hypertension. Pediatric pts had more frequent asthma/reactive airway disease, dactylitis, and splenic sequestration. The nature of sickle-related events varied between adult and pediatric pts (Table 2 ). Prior to study, adults had higher frequencies of pain crises, strokes, and priapism, while pediatric pts had more frequent infections and acute chest syndrome (ACS)/pneumonia. On study, a similar proportion of pediatric and adult pts (56.4% overall) were hospitalized, most frequently due to pain, fever, and ACS/pneumonia; a greater proportion of pediatric pts were hospitalized due to fever (P Conclusions Despite advances in care, SCD is associated with significant morbidity that contributes to high rates of hospitalization and absenteeism in both pediatric and adult pts. Continued follow-up in this registry will provide additional information about disease patterns and pt management. Disclosures: Heeney: Novartis: Consultancy, Research Funding; Eli Lilly: Research Funding. Off Label Use: Hydroxurea is indicated to reduce the frequency of painful crises and to reduce the need for blood transfusions in adult patients with sickle cell anemia. It is not approved for use in children. Mueller:Novartis: Research Funding. Adams-Graves:Novartis: Consultancy, Speakers Bureau. Paley:Novartis: Employment. Esposito:Novartis: Employment. Katie:Novartis: Employment. Vichinsky:Novartis: Consultancy, Research Funding; ApoPharma: Consultancy, Research Funding; ARUP: Research Funding, Research Laboratory, Research Laboratory Other.

Abstract 1010 Introduction: Sickle cell disease (SCD) is a clinically heterogeneous disease. Patients (pts) may present with severe symptoms in childhood, resulting in early morbidity and mortality, or may be asymptomatic until adulthood. Therapeutic goals include the prevention and treatment of complications across the spectrum of disease. We describe baseline and 12-mo assessments and therapies for pts in an ongoing registry documenting treatment patterns, natural history, and outcomes. Methods: Pts ≥2 years old with HbSS, HbS/β-thalassemia, or HbSC were enrolled from 57 US centers and assessed every 6 mos for up to 3 years. Differences between pediatric (age Results: In all, 498 pts completed the baseline visit. Most pts had HbSS disease (age Conclusions: This study showed differences in baseline characteristics and treatment patterns between adult and pediatric pts with SCD. Ongoing follow-up will provide further information on disease patterns, treatment practices, and outcomes, thus increasing our understanding of appropriate SCD management. Disclosures: Heeney: Novartis: Consultancy, Research Funding; Eli Lilly: Research Funding. Off Label Use: Hydroxurea is indicated to reduce the frequency of painful crises and to reduce the need for blood transfusions in adult patients with sickle cell anemia. It is not approved for use in children. Mueller:Novartis: Research Funding. Adams-Graves:Novartis: Consultancy, Speakers Bureau. Paley:Novartis: Employment. Esposito:Novartis: Employment. Vichinsky:Novartis: Consultancy, Research Funding; ApoPharma: Consultancy, Research Funding; ARUP Research Laboratory: Research Funding.

Abstract 3213 Introduction: There are few approved treatments for children with sickle cell disease (SCD) who experience painful vaso-occlusive crises (VOC). Evidence suggests a pathophysiologic role for platelets in SCD, in general, and in VOC, specifically. Thrombocytosis is common, and markers of platelet activation, are elevated in both children and adults with SCD. This activation may be mediated by adenosine diphosphate (ADP) released from hemolysis of sickled erythrocytes. Accordingly, platelets are a rational therapeutic target to explore with the aim to reduce the frequency and severity of VOC. Therefore, we studied prasugrel, an irreversible P2Y12 ADP receptor antagonist that inhibits platelet activation and aggregation, in children with SCD. The primary objective was to identify doses of prasugrel that produced a 30–50% inhibition of platelet activation for use in a pediatric phase 3 study of efficacy. Methods: We conducted a phase 2, open-label, multi-center, adaptive-design, dose-ranging, pharmacokinetic (PK) and pharmacodynamic (PD) study of prasugrel in children with SCD. Males and females 2–17 years of age (inclusive) with SCD (HbSS and HbSβ0-thalassemia genotypes) were eligible. Treatment was initiated with single prasugrel doses expected to have no effect and dosage was increased in accordance with an adaptive design for improved safety and dosage identification. Patients were to receive up to 3 single doses of prasugrel with the second and third doses being administered 14±4 days after the previous dose. Doses of prasugrel were increased or decreased based on the PD response to previous doses in the same patient and/or other patients. Demographic characteristics were also considered for dose assignment in order to balance age, body weight, and sex across the dose range that produced a 30–50% inhibition of platelet aggregation. Platelet inhibition was evaluated by the VerifyNow™ (VN) P2Y12 assay and the vasodilator-associated simulated phosphoprotein (VASP) phosphorylation assay. These PD measures were assessed at screening and 4 hours after each single prasugrel dose. Venous blood samples were collected 0.5 h, 1 h, 1.5 h, 2 h, and 4 h post-dose for PK analysis of prasugrel's active metabolite to calculate the area under the concentration-time curve (AUC). The PK/PD analyses were conducted to describe the relationship between prasugrel's active metabolite AUC and platelet inhibition. Spearman correlation statistics were used to assess the relationship between dose and PD parameters. Results: A total of 24 patients, ranging in age from 4 to 17 years of age, received at least one dose of prasugrel. The mean age was 11.0 years; 58.3% were female; 87.5% had HbSS and 12.5% had HbSβ0-thalassemia. Body weight ranged from 14.4 to 80.1 kg. Patients received single doses of prasugrel ranging from 0.03 to 0.60 mg/kg. A single-dose range of 0.30–0.50 mg/kg produced mean 30–50% inhibition in platelet activation by the VN assay whereas a single-dose range of 0.40–0.50 mg/kg produced mean 30–50% inhibition in the VASP assay. Minimal PD response was observed at single doses, up to approximately 0.25 mg/kg. Above 0.25 mg/kg, PD response increased with dose. The single 0.60 mg/kg dose produced >50% inhibition by both VN and VASP assays. In general, higher doses resulted in increased exposure to prasugrel's active metabolite. Increased variability in exposure and in PD response was seen at higher doses. The PD results demonstrate correlation between dose and 4-hour VN P2Y12 percent inhibition. Prasugrel appeared to be safe and well tolerated in this small patient sample. Three serious adverse events occurred in 2 patients; none of which were considered possibly related to prasugrel. No hemorrhagic events were observed, and no subjects discontinued participation due to an adverse event. Conclusions: These single-dose data will contribute to a population modeling strategy that targets the desired steady state range of platelet inhibition (30–50%) for a subsequent phase 3 clinical trial of the efficacy and safety of prasugrel for the reduction of VOC in children with SCD. Daily maintenance doses will be evaluated in a subsequent part of the current study. Disclosures: Styles: Eli Lilly and Company: Consultancy. Off Label Use: Inhibition of platelet activation by prasugrel in pediatric patients with sickle cell disease. Jakubowski:Eli Lilly and Company: Employment, Equity Ownership. Heiselman:Eli Lilly and Company: Employment, Equity Ownership. Heath:Eli Lilly and Company: Employment, Equity Ownership. Zhou:Eli Lilly and Company: Employment, Equity Ownership. Heeney:Novartis: Consultancy, Research Funding; Eli Lilly and Company: Research Funding; Pfizer: Consultancy. Redding-Lallinger:Eli Lilly and Company: Research Funding. Small:Eli Lilly and Company: Employment, Equity Ownership. Moser:Eli Lilly and Company: Employment, Equity Ownership. Winters:Eli Lilly and Company: Employment, Equity Ownership.

Abstract 1060 Introduction: The baseline characteristics and therapeutic management of sickle cell disease (SCD) can be quite variable depending on disease severity. This report describes baseline assessments and therapies of 420 patients in an ongoing registry designed to study current treatment patterns, natural history, and outcomes in patients with SCD. Methods: This registry trial involving 57 US hematology centers had a sequential recruitment target of 600 patients with SCD subdivided into 3 age groups (2- Results: Enrollment: 420 patients (254 Conclusions: These data indicate differences in baseline characteristics and treatment patterns between adult and pediatric patients with SCD in this registry. Karnofsky performance status was worse, and serum ferritin levels were higher in adult versus pediatric patients. The percentage of patients having regular transfusions was higher in pediatric patients, while adult patients had higher rates of intermittent transfusions. As expected, the lifetime chelation exposure was more frequent in adult patients. Hospitalizations and use of hydroxyurea were also greater in pediatric patients. In terms of medical history related to SCDs, pediatric patients had higher frequencies of asthma, abnormal TCDs, dactylitis and splenic sequestration, while adults had more frequent evidence of avascular necrosis, gall bladder disease, leg ulcers and pulmonary hypertension. Pain was the most common crisis type in both cohorts, although this accounted for a greater relative proportion of crises in the adult cohort. The relative proportion of ACS events was lower in the adult versus pediatric patients; all other crisis types occurred in similar relative proportions in both cohorts. This ongoing registry will provide information about disease patterns, current treatment practices, and outcomes, thus contributing to a better understanding of the appropriate therapeutic management of patients with SCD. Disclosures: Heeney: Novartis: Consultancy, Research Funding. Baltz:Novartis: Research Funding. Paley:Novartis: Employment. Esposito:Novartis: Employment. Sharma:Novartis: Employment. Vichinsky:Novartis, Apotex, Ferrokin: Research Funding.

Abstract 2141 Introduction: In patients with sickle cell disease (SCD), erythrocytes contribute to microvessel occlusion resulting in tissue damage and platelet activation. Platelet activation, aggregation, local thrombus formation and platelet activation-dependent leukocyte recruitment potentially amplify tissue ischemia. Antiplatelet therapy may therefore be useful in SCD. Here we evaluate levels of platelet activation markers in adolescents with SCD vs. normal controls and the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Methods: Blood was obtained from adolescents (10 – 18 yr) with SCD and healthy adult subjects. Platelet function was evaluated by: light transmission aggregation (LTA) in platelet-rich plasma with 20 μM ADP and in whole blood by VerifyNow P2Y12; Multiple Electrode Aggregometry (MEA) with 6.5 μM ADP; vasodilator stimulated phosphoprotein (VASP) P2Y12 assay; and whole blood flow cytometric analysis of basal and in vitro ADP-stimulated levels of platelet surface activated GPIIb-IIIa (reported by monoclonal antibody PAC1) and P-selectin, platelet-monocyte aggregates (PMA) and platelet-neutrophil aggregates (PNA). These endpoints were also evaluated after in vitro incubation of whole blood with R-138727 (0.1 – 10 μM). Results: In SCD patients compared with normal subjects, circulating PMA and PNA levels were significantly higher (76.5 ± 20.3% and 55.1 ± 21.8% vs. 20.1 ± 7% and 13.9 ± 4.2% [mean ± SD], respectively, p Treatment of whole blood in vitro with R-138727 resulted in a concentration-dependent inhibition of platelet function in both SCD patients and normal subjects. However, compared with normal subjects, the IC50 in SCD subjects was significantly lower for LTA but significantly higher for VerifyNow and VASP (Table). R-138727 inhibition of platelet function in SCD patients was similar to normal subjects as judged by MEA, whole blood flow cytometry for ADP-stimulated platelet surface P-selectin and activated GPIIb-IIIa expression, and % PMAs (Table). Sensitivity to R-138727 inhibition in SCD patient blood was greatest as measured by ADP-stimulated platelet surface P-selectin MFI, LTA, and MEA, less with ADP-stimulated platelet surface activated GPIIb-IIIa, and least with VASP, VerifyNow P2Y12 and % P-selectin-positive platelets (Table). Conclusions: 1) The markedly higher circulating PMA and PNA levels in SCD patients relative to normal donors demonstrates increased in vivo platelet activation in SCD patients and suggests that PMA and PNA may be useful markers of the in vivo pharmacodynamic effects of antiplatelet therapy in SCD patients. 2) Blockade of platelet P2Y12 with R-138727 produces dose-dependent inhibition of platelet function in SCD platelets. 3) Assay-dependent differences in IC50 values between SCD patients and normal donors suggest the presence of additional variables that affect these measures of platelet function. Further studies are needed to determine the relationship between platelet inhibition measured by these assays and clinical events in SCD patients. Disclosures: Frelinger: GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Consultancy, Research Funding; Takeda: Research Funding. Jakubowski:Eli Lilly and Company: Employment, Equity Ownership. Heeney:Lilly: Consultancy. Michelson:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Data Monitoring Committee for clinical trial, Research Funding; Takeda: Research Funding.

Abstract 845 Background: Transfusion therapy can effectively treat many complications associated with sickle cell disease (SCD), but iron overload will develop without iron chelation therapy. Despite long-term transfusion requirements, long-term data for iron chelation in SCD are limited. The oral iron chelator, deferasirox, effectively reduced iron burden in SCD patients aged ≥2 years during 1 year of treatment (Vichinsky et al. Br J Haematol 2007). This 5-year follow-up is the first report of long-term deferasirox treatment in SCD patients. Methods: Eligible patients that completed the 1-year core study (randomized to deferasirox [deferasirox cohort] or deferoxamine [crossover cohort]) entered the 4- year extension. All received deferasirox while continuing their regular transfusion regimen. Deferasirox dose in the extension was initially based on serum ferritin (SF) trends in the core study (deferasirox cohort) or transfusion requirements (crossover cohort). Dose adjustments were based on monthly SF and safety assessments (investigator-reported adverse events [AEs] and centrally processed lab parameters). Growth and sexual development (Tanner staging) were assessed annually. Results: Of the patients in the deferasirox (n=132) and crossover (n=53) cohorts that received ’1 deferasirox dose during the core or extension, 43 (33%) and 19 (36%) patients, respectively, completed the extension. Reasons for discontinuation included withdrawal of consent (n=44, 23.8%), loss to follow-up (n=17, 9.2%) and AEs (n=14, 7.6%). Three deaths occurred, all in the extension (intraventricular hemorrhage, n=2; intracranial bleed post liver transplantation, n=1); none considered by investigators to be deferasirox-related. Mean dose during the study was 18.7 ± 6.5 and 21.2 ± 5.3 mg/kg/day in the deferasirox and crossover cohorts, respectively. Investigator-assessed drug-related AEs (≥5% overall) included nausea (14.6%), diarrhea (10.8%), increased blood creatinine (5.9%) and vomiting (5.4%). Generally, these AEs were manageable and transient, and decreased in frequency over time. Serious AEs were reported in 70.8% of patients overall and were mostly related to the underlying disease. Serious investigator-assessed drug-related AEs were reported in 8 patients (6.1%) in the deferasirox cohort and 1 patient (1.9%) in the crossover cohort. In the deferasirox and crossover cohorts respectively, 9 (6.8%) patients and 1 (1.9%) patient had 2 consecutive serum creatinine level increases >33% above baseline and >upper limit of normal (ULN). Median creatinine clearance remained stable within normal range throughout the study. One patient from each cohort had alanine aminotransferase (ALT) >10 × ULN on 2 consecutive visits; both had ALT values ≤ULN at the start of deferasirox treatment. In 37 patients with data available before and after dose increases to ≥30 mg/kg/day, no clinically relevant differences were observed in AE profile or laboratory parameters before and after dose increase. Deferasirox had no adverse effect on pediatric growth and adolescent sexual development. Overall, median SF levels in patients who received deferasirox treatment for ≥4 years decreased significantly from 3410 to 3108 ng/mL at end of study (median absolute change, –591 ng/mL, P=0.027; n=67). Decreases in SF were more pronounced when mean deferasirox dose increased to >20 mg/kg/day (Figure 1). In the deferasirox cohort, the median absolute change in SF levels from start of deferasirox to end-of-study was greater in patients aged ≥16 than 2– Conclusions: This is the first study to report that deferasirox can significantly decrease SF levels over the long-term in SCD patients without evidence of renal toxicity. Treatment for up to 5 years was associated with a clinically manageable safety profile, without progressive decreases in median creatinine clearance, which remained within normal range. The modest, but statistically significant, decrease in SF is most likely due to under-dosing in the first 2 years and variability in SF related to disease factors. Patients titrated to an appropriate dose had a clinically relevant decrease in SF, highlighting the need to adjust dose, usually to >20 mg/kg/day, to achieve negative iron balance. Disclosures: Vichinsky: Novartis: Consultancy, Research Funding, Speakers Bureau; Apotex: Consultancy, Research Funding; Hemaquest: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bernaudin:Novartis: Investigator for SCD deferasirox (Exjade). Forni:Novartis: Research Funding. Gardner:Novartis: Research Funding. Hassell:Novartis: Research Funding. Heeney:Novartis: Research Funding. Kutlar:Novartis: Research Funding. Lane:Novartis: Research Funding. Mathias:Novartis: Research Funding. Porter:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Tebbi:Novartis: Speakers Bureau. Wilson:Novartis: Honoraria, Research Funding, Speakers Bureau. Griffel:Novartis: Employment. Deng:Novartis: Employment. Giannone:Novartis: Employment. Coates:Novartis: Research Funding, Speakers Bureau.

Abstract 629 Introduction: The peptide hormone hepcidin is a negative regulator of iron efflux from cells. In iron deficiency anemia (IDA) hepcidin levels are ordinarily undetectable, thereby stimulating the release of iron from macrophages, hepatocytes and duodenal enterocytes, promoting iron availability for erythropoieisis and increasing iron stores. Rare individuals and kindreds with lifelong hypoferremia, microcytic red blood cells and IDA unresponsive to enteral iron therapy and only an incomplete, transient response to parenteral iron supplementation—so-called iron refractory iron deficiency anemia—have been described. We previously reported that mutations in the hepatocyte-specific transmembrane serine protease TMPRSS6 cause IRIDA and that the physiology of the phenotype can be attributed to inappropriately elevated hepcidin levels (Nat Genet, 2008, 40:561). Here, we extend our description of the homozygous TMPRSS6-mutated IRIDA phenotype, identify disease-causing mutations in an additional probands, including the original family reported by Buchanan and Sheehan (J Pediatr, 1981, 98:72) and describe the phenotype of heterozygous TMPRSS6 deficiency. Methods: Individuals and kindreds with IRIDA were recruited in an IRB approved protocol and assessed for hematologic and biochemical markers of iron status, inflammation, urinary and plasma hepcidin and TMPRSS6 sequencing by previously described methods. Given that the transferrin saturation (TfSat) appears to have a central role in hepcidin regulation, an index relating the hepcidin to transferrin saturation, TfSat/log10hepcidin, was evaluated to attempt to distinguish inappropriate from appropriate hepcidin expression in probands and their family members. Results: Seven additional IRIDA probands had biallelic TMPRSS6 mutations identified including 9 novel mutations (L62fs, T182fs, Y191X, C245F, L281fs, Q238fs, C557S, L671fs, G706C, W775X) and 3 previously described mutations (G422R, E522K, R599X). Five additional probands each had only single mutations identified including 3 novel mutations (A80V, R93C, IVS4+1G>T) and 2 previously described mutations (E522K, IVS7+1G>A). All disease-associated missense variants were not present in the NCBI and Ensembl SNP databases as well as 100 control chromosomes. Each of these patients had elevated absolute urinary and/or plasma hepcidin concentrations. When hepcidin levels were normalized for serum transferrin saturation (‘Hepcidin Index’ = TfSat/log10hepcidin) probands with bialleleic mutations were easily distinguished from wild-type family members. Heterozygous family members had normal hematological and biochemical iron studies, but had an intermediate Hepcidin Index, indicative of a codominant phenotype. Several other individuals referred for a clinically milder IRIDA phenotype were found to have heterozygous mutations, suggesting that some heterozygotes may manifest clinically apparent disease. In addition, several individuals with a severe IRIDA phenotype were found to have high hepcidin levels in the absence of evidence of inflammation and TMPRSS6 mutations, raising the possibility that IRIDA is a genetically heterogeneous disorder. Discussion/Conclusions:TMPRSS6 mutations leading to inappropriately elevated hepcidin levels explain many, but not all, patients with the IRIDA phenotype. We add nine new mutations to previously described mutations in TMPRSS6. The Hepcidin Index (TfSat/log10hepcidin) can assist in distinguishing those with iron deficiency from those likely to haveTMPRSS6 mutations. Disclosures: Westerman: INTRINSIC LIFESCIENCES LLC: Consultancy, Employment, Equity Ownership.

Background: Many patients with SCD require chronic transfusion therapy to manage the complications of their disease (eg stroke prevention); as a consequence, secondary iron overload may develop. Controlled data from patients with SCD receiving long-term iron chelation therapy, particularly renal function, are lacking. Cumulative 3.5-year safety and efficacy data are presented for adult and pediatric patients with SCD with transfusional iron overload treated with deferasirox (Exjade®) in a 4-year extension to a 1-year comparative study (109). Methods: Study 109 demonstrated similar dose-dependent liver iron concentration (LIC) reductions with deferasirox and deferoxamine (DFO) in SCD patients with iron overload. Eligible patients entered a 4-year extension phase and received deferasirox only; dose adjustments were based on monthly serum ferritin (SF) levels and safety assessments (adverse events [AEs] and laboratory parameters). Patients with abnormal renal function were excluded. Results: 132 patients (mean age ± SD of 19.1 ± 10.7 years) who were initially randomized in the core study to receive deferasirox are included in this analysis. The median duration of exposure to deferasirox was 37.4 months (3.1 years) at a mean dose of 18.4 ± 6.2 mg/kg/day. Mean iron intake over this period was 0.3 ± 0.1 mg/kg/day. 72 patients (54.5%) continue to receive deferasirox. Reasons for discontinuations include: AEs (n=11), consent withdrawal (n=24), lost to follow-up (n=9), unsatisfactory therapeutic effect (n=4), and other reasons (n=11). There was also one death reported post-liver transplantation, which was not considered by the investigator to be related to the study drug. The most frequent drug-related (investigator-assessed) AEs were nausea (n=20; 15.2%), diarrhea (n=14; 10.6%), vomiting (n=8; 6.1%) and abdominal pain (n=6; 4.5%). Nine patients (6.8%) had two consecutive increases in serum creatinine that were both >33% above baseline and above the upper limit of normal (ULN); however, there were no progressive increases. Five patients (3.8%) had an increase in alanine aminotransferase >10xULN on at least one visit; baseline levels were already >ULN in two patients. Mean daily dose of deferasirox increased from 15.4 ± 6.9 mg/kg/day at month 1 to 22.3 ± 7.3 mg/kg/day at month 42. Overall baseline median SF level was 3439 ng/mL (n=132), which decreased by 651 ng/mL (P=0.0533, Wilcoxon signed rank test; n=49) by month 42 (Figure 1). SF decreases were dose-dependent (data not shown). Figure 1. Mean dose and median change in SF during deferasirox treatment Figure 1. Mean dose and median change in SF during deferasirox treatment Conclusions: Patients with SCD and transfusional iron overload receiving long-term deferasirox demonstrated continued reduction in their body iron burden (according to SF levels), without an exposure-associated increase in AE incidence, or evidence of progressive renal dysfunction.

Background: Many patients with SCD require acute or chronic transfusions to manage serious crises or prevent stroke. With age, there is also a risk of progressive renal dysfunction. A 1-year comparative study (109) in iron-overloaded patients with SCD demonstrated similar dose-dependent liver iron concentration (LIC) reductions with once-daily oral deferasirox and deferoxamine (DFO). This analysis presents cumulative long-term efficacy and safety data for patients with SCD receiving deferasirox treatment in a 4-year extension trial. Methods: In the 1-year core phase, deferasirox and DFO doses were assigned according to baseline LIC. In the extension study, patients either continued treatment with deferasirox (deferasirox cohort) or crossed over from DFO to deferasirox (crossover cohort). Patients with abnormal renal function were excluded. Dose adjustments during the extension phase were based on weight changes, serum ferritin (SF), creatinine, liver function tests and skin rash. Efficacy was determined by monthly SF; safety assessments included all adverse event (AE) monitoring, lab parameters, and ocular, auditory, and physical exams. Data were analyzed with a cut-off of 31 March 2007. Results: 185 patients entered the extension phase, 132 patients in the deferasirox cohort and 53 patients in the crossover cohort. Patients in the deferasirox cohort have received treatment for a median of 2.1 years. There have been 33 discontinuations (26 deferasirox, 7 crossover patients; 18%) due to: AEs (10), consent withdrawal (12), lost to follow-up (8), unsatisfactory chelation (2), and other (1). Only 1 patient has discontinued in the past 12 months due to pregnancy. No deaths have occurred during this study. In the deferasirox cohort, patients who initially received deferasirox 5/10 mg/kg/d had an increase from baseline in median SF levels at 12 months (+50 ng/mL; baseline 2805 ng/mL), which gradually declined and reached baseline at 24 months (−140 ng/mL) following a dose increase to approximately 20 mg/kg/d (starting at 12 months). SF was either maintained or reduced from baseline in the initial 20 and 30 mg/kg/d dose groups. Patients continue to receive deferasirox treatment in the extension phase trial and, as more data accumulate, the long-term effect of deferasirox at doses of 20–30 mg/kg/day will continue to be assessed. There were no significant changes in markers of liver or renal function in either cohort, and no cases of progressive increases in serum creatinine. The most frequent AEs were vomiting (n=10), nausea (n=23), and diarrhea (n=19); 9 patients experienced skin rash. Most AEs were mild and occurred during the core phase, with a steady decrease in incidence during the extension. No new AEs or safety concerns have been reported thus far in the extension study. Conclusions: Deferasirox demonstrates dose-dependent efficacy in patients with SCD, with a manageable tolerability profile and no new AEs reported over a median of 2.1 years of treatment.

The C-Data Project of the NIH-NHLBI funded Comprehensive Sickle Cell Centers Program was designed to establish a large and geographically diverse patient registry of persons with sickle cell disease regularly followed in the 10 comprehensive sickle cell centers. Opened to enrollment in March 2005 the database now contains current demographic, clinical, and health related quality of life information on 1,673 participants. Retrospective patient data are abstracted from the medical record at enrollment and updated from both patient interviews and medical record review at semi-annual contacts. Common sickle cell clinical events are defined in the context of modern diagnostic criteria so that as prospective data collection continues an unparalleled database of the burden of disease sickle cell syndromes may cause will emerge. While data are preliminary, interesting descriptive statistics have been derived. As of June 2006, 69% of participants are ≤17 yrs of age [peds], 63% have HbSS, 50% are female, and over half have been followed for 5 or more years in the same center. At least one episode of acute chest syndrome was reported by 56% of all subjects (51% peds, 76% adults) and of painful crisis by 69% (62% peds, 94% adults). CNS events occurred in 18% including ischemic stroke in 6% of peds and 11% of adults. Cholecystectomy was the most common surgical procedure reported in 19% (10% peds, 53% adults). Splenectomy was reported in 11% (10% peds, 16% adults), tonsillectomy in 12% (10% peds, 18% adults), and permanent central lines had been placed in 7% (5% peds, 15% adults). Adults reported acute and chronic renal failure in 6% and 3% respectively, and proteinuria in 11%. Transfusion during the past year was reported by 32%, and 54% of patients have received at least one transfusion during their lifetime. Iron overload was reported by 7% (5% peds, 14% adults). One or more hospitalizations within the last two years were reported by 62% (60% peds, 71% adults). Admissions were primarily for management of painful crisis in 39%, fever in 16%, and acute chest syndrome in 10%. Within the last year an MRI of the brain was performed in 16% and transcranial doppler (TCD) in 23% of pediatric patients. A cardiac echo was performed within the last year on 33% of adults. Hydroxyurea, the chemotherapy agent recommended for use by severely involved patients, was prescribed for 23% (18% peds, 41% adults). Twice daily prophylactic penicillin use in the last year was reported by 67% peds and 25% adults. At least 19% of patients had participated in at least one other research study. This database will be an invaluable tool for assessment of adherence to recommendations for care, heath services planning, and outcomes assessment. Year 1 data have demonstrated that CDATA will be able to facilitate the planning of multi-center research studies, provide a mechanism for identifying specific individuals within a center who are potentially eligible for participation in intervention studies, and provide the clinical data to support DNA based genotype-phenotype assessments.