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Introduction: Hydroxyurea is a potent therapeutic agent for sickle cell anemia (SCA), and treatment at maximum tolerated dose (MTD) is becoming the standard of care. Hydroxyurea exerts its disease-modifying effects primarily through induction of fetal hemoglobin (HbF), although the cellular and molecular mechanisms by which hydroxyurea increases HbF expression remain unclear. Children with SCA treated with hydroxyurea at MTD have substantial phenotypic variation, however, as some have higher HbF responses than others. We hypothesized that unknown quantitative trait loci modulate the pharmacological induction of HbF, so we performed a large genome wide association study (GWAS) of hydroxyurea-associated HbF responses for children with SCA treated prospectively with dose escalation to MTD. Methods: We analyzed genomic DNA from 831 children with SCA enrolled in pediatric research trials from the US (HUSTLE, SWiTCH, TWiTCH), the Caribbean (EXTEND, SACRED) and sub-Saharan Africa (REACH, NOHARM); all of these trials reported robust treatment responses with average HbF >20%. Study participants received hydroxyurea with dose escalation to MTD based on mild myelosuppression. Whole blood DNA was genotyped using the H3Africa SNP array (Illumina) with whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform. A transformed z-score for each study cohort gave a standardized measure of HbF induction relative to their steady-state level and their treatment HbF level at MTD. These standardized z-score HbF values were then used as a continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, using age, hydroxyurea dose at MTD, and sex as co-variates. We first performed an initial GWAS discovery using hydroxyurea response data from four distinct African populations (n=377). Single nucleotide variants (SNVs) with nominal significance (p Results: In the discovery GWAS step, no variant passed genome wide significance (p Conclusions: This large GWAS using global cohorts of children with SCA and robust prospective HbF phenotype data has identified genetic predictors of HbF hydroxyurea treatment responses. Three novel genetic loci, PTPRD, RPH3AL, and ELL2 have SNVs associated with lower HbF responses. PTPRD is a protein tyrosine phosphatase receptor involved in cellular processes such as cell growth and differentiation, while RPH3AL, a rabphilin 3A like protein, is known to be involved in calcium-ion-dependent exocytosis. ELL2 is an elongation factor for RNA polymerase II and could modify RNA processing under the cytostatic effects of hydroxyurea. These genes and variants will be investigated to determine how they impact individual HbF responses to hydroxyurea treatment. Disclosures Aygun: National Heart, Lung, and Blood Institute: Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Research Funding; National Institute of Nursing Research: Research Funding; Patient-Centered Outsomes Research Institute: Research Funding.

Background: Sickle cell anemia (SCA) is a monogenic disease resulting in polymerization of hemoglobin in hypoxic conditions. This leads to red blood cell (RBC) membrane damage and sickling, causing vaso-occlusion and hemolysis. Continual, excessive release of lysed RBC contents within the vascular space can activate the inflammasome, a multiprotein oligomer that promotes maturation and secretion of pro-inflammatory cytokines, including interleukin 1-beta (IL-1β). The intravascular inflammation associated with SCA, e.g., increased serum c-reactive protein (CRP) and absolute counts of neutrophils and monocytes, is predictive of long-term morbidity and mortality. Inflammation is a major component of many of the clinical complications of SCA, including vaso-occlusive pain episodes, acute chest syndrome, vascular-endothelial dysfunction, renal disease and other forms of end organ damage. Standard of care for SCA is hydroxyurea, which augments fetal hemoglobin levels and may have some anti-inflammatory effects by reducing neutrophil and monocyte counts. Canakinumab is a fully human monoclonal antibody targeting IL-1ß and blocking its downstream pro-inflammatory activities with potential to ameliorate the inflammatory complications of SCA. Objective:To clinically validate in pediatric and young adult SCA patients the hypothesis that IL-1ß blockade by canakinumab is safe and provides clinical benefits. Methods: A multi-center, randomized, parallel group, double-blind, placebo-controlled trial recruited SCA patients (HbSS or HbS/ß0thalassemia) with history of ≥2 major pain episodes/year, screening baseline detectable pain (using pain e-diaries) and serum high sensitivity CRP level ≥1.0 mg/L. Patients were randomized with 1:1 ratio to receive six monthly s.c. injections of either canakinumab 300 mg (4 mg/kg for patients ≤40 kg) or placebo. The concurrent use of hydroxyurea was a stratification factor at randomization. Outcomes were measured at baseline and at weeks 4, 8, 12, 16, 20, 24, after which all patients moved to open label canakinumab treatment for additional 6 months. Electronic patient reported outcomes included daily pain intensity with a 0-10 cm visual analog scale, school/work absences secondary to SCA, fatigue and analgesic use. The primary outcome was change from baseline in the 4-week average daily pain intensity at week 12. Other secondary and exploratory outcomes included daily activity measured by wrist actigraphy, rate of hospitalization and adverse events, serious adverse events, transcranial Doppler velocities, percent oxygen saturation and laboratory markers of inflammation and hemolysis. Results: A planned interim analysis for futility and safety was performed on the first 30 enrolled patients (canakinumab, n=16; placebo, n=14), of whom 26 patients completed the Week 12 assessments (canakinumab, n=14; placebo, n=12), and 13 patients completed the Week 24 assessments. Enrolled patients (median age 17 years, range 12-20; 19 male, 11 female) were evenly distributed between treatment arms. All except one patient were maintained on a stable hydroxyurea regimen. Baseline overall disease activity levels (median, [range]) included average daily pain 3.93 [0.29, 6.57]; high sensitivity CRP 3.93 mg/L [1, 64.7]; transcranial Doppler velocities 85.0 m/s [23, 267]; hemoglobin 94.8 g/L [73.5, 121]. Futility criteria were not met and no canakinumab-associated safety issues were identified in this first interim analysis. Conclusions: Canakinumab was well tolerated and not associated with any major side effects in SCA. Results from a second interim analysis of study outcomes for all currently enrolled patients (n=49) completing the blinded, 24-week treatment period will be available in November 2019. Disclosures Rees: Agios: Other: Grants; TauRx (methylene blue): Other: Data monitoring committees; Astra Zeneca (ticagrelor): Other: Data monitoring committees; Novartis: Other: Strategic advisory role,Principal investigator,sickle cell disease2.6 Investigator; Emmaus: Other: Strategic advisory role; Celgene: Other: Strategic advisory role; Global Blood Therapeutics: Other: Strategic advisory role; Alnylam: Other: Principal investigator. Dampier:Micelle Biopharma: Consultancy, Research Funding; Merck: Research Funding; Hudson Publishing Company: Consultancy; Global Blood Therapeutics: Consultancy; Ironwood: Consultancy; Epizyme: Consultancy; Modus Therapeutics: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mahlangu:Sanofi Genzyme: Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Novartis: Research Funding; Baxalta: Consultancy, Research Funding, Speakers Bureau; LFB: Consultancy; Biomarin: Research Funding; uniQure: Research Funding; Spark: Consultancy, Speakers Bureau; Chugai: Consultancy; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Freeline Therapeutics: Research Funding; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; World Federation of Haemophilia: Speakers Bureau. Mortier:Novartis Pharma AG: Employment. McNamara:Novartis Pharma AG: Employment. Li:Novartis Pharma AG: Employment. Oliver:Novartis Pharma AG: Employment, Equity Ownership. OffLabel Disclosure: canakinumab use in the treatment of sickle cell anemia.

Introduction: Primary cutaneous T cell lymphomas (PCTCL) including Mycosis fungoides (MF) and Sézary syndrome (SS) account for 75% of primary cutaneous lymphomas. The 5-year survival is 18-40% in patients with advanced-stage PCTCL. Management strategies and prognosis of PCTCL depend on the disease stage (affected body surface area, blood, visceral and nodal involvement). Allogeneic hematopoietic cell transplantation (allo-HCT) can be used to treat advanced stages in young adults who are otherwise in good health. However, post-transplant relapse is still an issue with no clear guidelines regarding its management. Here we describe the largest study investigating donor lymphocyte infusions (DLI) in patients who relapsed after allo-HCT for PCTCL. Patients and methods: We conducted an observational, retrospective, French multicenter study. Between January the 1st 2000 and December the 31st 2017, all patients who underwent an allo-HCT for PCTCL regardless of the subtype and who received DLI for a post-transplant relapse were included. Data were collected using the ProMISE database. As needed, centers were asked to provide additional data. Statistical analyses were carried out by the Lille University Hospital (CHRU Lille) Biostatistics Methodology Unit and were performed using SAS software (SAS Institute version 9.4). Results: All 13 patients who received DLI after allo-HCT for a PCTCL in France were enrolled in the study (Figure 1). Mean duration of follow-up was 718 days. See table 1 for study population characteristics. Four patients (30%) presented acute graft versus host disease (GVHD) following allo-HCT, of which no incidences were superior to grade 2. Those four patients relapsed at day 342, 463, 499 and 659 after allo-HCT. Five patients (38%) presented chronic GVHD of which three had an extensive presentation. Those three patients relapsed at day 1082, 1568 and 1861. Table 2 details relapses and relapse management in our cohort. Table 3 shows parameters relative to allo-HCT, post-therapeutic management, and follow-up. Objective response rates to DLI was 62% (n=8). Five patients (38%) showed complete response and three patients exhibited partial response (32%). Five patients (38%) did not respond to DLI. The median best response duration to DLI was 181 days. Six out of the eight patients who responded to DLI relapsed (75%); the median time before the relapse after DLI was of 405 days. The two patients who have received DLI and did not relapse on January the 1st 2018 had 321 and 1350 days follow-up. Progression-free survival (PFS) was 46% at 1 year and 19% at 5 years (Figure 2). Overall survival rates were 100% at 1 year and 59% at 5 years (Figure 3). Six patients (46%) presented GVHD after DLI of which three cases were chronic GVHD. Two of them was an extensive presentation. One patient had received an allo-HCT from a female donor. One patient received bone marrow transplant carrying a 9/10 mismatch. All other patients received peripheral blood stem cell (PBSC) transplantation; two of them received a geno-identical stem cell transplantation from sibling donors and three patients received non-sibling donor HSCT with a 10/10 mismatch. Only three patients received DLI following SFGM-TC guidelines. Four patients died before January the 1st 2018 in our cohort. One patient died because of direct complications of the HSCT and related treatments. Two patients died because of a disease relapse. One patient died from unrelated cause (severe pulmonary). Conclusion: With a 5-year survival rate of 59% from the date of post-transplant relapse, DLI appears to be an effective treatment in cases of patient relapse after allo-HCT for PCTCL. DLI should be considered in the management of post-transplant relapse whenever possible. To our best knowledge, this is the largest study cohort investigating DLI in the post-transplant setting Disclosures Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees; Actelion: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Peffault De Latour:Amgen Inc.: Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding.

Hydroxyurea has hematologic and clinical efficacy in sickle cell anemia (SCA), but its effects on transcranial Doppler (TCD) flow velocities remain undefined. Fifty-nine children initiating hydroxyurea therapy for clinical severity had pretreatment baseline TCD measurements; 37 with increased flow velocities (≥ 140 cm/s) were then enrolled in an institutional review board (IRB)–approved prospective phase 2 trial with TCD velocities measured at maximum tolerated dose (MTD) and one year later. At hydroxyurea MTD (mean ± 1 SD = 27.9 ± 2.7 mg/kg per day), significant decreases were observed in the right middle cerebral artery (MCA) (166 ± 27 cm/s to 135 ± 27 cm/s, P < .001) and left (MCA) (168 ± 26 cm/s to 142 ± 27 cm/s, P < .001) velocities. The magnitude of TCD velocity decline was significantly correlated with the maximal baseline TCD value. At hydroxyurea MTD, 14 of 15 children with conditional baseline TCD values improved, while 5 of 6 with abnormal TCD velocities whose families refused transfusions became less than 200 cm/s. TCD changes were sustained at follow-up. These prospective data indicate that hydroxyurea can significantly decrease elevated TCD flow velocities, often into the normal range. A multicenter trial is warranted to determine the efficacy of hydroxyurea for the management of increased TCD values, and ultimately for primary stroke prevention in children with SCA.

CXCR3 ligands were secreted by tissue fibroblasts and peripheral blood–derived mononuclear leukocytes in response to interferon-γ (IFN-γ) and Toll-like receptor (TLR) ligands. Subsequent purification and identification revealed the presence of truncated CXCL11 variants missing up to 6 amino acids. In combination with CD26/dipeptidyl peptidase IV, the metalloprotease aminopeptidase N (APN), identical to the myeloid cell marker CD13, rapidly processed CXCL11, but not CXCL8, to generate truncated CXCL11 forms. Truncated CXCL11 had reduced binding, signaling, and chemotactic properties for lymphocytes and CXCR3- or CXCR7-transfected cells. CD13/APN-truncated CXCL11 failed to induce an intracellular calcium increase but was still able to bind and desensitize CXCR3 for intact CXCL11 signaling. CXCL11 efficiently bound to CXCR7, but CXCL11 was not able to induce calcium signaling or ERK1/2 or Akt phosphorylation through CXCR7. CD26-truncated CXCL11 failed to attract lymphocytes but still inhibited microvascular endothelial cell (HMVEC) migration. However, further processing of CXCL11 by CD13 resulted in significant reduction of inhibition of HMVEC migration. Taken together, during inflammation or cancer, CXCL11 processing by CD13 may lead to a reduced number of tumor-infiltrating lymphocytes and in a more angiogenic environment.

Introduction: TCD with Transfusions Changing toHydroxyurea (TWiTCH Clinical Trials.gov NCT01425307), an NHLBI-sponsored multicenter trial, compared transfusion pluschelation (Standard Arm) tohydroxyurea (HU) plus phlebotomy (Alternative Arm) in children with sickle cell disease at high risk for stroke. Alternative arm patients underwent serial phlebotomy (10mL/kg, maximum 500mL) every 4 weeks after reaching maximal tolerated dose (MTD) of HU and discontinuing transfusions. Changes in liver iron concentration (dLIC), measured as mg Fe per gram dry weight liver, by both MRI R2 (FerriScan) and R2* were key secondary outcome measures. R2 and R2* are two, different MRI techniques that exploit the magnetic properties of tissue iron to estimate iron concentration. We previously reported significant differences between the two approaches at the baselinetimepoint. The purpose of this investigation was to determine the limits of agreement between measurements ofdLIC over a period of one year by R2 and R2* methods in both arms of the study. Methods: MRI R2 and R2* data were collected prior to randomization, and after 1 year (midpoint) and 2 years of therapy (study exit). dLIC between baseline to midpoint and midpoint to study completion was calculated for both R2 and R2* LIC values. Since LIC measurement variability increases with iron burden, each dLIC pair (R2 and R2*) were normalized to the patients iron burden at the start of the observation interval. That is, dLIC from baseline to midpoint was normalized to baseline LIC, while dLIC from midpoint to study end was normalized to midpoint LIC. The geometric mean of LICR2 and LICR2* was used to represent the baseline and midpoint LIC. Bland Altman analysis was performed on measurements of the percent change of dLICR2 and dLICR2* to determine the limits of agreement between the two techniques. Results: R2 measurements were performed in 104 patients at baseline, 94 at midpoint and 99 at study end, while R2* measurements were performed in 101, 87, and 89 patients, respectively. However, missing data limited Bland Altman comparisons ofdLIC to 74 patients between baseline to midpoint and 69 patients from midpoint to study end. Figure 1 (left) plots the measureddLIC using R2 (vertical axis) against the measureddLIC change by R2* (horizontal axis) for the Standard Arm participants. Dots represent LIC change over the first year and open circles represent LIC changes over the second year. The dotted line represents perfect agreement. Figure 1 (right) demonstrates the corresponding relationship for the patients in the Alternative Arm. Although the alternative arm appears to have greater disagreement, this represents an artifactcause by the transient increases in LIC that occurred as patients bridged from standard to alternative therapy. Iron chelationwas stopped when patients began hydroxyureabut patients required an overlap period of transfusions for stroke prophylaxis. Figure 2 demonstrates the difference in dLIC measured by R2 and R2*, expressed as a percentage of starting LIC, plotted against the starting LIC value. The standard arm (open circles) and alternative arms (dots) completely overlap. 95% limits of agreement between the two measures ofdLIC were -45.7% to 63.7% (light lines). At LIC values > 8.3 mg/g,dLIC predicted by R2 was larger than predicted by R2*, while the converse was true for LIC values below 8.3 mg/g, similar to our published baseline findings for LIC measurements. Conclusions: LIC by R2 and R2* tracked one another closely over time in patients in both study arms. These data indicate that either technique can be used with confidence to monitor patients on iron removal therapy (chelation or phlebotomy), but that the techniques should not be interchanged. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Wood: Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Vifor: Consultancy; Biomed Informatics: Consultancy; World Care Clinical: Consultancy; Ionis Pharmaceuticals: Consultancy; World Care Clinical: Consultancy; AMAG: Consultancy; AMAG: Consultancy; Celgene: Consultancy; Celgene: Consultancy; Biomed Informatics: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy. St Pierre:Resonance Health: Consultancy, Equity Ownership. Piccone:Novartis: Other: Speaker. Hankins:Novartis: Research Funding. Rogers:Apopharma: Consultancy. Ware:Bayer Pharmaceuticals: Consultancy; Global Blood Therapeutics: Consultancy; Biomedomics: Research Funding; Addmedica: Research Funding; Nova Laboratories: Consultancy; Bristol Myers Squibb: Research Funding.

Background: TCD With Transfusions Changing to Hydroxyurea (TWiTCH, ClinicalTrials.gov NCT01425307), an NHLBI-sponsored Phase III multicenter trial, compared transfusions to hydroxyurea for maintaining TCD velocities in children with sickle cell anemia who previously received transfusions for abnormal TCD velocities. Iron overload was treated with serial phlebotomy in children randomized to hydroxyurea. At the first scheduled interim analysis, non-inferiority of hydroxyurea was demonstrated and the study was terminated prematurely. Methods: Participants randomized to hydroxyurea received decreasing volumes of monthly transfusions during hydroxyurea dose escalation to maximum tolerated dose (MTD), averaging 6-7 months. During this transfusion overlap period, no chelation therapy was given. After hydroxyurea MTD was reached, transfusions were discontinued and children started monthly phlebotomy if their entry liver iron concentration (LIC) by MRI-R2 (FerriScan®) was ≥2 mg Fe/g dry weight liver (DWL). The prescribed phlebotomy volume was 10 mL/kg (maximum 500 mL) with adjustments for anemia (5 mL/kg for Hb 8.0-8.5 g/dL and held if Hb Results: Sixty children (mean age 9.7±3.2 years; range 5.2-19.0 years; 48% male) were randomized to the Hydroxyurea Treatment Arm. The average duration of previous transfusions was 4.5±2.8 years. Almost all (51/60, 85%) had previously received chelation, primarily deferasirox, and 48 (80%) were on chelation therapy at study enrollment. Hydroxyurea MTD was achieved in 57 children (95%), and 54 commenced phlebotomy (two had low iron burden with LIC A total of 18 Adverse Events (17 Grade 2 and one Grade 3) occurred in 14 participants in association with phlebotomy (2.3% prevalence). The most common complication was light headedness/near-syncope (6) followed by anemia (4), hypotension (3), headache (3), and pain at the venous access site (1). One subject had a syncopal episode followed by transient weakness, which was centrally adjudicated as TIA. An average of 53.6±21.8 mL/kg blood was administered in the hydroxyurea-treated arm, which calculates to an average iron loading of 40.1±16.3 mg Fe/kg, while an average of 112 mL/kg of venous blood was removed by phlebotomy, which calculates to an average iron unloading of 36.1±15.7 mg Fe/kg. For the 54 children who received phlebotomy, the average LIC was 12.0± 9.7 mg/g at study entry, 13.4±10.3 at midpoint reflecting overlap transfusions without chelation, and 9.7±8.9 at study exit reflecting serial phlebotomy, for an average net LIC decrease of 2.3±4.1 mg/g. Average serum ferritin at study entry was 3105±741 ng/mL and 1392±1542 ng/mL at study exit. For 39 children who completed all 24 months of treatment before study closure, the overall average LIC decrease was 3.2±3.8 mg/gram DWL and 10 had final LIC measurements Conclusions: In the TWiTCH trial, phlebotomy was a feasible, safe, well-tolerated, and effective treatment for transfusional iron overload in children with sickle cell anemia. Although initial overlap transfusions without chelation limited the phlebotomy effects, in children who reached hydroxyurea MTD and discontinued chronic transfusions, monthly phlebotomy led to net iron unloading and lower LIC, and significantly reduced iron burden. Disclosures Rogers: Apopharma: Consultancy. Kalfa:Baxter/Baxalta/Shire: Research Funding. Kwiatkowski:Sideris Pharmaceuticals: Consultancy; Luitpold Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Apopharma: Research Funding; Ionis pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Consultancy. Wood:World Care Clinical: Consultancy; Biomed Informatics: Consultancy; Biomed Informatics: Consultancy; Celgene: Consultancy; Celgene: Consultancy; AMAG: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy; AMAG: Consultancy; World Care Clinical: Consultancy; Vifor: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy. Ware:Global Blood Therapeutics: Consultancy; Biomedomics: Research Funding; Bayer Pharmaceuticals: Consultancy; Addmedica: Research Funding; Nova Laboratories: Consultancy; Bristol Myers Squibb: Research Funding.

Background: Value-based health care (VBHC) according to Porter et al. is a novel approach that aims to improve health care by identifying and systematically measuring both medical and patient-centered health care outcome. Thus, including the well-being of the patient as related to physical and mental status, social functioning, and occupational consequences. By applying generic and disease specific outcome measures, health care providers are able to benchmark their center with others in order to compare care strategies and outcome, and to make informed choices with regard to optimization of care, necessary investments and possible cost reductions. Erasmus University Medical Center aims to apply this system in 80% of the disease expert centers in the upcoming three years. SickleCell Disease (SCD) is a chronic hereditary disease characterized by severe anemia, unpredictable episodes of extreme pain, cumulative organ damage and high health care utilization rates. In addition, multiple psychosocial problems influence complexity of health care delivery. Therefore, SCD is an excellent model to explore the effects of VBHC. Our hospital has a large Comprehensive Sickle Cell Care Centre. Aim: To identify ten outcome measures in SCD patients and the validated tools to quantify these measures. This is not possible without definition of baseline patient characteristics of influence on patient outcome. Methods: In six well-prepared sessions, our multidisciplinary team, including both adult and pediatric care takers defined relevant patient outcome measures and most important patient characteristics, in live sessions and surveys in collaboration with patients and parents. Results: Ten outcome measures were identified (Table 1) and discussed with patients and parents. Eight patient characteristics were defined with effect on outcome (Table 2). Eight validated tools to quantify outcome measures were described (Table 3). Specific tools and evaluation modes will be integrated into electronic patient files (Table 3). Conclusions: We believe VBHC is a valuable strategy to optimize patient care and to facilitate informed decision making with regard to health care investments. We have made a first step by identification of patient outcome measures and important baseline patient characteristics according to VBHC methodology. The coming year, outcome will be measured, thus enabling comparisons in the near future with other (inter)national centers. Table 1. Overview of most relevant patient outcome measures Mortality Anxiety Veno-occlusive crises per year Admissions per year Pain related to veno-occlusive crises Caregiver burden Complications Quality of life Social functioning Self-efficacy Table 2. Overview of baseline patient characteristics Genotype Age Born < or >January 2007* Socio-economic status Hemoglobin level Communication Comorbidity Mode of treatment Treatment exclusively in University Medical Center *Neonatal screening for SCD was implemented >January 1st, 2007 Table 3. Overview of tools to quantify patient outcome measures Target group Tool Patient Outcome Measures All ages Electronic patient file · Mortality · Complications · Admissions per year · Veno-occlusive crises per year · 'Door-to-needle-time Emergency Department >18 yrs8-18 yrs0-4 yrs EQ-5D- 5 level version (EQ-5D-5L) Pediatric Quality of Life Inventory (PEDsQL)TNO-AZL Preschool children Quality of Life (TAPQOL) · Quality of life · Social functioning · Anxiety and insecurity 18 yrs Health Care Related Quality of Life (CAREQOL-7D) Long-term Orientation (LTO) · Caregiver burden All ages Visual Analogue Scale (VAS) · Pain related to veno-occlusive crisis >18 yr Hospital Anxiety and Depression Scale (HADS) · Anxiety and insecurity All ages Sickle Cell Self-Efficacy Scale (SCSES) · Self-efficacy Disclosures Cnossen: Pfizer: Other: travel funding, Research Funding; Baxter: Other: Travel Funding, Research Funding; Bayer: Other: travel funding, Research Funding; CSL Behring: Other: travel funding, Research Funding; Novo Nordisk: Research Funding; Novartis: Research Funding.

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.

Hydroxyurea has been shown to be efficacious for the treatment of sickle cell anemia (SCA), primarily through the induction of fetal hemoglobin (HbF). However, the exact mechanisms by which hydroxyurea can induce HbF remain incompletely defined, although direct transcriptional effects and altered cell cycle kinetics have been proposed. In this study, we investigated potential epigenetic and alternative molecular mechanisms of hydroxyurea-mediated HbF induction by examining methylation patterns within the Gγ-globin promoter and miRNA expression within primary CD71+ erythrocytes of patients with SCA, both at baseline before beginning hydroxyurea therapy and after reaching maximum tolerated dose (MTD). Using both cross-sectional analysis and paired-sample analysis, we found that the highly methylated Gγ-globin promoter was inversely correlated to baseline HbF levels, but only slightly altered by hydroxyurea treatment. Conversely, expression of several specific miRNAs was significantly increased after hydroxyurea treatment, and expression of miR-26b and miR-151-3p were both associated with HbF levels at MTD. The significant associations identified in these studies suggest that methylation may be important for regulation of baseline HbF, but not after hydroxyurea treatment, whereas changes in miRNA expression may be associated with hydroxyurea-mediated HbF induction. This study was registered at ClinicalTrials.gov (NCT00305175).

Hydroxyurea therapy has proven laboratory and clinical efficacies for children with sickle cell anemia (SCA). When administered at maximum tolerated dose (MTD), hydroxyurea increases fetal hemoglobin (HbF) to levels ranging from 10% to 40%. However, interpatient variability of percentage of HbF (%HbF) response is high, MTD itself is variable, and accurate predictors of hydroxyurea responses do not currently exist. HUSTLE (NCT00305175) was designed to provide first-dose pharmacokinetics (PK) data for children with SCA initiating hydroxyurea therapy, to investigate pharmacodynamics (PD) parameters, including HbF response and MTD after standardized dose escalation, and to evaluate pharmacogenetics influences on PK and PD parameters. For 87 children with first-dose PK studies, substantial interpatient variability was observed, plus a novel oral absorption phenotype (rapid or slow) that influenced serum hydroxyurea levels and total hydroxyurea exposure. PD responses in 174 subjects were robust and similar to previous cohorts; %HbF at MTD was best predicted by 5 variables, including baseline %HbF, whereas MTD was best predicted by 5 variables, including serum creatinine. Pharmacogenetics analysis showed single nucleotide polymorphisms influencing baseline %HbF, including 5 within BCL11A, but none influencing MTD %HbF or dose. Accurate prediction of hydroxyurea treatment responses for SCA remains a worthy but elusive goal.

Stroke is a devastating complication of sickle cell anemia (SCA), affecting 5% to 10% of patients before adulthood. Several candidate genetic polymorphisms have been proposed to affect stroke risk, but few have been validated, mainly because previous studies were hampered by relatively small sample sizes and the absence of additional patient cohorts for validation testing. To verify the accuracy of proposed genetic modifiers influencing stroke risk in SCA, we performed genotyping for 38 published single nucleotide polymorphisms (SNPs), as well as α-thalassemia, G6PD A− variant deficiency, and β-globin haplotype in 2 cohorts of children with well-defined stroke phenotypes (130 stroke, 103 nonstroke). Five polymorphisms had significant influence (P < .05): SNPs in the ANXA2, TGFBR3, and TEK genes were associated with increased stroke risk, whereas α-thalassemia and a SNP in the ADCY9 gene were linked with decreased stroke risk. Further investigation at these genetic regions may help define mutations that confer stroke risk or protection in children with SCA.

Background: Hydroxyurea has emerged as the primary disease-modifying therapy for both children and adults with sickle cell anemia (SCA). New guidelines from the National Heart, Lung, and Blood Institute include a recommendation that all children with SCA be offered hydroxyurea as young as nine months of age, regardless of the frequency or severity of clinical complications. The benefits of hydroxyurea are most evident when the dose is escalated to the maximum tolerated dose (MTD); however, a stable MTD may take 8-12 months to achieve. In this era of personalized medicine, individualized pharmacokinetics (PK) profiles can be used to predict appropriate medication dosing. Accordingly, we investigated hydroxyurea PK data in a large cohort of children with SCA at their first oral dose and at MTD, to understand inter-patient variability and to create an accurate dose prediction model for hydroxyurea MTD. Methods: The Hydroxyurea Study of Long-term Effects (HUSTLE, [NCT00305175][1]) is a longitudinal observational study for children with SCA receiving hydroxyurea therapy. Prospective investigation of hydroxyurea pharmacokinetics, pharmacodynamics, and pharmacogenomics is a primary study objective. HUSTLE includes two cohorts of children: a “New Cohort” with children initiating hydroxyurea therapy upon enrollment in the study, and an “Old Cohort” with children already taking hydroxyurea upon study entry. Children in the “New Cohort” received their first oral dose of hydroxyurea 20 mg/kg with formal first-dose PK data collection using peripheral blood analysis at baseline (t=0) followed by 15, 30, 60, 120, 240, and 480 minutes. PK analyses were performed at MTD for both the New and Old Cohorts. Hydroxyurea concentration was determined by a previously described color-based method, testing each sample in triplicate with a standard curve ranging from 0 to 1000mM, and sensitivity as low as 8mM. Results: A total of 97 children with SCA who enrolled in the New Cohort had first-dose hydroxyurea PK analysis, 65 of whom also had a paired PK study at MTD. Another 34 samples from the Old Cohort had PK analysis performed at MTD. | Patient samples | PK analysis timepoint | # of patients | Tmax (hr) | Cmax (mg/mL) | CL/F (L/hr) | t ½ (hr) | AUC (mg¥hr/mL) | | --------------- | --------------------- | ------------- | -------------------------- | -------------------------- | -------------------------- | -------------------------- | -------------------------- | | All | Baseline | 97 | 0.82 ± 0.47 (57.1) | 26.0 ± 6.60 (25.4) | 6.77 ± 3.14 (46.4) | 1.64 ± 0.55 (33.3) | 91.8 ± 23.0 (25.0) | | All | MTD | 99 | 0.80 ± 0.52 (65.1) | 31.2 ± 8.08 (25.9) | 7.27 ± 2.99 (41.2) | 1.94 ± 0.87 (44.6) | 115 ± 24.1 (20.9) | | Paired | Baseline | 65 | 0.82 ± 0.46 (56.4) | 25.3 ± 5.56 (22.0) | 7.11 ± 3.16 (44.4) | 1.73 ± 0.49 (28.5) | 93.1 ± 23.1 (24.8) | | Paired | MTD | 65 | 0.74 ± 0.47 (62.8) | 31.0 ± 7.82 (25.2) | 7.40 ± 3.13 (42.3) | 1.82 ± 0.89 (48.9) | 114 ± 22.3 (19.5) | Abstract 2707. Table 1. Baseline (first-dose) hydroxyurea pharmacokinetics revealed substantial interpatient variability, with a coefficient of variation ranging from ~25% for maximum concentration (Cmax) and area-under-the-curve (AUC) to ~50% for time to maximum concentration (Tmax) and apparent oral clearance (CL/F). At MTD, the PK parameters showed an expected increase in Cmax due to the higher average dose, but a convergence toward AUC of ~115 (mg¥hr/mL) with a reduced coefficient of variation. Based on this target AUC, predictive equations were generated that include PK parameters, but the simplest equation for the starting hydroxyurea dose is: Dose (mg) = 400 – [1000 x creatinine] + [21 x weight]. Conclusions: Individual hydroxyurea absorption patterns for young children with SCA can be used to predict MTD. The starting dose can be optimized by using predictive equations that reflect apparent oral clearance instead of body weight alone, and subsequently titrated by marrow suppression. AUC is the most relevant PK parameter, and our data show its variability is consistently lower at MTD, with a target AUC of 115 mg.hr/mL. As the use of hydroxyurea therapy expands, we plan a prospective study to tailor the starting dose using a pharmacometrics model involving weight, renal function, and selected PK parameters to rapidly achieve a safe and stable MTD. Disclosures Off Label Use: Hydroxyurea use for children with sickle cell anemia. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00305175&atom=%2Fbloodjournal%2F124%2F21%2F2707.atom

Hydroxyurea improves hematologic parameters for children with sickle cell disease (SCD), but its long-term efficacy at maximum tolerated dose (MTD) has not been determined. Between 1995 and 2002, hydroxyurea therapy was initiated for 122 pediatric patients with SCD including 106 with homozygous sickle cell anemia (HbSS), 7 with sickle hemoglobin C (HbSC), 7 with sickle/β-thalassemia (HbS/ β-thalassemia [6 HbS/β0, 1 HbS/β+]), and 2 with sickle hemoglobin OArab (HbS/OArab). Median age at initiation of therapy was 11.1 years. Hydroxyurea was escalated to MTD, with an average dose of 25.4 ± 5.4 mg/kg per day; the average duration of hydroxyurea therapy has been 45 ± 24 months (range, 6-101 months). Hydroxyurea was discontinued for 15 (12%) children with poor compliance. Mild transient neutropenia occurred, but no hepatic or renal toxicity was noted. Hydroxyurea therapy led to significant increases in hemoglobin level, mean corpuscular volume, and fetal hemoglobin (HbF) level, whereas significant decreases occurred in reticulocyte, white blood cell, and platelet counts and serum bilirubin levels. Children with variant SCD genotypes also had hematologic responses to hydroxyurea. HbF induction has been sustained for up to 8 years without adverse effects on growth or increased numbers of acquired DNA mutations. Long-term hydroxyurea therapy at MTD is well tolerated by pediatric patients with SCD and has sustained hematologic efficacy with apparent long-term safety.

Hydroxyurea is a safe and effective therapy for sickle cell disease (SCD), with the majority of its benefit correlating with the amount of fetal hemoglobin (HbF) produced at maximum tolerated dose, or MTD. There is substantial individual variability in HbF response to hydroxyurea, with baseline HbF levels accounting for approximately 40% of the observed variability in final HbF at MTD. Several genetic modifiers of baseline, or endogenous, HbF levels have previously been identified by genome wide association studies. These include certain beta-globin haplotypes, and polymorphisms at the BCL11A and HBS1L-MYB gene loci. The effect of these known genetic modifiers of baseline HbF on drug response has been investigated in a small (n=38) cohort of pediatric patients treated with hydroxyurea in several centers with different treatment guidelines. In this limited group, no association was found between the BCL11A or HBS1L-MYB variants and the change in HbF at MTD (ΔHbF; final HbF minus baseline HbF). Co-inheritance of alpha thalassemia has been reported to have a negative effect on HbF response to hydroxyurea. To independently verify these findings in a larger sample size, we tested the effect of four BCL11A single nucleotide polymorphisms (SNPs), three HBS1L-MYB SNPs, the XmnI polymorphism, and co-inheritance of α-thalassemia on ΔHbF at MTD in a cohort of 171 pediatric SCA patients, treated prospectively and uniformly on HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980) protocols. These patients represent the most accurate hydroxyurea phenotypes available, as all were meticulously titrated to MTD, and had complete laboratory data demonstrating compliance, such as absolute neutrophil count and absolute reticulocyte count within the therapeutic range. In our cohort, the BCL11A SNPs rs1427407, rs4671393 and rs11886868 were significantly associated with baseline HbF (Table 1). We saw no association between baseline HbF and any of the HBS1L-MYB SNPs, XmnI, α-thalassemia or BCL11A SNP rs7599488. In contrast to other reports, we found that coinheritance of α-thalassemia did not affect hydroxyurea treatment response (p=0.088). We found that BCL11A SNPs rs1427407, rs4671393 and rs11886868 were significantly associated with reduced ΔHbF following hydroxyurea treatment (Table 1), where individuals with the BCL11A SNPs had a smaller ΔHbF compared to individuals without the polymorphisms, as shown by the negative baseline β-value. For example, one BCL11A rs1427407 SNP is associated with a ΔHbF 3.46 percentage points lower than individuals without the SNP at MTD, with an additive, dose effect of the SNP at the second allele; homozygous individuals have higher baseline HbF, lower ΔHbF compared to heterozygotes or wild-type individuals (Figure 1). The other variants HBS1L-MYB SNPs, XmnI, α-thalassemia or BCL11A SNP rs7599488 did not significantly impact ΔHbF. None of the tested polymorphisms, including the BCL11A SNPs, were associated with a significant difference in final HbF levels. Individuals with higher baseline HbF due to BCL11A polymorphisms demonstrate a statistically significant lower rise in HbF in response to hydroxyurea than individuals without these polymorphisms. Identification of more variants associated with baseline and ΔHbF through next generation sequencing will help elucidate whether the negative effect of high baseline on ΔHbF is a BCL11A specific effect, or a manifestation of a general threshold effect, that there is a maximum amount of HbF an individual is able to achieve through hydroxyurea induction.Table 1Association between BCL11A SNPs and response to hydroxyurea.GeneSNP IDBaseline β-value (lnHbF)Baseline p-valueΔHbF β-value (%HbF)ΔHbF p-valueBCL11Ars14274070.3569.05x10-5-3.461.02x10-3BCL11Ars46713930.272.17x10-3-2.627.21x10-3BCL11Ars118868680.131.61x10-4-3.007.86x10-4BCL11Ars75994880.080.30-0.780.33Figure 1Effect of BCL11A rs1427407 on Hydroxyurea Response. Average baseline and ΔHbF values are shown for a sample BCL11A variant.Figure 1. Effect of BCL11A rs1427407 on Hydroxyurea Response. Average baseline and ΔHbF values are shown for a sample BCL11A variant. Disclosures: Off Label Use: Hydroxyurea is not FDA approved for use in pediatric sickle cell patients.

Abstract 1044 Background: Stroke With Transfusions Changing to Hydroxyurea (SWiTCH Clinical Trials.gov NCT00122980), an NHLBI-sponsored Phase III multicenter trial, compared chronic blood transfusions/chelation to hydroxyurea/phlebotomy for the reduction of recurrent stroke and improvement in iron overload management in children with sickle cell anemia (SCA) and history of overt stroke. To date, however, phlebotomy to manage iron overload has not been commonly performed in children, especially those with SCA. Objective: To describe the experience with SWiTCH phlebotomy procedures, including success rate, associated adverse events, and effect on liver iron stores. Methods: Quantitative liver iron concentration (LIC) was measured by liver biopsy at study entry. Only subjects with LIC > 5 mg Fe/gram dry weight liver (DWL) were eligible for randomization. Those randomized to hydroxyurea/phlebotomy received decreasing volumes of monthly transfusion during hydroxyurea dose escalation, which lasted 4–9 months. Phlebotomy was performed every 4±1 weeks after discontinuation of transfusions. The prescribed phlebotomy volume was 10 mL/kg (maximum 500 mL) for Hb ≥ 8.0 gm/dL, and 5 mL/kg for Hb 7.0–7.9 gm/dL. Phlebotomy was held if Hb was Results: Sixty-seven children (mean age 13.0 ± 4.0 years; range 5.2–19.0 years) with history of previous stroke and transfusion therapy for an average of 7.4 ± 3.8 years (range 1.5–15.5 years) were randomized to the hydroxyurea/phlebotomy arm. Most of them had also received chelation therapy: 47 (71%) with deferoxamine for an average of 4.8 ± 3.2 years, and 57 (86%) with deferasirox for 1.5 ± 0.8 years prior to study entry. Their average entry LIC was 16.5 ± 9.4 mg/gram DWL. Sixty of 67 children (90%) successfully transitioned to hydroxyurea after 7.2 ± 2.4 months of transfusion overlap; one subject had a stroke during overlap and six failed to demonstrate adequate response/compliance to hydroxyurea to safely discontinue transfusions. These 60 subjects received an average of 8 ± 3 transfusions providing 63 ± 44 mL/kg PRBCs before completing transition and commencing phlebotomy, and 3 ± 3 transfusions providing 19 ± 20 mL/kg PRBCs after starting phlebotomy, for various clinical indications. During the course of the study, a total of 935 phlebotomies were performed (mean 16 per subject) removing an average total volume of 127 ± 74 mL/kg per subject. The mean pre-phlebotomy Hb level on hydroxyurea (9.1 gm/dL) was not significantly different than the mean pre-transfusion Hb during the transfusion overlap period (9.0 gm/dL). Mean ferritin for these 60 subjects on the hydroxyurea/phlebotomy arm decreased from 3523 ± 2150 ng/mL at study entry to 2227 ± 1646 ng/mL (p Conclusions: Therapeutic phlebotomies were well-tolerated and did not result in worsening anemia or stroke recurrence in this cohort of children with SCA and previous stroke switched to hydroxyurea. Although ferritin levels decreased significantly, we did not demonstrate an overall decrease in LIC in this heavily iron overloaded cohort, most likely due to continued iron loading with transfusions in the overlap period and subsequent short duration of phlebotomy. Disclosures: Off Label Use: Use of hydroxyurea in children with sickle cell anemia.

Abstract 2131 Background: Over the past 15 years, numerous prospective and cross-sectional clinical trials have demonstrated that hydroxyurea has both laboratory and clinical efficacy for pediatric patients with sickle cell anemia (SCA). In infants, toddlers, children, and adolescents, hydroxyurea administered at maximum tolerated dose (MTD) leads to significant increases in hemoglobin (Hb) concentration, MCV, and %HbF along with simultaneous decreases in neutrophils, reticulocytes, total bilirubin, and serum lactate dehydrogenase. Clinical benefits include reduction in acute vaso-occlusive events (pain and acute chest syndrome), and emerging data suggest protection against chronic organ damage with a low risk of genotoxicity. For individual patients, however, the %HbF response to hydroxyurea and the MTD dose itself are highly variable. Currently there are no accurate predictors of the final %HbF or the MTD dose. Methods: To address the phenotypic variability, the Hydroxyurea Study of Long-Term Effects (HUSTLE, NCT00305175) was designed to capture prospectively first-dose (20 mg/kg) pharmacokinetics (PK) of hydroxyurea including maximum serum concentration (Cmax), area under the concentration curve (AUC), apparent oral clearance (CL/F), half-life (t1/2), and apparent volume of distribution (V/F). A consistent dose escalation schedule was then used to achieve a stable MTD, at which time PK studies were repeated and hydroxyurea responses (%HbF and MTD dose) were recorded along with other pharmacodynamics parameters. Results: After written informed consent, a total of 98 pediatric patients commenced hydroxyurea, 65 of whom reached MTD with complete paired PK information. In the majority of patients (39 of 65), a ‘fast/slow’ absorption phenotype identified at first-dose PK analysis was retained at MTD, supporting the concept of a genetic basis for this variation. Inter-individual PK variability was substantially greater than intra-individual variability; for example, the coefficient of variation (%CV) for CL/F was 44.4% on day 1 and 42.3% at MTD among all subjects, but averaged only 12.8% within the same subject. The CL/F was partly dependent on the rate of absorption but was most strongly influenced by subject weight and serum creatinine. At MTD, the average AUC was 114 ± 22.3 mg·h/mL, and the %CV of AUC was reduced from 24.8% on first-dose PK to 19.5% at MTD, likely reflecting dose titration toward a common degree of myelosuppression. In an attempt to predict the MTD dose toward this target AUC using data available at baseline, ‘best-fit’ equations were developed such as the following with or without PK data: Predicted MTD (mg/kg/day) = 31.9 - [17.1*Baseline Creatinine] - [0.14*Baseline BMI] + [0.0036*Baseline ARC] Predicted MTD (mg/kg/day) = 32.6 - [15.1*Baseline Creatinine] - [0.16*Baseline BMI] - [0.56*First-dose half-life] + [0.0034*Baseline ARC] - [0.48*PK phenotype (fast=0, slow=1)] A simpler equation for predicting MTD dose using only baseline creatinine and weight was then developed: Predicted MTD (mg) = 400 - [1000*Baseline Creatinine] + [21*weight] Conclusions: The relatively small intra-individual PK variability at hydroxyurea MTD compared to baseline PK studies, coupled with a similar degree of drug exposure when patients achieve a stable MTD, supports future investigation to predict the optimal hydroxyurea dose prior to the first dosing. Prediction equations of the MTD should be tested prospectively against standard dose-escalation schedules. Disclosures: Off Label Use: Hydroxyurea for children with sickle cell disease.

Abstract 1623 Introduction: Although the clinical course of sickle cell anemia (SCA) is highly variable, the level of fetal hemoglobin (HbF) is well-recognized as a critical laboratory parameter; lower HbF is associated with a higher risk of vaso-occlusive complications, organ damage, and early death. Hydroxyurea treatment has been shown to induce HbF, improve laboratory parameters, and ameliorate clinical complications of SCA but its mechanism of stimulating HbF expression remains incompletely defined. We examined the gene expression profiles of early erythroid cells to identify pathways or mechanisms by which hydroxyurea (a) affects red cell development and (b) induces the production of HbF. Methods: Peripheral blood was collected from patients enrolled in the Hydroxyurea Study of Long-term Effects (HUSTLE, ClinicalTrials.gov NCT00305175) and reticulocytes were purified by CD71+ positive selection. Total RNA was extracted from each sample and global gene expression was measured by Affymetrix Human HG-U133 Plus2Chip arrays. An average of 3000 transcripts/genes were detected for each patient RNA sample. Analysis of differential gene expression was then performed comparing: (1) paired baseline (pre-treatment) vs. maximum tolerated dose (MTD) hydroxyurea samples; (2) high vs. low HbF induced patient samples. Results: At MTD all 59 subjects had increased hemoglobin concentration, increased %HbF, along with decreased reticulocyte, neutrophil and WBC counts (all p30%). When the gene expression of these groups were compared, 123 genes were statistically related to optimum HbF induction. Gene ontology showed common themes for genes involved in nucleoplasm function, RNA binding, amino acid ligase activity and histone acetyltransferase activity. Subsequent quantitative PCR experiments confirmed the differential expression of 10 candidate genes; EIF1, ELOVL6, HBBP1, HSP90AA1, MAML3, OSBPL6, RAC2, RPL3 and SNRPN were all statistically different after hydroxyurea exposure; while H3F3A, HBBP1 and SNRPN were all statistically different in the high HbF responders (Table 1). Conclusions: Using reticulocyte RNA samples from patients with SCA, we have shown that hydroxyurea significantly alters gene expression in early circulating erythroid cells in vivo. Differentially expressed genes involved with translation elongation, ribosome assembly, and chromatin and chromosome organization likely reflect the cytostatic action of hydroxyurea. When genes related to HbF induction were examined, we identified several genes involved with RNA binding (e.g. SNRPN) and histone activity (e.g. H3F3A). Further investigation of these genes should greatly improve our understanding of HbF induction by hydroxyurea and potentially help predict patient clinical benefit prior to commencing hydroxyurea therapy. Genes were identified with expression differences related to hydroxyurea exposure or to HbF induction. For this subset of 10 genes, the change was seen by microarray and quantitative PCR (qPCR). The fold change is given as negative and positive for downregulated and upregulated genes, respectively. Disclosures: Off Label Use: The off-label drug use of hydroxyurea to treat the clinical complications of sickle cell anemia in children will be discussed.

Abstract 2639 Introduction: Stroke is perhaps the most catastrophic complication of sickle cell anemia (SCA), occurring in 11% of patients with SCA before 20 years of age. There is a definite need for biomarkers that could predict which children with SCA are at greatest risk for developing these irreversible cerebrovascular events. Many candidate genetic polymorphisms have been proposed to affect stroke risk but few have been validated, mainly due to the lack of additional patient cohorts. To validate the accuracy of published genetic modifiers, we genotyped polymorphisms in two large prospective cohorts. Methods: Pediatric patients with SCA and documented primary stroke (n=134, average age at stroke = 5.8 ± 2.8 years) were recruited through the Stroke With Transfusions Changing to Hydroxyurea (SWiTCH, NCT00122980) study. As a control non-stroke group, pediatric SCA patients (n=104, average age = 10.2 ± 3.5 years) enrolled in the Hydroxyurea Study of Long-Term Effects (HUSTLE, NCT00305175) were analyzed. All participants in the HUSTLE cohort were over 5 years old and without previous clinical stroke prior to beginning hydroxyurea treatment. We genotyped 38 single nucleotide polymorphisms (SNP's) with published associations for stroke risk, along with α-thalassemia trait, G6PD deficiency and the β-globin haplotype of each patient. Results: Only 5 of the 38 candidate SNPs were associated with stroke risk (Table 1). As previously reported the presence of α-thalassemia trait was also associated with stroke risk (p=0.009). In contrast, G6PD deficiency was not associated with stroke risk. The classical β-globin gene haplotypes were determined for all 238 subjects, resulting in alleles primarily representing the four classical African haplotypes including Benin (57.6%), Central African Republic (21.8%), Senegal (9.2%) and Cameroon (3.2%), as well as atypical haplotypes (8.2%). None of the classical β-globin haplotypes were associated with stroke. However, fine-mapping of the β-globin gene locus identified recombination events within the Aγ-globin gene region, which were significantly over-represented in the stroke versus non-stroke cohorts (n=26.5% vs. n=12.5%, p=0.0001). In particular, one haplotype we term BEN-Memphis has the classical Benin background haplotype but also has recombination between the promoter and intron 2 of the Aγ-globin gene. There were significantly more stroke subjects with this novel BEN-Memphis haplotype (n=9.3% vs. n=0.5%, p Conclusions: Our results confirm α-thalassemia trait is significantly protective against stroke in SCA. Fine-mapping of the β-globin gene locus identified novel recombinations within the β-globin gene locus that were associated with stroke risk. These variant haplotypes may be associated with altered γ- or β-globin gene expression. Of the other previously reported polymorphisms, only 5 of 38 SNPs were significantly associated with stroke risk (Table 1). These findings highlight the dangers of accepting non-validated genetic modifiers. The ADCY9 gene is highly expressed in the brain and is critical for neuronal signaling (Hacker BM et al., Genomics 1998). The TEK gene is an endothelial cell expressed tyrosine kinase that is crucial for prevention and recovery from stroke events (Bai Y et al., Neuroscience 2009). The ANXA2 gene has been proposed to affect the hypercoaguable state of SCA (Ling Q et al., J Clin Invest 2004). Finally, mutations in TGFBR3 have been linked with cerebrovascular disease (Santiago-Sim T et al., Stroke 2009). Further investigations at these genetic regions may help define the specific mutations that confer stroke risk or protection in children with SCA. The minor allele frequency (MAF) is given for each SNP. Significance between the control (HUSTLE, n=104) and stroke (SWiTCH, n=134) groups was tested using the Cochran-Armitage test. The HbA2 polymorphism is the Δ3.7kb α-thalassemia single gene deletion. Disclosure: Off Label Use: The off-label drug use of hydroxyurea to treat clinical complications of sickle cell anemia in children will be discussed.

Abstract 820 Introduction: Hydroxyurea therapy has proven laboratory and clinical efficacy for ameliorating the signs and symptoms of sickle cell anemia, primarily by increasing the level of fetal hemoglobin (HbF). Virtually all treated pediatric patients will have symptomatic benefit and improvement in laboratory parameters associated with disease severity, including increased HbF and decreased white blood cell (WBC) count and lactate dehydrogenase (LDH). However, significant inter-patient variability exists in the maximum tolerated dose (MTD) of hydroxyurea and some patients have exaggerated toxicity (excessive myelosuppression) at relatively low doses. Similarly, there is substantial variation in the degree of response for relevant laboratory parameters such as HbF and LDH. We hypothesized that genetic polymorphisms may play an important role in the observed inter-patient variability for both hydroxyurea response and toxicity. Methods: Pediatric patients enrolled in the prospective Hydroxyurea Study of Long-term Effects (HUSTLE, NCT00305175) were eligible for analysis, including 72 subjects who had reached hydroxyurea MTD based on standardized criteria with dose escalation to mild bone marrow suppression. Candidate genes were selected based on a presumed effect on hydroxyurea pharmacokinetics (PK) or pharmacodynamics (PD) such as putative HbF modifiers, ribonucleotide reductase, and urea transporters. For these candidate genes, sequencing was performed for single nucleotide polymorphism (SNP) discovery. In addition, published genetic modifiers were studied to validate the proposed effect on hydroxyurea responses. Each SNP was first tested for Hardy-Weinberg equilibrium and then tested for association with three variables: MTD HbF, MTD LDH, and MTD hydroxyurea dose (mg/kg/day) using Least Squares Regression in dominant, co-dominant, and recessive genetic models. Results: The average age (mean ± 1SD) at hydroxyurea treatment initiation was 8.4 ± 4.9 years, with 58% males. The average MTD dose was 25.8 ± 4.2 mg/kg/day (median 26.7, range 14.2 – 35.5 mg/kg/day). The average MTD laboratory values for this cohort included hemoglobin (Hb) = 9.7 ± 1.1 gm/dL, mean corpuscular volume (MCV) = 110 ± 12 fL, HbF = 24.5 ± 7.4%, and LDH 432 ± 158 U/L, all significantly different than baseline values (p Summary and Conclusion: In the prospective HUSTLE study that includes standardized hydroxyurea dose escalation to MTD, significant associations were identified between candidate gene variants and the MTD phenotype for HbF, LDH, and hydroxyurea dose. SNPs in candidate genes previously reported to be important in basal HbF regulation were significant for MTD dose and novel SNPs in the urea transporter UTB were also significantly associated with MTD HbF. These results support the hypothesis that genetic modifiers play an important role in the treatment and toxicity responses to hydroxyurea therapy for children with SCA. Disclosures: Off Label Use: Hydroxyurea for children with sickle cell anemia.

Abstract 263 Introduction: Glomerular hyperfiltration is an early manifestation of sickle cell nephropathy, and has been observed in children with sickle cell anemia (SCA) as young as 12 months of age with an age-dependent increase until the second decade of life. Over time, some children with SCA will develop microalbuminuria or frank proteinuria, but predictors of this complication are not currently available. The first aim of this study was to compare glomerular filtration rate (GFR) measured quantitatively by plasma clearance of injected 99-technetium diethylenetriaminepentaacetate (99mTC-DTPA) to GFR estimates using serum creatinine and cystatin C in children with SCA. A second aim was to identify predictors of elevated GFR and microalbuminuria/proteinuria among children with SCA. Methods: The Hydroxyurea Study of Long-Term Effects (HUSTLE) is a prospective observational study (NCT00305175) with the goal of describing the long-term cellular, molecular, and clinical effects of hydroxyurea therapy in SCA. At treatment initiation, patients have routine laboratory studies plus serum cystatin C, urine microalbumin, and GFR measurement by 99mTC-DTPA clearance. The GFR was also estimated using the following published equations: Correlations between continuous variables were measured using Pearson's Correlation Coefficient and medians between patient groups were compared using the Wilcoxon-Mann-Whitney test. Results: A total of 65 children with SCA (41 males) enrolled in the HUSTLE study before starting treatment with hydroxyurea. The mean age of the patients was 9.4 ± 4.6 years (range 2.0–18.0 years). All patients were normotensive with mean systolic and diastolic blood pressure measurements of 107 and 61 mmHg, respectively. The mean serum creatinine was 0.34 ± 0.10 mg/dL (range 0.1 – 0.6 mg/dL); mean serum cystatin C was 0.76 ± 0.14 mg/L (range 0.57–1.22 mg/L). The mean 99mTC-DTPA GFR value was elevated at 155.8 ± 38.9 mL/min/1.73m2 (range: 91–308 mL/min/1.73m2, normal for age 104 ± 20 mL/min/1.73m2). GFR values were not significantly different according to gender or age, although older teenagers had the lowest GFR values. Correlations between 99mTC-DTPA-GFR and the four estimated GFR values were all significant, but the best correlation was with the modified Schwartz formula (Pearson Correlation Coefficient = 0.45, p = 0.0014). The 99mTC-DTPA GFR values had a statistically significant negative correlation with serum cystatin C levels (Pearson Correlation Coefficient = −0.32, p = 0.0246) and a positive correlation with both systolic and diastolic blood pressure measurements (Pearson Correlation Coefficient = 0.27, p < 0.033 for both). A total of 63 patients had quantitative microalbumin measurements: 7 (11.1%) had microalbuminuria (30–299 mg protein/gm creatinine) and two (3.2%) had frank proteinuria (≥ 300 mg protein/gm creatinine). Mean 99mTC-DTPA GFR values were higher in the 9 patients with microalbuminuria or proteinuria as compared to 54 without microalbuminuria (184.2 versus 152.7 mL/min/1.73m2, p = 0.077). Conversely, cystatin C values were significantly lower in patients with microalbuminuria or proteinuria (0.63 mg/mL versus 0.78 mg/mL, p = 0.0028). The presence of microalbuminuria or proteinuria was also significantly associated with a higher systolic and diastolic BP, as well as lower WBC and ANC. Conclusions: This prospective study confirms elevated GFR values in children with SCA, but documents only modest correlations with published formulas for GFR estimation in the setting of glomerular hyperfiltration. GFR elevation is associated with lower cystatin C levels, and higher systolic and diastolic blood pressure measurements. Microalbuminuria/proteinuria was present in 14.3% of this pediatric cohort and was associated with lower cystatin C, higher blood pressure values, and lower WBC and ANC. Serial monitoring of cystatin C may be useful as an early marker of sickle cell nephropathy. Disclosures: Off Label Use: Hydroxyurea for children with sickle cell anemia.