ALBERT

Description: 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

Description: Hydroxyurea (HU) prevents many acute complications of sickle cell anemia (SCA) in adults and children, but its potential to delay or prevent chronic organ damage has not been defined. The objectives of this prospective IRB-approved study were to assess the safety and efficacy of HU in young children with SCA (age 18 mon–5 years) and to determine whether 2 years of therapy preserves renal function, reduces transcranial doppler ultrasound (TCD) values, and prevents development of brain ischemia as evidenced by magnetic resonance imaging/angiography (MRI/MRA). Fourteen children with SCA (11 male, 3 female; mean age 35±11 mon) enrolled and underwent evaluation including blood counts, %HbF measurement, determination of the glomerular filtration rate (GFR) by radionuclide DTPA clearance and Schwartz estimate, TCD mean cerebral artery (MCA) velocities, and brain MRI/MRA. HU was started at 20 mg/kg/day and escalated by 5mg/kg/day every 8 weeks to a maximum tolerated dose (MTD) or 30 mg/kg/day (mean dose 28±4 mg/kg). Children were evaluated initially every 4 weeks. All baseline tests were repeated at study exit (mean time 25±3 months). HU was tolerated well by all children. Hematological changes occurred as expected, with significant increases observed in hemoglobin concentration, MCV, and %HbF and significant decreases in reticulocytes, WBC, and neutrophils. The average GFR value did not rise as expected in this age range; the DTPA GFR decreased by 5.1 mL/min/1.73 m2 (p=0.26) with only 3 of 11 exit studies exceeding 150 mL/min/1.73 m2 and the Schwartz estimate increased by 16.5 mL/min/1.73 m2 (p=0.17). During HU therapy, the average TCD values significantly decreased with a mean decrease of 26±28 cm/sec in the right MCA (p

Description: 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

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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 p