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The Pediatric Hydroxyurea Phase 3 Clinical Trial (BABY HUG) was a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial of hydroxyurea in infants (beginning at 9-18 months of age) with sickle cell anemia. An important secondary objective of this study was to compare clinical events between the hydroxyurea and placebo groups. One hundred and ninety-three subjects were randomized to hydroxyurea (20 mg/kg/d) or placebo; there were 374 patient-years of on-study observation. Hydroxyurea was associated with statistically significantly lower rates of initial and recurrent episodes of pain, dactylitis, acute chest syndrome, and hospitalization; even infants who were asymptomatic at enrollment had less dactylitis as well as fewer hospitalizations and transfusions if treated with hydroxyurea. Despite expected mild myelosuppression, hydroxyurea was not associated with an increased risk of bacteremia or serious infection. These data provide important safety and efficacy information for clinicians considering hydroxyurea therapy for very young children with sickle cell anemia. This clinical trial is registered with the National Institutes of Health (NCT00006400, www.clinicaltrials.gov).

The most common form of neurologic injury in sickle cell anemia (SCA) is silent cerebral infarction (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial, we sought to identify risk factors associated with SCI. In this cross-sectional study, we evaluated the clinical history and baseline laboratory values and performed magnetic resonance imaging of the brain in participants with SCA (HbSS or HbSβ° thalassemia) between the ages of 5 and 15 years with no history of overt stroke or seizures. Neuroradiology and neurology committees adjudicated the presence of SCI. SCIs were diagnosed in 30.8% (251 of 814) participants who completed all evaluations and had valid data on all prespecified demographic and clinical covariates. The mean age of the participants was 9.1 years, with 413 males (50.7%). In a multivariable logistic regression analysis, lower baseline hemoglobin concentration (P < .001), higher baseline systolic blood pressure (P = .018), and male sex (P = .030) were statistically significantly associated with an increased risk of an SCI. Hemoglobin concentration and systolic blood pressure are risk factors for SCI in children with SCA and may be therapeutic targets for decreasing the risk of SCI. This study is registered at www.clinicaltrials.gov as #NCT00072761.

We evaluated spleen function in 193 children with sickle cell anemia 8 to 18 months of age by 99mTc sulfur-colloid liver-spleen scan and correlated results with clinical and laboratory parameters, including 2 splenic biomarkers: pitted cell counts (PIT) and quantitative Howell-Jolly bodies (HJB) enumerated by flow cytometry. Loss of splenic function began before 12 months of age in 86% of infants in association with lower total or fetal hemoglobin and higher white blood cell or reticulocyte counts, reinforcing the need for early diagnosis and diligent preventive care. PIT and HJB correlated well with each other and liver-spleen scan results. Previously described biomarker threshold values did define patients with abnormal splenic function, but our data suggest that normal spleen function is better predicted by PIT of ≤ 1.2% or HJB ≤ 55/106 red blood cells and absent function by PIT ≥ 4.5% or HJB ≥ 665/106. HJB is methodologically advantageous compared with PIT, but both are valid biomarkers of splenic function. This trial was registered at www.clinicaltrials.gov as #NCT00006400.

Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% ± 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% ± 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% ± 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% ± 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182.

Abstract 2134 BABY HUG randomly assigned children with sickle cell anemia ages 9 to 18 months at entry to a two-year double-masked treatment course of 20 mg/kg/d of hydroxyurea (HU) or placebo (PBO). The primary goal of this clinical trial was to determine if treatment with HU could reduce organ damage or dysfunction (spleen function assessed by 99mTc-sulfur colloid and renal function by 99mTc-DTPA clearance) by 50%. Children were seen at least monthly for blood and clinical evaluations. [Lancet 2011; 377(9778):1663–1672.] Between March 23, 2006 and June 1, 2006, the BABY HUG study was placed on “clinical hold” due to the omission of an expiration date on the treatment label for one lot of the study treatments. During the clinical hold, there were 94 children on active study treatment (47 HU, 47 PBO); four and six children, respectively, had finished their two-year follow up. Subjects continued regular visits and clinical event monitoring but received no study medication. To examine the impact of the clinical hold on toxicity and clinical events, the BABY HUG study period was divided into three intervals: Before, During, and After the clinical hold. Each period was offset by a one-month delay from the actual onset and end date to allow for wash-out and wash-in periods surrounding the clinical hold. The clinical hold produced both hold effects (effects produced by the hold that were consistent in the two treatment groups) and treatment-hold interaction effects (hold effects that were different by treatment). The interaction effects are presented below. The table presents the rate or prevalence of selected clinical events during each interval. The rate of pain, one of the clinical events shown to benefit from treatment with HU, was comparable to the PBO rate during the clinical hold but was significantly lower in the pre- and post-hold intervals. The prevalence of splenomegaly (defined by palpation ≥2 cm below the costal margin), which was slightly greater in the HU group than in the PBO group during the pre- and post-hold intervals, increased to a four-fold difference during the clinical hold. There was no difference between the groups in the rate of splenic sequestration in any interval.Clinical Events Before, During and After Clinical Hold+Before HoldDuring HoldAfter HoldHU 55.1*PBO 50.3*HU 8.3*PBO 7.8*HU 125.1*PBO 126.8*AENo.RateNo.RateNo.RateNo.RateNo.RateNo.RateDactylitis59.13467.6112.0338.51814.48667.8Pain3461.772143.114168.715192.3129103.1288227.1Splenomegaly^3054.42039.814168.7338.58366.36551.6Lab ResultsMeanMeanMeanMeanMeanMeanRetic Ct (k/mm3)191.5329.4300.5327.3237.0335.4Hgb (gm/dL)10.078.699.098.739.288.69MCV (fL)86.8481.9082.8279.8585.0080.00+Rates in bold are significantly different at p < 0.05 within each time period*Person Years of Exposure^Prevalence Hematologic changes showing a treatment-period interaction included a significantly larger difference in absolute reticulocyte count between the HU and placebo group During the Hold as compared to Before and After. The HU-induced increase in Hb and MCV was significantly diminished During the Hold. All measurements moved back towards their pre-hold differences after the hold was lifted. The clinical hold in BABY HUG provided an unforeseen opportunity to study the clinical and laboratory impact of cessation of HU therapy for a brief time period. No serious clinical effects or toxicities occurred during the clinical hold, perhaps due to its limited time span. Parents and physicians should be aware that stopping HU therapy may result in increased episodes of pain or splenic enlargement. Disclosures: Off Label Use: Hydroxyurea which is not approved for use in children with sickle cell disease. Casella:Adventrix: Honoraria.

Abstract 7 BABY HUG [Clinical Trials #NCT00006400], an NIH-NICHD sponsored randomized placebo-controlled trial showed that hydroxyurea (HU) administered to 9–18 month old children with sickle cell anemia (SCA) provides substantial clinical benefit. Benefits include a decrease in pain crises, acute chest syndrome events, need for transfusion and hospital admission; hematologic improvement include higher total and fetal hemoglobin concentration, larger red cell size, and lower WBC counts with toxicity limited to transient reduction in absolute neutrophil count (ANC) [Lancet 2011; 377:1663–72]. The parent or guardian of all 176 children who completed at least 18 months of randomized treatment were offered participation in an initial observational BABY HUG Follow-Up Study and 163 (93%) consented to participate. Clinical and laboratory data were collected every 6 months by structured abstraction of the medical record regarding use of clinically prescribed HU (dose escalation recommended), blood counts, clinical imaging, and sickle cell-related events. At the time of enrollment the family did not know their child's randomized study treatment assignment; 133 (82%) initially chose clinical prescription of open-label HU. Acceptance of HU has remained high through 36 months of follow-up; during each 6 month data collection period 68–75% of participants reported having taken HU. Only 2 patients have left the study (due to relocation) and more than 93% of expected data have been collected. Preliminary analyses as of May 2011, including 417 patient years (pt-yrs) of follow up, demonstrate that in comparison to participants not taking HU, children who continue to take HU have statistically lower rates of pain crises requiring emergency department (ED) visits, episodic transfusions, and hospital admissions for any reason, including acute chest syndrome or febrile illness (see table). The substantial decrease in acute chest syndrome episodes is similar to the effect demonstrated with HU use in the randomized BABY HUG trial in younger infants and consistent with published trials detailing the benefit of HU therapy in older children and adults. The decrease in the rate of admission for febrile events in HU-treated patients is also comparable to that in the randomized trial, but the reason for this benefit is uncertain. There was no difference in hospitalization rates for painful events including dactylitis. Two patients in the non-HU group had a stroke. There were no differences between groups in the frequency of a palpable spleen or rate of acute splenic sequestration crises. Through 36 months of follow up children taking HU had persistently higher hemoglobin and MCV, and lower WBC and ANC than those not taking HU. Results of these analyses including growth and development assessments will enhance our understanding of the impact of HU use in children with SCA starting at a very young age. The accruing data from the BABY HUG Follow-Up Study demonstrate a continuation of the substantial benefits of early HU therapy with no discernable additional toxicities. Ongoing follow up of this cohort is essential to fully define these benefits as children grow, and to observe for late toxicity.Event Rate per 100 pt-yrsHUNo HUp valueED visit for Pain Crisis28.853.60.004Episodic Transfusion18.334.00.010Hospital Admission (any cause)74.9133.20.001Acute Chest Syndrome (admission)9.522.30.0001Febrile Illness (admission)30.764.3 Disclosures: Off Label Use: Hydroxyurea is not indicated for treatment of children with sickle cell disease. Use of this medication was for clinical indications and not mandated by this observational study.

Abstract 846 Elevation of the serum level of secretory phospholipase A2 (sPLA2) has previously been reported to predict impending acute chest syndrome (ACS) in sickle cell disease (SCD) patients admitted with pain. The Sickle Cell Disease Clinical Research Network initiated the PROACTIVE Feasibility Study to 1) assess the feasibility of screening, randomizing and treating with simple transfusion eligible patients and 2) assess the predictive utility of sPLA2 in screening for imminent ACS, characterize the distribution of sPLA2 values in sickle cell patients hospitalized with pain, and define a cut-off point for predicting ACS to optimize its reliability, sensitivity and specificity in predicting ACS. All SCD patients (hemoglobin SS, SC and SBeta-thalassemia) admitted for pain at a participating center were to be screened for eligibility; patients with a diagnosis of ACS at entry were not eligible. sPLA2 levels were drawn daily on consenting patients for up to three days during the admission. All patients had at least one CXR (mandated at 72 hr if not done for clinical indications) during the course of their hospital stay to determine if they had developed a new infiltrate, considered diagnostic for ACS. Patients who developed T>38°C, a sPLA2 level > 100ng/ml and had a normal CXR were eligible for randomization to observation or simple transfusion; the remaining patients were followed in an Observation arm. Of 421 patients potentially eligible for screening, 238 were enrolled in the study with 10 of these patients randomized (4 to transfusion 6 to standard care). Of these, 203 patients had from one to three sPLA2 levels drawn prior to an ACS diagnosis or did not develop ACS; if transfused, they received blood only after a diagnosis of ACS was made. Twenty-two of these patients (11%) developed ACS. Analysis of the maximum sPLA2 levels prior to ACS diagnosis revealed that a threshold of 45 ng/ml was the most accurate level to predict ACS (accuracy: 73%, sensitivity: 73%, specificity: 73%). The positive predictive value (PPV: 25%) was low due to the relative infrequency of ACS events. When sPLA2 levels were analyzed in children versus adults, sPLA2 was more accurate for adults in predicting ACS with a level of 65ng/ml having accuracy: 88%, sensitivity: 44%, specificity: 95%, PPV: 57%. The addition of a requirement for fever did not improve the accuracy of sPLA2 in either adults or children. Further analysis of sPLA2 levels with various clinical and laboratory data is ongoing to improve the PPV of sPLA2. These results indicate that sPLA2 can predict ACS with good sensitivity, but may require additional parameters to be useful in clinical management of SCD patients admitted with pain and at risk for ACS. Supported by the NHLBI. Disclosures: Labotka: HemaQuest Pharmaceuticals, Inc: Research Funding.

Abstract 1631 Introduction: Infants with sickle cell anemia (SCA) are at risk for organ damage and clinical events. Risk may vary depending on steady state hemoglobin level. BABY HUG (ClinicalTrials.org, NCT00006400), a NHLBI-NICHD supported phase III randomized placebo-controlled trial, examined the ability of hydroxyurea (HU) to reduce end organ damage to the kidneys and spleen and attenuate other complications of SCA in infants. In this secondary study, we investigated whether placebo-treated subgroups defined by extremes of baseline hemoglobin level differed from one another in frequency of sickle cell-related complications and other laboratory findings, and then compared these data to the entire group of infants treated with HU. Methods: BABY HUG subjects randomized at ages 9 – 18 mo were treated with hydroxyurea (20 mg/kg/d) (N=96) or with placebo (N=97) for 2 years. Those randomized to placebo were classified according to their age-adjusted baseline hemoglobin level and subgroups in the lowest (n=24) and highest (n=24) quartiles compared. (Demarcating Hb values were: age 9 to 10.2gm/dL; age12 – 18 mo, 9.9gm/dL.) Results: BABY HUG primary endpoints of spleen (splenic uptake of 99mTc sulfur colloid on liver-spleen scan) and kidney (GFR by DTPA clearance) function did not differ in placebo group subjects who were in the lowest and highest quartile hemoglobin levels. However, those in the lowest hemoglobin quartile had a higher incidence of acute chest syndrome (ACS) than those in the highest quartile (0.31 vs. 0.02 events/person-year, RR 14.4, p=0.01) and this difference was significantly attenuated by HU (0.05 events/person-year). The relative risk for developing a pain crisis did not differ between the two placebo subgroups (2.2 vs. 2.1 events/person-year), but HU significantly reduced pain frequency compared to either subgroup (0.94 events/person-year, p Conclusions: Severe anemia in very young patients with SCA was associated with elevated WBC and platelet counts and higher TCD velocities. Interestingly, severe anemia also was associated with increased frequency of ACS, in contrast to the association of ACS with higher hemoglobin levels in older patients. Future studies will need to confirm this relationship and clarify to what extent associated findings (e.g., hyposplenia, elevated WBC or platelet count) contribute to this susceptibility. Severe anemia in young SCA patients is a negative prognostic factor that is significantly impacted by early hydroxyurea therapy. Disclosures: Off Label Use: Hydroxyurea is not approved by FDA for infants with sickle cell disease. Miller:NIH/NHLBI; Emmaus Med Inc, Novartis Pharmaceutical; St Jude Childrens Hospital: Research Funding.

Abstract 1301 Poster Board I-323 Background Neutralizing factor VIII (FVIII) alloantibodies (“inhibitors”) develop in about 15% of all patients with hemophilia A following replacement therapy. The pathogenesis of this important treatment-related alloimmune complication is complex and involves both genetic and environmental factors, and likely their interactions. The type of hemophilia-causing FVIII gene (F8) mutation influences the risk of developing inhibitors with large deletions conferring the greatest risk and subtypes of single-base substitution mutations the lowest. Additional genetic factors that include possible transcription-modulating polymorphisms in the promoter regions of the structural loci encoding tumor necrosis factor a (TNFA), interleukin 10 (IL10), and cytotoxic T-lymphocyte associated protein-4 (CTLA-4) were recently found to be associated with alloimmunization risk in the Malmo International Brother Study (MIBS). We recently demonstrated that mismatched FVIII replacement therapy due to the presence of common F8 nonsynonymous-single-nucleotide polymorphisms (ns-SNPs), which result in single amino acid changes in FVIII but do not cause hemophilia, may represent a novel genetic determinant of risk and contribute to the greater frequency of inhibitor development in black compared to white patients with hemophilia A (Viel et al. N Engl J Med. 360:1618-27, 2009). Because the implicated immune response gene variants described above were not controlled for in our initial study, however, we are now evaluating their influence on inhibitor development in this sample of black Americans with hemophilia A. Methods We used PCR and genomic DNA from 78 black hemophilia A patients to genotype a SNP in the TNFA promoter (-308 G>A) and a CA-dinucleotide repeat polymorphism in the IL10 promoter (IL10G) by direct sequencing and fragment-length analysis, respectively. The inhibitor status, baseline severity, age at enrollment, and biologic relationships of these study subjects were already known as was their hemophilic F8 mutations and the background haplotypes on which they reside. Analyses for genotype-specific associations with inhibitor status were performed with and without controlling for background F8 haplotype. Results Although we found the frequency of the 134bp IL10 risk allele (102bp in our modified assay) to be 20.3%, which is similar to that observed previously in the largely Euro-Caucasian MIBS cohort (26.8%), no obvious association between inhibitor risk and genotypes containing this allele was identified in the black hemophilia A patients studied, even after controlling for the background F8 ns-SNP haplotype. The frequency we identified for the -308A TNFA risk allele (8.8%) was similar to that observed previously in non-hemophilic African Americans (12%), but was far less frequent than observed in the MIBS (32.9%). Analogous to the IL10 results, we identified no association between inhibitor development and TNFA genotypes containing the risk allele (which was only G/A heterozygotes since no homozygous A/A individuals were identified) in this black patient cohort, even after controlling for their ns-SNP haplotypes. Conclusions We found no association between inhibitor development in black Americans with hemohilia A and promoter variants in two immune response genes recently implicated as risk genotypes in the MIBS. While we did not replicate the prior findings, the small sample size of our present study could explain these results if a true functional variant in one of these genes either has a small effect or is in poor linkage disequilibrium with the typed variant in this black patient population. Our findings therefore require confirmation in a larger study, which is currently under way. Disclosures Howard: Haplomics, Inc.: Equity Ownership, Patents & Royalties. Kempton:Biomeasure Inc: Consultancy; CSL Behring Foundation: Research Funding.

Abstract 262 Introduction: The most common cause of neurological injury in sickle cell anemia is silent cerebral infarcts (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) cohort, we sought to identify risk factors associated with SCI. Patients and Methods: In this cross-sectional study, we evaluated the clinical history, baseline laboratory values and performed magnetic resonance imaging of the brain. For those children with SCI-like lesions, a pediatric neurologist examined the child and neuroradiology and neurology committees adjudicated the presence of SCI. Children between the ages of 5 and 15 years with hemoglobin SS or S-beta° thalassemia and no history of overt strokes or seizure were evaluated. Results: A total of 542 children were evaluated; 173 (31.9%) had SCI. The mean age of the children was 9.3 years, with 280 males (51.7%). In a multivariate logistic analysis, two covariates were significant: a single systolic blood pressure (SBP) obtained during a baseline well-visit, p = 0.015 and hemoglobin F (Hgb F) level obtained after three years of age, p = 0.038. Higher values of SBP and lower values of Hgb F increased the odds of SCI; Figure. Baseline values of white blood cell count, hemoglobin level, oxygen saturation, reticulocytes, pain, or ACS event rates were not associated with SCI. Conclusion: SBP and Hgb F level are two previously unidentified risk factors for SCI in children with sickle cell disease. Modulation of SBP and Hgb F levels might decrease the risk of SCI. Disclosures: No relevant conflicts of interest to declare.

BABY HUG (NCT00006400) is an NHLBI-NICHD sponsored Phase III multi-center, randomized, double blind placebo controlled study of daily oral hydroxyurea in infants 9–17 months of age with sickle cell anemia. Subject retention and adherence are essential to maintain the power and validity of the trial. Initially, we hypothesized that families with higher socioeconomic status (SES) and advanced education would be better able to comply with the complex study procedures. We evaluated SES and other demographic factors which may influence adherence in the ongoing study. Both study drug adherence and visit adherence were examined. Study visits occurred every two weeks until a stable dose was reached and then every four weeks unless toxicity was encountered. Subjects remained on study for two years. Study drug adherence was monitored by measuring liquid in returned bottles at each visit. Bottles were included in the analysis if the subject was dispensed a bottle at a visit, returned it at the next scheduled visit with a non-zero volume, and did not have a drug stop order between the visits. From these bottles, each subject’s median drug adherence was calculated. Drug adherence (%) was defined as the amount consumed divided by the amount which should have been consumed. Visit adherence (%) was based on expected routine study visits. Subjects were withdrawn from the study upon family request or if they continually missed visits. We defined ‘good adherence’ as taking ≥80% study drug and attending ≥90% study visits. Adherence data was available on 186 of the 193 randomized infants. The median drug adherence was 101.7%, with 88.9% of subjects having drug adherence of ≥80%, and three infants having drug adherence >150%. The mean visit adherence was 97.3% ± 8.4 SD, with 82.3 % of subjects having no missed visits. Thirteen infants had a visit adherence of

Background: BABY HUG is an NHLBI/NICHD sponsored double-blind placebo-controlled trial (NCT00006400) testing the hypothesis that hydroxyurea therapy (HU), if started early in life, will prevent or postpone organ damage to the spleen and kidney in infants with sickle cell anemia (SCA). All BABY HUG subjects at entry have GFR measured by 99mTc-DTPA radioisotope clearance (DTPA) and estimated using the Schwartz formula, a calculation based on serum creatinine (Cr) and height of the patient (GFR = κ (height)/Cr); κ=0.55 for children age >1 to 13 yr or 0.45 for term infants to age 1 yr. Measurement of GFR in infants is problematic due to difficulties in obtaining urine specimens and venous access. While Cr is a well-established marker for GFR it is not independent of body mass and may be secreted by renal tubules, leading to an overestimate of GFR. Cystatin C (CysC) is a cysteine protease inhibitor produced by all human nucleated cells and freely filtered by the kidney. Serum levels are not affected by muscle mass or gender and stable from age 18 mo to 50 yr, with higher levels in newborns reflecting a lower GFR. CysC offers a more practical and perhaps more accurate measure of GFR than Cr, especially in children with SCA. Normal non-SCA values are 0.5 to 1.4 mg/L. Formulas are published to translate serum levels of CysC to GFR as conventionally reported. Our aim was to determine usual and mean CysC levels in infants with SCA and compare CysC to DTPA and Cr-based assessments of GFR. Methods: Sera obtained and frozen during the eligibility screening phase of BABY HUG were used to determine CysC levels by particle enhanced immune nephelometry. Results: A total of 152 sera from infants age 9–17 mo (mean 13.5) were available. CysC levels ranged from 0.532 to 1.369 mg/L (mean 0.92; median 0.907), approximating the normal range. CysC was strongly and inversely associated with GFR estimated by DTPA (R2=0.086; p=0.001). CysC was also significantly associated with age (inversely, p=0.02), Schwartz GFR (using κ=0.55) (inversely, p=0.007) and Cr (p=0.01), but not with Hb, HbF or WBC count. GFR determined using two CysC-based formulae and the Schwartz formula were compared to GFR by DTPA. GFR Formula GFR* R-square P-value Regression slope *(mean ± SD in ml/min/1.73m2);99mTc-DTPA GFR=123.67±33.72 ml/min/1.73 m2 GFR in non-SS infants age 1–1.5 yr by 51Cr-EDTA clearance: 91.5±17.8 ml/min/1.73 m2 (Piepsz A et al. Eur J Nucl Med Imag2006;33:1477) Schwartz, κ=0.55 191.11±57.85 0.062 0.036 0.141 Schwartz, κ=0.45 154.80 ± 54.06 0.052 0.0123 0.172 CysC (1) 84.45±19.11 0.18 0.0026 0.149 CysC (2) 102.78±25.09 0.16 0.0029 0.196 GFR determined using CysC-based formula 2: GFR = antilog {1.92 + [1.123 × log (1/CysC)]} gave mean values closest to DTPA and similar to published estimates of GFR in non-SCA infants. The Schwartz formula overestimated GFR, especially when the higher κ was used. While correlations are low due to high GFR variability, the four GFR determinations all have a significant association with DTPA. However, because the slopes of the regression lines are not close to 1 (line of equality), measurement agreement with DTPA is poor. Conclusion: The Schwartz formula overestimated GFR in BABY HUG subjects. CysC-based GFR formulae underestimate GFR compared to concurrent DTPA values, but results are similar to published norms. Reports of hyperfiltration, based on creatinine-based determination of GFR, may be exaggerated. The impact of hydroxyurea therapy on CysC levels, if any, will be apparent at completion of the trial in late 2009.

Introduction: Transcranial Doppler ultrasound (TCD) is used in children with sickle cell anemia (SCA) to detect stroke risk. TCD screening is mostly employed in children between 2–16 yrs of age; its use in infants Methods: All subjects underwent a baseline TCD using the Nicolet Companion (EME) 2-MHz pulsed Doppler. All infants were 7–17 months of age during screening, had no history of stroke and were not receiving chronic blood transfusion. Blood flow velocities were recorded using the Stroke Prevention Trial in Sickle Cell Anemia (STOP) protocol with the exception of reducing the standard sample volume to 4 mm. No sedation was used. The time averaged maximum mean velocity (TAMM) was measured to determine the highest velocity on either side to categorize the study as normal (highest velocity Results: TCD exams were attempted on 204 infants. Six exams were unsuccessful (no data) because of the subjects’ lack of cooperation and 11 TCD’s were inadequate. Of the remaining (187) TCD exams, 183 were normal and four included at least one conditional velocity. No subjects were found ineligible for the trial due to an abnormal TCD result. The mean velocity of the left MCA was 114.0 cm/sec ± 22.1 and that of the right MCA was 111.7 cm/sec ± 23.1. The top two deciles of the maximum TAMM reading for each child were 149 cm/sec and 141 cm/sec. Both age and total hemoglobin (Hb) were significantly associated (p Conclusions: Adequate baseline TCD evaluation was obtained on 187 of 204 (92%) subjects. All but 4 were normal by STOP criteria as compared to approximately 10% abnormal and 18% conditional in STOP screening of older children. Using multivariate analysis, baseline TCD velocities varied inversely with the degree of anemia, as expected, but in addition varied directly with age The lack of significant TCD abnormalities is interesting, given the presence of silent infarcts in 13% of this group of children (Pediatr Blood Canc, 2008). When post-treatment TCD data collection is completed, it may become apparent whether: lower TCD velocities reflect a lack of stenotic vascular disease in infants; infants with TAMMs in the upper deciles will be more likely to have abnormal/conditional velocities as they grow older; and HU will have an impact on TAMM.

A urine concentrating defect is quite common in sickle cell anemia (SCA), has its onset in early childhood, and may be reversible (with transfusion) until age 10 years. BABY HUG is an NHLBI-NICHD sponsored double-blind, placebo-controlled Phase III Clinical Trial (NCT00006400) designed to assess efficacy of hydroxyurea in preventing organ damage in young children with SCA (Hb SS or SβO thalassemia); primary endpoints are spleen function and glomerular filtration rate (GFR). Two hundred thirty-three infants, recruited without regard to disease severity, underwent eligibility screening. To assess urine concentrating ability as a secondary endpoint, parents were instructed to collect and save timed urine specimens from subjects after 4 to 10 hours of fluid deprivation (NPO) overnight for osmolality (OSM) determination. More prolonged deprivation was avoided due to safety concerns. A paired serum sample for OSM, urea nitrogen (BUN), and creatinine was obtained the next morning. All specimens were analyzed in a central laboratory. The analyses included 184 infants with a urine specimen and a reported period of fluid deprivation of at least four hours; 178 had concurrent sera. Mean age was 13.0±2.7 mo (range 7.5 – 17.9 mo) and mean duration of fluid deprivation was 7.4±2.4 hr (4–13 hr). Mean serum OSM was 286±6 mOsm/kg H2O and independent of age, height, weight, or duration NPO. Urine OSM (mean 410±152, median 433, range 58–794 mOsm/kg H2O) was significantly greater than serum osmolality (p [mean serum OSM + 1 SD]); twenty-two (12.0%) had urine/serum OSM ratio > 2 and 54 (29.4%) had urine OSM > 500 mOsm/L. In addition, five infants (2.7%) were isosthenuric (urine OSM within mean serum OSM ± 1 SD) and 37 (20.1%) hyposthenuric (> 1 SD below mean serum OSM) despite instructions to withhold fluid. Urine OSM was associated with increasing 99mTc-DTPA GFR (p=0.024) and BUN (p

Between 1995 and 2004, two NIH-sponsored studies (STOP/STOP II) showed that children with sickle cell disease (SCD) and abnormal transcranial Doppler blood flow measurements (high stroke risk) are protected from stroke with regular blood transfusions. Iron overload, which may lead to complications and requires iron removal therapy, was monitored by serum ferritin (SF). Liver iron concentration (LIC) measurement was not mandated by protocol and was performed at investigator discretion. Biopsy dates and lab values were captured during STOP/STOP II, providing an opportunity to validate SF against LIC. 75 LICs on 36 patients (19 female, 17 male) at 8 centers were obtained. No liver biopsy complications were reported. LICs were correlated with STOP/STOP II core laboratory SF and alanine aminotransferase (ALT) obtained within 180 days of LICs. Median age at first biopsy was 11.1 years (range, 4.5–17.8), median time from start of transfusion was 36 months (range, 2–100). Iron removal treatment was initiated a median 23 months (range, 4–108) from start of transfusion, with deferoxamine (n=27), and/or exchange transfusion (n=9). 21 pts (58%) had multiple LIC measures: 2 (n=9), 3 (n=8), 4 (n=2), 5 (n=2). Last LICs on iron removal therapy were obtained a median 72 months (range, 35–124) from start of transfusion. Correlation between SFs and LICs were r=-0.06 (n=18) for first LICs obtained prior to iron removal therapy, r=0.50 (n=17) for last LICs obtained on iron removal therapy, and r=0.51 for all LICs (n=60). Pts with single/last LIC >=15 mg/gram dry liver were significantly more likely to have ALTs >=45 IU/L compared to those with LICs =15 mg/gram and ALT >=45 IU/L tended to have higher SFs then those with normal ALT (mean SF 4927 ng/ml, 95% CI 1739–8115 vs. mean SF 2255 ng/ml, 95% CI 1599–2912). 37% (7/19) of pts with LIC >=15 mg/gram had SFs

Alloantibodies that inhibit the function of wildtype (wt) FVIII molecules constitute the most serious complication of replacement therapy for hemophilia (H)A patients. Although it is not possible to accurately predict which patients will develop inhibitors, ethnicity is a known risk factor as alloimmunization occurs ~2−fold more often in African Americans (AAs) than in Caucasians (Cs). Analysis of data from a resequencing study revealed that the FVIII gene (F8) contains four nonsynonymous−single nucleotide polymorphisms (ns−SNPs) that encode six distinct wt FVIII proteins designated haplotype (H)1−H6. While AAs express all but H6, only H1 and H2, the two recombinant (r)−FVIII proteins used clinically, are expressed by Cs. About 25% of AAs express H3, H4, or H5, proteins that differ structurally from r−FVIII at three ns−SNPs (R484H, D1241E, M2238V), two of which are located in dominant inhibitor epitopes and have AA−restricted minor−alleles. We designed the Pharmacogenetics of Inhibitor Risk (PIR) study to test the hypothesis that these ns−SNPs, when allelically mismatched in replacement therapy, are pharmacogenetic risk factors that contribute to the greater incidence of inhibitor development in patients of African−descent. We enrolled 94 of 223 total AA HA patients from collaborating treatment centers. Table 1 presents characteristics of the first 47 subjects (whose functional F8 regions have been resequenced entirely) including: FVIII haplotype (exposure) − H3/H4/H5 vs H1/H2; inhibitor status (outcome) − yes/no; F8 mutation − high−risk vs low−risk types; other covariates and potential confounders. A preliminary analysis demonstrated an odds ratio of 3.6 for inhibitors developing in the exposed subjects (P=0.10; 95% CI=0.7–17.6). Our finding suggests that pharmacogenetic factors may indeed contribute to the increased incidence of this alloimmune complication in AAs. To increase the power to detect a true association and allow adjustment for confounders, we are currently completing F8 resequencing in the next 47 patients. Table 1. Relevant characteristics of the first 47 African-American hemophilia-A PIR study patients*

There is a paucity of pharmacokinetic (PK) data about hydroxyurea (HU) in persons with sickle cell anemia (SCA) and none in very young children. HU clearance is predominantly renal. The kidney may be damaged during infancy in SCA, but the first abnormality is usually hyperfiltration which may lead to enhanced HU clearance. Following a 15 mg/kg oral dose of commercially available HU capsules, PKs in 7 adults with SCA and normal renal function have been reported to be a mean±SD half-life (T1/2) of 3.14±0.9 hrs, a maximal concentration (Cmax) of 28.32±11.0 ug/mL, and an area under the curve (AUC) of 81.66±15.5 ug•hr/mL. The Phase II Study of HU in adults concluded that initial 2 hour clearance of HU was not associated with either the maximum tolerated dose or fetal hemoglobin response to treatment. However, the AUC did predict HU toxicity and, in another study, the need for dose adjustment in adults with SCA and renal insufficiency. BABY HUG is an NHLBI-NICHD sponsored Pediatric Phase III Clinical Trial designed to critically assess the efficacy of a novel liquid HU preparation in preventing organ damage in young children with SCA. Forty-five African-American infants, 12 to 18 months (mo) of age (mean 14.7 mo) were recruited without regard to disease severity to the just completed randomized, double-blind, placebo-controlled BABY HUG Feasibility and Safety Pilot Trial. First dose PKs were obtained 0, 1, 2, and 4 hours after oral administration of 20 mg/kg of HU in 22 consecutive patients (mean age 14.5 mo), coincident with measurement of glomerular filtration rate (GFR) by nuclear medicine DTPA clearance. An additional PK sample was obtained between 4 and 8 hours in 15 of the 22 (68%). Samples were frozen at −70°C, shipped, and assayed by high resolution gas chromatography with mass spectrometric detection (limit of detection 0.5 ug/mL). The T1/2 (2.36±0.99 hrs), Cmax (19.81±5.8 ug/mL; 0.26±0.8 uM/L), and AUC (68.82±11.5 ug•hr/mL) were somewhat lower in BABY HUG patients than in the small group of adults with SCA from the literature. There were no apparent relationships between PKs and baseline GFR measured by DTPA or calculated from the Schwartz equation. Younger patients ≤15 mo (n=15) had a trend toward a shorter T1/2 (2.1 vs 2.8 hours; p=0.118) and a lower predicted measurable HU concentration at 8 hours (1.2 vs 2.1 ug/mL; p=0.056) than those 16–18 mo (n=7). There were no age related differences in AUC (67.4 vs 71.8 ug•hr/mL; p=0.421) or Cmax (20.5 vs 18.2 ug/mL; p=0.409). PKs of this novel liquid preparation of HU in young children with SCA may be different than that of adults with the standard capsule formulation. The BABY HUG Trial will continue to refine the PK model by additional evaluations including samples from patients as young as 9 mo of age, the new lower age of eligibility for the open study currently accruing patients.

Transcranial Doppler (TCD) is useful in children with sickle cell anemia (SCA) to detect increased risk of stroke. The use of TCD in infants less than 2 years of age is less well established, but has previously been shown to be feasible. As a secondary endpoint in the BABY HUG Trial, TCD is expected to provide useful information on the possible effects of hydroxyurea (HU) in babies with SCA. BABY HUG is an NHLBI-NICHD sponsored phase III clinical trial to compare hydroxyurea to placebo to ascertain effectiveness in preventing end organ damage of the spleen and kidney. Eligible subjects underwent a baseline TCD using the Nicolet Companion (EME) 2-MHz pulsed Doppler. All infants were 8–18 months of age at enrollment, had no history of stroke and were not receiving chronic blood transfusions. Blood flow velocities were recorded using the Stroke Prevention Trial in Sickle Cell Anemia (STOP) protocol with the exception of reducing the standard sample volume to 4 mm. No sedation was used. The time averaged maximum mean was measured to determine the highest velocity on either side to categorize the study as normal (all recordings

The development of neutralizing antibodies against plasma-derived or recombinant (r) molecules of wildtype (wt) factor (F)VIII protein is the most serious complication of replacement therapy for hemophilia A (hA) patients. Although the pathogenesis of these antibodies, termed inhibitors, is complex and poorly understood, ethnicity, a recently established risk determinant, is clearly involved since African-American (AA) patients experience this complication ~2-fold more often than Caucasians. In a previous study -- in which the FVIII genes (F8) from 137 unrelated healthy people, representing 7 ethnic groups, were resequenced -- we identified 4 common nonsynonymous single nucleotide polymorphisms (nsSNPs) and thereby demonstrated that FVIII is not a monomorphic protein in non-hemophiliacs. Interestingly, 5 of the 6 distinct wt proteins encoded by the allelic combinations or haplotypes (H) of these nsSNPs, designated H1, H2, ..., H5, are expressed by AA’s compared to only 2 in Caucasians (H1 and H2). Because H3, H4 and H5 are 1) partially defined by AA-restricted minor alleles of R484H and M2238V, 2 nsSNPs that substitute amino acids in the A2 and C2 major B-cell inhibitor epitopes, 2) represent the wt FVIII protein in ~27% of AA’s, and 3) differ from the rFVIII proteins used clinically, we designed the PIR study -- the 1st cross-sectional study of AA hA patients -- to determine if pharmacogenetic factors contribute to their greater inhibitor risk. Because the strongest known risk factor for inhibitors is the F8 mutation type, we are resequencing the known functional regions of F8 in the 1st 50 enrolled PIR subjects, out of the 223 total AA hA patients treated at the six participating Region IV South hemophilia treatment centers, to test the plausible alternative hypothesis that the higher inhibitor incidence is due to a different spectrum of molecular abnormalities than has been found in other ethnic subgroups of patients already examined at the DNA sequence level. We have 1) determined the plasma FVIII-activity and -antigen levels, 2) identified the causative mutation in 18 patients, several of which are novel, and 3) are currently performing RT-PCR analyses for detecting the common inversions in introns 1 and 22. Once completed, we will statistically compare the prevalence of AA F8 mutation subtypes to that in hA patients from other ethnic subgroups (detailed in the HAMSTeRS database), and the proportion of AA FVIII deficiencies that are 1) severe, moderate and mild, and 2) CRM-positive and -negative.

Recombinant factor VIII (r-fVIII) is the most widely-used and effective therapy for hemophilia A (hA). Many patients unfortunately become refractory when the wildtype(wt) protein is seen as foreign and is targeted by functionally neutralizing anti-fVIII antibodies termed inhibitors. While inhibitors occur most often in severe hemophiliacs with complex fVIII mutations and a complete circulating absence of the fVIII protein, recent studies show patients with missense-mutations (mMt) have a higher incidence than previously thought and demonstrate r-fVIII can be immunologically targeted even when differing by only a single amino acid. Because mMt represent the most frequent overall etiology of hA (~39% of cases), common non-hemophilic protein variants of fVIII may represent an important novel modulator of inhibitor development in this setting. To determine the extent of such variation, we resequenced all coding regions of the fVIII loci (F8) in 137 healthy subjects from 7 ethnic groups, including 86 Caucasians and 16 African-Americans (AA). We identified 5 common nonsynonymous single nucleotide polymorphisms (nsSNPs). Surprisingly, whereas 4 were polymorphic in AA only 2 were variable in Caucasians, despite having examined 6x the number AA X-chromosomes. Since recent studies show AA patients have a 2-fold higher inhibitor incidence than Caucasians, thus establishing ethnicity as a risk factor in this complication, we were intrigued to find that minor alleles for 2 nsSNPs are restricted to AA and substitute amino acids in major B-cell inhibitor epitopes located in the A2 and C2 domains. To confirm these findings and accurately define the number and frequency of human haplotypes (H) (eg. distinct combinations in which the alleles of these 5 nsSNPs are linked in vivo) we resequenced F8 in a second study group that included 75 additional healthy AA. Here we show there are at least 7 distinct wt forms of the human fVIII protein by defining 7 haplotypes (H) from the 5 nsSNPs: H1, H2, …, H7. H1 exists in all ethnic groups, is the most common overall form of fVIII and represents 2 of the 3 r-fVIII concentrates used clinically. While H2 is the most common form in AA (44%) and possibly represents the other therapeutic r-fVIII molecule, we found that at least 20% will have AA-restricted fVIII proteins; H4 (4%), H5 (12%) and H7 (4%). Only 2 forms of fVIII were found in Caucasians, H1 (90%) and H2 (10%), in contrast, and neither were ethnically restricted. In summary, when combined with reports of at least 3 other nsSNPs, our findings establish as inaccurate the long held view that fVIII is basically a monomorphic protein in non-hemophiliacs. We hypothesize that greater immunologic barriers exist when r-fVIII is infused into patients with mMt in endogenous fVIII molecules containing one or more minor alleles of these nsSNPs. Moreover, due to the number and frequency of AA-restricted wt fVIII variants, we predict these nsSNPs contribute pharmacogenetically to the higher incidence of inhibitors in this ethnic group. As such, we have established a 6-site multi-center study to test this hypothesis using AA hA patients as the optimal group. So far we have enrolled 34 of 223 total AA hA patients at the participating centers and are obtaining blood samples from each for fVIII:C and fVIII:Ag measurements and F8 mutation detection. Because the allelic basis of hA has not been studied in AA, this mutation scan is essential to rule out the plausible alternative hypothesis that their higher inhibitor incidence is due to a distinct spectrum of molecular abnormalities. Ultimately, all patients with mMt will be tested to determine the presence and titer of anti-fVIII, using both Bethesda and ELISA assays, and the H of their mutant fVIII will be defined. The number of patients with inhibitors and AA-restricted forms (H4, H5 or H7) will then be compared to those with inhibitors but whose mMt are in haplotypes found in r-fVIII molecules (H1 or H2).

Chronic anemia and intraparenchymal sickling within the kidney lead to intravascular volume expansion and an increased glomerular filtration rate (GFR) in sickle cell anemia (SCA). An elevated GFR is considered to be an early indicator of renal damage in SCA, and the pathophysiologic changes leading to sickle nephropathy and elevated GFR likely begin at a young age. The Pediatric Hydroxyurea Phase III Clinical Trial (BABY HUG), an NHLBI-sponsored multi-center double-blinded placebo-controlled study, compares hydroxyurea versus placebo in infants with SCA, with the primary goal of determining the efficacy of hydroxyurea for the prevention of organ dysfunction in the spleen and kidney. In the Feasibility and Safety Pilot, a primary objective is to assess GFR in infants with SCA between the age of 12 and 18 months by measuring plasma clearance of 99m Tc-DTPA (diethylenetriaminepentaacetic acid) and by estimating GFR using the Schwartz equation. The DTPA GFR was determined following administration of an IV bolus of 25–50 μCi/kg of the radiotracer, with venous blood samples obtained at 1, 2, and 4 hours. GFR was also calculated using the Schwartz equation: 0.55 x body length (cm) ÷ plasma creatinine (mg/dL). For both measurements, a logarithmic transformation was applied to improve linearity between the variables, and to stabilize the variance of the transformed data. To date, 17 infants with SCA (median age 13.2 months) have had GFR measurements, with no complications occurring. The geometric mean of the GFR (± SD) as measured by DTPA plasma clearance was 112 ± 14.6 mL/min/1.73m2 (range 53–178 mL/min/1.73m2). By regression analysis, the GFR was correlated with age, with an increase of approximately 10% per month (univariate p = 0.006, multivariate p = 0.02), and this correlation could not be ascribed to other age-adjusted changes in hemoglobin concentration (p = 0.35), % fetal hemoglobin (HbF, p = 0.67), white blood cell (WBC) count (p = 0.64), or platelet count (p = 0.76). The estimated GFR calculated by the Schwartz equation was not significantly correlated with age (univariate p = 0.12), and adjustments using hemoglobin, %HbF, platelet or WBC counts did not improve the correlation. There was a modest correlation between GFR determined by DTPA and the Schwartz equation (r = 0.44; p = 0.08). These data indicate that (1) GFR measurement using DTPA plasma clearance is feasible in one year-old infants with SCA; (2) renal damage as measured by an elevated DTPA GFR appears to be present early in life and to be increasing with age; (3) preliminary evaluation of the use of the Schwartz formula indicates only a modest level of correlation with results obtained using DTPA measurements; and (4) in the BABY HUG trial, further evaluation of the efficacy of hydroxyurea in preservation of renal function will likely require DTPA GFR measurements rather than GFR estimates using the Schwartz equation.