Abdullah Kutlar

Prevention of a First Stroke by Transfusions in Children with Sickle Cell Anemia and Abnormal Results on Transcranial Doppler Ultrasonography

BACKGROUND Blood transfusions prevent recurrent stroke in children with sickle cell anemia, but the value of transfusions in preventing a first stroke is unknown. We used transcranial Doppler ultrasonography to identify children with sickle cell anemia who were at high risk for stroke and then randomly assigned them to receive standard care or transfusions to prevent a first stroke. METHODS To enter the study, children with sickle cell anemia and no history of stroke had to have undergone two transcranial Doppler studies that showed that the time-averaged mean blood-flow velocity in the internal carotid or middle cerebral artery was 200 cm per second or higher. The patients were randomly assigned to receive standard care or transfusions to reduce the hemoglobin S concentration to less than 30 percent of the total hemoglobin concentration. The incidence of stroke (cerebral infarction or intracranial hemorrhage) was compared between the two groups. RESULTS A total of 130 children (mean [+/-SD] age, 8.3+/-3.3 years) were enrolled; 63 were randomly assigned to receive transfusions and 67 to receive standard care. At base line, the transfusion group had a slightly lower mean hemoglobin concentration (7.2 vs. 7.6 g per deciliter, P=0.001) and hematocrit (20.4 vs. 21.7 percent, P=0.002). Ten patients dropped out of the transfusion group, and two patients crossed over from the standard-care group to the transfusion group. There were 10 cerebral infarctions and 1 intracerebral hematoma in the standard-care group, as compared with 1 infarction in the transfusion group -- a 92 percent difference in the risk of stroke (P<0.001). This result led to the early termination of the trial. CONCLUSIONS Transfusion greatly reduces the risk of a first stroke in children with sickle cell anemia who have abnormal results on transcranial Doppler ultrasonography.

Hydroxycarbamide in very young children with sickle-cell anaemia: a multicentre, randomised, controlled trial (BABY HUG)

BACKGROUND Sickle-cell anaemia is associated with substantial morbidity from acute complications and organ dysfunction beginning in the first year of life. Hydroxycarbamide substantially reduces episodes of pain and acute chest syndrome, admissions to hospital, and transfusions in adults with sickle-cell anaemia. We assessed the effect of hydroxycarbamide therapy on organ dysfunction and clinical complications, and examined laboratory findings and toxic effects. METHODS This randomised trial was undertaken in 13 centres in the USA between October, 2003, and September, 2009. Eligible participants had haemoglobin SS (HbSS) or haemoglobin Sβ(0)thalassaemia, were aged 9-18 months at randomisation, and were not selected for clinical severity. Participants received liquid hydroxycarbamide, 20 mg/kg per day, or placebo for 2 years. Randomisation assignments were generated by the medical coordinating centre by a pre-decided schedule. Identical appearing and tasting formulations were used for hydroxycarbamide and placebo. Patients, caregivers, and coordinating centre staff were masked to treatment allocation. Primary study endpoints were splenic function (qualitative uptake on (99)Tc spleen scan) and renal function (glomerular filtration rate by (99m)Tc-DTPA clearance). Additional assessments included blood counts, fetal haemoglobin concentration, chemistry profiles, spleen function biomarkers, urine osmolality, neurodevelopment, transcranial Doppler ultrasonography, growth, and mutagenicity. Study visits occurred every 2-4 weeks. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00006400. FINDINGS 96 patients received hydroxycarbamide and 97 placebo, of whom 83 patients in the hydroxycarbamide group and 84 in the placebo group completed the study. Significant differences were not seen between groups for the primary endpoints (19 of 70 patients with decreased spleen function at exit in the hydroxycarbamide group vs 28 of 74 patients in the placebo group, p=0·21; and a difference in the mean increase in DTPA glomerular filtration rate in the hydroxycarbamide group versus the placebo group of 2 mL/min per 1·73 m(2), p=0·84). Hydroxycarbamide significantly decreased pain (177 events in 62 patients vs 375 events in 75 patients in the placebo group, p=0·002) and dactylitis (24 events in 14 patients vs 123 events in 42 patients in the placebo group, p<0·0001), with some evidence for decreased acute chest syndrome, hospitalisation rates, and transfusion. Hydroxyurea increased haemoglobin and fetal haemoglobin, and decreased white blood-cell count. Toxicity was limited to mild-to-moderate neutropenia. INTERPRETATION On the basis of the safety and efficacy data from this trial, hydroxycarbamide can now be considered for all very young children with sickle-cell anaemia. FUNDING The US National Heart, Lung, and Blood Institute; and the National Institute of Child Health and Human Development.

Stroke Prevention Trial in Sickle Cell Anemia

Stroke occurs in 7-8% of children with Sickle Cell Disease (Hb SS) and is a major cause of morbidity. Rates of recurrence have been reduced from 46-90% to less than 10% through chronic blood transfusions. Prevention of first stroke, however, would be preferable because even one stroke can cause irreversible brain injury. Transcranial Doppler (TCD) ultrasound can detect arterial blood flow rates associated with subsequent stroke risk. By combining TCD screening and a potentially effective treatment, first stroke may be prevented. The Stroke Prevention Trial in Sickle Cell Anemia (STOP) is the first stroke prevention trial in Hb SS and the first randomized, controlled use of transfusion in Hb SS. This multi-center trial is designed to test whether reducing sickle hemoglobin to 30% or less with periodic blood transfusions will reduce first-time stroke by at least 70% compared to standard care. Primary endpoints will be clinically evident symptoms of cerebral infarction with consistent findings on Magnetic Resonance Imaging and Angiography (MRI/MRA) or symptomatic intracranial hemorrhage. Secondary endpoints will be asymptomatic brain lesions detected by MRI in brain areas not involved in primary endpoints. The design calls for a 6-month start-up interval, 18 months of TCD screening and randomization, and observation for stroke from entry through month 54. Key features of the trial are standardized TCD and MRI/MRA protocols interpreted blindly, and blinded adjudication of endpoints. The sample size (60 per treatment group) is based on prospective data relating TCD velocity to risk of stroke. A time-averaged mean velocity of > or = 200 cm/sec is associated with a 46% risk of cerebral infarction over 39 months. The sample size is sufficient to detect 70% reduction in the primary endpoint at 90% power. This trial will determine if transfusion is effective in the primary prevention of stroke. Secondary aims may further the understanding of the effects of transfusion on the brain and guide future research into cerebrovascular disease in Hb SS.

Separation of normal and abnormal hemoglobin chains by reversed-phase high-performance liquid chromatography

A review is presented of the elution patterns on reversed-phase columns of the normal and abnormal globin chains of different hemoglobin types, including 16 beta-chain variants, 7 alpha-chain variants, 9 gamma-chain variants, and 4 variants with fusion or hybrid chains. Separations appear to be based primarily on differences in hydrophobicity. The method is ideally suited for the detection of abnormal globin chains, their quantitation and their isolation. Semi-quantitative data based on the calculation of the delta/non-alpha ratios allow the detection of beta-thalassemic conditions in situations where the quantitation of hemoglobin A2 by other procedures is impossible or complicated.

Association of genetic variation with systolic and diastolic blood pressure among African Americans: the Candidate Gene Association Resource study

The prevalence of hypertension in African Americans (AAs) is higher than in other US groups; yet, few have performed genome-wide association studies (GWASs) in AA. Among people of European descent, GWASs have identified genetic variants at 13 loci that are associated with blood pressure. It is unknown if these variants confer susceptibility in people of African ancestry. Here, we examined genome-wide and candidate gene associations with systolic blood pressure (SBP) and diastolic blood pressure (DBP) using the Candidate Gene Association Resource (CARe) consortium consisting of 8591 AAs. Genotypes included genome-wide single-nucleotide polymorphism (SNP) data utilizing the Affymetrix 6.0 array with imputation to 2.5 million HapMap SNPs and candidate gene SNP data utilizing a 50K cardiovascular gene-centric array (ITMAT-Broad-CARe [IBC] array). For Affymetrix data, the strongest signal for DBP was rs10474346 (P= 3.6 × 10−8) located near GPR98 and ARRDC3. For SBP, the strongest signal was rs2258119 in C21orf91 (P= 4.7 × 10−8). The top IBC association for SBP was rs2012318 (P= 6.4 × 10−6) near SLC25A42 and for DBP was rs2523586 (P= 1.3 × 10−6) near HLA-B. None of the top variants replicated in additional AA (n = 11 882) or European-American (n = 69 899) cohorts. We replicated previously reported European-American blood pressure SNPs in our AA samples (SH2B3, P= 0.009; TBX3-TBX5, P= 0.03; and CSK-ULK3, P= 0.0004). These genetic loci represent the best evidence of genetic influences on SBP and DBP in AAs to date. More broadly, this work supports that notion that blood pressure among AAs is a trait with genetic underpinnings but also with significant complexity.

Neuropsychological dysfunction and neuroimaging abnormalities in neurologically intact adults with sickle cell anemia

CONTEXT Sickle cell anemia (SCA) is a chronic illness causing progressive deterioration in quality of life. Brain dysfunction may be the most important and least studied problem affecting individuals with this disease. OBJECTIVE To measure neurocognitive dysfunction in neurologically asymptomatic adults with SCA vs healthy control individuals. DESIGN, SETTING, AND PARTICIPANTS Cross-sectional study comparing neuropsychological function and neuroimaging findings in neurologically asymptomatic adults with SCA and controls from 12 SCA centers, conducted between December 2004 and May 2008. Participants were patients with SCA (hemoglobin [Hb] SS and hemoglobin level < or = 10 mg/dL) aged 19 to 55 years and of African descent (n = 149) or community controls (Hb AA and normal hemoglobin level) (n = 47). Participants were stratified on age, sex, and education. MAIN OUTCOME MEASURES The primary outcome measure was nonverbal function assessed by the Wechsler Adult Intelligence Scale, third edition (WAIS-III) Performance IQ Index. Secondary exploratory outcomes included performance on neurocognitive tests of executive function, memory, attention, and language and magnetic resonance imaging measurement of total intracranial and hippocampal volume, cortical gray and white matter, and lacunae. RESULTS The mean WAIS-III Performance IQ score of patients with SCA was significantly lower than that of controls (adjusted mean, 86.69 for patients with SCA vs 95.19 for controls [mean difference, -5.50; 95% confidence interval {CI}, -9.55 to -1.44]; P = .008), with 33% performing more than 1 SD (<85) below the population mean. Among secondary measures, differences were observed in adjusted mean values for global cognitive function (full-scale IQ) (90.47 for patients with SCA vs 95.66 for controls [mean difference, -5.19; 95% CI, -9.24 to -1.13]; P = .01), working memory (90.75 vs 95.25 [mean difference, -4.50; 95% CI, -8.55 to -0.45]; P = .03), processing speed (86.50 vs 97.95 [mean difference, -11.46; 95% CI, -15.51 to -7.40]; P < .001), and measures of executive function. Anemia was associated with poorer neurocognitive function in older patients. No differences in total gray matter or hippocampal volume were observed. Lacunae were more frequent in patients with SCA but not independently related to neurocognitive function. CONCLUSION Compared with healthy controls, adults with SCA had poorer cognitive performance, which was associated with anemia and age.

Fetal hemoglobin in normal adults and β-thalassemia heterozygotes

SummaryA recently developed high performance liquid chromatographic (HPLC) procedure using a weak cation exchanger (PolyCAT) in columns of different sizes was used to quantify fetal hemoglobin (Hb F) in blood of normal adults and β-thalassemia (β-thal) heterozygotes with ten different types of mutations. Preparative PolyCATHPLC greatly facilitated the characterization of isolated Hb F, i.e., the determination of the relative quantities of the Gγ and Aγ chains. The method is accurate and allows quantitation of Hb F at the 0.5% level; preparative PolyCAT-HPLC allows isolation of (nearly) pure Hb F from blood samples with low (< 1%) Hb F. Adult Hb F levels were determined in 69 normal adults (including 24 diabetics); Hb F levels fell below 1% except for subjects with abnormal -Gγ-Gγ arrangement and a C→T mutation at position -158 relative to the Cap site of both Gγ genes. The effect of the same mutation in the normal-Gγ-Aγ-arrangement was variable. Certain β-thal mutations (namely, those at positions -29;-88; IVS-I-1; IVS-II-1) were associated with high Hb F levels in heterozygotes, while those at nucleotide (nt) positions IVS-I-6; IVS-I-110; codon 24; codon 39; codons 41/42; IVS-II-745 were not. Gγ values varied and often fell into two groups (high Gγ and low Gγ); high Gγ values were not associated with high Hb F values. The chromatographic procedure is ideally suited for Hb A2 quantitation. Average values of Hb A2 in β-thal heterozygotes with any one of nine of the ten mutations were twice that of normals; the one exception was the β-thal heterozygote with the IVS-I-6 (T→C) mutation with an average low Hb A2 value of 3.6%.

Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia

Senicapoc, a novel Gardos channel inhibitor, limits solute and water loss, thereby preserving sickle red blood cell (RBC) hydration. Because hemoglobin S polymerization is profoundly influenced by intracellular hemoglobin concentration, senicapoc could improve sickle RBC survival. In a 12-week, multicenter, phase 2, randomized, double-blind, dose-finding study, we evaluated senicapocs safety and its effect on hemoglobin level and markers of RBC hemolysis in sickle cell anemia patients. The patients were randomized into 3 treatment arms: placebo; low-dose (6 mg/day) senicapoc; and high-dose (10 mg/day) senicapoc. For the primary efficacy end point (change in hemoglobin level from baseline), the mean response to high-dose senicapoc treatment exceeded placebo (6.8 g/L [0.68 g/dL] vs 0.1 g/L [0.01 g/dL], P < .001). Treatment with high-dose senicapoc also produced significant decreases in such secondary end points as percentage of dense RBCs (-2.41 vs -0.08, P < .001); reticulocytes (-4.12 vs -0.46, P < .001); lactate dehydrogenase (-121 U/L vs -15 U/L, P = .002); and indirect bilirubin (-1.18 mg/dL vs 0.12 mg/dL, P < .001). Finally, senicapoc was safe and well tolerated. The increased hemoglobin concentration and concomitant decrease in the total number of reticulocytes and various markers of RBC destruction following senicapoc administration suggests a possible increase in the survival of sickle RBCs. This study is registered at http://clinicaltrials.gov as NCT00040677.

Hydroxycarbamide versus chronic transfusion for maintenance of transcranial doppler flow velocities in children with sickle cell anaemia - TCD with Transfusions Changing to Hydroxyurea (TWiTCH): A multicentre, open-label, phase 3, non-inferiority trial

Background For children with sickle cell anaemia and elevated transcranial Doppler (TCD) flow velocities, regular blood transfusions effectively prevent primary stroke, but must be continued indefinitely. The efficacy of hydroxyurea in this setting is unknown. Methods TWiTCH was a multicentre Phase III randomised open label, non-inferiority trial comparing standard treatment (transfusions) to alternative treatment (hydroxyurea) in children with abnormal TCD velocities but no severe vasculopathy. Iron overload was managed with chelation (Standard Arm) and serial phlebotomy (Alternative Arm). The primary study endpoint was the 24-month TCD velocity calculated from a general linear mixed model, with non-inferiority margin = 15 cm/sec. Findings Among 121 randomised participants (61 transfusions, 60 hydroxyurea), children on transfusions maintained <30% sickle haemoglobin, while those taking hydroxyurea (mean 27 mg/kg/day) averaged 25% fetal haemoglobin. The first scheduled interim analysis demonstrated non-inferiority, and the sponsor terminated the study. Final model-based TCD velocities (mean ± standard error) on Standard versus Alternative Arm were 143 ± 1.6 and 138 ± 1.6 cm/sec, respectively, with difference (95% CI) = 4.54 (0.10, 8.98), non-inferiority p=8.82 × 10−16 and post-hoc superiority p=0.023. Among 29 new neurological events adjudicated centrally by masked reviewers, no strokes occurred but there were 3 transient ischaemic attacks per arm. Exit brain MRI/MRA revealed no new cerebral infarcts in either arm, but worse vasculopathy in one participant (Standard Arm). Iron burden decreased more in the Alternative Arm, with ferritin difference −1047 ng/mL (−1524, −570), p<0.001 and liver iron difference −4.3 mg Fe/gm dry weight (−6.1, −2.5), p=0.001. Interpretation For high-risk children with sickle cell anaemia and abnormal TCD velocities, after four years of transfusions and without severe MRA vasculopathy, hydroxyurea therapy can substitute for chronic transfusions to maintain TCD velocities and help prevent primary stroke.BACKGROUND For children with sickle cell anaemia and high transcranial doppler (TCD) flow velocities, regular blood transfusions can effectively prevent primary stroke, but must be continued indefinitely. The efficacy of hydroxycarbamide (hydroxyurea) in this setting is unknown; we performed the TWiTCH trial to compare hydroxyurea with standard transfusions. METHODS TWiTCH was a multicentre, phase 3, randomised, open-label, non-inferiority trial done at 26 paediatric hospitals and health centres in the USA and Canada. We enrolled children with sickle cell anaemia who were aged 4-16 years and had abnormal TCD flow velocities (≥ 200 cm/s) but no severe vasculopathy. After screening, eligible participants were randomly assigned 1:1 to continue standard transfusions (standard group) or hydroxycarbamide (alternative group). Randomisation was done at a central site, stratified by site with a block size of four, and an adaptive randomisation scheme was used to balance the covariates of baseline age and TCD velocity. The study was open-label, but TCD examinations were read centrally by observers masked to treatment assignment and previous TCD results. Participants assigned to standard treatment continued to receive monthly transfusions to maintain 30% sickle haemoglobin or lower, while those assigned to the alternative treatment started oral hydroxycarbamide at 20 mg/kg per day, which was escalated to each participants maximum tolerated dose. The treatment period lasted 24 months from randomisation. The primary study endpoint was the 24 month TCD velocity calculated from a general linear mixed model, with the non-inferiority margin set at 15 cm/s. The primary analysis was done in the intention-to-treat population and safety was assessed in all patients who received at least one dose of assigned treatment. This study is registered with ClinicalTrials.gov, number NCT01425307. FINDINGS Between Sept 20, 2011, and April 17, 2013, 159 patients consented and enrolled in TWiTCH. 121 participants passed screening and were then randomly assigned to treatment (61 to transfusions and 60 to hydroxycarbamide). At the first scheduled interim analysis, non-inferiority was shown and the sponsor terminated the study. Final model-based TCD velocities were 143 cm/s (95% CI 140-146) in children who received standard transfusions and 138 cm/s (135-142) in those who received hydroxycarbamide, with a difference of 4·54 (0·10-8·98). Non-inferiority (p=8·82 × 10(-16)) and post-hoc superiority (p=0·023) were met. Of 29 new neurological events adjudicated centrally by masked reviewers, no strokes were identified, but three transient ischaemic attacks occurred in each group. Magnetic resonance brain imaging and angiography (MRI and MRA) at exit showed no new cerebral infarcts in either treatment group, but worsened vasculopathy in one participant who received standard transfusions. 23 severe adverse events in nine (15%) patients were reported for hydroxycarbamide and ten serious adverse events in six (10%) patients were reported for standard transfusions. The most common serious adverse event in both groups was vaso-occlusive pain (11 events in five [8%] patients with hydroxycarbamide and three events in one [2%] patient for transfusions). INTERPRETATION For high-risk children with sickle cell anaemia and abnormal TCD velocities who have received at least 1 year of transfusions, and have no MRA-defined severe vasculopathy, hydroxycarbamide treatment can substitute for chronic transfusions to maintain TCD velocities and help to prevent primary stroke. FUNDING National Heart, Lung, and Blood Institute, National Institutes of Health.

Fetal hemoglobin in sickle cell anemia: genetic determinants of response to hydroxyurea

The increase in fetal hemoglobin (HbF) in response to hydroxyurea (HU) varies among patients with sickle cell anemia. Twenty-nine candidate genes within loci previously reported to be linked to HbF level (6q22.3–q23.2, 8q11–q12 and Xp22.2–p22.3), involved in metabolism of HU and related to erythroid progenitor proliferation were studied in 137 sickle cell anemia patients treated with HU. Three-hundred and twenty tagging single nucleotide polymorphisms (SNPs) for genotyping were selected based on HapMap data. Multiple linear regression and the nonlinear regression Random Forest method were used to investigate the association between SNPs and the change in HbF level after 2 years of treatment with HU. Both methods revealed that SNPs in genes within the 6q22.3–23.2 and 8q11–q12 linkage peaks, and also the ARG2, FLT1, HAO2 and NOS1 genes were associated with the HbF response to HU. Polymorphisms in genes regulating HbF expression, HU metabolism and erythroid progenitor proliferation might modulate the patient response to HU.