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 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 participant's 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.

Therapeutic phlebotomy is safe in children with sickle cell anaemia and can be effective treatment for transfusional iron overload.

Serial phlebotomy was performed on sixty children with sickle cell anaemia, stroke and transfusional iron overload randomized to hydroxycarbamide in the Stroke With Transfusions Changing to Hydroxyurea trial. There were 927 phlebotomy procedures with only 33 adverse events, all of which were grade 2. Among 23 children completing 30 months of study treatment, the net iron balance was favourable (-8·7 mg Fe/kg) with significant decrease in ferritin, although liver iron concentration remained unchanged. Therapeutic phlebotomy was safe and well-tolerated, with net iron removal in most children who completed 30 months of protocol-directed treatment.

Whole exome sequencing identifies novel genes for fetal hemoglobin response to hydroxyurea in children with sickle cell anemia.

Hydroxyurea has proven efficacy in children and adults with sickle cell anemia (SCA), but with considerable inter-individual variability in the amount of fetal hemoglobin (HbF) produced. Sibling and twin studies indicate that some of that drug response variation is heritable. To test the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated phenotype-genotype associations using whole exome sequencing of children with SCA treated prospectively with hydroxyurea to maximum tolerated dose (MTD). We analyzed 171 unrelated patients enrolled in two prospective clinical trials, all treated with dose escalation to MTD. We examined two MTD drug response phenotypes: HbF (final %HbF minus baseline %HbF), and final %HbF. Analyzing individual genetic variants, we identified multiple low frequency and common variants associated with HbF induction by hydroxyurea. A validation cohort of 130 pediatric sickle cell patients treated to MTD with hydroxyurea was genotyped for 13 non-synonymous variants with the strongest association with HbF response to hydroxyurea in the discovery cohort. A coding variant in Spalt-like transcription factor, or SALL2, was associated with higher final HbF in this second independent replication sample and SALL2 represents an outstanding novel candidate gene for further investigation. These findings may help focus future functional studies and provide new insights into the pharmacological HbF upregulation by hydroxyurea in patients with SCA.

The clinical and laboratory spectrum of Hb C [ß6(A3)Glu→Lys, GAG>AAG] disease.

Newborn screening (NBS) provides early diagnosis of sickle hemoglobinopathies. After Hb S [ß6(A3)Glu→Val, GAG>GTG], Hb C [ß6(A3)Glu→Lys, GAG>AAG] is the most common hemoglobin (Hb) abnormality identified in the United States (1,2). Published data regarding children with Hb C disease are limited. This study was conducted to summarize a single institution's clinical and laboratory data for patients with Hb C disease, specifically homozygous Hb CC and its variants over a 10-year period. Forty-seven patients, whose mean age at diagnosis was 2.9 years (range 0.04 to 23 years), were identified. Twenty-nine had Hb CC and the remainder had compound heterozygous variants [10 Hb C/ß(+)-thalassemia (ß(+)-thal), four Hb C/ß(0)-thal, and one each with Hb C/Hb Hope or ß136(H14)Gly→Asp (GGT>GAT), Hb C/Hb Lepore (a hybrid δß-globin gene), Hb C/HPFH (hereditary persistence of fetal Hb) [probably a (G)γ HPFH-2 (the Ghanaian type)], and Hb C/Osu-Christiansborg or ß52(D3)Asp→Asn (GAT>AAT)]. All patients had mild microcytic anemia with reticulocytosis and frequent target cells on peripheral smear. Splenomegaly or cholelithiasis occurred in 2.6% of patients <8 years of age, however, these symptoms were more common (71.0%) in patients >8 years of age. No patient had serious infections or painful events resembling vasoocclusion. Accurate diagnosis and understanding of Hb C-related disorders helped to avoid confusion with sickle hemoglobinopathies and aided in proper clinical management.

Hydroxyurea treatment decreases glomerular hyperfiltration in children with sickle cell anemia.

Glomerular hyperfiltration and microalbuminuria/proteinuria are early manifestations of sickle nephropathy. The effects of hydroxyurea therapy on these renal manifestations of sickle cell anemia (SCA) are not well defined. Our objective was to investigate the effects of hydroxyurea on glomerular filtration rate (GFR) measured by (99m)Tc-DTPA clearance, and on microalbuminuria/proteinuria in children with SCA. Hydroxyurea study of long-term effects (HUSTLE) is a prospective study (NCT00305175) with the goal of describing the long-term cellular, molecular, and clinical effects of hydroxyurea therapy in SCA. Glomerular filtration rate, urine microalbumin, and serum cystatin C were measured before initiating hydroxyurea therapy and then repeated after 3 years. Baseline and Year 3 values for HUSTLE subjects were compared using the Wilcoxon Signed Rank test. Associations between continuous variables were evaluated using Spearman correlation coefficient. Twenty-three children with SCA (median age 7.5 years, range, 2.5-14.0 years) received hydroxyurea at maximum tolerated dose (MTD, 24.4 ± 4.5 mg/kg/day, range, 15.3-30.6 mg/kg/day). After 3 years of treatment, GFR measured by (99m)Tc-DTPA decreased significantly from 167 ± 46 mL/min/1.73 m² to 145 ± 27 mL/min/1.73 m² (P = 0.016). This decrease in GFR was significantly associated with increase in fetal hemoglobin (P = 0.042) and decrease in lactate dehydrogenase levels (P = 0.035). Urine microalbumin and cystatin C levels did not change significantly. Hydroxyurea at MTD is associated with a decrease in hyperfiltration in young children with SCA.

Hydroxycarbamide alters erythroid gene expression in children with sickle cell anaemia.

Sickle cell anaemia (SCA) is a severe debilitating haematological disorder associated with a high degree of morbidity and mortality. The level of fetal haemoglobin (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. Hydroxycarbamide treatment can induce HbF, improve laboratory parameters, and ameliorate clinical complications of SCA but its mechanisms of action remain incompletely defined and the HbF response is highly variable. To identify pathways of hydroxycarbamide activity, we performed microarray expression analyses of early reticulocyte RNA obtained from children with SCA enrolled in the HydroxyUrea Study of Long-term Effects (NCT00305175) and examined the effects of hydroxycarbamide exposure in vivo. Hydroxycarbamide affected a large number of erythroid genes, with significant decreases in the expression of genes involved in translation, ribosome assembly and chromosome organization, presumably reflecting the daily cytotoxic pulses of hydroxycarbamide. Hydroxycarbamide also affected expression of numerous genes associated with HbF including BCL11A, a key regulator of baseline HbF levels. Together, these data indicate that hydroxycarbamide treatment for SCA leads to substantial changes in erythroid gene expression, including BCL11A and other potential signalling pathways associated with HbF induction.

Stroke With Transfusions Changing to Hydroxyurea (SWiTCH): a phase III randomized clinical trial for treatment of children with sickle cell anemia, stroke, and iron overload.

BACKGROUND: Stroke occurs in 5-10% of children with sickle cell anemia (SCA) and has a high (>50%) risk of recurrence without therapy. Chronic monthly erythrocyte transfusions effectively prevent recurrent stroke, but their long-term use is limited by serious side effects, including iron overload. An alternative to transfusion for secondary stroke prevention in SCA is needed, especially one that also improves the management of iron overload. METHODS: Stroke With Transfusions Changing to Hydroxyurea (SWiTCH) is an NHLBI-sponsored Phase III multicenter randomized controlled clinical trial for children with SCA, stroke, and iron overload (NCT00122980). The primary goal of SWiTCH is to compare 30 months of alternative therapy (hydroxyurea and phlebotomy) with standard therapy (transfusions and chelation) for the prevention of secondary stroke and reduction of transfusional iron overload. DISCUSSION: SWiTCH has several distinctive study features including novel methodological and design components: (1) composite primary endpoint including both stroke recurrence rate and iron burden; (2) non-inferiority design with an "acceptable" increased stroke risk; (3) transfusion goals based on current academic community practices; (4) special oversight for the enrollment and randomization process; (5) overlap treatment period within the alternative treatment arm; (6) masking of the overall trial Principal Investigator to treatment results; (7) inclusive independent stroke adjudication process for all suspected new neurological events; and (8) periodic therapeutic phlebotomy program to alleviate iron overload. CONCLUSION: Investigation of alternative treatments in SWiTCH could lead to changes in the management of cerebrovascular disease for selected patients with SCA, stroke, and iron overload.

Pharmacokinetics, pharmacodynamics, and pharmacogenetics of hydroxyurea treatment for children with sickle cell anemia.

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.

Glomerular hyperfiltration and albuminuria in children with sickle cell anemia.

Early manifestations of sickle nephropathy include glomerular hyperfiltration and proteinuria, typically microalbuminuria. Over time, a subset of patients develops histologic changes, decreased glomerular filtration, and ultimately renal failure. This study was designed to determine the rate of glomerular hyperfiltration and prevalence of albuminuria in a cross-sectional analysis of untreated children with sickle cell anemia (SCA), and to identify correlates of both complications. Measured glomerular filtration rate (GFR) by plasma clearance of 99-technetium diethylenetriaminepentaacetate was compared to GFR estimates calculated from published formulas. Eighty-five children (mean age 9.4 ± 4.8 years) were studied; 76% had glomerular hyperfiltration with mean GFR = 154 ± 37 ml/min/1.73 m(2). GFR declined in teenage years and was significantly correlated with increased serum cystatin C levels and higher systolic blood pressure. Measured GFR had only modest correlations with GFR estimates (Pearson correlation coefficients ≤0.5). Albuminuria, usually microalbuminuria, occurred in 15.9% and was associated with higher diastolic blood pressure and lower white blood cell and absolute neutrophil counts. Cystatin C levels inversely reflect GFR changes and are associated with albuminuria; serial monitoring may provide a sensitive and accurate marker of nephropathy in children with SCA.

Genetic predictors for stroke in children with sickle cell anemia.

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 ß-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.

IgA-mediated autoimmune hemolytic anemia in an infant.

Autoimmune hemolytic anemia (AIHA) is characterized by the presence of autoantibodies, most frequently of the IgG isotype, directed against erythrocyte surface antigens. The direct antiglobulin test (DAT) is the critical laboratory test for the diagnosis of AIHA, but is negative in 3-11% of cases. In these cases of DAT negative AIHA, a wider spectrum of clinical data including more specialized testing for erythrocyte autoantibodies may be required. We describe the unique and challenging case of an infant with corticosteroid-responsive, DAT negative AIHA, in which specialized gel card testing identified an isolated IgA autoantibody on the erythrocyte surface.

The diagnostic dilemma of congenital unstable hemoglobinopathies.

Unstable hemoglobin variants represent a rare etiology of congenital hemolytic anemia. Without a high index of suspicion, plus proper laboratory testing and interpretation, the correct diagnosis can be elusive. We report on five children who were initially thought to have other congenital disorders such as hereditary spherocytosis or thalassemia, before ß-globin gene sequencing led to the definitive diagnosis. Recognizing the variable clinical presentation and laboratory data reported will aid clinicians in diagnosis of unstable hemoglobins variants in children with atypical forms of hemolytic anemia, particularly those with low pulse oximetry values or whose hemoglobin electrophoresis suggest ß-thalassemia trait.

Simultaneous acute splenic sequestration and transient aplastic crisis in children with sickle cell disease.

Acute splenic sequestration crisis (ASSC) is a hematological emergency in young children with sickle cell disease (SCD), characterized by worsening anemia and splenomegaly, usually with reticulocytosis and thrombocytopenia. Transient aplastic crisis (TAC) due to parvovirus B19 infection occurs in older children with SCD, and typically manifests as worsening anemia with reticulocytopenia and no splenomegaly. Five older children with SCD (4 HbSC, 1 HbSS on hydroxyurea) developed ASSC concurrent with TAC and had a severe clinical course. Our cases suggest that older children with SCD and acute parvovirus infection should be monitored closely for splenomegaly and multi-system dysfunction.

A pilot study of hydroxyurea to prevent chronic organ damage in young children with sickle cell anemia.

BACKGROUND: Hydroxyurea improves laboratory parameters and prevents acute clinical complications of sickle cell anemia (SCA) in children and adults, but its effects on organ function remain incompletely defined. METHODS: To assess the safety and efficacy of hydroxyurea in young children with SCA and to prospectively assess kidney and brain function, 14 young children (mean age 35 months) received hydroxyurea at a mean maximum tolerated dose (MTD) of 28 mg/kg/day. RESULTS: After a mean of 25 months, expected laboratory effects included significant increases in hemoglobin, MCV and %HbF along with significant decreases in reticulocytes, absolute neutrophil count, and bilirubin. There was no significant increase in glomerular filtration rate by DTPA clearance or Schwartz estimate. Mean transcranial Doppler (TCD) velocity changes were -25.6 cm/sec (P < 0.01) and -26.8 cm/sec (P < 0.05) in the right and left MCA vessels, respectively. At study exit, no child had conditional or abnormal TCD values, and none developed brain ischemic lesions or vasculopathy progression by MRI/MRA. Growth and neurocognitive scores were preserved and Impact-on-Family scores improved. CONCLUSIONS: These pilot data indicate hydroxyurea at MTD is well-tolerated by both children and families, and may prevent chronic organ damage in young children with SCA.

Hydroxyurea therapy lowers transcranial Doppler flow velocities in children with sickle cell anemia.

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 (> or = 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.

Prevention of secondary stroke and resolution of transfusional iron overload in children with sickle cell anemia using hydroxyurea and phlebotomy.

OBJECTIVE: Transfusions prevent secondary stroke in children with sickle cell anemia (SCA) but also cause iron overload. Alternatives for stroke prophylaxis with effective therapy to reduce iron burden are needed. STUDY DESIGN: For 35 children with SCA and stroke, transfusions were prospectively discontinued. Hydroxyurea was prescribed for stroke prophylaxis, and phlebotomy removed excess iron. Initial patients discontinued transfusions before hydroxyurea therapy, but later patients overlapped transfusions with hydroxyurea until tolerating full-dose therapy. RESULTS: Children received hydroxyurea for 42 +/- 30 months (range, 3-104 months). Hydroxyurea (26.7 +/- 4.8 mg/kg per day) led to mild neutropenia (3.9 +/- 2.3 x 10(9)/L) with significant increases in hemoglobin concentration, mean corpuscular volume, and fetal hemoglobin. Stroke recurrence rate was 5.7 events per 100 patient-years, but children receiving overlapping hydroxyurea therapy had only 3.6 events per 100 patient-years. For 26 children with >6 months of phlebotomy, 14,311 +/- 12,459 mL blood (315 +/- 214 mL/kg) was removed, with serum ferritin decreasing from a median of 2722 to 298 ng/mL. Among patients completing phlebotomy, liver biopsy documented normal histology and no excess iron deposition. CONCLUSIONS: For children with SCA and stroke, hydroxyurea effectively prevents secondary stroke and serial phlebotomy leads to complete resolution of transfusional iron overload.

Sustained long-term hematologic efficacy of hydroxyurea at maximum tolerated dose in children with sickle cell disease.

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/beta-thalassemia (HbS/ beta-thalassemia [6 HbS/beta0, 1 HbS/beta+]), 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.