ALBERT

Description: 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 Subjects were withdrawn from the study upon family request or if they continually missed visits.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

Description: The majority of pediatric ITP patients recover spontaneously within 6 months (acute ITP), while about 20% of the patients develop chronic ITP. The ability to distinguish patients at higher risk of developing chronic disease during the acute phase of the illness could give prognostic information and serve as a basis for clinical trials to study earlier or more aggressive interventions to prevent morbidity associated with long-term immunosuppression. We have previously described the gene expression pattern of pediatric ITP patients. This data set was used to search for genes which would predict progression to chronic ITP. We identified 8 samples from patients with acute self-limited ITP and 4 samples from patients who progressed to chronic ITP, all tested during the acute phase of the disease. At a false discovery rate of 0, two genes—VNN1 (vanin1) and AVIL (advillin) were the most significantly different between the groups. To validate these findings from microarray analysis, real-time PCR experiments were performed using unamplified whole blood RNA from the original cohort, with 4 additional patients (for a total of 9 samples in self-limited acute ITP group and 7 samples in progressed to chronic ITP group), and a control group of 5 normal children. All real-time data was normalized to housekeeping gene (GADPH) expression. Both VNN1 and AVIL expression were significantly increased in the group which developed chronic ITP compared to patients with acute self-limited ITP (VNN1 p=0.008, AVIL p=0.006) and normal controls (VNN1 p=0.044, AVIL p=0.009). Table 1 shows the medians and ranges of the normalized real-time PCR results of the three groups. The mechanism of these over-expressed genes in pediatric ITP patients who progress to chronic ITP is currently being investigated. VNN1 is a GPI anchored protein involved in T cell trafficking and has a pro-inflammatory role as an oxidative-stress response gene. AVIL, whose gene product is a member of the gelsolin/villin family of actin regulatory proteins, is involved in monocyte/macrophage phagocytosis and cytoskeletal remodeling. Thus both of these genes have functions consistent with recent reports implicating T cell regulation and trafficking as well as immune-mediated destruction of platelets in chronic ITP. These findings represent the first candidate biomarkers in predicting prognosis in pediatric ITP and at the same time open the door for further investigation of the molecular mechanism underlying the clinical transition from acute to chronic ITP. A prospective study to validate these findings is in progress. Table1: Normalized VNN1 (Vanin1) Normalized AVIL (Advillin) Median Range Median Range Self-limited Acute ITP group 0.461 0.170~0.813 8.535 0.953~17.395 Progress to chronic ITP group 1.827 0.326~4.723 25.601 6.031~44.296 Normal control group 0.350 0.202~1.029 5.491 1.449~8.585

Description: Abstract 2077 Introduction: Pulmonary arterial hypertension (PAH) has been accepted to be a clinical entity associated with hemoglobinopthies, mostly reported in sickle disease and thalassemia intermedia (TI). Anecdotal cases of PAH in other chronic hemolytic disorders have been reported; many of which have been associated with a prior splenectomy. The pathogenesis of PAH in the non sickle-cell RBC hemolytic disorders has not been well defined and the large variability in its frequency and severity are not clear. We aimed to characterize biomarkers in thalassemia and in other RBC disorders and evaluate the specific role of splenectomy (spleen.) in these diseases. Patients and Method: A total of 106 patients were analyzed: Forty two were regularly transfused thalassemia major (TM), 34 had TI (16 with Hb E/beta thalassemia, 11 with beta thalassemia, and 7 with hemoglobin H-Constant Spring (Hb H-CS); 18 with hereditary spherocytosis (HS) or pyruvate kinase (PK) deficiency, 2 with an unstable Hb (Hb Koln), and 10 with a history of splenectomy for non-RBC related reasons (trauma or chronic ITP). PAH was determined by using a Doppler echocardiogram showing a tricuspid regurgitation jet velocity (TRV) of 〉2.5m/s. Markers of platelet activation (P- selectin, soluble CD40 (sCD40L); abnormal exposure of Phosphatidyl Serine (PS) on RBCs, thrombin generation (thrombin-anti-thrombin, TAT); increased ventricular pressure strain (brain natriuretic peptide (BNP); plasma free Hb, LDH and NOx, and endothelin-1, soluble vascular cell adhesion molecule (sVCAM) were analyzed. Result: Nineteen (56%) of the TI patients (15 with beta-TI and 4 with Hb H-CS) had abnormally elevated TRV (Mean 3.0±0.5). The 2 patients with Hb Koln had increased TRV. There were no PAH cases detected among patients with membrane or RBC enzyme deficiencies, neither was there an increased TRV in patients who had been splenectomized without a RBC disorder. There were also no cases of increased TRV in the transfused TM cohort. Patients with TI, in addition to a prior splenectomy and increased hemolysis had increased platelet activation, abnormal RBC PS exposure and anemia. TI patients also had an increase in ET-1 levels, a potent vasoconstrictor. The 2 patients with Hb Koln had increased thrombin generation in addition to hemolysis and absence of spleen. Conclusions: PAH was not detected in splenectomized patients with only a mild chronic hemolytic disorder or in those without a RBC disorder. In addition to the absence of spleen, a higher rate of hemolysis, resulting in NO scavenging and increase in ET-1 and the involvement of platelet activation or thrombin generation are likely required for the development of dysregulation of the pulmonary vascular function. PAH is rare in TI patients which have not undergone splenectomy; absence of spleen likely enhances the effects of thrombocytosis and platelet activation. Our data indicates that PAH is common in patients with Hb H-CS, mostly splenectomized, and should be screened for. In addition our data shows that blood transfusions reduce the elements of anemia, hemolysis and coagulation activation and prevent the development of PAH in splenectomized and non-splenectomized patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3681 Background: Pediatric Immune Thrombocytopenia (ITP) has an incidence of 4–6/100,000 with 1/3 of cases becoming chronic. Treatment choice is arbitrary, because few studies are powered to identify predictors of therapy response. Increasingly, rituximab is becoming a treatment of choice in those refractory to other therapies (Neunert CE, et al. Pediatr Blood Cancer 2008; 51(4):513). Previous studies in ITP have not examined predictors of response to rituximab or whether response to prior treatments predicts response. Objective: To evaluate univariate and multivariable predictors of platelet count response to rituximab. Methods: After local IRB approval, 550 patients with chronic ITP enrolled in the longitudinal, North American Chronic ITP Registry (NACIR) between January 2004 and June 2010. Eligibility included: ages 6 months-18 years at ITP diagnosis, clinical diagnosis of ITP, and ITP duration 〉6 months. Primary ITP was defined as isolated thrombocytopenia without associated conditions. Secondary ITP included those patients with immune thrombocytopenia associated with other immune-mediated medical conditions, including Evans Syndrome. Treatment response was defined as a post-treatment platelet count ≥50,000/uL within 16 weeks of rituximab and within 14 days of steroids. Steroids were prescribed as 1–4 mg/kg prednisone or adult equivalent over 4–14 days with or without taper. The NACIR captured treatment responses both retrospectively prior to enrollment and then prospectively, and both periods were included in this analysis. The multivariable logistic regression modeling process utilized SAS 9.1 using binary variables which were either significant in the univariate analysis or clinically important. A backwards elimination procedure was used to select the final model. Results: Seventy-six (13.8%) patients were treated with rituximab. Demographics of the patients treated with rituximab include: 42% male; 81% Caucasian, 17% Black, and 2% Asian. The mean age at diagnosis of ITP was 8.4 ± SD 5.1 years. The median platelet count at diagnosis of acute ITP was 10,000/uL (IQR 5,000-20,000/uL). 19 (25%) patients had secondary ITP or Evans syndrome. Treatment with rituximab had an overall response rate of 63.2% (48/76). Univariate predictors of response to rituximab are shown in Table I. The strongest univariate predictor of response to rituximab was response to steroids. Gender, ethnicity, and race were not predictive of response to rituximab. Furthermore, other variables which did not predict rituximab response include: history of a bleeding score ≥3 (Buchanan and Adix, J Pediatr 2002; 141: 683), symptoms ≥1 month prior to ITP diagnosis, older age (age 〉5 years), platelets ≥20,000/uL at acute ITP diagnosis, and a positive ANA. In multivariable analysis, response to steroids remained a strong predictor of response to rituximab with an OR 6.2 (95% CI 1.8–21.3, p=0.004). Secondary ITP also remained a strong a predictor of a positive response to rituximab with an OR 5.9 (95% CI 1.2–33.3, p=0.03). Conclusion: In the NACIR, response to steroids and secondary ITP were strong predictors of response to rituximab, a finding not previously reported in children or adults. Although this finding requires further validation, this result may provide evidence that rituximab should be most considered in patients previously responsive to steroids. Disclosures: Off Label Use: Rituximab for chronic ITP. Lambert:Cangene: Membership on an entity's Board of Directors or advisory committees. Klaassen:Novartis: Research Funding; Cangene: Research Funding. Neufeld:Novartis, Inc: Research Funding.

Description: In order to explore possible mechanisms for idiopathic neutropenia, molecular and functional analyses on neutrophils and plasma of pediatric neutropenic patients were performed. 22 subjects, 8 acute and 14 chronic neutropenia, aged 5 months to 12 years, were recruited through Lucile Salter Packard Children’s Hospital hematology clinics with an IRB-approved consent. Patients with known neutrophil elastase mutations were excluded. We first examined whether the neutrophils of neutropenia patients had increased expression of molecules associated with cell death. QT-PCR was performed and transcript levels in neutrophils of neutropenia subjects were compared against those of normal subjects. In addition, to assess if a factor(s) in the plasma of neutropenia patients induced neutrophil cell death, neutrophil survival assays were done by incubating neutrophils isolated from normal healthy controls with 200 ul of undiluted plasma from chronic neutropenic subjects. Neutrophil death was then analyzed via Annexin V-PI staining. Finally, to identify specific plasma proteins, which may have a potential role in neutrophil-mediated cell death, 2D gels were run on neutropenic and control plasma. Results: Due to limited sample size, assays were not run on all subjects. Acute and chronic neutropenic patients (n=10) experienced an increase in FAS, an apoptotic marker and protein known to be associated with neutrophil cell death. There were no significant differences in fold expression between acute and chronic patients. Neutrophil survival assays (n=9) revealed a median of 6.5 and mean 6.8 fold (range 2–24 fold) higher level of plasma-mediated cell death with neutropenic plasma. Preliminary studies of 2D gels on plasma revealed differential expression of inflammatory proteins in neutropenia patients compared to normal healthy controls. Further investigation on these proteins is ongoing. In summary, rapid cell death can be induced in normal neutrophils through neutropenia plasma incubation. Further identification of these apoptosis-associated plasma proteins is in process. In addition, the increase in certain apoptosis-associated chemokines and cytokines could lead to further target identification for diagnostic and/or treatment purposes of neutropenia. Table 1 Patient Number Trascripts Expressed in Neutrophils (Fold Over Trancripts from Neutrophil of Normal, Healthy Control *= acute neutropenic patient TNF-α R1 FAS 1 2 13 2* 1.5 12.5 3 2 10 4 1 14 5* 2 12 6 2 13.5 7* 1 11 8* 2 10.5 9 2 7 10 1.5 10

Description: Background: The indications for treating children with immune thrombocytopenia (ITP) remain controversial. A valid and reliable bleeding assessment tool could assist in objectively quantifying bleeding and influence treatment decisions. Both the ITP Bleeding Score (IBLS) and the ITP-Bleeding Assessment Tool (BAT) have been developed to assess bleeding severity in ITP. The IBLS scores bleeding severity using an 11-item tool with grading from 0 to 2 and the 18-item BAT grades from 0 to 3 or 4. The BAT includes some types of bleeding not represented on the IBLS, such has intramuscular hematomas. To date, no data describe how these two measures compare when measuring bleeding associated with ITP. Objective: To describe and compare bleeding as assessed by both the IBLS and BAT and to correlate bleeding severity with platelet counts in a cohort of children with ITP. Methods: A longitudinal observational cohort of children ages 〉1 and 〈 18 years with ITP, were enrolled from 2013-2015 in the Pediatric ITP Consortium of North America ICON1 trial. All children were enrolled prior to starting a new second line monotherapy (not IVIG, steroids or anti-D). At enrollment, bleeding was assessed using the IBLS in all children. A subset of children also underwent a BAT assessment. Grades of bleeding were described and compared between tools and agreement in grading was assessed. Severity was correlated with platelet count using Spearman's correlation calculation. Results: 118 children were enrolled from 21 ICON centers. 54% had chronic ITP and the median age was 11.4y (range 1.2-17.8). The mean platelet count was 28 x 109/l (SD 57) and 88% had a baseline platelet count of

Description: Accurate and timely diagnosis of inherited bone marrow failure syndromes and inherited myelodysplastic syndromes (iBMFS/iMDS) is essential to guide optimal medical management and treatment. Their early diagnosis allows appropriate clinical monitoring to initiate hematopoietic stem cell transplantation (HSCT) prior to the development of leukemia, which carries a poor prognosis in these patients. Additionally these inherited syndromes typically do not respond to standard medical therapies for aplastic anemia (i.e. ATG and cyclosporine) and are associated with excessive toxicity with standard HSCT conditioning regimens. The recognition of an inherited disorder also informs donor selection as it allows unambiguous identification of affected siblings. Currently, testing of individual genes for iBMFS/iMDS is guided by clinical suspicion. Therefore, diagnosis is easily missed in patients with cryptic presentations. Moreover, sequential genetic testing is often cost-prohibitive and may not be completed within a clinically useful timeframe. To address these clinical challenges, we developed a targeted gene capture pool coupled with next generation sequencing for the exons and flanking intronic sequences of 108 genes implicated in inherited and acquired marrow failure and MDS. Median base coverage was 192X, with 94.5% of targeted bases having 〉50x coverage and 99.6% of targeted bases having 〉10X coverage. This level of coverage together with our bioinformatic pipeline enabled identification of mutations spanning all classes (point mutations, small insertions and deletions, copy number variants, and genomic rearrangements). Rigorous criteria were applied to identify functionally deleterious mutations. Mutations were validated by conventional Sanger sequencing. Both germline and acquired mutations could be discerned, depending on DNA source (fibroblasts, peripheral blood or marrow mononuclear cells). The assay was validated with a blinded analysis of 12 patients harboring known mutations spanning different mutation classes. All mutations were successfully identified, including discerning mutations in genes from those in cognate pseudogenes. We next tested for cryptic presentations of iBMFS/iMDS in 71 patients with idiopathic marrow failure. Fifty-nine subjects were drawn from the pediatric clinic and 12 from the adult clinic. Patients previously diagnosed with a known iBMFS/iMDS were excluded from this analysis. Twenty seven of the 71 subjects (38%) had a family history suggestive of an inherited marrow failure syndrome. Diagnoses of iBMFS/iMDS were made in 7 patients (10%), of whom only 2 patients had a history suggestive of familial marrow failure. An additional 4 patients had possible mutations in iBMFS/iMDS genes (TINF2 and SRP72) with functional validation pending. Deleterious mutations in GATA2 were identified in 4 patients (6%). Four additional patients had deleterious mutations in RUNX1 of which 2 were constitutional and 2 were somatically acquired. The diagnosis had previously been missed by clinical laboratory testing of these genes in 2 patients, possibly due to technical limitations of standard approaches. One additional patient with marrow failure had pathogenic mutations in a DNA repair gene not previously reported to cause iBMFS/iMDS. These data demonstrate the utility of this unbiased approach to diagnose patients irrespective of prior clinical preconceptions. The diagnosis of iBMFS/iMDS in 2 of the 12 adult patients highlights the need to consider inherited syndromes in adults with idiopathic marrow failure. More broadly, our results draw attention to the large number of patients (85-90%) remaining genetically undefined despite comprehensive screening for mutations in known iBMFS/iMDS genes. In summary, we have developed and applied a comprehensive genomic approach to diagnose iBMFS/iMDS in 10% of patients presenting with idiopathic marrow failure. Mutations in GATA2 or RUNX1 were the most common causes of idiopathic marrow failure. Testing should be considered in both pediatric and adult patients even in the absence of prior family history. This unbiased diagnostic approach has revealed previously unexpected phenotypic features of these disorders. This efficient and cost-effective (

Description: Introduction: Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by the accumulation of CD1a+ CD207+ histiocytes. Hemophagocytic lymphohistiocytosis (HLH), a non-malignant histiocytic disorder, is typified by the accumulation and activation of CD8+ T cells and macrophages, which secrete high levels of pro-inflammatory cytokines. The co-existence of LCH and HLH has been reported, albeit rarely, and is believed to be associated with a poorer outcome. To better understand the relationship between these two conditions, in this study we sought to describe the incidence, risk factors for development, and outcome of HLH when it develops in children and young adults with multisystem-LCH (MS-LCH). Methods: We conducted a retrospective study involving 14 centers and collected data on 384 MS-LCH patients aged less than 30 years and who were diagnosed between year 2000 and 2015. Data collected on the eligible patients included clinical information at the time of LCH diagnosis, clinical and laboratory parameters at HLH diagnosis (for those who developed HLH), treatment and disease outcome. Patients who developed HLH were classified as having "true-HLH", which was defined as disease fulfilling 5 of 8 HLH-2004 diagnostic criteria or as "HLH-like" disorder, which was defined as fulfilling 7 days before or after LCH diagnosis. The 3-year cumulative incidence of HLH (true or HLH-like) in MS-LCH was 16.8%. The 5-year overall survival of LCH patients without HLH was 98 ± 9%, while survival for those with an HLH-like disorder or true-HLH was 75 ± 12% and 70 ± 14%, respectively (P

Description: Abstract 3319 Background: An unpredictable subset of patients (∼20–30%) with pediatric immune thrombocytopenia (ITP) progress to chronic ITP; this increases the risk of morbidity and mortality from bleeding, long-term immunomodulation, and/or splenectomy. Furthermore, treatments such as chronic steroid therapy often result in intolerable side effects, raising the need for targeted therapies. We previously tested a novel list of genes that might predict progression to chronic ITP (Zhang et al Blood 2011). Oxidative stress (OS)-related pathways were among those most significantly perturbed in chronic ITP. For further evaluation of the role of OS in ITP, we measured glutathione as a marker of redox capacity and protein carbonyl content as a marker of oxidative cell damage. Methods: Pediatric patients with primary ITP were included, with exclusion of subjects with secondary thrombocytopenia, other autoimmune disorders (ie, lupus), or other chronic illnesses. Healthy pediatric volunteers were recruited as controls. Patients had blood draws within 1 month from ITP diagnosis. Reduced (GSH) to oxidized (GSSG) glutathione ratios were measured from whole blood by tandem mass-spectrometry. Protein carbonyl content (PCC) levels were measured from platelet-rich plasma by enzyme-linked immunosorbent assay (ELISA). Subjects were followed up to 15 months from diagnosis and monitored for disease resolution or progression. Chronic ITP was defined as thrombocytopenia (platelets

Description: Background: Children with ITP who are prescribedsecond line treatments vary in terms of their clinical phenotype, prior treatments, and health related quality of life (HRQoL). Objective: To describe the clinical characteristics and HRQoL of North American pediatric patients with ITP initiating second line treatments. Methods: A longitudinal observational cohort of 118 children with ITP starting second line treatments was enrolled from 2013-2015 at 21 ICON centers. Enrollment requirements included age 1-17y and starting a second line treatment (not IVIG, steroids or anti-D) as monotherapy. Baseline demographic and clinical characteristics were recorded, including response to prior treatments, worst bleeding scores, and baseline platelet counts. Fisher's exact test was used to compare treatment with phase of ITP, age cohort, and gender. HRQoL was measured by patient/caregiver report using the Kids ITP Tool (KIT) where 0 is worst and 100 is best, while physicians assessed the perceived effect of ITP on patient HRQoL using a 5-point scale. Spearman correlations were used to test for association between bleeding, HRQoL, and duration of ITP. ANOVA was used to compare the mean KIT scores of the treatment groups. Results: The clinical characteristics of the cohort are shown in the Table. Median age at enrollment was 11.4 y and 15% had newly diagnosed ITP, 31% had persistent ITP, and 54% had chronic ITP. The median number of prior treatments was 3 (range: 1-9). The prior response rate (platelet 〉30 x 109/µL and no bleeding) to prednisone was 59% and IVIG 66%. Fifty-five (47%) patients had received at least one prior second line treatment. At enrollment, physicians reported that ITP had impacted the patients' HRQoL severely in 15%, significantly in 44%, moderately in 38%, and almost not at all in 3%. The mean score of the child KIT report was 71.4 (SD 17.2), the parent proxy KIT was 64.7 (SD 16.4), and the parent impact KIT was 36.1 (SD 19.2). The physician's assessment of the patient's HRQoL significantly correlated with the child report (p