ALBERT

Description: Background: The Stroke Prevention Trial in Sickle Cell Anemia (STOP) and Optimizing Primary Stroke Prevention in Sickle Cell Anemia (STOP 2) established routine transcranial Doppler ultrasound (TCD) screening with indefinite chronic red cell transfusions (CRCT) for children with abnormal TCD as standard of care. To identify children at high-risk of stroke, annual TCD screening is recommended from ages 2 to 16 years, with more frequent monitoring if the result is not normal. A reduction in stroke incidence in children with SCD has been reported in several clinical series and analyses utilizing large hospital databases when comparing rates before and after the publication of the STOP study in 1998. We sought to determine the rate of first ischemic stroke in a multicenter cohort of children who had previously participated in the STOP and/or STOP 2 trials and to determine whether these strokes were screening or treatment failures. Subjects and Methods: Between 1995 and 2005, STOP and STOP 2 (STOP/2) were conducted at 26 sites in the US and Canada. These studies included 3,835 children, ages 2 to 16 y with SCD type SS or S-beta-0-thalassemia. Participation in STOP/2 ranged from a single screening TCD to randomization. STOP 2 also had an observational arm for children on CRCT for abnormal TCD whose TCD had not reverted to normal. The Post-STOP study was designed to follow-up the outcomes of children who participated in one or both trials. 19 of the 26 original study sites participated in Post-STOP, contributing a total of 3,539 (92%) of the STOP/2 subjects. After exit from STOP/2, these children received TCD screening and treatment according to local practices. Data abstractors visited each clinical site and obtained retrospective data from STOP/2 study exit to 2012-2014 (depending on site) including follow-up TCD and brain imaging results, clinical information, and laboratory results. Two vascular neurologists, blinded to STOP/2 status and prior TCD and neuroimaging results, reviewed source records to confirm all ischemic strokes, defined as a symptomatic cerebral infarction; discordant opinions were resolved through discussion. For the first Post-STOP ischemic stroke, prior TCD result and treatment history subsequently were analyzed. Results: Of the 3,539 subjects, follow-up data were available for 2,850 (81%). Twelve children who had a stroke during STOP or STOP2 were excluded from these analyses resulting in data on 2,838 subjects. The mean age at the start of Post-STOP was 10.5 y and mean duration of follow-up after exiting STOP/2 was 9.1 y. A total of 69 first ischemic strokes occurred in the Post-STOP observation period (incidence 0.27 per 100 pt years). The mean age at time of stroke was 14.4±6.2 (median 13.8, range 3.5-28.9) y. Twenty-five of the 69 patients (36%) had documented abnormal TCD (STOP/2 or Post-STOP) prior to the stroke; 15 (60%) were receiving CRCT and 9 (36%) were not (treatment data not available for 1 subject). Among the 44 subjects without documented abnormal TCD, 29 (66%) had not had TCD re-screen in the Post-STOP period prior to the event; 7 of these 29 (24%) were 16 y or older at the start of Post-STOP, which is beyond the recommended screening age. Four of the 44 (9%) patients had inadequate TCD in Post-STOP (1 to 10.7 y prior to event). Six (14%) had normal TCD more than a year before the event (1.2 - 4 y); all but one of these children were younger than 16 y at the time of that TCD. Only 5 (11%) had a documented normal TCD less than 1 year prior to the event. Conclusions: In the Post-STOP era, the rate of first ischemic stroke was substantially lower than that reported in the Cooperative Study of Sickle Cell Disease, prior to implementation of TCD screening. Many (39%) of the Post-STOP ischemic strokes were associated with a failure to re-screen according to current guidelines, while only 11% occurred in children who had had recent low-risk TCD. Among those known to be at high risk prior to stroke, treatment refusal or inadequate treatment may have contributed. While TCD screening and treatment are effective at reducing ischemic stroke in clinical practice, significant gaps in screening and treatment, even at sites experienced in the STOP protocol, remain to be addressed. Closing these gaps should provide yet further reduction of ischemic stroke in SCD. Disclosures No relevant conflicts of interest to declare.

Description: A novel glass needle–mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix–coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34+, CD34+/CD38−, and CD34+/CD38−/Thy-1lo human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% ± 8.4% of CD34+ cells and 42% ± 14% of CD34+/CD38−cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34+ and CD34+/CD38−cells, and retention of the ability of CD34+/CD38− cells to generate progenitor cells. Delivery of DNA constructs (currently ≤ 19.6 kb) and fluorescently labeled proteins into CD34+ and CD34+/CD38− cells was achieved with transient expression of green fluorescent protein observed in up to 75% of injected cells. These data indicate that glass needle–mediated delivery of macromolecules into primitive hematopoietic cells is a valuable method for studies of stem cell biology and a promising method for human blood stem cell gene therapy.

Description: Background: Pulmonary function is particularly susceptible to acute and chronic injury occurring in patients with sickle cell anemia (SCA). Acute Chest Syndrome (ACS), a common and potentially fatal complication of SCA, can be the cause or the consequence of abnormal pulmonary function (Siddiqui & Ahmed, 2003). Use of hydroxyurea therapy (HU) is increasing for children with recurrent pulmonary complications, following the findings that HU reduces risk of ACS in adults (Charache et al, 1995). How HU may improve pulmonary function in children with SCA is still ill-defined. The purpose of this study was to assess the change observed on serial pulmonary function tests (PFT) for children prescribed HU for abnormal pulmonary function. Methods: Over 240 children with pulmonary complications, such as acute chest syndrome, reactive airway disease, and chronic hypoxia, have been evaluated in the Sickle Cell Pulmonary Clinic at Children’s Healthcare of Atlanta since beginning in July 2000. As part of the Clinic’s standard of care, PFT were routinely attempted on children over the age of 5. A retrospective review of PFT results was done for the children with SCA and prior abnormal PFT. Children were included in the HU cohort (+HU) if repeat PFTs were available following 〉 3 months on HU therapy and if time on HU did not include frequent RBC transfusions. Children without history of HU therapy (−HU) were selected for comparison. PFTs were performed on a standard plethysmograph. Paired t-test was used to evaluate observed differences. Results: Thirty-one children with prior abnormal PFT had test before and following prescription of HU. Their mean age was 12.6 (range 6–20) years and mean duration of HU was 21 (range 4–47) months at the time of the repeat PFT. Hematologic changes expected on HU occurred for all 31 children. Twenty-four children followed for abnormal PFT had no history of HU (−HU) and matched the +HU group by gender, age (mean 12.3, range 7–19 years), duration followed at time of repeat PFT (mean 19, range 3–66 months; p=0.36), and initial hematologic parameters. Spirometry findings changed for +HU group, and remained stable for −HU group. Mean FVC and FEV1 values improved significantly on HU, when compared to initial (PreHU) PFT and to −HU controls. Conversion to a normal PFT, as interpreted by a pediatric pulmonologist masked to treatment status, occurred in 18 (58%) and 2 (8%) children in the +HU and −HU groups, respectively. Table 1: Serial PFT and hematologic parameters according to HU exposure −HU (n=24) +HU (n=31) Initial Repeat # PreHU Repeat 1 % predicted value for age, sex and height of subject. 2Mean (SD) #No significant changes compared to Initial, p 〉 0.05. *P value 〈 0.001 compared to PreHU; ++P value 〈 0.001 compared to −HU repeat. PFT parameter 1 TLC 88 (13.6)2 85 (11.7) 85 (13.4) 90 (13.5) FVC 78 (9.8) 79 (9.9) 75 (12.6) 90 (13.2)*,++ FEV1 75 (9.6) 74 (12.2) 72 (11.4) 86 (10.2)*,++ FEF 25-75 75 (22) 72 (28.0) 77 (23.4) 79 (26.8) FEV1/FVC (%) 85(6.7) 82 (9.8) 87 (11.8) 86 (11.1) Pulse oximetry (%) 95 (3.1) 96 (2.4) 94 (4.4) 97 (2.8)* WBC (x103/ul) 14 (3.8) 13 (2.9) 13.02 (2.9) 9.0 (2.1) *,++ Hb (g/dl) 8.0 (0.1) 8.0 (0.9) 7.8 (1.1) 9.1 (1.3) *,++ MCV (fl) 84 (9.2) 85 (10.0) 87.3 (8.1) 101.2 (10.1) *,++ HbF (%) 4.3 (1.5) 5.2 (5.2) 6.0 (3.8) 14.0 (7.3) *,++ Conclusions: Serial PFTs showed improved pulmonary function, following initiation of HU therapy in children with prior abnormal PFT. Routine PFT assessments of children prescribed HU may serve as an objective measure of clinical response in children with SCA and pulmonary complications. These results will help in the design of future prospective studies examining the clinical benefits of HU therapy for children at risk for long-term pulmonary complications.

Description: Abstract 1923 Background: Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells for transplantation and regenerative medicine applications. UCB collection bags must contain an anticoagulant to prevent coagulation and to maintain viability during transport to the processing facility. Although Citrate Phosphate Dextrose (CPD) has been widely used based on the commercial availability of pre-filled collection bags, heparin is also accepted as an anticoagulant for processing hematopoietic stem cells. Data is lacking on the optimal anticoagulant for UCB collections particularly in the volume range of cord blood collected for family banking purposes. CPD was originally formulated for use in whole blood collections at a dilution ratio of 1:7 CPD to blood. Commercially available UCB bags contain 35 mL of CPD and are intended to allow a maximum collection volume of 210 mL in a 250 mL bag, creating up to a 1:6 dilution. However, typical cord blood collections rarely reach this volume. Given the wide variability in UCB collection volumes and the standard volume of 35 mL CPD pre-loaded in each bag regardless of collection volume, dilution ratios for UCB collections could drop as low as 1:1 CPD to blood. Concentrated exposure to CPD could adversely impact cell viability and blood chemistry. Methods: UCB collections from 15 individual donors were each approximately equally divided (p〉0.05) between a 300 mL collection bag containing 500 units of lyophilized heparin (LH) and a 250 mL bag with 35 mL liquid CPD. Samples were placed on an orbital mixer at room temperature for a simulated shipping time of 48 hours. Aliquots of the samples were assessed for total nucleated cell (TNC) count and cell viability at time of collection, and at 24 and 48 hour time points. Viable TNC were obtained utilizing acridine orange/propidium iodide staining. Total cell counts were obtained using a Sysmex hematology analyzer. The values at each time point were compared to the previous measurement to detect changes over time. Results: Shown in Table 1. Samples collected in CPD showed a statistically significant decrease in TNC yield at 24 hours (p=0.004) and again at 48 hours (p=0.002). There was a significant but lesser decrease in TNC yield in samples collected in LH between hours 0 and 24 (p=0.04) and no significant difference thereafter. There was a significant decrease in TNC viability in CPD samples at both time points. The viability of samples collected in LH vs. CPD was 96.6±3.0% vs. 85.9±9.0% (p

Description: Thrombotic thrombocytopenic purpura (TTP) is a rare disorder associated with microangiopathic haemolytic anaemia, thrombocytopenia and multisystem microvascular thrombosis. It results from a deficiency of ADAMTS 13, required to cleave Ultra large von Willebrand factor multimers. An underlying autoimmune basis is suggested; indeed in up to 90% of idiopathic cases, antibodies, primarily IgG, to ADAMTS 13 can be demonstrated during an acute episode. Joseph et al( Am J Hematology 1994) reported that HLA-DR53 protects against TTP. In this study, twenty patients with idiopathic TTP, 6 male and 14 female, were typed for HLA-DR and DQ by PCR-SSP. Thirteen had recurrent (〉3) episodes of TTP and all patients had ADAMTS 13 activity

Description: Using a new magnetic resonance method that separately estimates the two principal forms of storage iron, ferritin and hemosiderin, in the heart, we examined the relationship between myocardial storage iron fractions and left ventricular function in thalassemia major. In patients with iron overload, the amount of iron in functional and transport pools changes only slightly. Virtually all of the excess is sequestered in storage forms of iron, as ferritin, a diffuse, soluble fraction, and as hemosiderin, an aggregate, insoluble fraction. The two storage forms of iron strongly affect signal intensity in both T2 and T2* weighted images but influence MRI signal decay through different means because of their differences in solubility and in intracellular distribution (Magn Reson Med2002; 47:1131–8). Separate estimates of the iron concentrations of the two forms of storage iron may be obtained by measuring two distinct relaxation parameters, the “ferritin iron index” (“reduced” transverse relaxation rate) RR2, and the “hemosiderin iron index”, A. We studied 14 patients with thalassemia major, all being treated with subcutaneous deferoxamine. Study participants were examined with a Philips 1.5 T Intera scanner using three Carr-Purcell-Meiboom-Gill (CPMG)-like multi-echo spin echo sequences with varied inter-echo times, using electrocardiographic triggering and respiratory navigator gating to estimate RR2 and A. The left ventricular shortening fraction was measured using standard echocardiographic methods. The Figure shows the relationship (R=0.91, p

Description: Abstract 1690 Background – Acute Myeloid Leukaemia (AML) has a worldwide incidence of approximately 3.5 per 100,000 population per year with most cases occurring in adults. Survival at 12 months is less than 30% and at 5 years is less than 10% (NCI, 2010). AML is a complex disease that demonstrates marked heterogeneity morphologically, cytogenetically and molecularly. The largest cytogenetic subgroup of AML has normal cytogenetics and accounts for approximately 45% of de novo AML cases. Furthermore, the response to treatment and survival outcomes of cytogenetically normal AML (cn-AML) is remarkably heterogeneous. Aims – – To undertake whole-genome and exome sequence analysis of a cn-AML case at diagnosis and at relapse to identify subtle, potentially oncogenic mutations at the molecular level that may initiate AML and those that may be responsible for drug resistance or relapse. Specific Aims: i. –Comparison of the AML genomes at diagnosis, remission and relapse to identify genomic regions with changes in copy number and loss of heterozygosity, using single nucleotide polymorphism (SNP) microarray analysis. ii. Identify chromosomal rearrangements such as small insertions, duplications, inversions and deletions, (“indels”) and translocations that may contribute to AML initiation or relapse, using massively parallel, ultra-high throughput (“next generation”), paired-end sequencing of total genomic DNA at low depth. iii. Identify DNA point mutations potentially affecting protein function that may contribute to AML initiation or relapse by using next generation sequencing at high depth of the captured exomes (putative coding regions) of the AML and control genomes. Methods – The PwC (Price-Waterhouse-Coopers) Leukaemia and Lymphoma Tissue Bank, a joint initiative of the Australasian Leukaemia and Lymphoma Group and the Leukaemia Foundation, provided samples from an AML patient with normal cytogenetics and a blast count at diagnosis of 70% and at relapse of 85%. Genomic DNA extracted from autologous mesenchymal stem/stromal cells (MSCs) was used to represent non-leukaemic, germline, control DNA. Primary cell culture of MSCs from cryopreserved bone marrow aspirate cells (50 × 106) of our test patient at remission was achieved with standard tissue culture methods. High molecular weight DNA was extracted from the patient's MSCs and marrow cells at diagnosis, remission and relapse samples. Sonication (Covaris) and libraries appropriate for paired-end high-throughput sequencing (Illumina Genome Analyzer II instrument) were prepared from gel purified DNA fragments approximately 200 bp in size. Results and Discussion – Primary cell culture of MSCs from bone marrow aspirates proved to be a robust source of germline genomic DNA with several advantages over skin. Firstly, tissue banked samples, will not include biopsies of normal skin. Furthermore, skin can be contaminated by circulating leukaemic cells, which is problematic with low-depth genomic sequencing. The homogenous immunophenotype of the cultured MSCs indicate their purity. Preliminary SNP microarray analysis identified a large region of uniparental disomy (copy number neutral loss of heterozygosity) involving most of chromosome 13q, which was not identified by standard cytogenetic analysis. Low-pass or shallow paired-end genomic DNA sequencing has generated the following outputs. MSC genome: 10.7 Gb (3.6X haploid genome coverage) resulting from 152 × 106 paired sequence reads, AML_diagnosis genome: 21.6 Gb (7.2X) resulting from 142 × 106 paired sequence reads, AML_relapse genome: 26.8 Gb (8.9X) resulting from 177 × 106 paired sequence reads. The capture baits representing the human exome span 26,225,870 bp (approximately 0.9% of haploid human genome). Exome capture libraries were prepared from the three sources of DNA (MSC, AML_diagnosis and AML_relapse) and sequenced using a single lane for each library. The outputs for all three were very similar, approximately 23 × 106 paired sequence reads, representing 1.3 Gb (49X haploid exome coverage). The reads for whole genome or exome capture were of high quality and more than 98% could be unambiguously aligned to the human reference genome in the correct orientation and interval distance. Here we will present MSC enrichment results and sequencing output results, quality control and preliminary analysis of genomic alterations and exonic mutations. Disclosures: No relevant conflicts of interest to declare.

Description: Platelet transfusion refractoriness results in adverse outcomes and increased healthcare costs. Managing refractoriness due to HLA alloimmunization necessitates the use of HLA antigen matched platelets, but requires a large platelet donor pool, and does not guarantee full matching. We report the first ever randomized, double-blind, non-inferiority, cross-over trial comparing HLA epitope-matched (HEM) platelets with HLA standard antigen-matched (HSM) platelet transfusions. Eligibility criteria were alloimmunized, platelet refractory, thrombocytopenic patients with aplastic anemia, myelodysplastic syndrome or acute myeloid leukemia. HEM platelets were selected using HLAMatchMaker epitope (specifically eplet) matching. Patients received up to eight prophylactic HEM and HSM transfusions provided in random order. Primary outcome was one-hour post transfusion platelet count increment (PCI). 49 patients were randomized at 14 UK hospitals. For intention-to-treat, number of evaluable transfusions was 107 and 112 for HEM and HSM, respectively. Unadjusted mean (SD) PCI for HEM and HSM was 23.9 (15) and 23.5 (14.1) respectively (adjusted mean difference -0.1, 95% CI -2.9, 2.8). As the lower limit of 95% CI was not greater than pre-defined non-inferiority limit, HEM was declared non-inferior to HSM. There were no differences in secondary outcomes of platelet counts, transfusion requirements and bleeding events. Adequate one-hour PCI was more frequently observed with a mean number of 3.2 of epitope mismatches compared to 5.5 epitope mismatches for inadequate one-hour increments. For every additional one epitope mismatch, the likelihood of an adequate PCI decreased by 15%. Epitope matched platelets should be considered to support HLA alloimmunized patients. Funded by NHS Blood and Transplant, ISRCTN23996532.

Description: Aberrant mRNA processing is known to drive the pathogenesis of chronic lymphocytic leukemia (CLL). Recurrent gene mutations in the RNA splicing factor SF3B1 and widespread RNA intronic polyadenylation impact genome-wide gene expression and inactivate tumor suppressors, respectively. Nevertheless, how mRNA processing is regulated and exerts its function in CLL remain elusive. To comprehensively characterize the role of mRNA processing in CLL, we performed RNA sequencing (RNA-seq) and Tandem Mass Tag (TMT) proteomics using normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=22). We detected 328 proteins differentially expressed between normal and CLL B cells (|Log2FC|〉0.58, q

Description: The nuclear receptors (NR) retinoid X receptors (RXRs) exert immunomodulatory functions to control inflammation and metabolism via homodimers and heterodimers with several other NRs including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers but not heterodimers. Here, we show that in vivo IRX4204 was compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T cell proliferation, reducing T helper 1 differentiation and promoting regulatory T cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating pro-inflammatory pathways. In vitro, inducible Treg differentiation from naïve CD4+ T cells was enhanced by IRX4204; in vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrate that IRX4204 supported Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be used as a novel approach to prevent acute GVHD in the clinic.