bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene Modification of Human Primary Natural Killer Cells by Electroporation with mRNA or DNA Coding for An Anti-GD2 Chimeric Antigen Receptor.
Authors: Somanchi Srinivas S. , Denman Cecele J , Amritkar Atharva , Senyukov Vladimir , Olivares Simon , Cooper Laurence J.N , Lee Dean A. 
Published: October 2019

Abstract
Abstract 2467
Poster Board II-444
Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. The main restrictions to developing NK cells for immunotherapy are the limited quantity of cells available for adoptive transfer and their relative resistance to gene transfer by any method. We have developed an efficient method to expand CD3-CD56+ primary NK cells in vitro using K562 artificial APCs expressing membrane-bound IL21. Here we have investigated the potential of these expanded human NK cells to be gene modified through electroporation of DNA and mRNA. Expanded NK cells were electroporated (Amaxa Nucleofector device, program X-01) with DNA or mRNA coding for the GFP reporter gene, and expression of the transgene was evaluated by flow cytometry. Analysis at 48 hours post electroporation revealed that the viability of NK cells electroporated with GFP mRNA was 78% and those electroporated with GFP DNA was 69%. When electroporated with DNA, 32% of the viable NK cells were positive for GFP but had heterogeneous expression level, whereas 98% of viable cells were positive for GFP following mRNA electroporation, with much more homogeneous GFP expression (Figure).
Based on this success we further investigated the potential of expanded NK cells to be gene modified with a Sleeping Beauty transposon/transposase vector system carrying the transgene for a second-generation Chimeric Antigen Receptor (CAR) against the GD2 ganglioside antigen, with signaling via the CD28 and CD3z endodomains. The GD2 antigen is abundantly expressed in neuroblastoma and melanoma and is therefore a relevant target for adoptive immunotherapy. Electroporation of expanded NK cells with the GD2-CAR transposon alone yielded 25% electroporation efficiency, with a viability of 55%. Electroporation of expanded NK cells with the GD2-CAR transposon and the transposase plasmid decreased the transfection efficiency to 14%. Nonetheless, expanded NK cells modified with the GD2-CAR showed improved killing of the target cell CHP-134 using the calcein AM release assay, as compared to unelectroporated expanded NK cells from the same donor. While freshly isolated human NK cells are highly resistant to gene modification, in this study we show that expanded human NK cells can be efficiently electroporated with both DNA and mRNA. NK cells modified with DNA to express CAR gain improved cytotoxic function against target cells, but viability and gene transfer efficiency are low. Since electroporation of GFP mRNA resulted in increased transduction efficiency and viability, we are now evaluating electroporation of expanded NK cells with GD2-CAR mRNA.

Disclosures:
No relevant conflicts of interest to declare.
Membrane Raft Localization Is Critical for Erythropoietin Receptor Signaling.
Authors: Rocha Kathy , Fuhler Gwenny , Johnson Joseph , Clark Justine , Caceres Gisela , Sokol Lubomir , List Alan F. 
Published: October 2019

Abstract
Abstract 2506
Poster Board II-483
Erythropoietin (EPO) is the principal regulator of erythroid progenitor proliferation, differentiation, and survival. Upon ligand engagement, the EPO-receptor (R) homodimerizes to activate the tyrosine kinases, Janus kinase-2 (JAK-2) and Lyn, which in turn phosphorylate the signal transducer and activator of transcription (STAT)-5. Although recent investigations have identified key negative regulators of the EPO-R signal, little is known about the membrane localization and dynamic control of receptor signal fidelity. Here we show a critical role for membrane raft microdomains in the creation of signaling platforms that are essential for signal integrity. Using UT-7 cells, we showed that recombinant human EPO (rhuEPO) stimulation rapidly induced raft formation and aggregation. Confocal microscopy quantitation of the raft ganglioside GM-1 fluorescence showed that raft aggregates increased from a mean of 4.3 ± 1.4 (SE) per cell to 25.6 ± 3.2 aggregates per cell after cytokine stimulation (p≤0.001), accompanied by a greater than 3-fold increase in cluster size (mean stimulated to untreated aggregate size ratio: 3.33 ± 0.11, p≤0.001). Isolation and immunoblot analysis of detergent insoluble raft fractions showed that the EPO-R translocated to lipid rafts after EPO stimulation of UT-7 cells. Confocal microscopy confirmed translocation of the EPO-R into membrane rafts in EPO-stimulated UT-7 cells and normal erythroid bursts. Receptor recruitment into rafts was accompanied by Jak2, Lyn and STAT5 loading into membrane raft fractions upon EPO stimulation. Treatment with methyl-β-cyclodextrin (MBCD) to deplete raft cholesterol and disrupt raft integrity extinguished EPO-induced STAT5 phosphorylation in UT-7 cells and human bone marrow progenitors. Similarly, membrane cholesterol-sequestration by nystatin markedly reduced EPO induced STAT5 phosphorylation in UT-7 cells. MBCD pretreatment of these cells prior to stimulation with phorbol 12-myristate 13-acetate (PMA) did not alter mitogen-activated protein kinase (MAPK) phosphorylation, indicating preservation of non-receptor, non-raft signal integrity despite depletion of cholesterol rich microdomains. These data establish a critical role for membrane raft microdomains in the recruitment and physical assembly of the EPO-R and its signaling intermediates into discrete platforms necessary to optimize signal integrity

Disclosures:
No relevant conflicts of interest to declare.
Chromatin Profiling of the Globin Loci of Human ES Cells and ES-Derived Erythroid Cells.
Authors: Fang Xiangdong , Chang Kai-Hsin , Bates Daniel , Diegel Morgan , Sandstrom Richard , Weaver Molly , Dorschner Michael O. , Sabo Peter J. , Papayannopoulou Thalia , Stamatoyannopoulos George , Stamatoyannopoulos John 
Published: October 2019

Abstract
Abstract 2535
Poster Board II-512
We used a high throughput approach to determine the chromatin profiles of the human β and α globin loci and their upstream and downstream regions in human undifferentiated ES cells, ES cell-derived erythroid cells, human fetal and adult origin erythroid cells and in primary cells and cell lines of endo-meso and ectodermal origins. All DNase I hypersensitive sites of the b-locus were absent in undifferentiated human ES cells except for HS2 of the b-globin locus control region. The chromatin profiles of the β and α globin loci of ES cell-derived erythroid cells were identical to those of fetal liver erythroid cells except that the hypersensitive site of the embryonic globin gene was more prominent. DNase I hypersensitive site 2 of the b-globin LCR, a potent enhancer, was present in all the cell lines and primary lineages we studied, providing direct evidence that it is ubiquitous. Several new erythroid specific DHSs were detected upstream of 5′HS7 of the β-LCR, raising the possibility that they play a role in the regulation of the β globin locus. The region downstream to 3′HS1 was depleted of DHSs except for the previously identified DHS mapping near the breakpoint of HPFH 1. Since DHSs are absent near the breakpoints of deletional HPFHs and db thalassemias and since enhancers are typically DHS positive, our results argue against the hypothesis of imported enhancers in the pathogenesis of deletional HPFH and db thalassemia mutants. All the previously identified erythroid specific DHSs of the α globin locus were absent in human ES cells. The α globin locus of ES cells, however, displayed three very prominent DHSs, which were located almost symmetrically about 40 Kb apart from each other and they were constitutively formed in all the lineages and cell lines we have studied; the 3′ and 5′ DHSs carried CTCF sites by ChIP-Seq assay raising the possibility that they mark the sites of chromatin insulators. Overall these results demonstrate the power of the new high throughput chromatin profiling approaches and their ability to uncover features of chromatin that may be of regulatory relevance.

Disclosures:
No relevant conflicts of interest to declare.
CD41+ Progenitors Are Critical for the Engraftment of Hematopoietic Cells Derived From Embryonic Stem Cells.
Authors: Aggarwal Manish , Bonde Sabrina , Zavazava Nicholas 
Published: October 2019

Abstract
Abstract 2548
Poster Board II-525
Bone marrow cells are by far the most transplanted cells. However, their high immunogenicity requires severe immunosuppressive regimens that can be toxic and lethal to patients. Previous studies from our lab have shown the potential of embryonic stem cells to generate hematopoietic progenitor cells (HPCs), which can reconstitute bone marrow. These cells are uniquely qualified for transplantation due to their poor immunogenicity. Murine embryonic stem cells were differentiated in vitro over 26 days using embryoid body formation and a cocktail of hematopoietic cytokines and growth factors. After differentiation, the HPCs are a mixture of progenitor subpopulations that express different surface markers. The majority of these cells are, however, CD45+. Recent evidence in the literature has pointed to CD41+ cells as an earlier progenitor population in both the bone marrow and HPCs. Here, we hypothesize that CD41 expressing progenitors generated from murine embryonic stem cells engraft more efficiently in immunodeficient mice than the CD45+CD41− cells. This concept was borne out of our observations that CD41 expression peaks at day 14 during the differentiation process decreasing thereafter. In contrast, CD45 peaks at the 26-day mark, when the differentiation process ends. Indeed when these cell populations were transplanted respectively in Rag2−/−γc−/− mice, higher degrees of chimerism were achieved with the CD41+ cells. To study this phenomenon more definitively, Rag2−/−gc−/− mice were transplanted with either the CD41+ or the CD45+ cells. The CD41+ cells achieved a higher level of mixed chimerism, 35.1%±18.1%, compared with only 5.1%±0.1% observed in animals transplanted with CD45+ cells. We further observed that CD41+CD45− cells are a transient cell population that quickly becomes CD45+ in culture. Surprisingly, the CD45+ cells formed many more colony forming units as those detected in the CD41+ cell cultures, challenging the long-held paradigm that CFUs are a true reflection of hematopoiesis potential. Thus, our data here allow the conclusion that transplanting CD41 expressing derived from embryonic stem cells might be preferable to achieving more robust bone marrow reconstitution.

Disclosures:
No relevant conflicts of interest to declare.
Sickle Cell Leg Ulcers Are Associated with Hyperuricemia, Hemolysis, Pulmonary Hypertension and Death.
Authors: Minniti Caterina P. , Hildesheim Mariana , Sachdev Vandana , Allen Darlene , Castro Oswaldo , Taylor James G , Kato Gregory J. 
Published: October 2019

Abstract
Abstract 2583
Poster Board II-560

Background:
Leg ulceration is a common, debilitating complication of sickle cell disease (SCD), affecting 8 to 50% of patients, and recently found to be associated with the hemolytic phenotype. We evaluated the relationship of leg ulceration history with estimated pulmonary artery systolic pressure, hemolytic rate and other clinical characteristics in a cohort of 396 adults with SCD.

Materials and methods:
All SCD patients were enrolled in a NHLBI-approved protocol and were screened for pulmonary hypertension with echocardiography at steady state. We collected a detailed past medical history, as well as a comprehensive set of laboratory tests. Comparisons between patients with and without a history of leg ulcers were made using Wilcoxon rank sum tests to compare medians of continuous variables. Associations between categorical variables and leg ulcer history in two groups were tested using the chi-square test of independence.

Results:
Eighteen % of all subjects had a history of leg ulceration. Patients affected were older, predominantly had homozygous SCD, and had markers of significantly more severe hemolysis, including low hemoglobin and high reticulocyte counts, LDH and AST. They also had a significantly higher prevalence of elevated tricuspid regurgitation velocities (TRV≥2.5 m/sec, 56% vs. 40%, p=0.02; TRV≥3 m/sec, 22% vs. 12%, p=0.006). High serum uric acid and lower serum albumin were significantly associated with a history of leg ulcers. A self-reported history of hepatitis also was associated with leg ulceration. None of the other parameters evaluated were significantly associated with leg ulceration, including history of pain, acute chest syndrome, stroke or priapism. Significantly, patients with a history of leg ulcers were more likely to have died by the time of data analysis (21% vs. 9%, P=0.02).

Discussion:
These data in SCD patients with a history of leg ulcers provide the first demonstration of an association with elevated serum uric acid and confirmation of published associations with elevated pulmonary pressures and markers of hemolytic severity. The uric acid association is more significant than that for serum creatinine or urea nitrogen, suggesting that uric acid is more than simply a marker of renal dysfunction. In patients without SCD, there is a growing literature implicating uric acid as a possible cause of hypertension and a marker of risk for cardiovascular disease, pulmonary hypertension, and early mortality. This is particularly interesting, in view of the epidemiological relationship between leg ulcers and pulmonary hypertension demonstrated here and previously by others. The results continue to support linkage of leg ulcers and pulmonary hypertension to a hyperhemolytic -vasculopathy subphenotype of SCD.

Disclosures:
No relevant conflicts of interest to declare.
Amplification of AML1 Does Not Impact Early Outcome of Children with Acute Lymphoblastic Leukemia (ALL) Treated with Risk-Directed Chemotherapy: A Report From the Children's Oncology Group (COG).
Authors: Heerema Nyla A. , Carroll Andrew J. , Borowitz Michael J. , Devidas Meenakshi , Larson Eric C. , Loh Mignon , Mattano Leonard A. , Maloney Kelly , Raetz Elizabeth , Wood Brent , Winick Naomi J. , Hunger Stephen P , Carroll William L. 
Published: October 2019

Abstract
Abstract 2598
Poster Board II-574

Amplification of a region of chromosome 21 (which includes the AML1 (RUNX1) locus (amp(AML1)) has been reported as a recurring abnormality in pediatric ALL that is associated with increased age (median 9 years), a low white blood cell (WBC) count, and a poor outcome. (Moorman et al Blood 109:2327, 2007; Attarbaschi et al JCO 26:3046, 2008). Early results in these studies indicated approximately a 75% event-free survival (EFS) at 2 years. Amp(AML1) can be reliably detected only by fluorescence in situ hybridization (FISH), and can be recognized when blasts are tested by FISH for the presence of the TEL(ETV6)/RUNX1 translocation. We reviewed the clinical features and outcome of children with ALL and amp(AML1) treated on current generation COG trials from 2003–2009 (median follow up 1.2 years, range: 0.03–3.83 yrs). Diagnosis of amp(AML1) required at least 5 copies of AML1, with 4 copies on a single chromosome. Children with B-precursor ALL received a 3- or 4-drug induction based on NCI risk group, with post-induction therapy based on further risk stratification variables measured during the first month of induction. Patients with a poor early response to therapy (day 15 bone marrow (BM) with >5% blasts or day 29 BM minimal residual disease (MRD) >0.1%) were non-randomly assigned to receive augmented chemotherapy. Treatment was not altered for patients with amp(AML1). Since June 1, 2007, ETV6/RUNX1 FISH was required on all children enrolled on this study. From Dec 29, 2003 to June 1, 2007, FISH was not required on all patients so ascertainment of amp(AML1) may not be complete. There were 89/5470 (1.6%) children with B-precursor ALL in whom amp(AML1) was detected. Similar to previous reports (see Table), their median age was 9.1 years, median WBC was 4.5×109/l; and 55.1% were female, a higher proportion than previously reported (43%–48%). While the distribution of day 29 MRD by flow cytometry was different, NCI risk group distribution, 2-year EFS and overall survival (OS) were similar between patients with and without amp(AML1). Of the 10 events that occurred in patients with amp(AML1), there were 3 induction failures, 3 induction deaths, and 4 relapses. Thus, early response to risk-adapted therapy is similar in children with and without amp(AML1).
We conclude that, using risk-adapted therapy, children with ALL and amp(AML1) have an early outcome similar to those lacking this feature; however, longer follow up is needed to determine the full impact of amp(AML1) on eventual outcome.

Disclosures:
No relevant conflicts of interest to declare.
Elevated Systolic Blood Pressure and Low Fetal Hemoglobin Are Risk Factors for Silent Cerebral Infarcts in Children with Sickle Cell Anemia.
Authors: Sarnaik Sharada A. , Casella James F. , Barton Bruce A , Afif Michele , Airewele Gladstone , Berman Brian W. , Bernaudin Francoise , Coates Thomas , Fuh Beng , Hartung Helge , Heiny Mark E. , Howard Thomas H. , Inusa Baba , Iyer Rathi V. , Kalinyak Karen , King Allison , Kirby-Allen Melanie , Kirkham Fenella J , Kwiatkowski Janet L , Miller Scott T. , Panepinto Julie A. , Quinn Charles T. , Redding-Lallinger Rupa , Rhodes Melissa M. , Sabio Hernan , Saccente Suzanne , Scher Charles , Telfer Paul , Thompson Alexis A , Woods Gerald M , DeBaun Michael R. 
Published: October 2019

Abstract
Abstract 262

Introduction:
The most common cause of neurological injury in sickle cell anemia is silent cerebral infarcts (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) cohort, we sought to identify risk factors associated with SCI.

Patients and Methods:
In this cross-sectional study, we evaluated the clinical history, baseline laboratory values and performed magnetic resonance imaging of the brain. For those children with SCI-like lesions, a pediatric neurologist examined the child and neuroradiology and neurology committees adjudicated the presence of SCI. Children between the ages of 5 and 15 years with hemoglobin SS or S-beta° thalassemia and no history of overt strokes or seizure were evaluated.

Results:
A total of 542 children were evaluated; 173 (31.9%) had SCI. The mean age of the children was 9.3 years, with 280 males (51.7%). In a multivariate logistic analysis, two covariates were significant: a single systolic blood pressure (SBP) obtained during a baseline well-visit, p = 0.015 and hemoglobin F (Hgb F) level obtained after three years of age, p = 0.038. Higher values of SBP and lower values of Hgb F increased the odds of SCI; Figure. Baseline values of white blood cell count, hemoglobin level, oxygen saturation, reticulocytes, pain, or ACS event rates were not associated with SCI.

Conclusion:
SBP and Hgb F level are two previously unidentified risk factors for SCI in children with sickle cell disease. Modulation of SBP and Hgb F levels might decrease the risk of SCI.

Disclosures:
No relevant conflicts of interest to declare.
Clinical Relevance of Ubiquitin-Proteasome System Profiling in Acute Leukemias.
Authors: Ma Wanlong , Kantarjian Hagop M. , Zhang XI , Wang Xiuqiang , Estrov Zeev , O'Brien Susan , Albitar Maher 
Published: October 2019

Abstract
Abstract 2633
Poster Board II-609
The ubiquitin-proteasome system (UPS) plays a major role in the homeostasis of cellular proteins: chymotrypsin-like (Ch-L), caspase-like (Cas-L), and trypsin-like (Tr-L). The UPS system is involved in most critical cellular processes and its role in oncogenesis is well established. Here we compared UPS protein levels (proteasome and ubiquitin) and proteasome enzymatic activities in peripheral blood plasma from newly diagnosed patients with acute myeloid leukemia (AML; n=147), acute lymphoblastic leukemia (ALL; n=34?), or myelodysplastic syndrome (MDS ; n=27), and 99 apparently healthy control subjects. Proteasome and ubiquitin were measured using immunoassays based on electro-chemiluminescence technology. The proteasome enzymatic activities were measure using a standard enzymatic assay utilizing fluorogenic peptide-AMC substrate. By normalizing these enzymatic activities to the levels of proteasome protein in plasma, we also determined specific proteasome enzyme activities (Ch-L/p, Cas-L/p, and Tr-L, respectively). AML, ALL, and MDS patients all had high levels of proteasome protein, ubiquitin, and enzymatic activities relative to control subjects. However, each specific enzyme activity in general was lower in all 3 disease groups than in control subjects, which suggests that each proteasome has lower enzymatic activity in blast cells but we cannot rule out that the number of proteasomes might be increased. Despite these similarities, AML, ALL, and MDS each exhibited a specific profile of UPS protein and enzymatic activities . Proteasome protein levels and enzymatic activities also correlated with clinical behavior. In AML, proteasome protein level was a strong predictor of survival as a both a continuous (P<0.00001) and a dichotomous (P=0.04) variable, independent of cytogenetics, performance status, and age. In ALL, Ch-L/p showed a significant negative correlation with survival (P=0.0015). In conclusion, these data demonstrate that each disease has a unique UPS profile and confirm that the UPS system plays a major role in the leukemic process. In addition, profiling the UPS in leukemias using plasma provides valuable biomarkers that can be used to help predict clinical behavior and may ultimately help manage disease.

Disclosures:
No relevant conflicts of interest to declare.