bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of T-Cell Lymphoblastic Leukemia Genetic Etiology
Authors: Drury Luke , Brett Benjamin , Sheetz Todd , Dupuy Adam J 
Published: October 2019

Abstract
Abstract 5128

Introduction.
T-cell lymphoblastic leukemia (T-ALL) is inflict nearly 1500 children annually in the US. We recently reported using Sleeping Beauty mutagenesis as a forward genetics approach to model two major subtypes of T-ALL, typical and early T-cell precursor (ETP). The genetic mutations that define these subtypes is incomplete. Moreover, identifying driver from passenger mutations within these tumors is paramount for developing targeted therapeutics to specific genetic lesions.

Method.
T-ALL tumors were generated using a Cre-inducible Sleeping Beauty system to drive mutagenesis at either early (Vav-iCre) or late (CD4-Cre) stage of T-cell development. Synthetic gene standards were spiked into samples at known concentrations to permit quantitation of transposon insertion events within individual tumors. Samples were prepared by ligation mediated PCR and sequenced using an Illumina HiSeq. Using the standard as a barometer, common insertion sites (CIS) were defined as initiating, transition, or progression based on their abundance. We further developed a network analysis pathway to identify CISs within similar signaling axes.

Results.
Spiked standards were reproducibly seen at expected abundances in several replicated tumors. Further, we are able to distinguish insertions found at high abundances in tumors from lower occurring or background events. Together this allowed us to identify the order of mutations in each tumor and characterize mutations as initiators or progression events. Using our network analysis pathway we found Myc and Stat5b signaling axes driving the majority of ETP-ALL and Notch1 signaling driving typical T-ALL.

Conclusions.
Our data are the first to utilize NextGen sequencing techniques to quantitate CIS events to understand the genetic etiology of T-ALL. Coupled with our network analysis software, we are able to more accurately identify driving pathways in individual T-ALL tumors induced by Sleeping Beauty mutagenesis.

Disclosures:
No relevant conflicts of interest to declare.
Platelet Activation and Inhibition in Sickle Cell Disease (PAINS) Study
Authors: Frelinger Andrew L. , Jakubowski Joseph A. , Brooks Julie K. , Zayas Sabrina L. , Berny-Lang Michelle A. , Barnard Marc R. , Heeney Matthew M. , Michelson Alan D. 
Published: October 2019

Abstract
Abstract 5147
Platelet activation/aggregation in sickle cell disease (SCD) may promote tissue ischemia, suggesting antiplatelet therapy may be useful. However, assessing platelet function and the effect of antiplatelet therapy in blood from SCD patients may be confounded by hemolysis with release of ADP. Here we evaluate levels of platelet activation markers in SCD adolescents vs. normal controls and compare, by multiple methods, the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Platelet activation markers in blood from SCD adolescents (n=15) and healthy adults (n=10), and the effect of R-138727 (0. 1 – 10 μM) added in vitro, were evaluated with and without ADP stimulation. Circulating levels of platelet-monocyte and platelet-neutrophil aggregates were significantly higher (p <0. 01) in SCD patients than in healthy controls. R-138727, in a concentration-dependent manner, inhibited platelet function in both SCD patients and healthy subjects as judged by ADP-stimulated light transmission aggregation, VerifyNow P2Y12 assay, multiple electrode aggregometry, and flow cytometric analysis of platelet vasodilator-stimulated phosphoprotein, activated GPIIb-IIIa and P-selectin. The R-138727 IC50s for each assay were not significantly different in SCD vs. healthy subjects. In summary: 1) The high circulating levels of platelet-monocyte and platelet-neutrophil aggregates demonstrate in vivo platelet activation in SCD and may be useful as markers of the in vivo pharmacodynamic efficacy of antiplatelet therapy in SCD. 2) The similar in vitro R-138727 IC50s in SCD and healthy subjects suggest that the prasugrel dose-dependence for platelet inhibition in SCD patients will be similar to that previously observed in healthy subjects.

Disclosures:
Frelinger: Eli Lilly: Consultancy, Research Funding; Daiichi Sankyo: Research Funding; GLSynthesis: Research Funding. Jakubowski:Eli Lilly: Employment. Heeney:Novartis: Consultancy, Research Funding; Eli Lilly and Company: Research Funding; Pfizer: Consultancy. Michelson:Eli Lilly: Data monitoring committee and idependent external monitor of clinical trials, Research Funding; Takeda: Research Funding; Oxygen Biotherapeutics: Research Funding; Alexion: Research Funding; Omthera: Research Funding.
Hereditary Hemolytic Anemia in Korea From 1997 to 2011: A Study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology
Authors: Park Eun Sil , Jung Hye Lim , Cho Hee Soon , Hah Jeong-Ok , Park Sung Sup , Koo Hong Hoe , Kim Moon Kyu , Kim Yeo-Kyeoung , Kim Young Dae , Kim Hee Jin , Kim Heung Sik , Kim Soon Ki , Seo Jong Jin , Seong Moon-Woo , Song Sang Hoon , Shin Hee Young , Ahn Hyo Seop , Yoo Eun Sun , Lyu Chuhl Joo , Yoon Hoi Soo , Lee Kun Soo , Lee Mee Jeong , Lim Young Tak , Im Ho Joon , Jeong Dae Chul , Choi Eun Jin , Choi Hyoung Soo , Han Dong kyun , Chueh Hee Won 
Published: October 2019

Abstract
Abstract 5157

Background:
With the development of diagnostic technique, an accurate diagnosis of hereditary hemolytic anemia (HHA)- red blood cell (RBC) membranopathy, hemoglobinopathy, RBC enzymopahty – have been made. Therefore, we surveyed the prevalence and characteristics of patients diagnosed as HHA during recent five years in Korea.

Methods:
Through the use of questionnaires, information on the clinical and laboratory findings of HHA diagnosed from 2007 to 2011 in Korea was collected. The globin gene analysis (direct sequencing) and RBC enzyme analysis was performed at the representative laboratories. A total of 203 cases were collected in this study by the Korean Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology.

Results:
Patients number of RBC membranopathy, hemoglobinopathy, and RBC enzymopahty was 125, 47, and 31, respectively. Percentage of patients with dominant family history was 57% in patients with hereditary spherocytosis (n=116) and dominant symptoms were anemia, jaundice, splenomegaly and gallstones. Osmotic fragility test and flow cytometric method for detection of RBC membrane defect were performed about 60% of patients. RBC membrane protein analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on 59 patients. Of the 47 cases of hemoglobinopathies, 36 cases (77%) were β-thalassemia minor, 10 cases (21%) were α-thalassemia minor and one case (2%) was unstable Hb, Hb M-Saskatoon (beta 64 His-→Tyr). Median age at diagnosis was 7 years (range: 6 months–58 years). Eleven of 47 cases (23%) had family history of HHA. As all thalassemia patients were thalassemia minor, they presented with mild jaundice or pallor. Of the 31 patients were diagnosed as RBC membranopathy, pyruvate kinase deficiency was 3 cases and glucose-6 phosphate dehydrogenase deficiency was 2, and other various forms were reported.

Conclusions:
We could confirm that accurate diagnosis has been made in more patients using elegant diagnostic technique. However, more defined diagnostic approaches were needed in this rare disease and further systematic supporting systems for patients and their families were warranted in public health aspect.

Disclosures:
No relevant conflicts of interest to declare.
The Ischemic Disease: A Joke Where There's Nothing to Be Gained
Authors: Corinaldesi Giorgio 
Published: October 2019

Abstract
Abstract 5198
This study explored the hypothesis that the ischemic disease (ID) is associated with systemic alterations of the blood coagulation system: platelet activation (sCD40L: determined by ELISA) and release of various proteins (CRP: determined by nephelometry, IL-6: determined by ELISA).
Plasma concentration of these factors were higher in patients with ischemic disease: sCD40L (ID: 724. 2 +/− 58. 4 vs controls 212 +/− 8. 6 pg/ml; p<0001), CRP (ID: 5. 76+/−1. 8 vs controls 2. 54 +/− 0. 3g/l; p<001), IL-6 (ID: 6. 36 +/− 1. 32 vs controls 2. 02 +/− 064 pg/ml; p<001).
Platelet activation predicts the risk of ischemic events, in fact CD40L binds and activates alfa IIb/beta 3, and increases PMC (platelet – monocyte complexes), and PDMP (platelet derived microparticle), it also promotes the release of several chemokines (FP4, RANTES, CxCL, ENA 78, NAP-2) that may play an important role in plaque destabilization; high levels of CD40L predict cardiovascular risk of death or myocardial infarction, or stroke within 8 to 12 month in patients with asyntomatic low grade stenosis (carotid or coronary).
Several data suggest that CRP has a prognostic value in patients with negative troponin and it is predictive for the development of CAD and refractory ischemic disease, plaque destabilization, and future adverse events, or for the development of severe disease.
IL-6 was strongly associated with markers of inflammation (CRP, fibrinogen, white cells count), plasma viscosity, elevated markers of coagulation (fibrin, D-Dimer, FVIII, FIX); moreover IL-6 is associated with insulin resistance, and its levels are high in metabolic syndrome; it is significantly correlated with age, BMI, white cell count; IL-6 increases dendritic platelet forms and acts synergistically with thrombopoietin and stem-cell factor in stimulating megakaryocytopoiesis.
Although it has been controversial, IL-6, PCR, and CD40L appear now to be very promising for quantifying the degree of ischemic disease and to monitor or to identify the efficacy and the response to the therapy.

Disclosures:
No relevant conflicts of interest to declare.
The G-CSF Produces Long-Term Changes in Gene and Mirnas Expression Profiles in CD34+ From Healthy Donors
Authors: Báez Alicia , Prats-Martín Concepción , Álvarez-Laderas Isabel , Garcia Estefania , Sanchez-Abarca Luis Ignacio , Martin-Antonio Beatriz , Urbano-Ispizua Alvaro , Simón Jose Antonio Pérez 
Published: October 2019

Abstract
Abstract 588

Introduction:
The G-CSF (granulocyte colony-stimulating factor) is the cytokine most commonly used for the mobilization of hematopoietic progenitor cells (HPCs) from healthy donors used in allogenic transplantation (allo-HSCT). Although the administration of G-CSF is considered safe, the knowledge about its long-term effects, especially in HPCs, is limited. The aim of this study was to analyze whether G-CSF induces changes in gene and miRNAs expression profiles in HPCs from healthy donors, and determine whether or not these changes persist at the long term.

Materials and Method:
CD34 + cells were isolated by immunomagnetic separation and sorting from 5 healthy donors before mobilization with G-CSF and at afterwards at 5, 30 and 365 days after mobilization. A pool of samples from PB not mobilized was used as reference group. We analyzed the expression of 375 miRNAs using TaqMan MicroRNA Arrays Human v2.0 (Applied Biosystems), and the gene expression profile using Whole Human Genome Oligo microarray kit 4×44K (Agilent). The expression levels of genes and miRNAs were obtained by the 2-ΔΔCTmethod. From expression data hierarchical clustering was performed using the Euclidean distance. To identify genes and miRNAs differentially expressed due to the effect of G-CSF at different time-points a cut-off value of level expression 2,5 above or below the control values was used and non-parametric Mann-Whitney test was applied. All analysis were performed using the Multi-experiment Viewer 4.7.1. The function of the miRNAs and genes of interest was determined from the various databases available online (TAM database, Gene Ontology, TargetScan Human).

Results:
Seven miRNAs were differentially expressed in HPCs at 5, 30 and 365 days after mobilization with G-CSF, as compared to HPCs obtained from not mobilized PB. In addition, 67 genes were also differentially expressed after administration of G-CSF, whose expression profiles remained abnormal 1 year after mobilization.
These genes are involved in biological processes such as hematopoietic cell proliferation, ribosomal protein synthesis, cell metabolism and transmembrane transportation. Interestingly, 8 of these genes are target of the miRNAs also identified in the current study, which suggests that their expression might be regulated by these miRNAs.

Conclusion:
The G-CSF modifies gene expression profiles and miRNAs in HPCs from healthy donors. Remarkably, these changes were observed from early time-points and persisted at least 1 year after exposure to the drug. This effect of G-CSF on HPCs have not been previously reported and might be clinically relevant.

Disclosures:
No relevant conflicts of interest to declare.
Experience with 2 Dose Schedules of Inotuzumab Ozogamicin, Single Dose, and Weekly, in Refractory-Relapsed Acute Lymphocytic Leukemia (ALL)
Authors: O'Brien Susan , Thomas Deborah A. , Jorgensen Jeffrey L. , Kebriaei Partow , Ravandi Farhad , Kwari Monica , Faderl Stefan , Cortes Jorge E. , Jabbour Elias J. , York Sergernne , Garris Rebecca , Kantarjian Hagop M. 
Published: October 2019

Abstract
Abstract 671

Background:
CD22 expression occurs in more than 90% of patients with ALL. Inotuzumab ozogamicin, a CD22 monoclonal antibody bound to calicheamicin, is active in ALL.

Study aims:
Evaluate the efficacy and toxicity profiles of two schedules of inotuzumab, single-dose and weekly, in refractory-relapsed ALL.

Patients and methods:
Patients with refractory-relapsed ALL were eligible. Pediatric patients were allowed on therapy after 10 adults were treated. Eligibility criteria were standard including normal organ functions (renal, hepatic). The original single dose schedule gave inotuzumab at 1.3–1.8 mg/m2 IVq 3–4 weeks. After the initial 49 patients were treated, the schedule was modified to inotuzumab weekly, 0.8 mg/m2 D1, 0.5 mg/m2 D8 and 15, q 3–4 weeks. The latter schedule was based on higher invitro efficacy with more frequent exposure, and possible lower clinical toxicity.

Results:
83 patients were treated, 49 with single dose, and 34 with weekly dose. 29% were ≥ 60 yrs; 71% were in salvage 2 or beyond; only 8 (10%) were in salvage 1 with CRD1>12 months; 28% had Ph+ or t (4; 11); 10 (12%) had prior allogeneic SCT. Overall 14 patients (17%) achieved CR, 23 (28%) had CRP (no platelet recovery), and 9 (11%) had marrow CR (no recovery of counts), for an overall response rate of 46/83=55%. Response rate was 28/49 (57%) with single dose and 18/34 (53%) with weekly dose. Only 4 patients died in the first 4 weeks of therapy. Among 28 patients with cytogenetic abnormalities who achieved response, 25 (89%) achieved completed cytogenetic response. Among 44 patients who achieved response and had MRD studies by multi-parameter flow cytometry, 28 (64%) became MRD negative. The median survival was 5.4 months, 5.0 months with the single dose and 6.3 months with the weekly dose. Reversible grade 1–2 and 3–4 bilirubin elevations were observed in 24% and 4% respectively on single-dose and in 3% and 0% on weekly dose. Reversible grade 1–2 and 3–4 liver enzyme elevations were observed in 55% and 2% respectively on single-dose and in 21% and 6% respectively on weekly dose. Adverse factors for response included: salvage 2 or later vs. salvage 1 (49% vs. 71%); Ph+ and t(4; 11) abnormalities vs. others (39% vs. 62%). Allogeneic SCT was performed 22/49 patients (49%) on single-dose and in 9/34 (26%) so far (shorter follow-up) on weekly schedule: veno-occlusive disease was observed in 5/22 with single-dose (23%) and in 1/9 with weekly dose (11%).

Conclusions:
Inotuzumab is highly active in refractory relapsed ALL. Weekly dose appears to be equally effective and less toxic than single dose.

Disclosures:
Cortes: Pfizer: Consultancy, Research Funding. Jabbour:Pfizer: Honoraria.
Loss of Wild-Type Jak2 Allele Enhances Myeloid Cell Expansion and Accelerates Myelofibrosis in Jak2V617F Knock-in Mice
Authors: Akada Hajime , Akada Saeko , Yan Dongqing , Hutchison Robert , Mohi Golam 
Published: October 2019

Abstract
Abstract 809
The activating JAK2V617F mutation is the most common mutation found in Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p involving JAK2 has been observed in ∼30% of patients with MPNs particularly in PV and PMF. JAK2V617F homozygosity through 9pLOH has been linked to more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the role of wild-type JAK2 in MPNs induced by JAK2V617F, we have utilized conditional Jak2 knock-out and Jak2V617F knock-in alleles and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like disease in mice, loss of wild-type Jak2 allele in hemizygous or homozygous Jak2V617F mice results in a significantly greater increase in reticulocytes, white blood cells, neutrophils and platelets in the peripheral blood and larger spleen size. We also have found that hemizygous or homozygous Jak2V617F expression significantly increased megakaryocyte-erythroid progenitors in the bone marrow and spleens and marked infiltration of neutrophils in the liver compared with heterozygous Jak2V617F. More importantly, hemizygous or homozygous Jak2V617F mice show accelerated myelofibrosis compared with heterozygous Jak2V617F-expressing mice. Thus, loss of wild type Jak2 allele increases myeloid cell expansion and enhances the severity of the MPN. Together, these results suggest that wild-type Jak2 serves as a negative regulator of MPN induced by Jak2V617F.

Disclosures:
No relevant conflicts of interest to declare.
The Viral Oncoprotein vFLIP Encoded by KSHV/HHV-8 Is Responsible for the Unique Transcriptional Signature and Phenotypic Features of Primary Effusion Lymphoma
Authors: Guasparri Ilaria , Hassane Duane C , Roshal Mikhail , Cesarman Ethel 
Published: October 2019

Abstract
Abstract 900

Background:
Primary effusion lymphomas (PELs) are caused by the Kaposi's sarcoma herpesvirus (KSHV, also called human herpesvirus 8, HHV-8). PELs have a distinctive immunophenotype, lacking expression of most B cell antigens. In addition, PELs have a very distinct gene expression signature when compared to that of other lymphoma subtypes. While KSHV encodes almost 100 proteins, only a handful is expressed in latently infected PEL cells. One of these is vFLIP, which activates NF-kB and inhibits apoptosis and autophagy, and causes tumors in mice. In this study we evaluated the global effect of ectopic expression of vFLIP in a B cell lymphoma cell line, as well as that of suppression of vFLIP expression in a PEL cell line.

Methods:
vFLIP expression was knocked down with siRNA in the PEL cell line BC-3. Namalwa cells were stably transfected with a tetracycline-inducible vFLIP, and vFLIP expression was induced with doxycycline for 48 hours. The Namalwa cell line was selected because it contains EBV with a type I latency pattern, also a feature of most PELs. We evaluated RNA extracted from these vFLIP+ and FLIP– cells in a PEL and Burkitt lymphoma background for gene expression array analysis using the Illumina Direct Hybridization Assay. Results were validated by immunohistochemistry and flow cytometry.

Results:
The vFLIP signature in a BL cell line was analyzed by gene set enrichment analysis (GSEA), which is an analytical method to determine how gene sets from other public studies compare to our signature. 1,445 public gene signatures were compared to a rank ordered list of genes that were altered by expression of vFLIP. The top match among these previous gene signatures, for both vFLIP upregulated and downregulated genes, was the PEL signature as compared to other lymphoma subtypes and normal B cell subsets (Kelin et al, Blood 101, 2003). Thus, expression of vFLIP in BL leads to a switch from a BL expression signature to that of PEL. Furthermore, expression of vFLIP in BL cells let to an almost complete downregulation of several B cell antigens, including surface Ig light chains, CD19, as well as CD10, in conjunction with upregulation of CD138 expression.

Conclusion:
The unique phenotype of PEL has long been an intriguing feature of this disease. We have found that a single gene encoded by KSHV, vFLIP, can induce a transcriptional signature of PEL, as well as the phenotype characteristic of this disease.

Disclosures:
No relevant conflicts of interest to declare.
Pre-mRNA Splicing Mechanisms, Misregulation in Disease, and Therapeutic Strategies
Authors: Krainer Adrian 
Published: October 2019

Abstract
Abstract SCI-13
Most eukaryotic protein-coding genes have one or more introns, and their transcripts can undergo alternative splicing, giving rise to multiple isoforms. Accurate splicing is essential for normal gene expression, and alternative splicing is a key mechanism for expanding the proteome and regulating the expression of diverse protein isoforms. This session will review the general mechanisms of pre-mRNA splicing and the regulation of alternative splicing. In addition, the process of how abnormal splicing arises as a result of intronic or exonic mutations in particular genes, or more globally as a result of splicing-factor misregulation, as well as the contribution of splicing misregulation to cancer, will be described. Lastly the current status of targeted therapeutics development, focusing on antisense approaches to correct abnormal splicing of specific genes or to modulate alternative splicing, will be discussed.

Disclosures:
Krainer: ISIS Pharmaceuticals: Consultancy, Patents & Royalties, Research Funding.