bluuebunny/crossref_metadata_2025_split huggingface dataset:
Soluble Monomeric Von Willebrand Factor A1A2A3 (R1450E) Mutant Fragment Enhances Shear-Induced Platelet Aggregation and Platelet Adhesion to Fibrin(ogen) Via αIIbβ3 Under High Shear Stress.
Authors: Cruz Miguel A. , Sowa Katie E. , Smith Scott M. 
Published: October 2019

Abstract
Abstract 4025
Poster Board III-961
Recently, we described that the gain of function mutation R1450E in the A1 domain of von Willebrand factor (VWF) eliminates the formation of catch bond with glycoprotein (GP)Ibα, prolonging the bond lifetimes at low forces. Because those studies were performed with the mutant immobilized on a plastic surface, we further characterize the effect of this mutant on platelet function in solution and under shear stress. Both wild type (WT) and mutant A1A2A3 proteins were expressed in HEK293 cells and purified to homogeneity. The monomeric state of A1A2A3 proteins were assessed by gel filtration chromatography and neither of the proteins had formed dimers or any higher order aggregates. The recombinant A1A2A3 mutant bound spontaneously to GPIbα without the modulator ristocetin with a half-maximal binding observed at 65 ± 8 nM. This apparent dissociation constant was comparable to that of WT (50 ± 10 nM) in the presence of ristocetin. The mutant failed to induce spontaneous platelet aggregation under stirring conditions, and blocked 100% ristocetin-induced platelet agglutination (RIPA) at concentration of 250 nM. At the same concentration, the mutant increased shear-induced platelet aggregation (SIPA) at 500s-1 and 5000s-1 shear rates, reaching 42% and 66%, respectively, while SIPA did not exceed 18% in the presence of WT. The anti-αIIbβ3 antibody 7E3 blocked the effect of the mutant on SIPA. Blood was then incubated with the mutant (250 nM) and perfused over a surface coated with fibrin(ogen) at different shear rates. Blood containing WT resulted in <10% surface coverage by platelets after 1.5 minutes while platelets from blood containing the mutant rapidly bound covering 100% of the fibrin(ogen) surface area at 1500s-1. At shear rate of 2500s-1, surface coverage was 20% for the mutant and 0% for WT fragment. EDTA and antibodies 6D1 (GPIbα) and 10E5 (αIIbβ3) effectively blocked mutant-mediated platelet adhesion and thrombus formation under high shear rates. The addition of ristocetin (0.5 mg/ml) to whole blood prior perfusion reproduced the effect of the mutant. Here, we describe an A1A2A3 mutant that bound spontaneously to GPIbα but affected differently RIPA and SIPA. These results suggest that hydrodynamic forces directly act on the GPIbα-mutant A1A2A3 complex, regulating signaling. In addition, platelet activation induced by the binding of soluble mutant A1A2A3 or plasma VWF results in αIIbβ3-mediated platelet adhesion to fibrin(ogen) under high shear rates.

Disclosures:
No relevant conflicts of interest to declare.
Transfection of Human Platelets Down-Regulates Endogenous mRNA.
Authors: Kondkar Altaf A , Hong Wei , Bergmeier Wolfgang , Herman Jay H. , Jin Ying , Nagalla Srikanth , Bray Paul F. 
Published: October 2019

Abstract
Abstract 4026
Poster Board III-962
Platelets are anucleate and, as such, lack the capacity for transcribing and expressing exogenously introduced genes or cDNAs. However, platelets contain megakaryocyte-transcribed mRNAs and respond to physiological stimuli by translating mRNA into protein. Compared to the megakaryocyte, the relative contribution of platelet translation to the platelet proteome is unknown. However, it is noteworthy that platelets in Blood Bank storage conditions increase their levels of integrin β3 over time and that the half-life of some mRNA is longer in platelets than in nucleated cells (Thon et al, Transfusion 2009). Considering the potential clinical and research utility of manipulating platelet gene expression, we tested the feasibility of transfecting platelets in-vitro with a fluorescently (FAM)-labeled siRNA for GAPDH as assessed by flow cytometry. We tested both electroporation and Lipofectamine with both washed platelets and platelet-rich plasma (PRP) using a variety of conditions. Our studies demonstrated optimal transfection efficiency at 24 hours using 2 × 108/mL washed platelets, 6 μL Lipofectamine and 400 pmoles siRNA. These initial studies demonstrated that 8.4% of platelets were transfected with the FAM-labeled siRNA compared to control platelets without Lipofectamine. Transfected platelets were then isolated by cell sorting. Changes in GAPDH mRNA levels were determined by TaqMan gene expression assays using18S RNA as an endogenous control. Two independent experiments demonstrated a 40% reduction in GAPDH mRNA in platelets transfected with GAPDH siRNA as compared to a FAM-labeled scrambled siRNA. These results offer proof-of-concept that nucleic acids can be introduced into platelets by non-viral methods and that these nucleic acids can modify gene expression. Future studies to increase platelet transfection efficiency may be valuable for manipulating platelet qualities in stored platelet products or to use platelets as vehicles for delivering products to the sites of vascular damage.

Disclosures:
No relevant conflicts of interest to declare.
Role of KLF4 and KLF12 in γ-Globin Gene Regulation.
Authors: Kalra Inderdeep S , Li Wei , Muralidhar Shalini , Pace Betty 
Published: October 2019

Abstract
Abstract 4075
Poster Board III-1010
Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in gene promoters. The CACCC element is critical for the developmental regulation of the human γ-globin and β-globin genes and studies are being done to ferret out various factors that bind this region and modulate gene activity. We recently identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased based on microarray-based gene profiling, concomitantly with decreased γ-globin expression during erythroid maturation. Decreased expression of both factors was further confirmed using quantitative PCR (qPCR) analysis. KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively by day 28 compared to levels in day 7 erythroid progenitors. We next determined if KLF4 and KLF12 bind the γ-globin CACC box by electrophoretic mobility shift assay (EMSA) using nuclear proteins extracted from K562 cells and a [γ-32P] labeled γ-CACC probe located between -155 to -132 relative to the γ-globin gene cap site. Three DNA-protein complexes were observed. The specificity of these interactions was confirmed by competition reactions in which preincubation with excess unlabelled γ-CACC oligonucleotide effectively abolished the formation of all DNA/protein complexes; addition of nonspecific oligonucleotide had no effect on binding activity. Addition of polyclonal KLF4 or KLF12 antibodies to the EMSA reaction resulted in a marked decrease in intensity of all DNA-protein complexes suggesting both KLF4 and KLF12 are present. Additional studies were performed to determine the effect of the known fetal hemoglobin inducer hemin on KLF gene expression in K562 cells. Hemin stimulated γ-globin transcription while increasing KLF4 and KLF12 66-fold and 4-fold respectively (p<0.05). Hemin treatment in KU812 erythroleukemia cells which actively transcribe both γ- and β-globin, also produced a 10-fold increase (p<0.05) in KLF4; KLF12 levels were not changed. Our preliminary data suggest these KLFs might play a role in γ-globin regulation. siRNA mediated gene silencing studies are underway to determine if KLF4 and/or KLF12 play a direct role in γ-globin gene regulation. This mechanism could provide important molecular targets for fetal hemoglobin reactivation. This will be highly significant towards developing therapeutic strategies for hemoglobinopathies like sickle cell anemia and β-thalassemia.

Disclosures:
No relevant conflicts of interest to declare.
Outcomes of CAG Regimen for Refractory Biphenotypic Acute Leukemia Patients.
Authors: He Guangsheng , Zhang Xiang , Depei Wu , Sun Aining , Jin Zhengming , Qiu Huiying , Miao Miao , Tang Xiaowen , Fu Chengcheng , Han Yue 
Published: October 2019

Abstract
Abstract 4111

Objective
Biphenotypic acute leukemia (BAL) involves both myeloid and lymphoid cells, and there is lack of uniformity in treatment at present. Optimal approach for therapy of BAL is unknown, and BAL usually becomes refractory to conventional chemotherapy. It was found that CAG regimen [low-dose cytosine arabinoside (Ara-C) plus aclarubicin with concurrent administration of granulocyte colony-stimulating factor (G-CSF)] is effective for both myeloid leukemia and refractory acute lymphoblastic leukemia. To explore the efficiency of CAG regimen for refractory BAL, 12 BAL patients who were failed to daunorubicin/mitoxantrone, cytosine arabinoside, vincristine, prednisone (D/MAOP) regimen, were treated by CAG. 8 were males and 4 was female, with ages ranging from 20 to 43 years (median=34 years). Immunophenotype of B lymphoid lineage/myeloid lineage showed in 8 patients, and T lymphoid lineage/myeloid lineage showed in 4 patients. The patients whose blast cells did not decrease 50% after induction regimen with lymphoid and/or myeloid drugs were defined as refractory cases.

Methods
12 refractory BAL patients were treated by CAG regimen (cytosine arabinoside 10 mg/m2 subcutaneously every 12 hours, day 1-14; aclarubicin 5-7 mg/m2 intravenously daily, day 1-8; and concurrent use of granulocyte colony-stimulating factor 200μg·m-2·d-1 subcutaneously) from November 2002 to April 2009. The efficacy of the regimen was evaluated by response rate, and the side-effects was also measured.

Results
The major chemotherapy toxicity was bone marrow depression, mainly showing as pancytopenia. Median duration of absolute neutrophil count (ANC)<0.50×109/L and of platelet (PLT) count<20×109/L was 13 (range, 1-17) days and 1 day (range, 0-5 days), respectively. Median transfusion of red cells and PLT was 4 (range, 1.5-12) U and 20 (range, 0-40) U, respectively.
Besides bone marrow depression, other side-effects such as infection, bleeding, nausea and vomiting also occurred during therapy. According to chemotherapy toxicity assessment of WHO, there were 7 patients confronting infection, among whom only 2 patient occurred III-‡W infection. No serious nausea vomiting or hepatic function damage happened.

Disclosures:
No relevant conflicts of interest to declare.
Induction of REMISSION with Clofarabine IN CHILDREN with Relapsed Acute LYMPHOCYTIC Leukemia.
Authors: Moschovi Maria M , Trimis Georgios G , Karamolegou Kaliopi , Tourkantoni Niki , Tzanoudaki Maria , Divane Anna , Botsa Eleni , Bouzios Elias , Koultouki Eleftheria , Tzortzatou-Stathopoulou Fotini F 
Published: October 2019

Abstract
Abstract 4112
New treatment protocols have improved significantly the survival of patients with acute lymphocytic leukemia (ALL). However, there is a risk of relapse, which depends on disease classification and the protocol of chemotherapy administered. In case of relapse, new remission has to be achieved to proceed to bone marrow transplantation (BMT). For this reason, new therapies for remission induction, except standard chemotherapy, are under investigation. Clofarabine, a new class agent, is a nucleoside analogue of purine which inhibits DNA synthesis and repair by blocking ribonucleoside reductase and DNA polymerase. It also disrupts mitochondrial membrane, driving to cell apoptosis. We describe two children with B-cell ALL, in whom clofarabine was administered because of disease resistance. Case 1: Girl, 12 years old, with ALL, presented a second bone marrow (BM) relapse. The first relapse was also in BM and was presented 30 months after the end of primary chemotherapy. Remission was achieved with chemotherapy based on protocol ALL-REZ-BFM 96. In the second relapse, that occurred one year after the end of ALL-REZ-BFM 96, conventional cytogenetics analysis showed an apparently normal female karyotype 46,XX, while molecular cytogenetics analysis (FISH) showed 3 and 4 copies of AML1 gene in 10.7% and 82.4%, of analyzed nuclei respectively. Clofarabine was administered in 52mg/m2/24h for 5 days, every 4-6 weeks. It was well tolerated. Moderate myelotoxicity presented, early after the end of regimen that was uncomplicated. Mild and transient elevation of transaminases was detected in the peripheral blood. Reevaluation after 3 cycles of clofarabine, revealed morphologic and cytogenetic remission. The patient underwent successful heterologous BMT. Case 2: Boy 18 years old with ALL presented a first BM relapse 22 months after the end of initial chemotherapy. Conventional cytogenetics analysis showed an apparently normal male karyotype 46,XY, while molecular cytogenetics analysis (FISH) was negative for clonal rearrangement involving the MLL gene and TEL/AML1, or BCR/ABL hybrid genes. Chemotherapy was administered based on protocol ALL-REZ BFM 96, but remission was not achieved. Clofarabine in 52mg/m2/24h for 5 days was administered. The patient presented mild uncomplicated myelotoxicity. Remission was achieved after the first cycle of clofarabine and the patient underwent successful heterologous BMT.

Conclusion
Clofarabine administration in children with refractory ALL seems to achieve disease remission and is well tolerated. More studies are needed to establish clorafabine as first line therapy in patients with relapsed ALL, prior to bone marrow transplantation.

Disclosures:
No relevant conflicts of interest to declare.
VEGF Autocrine System Is Demonstrated in Some of Acute Promyelocytic Leukemia Cases: Relationship to WBC Count at the Disease Onset.
Authors: Sugao Toshihiko , Oka Yoko , Tashiro Haruko , Shirasaki Ryosuke , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki 
Published: October 2019

Abstract
Abstract 4127

Aims:
In acute promyelocytic leukemia (M3), a prominent production of vascular endothelial growth factor (VEGF) is reported, and the obvious angiogenesis is observed in bone marrow specimens; however, VEGF receptors (VEGFRs) are not expressed in M3 cells in general. We analyzed VEGF systems in M3 blasts.

Materials and Methods:
Bone marrow cells were obtained from informed M3 patients, whose mononuclear cells were prepared with density-sedimentation method. Cells were cultured for one day for the elimination of an adherent cell-fraction. RNA was extracted from the non-adherent mononuclear cell-fraction, and cDNA was synthesized. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the expression of VEGF and VEGFRs. When the expression of VEGFR type-1 and -2 was demonstrated with RT-PCR, cells were analyzed on the protein level with FACS. VEGF-A levels in sera and in the conditioned media from the cultured M3 cells were determined with ELISA kit. When M3 blasts expressed VEGFR type-1, -2, and also secreted VEGF-A, a growth-inhibition by anti-VEGF antibody was assayed in in vitro cultures.

Results and Discussion:
In all 25 cases examined VEGF-A production was observed, in which VEGFR type-3 was not expressed in any cases. VEGFR type-1 and -2 were expressed in 3 cases, in all of which WBC count at the onset of the disease was above 20,000/μl. When VEGF antibody was added to the blast cell-cultures, the cell-growth was inhibited significantly. These observations indicate that VEGF system works on proliferation in a few of M3 cases with hyper-leukocytosis.

Disclosures:
No relevant conflicts of interest to declare.
CD33-Negative Acute Myeloid Leukemia (AML) Has Similar Characteristics to CD33-Positive Disease.
Authors: Arana Yi Cecilia Y. , Tan Wei , Wilding Gregory , Ford Laurie Ann , Sait Sheila N.J. , Block AnneMarie W. , Deeb George , Wallace Paul K. , Starostik Petr , Thompson James E. , Wang Eunice S. , Wetzler Meir 
Published: October 2019

Abstract
Abstract 4129
Several immunophenotypic subgroups within AML are associated with specific disease characteristics, e.g., CD19-positive AML is associated with t(8;21)(q22;q22) and CD56 expression is associated with adverse outcome. CD33 is a sialic acid-binding immunoglobulin-like lectin (Siglec) located on chromosome 19q13.3. It is highly expressed on early multilineage hematopoietic progenitors but absent from the pluripotent hematopoietic stem cells. It contains two tyrosine phosphorylation motifs whose phosphorylation depends on Src and is the target of gemtuzumab ozogamicin. We asked if lack of CD33 expression (&lt;10%) on the surface of AML blasts was associated with different disease characteristics. We identified 15 such newly diagnosed AML cases and compared their characteristics with 45 (1:3 ratio) cases matched by age and French-American-British subtypes (only cases with M0, M1 and M2 were included). The mean white blood cell count at diagnosis (18.3 ×109/L, 41.7×109/L; p=0.149) and percent of secondary AML [either antecedent hematologic disorder (13%, 18%; p=1) or therapy-related (7%, 16%; p=0.6657)] among the CD33-negative group was similar to the CD33-positive cohort. The percent of unknown (21%, 19%), intermediate (64%, 51%) and unfavorable (14%, 30%) karyotype (p=0.5484) and the percent of cases with FLT3 (20%, 18%; p=0.6058) and NPM1 (7%, 22%; p=0.1562) was also similar among the CD33-negative and the CD33-positive cohorts. The patients were treated with available protocols. There was no significant difference between CD33-negative and CD33-positive treatment assignment. A total of 50% of the CD33-negative cases achieved complete remission compared to 48% of the CD33-positive cases. The median overall survival was 17.1 [95% confidence interval (CI) 10.3, 22.5] versus 10.6 (95% CI 7.1, 16.9) months (p=0.7488) for the CD33-negative compared with the CD33-positive cases. Finally, disease-free survival was also similar between the two groups; 11.0 (95% CI 6.2, 22.6) months for the CD33-negative cohort versus 9.5 (95% CI 4.5, 118.2) months for the CD33-positive group (p=0.966). In summary, CD33 expression does not alter disease characteristics and does not appear to affect outcome in AML unless treatment with gemtuzumab ozogamicin is considered.

Disclosures:
No relevant conflicts of interest to declare.
Immunophenotypic Profile of Acute Myeloid Leukemia in Elderly Patients: a Tertiary Center Experience in Brazil.
Authors: Perini Guilherme F , Santos Fabio P S , Seguro Fernanda , Bacal Nydia , Velloso Elvira , Hamerschlak Nelson 
Published: October 2019

Abstract
Abstract 4137
Acute myeloid leukemia (AML) has an extremely poor prognosis in elderly patients. However, AML is considered a heterogeneous disease, and few reports have analyzed the immunophenotypic profile of AML in elderly patients. We thus analyzed the immunophenotypic markers of newly diagnosed AML in elderly patients in order to determine the incidence of anomalous markers and the correlation with cytogenetic findings. We analyzed samples from 127 patients older than 65 years old with newly diagnosed adult AML seen at our institution from January, 2002 until December, 2007. Markers were assessed by flow cytometry, and the cut-off for positivity was 20%. The expression of CD33 and CD13 was positive in 82.7% and 88.2% of the samples, respectively. HLA-DR was found in 73.2% of cases. The most common aberrant lymphoid markers were CD56 (22.2%), CD7 (16.5%) and CD2 (6.3%). The expression of CD4 and CD11b was correlated with an abnormal karyotype (p<0.05). CD36 was significantly more expressed in patients younger than 75 years of age (p=0.005). AML in elderly patients is a biologically heterogeneous disease, and the immunophenotypic profile reflects this. Future studies should analyze the prognostic value of immunophenotypic abnormalities in this population.

Disclosures:
No relevant conflicts of interest to declare.
Hepatotoxicity in Patients Treated with Gemtuzumab Ozogamicin - Specific Targeting of Hepatocytes?
Authors: Maniecki Maciej Bogdan , Hasle Henrik , Bendix Knud , Møller Holger Jon 
Published: October 2019

Abstract
Abstract 4150
The immunotoxin gemtuzumab ozogamicin (Mylotarg‘, GO) comprises a potent cytotoxic agent, calicheamicin, and a recombinant humanized monoclonal antibody directed against CD33. GO is increasingly used for treatment of acute myeloid leukemia (AML) as CD33, a trans-membranous glycoprotein, is highly expressed on most leukemic blast cells, but, in normal cells confined to early multilineage hematopoietic progenitors and myelomonocytic precursors. Adverse effects of GO treatment are primarily related to myelosuppression (infections, fever, bleedings). However, severe sinusoidal obstructive syndrome (SOS) and hepatotoxicity have also been reported. Since the introduction of GO treatment into routine clinical practice, the incidence of SOS has been reported to span from 0% to 33%, but, the direct cause of the liver-associated effects remain unknown. Using immunohistochemistry we show that the CD33 receptor is widely distributed in the liver tissue and highly expressed on hepatocytes making them susceptible for targeted CD33 chemotherapy by GO. Moreover, utilizing CD163 as a specific marker for macrophages, double immunofluorescence staining revealed that Kupffer cells strongly express CD33. Therefore, the reported hepatoxicity associated with GO treatment may be due to accumulation of antibody-toxin conjugates in hepatocytes and Kupffer cells causing calicheamicin-induced damage.

Disclosures:
No relevant conflicts of interest to declare.
RASGRP1:APTX Gene Expression Ratio Enriches for Responders to the Combination of Tipifarnib and Etoposide in Elderly Newly Diagnosed AML.
Authors: Raponi Mitch , Vener Tatiana , Derecho Carlo , Greer Jacqueline , Malek Sami , Palma John , Karp Judith E 
Published: October 2019

Abstract
Abstract 4154
A 2-gene ratio (RASGRP1:APTX) has previously been shown to predict response to the single agent farnesyltransferase inhibitor, tipifarnib, in patients with both newly diagnosed or relapsed and refractory acute myeloid leukemia (AML) (Raponi et al. Blood 2008, 111:2589). Here we investigated the utility of the 2-gene ratio to predict response to the combination of tipifarnib and etoposide (VP16) in a phase 1 study (Karp et al. Blood 2009, 113:4841) and continuing phase 2 study of elderly patients with newly diagnosed AML. A Taqman quantitative RT-PCR assay was performed on leukemic blasts from a total of 81 patients who were evaluable for response. A receiver operator characteristic analysis indicated that the predictive value of the 2-gene ratio was 76% for predicting overall response (CR+PR) in the Phase 1 study (51 patients). When using an optimized cutoff the overall response rate doubled from 25% to 50% with a negative predictive value of 88% (p = 0.008). These data indicate that the RASGRP1:APTX gene ratio has utility in predicting response to the combination of tipifarnib and etoposide in elderly newly diagnosed AML. This assay could find broader utility in enriching for responders to tipifarnib combination therapy.

Disclosures:
Malek: Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding.