bluuebunny/crossref_metadata_2025 huggingface dataset:
DNMT1 Cxxc Domain Can Functionally Substitute In An MLL Fusion Protein
Authors: Risner Laurie E. , Kuntimaddi Aravinda , Bushweller John H. , Zeleznik-Le Nancy J.
Published: October 2019

Abstract 4193

The MLL gene encodes a multi-domain protein that is involved in the maintenance of Hox gene expression during development and hematopoiesis, and was first identified through its involvement in chromosome translocations that cause leukemia. The CXXC domain of MLL, which is retained in leukemic MLL fusion proteins, is a cysteine rich DNA binding domain, with specificity for binding nonmethylated CpG-containing DNA, and is essential for MLL fusion proteins' oncogenic properties.

We performed domain swap experiments in which CXXC domains from other proteins were swapped in to replace MLL's CXXC domain in the context of an oncogenic MLL fusion. CXXC domains from DNA methyltransferase 1 (DNMT1), CpG binding protein (CGBP), and methyl-CpG binding domain protein 1 (MBD1), as well as a methyl binding domain (MBD) from MBD1 were swapped into the MLL-AF9 fusion. These particular domains were chosen because their described CpG DNA binding capacity is either similar or different from that described for MLL. In vitro colony assays on isolated murine bone marrow progenitor cells infected with domain swapped or wild type MLL-AF9 fusion genes were performed in order to determine whether CpG binding domains from other proteins would affect the ability of MLL-AF9 to give an enhanced proliferative capacity to bone marrow progenitor cells. In vivo murine studies determined whether the different CpG binding domains alter the ability of MLL fusion proteins to cause leukemia. We predicted that the different CpG binding domains would change the strength or specificity of MLL binding to DNA, which would affect the ability of MLL-AF9 to cause leukemia.

The results of both in vitro replating assays and in vivo leukemogenesis experiments have shown significant differences between the ability of various CpG DNA binding domains to function in the context of an MLL-AF9 fusion protein. MLL-AF9 containing the DNMT1 CXXC domain shows robust in vitro colony forming activity and in vivo leukemogenesis activity, similar to the oncogenic MLL-AF9 fusion. However, MLL-AF9 containing either the CXXC domain from CGBP or MBD1, or the MBD domain of MBD1 all show reduced colony forming ability and leukemogenicity in vivo. In vitro DNA binding experiments are currently being performed to directly measure and compare the DNA binding affinity of the CXXC domain from MLL to the other domain swap proteins. Preliminary data suggests that MLL CXXC has a stronger DNA binding affinity to non-methylated DNA compared to the other CXXC domains. Furthermore, the DNMT and CGBP CXXC domains both show lower affinity DNA binding compared to MLL CXXC, but they have different effects in MLL-AF9. This suggests that CXXC domain properties in addition to DNA binding affinity, perhaps including protein recruitment, also contribute to an MLL fusion protein's leukemogenic properties.

Disclosures:

No relevant conflicts of interest to declare.
Candidate Gene Polymorphisms and the Risk for Pregnancy Related Venous Thrombosis
Authors: Dahm Anders E. A. , Bezemer Irene D. , Bergrem Astrid , Jacobsen Anne Flem , Jacobsen Eva Marie , Skretting Grethe , Rosendaal Frits , Sandset Per Morten
Published: October 2019

Abstract 4203

Background:

Venous thrombosis (VT) is the leading cause of maternal death in the western world. Genetic causes of pregnancy related VT are insufficiently understood.

Objectives: To investigate if recently discovered associations between single nucleotide polymorphisms (SNPs) and thrombotic disease influenced the risk for pregnancy related VT. To investigate candidate genes for new associations between SNPs and pregnancy related VT.

Methods:

A hospital based case control study. Cases were women with objectively verified VT during pregnancy or puerperium, controls were women giving birth without having VT. We selected 49 candidate genes involved in coagulation (35 genes), inflammation (4 genes), and hormonal metabolism (10 genes). In these genes, 463 tag-SNPs were selected from the CEU population in Hapmap and analyzed in 313 cases and 353 controls. Odds ratios for VT were calculated with the most common genotype as the reference genotype for each SNP. P-values were adjusted for multiple testing to false discovery rates.

Results:

Of the SNPs previously shown by others to be associated to thrombotic disease, rs2289252 in the FXI gene (F11), rs3917643 in the tissue factor gene (F3), rs1613662 in the glycoprotein VI gene, rs2001490 in NAT8B, rs4524 in the FV gene (F5), and rs3087505 in the prekallikrein gene were associated to pregnancy related VT. After analyzing all 463 tag-SNPs, 13 SNPs were associated to total pregnancy related VT with a false discovery rate ≤ 0.20. These SNPs belonged to F5, SELP (coding for P-selectin), F3, F8 (coding for factor VIII), and F11. When we studied antenatal and postnatal VT separately, 17 SNPs were associated with antenatal VT, while only 1 SNP was associated with postnatal VT. Thirteen SNPs in F5, SELP, or the gene for E-selectin were associated with thrombosis. After excluding carriers of factor V Leiden, 4 of these SNPs remained associated. Two SNPs in the gene for epidermal growth factor receptor (EGFR) were associated to antenatal thrombosis.

Conclusion:

Genetic variation seems more important for antenatal than for postnatal VT. We confirmed the importance of some SNPs for the risk for pregnancy related VT, in particular rs2289252 in F11. The EGFR-gene appears to be a new genetic locus associated with pregnancy related VT.

Disclosures:

No relevant conflicts of interest to declare.
Sub-Optimal Inhibition of Thrombus Formation by Aspirin (as measured by RTTP analysis) In Primary Thrombocythemia
Authors: Stephens Gillian , Tauscher Jan , Siegel Fabian , Phillips David R. , Andre Patrick , Petrides Petro E.
Published: October 2019

Abstract 4218

Primary thrombocythemia (PT) is a myeloproliferative disease with a high incidence of thrombotic complications. First-line therapy is the inhibition of platelet aggregation with aspirin (via COX-1 inhibition) for the prevention of thromboembolic complications. However, this does not account for the potential for COX-2 expression in newly synthesized platelets. The purpose of the present study was 1) to determine whether aspirin reduces the thrombotic potential of PT patients who did not undergo cytoreductive therapy to the levels of those achieved in healthy volunteers (HV), and 2) to determine whether the presence of the JAK2V617F mutation is responsible for the elevated thrombotic potential in PT patients.

We determined the thrombotic potential of a cohort of 16 PT patients and compared these values to those of 10 healthy volunteers all treated with 100mg aspirin QD. Thrombotic potential was determined using a custom built Real Time Thrombosis Profiler (RTTP). Whole blood anticoagulated with a Factor Xa inhibitor (to preserve physiological Ca++ concentration) with Rhodamine 6G-labeled platelets was perfused over a fibrillar collagen surface at shear rates approximating those in veins (100s-1), arteries (600 s-1) and moderately stenosed arteries (1600s-1). The deposition of fluorescently labeled platelets on the collagen surface was monitored in real time by fluorescence microscopy in the RTTP. Endpoint thrombosis (size of thrombi at t=300sec expressed as Mean Fluorescent Intensity/Area of coverage) and rate of thrombus growth (initial rate of platelet deposition) were recorded for each individual as measures of thrombotic potential and data are expressed as mean ± SEM (statistics performed using GraphPad Prism v4.03 using unpaired, 2-tailed Students t-test).

Despite aspirin therapy, PT patients were characterized by significant increases in thrombotic potential at venous and arterial shear rates (Table 1) with a greater than 2-fold increase in the rate of platelet deposition. Interestingly only the size of thrombi formed but not the rate of formation was elevated in PT patients at moderately stenosed shear, likely a reflection of the increasing contribution of shear and decreasing dependence on absolute platelet count and other individual cellular factors in whole blood. The

Disclosures:

No relevant conflicts of interest to declare.
Addressing the Issue of the Pathogenesis of Deep Vein Thrombosis In Cancer
Authors: Thomas Grace M , Brill Alexander , Schatzberg Daphne , Wagner Denisa D.
Published: October 2019

Abstract 4220

The risk of thrombotic complications during tumor development has been the focus of several clinical studies over the past decades. Thrombotic disease is a common cause of death in cancer patients and almost one in five of all symptomatic cases of Deep Vein Thrombosis (DVT) are thought to be cancer related. Tumor cells generate and profit from a local and systemic hypercoagulable state, which promotes tumor growth, angiogenesis, and metastasis. The resulting procoagulant state is responsible for the heightened risk for thrombotic complications and the etiology of the resulting DVT event. However, despite the fact that some molecules, such as tissue factor, have recently been described as potential modulators of clot formation in DVT, the true trigger of DVT in cancer patients remains unknown. To determine whether the presence of cancer cells can trigger formation of a thrombus in a deep vein in vivo, we combined a murine model of Trousseau's Syndrome with a model of DVT. First, we induced the development of a tumor by subcutaneous injection of syngeneic pancreatic tumor cells in C57BL/6 mice. It was previously demonstrated that these mice have significantly reduced tail bleeding time and time to occlusion in injured mesenteric arteries and veins compared to control vessels. After 3 hours of partial flow restriction of the inferior vena cava, we observed that all tumor-bearing mice developed an occlusive thrombus (n=8) while only 29% of the control mice (n=7) formed occlusive thrombi. Interestingly, perfusion of cancer-derived microparticles in mice also induced thrombus formation in this DVT model, reproducing the tumor-bearing mice phenotype. This new approach showed that microparticles shed by cancer cells have a key role in the pathogenesis of cancer-associated DVT, and they may become a reasonable pharmaceutical target to prevent thrombosis associated with cancer development.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Analysis of the SEC23B Gene In Patients Affected by Congenital Dyserythropoietic Anemia Type II (CDAII)
Authors: Bianchi Paola , Schwarz Klaus , Fermo Elisa , Heinrich Katja , Vercellati Cristina , Marcello Anna Paola Maria Luisa , van Wijk Richard , Zwieten Rob van , Banov Laura , Sau Antonella , Wuillemin Walter A. , Opat Stephen , Barcellini Wilma , Heimpel Hermann , Zanella Alberto
Published: October 2019

Abstract 4227

CDAII, the most frequent type of congenital dyserythropoietic anemia, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblast morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Last year we and others identified SEC23B as the gene responsible for CDAII (Schwarz et al, 2009, Bianchi et al, 2009). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles from the endoplasmic reticulum to the Golgi apparatus. The gene, localized on chromosome 20p11, is split in 20 exons and codifies for a 767 aa protein. The aim of the study was to characterize the molecular defect in a large series of CDAII patients of Caucasian origin. Fifty-one CDAII patients from 49 unrelated families (17 Italians, 20 German or Swiss, 4 Dutch, 2 British, 2 Czech and 1 Greek, Turkish, Bulgarian and Irish origin) were analyzed by direct exon sequencing. We identified 36 different mutations, 25 of which were not described before. Eighteen were disruptive and 18 were missense mutations. The patients' molecular data are summarized in the Figure (novel mutations are reported in bold). All the missense mutations affected highly conserved aminoacids and were not found in 200 normal alleles examined. The c.325G>A mutation was identified in 15 homozygous patients and in two cases in combination with other mutations. The change c.40C>T was detected in 16 unrelated patients as heterozygous mutation and, for the first time, at the homozygous level in one patient only. Considering the entire series of patients (including previously published cases) characterized by our groups (86 CDAII patients from 77 unrelated families) c.325G>A and c.40C>T mutations account for 49% (76/154) of the unrelated mutated alleles and therefore should be firstly screened during molecular diagnosis of CDAII. A c.325G>A mutation usually results in a mild to moderate clinical picture at homozygous level, whereas it may cause a very severe clinical pictures when combined with other mutations (Fermo, et al 2010). Despite the sequencing of all exons and flanking intronic regions, 5 patients displayed only one mutation, suggesting the possibility that mutations could be located in regulatory regions or that a second gene could be involved in the pathogenesis of CDAII.

P. Bianchi and K. Schwarz contributed equally to this work.

Disclosures:

No relevant conflicts of interest to declare.
Single ROI Versus Multislice T2* MRI Approach for the Quantification of Hepatic Iron Overload
Authors: Meloni Antonella , Luciani Antongiulio , Positano Vincenzo , Marchi Daniele De , Valeri Gianluca , Restaino Gennaro , Roccamo Gaetano , Armari Sabrina , Dell'Amico Maria Chiara , Favilli Brunella , Lippi Alma , Lombardi Massimo , Pepe Alessia
Published: October 2019

Abstract 4263

Introduction.

Precise and effective measurements of iron overload in the liver, where iron deposition seems to be primarily noticeable (Noetzli LJ et al, Blood 2008), are important for the early diagnosis, treatment and follow-up of patients with hemochromatosis or hemosiderosi. T2* Magnetic resonance imaging (MRI) represents the most available noninvasive technique to assess hepatic iron content and shows a good correlation with biopsy results (Wood JC et al, Blood 2005). In the clinical practice, a single hepatic slice is acquired and T2* measurement is performed in a single region of interest (ROI) of the parenchyma (Pepe A et al, Eur J Haematol 2006). This approach may mask an heterogeneous iron distribution. Thus, the aims of this study were to set up a MRI acquisition technique for the detection of the iron burden in the whole liver and to evaluate the effectiveness of the single ROI technique.

Methods.

Five transverse hepatic slices were acquired by a T2* gradient-echo sequence in 101 thalassemia major (TM) patients (48 males, mean age 29 ± 8 years, mean serum ferritin level 1413 ± 1209 ng/mL, mean pre-transfusion hemoglobin 9 ± 1 g/dl.) and 20 healthy subjects. The T2* value was calculated in a single ROI defined in the medium-hepatic slice. Moreover, the T2* value was extracted on each of the eight ROIs defined in the functionally independent segments according to Couinaud classification. The mean hepatic T2* value was obtained by averaging all segmental values.

Results.

For patients the mean T2* values over segments VII and VIII were significantly lower than the other segments (P<0.0001) (figure 1). This pattern was substantially preserved in the two groups identified considering the T2* normal cut-off (Ramazzotti A. et al J Magn Reson Imaging 2009) (group A: single ROI T2* < 15.8 ms and group B: single ROI T2* ≥ 15.8 ms). All mean segmental T2* values as well as the mean hepatic T2* value were strongly correlated with the single ROI T2* value. After the application of a correction map based on T2* fluctuations in the healthy subjects, no significant differences were found in the segmental T2* values for the whole patient population (P=0.251), for both the previously defined groups (Group A: P = 0.073 and Group B: P=0.476) as well as for the Group A patients (N = 23) with chronic hepatitis (P=0.111).

Conclusions.

Hepatic T2* variations are low and due to artifacts and measurement variability. The single ROI approach can be adopted in the clinical arena, taking care to avoid the susceptibility artifacts, occurring mainly in segments VII and VIII.

Disclosures:

No relevant conflicts of interest to declare.
Maturation of Monocyte-Derived Dendritic Cells Induced by Dual Targeting of Endocytosis Receptor and CD40
Authors: Staudinger Matthias , Catenhusen Inga Rebekka , Peipp Matthias , Kellner Christian , Repp Roland , Gramatzki Martin
Published: October 2019

Abstract 4293

Dendritic cells (DC) are professional antigen presenting cells, which can induce and regulate adaptive immune responses. Hence, DC are attractive candidates for anti-tumor vaccination strategies. The aim of this study was to create recombinant bispecific Fab to target and activate DC, by using derivates of monovalent agonistic CD40 scFv and endocytosis receptors FcγRI (CD64), FcαR (CD89), DEC205 (CD205), as well as DC-SIGN (CD209), respectively. The recombinant bispecific molecules were expressed in the eukaryotic cell line Hek 293T, enriched to homogeneity by affinity chromatography and analyzed for specific binding to the targeted receptors. Functional analysis of these molecules indicated, that concurrent engagement of CD40 and endocytosis receptors expressed by immature IL-4/GM-CSF cultured monocytes-derived DC induced maturation of these DC. This was suggested by changes of DC phenotype, especially enhanced surface expression of co-stimulatory molecules like CD80 and CD86, and gain of CD83 expression as measured by flow cytometry. To particularly analyze the T cell activation properties of these DC, MACS enriched T cells of autologous donors were co-cultivated with antigen-loaded and subsequently maturated DC for seven days. After restimulation of T cells, flow cytometric detection of intracellular IFN-γ levels and surface expression of CD69 used as markers for T cell activation implies, that these DC exhibit elevated properties to activate antigen specific T cells. In contrast, a bispecific [CD64scFv × CD40scFv] Fab used as control failed to induce maturation of DC lacking CD64 expression, which may be due to abrogation of bivalent binding of this molecule and therefore CD40 cross-linking. Moreover, dual targeting with these novel immunoconstructs when linked to peptides of a tumor-associated antigen may allow direct antigen delivery to DC in combination with DC activation, and thus anti-tumor vaccination in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of Endothelial Cell Tube Formation by ADAMTS13 Occurs Via Interaction with VEGF
Authors: Rodansky Eva S. , Smith Justin K. , Fryer Hugh J.L. , Greenfield Robert S. , Rodgers George M.
Published: October 2019

Abstract 4307

The von Willebrand factor-cleaving protease, also known as ADAMTS13, is synthesized, in part, by endothelial cells (EC). We previously reported that proliferating EC secreted ∼3-fold more ADAMTS13 (antigen and activity) than confluent EC, and that this synthesis was transcriptionally regulated (SJ Kling et al, Pathophysiol Haemost Thromb 2008; 36: 233.) Thrombospondin domains, a defining feature of the ADAMTS protease family, in other ADAMTS family members mediate inhibition of angiogenesis. In particular, ADAMTS1 inhibits angiogenesis by sequestering vascular endothelial growth factor (VEGF) (A Luque et al, J Biol Chem, 2003; 278: 23656). Since ADAMTS13 also has thrombospondin domains, we hypothesized that ADAMTS13 might also mediate anti-angiogenic activity. Human umbilical vein EC angiogenesis was quantified by Angioquant analysis of fluorescence microscopy tube images in a Matrigel assay. Treatment of EC with inhibitors of angiogenesis (anti-VEGF, vasostatin) did not alter EC production of ADAMTS13. However, exogenous recombinant ADAMTS13 inhibited angiogenesis in a dose-dependent manner (Figure 1), in media containing VEGF. In VEGF-free media, ADAMTS13 had no effect on EC tube formation. Preincubation of ADAMTS13 with an antibody to the ADAMTS13 thrombospondin domains 5–7 partially reversed the inhibition of tube formation, implicating these domains in the anti-angiogenic interaction (Figure 2). We also found that VEGF co-immunoprecipitates with ADAMTS13, providing strong evidence that these two proteins interact. When EC lysates were crosslinked with DTSSP prior to immunoprecipitation, anti-ADAMTS13 immunoprecipitated as much VEGF as did anti-ADAMTS1, while an antibody to ADAM17, a similar protein that lacks thrombospondin domains, failed to co-immunoprecipitate VEGF (Figure 3). These data indicate that as with other ADAMTS family members, ADAMTS13 inhibits tubule formation - a parameter of angiogenesis – through its interaction with VEGF, an effect likely mediated by its thrombospondin domains. Inhibition of angiogenesis adds to the expanding roles of ADAMTS13 in down-regulation of thrombosis and inflammation.

Fig. 1. Fig. 1.

Fig. 2. Fig. 2.

Fig. 3. An antibody to ADAMTS13 immunoprecipitates VEGF in similar amounts as anti-ADAMTS1 when proteins from HUVEC lysates are crosslinked prior to co-immunoprecipitation. Nonspecific IgG fails to immunoprecipitate VEGF, as does anti-ADAM17. (A1 = ADAMTS1; A13 = ADAMTS13) Fig. 3. An antibody to ADAMTS13 immunoprecipitates VEGF in similar amounts as anti-ADAMTS1 when proteins from HUVEC lysates are crosslinked prior to co-immunoprecipitation. Nonspecific IgG fails to immunoprecipitate VEGF, as does anti-ADAM17. (A1 = ADAMTS1; A13 = ADAMTS13)

Disclosures:

Fryer: American Diagnostica Inc: Employment. Greenfield:American Diagnostica Inc: Employment.
Longitudinal Analysis of VWF Multimerization Patterns In a well-defined TTP Cohort
Authors: Yang Shangbin , Haven Sara , Jin Ming , Geyer Susan , Cataland Spero R , Wu Haifeng
Published: October 2019

Abstract 4308

VWF function has long been implicated in TTP's pathogenesis. Initial study reported unusually large (UL)VWF multimers in patients with relapsing TTP. Later, ULVWF were demonstrated in TTP patients during remission that are acutely consumed at the onset of disease. However, many detailed steps concerning the timely relationship between VWF multimerization and clinical courses such as disease onset, mortality, disease refractoriness, and treatment response are still missing. In this study, we performed VWF multimeric analyses in over 400 serial samples longitudinally collected from more than 20 TTP patients at various clinical stages, including initial presentation, acute treatment, clinical remission, and clinical visits prior to TTP relapses. At the time of acute presentation, approximately 50% of cases show presence of ULVWF multimer patterns, while around 35% demonstrate acute consumption of UL and high molecular weight VWF multimers. The other 15% of cases however reveal normal VWF multimer patterns at the time of disease presentation. For the TTP patients who normally respond to plasma exchange (PE) therapy, a mild ULVWF multimer pattern may be present after discontinuation of daily PE therapy. Upon restoration of ADAMTS13 activity and achievement of sustained remission, VWF multimer patterns are normalized. During the period of daily plasma exchange for acute episodes of TTP, a persistent presence of ULVWF multimer patterns appears indicative of disease refractoriness and is associated with disease mortality. In the course of this study, ADAMTS13 activity levels (reportable range of 0.5 % –100%) in these longitudinal samples were also precisely determined using SELDI-TOF mass spectrometry to establish the correlation between ADAMTS13 activity measured in vitro and VWF multimeric patterns formed in vivo. Our study for the first time defines the dynamic changes of VWF multimeric patterns in relation to the evolution and natural history of TTP in a large well-defined clinical cohort. These novel observations provide new implications into the pathobiology of TTP and may help to develop new biomarkers for monitoring therapeutic responses and for screening for TTP relapses.

Disclosures:

No relevant conflicts of interest to declare.
Kruppel-Like Factor 10 (KLF10)-Deficient Mice Have Marked Defects In EPC Differentiation, Function, and Angiogenesis
Authors: Wara Akm Khyrul , Croce Kevin , Foo ShiYin , Sun Xinghui , Icli Basak , Tesmenitsky Yevgenia , Cao Zhuoxiao , Esen Fehim , Subramaniam Malayannan , Kutok Jeffery , Rodig Scott J. , Spelsberg Thomas , Rosenzweig Anthony , Feinberg Mark
Published: October 2019

Abstract 4314

Background:

Emerging evidence demonstrates that endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of being recruited to sites of ischemic injury and contribute to neovascularization. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood.

Methods and Results:

We previously identified that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) can preferentially differentiate into EPCs and possess high angiogenic activity under ischemic conditions compared to megakaryocyte-erythrocyte progenitors (MEPs), hematopoietic stem cells (HSCs), and common lymphoid progenitors (CLPs). Herein, we identify that a TGF-β1-responsive Kruppel-like Factor, KLF10, is robustly expressed in EPCs derived from CMPs and GMPs, compared to progenitors lacking EPC markers. KLF10–/– mice have marked defects in circulating EPCs (–23.6% vs. WT, P<0.004). In addition, EPC differentiation and TGF-β induced KDR responsiveness is markedly impaired (CMPs: WT 22.3% vs. KO 8.64%, P<0.0001; GMPs: WT 32.8% vs. KO 8.97%, P<0.00001). Functionally, KLF10–/– EPCs derived from CMPs and GMPs adhered less to fibronectin-coated plates (CMPs: WT 285 vs. KO 144.25, P< 0.0004; GMPs: WT 275.25 vs. KO 108.75, P <0.0003) and had decreased rates of migration in transwell Boyden chambers (CMPs: WT 692 vs. KO 298.66, P<0.00004; GMPs: WT 635.66 vs. KO 263.66, P<0.00001). KLF10–/– mice displayed impaired blood flow recovery after hindlimb ischemia (day 14, WT 0.827 vs. KO 0.640, P <0.009), an effect completely rescued by WT EPCs, but not KLF10–/– EPCs. Matrigel plug implantation studies demonstrated impaired angiogenesis in KLF10–/– mice compared to WT mice (WT 158 vs. KO 39.83, P<0.00000004). Overexpression studies revealed that KLF10 rescued EPC formation from TGF-β1+/– CMPs and GMPs. Mechanistically, TGF-β1 and KLF10 target the VEGFR2 promoter in EPCs which may underlie these effects.

Background:

Collectively, these observations identify that TGF-β1 signaling and KLF10 are part of a key signaling pathway that regulates EPC differentiation from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.

Disclosures:

No relevant conflicts of interest to declare.
Cell Adhesion Molecule 1 (CADM1), a Novel Venous Thrombosis Risk Factor, Is Ubiquitously Present In Vascular Endothelium and Smooth Muscle Cells
Authors: Tatsumi Kanayo , Taatjes Douglas J , Bouchard Beth A , Trotman Winifred , Bovill Edwin G
Published: October 2019

Abstract 4316

We recently demonstrated that CADM1, interacting with protein C deficiency, is a novel risk factor for venous thrombosis (VT) Blood 114:3084-3091, 2009. CADM1 likely plays a role in maintaining endothelial barrier function. Prior to this study CADM1 had not been identified in endothelial cells. In this study we have determined the distribution of CADM1 in human vasculature.

Methods:

Human tissue samples representing all organs as well as large vessels were accessioned from archived paraffin blocks from the surgical pathology division of FAHC. Tissue sections were processed for immunoflourescence using as primary antibodies a chicken monoclonal anti-CADM1 IgY antibody (anti-SynCAM/TSLC1 clone 3E1, MBL Inc, Woburn, MA) and anti-smooth muscle actin (SMA) clone 1A4 (Sigma, St. Louis, MO), anti-von Willebrand factor (vWF) rabbit polyclonal antibody (DAKO Inc., Glostrup, DK). vWF staining was used to identify endothelial cells. The secondary antibodies for CADM1, SMA, and vWF were donkey anti chicken alexa 488, donkey anti mouse alexa 647, and donkey anti rabbit alexa 647, respectively. Appropriate negative and positive controls were run. Intensity of staining was rated as absent, trace or present by three observers (TK, DJT, and EGB). Immunoelectron microscopy (IEM) was performed on cultured human umbilical vein endothelial cells (Allcells LLC, Emeryville, CA) using a post embedding procedure with protein-A gold. Peripheral blood leukocytes were evaluated for the presence of CADM1 using the same antibody following cytospin preparation.

Results

CADM1 was found ubiquitously in the macro- and micro-vasculature of all organs as well as the aorta and saphenous vein with intensity of staining showing modest variability from organ to organ. CADM1 staining of SMA was observed in both arterial and venous vessels but was considerably stronger on the arterial side. IEM demonstrated cytoplasmic immunogold staining associated with elements of rough endoplasmic reticulum and actin filaments as well as at the membranes of filopodia. In contrast, peripheral blood monocytes, lymphocytes and granulocytes were negative for CADM1expression.

Conclusions:

CADM1 is expressed ubiquitously in endothelial and smooth muscle cells of the macro-and micro-vasculature with IEM evidence for its presence in filopodial cytoplasm and membranes. Peripheral blood leukocytes did not show evidence of CADM1 expression. This is the first report of CADM1 positivity in vascular smooth muscle cells. The biological role of CADM1 remains to be determined but its ubiquitous expression in the endothelium of the macro- and micro-circulation, together with an apparent role in endothelial cell motility, suggests an important role in endothelial homeostasis.

Disclosures:

Bovill: Haemotologic Technologies, Essex Jct., VT: Equity Ownership.
Circulating Plasma Levels of Connective Tissue Growth Factor (CTGF) Are Elevated In Patients Afflicted with Rheumatoid Arthritis
Authors: Del Carpio-Cano Fabiola , Rico Mario C , Kunapuli Satya P. , Safadi Fayez , De La Cadena Raul A.
Published: October 2019

Abstract 4320

Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA are at an increased risk to develop cardiovascular diseases and the precise mechanism(s) of action remain obscure. This laboratory has proposed the existence of a pro-inflammatory axis in RA comprised by thrombospondin-1 (TSP1), transforming growth factor-beta (TGF-b) and CTGF. The present study evaluated plasma levels of TSP1, TGFb and CTGF in patients with RA by ELISA as well as specific cytokines and chemokines. CTGF plasma levels in RA patients (9.2 pg/ml mean, range 3.44–17.08) were found significantly increased (P<0.047) when compared to control subjects (mean 4.43 pg/ml, range 1.62–7.35). This is the first report in the medical literature documenting an augmented human plasma circulating levels of CTGF in RA. TSP1 circulating levels were found elevated in RA patients when compared to control subjects (mean 315 ng/ml vs. 25.4 ng/ml respectively, P<0.039). TGFb showed a trend for higher levels in patients with RA (10.1 ng/ml vs. 4.5 ng/m, P<0.095). The source CTGF in plasma could not be determined precisely but there is evidence of platelet activation in RA (Platelets 19:146, 2008), and TSP1 is the major content of the platelet a-granule (25%). CTGF can attract monocytes and both have been found co-localized in atherosclerotic plaques (Circulation 95, 831, 1997). IL-6 (P< 0.05), IL-12p70 (P<0.04), IP-10 (P<0.03) and MIP1b (P<0.016) were increased in the RA patients when compared to controls. Recent findings have placed platelet microparticles (Science 327; 580, 2010) as playing a major role (arsonists) in RA via monocytes and neutrophils. In summary, our results provide evidence that a pro-inflammatory axis is active in RA potentially contributing to cytokine changes as well as the cardiovascular disease associated with RA since TSP1 can induced IL-6 generation from monocytes and can activate inert TGFb on cell surfaces (fibroblasts and synoviocytes) with the subsequent up-regulation of CTGF. Disruption of the axis in experimental models of RA may prove to be an emerging therapeutic target in RA. Elucidation of the source of CTGF should provide as well key information for cell-targeting to prevent CTGF secretion into the blood milieu.

Disclosures:

No relevant conflicts of interest to declare.
Irradiation-Induced Growth Hormone Neurosecretory Disfunction In ALL Patients
Authors: Biceroglu Gulay , Akcay Teoman , Aydogan Gonul , Akcay Arzu , Akici Ferhan , Tugcu Deniz , Salcioglu Zafer , Sayilan Hulya , Ayaz Nuray
Published: October 2019

Abstract 4329

With the recent developments in the treatment of ALL life expectancy is prolonged and complications due to therapy are increased. Combined chemotherapy may be applied in combination with distinct doses and schema of cranial radiotherapy according to the risk groups of patients. CRT damages the hypothalamus-hypophysis axis and affects the secretion of growth hormone (GH) at first and the other anterior hypophysis hormones also. At high doses of CRT, GH deficiency might occur in the long term follow-up, at lower doses the secretion pattern of GH may change. In this study, GH axis of the ALL patients treated with prophylactic CRT were evaluated.

Thirty-two children (14 girls and 18 boys), diagnosed as ALL and applied prophylactic cranial therapy were enrolled to the study. Physical findings, weight and height measurements, Tanner stagings and bone age evaluations were performed to all patients. In order to interpret the functions of anterior and posterior hypophysis morning basal IGF-1, IGFBP-3, thyroid hormones, cortisol, prolactin and dansity of urine were measured. Gonodotrophins were not tested because none of our patients had the signs of early or late puberty. In order to evaluate the growth rate, weights and heights of the patients were measured after the first year. Even though the growth rate of some patients were normal, GH stimulation tests with clonidine were done to all cases, due to the fact that GH deficiency and neurosecretory dysfunction might be present pharmacologically. Second GH stimulation test with L-dopa were applied to patients with peak GH levels of <10 ng/ml. If the level was low in the second test, patients were accepted as GH deficient. For patients with low growth rate, low IGF-1/IGFBP-3 according to age and sex, but normal GH levels, night GH secretion pattern were tested. Patients with inadequate response to night secretion test were accepted as neurosecretory dysfunction.

In our study, 25% of the patients were found to have complete and 21,7% incomplete GH deficiency. In 9.3% of cases there was GH-neurosecretory dysfunction (GH_NSD). There was no statistically significant relationship between the time passed after radiotherapy and GH axis dysfunction degree. IGF-BP3 was found to be more reliable than IGF-1 in estimating GH deficiency/GH-NSD.

As a result, it is clear that prophylactic CRT affects the GH axis negatively in ALL patients, so their close follow-up is precious and mandatory.

Disclosures:

No relevant conflicts of interest to declare.
Hypercvad+Bortezomib Is a Well Tolerated Salvage Regimen for Relapsed/Refractory Adult Acute Lymphoblastic Leukemia
Authors: Raychaudhuri Jyotishankar , Ahmad Mudhasir , Kalra Manas , Dua Harsh , Chopra Rakesh , Rawat Sangeeta , Thakur Rashmi
Published: October 2019

Abstract 4345

We report the excellent tolerability of a regimen of HyperCVAD+Bortezomib for relapsed/refractory ALL in adults. The regimen consisted of addition of two doses of Bortezomib 2 mg(1mg/m2) on day 1 and day 4 of the HyperCVAD regimen(arm A). The doses of bortezomib were given an hour after the second dose of cyclophosphamide on day 1 and on day 4. GCSF 10 mcg/kg was used from nadir to recovery of ANC > 500. Two patients, one with relapsed ALL and the second with primary induction failure were treated with this regimen after informed consent. The first patient was a 38 year male who was diagnosed with Ph+ ALL four years ago and had relapsed three years later on maintenance treatment. He was subsequently treated with Adriamycin, Etoposide and Cyclophosphamide with minimal response. Last bone marrow showed 35–40% blasts. The second patient was 40 year female with CALLA + Pre-B cell ALL with primary induction failure with a BFM regimen. Both patient reached nadir counts by day 10–11 and recovered the absolute neutrophil counts > 500 by day 16. There was minimal blood product requirement and minimal episodes of neutropenic fever which were treated as per institutional protocol. All cultures were negative. Isolation was not required. Strict aseptic precautions were maintained. Minimal grade I mucositis occurred in the first patient. Minimal diarrhea with nausea and vomiting occurred in the second patient on day 15. No neuropathy was noted. On day +21, the first patient, who was resistant to multiple chemo agents still has 40% blasts in the marrow but also shows recovery of the normal marrow elements. The second patient, who had primary induction failure is in complete remission in the marrow. One report of a phase I trial by Messinger et al from Minnesota showed the feasibility and tolerability of Bortezomib added to VXLD in pediatric ALL. Horton et al from a COG phase I study did not find any objective responses to single agent Bortezomib in resistant leukemia even though it suppressed NfKB activity. This is the first report to our knowledge on the use of salvage bortezomib based regimen in relapsed/refractory adult ALL. We used low dose bortezomib(1 mg/m2) after the administration of chemotherapy on day 1 and day 4. The timing of bortezomib just after chemotherapy is probably important. One ASH abstract #949(2007) by Lonial et al suggested that Bortezomib following high dose melphalan in multiple myeloma results in increased apoptosis of MM cells than if Bortezomib precedes melphalan. We plan to proceed to a phase II trial to test this regimen in relapsed/refractory ALL.

Disclosures:

Off Label Use: Bortezomib as chemosensitizer with Hyper CVAD in ALL.
Protein Degradation and Resultant Hsp90 Dephosphorylation as An Alternative C-KIT Kinase Inhibition Strategy
Authors: Zhang Bo , Fang Haitong , Pan Xiaofen , Xie Jun , Chen Yonglong , Chen Sai-Juan , Zhou Guangbiao
Published: October 2019

Abstract 4361

Occupying the ATP binding pocket represents a common strategy for tyrosine kinase inhibition, exemplified by imatinib mesylate in BCR-ABL positive leukemia and gefitinib in EGFR mutated lung cancer. Here we report that the natural compound triptolide and proteasome inhibitor Bortezomib induce C-KIT turnover in cancer cells. Bortezomib triggers a clathrin-mediated endocytosis and lysosomal degradation of KIT in t(8;21) leukemia. While phosphorylation of heat shock protein 90 (Hsp90) by KIT is required for Hsp90-caused suppression of apoptosis, Bortezomib causes dephosphorylation of Hsp90 and lease of Apaf-1/cytochrome C, leading to reactivation of caspase-3 which cleaves the t(8;21)-generated AML1-ETO/AML1-ETO9a oncoproteins with production of cleavage fragments. The cleavage fragments antagonize the parental proteins in vitro and in vivo. Intriguingly, Bortezomib shows potent anti-leukemia efficacy in two murine models. These data indicate that degradation of C-KIT/dephosphorylation of Hsp90 represents a novel strategy for kinase inhibition, and drugs inhibiting both transcription factor and kinase could be a powerful therapeutic regimen for human leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Comparing Induction and Consolidation with or without Cytarabine In the Treatment of Acute Promyelocytic Leukaemia In Hong Kong Chinese
Authors: Law Man Fai , Yip Sze-Fai , Chan Hay Nun , Yeung Yiu Ming , Choi Wai
Published: October 2019

Abstract 4366

Previous studies suggested that cytarabine was not required in the treatment of newly diagnosed acute promyelocytic leukaemia (APL). They suggested that omitting cytarabine in the treatment of APL could reduce treatment toxicity without increasing relapses and affecting survival. No previous study assessed the effect of cytarabine in the treatment of Chinese APL patients.

We compared the outcome of APL patients with or without cytarabine in induction and consolidation therapy in Hong Kong Chinese in a local hospital.

Method

It was a retrospective study of newly diagnosed APL patients from Jan 1996 to Dec 2009. They were divided into two groups. One group was given ATRA (All-trans-retinoic acid) 45mg/m2/day combined with daunorubicin 60mg/m2/day for 3 days plus cytarabine 200mg/m2/day for 7 days as induction therapy. It was followed by two courses of consolidation with daunorubicin and cytarabine and then 2-year maintenance with low dose chemotherapy and intermittent ATRA. Another group was given the same treatment without cytarabine. The remission rate, relapse rate, overall survival and event-free survival were compared in the two groups of patients.

Results

Eighteen patients with median age of 41 (range 24–62) received cytarabine. 22% of them had initial WBC count >10,000/uL. Eight patients with median age of 42 (range 16–57) received no cytarabine. 25% of them had WBC count >10,000/uL. The complete remission rates were 100% in both groups. The two-year relapse rate was 5.5% (1/18) for cytarabine group and 62% (5/8) for no cytarabine group (p=0.004, Fisher's exact test).

The two-year event-free survival was 82% for cytarabine group and 37% for no cytarabine group (p=0.0017). The two-year overall survival was 89% for cytarabine group and 75% for no cytarabine group (p=0.18). The adverse effects profile was similar in both groups.

Conclusion

The results support a role of cytarabine in addition to ATRA and daunorubicin in the treatment of newly diagnosed APL. The relapse rate was much lower in patients receiving cytarabine. The two-year event-free survival and two-year overall survival were also significantly better in the cytarabine group.

Disclosures:

No relevant conflicts of interest to declare.
Incidence and Clinical Features of Acute Promyelocytic Leukemia In a Population-Based Canadian Cohort
Authors: Paulson Kristjan , Szwajcer David , Ahsanuddin Arshad , Lambert Pascal , Turner Donna , Rubinger Morel , Kumar Rajat , Seftel Matthew D.
Published: October 2019

Abstract 4374

Introduction:

Acute promyelocytic leukemia (APL) is a rare malignancy whose causes and epidemiology are not well known. The incidence of APL in the United States was estimated to be 0.22 cases per 100,000 population, and a higher incidence has been suggested in specific geographic or ethnic sub-groups. However, clinical and epidemiological data on APL arise primarily from clinical trials and single centre experiences rather than population-based data, which may lead to selection bias. We studied a cohort of APL cases in a well defined population using the diagnostic gold standard of polymerase chain reaction (PCR) testing.

Methods:

The Canadian province of Manitoba, with a population of 1.2 million, has a single referral centre for the definitive diagnosis and clinical care of all acute leukemia patients. Only one laboratory is capable of performing PCR based testing for the PML-RARA fusion gene product. We examined consecutively diagnosed patients with APL based on PCR testing from January 1999 until July 31 2010 in order to determine the incidence, clinical characteristics, and outcomes of patients with APL.

Results:

Over the 11.5 year period of observation, 24 patients were diagnosed with APL. Based on the 2006 Canadian census, the annual incidence of APL in Manitoba is thus 0.18/100,000 population. These cases did not appear to be distributed evenly, with an average of 1.1 cases diagnosed annually between 2002 and 2008 compared to six cases diagnosed in the most recent period between 2009 and 2010 (relative to 2002–2008, incident rate ratio (IRR) 2.61, p = 0.075) and ten cases diagnosed between 1999 and 2001 (IRR 2.94, p = 0.023). Symptomatic CNS disease at diagnosis was present in 3 (12.5%) patients. Thirteen patients developed differentiation syndrome during the course of their therapy (54.1%). In all but one patient, ATRA was able to be successfully resumed. Ten patients presented with DIC (41.7%).

Conclusions:

In this population-based study, we noted episodic clustering of new cases and a relatively high proportion of CNS disease at diagnosis. While these observations may be due to normal fluctuations in the patterns a rare disease, this could also suggest an underlying environmental trigger. Additional investigation is required to confirm or refute this hypothesis, due to the relatively small numbers in this cohort. The high rate of CNS disease suggests that this finding maybe more common than previously thought. As the pathogenesis of APL remains unclear, these observations deserve additional epidemiological investigation in larger cohorts.

Disclosures:

Kumar: Celgene: Membership on an entity's Board of Directors or advisory committees.
Dynamics of Procalcitonin and Bacteremia In Adult Acute Myeloid Leukemia with Chemotherapy-Induced Neutropenia
Authors: Gac Anne-Claire , Parienti Jean-Jacques , Chantepie Sylvain , Leclercq Roland , Reman Oumedaly
Published: October 2019

Abstract 4378

Sensitive markers of infection are rare or of limited validity in neutropenic patients. Procalcitonin (PCT), a precursor protein of calcitonin, is a specific and sensitive marker of severe bacterial infections during short-term neutropenia. Because the value of PCT measurements among patients undergoing long periods of neutropenia remains uncertain and because several mechanisms, such as bacterial or fungal infections, reactions to drugs or blood products or tumor-associated events, can cause fever, we described the dynamics of PCT in 29 acute myeloid leukemia (AML) patients with 39 instances of chemotherapy-induced neutropenia. Plasma levels of PCT were determined prospectively by an immunoluminometric assay every four days starting at the onset of chemotherapy and continuing until the resolution of fever. We found that bacteremia did increase PCT levels above 0.5 ng/mL and these levels predicted bacteremia at day 15 of chemotherapy. This finding may be relevant in the decision to alter antibiotic regimens to decrease toxicity and cost when patients remain febrile at day 15.

Disclosures:

No relevant conflicts of interest to declare.