bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Myeloid Differentiation-Associated Autophagy In Combination with ATRA-Therapy Enhances Neutrophil Differentiation of AML Cells.
Authors: Humbert Magali , Britschgi Adiran , Simon Hans-Uwe , Tobler Andreas H. , Yousefi Shida , Fey Martin F. , Tschan Mario P.
Published: October 2019

Abstract 1046

Autophagy is a self-digesting recycling mechanism for cellular organelles and macromolecules with cytoprotective functions. On the other hand, autophagy might also lead to caspase-independent, type II cell-death. Of interest, there is accumulating evidence for non-metabolic, cell specific functions of autophagy in mature hematopoietic cells but also during hematopoietic differentiation. We therefore aimed at investigating the involvement of autophagy in the molecular pathogenesis of acute myeloid leukemia (AML) and, in particular, in acute promyelocytic leukemia (APL). We found significantly increased autophagic activity during ATRA-induced neutrophil differentiation of NB4 and HL60 AML cells as evidenced by increased autophagosome formation (appearance of LC3-GFP dots), LC3-I to -II conversion and degradation of p62 (SQSTM1), which is degraded in the autophagosome. Most importantly, ATRA treatment induced a novel type of autophagy that was independent of the key autophagy gene Beclin-1 as opposed to autophagy induced by starvation or chemotherapeutics. Moreover, autophagy seen during neutrophil differentiation of AML cells was clearly less intense than, for example, starvation-induced autophagy as measured by the number of LC3-GFP dots per cell. We have previously shown that the autophagy gene ATG5 as well as death-associated protein kinase 2 (DAPK2) are crucial in all-trans retinoic (ATRA)-induced neutrophil differentiation. Since it was found earlier that overexpression of DAPK2 can induce autophagy in epithelial cells, we were asking whether there might be a direct link from DAPK2 to ATG5. Indeed, we found direct DAPK2-ATG5 binding when overexpressed in 293T HEK cells. Moreover, we were able to pull down endogenous DAPK2 together with ATG5 in ATRA-treated NB4 APL cells. Using DAPK2 overexpressing NB4 cells, we found increased DAPK2-ATG5 interaction with progressive neutrophil differentiation. Further, knocking down DAPK2 caused decreased ATG5-ATG12 complex formation during AML differentiation. Lastly, we found that pharmacological activation of autophagy in combination with ATRA significantly boosted AML differentiation, representing a potential new strategy for APL therapy. In summary, we show that autophagy is needed for neutrophil differentiation of AML cells, possibly via the newly identified DAPK2-ATG5 pathway, and would like to refer to this new type of Beclin-1-independent, non-canonical autophagy as myeloid differentiation-associated autophagy (MDAA).

Disclosures:

No relevant conflicts of interest to declare.
Mesenchymal Stromal Cells From AML Bone Marrow Are Abnormal by Gene Expression Profiling.
Authors: Ma Wencai , Davis R. Eric , Jacamo Rodrigo , Konopleva Marina , Garzon Ramiro , Croce Carlo M. , Andreeff Michael
Published: October 2019

Abstract 1055

Cytogenetic and other evidence suggests that the mesenchymal stromal cell (MSC) is abnormal in bone marrow (BM) affected by acute myelogenous leukemia (AML). To gain further insight into molecular and physiologic abnormalities, we used Affymetrix HG-U133 Plus 2 microarrays to compare gene expression between BM-MSCs from 12 AML patients and BM-MSCs from 4 normal donors (ND). BM-MSCs were purified by in vitro culture as adherent cells with a purity of over 95%. Comparison at the single-gene level between AML and ND samples found only one differentially-expressed probe by t tests at a false-discovery rate (FDR) of 0.1. Comparison by the gene set enrichment analysis (GSEA) method of Subramanian et al., which is a more powerful way to find small differences that are significantly enriched within sets of biologically-related genes, first found that many enriched gene sets were predominantly the result of data from one AML sample. After excluding this sample, GSEA at an FDR of 0.25 found 115 downregulated gene sets for AML BM-MSCs from the Gene Ontology-based “C5” category of the mSigDB collection of gene sets. 19 of the 20 most significantly enriched downregulated gene sets were related to cell cycle progression, indicating that BM-MSCs are less proliferative in AML than in normal BM. An upregulated enriched gene set in AML BM-MSCs, from the “C2” category of curated gene sets, was composed of extracellular matrix genes for keratins, collagen, and laminin; while surprising, this is consistent with reports of BM-derived MSCs differentiating into epithelial cells after autografting, and suggest that BM-MSCs in AML may remodel the extracellular matrix. Overall, these results indicate that BM-MSCs in AML patients are substantially different from normal BM-MSCs. These and other differences could have substantial effects on the BM microenvironment and therapy response in AML, and should be studied further.

Disclosures:

No relevant conflicts of interest to declare.
Meta-Analysis of CAG (cytarabine, aclarubicin, G-CSF) Regimen for the Treatment of 580 Patients with Acute Leukemia In China and Japan.
Authors: Wei Guoqing , Liu Delong
Published: October 2019

Abstract 1064

Background:

Current therapy is still unsatisfactory in patients with high-risk AML (elderly, relapsed, refractory, and secondary). A CAG regimen has been commonly used in China and Japan for the treatment of this type of patients (pts). The CAG regimen consists of low-dose cytarabine 10 mg/m2 q12 SQ on d1–14, aclarubicin 7 mg/m2, QD on d1–8, or 14 mg/m2, QD on d1–4, and G-CSF 200 μg/m2, QD on d1–14. The aim of this study is to summarize the data and to analyze the efficacy as well as the toxic effects of CAG regimen in acute leukemia (AL) patients.

Methods:

A meta-analysis of 15 trials with a total of 580 AL pts was performed to summarize and analyze the efficacy of CAG regimen as well as the toxic effects. The literature search was conducted in PubMed, Google Scholar, and Medline, as well as ASH and ASCO meeting abstracts.

Results:

Among the 580 pts treated with CAG, 456 pts were AML, 100 pts were MDS/tAML,19 pts were ALL, 5 pts were BAL. 163 pts were newly diagnosed AML, 141/75 pts were relapsed/refractory (R/R). The CR rates of CAG in newly-diagnosed, relapsed, refractory, elderly AML patients were 63.9% (49%-67.6%), 83% (40%-86%), 30.4 (12.5%-48.4%), and 53.5% (31.5%-67%), respectively. The median OS in new, relapsed, elderly AML patients were 14.5m (9m-17m), 16m (15m-17m), and 8m (7m-9m), respectively. Data available from 4 trials which studied the efficacy of CAG in MDS/tAML patients showed that CR rate was 40.5% (38.5%-46.4%). There were only two small studies of CAG in ALL and in BAL that showed ORR of 100%. The toxicity of CAG in all reports were mild and the CAG regimen were well tolerated.

Conclusion:

This low-intensity CAG regimen appears to be very well tolerated with clear activity in high-risk AL pts. Randomized study may be warranted.

Disclosures:

No relevant conflicts of interest to declare.
Recombinant Human Soluble Thrombomodulin Enhances the Anti-Fibrinolytic and Anti-Leukemia Effects of All-Trans Retinoic Acid In Acute Promyelocytic Leukemia Cells.
Authors: Ikezoe Takayuki , Yang Jing , Nishioka Chie , Isaka Mayuka , Iwabu Naomi , Sakai Mizu , Taniguchi Ayuko , Togitani Kazuto , Honda Goichi , Yokoyama Akihito
Published: October 2019

Abstract 1079

Recombinant human soluble thrombomodulin (rTM) is composed of the active, extracellular domain of thrombomodulin, and inactivates coagulation by binding to thrombin. In addition, the thrombin-rTM complex activates protein C to produce activated protein C (APC), which in the presence of protein S, inactivates factors VIIIa and Va, thereby inhibiting further thrombin formation. Use of rTM for treatment of disseminated intravascular coagulation (DIC) associated with haematological malignancies or infections has been approved in Japan, although its effects on hyperfibrinolysis caused by acute promyelocytic leukemia (APL), as well as all-trans retinoic acid (ATRA)-mediated anti-fibrinolytic activity, remain to be fully elucidated. This study examined the effects of rTM on the plasmin activity in APL NB4 cells in vitro and found that rTM (100 nM, 48 h) alone inhibited the plasmin activity by approximately 35% when compared to the control diluent-treated NB4 cells. ATRA (100 nM, 48 h) also inhibited the plasmin activity by 40%, and when ATRA (100 nM, 48 h) was combined with rTM (100 nM, 48 h), the plasmin activity was inhibited by 60%. In further studies, neither anti-proliferative nor pro-differentiative activity was noted in NB4 cells after exposure to rTM (3-100 nM, 48 h). However, rTM significantly enhanced the ability of ATRA to induce growth arrest, differentiation and apoptosis in NB4 cells, as measured by colony forming assay, quantification of CD11b cell surface antigens and nitroblue tetrazolium reduction test, respectively. Western blot analyses showed that ATRA (30 or 100 nM, 48 h) increased nuclear levels of CCAAT/enhancer binding protein e in NB4 cells, a critical transcription factor for myeloid differentiation, which was further increased in the presence of rTM (3-100 nM, 48 h). Furthermore, we treated individuals with DIC caused by APL (n=4) with rTM (380 U/kg) in combination with ATRA (45 mg/m2) and chemotherapy (idarbicine and cytarabine), and compared their clinical outcomes with historical control patients (n=6) treated with ATRA and/or chemotherapy (idarbicine and cytarabine). Notably, treatment with rTM rescued patients from DIC earlier than historical controls (8.3 ± 4.5 days vs 12.5 ± 5.2 days in historical controls) and significantly reduced the transfusion of fresh frozen plasma to maintain plasma levels of fibrinogen at more that 150 mg/dl (0.13 ± 0.25 U/day vs 3.93 ± 1.18 U/day in historical control, p=0.0131). Taken together, the present results suggest that administration of rTM be considered in the treatment of individuals with DIC associated with APL.

Disclosures:

Honda: Asahi Kasei Pharma: Employment.
Determination of the International Sensitivity Index for Rivaroxaban Using the WHO RBT/90 Thromboplastin as a Calibrant for Other Thromboplastin Reagents.
Authors: Harenberg Job , Giese Christina , Marx Svetlana , Kraemer Roland
Published: October 2019

Abstract 1089

Patients on treatment with vitamin-K-antagonists (VKA) are monitored by the international normalized ratio (INR). The international sensitivity index (ISI) standardizes the differences between prothrombin assays (PT) through a comparison with a WHO thromboplastin reagent. Rivaroxaban prolongs PT assays dose dependently. The steepness of these curves differs between the PT assays. PT assays have not been standardized for rivaroxaban using a WHO Thromboplastin reagent.

Rivaroxaban was extracted from commercially available drug with dichloromethane. Identity and purity of the isolated compound were confirmed by mass spectrometry (exact mass 436,0734), elementary analysis (carbon, hydrogen, nitrogen content) and 1H-NMR spectroscopy. Pooled plasma from healthy persons was spiked with 25 ng/ml to 900 ng/ml rivaroxaban. Thromboplastin reagents were WHO RBT/90, neoplastinPlus, recombiplastin, innovin and thromborel. All assays were performed with the manual kolla-hook method and with the KC coagulometer.

Increasing concentrations of rivaroxaban prolonged coagulation values of all PT assays linearly (coefficient of correlation between r=0.97 and r=0.99). The steepness of the dilution curves differed substantially between assays. Accordingly, the ratios for the rivaroxaban concentrations varied (ratio=PTriva(xi)/PTriva(i0)). The coefficient of variation CV of the ratios between methods ranged from 7% to 33%. The ISI for rivaroxaban was calculation for each method by x=(y-a)/b (x=ratio WHO-kolla hook manual method, y=ratio method new, a=intercept, b=steepness). The ISIRiva was 1 for the WHO reagent using the manual method and ranged from 0.94 to 1.67 for the other PT-methods compared to the WHO RBT/90 PT reagent. The equation INRriva=ratiorivaISIriva was used to calculate INRriva. The CVs of INRriva between methods decreased to 1.1% to 7.2% (mean 3.9%) as compared to the ratio without ISIRiva.

The differences of the CV between TP-reagents were reduced to about 4% using the WHO RBT/90 thromboplastin reagent manual method as standard and an ISI for rivaroxaban.

Disclosures:

No relevant conflicts of interest to declare.
Generic Versions of Low Molecular Weight Heparin May Not Have the Same Safety-Efficacy Profile as the Branded Low Molecular Weight Heparins In Acute Coronary Syndrome.
Authors: Hoppensteadt Debra , Jeske Walter , Litinas Evangelos , Iqbal Omer , Walenga Jeanine M. , Lewis Bruce E , Kahn Hussein , Fareed Jawed
Published: October 2019

Abstract 1098

LMWHs lp;&5q;1such as enoxaparin and dalteparin are widely used for the management of ACS. Recently, several generic versions of enoxaparin and dalteparin have been approved in Asian and South American countries for all of the branded product's indications. However, no data on their clinical equivalence in ACS is available. Since generic versions of enoxaparin and dalteparin are manufactured by different processes and may use starting material from different sources, these drugs may differ in their pharmacological profile in simulated ACS settings. A branded version of enoxaparin was compared with three of the generic versions in a primate model at a dosages of 1 mg/kg IV. Such pharmacodynamic parameters such as TFPI release, TAFI modulation, vWF release, and TF mediated platelet activation. In addition, the anticoagulant effects of these drugs were also measured after IV administration using iSTAT ACT and aPTT. Simulated catheter related thrombosis studies were also carried out to differentiate each agent in contact, intrinsic and extrinsic clotting systems. The generic versions of enoxaparin namely, Cutenox (Gland Pharma, India), Fibrinox (Sandoz, Argentina), Versa (EuroPharma, Brazil) exhibited product based pharmacodynamic differences compared with the branded product and the results are provided in the following table.

Drug TFPI (ng/ml) TAFI (% NHP) VWF (% NHP) Enoxaparin 210+10 65+4.8 138+12 Fibrinox 160+10 74+8 175+16 Versa 205+16 59+7 148+12 Cutenox 180+11 60+4 110+12

Each of the generic products exhibited its own specific pharmacologic profile despite comparable molecular weight distributions and anti-Xa potencies. The ACT values at a 10 ug/ml varied from 172–200 seconds among the four products. The ED 50 values in the catheter related thrombogenesis model varied considerably. Significant differences were noted in the anticoagulant effects of each of these agents as compared with the branded products. Differences were also noted in HIT antibody mediated aggregation studies. The HIT antibody mediated aggregation of platelets varied from 17–28%. The pharmacodynamic differences between the branded and generic versions of LMWHs may be due to the higher dosages used and the potential IV administration which leads to higher circulating levels of these agents. These observations suggest the need for additional animal studies and clinical trials to validate the use of generic versions of LMWHs in ACS.

Disclosures:

No relevant conflicts of interest to declare.
Covalent End-Labeling of Polyphosphate Facilitates Studies of Its Procoagulant Activities and Development of Enhanced Agents to Treat Bleeding.
Authors: Choi Sharon H , Davis-Harrison Rebecca L , Smith Stephanie A. , Collins Julie N.R. , Rienstra Chad M , Morrissey James H.
Published: October 2019

Abstract 1138

Introduction:

Inorganic polyphosphates (polyP) are negatively charged, linear phosphate polymers that are abundant in platelet dense granules and secreted upon platelet activation. We recently reported that polyP may be the long-sought (patho)physiologic activator of the contact pathway with important roles in inflammation and thrombosis. We also reported that polyP opposes the action of many anticoagulant drugs and thus has potential as a general procoagulant agent to treat bleeding. Studies of the role of polyP in blood clotting would be facilitated by being able to covalently attach probes – including fluorophores and biotin – to the ends of polyP. For therapeutic applications, it would also be advantageous to covalently immobilize polyP onto solid supports like collagen sponges and wound dressings. We now report that a wide variety of primary amine-containing compounds can be covalently attached to the terminal phosphates of polyP via phosphoramidate linkages. This allows essentially the full armamentarium of protein chemistry to be employed in modifying polyP.

Methods:

We have developed and optimized reaction conditions under which EDAC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide) efficiently promotes the covalent coupling of compounds with primary amines to polyP via the formation of stable phosphoramidate linkages with the terminal phosphate groups (see figure).

Results & Conclusions:

Using 31P NMR, we have confirmed that EDAC-mediated reaction between primary amines and polyP results in stable phosphoramidate linkages with the terminal phosphate groups. We have used this chemistry to efficiently float polyP onto amine-derivatized microtiter plates and chromatography beads, and have used this presentation of polyP to quantify the binding affinities of thrombin, kallikrein, and factor XIa for polyP. We have also successfully attached fluorescent probes to the termini of polyP and thereby visualized the incorporation of polyP into fibrin clots. We have also demonstrated that polyP covalently attached to solid supports via phosphoramidate linkages retains potent procoagulant activity. And finally, we have found that attaching small organic molecules to the terminal phosphates of polyP protects polyP from degradation by exopolyphosphatases such as alkaline phosphatase, which should prolong its in vivo half-life considerably. These findings facilitate more extensive studies of the biological role(s) of polyP, as well as development of enhanced polyP-based treatments for bleeding.

Disclosures:

Smith: University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.
A Novel Dimeric Inhibitor That Targets Dimeric ß2GPI In ß2GPI/Anti-ß2GPI Antibody Complexes.
Authors: Beglova Natalia , Kolyada Alexey , Lee Chang-Jin , De Biasio Alfredo
Published: October 2019

Abstract 1142

ß2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome, an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of ß2GPI generated by anti-ß2GPI antibodies is pathologically important, in contrast to monomeric ß2GPI which is abundant in plasma. Several cell-surface molecules including anionic phospholipids and ApoER2, a member of the lipoprotein receptor family, are implicated in the pathology of antiphospholipid syndrome.

We created a dimeric inhibitor, A1-A1, to selectively target ß2GPI in ß2GPI/antibody complexes. This inhibitor consists of two ligand-binding A1 modules from ApoER2 connected by a flexible linker. A1-A1 disrupts the binding of ß2GPI/antibody complexes with anionic phospholipids and lipoprotein receptors. We compared the potency of A1-A1 to monomeric A1 for inhibition of the binding of ß2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of ß2GPI present in human serum, ß2GPI purified from human plasma and domain V of ß2GPI. We demonstrated that when ß2GPI/antibody complexes are formed, A1-A1 is more effective than A1 in inhibition of the ß2GPI binding to cardiolipin, regardless of the source of ß2GPI. Half maximum inhibition of ß2GPI in human serum in the presence of anti-ß2GPI antibodies was achieved at 9 μM of A1-A1 and 218 μM of A1. Similarly, ß2GPI purified from plasma was inhibited to 50% by 26 μM of A1-A1 and 191 μM of A1. Also, A1-A1 strongly inhibited the binding of dimerized domain V of ß2GPI to cardiolipin compared to monomeric A1. Concentration of A1-A1 and A1 at half maximum inhibition of the binding of ß2GPI domain V to cardiolipin in the presence of dimerization antibodies was 29 μM and 204 μM, respectively. In the absence of anti-ß2GPI antibodies, A1-A1 and A1 were equally inefficient in the inhibition of the binding of monomeric ß2GPI to cardiolipin. The concentration of inhibitor at half-maximum inhibition of the binding of ß2GPI in human serum to cardiolipin was 189 μM for A1-A1 and 176 μM for A1.

A novel inhibitor A1-A1 may be a starting point in the development of an effective therapeutic for antiphospholipid syndrome.

Disclosures:

No relevant conflicts of interest to declare.
Effects of Rabbit Anti-Mouse Thymocyte Globulin Treatment in a Splenocyte-Induced Model of Aplastic Anemia
Authors: Ruzek Melanie C , Phillips Kathleen , Richards Susan , Mamlouk Khalid , Williams John , Garman Richard
Published: October 2019

Abstract 1172

Acquired aplastic anemia is an immune-mediated disease where destruction of hematopoietic stem cells (HSCs) in the bone marrow results in severe and life-threatening pancytopenia. Thymoglobulin® is often used as immunosuppressive therapy in this disease with up to 80 percent of patients responding to a combination of Thymoglobulin and cyclosporine. In an effort to better understand the activities and mechanism of action of Thymoglobulin we developed a mouse model of immune-mediated aplastic anemia and evaluated a murine surrogate of Thymoglobulin®, rabbit anti-mouse thymocyte globulin (mATG) in this model. We modified a graft-versus-host (GVH)-induced model described in the literature (Bloom, et al., 2004) utilizing HSC-depleted spleen cells transferred from C57BL/6 into CByB6F1 mice instead of lymph node cell transfer. Our modified model shows a cell dose-dependent increase in pancytopenia and lethality. Mice receiving a high dose (100×106) of HSC-depleted splenocytes experienced severe pancytopenia and rapid death occurring around day 21 whereas mice receiving lower doses (70×106, 35×106 and 17×106) of cells showed progressively less pancytopenia and lethality as the dose of cells decreased. Histopathology also showed marked loss of hematopoietic progenitor cells in the bone marrow with little evidence of GVHD in other tissues. Prophylactic administration of mATG (25mg/kg, 2x, day 0 and 3) to mice given high doses of HSC-depleted splenocytes (100×106) resulted in a significant improvement in pancytopenia and survival (70%) in this model. Interestingly, therapeutic administration of mATG was more effective when given later relative to disease induction. Delivery of mATG (25mg/kg, 2x, three days apart) starting on day 3 showed some delay in disease progression (day 30 vs day 21) and mATG started on day 6 slightly increased survival (40%). However, mice receiving mATG starting on days 10 or 14 showed a much greater overall survival of 100% and 60%, respectively, with full rebound of hematopoietic cells in the blood to normal levels. The complete response observed with later mATG administration (day 10 or day 14) mimics the treatment and response of patients given Thymoglobulin®. In summary, we have established a novel model of HSC-depleted splenocyte induction of bone marrow failure in mice that is responsive to therapeutic ATG administration. Studies in this model will aid in further understanding the mechanism of ATG in aplastic anemia and may contribute to the development of potential new therapies.

Disclosures:

Ruzek: Genzyme: Employment. Phillips:Genzyme: Employment. Richards:Genzyme: Employment. Mamlouk:Genzyme: Employment. Williams:Genzyme: Employment. Garman:Genzyme: Employment.
Laser Transmyocardial Revascularization (TMLR) Combined with Autologous Bone Marrow Cells (ABMC) for Diffuse Coronary Disease (DCD). Results In 19 Cases
Authors: Alegre Adrian , Aguado Beatriz , Alfonso M. Perez , Muñoz C. , de Landázuri M. Ortiz , Sanchez F. Madrid , Cámara C. , García-Fernández N. , Bascones L. , Martinez S. , García-León N. , Roig M. , Duarte J. , Reyes G.
Published: October 2019

Abstract 1181

Introduction:

Stem cells (SC) show a potential of cellular therapy in diffuse coronary disease with controverted results. We present here an analysis of a group of patients treated with TMLR plus ABMC. (Caiber Program Advanced Therapy Proyect FIS 2009).

Patients and methods:

19 patients with DCD were included. Age 66 (range: 52–78). ABMC were obtained by aspiration and processed using HARVEST-TM system -TALEX (Harvest Technologies Inc. GMBH, Munich) to obtain 20 ml. Surgery consisted of anterior thoracotomy incision at the fourth and fifth intercostal space. Between 15–30 transmyocardial channels were realized with TLMR PHOENIX-TM (Cardiogenesis, Irvine, CA, USA) and CrystalFlex 1 mm optical fibre. 0.5–2 cc of MO were administered in each channel. Total number CMN were determined by automatic cell counter and flow cytometry and were typified specific cell populations such as CD34+ and CD133+.

Results:

The duration of stem cell collection was less than 30 minutes. All the procedure was performed on surgery in 1 step. Any complications were observed with this technique. Correlation between the cellular count and demographic data was not observed. After a follow up of 22 months (5-36) only one patient has died (20m) of DCD. Initial clinical results are satisfactory with a significant reduction in the functional grade of angina.

Comments and conclusions:

Our data show that TMLR plus ABMC is a safe method with clinical efficacy in patients with DCD that are not candidate to other therapy options. The described method offers efficiency and simplicity to obtain cells. More cases and follow-up are required, as well as later functional evaluations.

Disclosures:

Alegre: Celgene: Honoraria. Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma.
Isolation of G-CSF Mobilized Human Mesenchymal Stem Cells by Elutriation.
Authors: Jiang Yajuan , Leary Elizabeth , Yavanian Gregory , Anbazhagan Rajesh , Salerno Anthony , Marasco Wayne , Rodgerson Denis
Published: October 2019

Abstract 1183

Mesenchymal stem cells (MSCs) are a population of adult stem cells that constitute a promising source for therapeutic applications. MSCs exist in various adult tissues, such as bone marrow and umbilical cord blood. The most common method for MSC isolation relies on plastic adherence, however, this isolation procedure is hampered by the unpredictable influence of other co-cultured adhering cells. Phenotypic characterization of MSCs is usually carried out by FACS analysis of cell surface marker expression, but no unique marker has yet been identified. To circumvent these limitations, using granulocyte-colony stimulating factor (G-CSF) mobilized adult human peripheral blood (PB), we explored the use of Elutra® Cell Separation System (Caridian BCT) to isolate MSC enriched fractions based on their size and density.

A group of 28 donors were studied after mobilization for two consecutive day of G-CSF injection (480μg/day subcutaneously). Total nucleated cells (TNC) were collected by apheresis on the third day. An average of 6-fold increase in MSCs in the post-mobilized sample was observed. At least 1 million TNC in pre- and post-mobilized PB were analyzed by FACS for the presence of MSCs by cell surface staining with antibodies to CD105, CD90, CD73, CD31, CD45 and CD34. Circa 3 × 1010 TNC were loaded onto the Elutra to fractionate the apheresis product into several distinct cell fractions. Typically, the MSCs were enriched in 2 fractions, and yielded about 5–10% of the initial loaded cell number. Following culturing and passage, cells from these 2 fractions were grown on plastic with low contamination by other cell types. The cultured cells showed spindle-shaped morphology. Cells were further characterized by FACS analysis and shown to stained positive for mesenchymal specific markers CD90 and CD105; and negative for hematopoietic markers of CD45 and CD34. Furthermore, after cells from all fractions were plated for colony forming unit-fibroblast assay (CFU-F), no colony formation were observed from other than the 2 fractions.

In summary, we demonstrate that MSCs are mobilized into PB by G-CSF and can be collected by apheresis. Size-based Elutra fractionation of apheresis product in a closed system could provide a novel strategy for the large-scale isolation and expansion of human MSCs for autologous cell therapy *Dr. Marasco is a paid consultant for NeoStem and an equity holder.

Disclosures:

No relevant conflicts of interest to declare.
Arsenic Sulfide Binds c-CBL to Promote BCR-ABL Ubiquitination and Degradation In Chronic Myelogenous Leukemia.
Authors: Mao Jian-Hua , Sun Xiao-Yan , Liu Jian-Xiang , Zhang Qun-Ye , Liu Ping , Huang Qiu-Hua , Li Ke-Qin , Zhang Xiao-Wei , Chen Quan , Chen Zhu , Chen Sai-Juan
Published: October 2019

Abstract 1200

Using immunoprecipitation (IP)-2D-nano-HPLC-MALDI-MS-MS, we identified c-CBL in association with BCR-ABL in a multi-protein complex in K562 cells. In vitro ubiquitination and mutagenesis analyses show that c-CBL serves as a specific E3 ligase for ubiquitination of BCR-ABL at K1517. Arsenic sulfide (As4S4) treatment results in increased c-CBL protein level, which promotes ubiquitination and subsequent degradation of BCR-ABL and apoptosis of K562 cells. Elevated c-CBL is necessary and sufficient to recapitulate the effect of As4S4. Interestingly, arsenic directly binds the RING finger domain of c-CBL, inhibiting its self-ubiquitination and degradation, thus leading to accumulation of c-CBL. However, this interaction between As4S4 and c-CBL does not interfere with its E3 ligase activity towards BCR-ABL. Increased c-CBL protein and BCR-ABL degradation are also observed in vivo after As4S4 administration in BCR-ABL leukemia mice. These findings provide insights into the molecular mechanisms of arsenic and its potential therapeutic applications in CML.

Disclosures:

No relevant conflicts of interest to declare.
Distinct Roles for the NF-κB Pathway In Myeloid and Lymphoid Transformation and Leukemogenesis by BCR-ABL.
Authors: Hsieh Mo-Ying , Van Etten Richard A.
Published: October 2019

Abstract 1225

The BCR-ABL tyrosine kinase, product of the t(9;22) Ph chromosome, activates multiple signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have shown that NF-κB is activated in BCR-ABL-expressing cell lines and contributes to transformation of primary B-lymphoid cells by BCR-ABL (Reuther et al., Genes Dev. 1998;12:968), but the mechanism of activation has not been defined (Kirchner et al., Exp. Hematol. 2003;31:504), and importance of NF-kB to myeloid and lymphoid leukemogenesis by BCR-ABL is unknown.

To interrogate the role of NF-κB in BCR-ABL-mediated transformation, we utilized a super-repressor mutant form of IκBα (IκBαSR), which has been used to block NF-κB nuclear localization and transactivation by constitutively sequestering NF-κB in the cytoplasm. Using retrovirus co-expressing BCR-ABL and IκBαSR, we found that IκBαSR blocked nuclear p65/RelA expression and inhibited the IL-3 independent growth of Ba/F3 cells and primary B-lymphoid cells transformed by BCR-ABL. The effect of NF-κB inhibition was primarily on proliferation rather than on cell survival, as there was no increase in apoptosis in cells expressing IκBαSR. When primary bone marrow cells were transduced and transplanted under conditions favoring induction of B-ALL or CML-like myeloproliferative neoplasm in recipient mice, co-expression of IκBαSR significantly attenuated disease development and prolonged survival of diseased mice. Molecular analysis of these leukemias demonstrated that NF-κB inhibition decreased the frequency of leukemia-initiating (“stem”) cells in the CML model, but not in the B-ALL model, and was associated with decreased expression of c-Myc, an NF-κB target. To clarify the mechanism of activation of NF-κB in BCR-ABL-expressing cells, we targeted two upstream kinases that negatively regulate IκBα, IKKα/IKK1 or IKKβ/IKK2. To accomplish this, we engineered retroviruses co-expressing BCR-ABL and kinase-inactive, dominant-negative mutants of IKK1 (IKK1KM) or IKK2 (IKK2KM). Co-expression of either IKK mutant inhibited both B-lymphoid transformation and leukemogenesis by BCR-ABL, as well as induction of CML-like MPN, with IKK1 inhibition more effective than IKK2.

Together, these results demonstrate that NF-κB is activated in part through the canonical IKK pathway in BCR-ABL-expressing leukemia cells, and that NF-κB signaling plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL. In CML, NF-κB may play a role for in generation and/or maintenance of leukemic stem cells. These results validate IKKs as targets for therapy in Ph+ leukemias, and motivate the evaluation of small molecule IKK inhibitors in these diseases.

Disclosures:

No relevant conflicts of interest to declare.
Adherence to Treatment In Patients with Chronic Myelogenous Leukemia During a 10-Year Time Period: A Medical Record Review.
Authors: Yood Marianne Ulcickas , Oliveria Susan A , Hirji Ishan , Cziraky Mark , Davis Catherine
Published: October 2019

Abstract 1235

Introduction:

Over the past 10 years, oral treatment options for chronic myelogenous leukemia (CML) have provided patients autonomy over treatment administration. Low adherence to long-term maintenance therapy is a known challenge in a number of chronic diseases. The correlation between poor adherence and poor clinical outcomes has been previously demonstrated among CML patients in a clinical study setting. Few studies have evaluated adherence to CML treatments in a real-world setting and none have assessed treatment and adherence trends over time in a contemporary cohort of patients. Further, no studies have used medical record review to obtain comprehensive CML diagnosis and treatment information.

Method:

Medical and pharmacy claims from HealthCore Integrated Research Database™ (8.5 million covered US lives during study time period) were used to identify patients with CML (2001 – 2005). Review of medical records validated diagnosis and treatment exposure. Currently, additional data are being collected on this cohort to extend follow-up through 2010, and expand the number of CML patients to include data on the newer CML treatments. To measure adherence we used medication possession ratio (MPR) (number of days' supply of prescription divided by 365) and treatment interruptions (TI) (failure to refill prescription within 30 days of end of supply from previous prescription or clinician-directed discontinuation). For the current analysis, adherence to imatinib treatment was assessed for one year following treatment initiation.

Result:

In the initial study time frame (2001-2005), during which imatinib was indicated as first-line treatment, 216 CML patients treated with imatinib were identified. Mean age at imatinib initiation was 51 years and 42.6% were female. Fifty-one percent of patients had a MPR <85% for the one year period after imatinib initiation and 57% of patients experienced at least one TI during the follow up.

Conclusion:

Our preliminary findings indicate that adherence to imatinib treatment is low in CML patients for the time period studied. This is the first study to use medical record review to validate exposure and adherence by obtaining comprehensive imatinib treatment information on CML patients in a real-world setting and indicates that adherence may be even lower than previously reported. Adherence data from ongoing analyses which includes longer patient follow-up (up to 10 years) and second-generation tyrosine-kinase inhibitors will be presented.

Disclosures:

Hirji: Bristol-Myers Squibb: Employment. Davis:Bristol-Myers Squibb: Employment.
Involvement of Ets Factor Spi-B and Id2 In Waldenström's Macroglobulinemia
Authors: Zhou Yangsheng , Liu Xia , Xu Lian , Hunter Zachary , Sun Jenny , Yang Guang , Ciccarelli Bryan , Patterson Christopher , Treon Steven P.
Published: October 2019

Abstract 129

Waldenström's macroglobulinemia (WM) is an incurable disorder with a lymphoplasmacytic infiltrate in the bone marrow (BM) and IgM monoclonal gammopathy. WM tumor cells show variable differentiation, ranging from mature B-cells to plasma cells, which likely results from failure to fully undergo differentiation. Here we analyzed the expression of several genes involved in B cell differentiation by real time RT-PCR, such as Ets factors, the basic helix-loop-helix (bHLH) E proteins, as well as the inhibitors of DNA binding (Id) proteins which antagonize E protein activity. Comparison of bone marrow CD19+ B cells obtained from 12 untreated WM patients with 15 age-matched healthy donors showed that expression of the Ets factor Spi-B was increased four-fold, while Id2 was decreased three-fold. However, transcript levels of E proteins were similar between these two groups. Furthermore, along with differentiation of primary human CD19+ cells from peripheral blood into CD38+CD20− plasmablasts, Spi-B and Id2 expression levels were significantly decreased and increased, respectively. Ectopic expression of Spi-B in primary human CD19+ cells inhibited the plasma cell differentiation associated with decreased transcription levels of BLIMP1, XBP-1 spliced form, and IRF4. In addition, overexpression of Spi-B in BCWM.1 WM cells also resulted in repressed expression of BLIMP1, XBP-1 spliced form, and IRF4. Conversely, knocking down Spi-B in BCWM.1 WM cells increased IRF4 and Id2 expression. Importantly, in primary WM bone marrow CD19+ cells, knocking down of Spi-B induced CD38+CD20- plasma cell formation and increased expression of BLIMP1, XBP-1 spliced, IRF4, and Id2. Moreover, knocking down Spi-B in primary WM cells decreased Bcl-2 expression. Collectively, our results suggest that Spi-B overexpression plays an essential role in the pathogenesis of WM by repressing factors involved in plasma cell differentiation while promoting tumor cell survival through Bcl-2.

Disclosures:

No relevant conflicts of interest to declare.
Total Therapy 2 (TT2) for Multiple Myeloma (MM): Contributions to Survival Outcomes of Dosing of Thalidomide (T), Dexamethasone (D) and Interferon (I) Maintenance Components.
Authors: van Rhee Frits , Barlogie Bart , Szymonifka Jackie , Anaissie Elias J. , Nair Bijay , Waheed Sarah , Alsayed Yazan , Petty Nathan , Shaughnessy John , Hoering Antje , Crowley John
Published: October 2019

Abstract 1356

Background:

We have previously reported on the roles of VTD (bortezomib, thalidomide, dexamethasone) maintenance components in Total Therapy 3 (TT3), noting the important contributions of cumulative dosing of VTD components for overall survival (OS) and, especially, a detrimental effect of pre-mature drug discontinuation (PMDD) in the case of V (van Rhee et al: Blood 2010). Here we examine in a similar fashion the roles of T, D and I in TT2 toward OS.

Patients and Methods:

TT2 accrued a total of 668 patients, of whom 323 were randomized to the T arm and the remainder to the control arm. The cumulative dosing effects of T and D during induction, transplantation, consolidation and maintenance phases were evaluated while, in the case of I, its contribution in the maintenance phase only was examined. We also examined outcomes according to the number of induction and consolidation cycles applied prior to and after transplantation and according to the number and timeliness of administering both transplant interventions. In order to utilize the entire patient population, post-baseline treatment interventions and responses were treated as time-dependent variables. Multivariate analyses were performed to examine the collective impact on OS and EFS of baseline characteristics, treatment compliance and type of and time to salvage therapy.

Results:

Randomization to T prolonged both OS and EFS, as did timely completion of second transplant and first cycle of consolidation chemotherapy. During most protocol steps, cumulative dosing of both D and T benefited OS or EFS while pre-mature discontinuation of D shortened both OS and EFS; this was upheld in multivariate analyses, whether or not GEP data were included and also when time to salvage therapy was considered. D dosing prior to relapse was an independently favorable variable for post-relapse survival, which also was longer when disease progression did not occur within 2 years. D's dosing role was apparent only in the subset of patients presenting with lowest tertile gene expression levels of the glucocorticoid receptor gene, NR3C1.

Conclusion:

In addition to confirming well-established variables (beta-2-microgobulin, lactate dehydrogenase, cytogenetic abnormalities, gene expression profiling [GEP]-derived risk and molecular subgroups), randomization to T and timely applications of second transplantation consolidation and maintenance phases favorably affected outcomes. We conclude that D dosing, especially in low NR3C1-level myeloma, is an important treatment component that favorably affects global survival.

Disclosures:

No relevant conflicts of interest to declare.
Assessing Haemostatic Efficacy In VWF/FVIII Replacement Therapy Trials Utilizing Subjective Vs. Objective Criteria – a Way to Improve Comparability and Accuracy of Clinical Outcome.
Authors: Kessler Craig M. , Schwartz Bruce A. , Hegener Oliver , Walter Olaf , Knaub Sigurd
Published: October 2019

Abstract 1424

Objective:

The assessment of the clinical efficacy during the conduct of a clinical trial is critical to the potential acceptance of that product to be a viable therapeutic option. This is especially true in the conduct of clinical trials involving rare bleeding disorders such as VWD, where there is predictably small patient enrollment and limited use of a control group. A 4-point Likert hemostatic efficacy scale with mostly subjective general criteria (excellent, good, moderate, none) has been used previously in most clinical trials published to date in the VWD field.

Methods:

A validated and more objective set of criteria, intended to more specifically assess the therapeutic efficacy of VWF/FVIII replacement therapy was developed. These objective criteria include: quantization of transfusions of blood components; the need for salvage therapy utilizing alternative VWF/FVIII concentrates or other hemostatically active products; the requirement for significant dosing increases of replacement products; etc.

Patients in a VWD clinical trial with a new VWF/FVIII replacement product, wilateÒ (Octapharma AG, Lachen, Switzerland), were assessed for clinical hemostatic efficacy in the treatment of their acute bleeding episodes, using both the traditional 4 point Likert scale, as assessed by the treating physician, and the newly proposed set of objective success and failure criteria for bleeding episodes during treatment, as determined by evaluation of the actual clinical data generated for each individual patient.

Results of the analysis using the new objective criteria when compared to the previous subjective efficacy assessment approach showed a comparable efficacy profile. This proposed objective hemostatic efficacy assessment examined specific clinical criteria that could be considered as indicative of a more accurate status of treatment.

Conclusions:

This objective approach avoids the potential pitfalls of observer bias and inconsistency, which form the basis of criticism of the previously used subjective assessment tools. Furthermore, this more objective type of assessment approach has been requested and accepted in recent registration trials by FDA in the US.

Disclosures:

Kessler: Grifols S.A.: Research Funding; Baxter-Immuno: Research Funding; NovoNordisk: Research Funding. Schwartz:Octapharma: Employment. Hegener:octapharma: Employment. Walter:Octapharma: Employment. Knaub:Octapharma: Employment.
Romiplostim (N-Plate) In Children with Chronic Refractory ITP.
Authors: Elalfy Mohsen Saleh , Abdelmaksoud Abeer Ahmed , Eltonbary Khadiga Yehia
Published: October 2019

Abstract 1443

Background:

Chronic immune thrombocytopenic purpura (ITP) is characterized by platelet deficiency due to platelet destruction and/or inadequate production. Romiplostim stimulates thrombopoietin receptor to increase platelet production in chronic ITP. This study aimed to evaluate the safety and short term efficacy of Romiplostim (N-Plate) in children with chronic refractory ITP.

Design and Methods:

Eighteen patients less than 16 years old suffering from refractory chronic ITP for more than a year who failed to maintain response on more than two therapeutic modalities (steroids, IVIG, Anti-D and/or immunosuppressive therapy) were enrolled. Written informed consent was taken from all parents and consent or ascent form from patients when applicable. Patients were randomized (2:1) to receive Romiplostim or placebo for 12 weeks, initiated at 1μg/kg/week escalated weekly to 5μg/kg/week; then tapered based on weekly platelet count and were followed up to week fifteen.

Results:

The median age of the studied group was 8.5 years, median duration of thrombocytopenia was 3.5 years and the median baseline platelet count (PC) was 10.5 × 109/L. The median weekly dose of Romiplostim was 2μg/kg. Fifty percent of patients in both the Romiplostim and placebo arms had at least one AE with no serious AEs. Ten patients (83.3%) on Romiplostim achieved efficacy endpoint (platelet count of > 50 × 109/L for 2 consecutive weeks without rescue therapy at any point during the treatment period) and 6 patients (50%) maintained that response 3 weeks after treatment discontinuation in contrast to non in the placebo group. Four of the patients on Romiplostim reached the targeted range (>50 × 109/L) after the 2nd week and 11 patients reached the targeted range after the 5th week. None of our patients experienced thrombocytosis or rebound thrombocytopenia. The peak PC was reached in the Romiplostim arm by the 5th week of treatment at a dose of 5ug/kg with median PC of 73.50 ×109/L; compared to 18.50 ×109/L for the placebo group. Rescue medication was administered to 1/12 (8.3%) patient on Romiplostim due to accidental head trauma compared to 2/6 (33.3%) patients on placebo. Number of patients in the Romiplostim arm with grade 3 bleeding severity decreased from 33.3% (prior to study) to 0% (during the study and then after) and from 50% to 16.6% for grade 2.

Conclusion:

Romiplostim is well tolerated in children with refractory chronic ITP with no serious AEs. It was efficient in treating most of them with minimal bleeding during the study period while using lower doses of N-Plate compared to previous published data.

Abbreviations:

ITP; immune thrombocytopenia. PC; platelet count. AE; adverse event.

Disclosures:

No relevant conflicts of interest to declare.