bluuebunny/crossref_metadata_2025_split huggingface dataset:
Whole Kinome Profiling In Pediatric Acute Leukemia.
Authors: Gombert Michael , Krell Pina Fanny Ida , Linka René Martin , Knapp Stefan , Borkhardt Arndt 
Published: October 2019

Abstract
Abstract 1029
With almost 130.000 entries in PubMed by today, the kinase family represents one of the gene families with the best documented history in malignant diseases.
Acute myeloid leukemias account for 4.7% of all childhood cancers and 13.7% of all childhood leukemias. Recent german statistics (Kinderkrebsregister Mainz) indicate a 5-year survival rate of only 66%, emphasizing the need to discover novel therapeutic targets and prognostic markers. Here we present an adaptable workflow to detect mutations in kinase family members on genomic and expression level as well as in silico assessment of their functional consequences.
To undertake the venture of resequencing the human kinome of leukemia patients, we constructed a custom SequenceCapture microarray (NimbleGen) based on all exons of all 518 members of the kinase family previously reported by Greenman and colleagues (Greenman et al, Nature, 2007) as well as a set of TaqMan® low density arrays (Life Technologies) to confirm the expression of potential candidates in tumor material. Genomic DNA from the initial tumor sample and germline material from a 16 year old female patient diagnosed with a AML FAB1 with Auer bodies, 93% blasts, CD33+ CD117+ CD38+ and partial CD15+ and from a 16 year old female patient with a bilinear CD22+ CD33+ leukemia was enriched on SequenceCapture arrays and sequenced on the Roche GS FLX Titanium platform. Acquired sequences were mapped against the current human reference (hg19/GRCh37). RNA derived from the initial leukemic blasts was used for kinome-wide expression profiling on TaqMan low density arrays.
From sequencing the enriched DNA, we recieved between 85 and 129 megabases of tumor and germline sequence, of which 97.7–99.4% mapped to the human reference. About 90% of the targeted regions were covered at least once. Mapping and comparison to the germline material led to 28 and 46 tumor specific non-synonymous single nucleotide variants sequenced at a depth of ≥10x that were not previously reported as SNPs (dbSNP130). Analyzing these candidates with UniProt revealed that 6 and 12 were located in or very close to important functional domains of their respective protein. Structural protein models of one patients remaining mutations underscore the potential influence of detected mutations on the protein function. TaqMan® expression profiling of all kinases present on the SequenceCapture Array identified 3 and 4 of the remaining genes to be expressed in the tumor on mRNA level. In conclusion, starting from 518 candidates, we present a powerful workflow for high throughput analysis of the whole kinome in leukemic samples as well as CDK4, DYRK1B, GSG2 (pt1) and MASTL, MARK3, RP6KA5, CLK4 (pt2) as potential targets for further functional analysis.

Disclosures:
No relevant conflicts of interest to declare.
Pharmacokinetic, Pharmacodynamic and Pharmacogenetic Determinants of Osteonecrosis In Children with Acute Lymphoblastic Leukemia.
Authors: Kawedia Jitesh D. , Kaste Sue , Pei Deqing , Panetta John C , Cai Xiangjun , Cheng Cheng , Neale Geoffrey A. , Howard Scott C , Evans William E. , Pui Ching-Hon , Relling Mary V. 
Published: October 2019

Abstract
Abstract 1033
Osteonecrosis is a debilitating corticosteroid-induced toxicity in patients treated for acute lymphoblastic leukemia (ALL). Our goal was to ascertain genetic and non-genetic risk factors for this complication. In St. Jude Total XV protocol for children with newly diagnosed ALL, we prospectively screened 365 patients with magnetic resonance imaging of hips and knees, irrespective of symptoms. We determined whether age, race, sex, ALL treatment arm, body mass index, serum lipids, albumin and cortisol levels, dexamethasone pharmacokinetics and genome-wide germline genetic polymorphisms were associated with symptomatic (grade 2–4) osteonecrosis. Sixty-eight patients developed symptomatic osteonecrosis with a cumulative incidence of 17%. Age greater than 10 years (odds ratio 4.8; 95 % confidence interval [CI], 2.5–9.2; p = 0.00001) and treatment arm (standard/high risk vs. low risk) (odds ratio 2.5; 95% CI 1.2–4.9; p = 0.011) were associated with increased risk of osteonecrosis, and were included as covariates in all other analyses. Lower serum albumin (p = 0.04) and higher serum cholesterol levels (p = 0.02) were associated with symptomatic (grade 2–4) osteonecrosis. In addition, higher dexamethasone plasma exposure (area-under-the-curve) was associated with severe (grade 3–4) osteonecrosis (p = 0.0007). After adjusting for clinical features (age and treatment arm), we identified several single nucleotide polymorphisms (SNPs) associated with the risk of developing osteonecrosis. These include: ACP1 (rs12714403, p = 1.03 × 10-5; odds ratio 4.8; 95% CI 2.4–9.6), a gene involved in regulation of lipids and osteoblast differentiation; and SH3YL1 (rs4241316, p = 5.7 × 10-6; odds ratio 5.0; 95% CI 2.5–10.1). These SNPs were also associated with lower albumin and elevated serum cholesterol levels. In conclusion, older age, lower albumin levels, increased dexamethasone systemic exposure, and higher cholesterol levels were associated with osteonecrosis. Inherited genomic variation that predisposes to osteonecrosis may do so via pleiotropic effects on dexamethasone pharmacokinetics, serum cholesterol and serum albumin.

Disclosures:
Pui: EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria. Relling:St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding.
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
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
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
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
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
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
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
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.