bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ten Years and Over 3600 Cases of Recombinant Activated Factor VII Use: What Does the Haemostasis Registry Data Tell Us?
Authors: Phillips Louise E , Zatta Amanda J , Isbister James , Dunkley Scott , McNeil John , Cameron Peter 
Published: October 2019

Abstract
Abstract 3352

Background:
Recombinant activated factor VII (rFVIIa, NovoSeven®) is approved for the treatment of spontaneous and surgical bleeding in patients with haemophilia A or B and with antibodies to either factor VIII or factor IX. However rFVIIa has increasingly been used for indications outside the approved areas as a last resort treatment in trauma, cardiac surgery and other critical bleeding episodes despite the absence of conclusive randomized clinical trial (RCT) data.

Aims:
The aim of the Haemostasis Registry was to collect data (including dose, adverse events and outcome) on the off-license use of rFVIIa in Australia and New Zealand.

Methods:
Monash University established the Haemostasis registry in 2005 with an educational grant from Novo Nordisk Pharmaceuticals. More than 100 hospitals (including all major users of rFVIIa in Australia and New Zealand) contributed data to the Registry covering a ten year period (2000 to 2010).

Results:
More than 3600 off-license rFVIIa cases have been reported to the Registry. 65% of cases were male and the median age was 56 years. Major areas of use were cardiac surgery (∼43%), other surgery (18%) and trauma (13%). The majority (∼77%) of patients received a single dose of rFVIIa with a median (IQR) dose of 91 (73-103) mcg/kg. Seventy-four percent of cases documented a response (decrease or cessation of bleeding) following rFVIIa. Patient mortality rate at 28 days was lower if bleeding was reportedly stopped or decreased (88% or 82% vs. 39% no change, respectively) (χ22=533, p<0.001). Stepwise logistic regression analysis identified the number of units of RBC's, pH and context of bleeding as significant independent predictors of patient response to rFVIIa administration and pH, context of bleeding and units of FFP were included in the best predictive model of patient outcome at 28 days. Thromboembolic adverse events were reported in 7.5% of all patients however risk-adjusted adverse event rates in Haemostasis Registry cardiac patients were not significantly different from those seen in non-rFVIIa treated cardiac patients recorded in the ASCTS registry (www.ascts.org).

Conclusions:
The Haemostasis Registry is the largest dataset of its kind and provides critical observational data on the importance of pH, temperature and coagulopathic state in determining the response to rFVIIa. Although the role of rFVIIa in critical bleeding is still not clear, the Haemostasis Registry has been an invaluable resource for rigorously tracking adverse events and helping to guide clinical practice in an arena unlikely to be supported by new RCT data.

Disclosures:
Phillips: Novo Nordisk: Educational Grant. Off Label Use: rFVIIa; used for the treatment of critical bleeding in trauma, cardiac surgery, liver and obstetrics. Zatta:Novo Nordisk: Educational Grant. McNeil:Novo Nordisk: Educational Grant. Cameron:Novo Nordisk: Educational Grant.
Hypomorphic Lig4 Impacts on Igh Class Switching and Translocations.
Authors: Troppito Logan , Muniandy Parames , Hedouin Sabrine , Silva Joshua , Feldman Lauren , Wu Christine , Yan Catherine T. 
Published: October 2019

Abstract
Abstract 3373
The DNA Ligase IV (Lig4) protein is an essential component of the non-homologous end-joining (NHEJ) repair pathway that plays a key role in the repair of general DNA double strand breaks (DSBs) and those generated during V(D)J recombination and IgH Class Switch Recombination (CSR) in B lymphocytes. Hypomorphic mutations in the Lig4 gene in humans underlies the DNA Lig4 Syndrome, which is associated with radiosensitivity, developmental delay, growth defects, facial dysmorphism and variable degrees of immunodeficiencies. We recently described a mouse model carrying a specific Lig4 hypomorphic mutation in both copies of the Lig4 coding exon that generates an arginine to histidine amino acid #278 (R278H) replacement that corresponds to the first Lig4 mutation reported in humans. Our studies revealed that similar to human patients, the phenotype of homozygous mutant mice Lig4R278H/R278H (Lig4R/R) includes growth retardation, significant decreased life span, severe cellular sensitivity to ionizing radiation, and a severe but incomplete block in T and B cell development. Peripheral T lymphocytes show an activated and anergic phenotype, reduced viability, and a restricted repertoire, reminiscent of human leaky SCID, and high levels of genomic instability associated with increased incidence of thymic and peripheral T lymphomas. Severe defects in B lymphocyte developmental functions include repertoire restriction during aging, spontaneous low affinity antibody production and inability mount high affinity antibody responses. Our studies now reveal that introduction of IgH and IgL knock-in (HL) variable region exon into the R/R mice leads to significant increase in mature B lymphocytes in peripheral lymphoid organs, directly demonstrating that the overall severe reduction in B lymphocytes in the R/R mice is in part due to a block in their developmental progression. We further observe a mild reduction in IgH class switching in the in vitro CSR activated R/RHL B lymphocytes, accompanied by an unusual increase in IgH specific DSBs and translocations, at levels significantly higher than what we previously observed in Lig4 or XRCC4 deficient peripheral B cells, and lower than expected general DSBs. The implications of our findings in the context of B lymphocyte development and CSR, potential altered utilization of alternative end-joining DSB repair mechanisms and on B lymphomagenesis will be discussed.

Disclosures:
No relevant conflicts of interest to declare.
Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.
Authors: Gioia Romain , Leroy Cedric , Drullion Claire , Lagarde Valérie , Roche Serge , Mahon Francois-Xavier , Pasquet jean-Max 
Published: October 2019

Abstract
Abstract 3376
Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML.

Disclosures:
No relevant conflicts of interest to declare.
Effects of the Second Generation Tyrosin Kinase Inhibitors on Osteogenic Differentiation.
Authors: Tibullo Daniele , Barbagallo Ignazio , Giallongo Cesarina , Cava Piera La , Branca Antonio , Conticello Concetta , Stagno Fabio , Chiarenza Annalisa , Palumbo Giuseppe A , Raimondo Francesco Di 
Published: October 2019

Abstract
Abstract 3386
It has been demonstrated that long term treatment of CML patients with Imatinib (IM) is associated with altered bone and mineral metabolism. The mechanisms that are responsible for this effect are not fully understood but an inhibition of the PDGF-Rβ (Platelet Derived Growth Factor- Receptor beta) axis has been documented. In order to evaluate if stimulation of osteoblastogenesis is a common feature of other tyrosine kinase inhibitors (TKIs) approved for the treatment of patients with CML, we evaluated the osteoblatic differentiation of Mesenchymal Stem Cells derived from adult bone marrow donors (BM-MSCs) after in vitro treatment with Dasatinib (DA), Nilotinib (NI) or Bosutinib (BO). BM-MSCs were induced to differentiate in osteoblastic cells by treatment with osteogenic medium (OM) with or without DA, NI or BO. BM-MSCs was induced to differentiate in osteoblastic cells by treatmet with osteogenic medium (0.2 mM ascorbic acid, 0.1 μ m dexamethasone and 10 mM β-glycerophosphate, OM) with or without DA 2nM, BO 5nM or NI 100nM. After 21 days of treatment, in the cultures treated with DA e NI for 21 days we have observed a significant increase of extracellular mineralization and of osteogenic markers such as bone morphogenetic protein (BMP2) (p<0.005), osteocalcin (OCN) (p<0.002), and Runx2/cfba1, (p<0.002) measured by real time PCR. OPG/RANKL ratio (measured by ELISA) was also increased in surnatant of cultures treated with DA and NI (p<0.002). All these phenomena were not observed when BO was added to the cultures.
In conclusion, we show that besides IM, other TKIs such as DA and NI, but not BO, increase osteogenic markers in hBM-MSCs. Since BO differs from the other TKIs because of its low affinity to PDGF-R, these experiments suggest that inhibition of PDGF-R is a major pathway in the induction of osteoblastogenesis by TKIs. In addition, a perturbation of bone metabolism induced by TKi may interfere with physiological growth and have negative consequences in children and adolescents. In this subset of patients, treatment with BO could became the first choice because we have demonstrated that this drug does not affect osteoblastogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Role of Stromal Microenvironment In Non-Pharmacological Resistance of CML to Tyrosine Kinase Inhibitors through Lyn/CXCR4 Interactions In Lipid Rafts.
Authors: Tabe Yoko , Jin Linhua , Yixin Zhou , Ichikawa Naoki , Iwabuchi Kazuhisa , Miida Takashi , Cortes Jorge , Andreeff Michael , Konopleva Marina 
Published: October 2019

Abstract
Abstract 3390
In chronic myeloid leukemia (CML), the mechanisms of resistance to tyrosine kinase inhibitors (TKIs) beyond the Bcr-Abl mutations are not well understood. We have previously reported that TKI imatinib induces cell-surface expression of the chemokine receptor CXCR4, which results in enhanced migration towards CXCL12-producing BM stromal cells, promotes cell quiescence and development of the microenvironment-mediated, non-pharmacological drug resistance (Jin, Mol Cancer Ther 2008;7:48). Bcr-Abl tyrosine kinase directly activates Src-related kinase Lyn known to frequently localize in lipid raft plasma membrane microdomains and interact with CXCL12/CXCR4 signaling and is directly activated by p210Bcr-Abl. In this study, we investigated the effects of TKIs on the localization and interaction of CXCR4 and Lyn in the lipid rafts, and the role of lipid rafts as the signal transduction platform for CML cell migration. Confocal microscopy and discontinuous sucrose density gradient fractionation demonstrated that in CML cells CXCR4 primarily localized in the non-raft cell surface regions, while Lyn was present both in the lipid raft and non-raft fractions. In turn, the active, phosphorylated form (p-)LynTyr396 is present within the lipid rafts, while inactive p-LynTyr507 in non-raft fractions. Imatinib treatment under co-culture with mesenchymal stem cells (MSC) induced CXCR4 clustering in lipid raft fractions, which was directly co-immunoprecipitaed with Lyn. Under these culture conditions, imatinib repressed p-LynTyr507, but failed to deplete p-LynTyr396. Knock-down of Lyn by siRNA, Src inhibitor treatment, or lipid raft destruction by methyl-b cyclodextrin (MbCD) abrogated imatinib-induced KBM5 migration to MSCs and CXCL12 without affecting CXCR4 surface expression. Consistent with its effects on Src, dual Src/Abl kinase inhibitor dasatinib induced significantly less migration of CML cells to CXCL12 compared with imatinib or nilotinib (p =0.04). In summary, our data indicate that stromal cells interfere with inhibitory effects of TKI on active Lyn (p-Lyn)Tyr396 in CML cells and promote clustering of CXCR4 in lipid rafts where it co-localizes with p-LynTyr396 and facilitates migration of CML cells to the MSC monolayer. Lipid raft disruption by cholesterol depletion inhibit CML cells migration, suggesting that lipid rafts represent one of the key signaling modules responsible for interactions of CML cells with cells of BM niche. We propose that pharmacological disruption of lipid rafts may eliminate BM-resident CML cells through interference with microenvironment-mediated resistance.

Disclosures:
No relevant conflicts of interest to declare.
Role of Protein Disulfide Isomerase In Thrombus Formation In a Collagen-Induced Pathway of Thrombus Formation
Authors: Martin Lola Bellido , Furie Bruce , Furie Barbara C. 
Published: October 2019

Abstract
Abstract 345
We have demonstrated that platelet activation in vivo can take place via two pathways, one initiated by the generation of thrombin and the other initiated by the exposure of collagen on the injured vessel wall (Furie & Furie, 2008). In our laser-induced model of thrombosis, in which the endothelium is activated but intact, platelet activation by thrombin dominates and collagen is not required. In the widely used ferric chloride model of thrombosis the endothelium is denuded exposing collagen which leads to initial platelet activation. Using our laser-induced thrombosis model we previously demonstrated that protein disulfide isomerase is expressed on the vessel wall and within the platelet thrombus at the site of injury. Both bacitracin A, a non-specific inhibitor of thiol isomerases, and an inhibitory antibody specific for protein disulfide isomerase (RL90) block platelet thrombus formation and fibrin generation. Here we extend our study of the role of protein disulfide isomerase in thrombus formation to the ferric chloride model of thrombosis. We used intravital fluorescence microscopy in mouse arterioles exposed to filter paper saturated with 10% ferric chloride for 3 minutes. Protein disulfide isomerase, detected with a non-inhibitory polyclonal anti-protein disulfide isomerase antibody, accumulated in ferric chloride-induced platelet thrombi in cremaster arterioles. Bacitracin A (5 mg/mouse) delayed initiation of thrombus formation in mesenteric arterioles. Median time to initial platelet accumulation increased from 1 min in the absence of inhibitor to 4 min in the presence of inhibitor. In 4 out of 8 mice treated with 7 mg of bacitracin A platelet accumulation was completely inhibited. Similarly, bacitracin A prolonged the time to occlusion of ferric chloride-injured arterioles. Less than 50% of injured arterioles in mice treated with 5 mg of bacitracin A and only 25% of injured arterioles in mice treated with 7 mg of bacitracin A occluded after 30 minutes compared to a 100% of arterioles occluded in control saline treated mice. Pretreatment of mice with RL90 at 0.3, 1 or 3 μg/g mouse delayed the appearance of the first aggregates of platelets. Median time to initial platelet accumulation was prolonged from 0.6 min in the presence of isotype-matched control antibody (1 ug/g mouse) to 1.5 min in the presence of 0.3 mg/g mouse of RL90. Platelet accumulation was not observed in 1 out of 7 animals treated with RL90 at 1 mg/g mouse and in 3 out of 7 animals treated with RL90 at 3 mg/g mouse. RL90 also inhibited fibrin deposition after ferric chloride injury. Minimal fibrin was detected in the presence of RL90 at 1 μg/g mouse while fibrin appeared rapidly in mice treated with a control antibody. These data indicate that PDI is a component of a general regulatory pathway for initiation of thrombus formation.

Disclosures:
No relevant conflicts of interest to declare.
Chronic Kidney Disease and That Risk Factor In Patients Alive More Than 10 Years After Allogenic Hematopoietic Stem Cell Transplantation.
Authors: Shimoi Tatsunori , Ando Minoru , Kobayashi Takeshi , Kakihana Kazuhiko , Yamashita Takuya , Ohashi Kazuteru , Akiyama Hideki , Sakamaki Hisashi 
Published: October 2019

Abstract
Abstract 3463

Introduction:
Chronic kidney disease (CKD) is common in survivors of hematopoietic stem cell transplantation (SCT). However, evolution over time of kidney dysfunction and its association with post-SCT acute kidney injury (AKI) are unclear.

Methods:
A retrospective cohort study was performed in 86 myeloablative allogenic SCT patients who received SCT between 1990 and 1999 and lived without relapse for 10 years or more. CKD was defined as a sustained decrease in estimated GFR less than 60 ml/min/1.73 m2 at least for a period more than 3 months. Post-SCT AKI was classified into three stages according to the acute kidney injury network (AKIN) criteria within 100 days after SCT. Incidence of new-onset CKD was studied by 1-year interval along the course of follow-up. Cumulative CKD incidence was evaluated by the Kaplan-Meier analysis. The factors associated with CKD at the time of 10 years after SCT were examined using Cox regression analysis.

Results:
The incident of new CKD was the highest (10.5%) at the first year after SCT and then remained almost constant (2.3 to 3.5%) (Figure 1). The prevalence of CKD increased along the follow-up time (Table 1). The cumulative incidence of CKD increased according to increasing AKI stages with significant difference between stages ≥1 and no AKI (Figure 2). Cox regression showed that each AKIN stage was a significant predictor of CKD: stage 3: hazard ratio (HR) 12.7, 95% confidence interval (CI) 2.42–97.6; stage 2: HR 7.75, 95% CI 1.83–53.6; and stage 1: HR 4.36, 95% CI 1.06–29.5. Other predictors included total body irradiation (TBI) (HR, 4.00; 95% CI, 1.63–10.5) and age on SCT (HR, 1.08; 95% CI, 1.03–1.13).

Conclusions:
CKD accumulated among long-term survivors receiving myeloablative allogenic SCT. Post-SCT AKI, regardless of the AKIN stages, is the most significant risk of CKD in such SCT population.

Disclosures:
No relevant conflicts of interest to declare.
Acute Renal Failure Is a Common Presentation of Engraftment Syndrome In Light Chain Amyloidosis (AL) Patients After Autologous Stem Cell transplantation.
Authors: Irazabal Maria V , Eirin Aflonso , Gertz Morie Abraham A. , Dispenzieri Angela , Kumar Shaji , Buadi Francis , Lacy Martha , Hayman Suzanne R. , Dingli David , Hogan William J , Leung Nelson 
Published: October 2019

Abstract
Abstract 3468
Engraftment syndrome (ES) is a complication of hematopoietic stem cell transplantation classically characterized by fever, rash, diarrhea, weight gain and non-cardiogenic pulmonary edema. Renal failure has been observed but is not considered to be a major part of the syndrome. We have noted that acute renal failure (ARF) is common around the time of leukocyte engraftment in light chain amyloidosis (AL) patients after autologous stem cell transplant (ASCT). We suspect this is due to ES and this retrospective review was conducted to further study this entity. Data were collected from 377 AL patients who underwent ASCT from 7/1997 to 10/2009. Patients who experienced an elevation of serum creatinine (Scr) > 0.5 mg/dl within 4 days of leukocyte engraftment were selected. Patients who carried a diagnosis of ES were also included regardless of Scr elevation. Nephrotoxic insults were reviewed. Vitals were collected. Forty-one patients met criteria. Four patients were found not to have ES, 2 sepsis, 1 allergic interstitial nephritis (AIN), 1 acute cellular rejection. ES was noted in 37 (9.8%) patients, 2 presented with mainly pulmonary ES and 8 had both pulmonary and renal involvement. The degree of renal failure varied from mild and self-limited to severe and requiring dialysis. The renal failure is usually preceded by fever, rash, increasing edema, relative hypotension and low urine sodium (Figure 1). Treatment with glucocorticoids (60 mg/d) was successful in reversing the renal failure in most patients but those with pulmonary involvement required higher doses. Our study showed that ES presents more commonly with renal failure than pulmonary involvement in AL patients. Further complications may be prevented by early treatment with corticosteroids once infection is ruled out. Dosing depends on concurrent pulmonary findings. Distinguishing ES with AIN is difficult as presentations are similar but AIN will continue until the offending drug is removed while ES generally responds quickly to steroids alone.
Figure 1. Figure 1.

Disclosures:
Dispenzieri: Celgene: Honoraria, Research Funding; Binding Site: Honoraria. Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.