bluuebunny/crossref_metadata_2025 huggingface dataset:
No Thrombotic Risk in Patients with Asymptomatic Antiphospholipid Antibodies
Authors: Zawilska Krystyna M , Kopydlowska Agata M
Published: October 2019

Abstract 5254

Aim of the study.

Antiphospholipid antibodies (APA) are well known risk factor for thromboembolic events and/or obstetric complications. They may be found incidentally in patients without thrombotic complications (asymptomatic APA), and they often lead to an incorrect suspicion of hemorrhagic diathesis, as well as to an unnecessary disqualification from surgical procedures or withdrawal from a proper antithrombotic prophylaxis during these procedures. The aim of the study was to register venous and/or thrombotic events in a group of patients with asymptomatic APA, diagnosed according to the international guidelines (Myakis et all. 2006).

Material and methods.

The study group consisted of 25 patients (18 women and 7 men) of the mean age of 46 years (20 – 75 years). Concomitantly 9 of them had other autoimmune disorders (no systemic lupus erythematosus) and 4 had neoplasms. Among risk factors for arterial thrombosis 5 patients had hypercholesterolemia, 5 – hypertension, 4 were smokers and 4 were obese (BMI >30 kg/m2). None of the patients had hereditary thrombophilia (antithrombin, protein C or protein S deficiency, factor V Leiden, prothrombin G20210A mutation, increased activity of factor VIII). Family history of venous thromboembolic disease has been noticed in 4 patients and of arterial thrombosis in 6 patients. The observation lasted for 3 to 127 months (mean 35 months).

Results.

The number and percent of patients (n = 25) with abnormal results 997337of different laboratory diagnostic assays for APA:

aPTT-activated partial thromboplastin time; dRVVT - diluted Russell viper venom time; ACA-anticardiolipin antibodies; β2-GPI – anti-β2–glycoprotein I antibodies

Abnormal results of more than one assay in any combination were found in 16 patients, lupus anticoagulant alone in 9 patients. Only 4 out of 25 patients have taken aspirin - 75mg daily, in the other 4 a prophylactic dose of low molecular weight heparin was administered temporarily because of surgery.

During observation time no venous or arterial thrombotic events occurred in the study group. On a basis of a limited number of previous studies that predominantly included systemic lupus erythematosus patients, aPL-positive patients without previous thrombosis have a 0% to 3.8% annual incident thrombosis risk (Barbhaiya M, Erksan D. Curr Rheumatol Rep. 2011; 13: 59–69).

In two patients asymptomatic APA disappeared.

Conclusion:

independently of the type and quantity of asymptomatic antiphospholipid antibodies, there were no venous or arterial thromboembolic events in the group of patients observed for meanly 35 months.

Disclosures:

No relevant conflicts of interest to declare.
The Risk of Malignancy Following Idiopathic Venous Thromboembolism: A Population-Based Cohort Study
Authors: Kondal Dimple , Kahn Susan R. , Tagalakis Vicky
Published: October 2019

Abstract 5255

Background:

Venous thromboembolism (VTE), encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE), can be the earliest manifestation of an occult malignancy.

Objectives:

We used the hospital discharge (MED ECHO) database of the province of Quebec, Canada to determine the risk of a new cancer diagnosis in patients with idiopathic VTE.

Methods:

Using data from Med-Echo which systematically records information on all hospital admissions in Quebec, we constructed a cohort of all individuals with a first-time diagnosis of DVT or PE between January 1, 1996 and December 31, 2004 and no cancer diagnosis preceding the VTE. Subjects were excluded if there was a surgical procedure, pregnancy, trauma or hospitalization in the 3 months prior to the VTE. Subjects were followed for 12 months after VTE for a first diagnosis of cancer (except non-melanoma skin cancer). VTE and cancer were defined using the 9th edition International Classification of Diseases codes.

Results:

In all, 20 740 patients with idiopathic VTE and without previous cancer were identified. The mean age was 65.9 years (SD 12.6) and 58% were female. Overall, 696 (3.4%) patients were diagnosed with cancer in the 12 months following VTE. The risk was lowest among subjects aged 50 years or less (1.0%) and highest among patients aged 80 years or more (5.5%). Lung (25%) and gastrointestinal tumours (21%) were the most common diagnoses.

Conclusions:

In this large population of patients with idiopathic VTE we found that 3.4% of patients were diagnosed with cancer in the first year following VTE, and that older patients had a higher risk of cancer compared to younger patients. This risk is less than previously reported estimates of 4–12%. The value of occult cancer screening in patients with idiopathic VTE is uncertain.

Disclosures:

Tagalakis: Sanofi Aventis: Research Funding; Pfizer: Research Funding.
Insulin-Like Growth Factor and Platelet Activation the Role in Arterial Injury and Restenosis
Authors: Corinaldesi Giorgio
Published: October 2019

Abstract 5267

Insulin-like growth factor (IGF-1) is an activator of the PI3K/AKT (serine/threonine protein kinase) signalling pathway. It can also stimulate cell growth (PDGF, PDAF, VEGF, TGF-alfa and beta), and cellular proliferation, and is a powerful stimulator of directed migration of VSMC (vascular smooth muscle cells). The deregulated action of IGF-1 influences coronary artery disease (CAD) by promoting neo-intimal hyperplasia and leukocyte recruitment, pro-mytotic activity (MAPK: mitogen-activated protein kinases, serine/threonine kinase), and extracellular matrix synthesis in atherosclerotic lesions; it also increases platelet activity, and the expression of P-selectin, IL-6 and platelet CD62. This growth factors may also promote chemotaxis, LDL-cholesterol uptake, and the release of pro-inflammatory cytokines, finally it is a strong inhibitor of apoptosis (BAX, Fas/FasL); in other words, a high level of IGF-1 is a strong risk marker for ischemic heart disease.

We have studied a group of 140 patients (92 males and 48 females, mean age was 58 years, range 46–68 years); 72 patients with CAD in early stage of restenosis and 68 control. Patients with arterial restenosis have significantly high levels of IGF-1 (142.8 ng/ml +/− 24.2 ng/ml) that appear closely related to recurrence, than control subjects (mean level of IGF-1: 98.6 ng/ml +/− 14.4 ng/ml).

Additional investigations are needed to determine the exact weight of IGF-1 from a slightly clinical point of view, as this marker showed interestingly to have a key role in the pathogenesis of atherosclerosis.

Disclosures:

No relevant conflicts of interest to declare.
Molecular characterization of the First Italian Variant of Phosphoglycerate Kinase Deficiency
Authors: Bianchi Paola , Fermo Elisa , Mandarà Luana , Vercellati Cristina , Marcello Anna Paola Maria Luisa , Cortelezzi Agostino , Barcellini Wilma , Zanella Alberto
Published: October 2019

Abstract 5270

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate (1,3-BPG) to ADP to form 3-phosphoglycerate (3-PG) and ATP. It is a small monomeric molecule characterized by two hinge-bent domains, with a highly conserved structure. The N-terminal domain binds 1,3-BPG or 3-PG, whereas the C-terminal domain binds Mg-ADP or Mg-ATP. Two isozymes, PGK1 and PGK2, are present in humans, encoded by two distinct genes. Whereas PGK2 is a testis-specific enzyme, PGK1 is expressed in all the somatic cells.

The PGK1 gene is located on the Xq-13.1 chromosome, and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in enzyme deficiency characterized at clinical level by mild to severe hemolytic anemia, neurological dysfunctions and myopathy. Patients rarely exhibit all three clinical features. Since the first description by Kraus et al, nearly 40 patients with PGK deficiency have been reported, 27 of them characterized at the DNA or protein level. To date, 20 different mutations with worldwide distribution have been described.

The aim of the study was to characterize the molecular defect in an Italian patient affected by phosphoglycerate kinase deficiency.

The patient, born from unrelated parents with negative family history of neurological defects, showed at birth neonatal jaundice. At the age of four years, in concomitance of an infective episode, he displayed hemolytic anemia (Hb 8.6 g/dL, reticulocytes 19%, unconjugated bilirubin 0.91 mg/dL, LDH 445 u/l, aptoglobin absent) and increased CPK values (2483U/L). The patient showed respiratory distress. The study of red cell glycolytic enzymes displayed a drastic reduction of PGK activity (41.8 UI/gHb ref. values 287–392). We examined again the patient at the age of 25 yrs in occasion of his sister's first pregnancy. The patient displayed compensated hemolytic anemia (Hb 14.1 g/dL, reticulocytes 6.6%) and severe myopathy.

Sequencing analysis of the entire coding region and flanking intronic sequences of PGK1 gene showed the presence of a novel missense mutation c.1112 (ATA>AAA) responsible for amino acid substitution Ile371Lys. Although the mutation falls in the third last nucleotide of exon 9, it doesn't alter the splicing as confirmed by patient cDNA analysis that showed a normal transcript. The new mutation is located in a highly conserved region among species close to the ATP binding site and it was not found among the 100 normal alleles examined thus excluding the possibility of a polymorphism. Family study performed in the parents, the two healthy sisters and maternal uncle confirmed the X-linked transmission of the disease.

Disclosures:

No relevant conflicts of interest to declare.
An ANK1 Mutation Study in Chinese Patients with Hereditary Spherocytosis
Authors: So Jason CC , Wong Patricia , Chan Purdy , Chiang Alan KS , Chan Li Chong
Published: October 2019

Abstract 5277

Hereditary spherocytosis (HS) is the commonest hereditary hemolytic anemia due to a red cell membrane defect. Multiple proteins of the vertical component of the cytoskeleton are implicated. They include ankyrin-1, protein 4.2, band 3 protein, alpha and beta spectrin. Previous studies in Caucasians (Eber et al Nat Genet 1996) and Japanese (Nakanishi et al Int J Hematol 2001) showed that mutations in ANK1, the gene coding for ankyrin-1, was the most prevalent defect in HS. There is no published data on mutations in Chinese patients with HS.

Thirteen Chinese HS patients from 9 unrelated families and 28 normal Chinese subjects were studied. Fourteen of the 42 exons of ANK1 (exon 1, 2, 4, 5, 6, 16, 17, 20, 21, 22, 26, 33, 38, 39) were screened for mutation using direct nucleotide sequencing. Selection of exons for study was based on the frequencies of mutation detected in a large study in an Asian population (Nakanishi et al Int J Hematol 2001). Two novel nonsense mutations were detected in 2 unrelated patients in heterozygous state – codon 968 GAG>TAG and codon 1762 TGG>TAG. Family studies showed that 1 was a de novo mutation and the other was autosomal dominant. The overall prevalence of ANK1 mutations in this cohort was 22%. The actual prevalence may be higher as pathogenic mutations may also lie in the unscreened promoter and exons. Ten benign polymorphisms were detected in 8 of the 14 exons screened. Nine were synonymous mutations and 1 was a reported missense mutation. All but 1 were seen in our normal controls, including the missense mutation.

Our study shows that ANK1 mutation is a genetic cause of HS in Chinese but other candidate genes are definitely implicated. In consistence with published data in Caucasians pathogenic mutations are usually private. Many benign polymorphisms exist and they are commonly found in the general population. These features present significant challenges for screening of mutations and their interpretation in HS patients.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro GDF15 Expression During Thalassemic Erythroid Differentiation and Maturation
Authors: Colancecco Alessandra , Ronzoni Luisa , Duca Lorena , Sonzogni Laura , Nava Isabella , Graziadei Giovanna , Cappellini Maria Domenica
Published: October 2019

Abstract 5285

INTRODUCTION:

The growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-β superfamily, is thought to be related to ineffective or apoptotic erythropoiesis. GDF-15 levels were found to be significantly elevated in sera of patients with β thalassemia major (TM), however little is known about the GDF15 expression during thalassemic erythropoiesis. Non-transfusion dependent thalassemia intermedia (NTDT) represents the model of thalassemic erythropoiesis not affected by transfusions.

AIM:

To determine the GDF15 gene expression profile during normal and thalassemic erythroid differentiation in standard cultures and under different iron conditions, from CD34+ of normal and thalassemia intermedia subjects.

METHODS:

After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with NTDT by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. Each culture was split in 3 flasks: standard condition, with addition of deferoxamine (DFO 4 mM) as iron chelating agent and ferric ammonium citrate (FAC 100 mM) at day 0 of culture. The expression profiling of GDF15 gene was evaluated at baseline, day 7 and day 14 by real-time PCR (2̂-dCt). GDF15 concentrations in culture supernatants were also evaluated by enzyme-linked immunosorbent assay using DuoSet Sandwich ELISA Kit (R&D Systems, Minneapolis, MN).

RESULTS:

GDF15 expression and secretion increased significantly during erythroid differentiation either in normal and in NTDT cultures. At day 14 in thalassemia intermedia cultures GDF15 expression as well as the concentrations in supernatant were higher (althoug not statistically significant) compared to control (Table 1). At day 14 in control cultures GDF15 expression is up-regulated by DFO and down-regulated by FAC addition. In NTDT GDF15 expression was influenced by iron addition in cultures, but was not increased by iron depletion (Table 2).

CONCLUSIONS:

GDF15 levels in erythroid cultures are related to the erythropoietic stage of differentiation. In NTDT cultures the GDF15 gene profile and protein levels in supernatants are higher than in normal cultures. GDF15 levels seem to be modulated by iron in normal cultures whereas in NTDT cultures they seem to be independent from iron availability. This in vitro study supports that signals different than iron, such as erythropoietic stress, could be the major factor regulating GDF15 expression.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Ferroportin Expression in Thalassemia Intermedia During Erythroid Differentiation
Authors: Ronzoni Luisa , Colancecco Alessandra , Sonzogni Laura , Graziadei Giovanna , Cappellini Maria Domenica
Published: October 2019

Abstract 5287

INTRODUCTION:

Ferroportin (FPN) is the sole mammalian iron exporter protein known and it plays a critical role in iron metabolism. It is expressed in various types of cells including duodenal enterocytes, hepatocytes, erythroblasts cells, syncytiotrophoblasts and reticuloendothelial macrophages. Ferroportin is expressed in multiple alternative transcripts: with (FPN1A) or without (FPN1B) an iron-responsive element (IRE). The expression of one form rather than the other depends on cell type and iron availability. The expression of ferroportin in thalassemia intermedia (TI), characterized by iron overload, is not yet fully elucidated.

AIM:

To investigate the different expression profile of ferroportin isoforms during erythroid differentiation in control and TI cell cultures.

METHODS:

After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with thalassemia intermedia by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. The expression profiling of FPN1A and FPN1B was evaluated at baseline, day 7 and day 14 of culture by real-time PCR (2̂-dCt).

RESULTS:

In control cultures, FPN1A isoform was highly expressed at erythroid progenitors stage (day 0 of culture), decreased at early erythroblasts stage (day 7) and increased again at late erythroblasts stage (day 14). In TI cultures, the FPN1A isoform expression remained high even in early erythroblasts (Table 1). In control cultures the FPN1B isoform expression was very low at any stage of erythroid differentiation, whereas in TI cultures it was highly expressed at baseline and, althoug decreased during differentiation, remained always higher than control (Table 2).

CONCLUSIONS:

In thalassemic conditions the FPN1B is the major expressed ferroportin isoform, possibly contributing to iron overload. In control cultures, FPN1A was mainly expressed in undifferentiated erythroid progenitors and in mature erythroblasts, suggesting a functional role at these stages of erythroid differentiation. In TI cultures, the persistent expression of FPN1A at early erythroblasts stage was probably due to thalassemic erythropoiesis. These data suggest that in TI condition other signals, such as the erythropoiesis status, can override iron overload in regulating ferroportin expression.

Disclosures:

No relevant conflicts of interest to declare.
Right Ventricular Volumes and Function Normalized to Body Surface Area, Age and Sex in a Large Cohort of Well-Treated Thalassemia Major without Myocardial Iron Overload
Authors: Meloni Antonella , Aquaro Giovanni , Ait-Ali Lamia , Campisi Saveria , D'Ascola Domenico Giuseppe , Quarta Antonella , Bitti Pier Paolo , Roccamo Gaetano , Bisconte Maria Grazia , Piraino Basilia , Lai Maria Eliana , Peluso Angelo , Renne Stefania , De Franceschi Lucia , Positano Vincenzo , Lombardi Massimo , Pepe Alessia
Published: October 2019

Abstract 5293

Introduction.

Cardiovascular Magnetic Resonance (CMR) has provided the opportunity to quantify right ventricular (RV) parameters with excellent reproducibility and accuracy. The role of the RV is gaining ground in thalassemia major (TM) patients and this population could experience different “normal” RV values due to chronic anemia and eventually pre-existing iron burdens. The aim of this study was to establish the ranges for normal RV volumes, mass and ejection fraction (EF) normalized to the influence of body surface area (BSA), age and sex from CMR in a large cohort of well-treated TM patients without myocardial iron overload.

Methods.

Among the 923 TM patients enrolled in the Myocardial Iron Overload (MIOT) network who underwent CMR for the assessment of cardiac iron overload, function and fibrosis, we selected 142 patients with no known risk factors or history of cardiac disease, normal electrocardiogram, no myocardial iron overload (all the cardiac segments with a normal T2* value) and no myocardial fibrosis. All patients had been regularly transfused and chelated since early childhood. Moreover, we studied 71 healthy subjects matched for age and sex. RV function parameters were quantitatively evaluated in a standard way by SSFP cine images using MASS® software. RV end-diastolic volume (EDV), end-systolic volume (ESV) and stroke volume (SV) were normalized by body surface area (EDVI, ESVI, SVI).

Results.

The table shows the comparison of the CMR parameters with differentiation for sex and age in TM patients and healthy subjects and the cut-off of normality defined as mean – 2 standard deviation (SD). TM patients showed significantly lower BSA than the controls (P<0.0001). TM males (except age group 14–20 yrs) showed significantly higher RV EF compared to controls. In TM patients all LV volumes indexes were significantly larger in males than in females (P<0.0001 in all age groups). The EF was not different between the sexes. In males as well as in females the RV volumes were no significant different among the age groups, while in males the EF was significant different (P=0.004).

Conclusion.

In a large cohort of well-treated TM patients males showed significantly higher RV EF compared to controls. Due to the influence of BSA, sex and age, appropriate “normal” reference ranges normalized to these variables should be used to avoid misdiagnosis of cardiomyopathy in the clinical arena in TM patients.

Disclosures:

No relevant conflicts of interest to declare.
Relationship of Being a Beta Globin Gene Carrier with Insulin Resistance
Authors: Bahar Adeleh , Kashi Zahra , Sohrab Mehrnoush , Kowsarian Mehrnoush , Janbabaei Ghasem , Ghavamzadeh Ardeshir
Published: October 2019

Abstract 5305

Objective:

There was a paper in favor of beta-globin gene carrier and insulin resistance. Considering the high prevalence of these individuals in north of Iran, the present study was designed to assess the relationship between a beta-globin gene carrier and developing insulin resistance.

Methods:

This historical cohort study was conducted on 164 people, including 82 healthy controls and 82 individuals with thalassemia minor. The two study groups were matched for age, body mass index(BMI) and family history of diabetes mellitus. Blood samples were taken for; CBC, fasting blood sugar(FBS), liver enzymes (AST, ALT), high sensitive C- reactive protein(CRP), serum insulin and a standard OGTT were performed in all. Insulin resistance was diagnosed based on homeostasis model assessment method(HOMA). TM was diagnosed if microcytic(MCV<80 fl) hypochromia(MCH<25 pg) was detected on CBC and HbA2 ≥3.5% using HPLC method. Controls had negative past medical history and normal CBC. Student T- Test and and Chi-Square test were used to compare demographic data. Relative Risk was measured to test the hypothesis. P<0.05 was considered as significant.

Results:

Age, gender, BMI, were similar. CRP, and AST were significantly higher in case group. (p value <0.05). The Relative Risk for diabetes mellitus and insulin resistance in the cases with minor thalassemia was 2 (CI % 95:1.8–2.5) and 2.02 (CI % 95:1.7–2.4), respectively.

Conclusion:

The risk of developing diabetes and insulin resistance in patients with thalassemia minor is two times greater than the general population. Considering the high serum levels of CRP in these cases, the inflammation noted in liver cells could be considered as the underlying cause of insulin resistance, impaired glucose tolerance and diabetes in these patients.

Disclosures:

No relevant conflicts of interest to declare.
Nitrite Oxidase Activities of Cytochrome P450 and Mitochondria
Authors: Curtis Erin , Hsu Lewis L , Hon Yuen Yi , Geary Lisa , Noguchi Audrey C , Shiva Sruti
Published: October 2019

Abstract 5310

Once dismissed as an inert byproduct of nitric oxide (NO) auto-oxidation, nitrite (NO2−) is now accepted as an endocrine reserve of NO that elicits a number of fundamental biological responses in all major organ systems. While it is known that tissue nitrite is derived from both oxidation of NO and from dietary nitrite and nitrate, much less is known about how nitrite is metabolized by tissue or about the factors that influence this metabolism. Here we investigate the rates and mechanisms by which nitrite is metabolized by tissue over a range of oxygen tensions in rats and mice. We show that the rate of nitrite metabolism differs in heart, liver, lung and brain tissue. Further, oxygen regulates the rate and products of nitrite metabolism in each of these tissues. In hypoxic tissue, nitrite is predominantly reduced to NO, with significant formation of iron-nitrosyl heme proteins and S-nitrosothiols. Interestingly, this hypoxic nitrite metabolism is mediated by different sets of nitrite reductase enzymes in each tissue. In contrast, tissue consumption of nitrite is more rapid in normoxia and the major end product is nitrate. While cytochrome P450s and myoglobin contributed in the liver and heart respectively, mitochondrial cytochrome c oxidase played a significant role in this normoxic nitrite oxidation, which could be completely inhibited by cyanide in all tissues. We used cyanide-based nitrite preservation solution to measure the pharmacokinetics of oral and intraperitoneally administered nitrite in vivo. Using this methodology, we measured basal levels of nitrite in the major tissues and confirm that the heart contains the greatest concentration of nitrite, followed by the liver and finally the lung. We demonstrate that intraperitoneal administration of nitrite to mice increases nitrite levels most significantly in the liver and heart, where nitrite uptake is rapid (5–10 min) and steadily decreases thereafter, such that levels are back to baseline by 30 min. Little to no increase was observed in the lung. In these studies, changes in nitrate were difficult to detect due to the high levels of basal nitrate present in vivo and low concentration of nitrite administered. However, the rapid metabolism of nitrite in the tissue suggests that oxidation is at least partially responsible. In contrast to intraperitoneal nitrite, oral nitrite increased nitrite levels in all organs of mice, an effect which peaked in all organs except liver at 3 days. Collectively, these data provide insight into the fate of nitrite in tissue, the enzymes involved in hypoxic and normoxic nitrite metabolism and the role of oxygen in regulating these processes.

Disclosures:

No relevant conflicts of interest to declare.
Deficiency of Superoxide Dismutase Impairs Generation of Activated Protein C and Enhances Susceptibility to Experimental Thrombosis in Mice
Authors: Dayal Sanjana , Wilson Katina M , Hutchins Ryan , Lentz Steven R.
Published: October 2019

Abstract 535

In vitro studies have suggested that reactive oxygen species such as superoxide can produce several potentially prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin that impairs its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo. Using a murine model that is genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), we tested the hypothesis that lack of superoxide dismutase enhances susceptibility to thrombosis. Additionally, we investigated the mechanisms of superoxide-enhanced thrombosis. First, we examined the susceptibility to carotid artery thrombosis in a photochemical injury model. We found that Sod1−/− mice formed stable occlusions significantly faster than Sod1+/+ or Sod1+/− mice (P<0.05). Further, using an inferior vena cava (IVC) stasis method we observed that Sod1−/− mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 compared with Sod1+/+ or Sod1+/− mice). These findings suggest that deficiency of SOD1 leads to increased susceptibility to both arterial and venous thrombosis in mice. To address the mechanism of accelerated thrombosis, we first examined activation of platelets in response to multiple agonists using flow cytometry. After activation by thrombin (0.5 U/ml) and convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen to activated platelets were observed between Sod1−/−, Sod1+/+, or Sod1+/− mice, suggesting that increased susceptibility to thrombosis in Sod1−/− mice is not platelet mediated. Next, we measured expression of TF mRNA in lung by real time qPCR. TF mRNA levels in Sod1−/− mice were similar to those in Sod1+/+ mice, suggesting that deficiency of SOD1 does not influence TF expression in mice. Finally, we measured the activation of protein C in vivo in response to infusion of thrombin (40 U/Kg). Generation of activated protein C was significantly lower in Sod1−/− mice compared with Sod1+/+ mice (P<0.05). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1−/− mice compared with Sod1+/+ mice (P=0.4 and 0.6 respectively), suggesting that altered generation of activated protein C in Sod1−/− mice may be related to a direct oxidative effect on thrombomodulin rather than to decreased expression of thrombomodulin or EPCR. We conclude that lack of SOD1 in mice accelerates thrombosis and impairs the protein C anticoagulant response to thrombin.

Disclosures:

No relevant conflicts of interest to declare.
Microfluidic and Flow Cytometric Studies Support a Role for Monocytes and Coated Platelets in the Prothrombotic State in Heparin-Induced Thrombocytopenia (HIT)
Authors: Tutwiler Valerie , Ahn Hyun Sook , Cines Douglas B. , Camire Rodney M. , Poncz Mortimer , McKenzie Steven , Rauova Lubica
Published: October 2019

Abstract 539

HIT is an immune thrombocytopenia associated with a high risk of developing thrombosis. A passive immunization murine model of this disorder has provided important insights into the underlying pathogenesis of this disease, but is limited by its inability to study human cells and limited ability to define the contribution of various hematopoeitic and vascular cells to the prothrombotic state. We used a microfluidic system in conjunction with flow cytometry to further our understanding of the prothrombotic nature of HIT. Platelet adhesion and aggregation was studied in whole blood labeled with Calcein AM, perfused through a microfluidic channel (BioFlux 200 system, Fluxion) coated with von Willebrand factor (vWf) at shear stress of 20 dyne/cm2 at 37°C. A 40–60% increase in platelet adhesion (relative area covered by platelets) with up to a 4 fold increase in average aggregate size was seen in the presence of the pathogenic HIT-like monoclonal antibody (moAb) KKO (50 μg/ml) in conjunction with PF4 (10 μg/ml) when compared to control samples with PF4 only or with PF4 plus a non-pathogenic anti-PF4 moAb RTO (p <0.01). Monocyte-depletion decreased platelet aggregation by 20 – 40% relative to whole blood or after monocyte-repletion (P<0.0001). In HIT, thrombin plays a key role in the formation of platelet aggregates. Addition of thrombin inhibitor PPACK to the whole blood stimulated by KKO and PF4 decreased thrombus formation in the microfluidic chamber by 40% (p<0.001). Coated platelets are prothrombotic and characterized by phosphatidylserine (PS) exposure and binding of FVa and FXa. This activated state requires dual stimulation via thrombin and ITAM receptors. Flow cytometric studies of annexin V and FXa binding showed extensive induction of coated platelets in whole blood by KKO plus PF4 in contrast to PF4 or PF4 plus RTO (annexin V: p<0.0001; Factor Xa p<0.01). These new studies, focused on human blood, support our finding in the passive murine HIT model as to the importance of monocytes to thrombus formation and suggest that the prothrombotic nature of HIT may also be promoted by the generation of coated platelets. Identification of coated platelets may also lead to new diagnostic tests and new therapeutic interventions in HIT.

Disclosures:

No relevant conflicts of interest to declare.
Enrichment of Allogeneic Tumor Antigen-Specific T Cells From Bone Marrow (BM) of Patients Treated with High-Dose Post-Transplant Cyclophoshamide (Cy) – A Novel Approach to Adoptive Immunotherapy
Authors: Noonan Kimberly , Luznik Leo , Borrello Ivan M.
Published: October 2019

Abstract 647

Background:

Donor lymphocyte infusions (DLI) has limited efficacy and is associated with substantial graft vs host disease (GVHD) in treating relapse after allogeneic BMT (alloBMT) in large part due to the absence of significant tumor specificity. Thus, novel strategies are required that increase the tumor specificity with reduced alloreactivity. High-dose Cy post-BMT effectively reduces GVHD through early elimination of allo-reactive T cells and enables immune reconstitution free of ongoing pharmacologic immunosuppression. Since the BM is both the site of disease in most hematologic malignancies and a reservoir of tumor specific T cells, we hypothesized that marrow infiltrating lymphocytes (MILs) collected after alloBMT in patients treated with high-dose Cy could be a source of tumor-specific T cells for adoptive immunotherapy.

Methods:

BM was obtained from patients 2 –12 months after HLA-matched alloBMT on the clinical trial using high-dose post-transplant Cy as a single agent GVHD prophylaxis. The MILs were activated and expanded with CD3/CD28 antibody-coated beads.

Results:

Allo-MILs can be reproducibly expanded (574-fold avg expansion; 14–2000) at all time points tested. The activated allo-MILs are not anergic, exhibit anti-HLA-reactivity against third party allo-antigens but do not respond to recipient allo-antigens. Tumor-specific MILs were significantly expanded as determined by reactivity to HL60/K562 myeloid cell lysates (P<0.0001; pre- vs. post-activation). In contrast, no change was observed in cultures pulsed with irrelevant antigens, thus suggesting the specificity of the response. The ability to expand allogeneic antigen-specific MILs was also confirmed with a 15.5 fold expansion of PR-1 specific (HLA) A*0201-restricted CD8+ T-cells using tetramers.

Conclusions:

We can augment the tumor-specificity of MILs obtained post-transplant with minimal allo-reactivity in patients treated with post-BMT high-dose Cy. This could represent a novel approach to highly tumor-specific DLI. A phase I/II trial is planned to assess the safety and feasibility of administering ex vivo expanded post-transplant, allogeneic MILs to patients with relapsed hematologic malignancies after alloHSCT.

Disclosures:

No relevant conflicts of interest to declare.
Genetic Susceptibility Loci and Risk of Infant Leukemia by Subtype and MLL status: A Children's Oncology Group Report
Authors: Ross Julie A. , Linabery Amy M. , Blommer Crystal , Langer Erica , Spector Logan G. , Hilden Joanne M. , Heerema Nyla A. , Tower Richard L. , Davies Stella M.
Published: October 2019

Abstract 749

Introduction.

Leukemia in infancy is extremely rare and etiologically distinct from leukemia arising in older children. Infant leukemia cases likely arise in utero, typically involve rearrangements of the mixed lineage leukemia (MLL) gene, and have a much poorer survival than older children with leukemia. Since genetic determinants of susceptibility to infant leukemia are likely different from those in older children, we aimed to evaluate genetic variants previously identified by genome wide association studies of childhood acute lymphoblastic leukemia (ALL).

Methods.

We genotyped candidate susceptibility loci in 171 Caucasian infants less than 12 months of age with acute leukemia (including 102 ALL, 67 acute myeloid leukemia (AML), and 2 biphenotypic cases) diagnosed during the period 1996–2006. Controls consisted of 384 healthy Caucasian blood donors. Associations between genetic polymorphisms in IKZF1, ARID5B, and CEBPE and infant leukemia were evaluated using logistic regression and were further stratified by leukemia subtype and presence or absence of MLL gene rearrangements.

Results.

We observed a statistically significant association with two copies of the IKZF1 rs11978267 variant allele in infant leukemia overall (Odds Ratio (OR)=2.3, 95% Confidence Interval (CI)=1.3-4.2), but not with the other susceptibility loci. The association with IKZF1 was concentrated in the AML group, with an increased risk associated with two copies of the variant allele (OR=3.9, 95%CI=1.8-8.4), but not one copy. The association was similarly strong in those with and without a MLL gene rearrangement. In contrast, a strong association with the IKZF1 variant was seen in infants with ALL lacking a MLL rearrangement (OR=5.1, 95%CI=1.8-14.5), but not in those with MLL rearrangements (OR=0.7, 95%CI=0.2-2.2). Moreover, infants with ALL and no MLL rearrangement had a strong association with a genetic variant in the gene, ARID5B (rs10821936, OR=7.2, 95%CI=2.5-20.6), which was also not seen in those with ALL and a MLL rearrangement.

Discussion.

IKZF1 (IKAROS)is expressed in early hematopoiesis, and its role in lymphopoiesis and lymphoid leukemia is well-described. IKAROS is also expressed in precursor myeloid cells, and our report is the first evidence that IKZF1 is important in the etiology of infant myeloid leukemia, irrespective of MLL gene rearrangements. Moreover, our data indicate striking differences in genetic susceptibility to infant ALL with and without rearrangements of the MLL gene. This knowledge could provide important new etiologic insights into this extremely rare but heterogeneous hematopoietic malignancy. Supported by NIH CA079940, T32 CA099936, K05 CA157439 and the Children's Cancer Research Fund, Minneapolis, MN.

Disclosures:

No relevant conflicts of interest to declare.
MEK Inhibition Modulates the Growth of Nf1 mutant Hematopoietic Cells and Induces Clinical Improvement in a Murine Model of Juvenile Myelomonocytic Leukemia (JMML)
Authors: Chang Tiffany Y. , Krisman Kimberly , Harding-Theobald Emily Rose , Braun Benjamin S. , Shannon Kevin M.
Published: October 2019

Abstract 797FN2

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder that is caused by germ line mutations in the NF1 tumor suppressor gene. Children with NF1 have a 200- to 500-fold increase in the risk of developing malignant myeloid disorders, particularly JMML, an aggressive myeloproliferative neoplasm (MPN) characterized by over-production of myeloid lineage cells that show extensive tissue infiltration, anemia, and thrombocytopenia. The only curative therapy for JMML is hematopoietic stem cell transplantation, and 50% of children relapse after HSCT and die of recurrent JMML or of transformation to acute myeloid leukemia (AML). Somatic inactivation of Nf1 in hematopoietic cells causes a progressive MPN in Mx1-Cre, Nf1flox/flox mice that closely models JMML. We administered the MEK inhibitor PD0325901 (901) to Mx1-Cre, Nf1flox/flox mice with MPN to investigate the therapeutic effects of inhibiting Raf/MEK/ERK signaling in vivo and to interrogate the underlying mechanism of any observed response. Mx1-Cre, Nf1flox/flox mice (n = 35) and WT littermates (n = 38) were randomized to receive 901 (5 mg/kg/day) or control vehicle at 6 months of age. Treatment was continued for 10 weeks or until the mice became moribund. Mx1-Cre, Nf1flox/flox mice that received 901 demonstrated a rapid reduction in leukocyte counts and enhanced erythropoiesis. In addition, spleen size was markedly reduced with correction of aberrant proliferation and differentiation of progenitor cells. Flow cytometric analysis showed that 901 treatment restored a normal pattern of erythroid differentiation and greatly reduced splenic hematopoiesis. Remarkably, genetic studies revealed persistence of Nf1-deficient hematopoietic cells despite dramatic improvement in hematologic abnormalities. Based on these data, we conclude that treatment with 901 rebalances the proliferation and differentiation of Nf1 mutant cells in vivo rather than eliminating them. These preclinical data and a recent study in Kras mutant mice with MPN (Lyubynska N, et al. Science Trans Med 2011; 3: 76ra27) support clinical trials of MEK inhibitors in JMML and chronic myelomonocytic leukemia and suggest differentiation of malignant cell as a potential mechanism of response to this class of targeted agents.

Disclosures:

No relevant conflicts of interest to declare.
Inotuzumab Ozogamycin (I0), a CD22 Monoclonal Antibody Conjugated to Calecheamicin, Is Active in Refractory-Relapse Acute Lymphocytic Leukemia (R-R ALL)
Authors: o'Brien Susan , Thomas Deborah A. , Ohanian Maro , Jorgensen Jeffrey L. , Kebriaei Partow , Jabbour Elias , Rytting Michael E. , York Sergernne , Ravandi Farhad , Kwari Monica , Faderl Stefan , Rios Mary Beth , Cortes Jorge E. , Fayad Luis , Tarnai Robert , Wang Sa A. , Champlin Richard , Advani Anjali S. , Kantarjian Hagop M.
Published: October 2019

Abstract 875

Background:

Patients (pts) with R-R ALL have a poor prognosis. CD22 is highly expressed in ALL. IO is active in CD22-positive lymphomas. Methods: Pts with R-R ALL received IO 1.8 mg/m2 IV over 1hr every 3 weeks. First 3 pts received 1.3 mg/m2 in course 1. Children were eligible for study after 10 adults were treated; first 3 children received 1.3 mg/m2. Study started June 2010. Results: 49 pts were treated. Median age 36 yrs (range 6–80); 12 pts ≥ 60 yrs; Ph+ in 7, t(4:11) in 5, other abnormalities in 25. CD22 expressed in > 50% blasts in all pts, > 90% in 28. Median number courses so far 2 (1–5). 13 pts received IO as salvage 1 (S1), 24 as salvage 2 (S2), 12 as ≥ salvage 3 (S3). 7 pts had allo SCT before IO. Overall, 9/49 pts (18%) achieved CR, 5 had marrow CR (10%), 14 had CRp (29%), 21 were resistant, 2 died within 4 wks. Overall, CR+mCR+CRp (OR) was 57%: S1 9/13 (54%); S2 11/24 (46%); S3 8/12 (67%). A lower response rate was noted in Ph-positive ALL (3 of 7 = 43%) and in t (4;11) (1 of 5 = 20%) otherwise there were no clear correlations between OR and pts/ALL characteristics or with CD22 expression ≥ vs. < 90%. In 24 pts with evaluable pharmacokinetics studies: 3-hr I0 level > 100 ng/ml associated with OR in 8/9 vs. 5/15 if I0 level < 100 ng/ml. Median survival 5.1 months: S1 not reached (65% at 8 mos), S2 4.1 mos, ≥ S3 6.7 mos. Median response duration, CR+mCR+CRp (OR), not reached (estimated 70% at 8 mos). Median survival in 28 responders 7.9 mos. Fever and mild hypotension occurred in most pts on days 1–2 and resolved with support. Liver function abnormalities (LFA) were noted as IO related in 32 (67%), severe in 4 (13%). 2 pts had liver biopsies showing periportal fibrosis (after 1 and 4 courses). 20 pts were able to proceed to allo SCT (19 in remission, 1 with active ALL); 5 pts post-allo SCT developed veno-occlusive disease, 2 receiving second allo SCT, 4 receiving thio-TEPA + clofarabine as part of their preparative regimen. Conclusions: IO is highly active in R-R ALL. LFAs occur and are reversible. Study of IO weekly schedule is ongoing. IO and chemotherapy combinations should be pursued.

Disclosures:

Cortes: Pfizer: Consultancy, Research Funding. Kantarjian:Novartis: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding.
Iron Containing Compound Stimulates Expression of Pulmonary Hypertension Promoting Factor PlGF
Authors: Wang Xunde , Kato Gregory J. , Mendelsohn Laurel
Published: October 2019

Abstract 900

We have previously reported elevated plasma levels of placental growth factor (PlGF) and endothelin-1 (ET-1) in patients with sickle cell disease with high estimated pulmonary artery systolic pressure, a marker of pulmonary hypertension, and gene transfer experiments in mice document that PlGF stimulates ET-1 expression and pulmonary hypertension. Among other markers, PlGF in SCD subjects is correlated with markers of excessive iron burden, including serum ferritin and transferrin, markers that have been previously associated with pulmonary hypertension in SCD in many studies. We therefore hypothesized that iron stimulates expression of PlGF. Because tissue postnatal expression of PlGF in vivo is restricted primarily to early erythroid cells, we chose K562 erythroleukemia cells as a cell culture model. We find that heme-bound iron (hemin) induces PlGF mRNA robustly in a dose-dependent and time-dependent fashion. The PlGF transcript rises robustly within hours of hemin stimulation, and reaches levels of 40 to 80-fold over baseline in a real time PCR assay. Heme analog compound, mesoporphyrin without iron fails to induce PlGF, but as a control induces hemeoxygenase-1 mRNA. Iron plus mesoporphyrin induces PlGF strongly, indicating a critical requirement for iron in inducing PlGF transcript. In promoter-reporter luciferase constructs transfected into K562 cells, hemin induces the human PlGF promoter significantly, and a minimal promoter construct contains binding sites for erythroid Kruppel-like factor (KLF-1). KLF-1 mRNA levels rise in hemin-stimulated K562 cells in parallel with PlGF, and in peripheral blood mRNA from SCD subjects and healthy controls, the level of KLF-1 transcript correlates closely with PlGF transcript (r=0.82, p<0.0001). Additional studies of KLF-1 are under way.

Our results indicate for the first time a specific mechanistic pathway induced by iron that is linked in humans with SCD and in mice to vasculopathy and pulmonary hypertension. Rather a simple epiphenomenon of frequent transfusion or source of nonspecific oxidative stress, iron may trigger a KLF-1/PlGF/ET-1 linear mechanistic pathway to stimulate pulmonary hypertension. This suggests that PlGF levels are linked to the iron overload in patients with SCD. Further details in this pathway are under investigation.

Disclosures:

No relevant conflicts of interest to declare.
Megakaryocyte Lineage Commitment in Hematopoietic Stem Cells
Authors: Yamamoto Ryo , Morita Yohei , Ema Hideo , Ooehara Jun , Hamanaka Sanae , Onodera Masafumi , Nakauchi Hiromitsu
Published: October 2019

Abstract 909

Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into all blood lineages. Lineage commitment can take place at any stage from daughter cells of HSCs to multipotent progenitors. We have previously suggested that lineage commitment takes place at the level of HSCs (Takano et al. 2004). According to our asymmetric model, one HSC gives rise to two daughter cells consisting of one HSC and one lineage-committed progenitor. To verify this model, we performed a series of single-cell transplantation, followed by paired daughter cell experiments. The Ly5 congenic system has been commonly used to distinguish test donor cells from recipient cells and competitor cells. Since the Ly5 antigen is only expressed in leukocytes, platelets and erythrocytes have not been examined for evaluation of multi-lineage reconstitution. To assess lineage contribution more precisely, we have recently generated a transgenic mouse line in which Kusabira Orange (KuO) is expressed in all blood lineages including platelets and erythrocytes. CD150+CD41−CD34−/lowc-Kit+Sca-1+Lin− (CD150+CD41−CD34−KSL) cells, in which HSCs are highly enriched (Kiel et al. 2005, Morita et al. 2010), and CD150−CD41−CD34−KSL cells were individually isolated from KuO transgenic mice (B6-Ly5.1), and were transplanted into lethally irradiated mice (B6-Ly5.2), along with 2×105 bone marrow cells (B6-Ly5.1/Ly5.2). For secondary transplantation, 1×107 bone marrow cells from recipient mice were transplanted to lethally irradiated mice. Peripheral blood cells of primary and secondary recipient mice were periodically analyzed after transplantation. In some mice, full-lineage reconstitution was detected after transplantation with single CD150+CD41−CD34−KSL cells. In others, only common myeloid progenitor-type reconstitution was detected whereas common lymphoid progenitor-type reconstitution was not detected at all. Interestingly, megakaryocyte lineage (Meg)-restricted reconstitution and Meg/erythrocyte lineage-restricted (ME) reconstitution were observed after transplantation with single CD150+CD41−CD34−KSL cells, but not after transplantation with single CD150−CD41−CD34−KSL cells. These results suggest that Meg and ME progenitors are closely related to HSCs in their development. Furthermore, we performed paired daughter cell assays combined with single cell transplantation. Following division of CD150+CD41−CD34−KSL cells in culture, individual two daughter cells were separated by micromanipulation techniques and were transplanted into lethally irradiated mice with 2 × 105 competitor cells. Preliminary data showed in effect that the recipient mice of each single daughter cell derived from one CD150+CD41−CD34−KSL cell exhibited asymmetric differentiation; one with full-lineage reconstitution and the other with Meg-restricted reconstitution. Our findings offer a new perspective of HSC differentiation pathways.

Disclosures:

No relevant conflicts of interest to declare.