bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Integrins aMb2 (CD11b/CD18) and aDb2 (CD11d/CD18) in Macrophage Fusion.
Authors: Wu YiFei , Kushchaeva Evgeniya , Ugarova Tatiana
Published: October 2019

Abstract 2138

Macrophage fusion leading to the formation of multinucleated giant cells (MGCs) is a hallmark of many chronic inflammatory reactions. MGCs are an invariable constituent of tuberculoid lesions and also found in a variety of conditions leading to granulomatous inflammation as well as the foreign body reaction. Despite the prominent phenotype, the molecular mechanisms underlying macrophage fusion are not well understood. MGCs originate from macrophages that are recruited to sites of chronic inflammation. The major myelo-monocytic integrin αMβ2 (CD11b/CD18, Mac-1), together with a related integrin αDβ2 (CD11d/CD18), mediate critical adhesive reactions of monocyte/macrophages. The function of β2 integrins in macrophage fusion remains controversial. Some studies using function blocking antibodies implicated αMβ2 in the cell/substrate adhesive interactions that are required for MGC formation, whereas one recent report indicated that this integrin plays a minor role. Moreover, the contribution of αDβ2, a receptor with recognition specificity overlapping that of αMβ2, to macrophage fusion is unknown. To evaluate the role of αMβ2 and αDβ2 in MGC formation, we examined fusion of inflammatory peritoneal macrophages isolated from wild-type mice and mice with deficiency of αM or αD integrin subunits. Macrophages were isolated at day 3 after thioglycollate injection and cultured for 24–72 hours in the presence of IL-4 to induce fusion. Percentage fusion was quantified as the number of giant cell nuclei (≥2 nuclei) to the number of total nuclei. The number of fused macrophages isolated from wild-type mice gradually increased and ∼40–50% macrophages formed MGCs after IL-4 stimulation by day 3. Analyses of fusion of αMβ2-deficient macrophages demonstrated that fusion was significantly reduced. By day 3, macrophage fusion of αMβ2-deficient macrophages was 23 ± 2% of wild-type macrophages. Fusion of αDβ2-deficient macrophages was also decreased and the change was statistically significant, albeit to a smaller degree (75 ± 4%). Using a mouse model of sterile peritonitis induced by thioglycollate injection, we also examined the formation of MGCs in vivo. In wild-type mice, the number of MGCs gradually increased from 2.5 ± 0.3% at day 0 (resident macrophages) to 17 ± 2% at day 18 (the resolution phase of inflammation). Moreover, expression of αMβ2 and αDβ2 on peritoneal macrophages increased by ∼2 and 1.6-fold, respectively, on 18th day after induction of inflammation. In αMβ2-deficient mice, the number of MGCs was reduced by 2.1-fold compared to wild-type mice. Furthermore, while the size of wild-type and αMβ2-deficient giant cells was the same, the number of cells with 3 and more nuclei in fused αMβ2-deficient MGCs was 4-fold less than in wild-type cells. The results indicate that both αMβ2 and αDβ2 integrins support macrophage fusion with αMβ2 playing a dominant role.

Disclosures:

No relevant conflicts of interest to declare.
Safety and Value of Bronchoalveolar Lavage and Lung Biopsy in Children with Leukemia: A Single Institution Experience.
Authors: Montoya Libby , Avent Yvonne , Wilder Keith , Pei Deqing , Cheng Cheng , Jeha Sima , Morrison R Ray , Elbahlawan Lama
Published: October 2019

Abstract 2153

Patients with hematological malignancies are at risk of developing life threatening opportunistic pulmonary infections. The value of bronchoalveolar lavage (BAL) and lung biopsy (BX) in the management of these patients is debatable. While some believe these procedures are relatively safe and often provide valuable information that can guide management in these patients, others question the benefit of the procedures or are concerned about the potential complications of the procedures. We reviewed the St Jude experience with bronchoalveolar lavage and lung biopsy in children with leukemia to assess the benefit and safety of these 2 procedures. Review of medical records from January 1, 1997 through December 31, 2007 identified 183 potential episodes of BAL or BX in patients with leukemia who were younger than 22 years at time of procedure. In 30 patients who had multiple procedures, only the initial procedure was included.

A total of 64 patients were included, 36 (56.3%) of which had lung biopsy and 28 (43.7%) who had BAL. (See Table 1 for Demographics). There was a trend of better yield with BAL as compared to BX (67% versus 44%, p=0.079). Better BAL yield was observed in: males versus females (88% versus 42%, p=0.017), ALL versus AML (79% versus 44%, p=0.097), non-neutropenic versus neutropenic children (90% versus 56%, p=0.098). Both procedures resulted in change in antimicrobial coverage (78% in lung biopsy, 89% in BAL). Although both procedures were relatively safe, patients with BX had more complications as compared to BAL. Bx resulted in new oxygen need in 4 patients, increased oxygen need in 1 patient, intubation in 2 patients, and bleeding in 1 patient. BAL resulted in new oxygen need in 2 patients and increase in need in 7 patients, but no intubation or bleeding was related to this procedure.

In summary, BAL and BX have a reasonable safety profile and contributed to the management of pulmonary infections in children with leukemia. Compared to BX, BAL was associated with higher yields and less complications.

Table 1. Demographics Variable BAL (28) BX (36) Sex M/F 16/12 19/17 Mean Age 11 years 11 years ALL patients 19 12 AML patients 9 24 Neutropenia (ANC≤500) 18 21 Intubated prior to procedure 13 4

Disclosures:

No relevant conflicts of interest to declare.
ADAMTS13 TSP1 Repeat Domains Promote HUVEC Angiogenesis Via Phosphorylation of VEGFR2.
Authors: Lee Manfai , Xiao Juan , Zheng X. Long , Baza Jonathan , Hoyt Courtney , Rodgers George M
Published: October 2019

Abstract 2182

The primary role of ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, 13) is to cleave unusually large von Willebrand factor (ULVWF) multimers under shear stress. Recently, we reported that ADAMTS13 may be a potent mitogen and chemoattractant, and it modulates angiogenesis in vitro (Microvas Res. 2012, 84, 109–115). However, the structural components and mechanism of ADAMTS13 modulating angiogenesis are not understood. Herein, we report the effect of ADAMTS13 variants on cell proliferation, migration, and, tube formation of human umbilical vein endothelial cells (HUVEC). In addition, we determined the signaling pathways by which ADAMTS13 promotes angiogenesis.

ADAMTS13 fragments containing TSP1 repeat (i.e. MDT, MDTCS, TSP1 2–8, TSP1 5–8 plus CUB, and TSP1 2–8 plus CUB) were used in this study. In the proliferation model, TSP1 2–8 at the concentration of 34.6 ng/mL (651 pM) increased endothelial cell proliferation by 267 %. In the chemotaxis assay, TSP1 2–8 at the concentration of 27.7 ng/mL (521 pM) increased endothelial cell migration across a gelatinized polycarbonate membrane by 71 %. Similarly, TSP1 2–8 at the concentration of 55.4 ng/mL (1.0 nM) induced endothelial cell tube formation in Matrigel by 45 %. In all three models, the TSP1 2–8 induced angiogenic responses with similar efficacy to full-length ADAMTS13. MDT and MDTCS fragments did not affect proliferation, migration, or tube formation significantly, as compared to the negative control.

To determine the mechanism by which ADAMTS13 induces angiogenesis, we incubated endothelial cells with ADAMTS13 at the concentration of 147 ng/mL (1.0 nM). We showed that ADAMTS13 increased the phosphorylation of VEGFR2, Akt, and, P44/42 MAPK, which may trigger downstream activation to promote cell proliferation and migration. Addition of anti-VEGF antibody in the culture system significantly blocked the ADAMTS13-induced effect, indicating that ADAMTS13 plays a role in promoting angiogenesis by inducing VEGF secretion from endothelial cells (Fig 1).

The biological role of ADAMTS13 in angiogenesis was further demonstrated in a chick embryo model. Collagen onplants supplemented with EBM-2 (as negative control), 40 ng/mL VEGF165 (2.1 nM) (as positive control), and 306 ng/mL ADAMTS13 (2.2 nM) were placed on the chorioallantoic membrane of day 8 fertilized white leghorn chicken embryos. Localized and sustained release of VEGF and ADAMTS13 over a course of 72 hours resulted in 8-fold increase in capillary migration into the collagen onplants. Together, our findings suggest that the TSP1 repeats of ADAMTS13 metalloprotease promote angiogenesis by inducing VEGF secretion and VEGFR2 phosphorylation.

Disclosures:

No relevant conflicts of interest to declare.
Fut2
−/− (Non-Secretor) Mice Exhibit High Vwf Levels.
Authors: Johnsen Jill M. , Yohannes Ermias , Walsh Thomas W
Published: October 2019

Abstract 2184

Levels of the critical adhesive glycoprotein von Willebrand Factor (VWF) are widely variable in human populations. Two-thirds of VWF variation is heritable, of which one-third is due to the carbohydrate blood group ABO. Another closely related carbohydrate blood group, Secretor (FUT2), has also been reported to influence VWF levels, but this association is controversial. In humans, FUT2 nonsense mutations are common and result in the loss of FUT2 enzyme activity and the absence of Lewis and ABH sugars in mucosal secretions, hence the designation of FUT2 null individuals as “non-Secretors”. The FUT2 (Secretor) fucosyltransferase makes H antigen (fucalpha1à2betagal) in mucosal tissues, which can subsequently be fucosylated by other Lewis enzymes (FUT3, FUT6, etc.) to generate the variations within the Lewis blood groups system, or serve as a substrate for the ABO glycosyltransferase to generate ABH structures. To investigate the hypothesis that Secretor influences VWF, we examined Vwf levels in Fut2 knock-out (non-Secretor) mice. The Fut2 knock-out mouse line backcrossed >20 generations to C57BL6/J was recovered from cryopreserved stock at the Jackson Laboratory. Eight to 12 week old male mice were selected for study to avoid the potential effects of estrous and aging on Vwf levels. Platelet-poor plasma was obtained from Fut2−/− (non-Secretor, n=7) and their Fut2+/+ (Secretor, n=5) littermates and assayed for Vwf antigen (Vwf:Ag) by sandwich ELISA using polyclonal anti-VWF antibodies [rabbit anti-human VWF (Dako); sheep anti-human VWF (Abcam)] and a pooled C57BL6/J plasma standard. Non-Secretor (Fut2−/−) animals exhibited Vwf:Ag levels nearly double those of their Secretor (Fut2+/+) littermates (p<0.01, Table 1). These data show that Secretor (Fut2) plays a significant role in VWF homeostasis in mice, consistent with previous reports suggesting that human Secretors have lower VWF levels than non-Secretors. We speculate that the large effect size of Fut2 on Vwf:Ag observed in this study is due to the absence of the confounding effects of variation in ABO and vascular H expression common amongst humans. Though Fut2 is expressed primarily at mucosal surfaces, FUT2-derived H antigen is thought to be transferred to red cells via adsorption of glycolipids. This work offers new clues into the complexities of VWF homeostasis by implicating carbohydrate-dependent mechanisms other than those of classical intracellular ER-golgi post-translational glycosylation as a means to influence VWF.

Vwf:Ag (+/− 1SD) Secretor (Fut2+/+) 103 +/− 39 Non-Secretor (Fut2−/−) 196 +/− 65 p=0.01, Student's unpaired two-tailed ttest.

Disclosures:

No relevant conflicts of interest to declare.
Clinical Features of Severe Acquired ADAMTS13 Deficiency in Thrombotic Thrombocytopenic Purpura: Second Report of the Korean TTP Registry.
Authors: Oh Doyeun , Jang Moon Ju , Kim Inho , Bang Soo-Mee , Jung Chul-Won , Lee Jong-Wook , Eom Ki-Seong , Lee Hong Ghi , Kim Yeo-Kyeoung , Kim Byung Soo , Kim Jin-Seok , Lee Young , Yhim Ho-Young , Sohn Sang Kyun , Yoon Hwi-Joong , Jang Seongsoo , Jo Deog-Yeon , Cho Goon Jae
Published: October 2019

Abstract 2188

The clinical utility of ADAMTS13 activity for TTP has been extensively studied for last years. However, the clinical significance of ADAMTS13 activity for response to treatment, mortality rate, recurrence, and prognosis is still unclear. We previously reported the characteristics of severe ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP) and patients with severe ADAMTS13 deficiency had lower serum creatinine levels than patients with non-severe ADAMTS13 deficiency using 66 patients enrolled from January 2005 to December 2008. (Jang MJ et al, Int J Hematol 2011;93:163–9). In this second report, we enrolled 65 additional patients from January 2009 to June 2012 and analyzed 131 TTP patients using same methods. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels (P=0.001), lower platelet counts (P<0.0001), and high total bilirubin levels (P=0.018) at presentation. However, as same as previous results, treatment outcomes did not differ significantly between severe and non-severe ADAMTS13 deficiency groups in response rate (82 vs. 65%, P = 0.256), remission rate (70 vs. 63%, P = 0.781), and mortality rate (23 vs. 18%, P = 0.820). After adjusting for clinical and laboratory features, multivariate analysis did not reveal any independent risk factors for TTP-associated mortality. In conclusion, although TTP with severe ADAMTS13 deficiency is a unique subgroup characterized by lower platelet count and relatively good renal function, the prognostic significance of ADAMTS13 is still unclear and further study would be required to clarify it.

Disclosures:

No relevant conflicts of interest to declare.
Preclinical Efficacy Testing of Baxter's Recombinant ADAMTS13 in a Mouse Model of TTP.
Authors: Schiviz Alexandra , Resch Marlene , Farnleitner Elisa , Rottensteiner Hanspeter , Scheiflinger Friedrich , Schwarz Hans Peter , Hoellriegl Werner , Muchitsch Eva-Maria
Published: October 2019

Abstract 2216

Baxter is currently developing a recombinant ADAMTS13 (rADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) product for potential prophylaxis and treatment of thrombotic thrombocytopenic purpura (TTP). We established a disease model in ADAMTS13 ko mice (B6.129-ADAMTS13tm1Dgi), in which the animals simultaneously and consistently develop TTP-like symptoms by challenge with a high dose of human recombinant von Willebrand factor (rVWF) containing ultra-large VWF multimers.

To test the prophylactic efficacy of Baxter's rADAMTS13, groups of 10 ADAMTS13 ko mice received single doses of rADAMTS13 (1, 5, 40, 80 or 200U/kg) before challenge with rVWF. Doses of 1 and 5mL/kg fresh frozen plasma corresponding to 1 and 5U/kg ADAMTS13 served as positive controls. In a second study, the prophylactic and therapeutic efficacy of Baxter's rADAMTS13 was tested over time. Groups of 10 ADAMTS13 ko mice received a single dose of 200 FRETS-U/kg rADAMTS13 5min-120h before, or 15–180min after challenge with rVWF. In both studies, buffer was used as a negative control. Efficacy was defined as the degree of prevention of platelet drop and LDH increase. Schistocytosis and organ damage were also assessed.

Morbidity in negative controls was 100%: all animals receiving rVWF containing ultra-large VWF multimers were severely thrombocytopenic, showed increased LDH levels, schistocytosis and organ damage.

Prophylactic treatment with rADAMTS13 prevented LDH increase (p<0.0001), and dose-dependently prevented thrombocytopenia (p≤0.0003), schistocytosis and alleviated organ damage compared to treatment with buffer.

Efficacy of rADAMTS13 was treatment interval-dependent in both studies. Platelet count at termination of all rADAMTS13-treated animals was statistically higher than that of buffer-treated controls (p≤0.0001). However, animals receiving prophylactic treatment 120h before administration of rVWF showed severe thrombocytopenia; clinically relevant protection was only seen with treatment intervals ≤72h. Therapeutic treatment with rADAMTS13 stabilized platelet count and prevented further thrombocytopenia. LDH, schistocytosis and organ damage confirmed the treatment interval-dependent improvement in platelet count.

In summary, Baxter's rADAMTS13 was effective in an rVWF-induced animal model closely mimicking the situation in patients with hereditary TTP. Comparison with FFP, the current standard of treatment of TTP, showed Baxter's rADAMTS13 to have similar or slightly improved efficacy.

Disclosures:

Schiviz: Baxter Innovations GmbH: Employment. Resch:Baxter Innovations GmbH: Employment. Farnleitner:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.
Structural Characterization of Baxter's Recombinant Human ADAMTS13 Drug Candidate.
Authors: Foettinger-Vacha Alexandra , Kaliwoda Martin , Matthiessen Peter , Hasslacher Meinhard , Rottensteiner Hanspeter , Turecek Peter L , Mitterer Artur , Scheiflinger Friedrich
Published: October 2019

Abstract 2236

Baxter has developed a human recombinant (r) ADAMTS13 for treatment of patients with hereditary TTP. The protein is produced in a CHO cell-line using serum and protein-free fermentation technology. The purification process does not use immune-affinity chromatography, and includes two viral reduction steps. The final drug product is formulated without proteins of animal or human origin.

Here we characterize preclinical and clinical lots of rADAMTS13 with respect to their structural properties. The molecular weight of rADAMTS13 was determined by MALDI TOF as ∼173 kDa. SDS-PAGE showed one band at ∼190 kDa; no additional distinct protein bands corresponding to other proteins or product related degradation products were visible in the gels. Investigation of the primary structure of rADAMTS13 by peptide mapping revealed a sequence coverage of 98% compared to the theoretical amino acid sequence, and no peptides from the signal or propeptide. Peptide mapping was also used to determine posttranslational modifications of rADAMTS13. Seven O-glycosylation sites were identified in addition to the predicted N-glycosylation, O-fucosylation and C-mannosylation sites. The relative monosaccharide and N-linked oligosaccharides composition was consistent in all preclinical and clinical rADAMTS13 batches. The main N-glycan structure found on rADAMTS13 is a biantennary, core fucosylated complex type oligosaccharide with one or two sialic acids. Quantification of sialic acid determination showed consistent amounts of sialic acid on the terminal ends of the glycans in preclinical and clinical rADAMTS13 samples. N-glycolylneuraminic acid, a potentially immunogenic variant of sialic acid, was <1% the amount of total sialic acids.

Higher order structure analysis using dynamic light scattering showed a similar hydrodynamic diameter for all preclinical and clinical lots. Finally, FT-IR and CD analysis showed comparable amounts of α-helix and β-sheet in preclinical and clinical lots.

Taken together, similarity was shown between the preclinical and clinical lots of rADAMTS13 in all assays. A phase I clinical trial using rADAMTS13 for the treatment of hereditary TTP patients is expected to start soon.

Disclosures:

Foettinger-Vacha: Baxter Innovations GmbH: Employment. Kaliwoda:Baxter Innovations GmbH: Employment. Matthiessen:Baxter Innovations GmbH: Employment. Hasslacher:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.
Effects of Multiple Myeloma (MM) Therapy and Type of Thromboprophylaxis On the Incidence and Timing of Venous Thromboembolism (VTE) and Factors Predictive of VTE.
Authors: Shah Jatin J. , Hegde Aparna , Zhou Xiao , Thomas Sheeba K. , Wang Michael , Weber Donna M. , Alexanian Raymond , Orlowski Robert Z. , Vadhan-Raj Saroj
Published: October 2019

Abstract 2244

Background:

Patients (pts) with MM are at increased risk for VTE due to various risk factors related to the host, disease, and treatment. Immunomodulatory drugs (IMiDs) such as thalidomide and lenalidomide have further increased the risk of VTE. Several studies have shown the VTE risk can be reduced with the use of low molecular weight heparin (LMWH) or aspirin thromboprophylaxis. Based on these findings, VTE thromboprophylaxis has been recommended in pts receiving IMiDs + Dexamethasone (Dex), but the impact of these guidelines on patient outcomes in clinical practice is unclear. The objective of this observational study was to evaluate the incidence, timing and risk factors of VTE and the impact of different types of thromboprophylaxis on the incidence of VTE.

Methods:

This was a retrospective cohort study, and included all MM pts newly referred to the M.D. Anderson Cancer Center in 2006. Medical records of these pts were reviewed for the type and site of VTE, the incidence and timing of VTE during the five-year period from the referral date, and the risk factors, including pt demographics, co-morbidities, baseline laboratory values, types of MM and treatment, and types of thromboprophylaxis. Univariate and multivariate proportional hazard models were fitted to find the independent risk factors predictive of VTE. The stepwise selection method was employed to build a multivariate model using variables with p<0.15 in univariate analysis.

Results:

The cumulative incidence of VTE was 24% (38/159 pts) during the 5-year follow up period. Of the 38 pts with VTE, 25 (66%) had deep vein thrombosis (DVT), 11 (29%) had pulmonary embolus (PE), and 2 had concurrent DVT and PE. Most of the pts (32/38, 84%) had VTE within 1 year from the referral date. The incidence of recurrent VTE among these pts was 27.5% (11/38 pts), for a total of 52 episodes. Since the majority of VTEs and recurrences were within one year, we examined the risk factors for VTE during this period. Treatment with IMiDs + Dex and thromboprophylaxis with LMWH or Coumadin were independent predictive factors as shown below.

The incidence of VTE was highest in pts exposed to IMiDs + Dex (30/38 pts), even after discontinuation of treatment, with most episodes (17/30) occurring during the preparation (7/30) or within 30 days (10/30) following hematopoietic stem cell transplantation (HSCT), when most (16/17) pts were not receiving anti-coagulants.

Conclusions:

These findings suggest that patients treated with IMiDs + Dex are at high risk for VTE, even after discontinuation of this treatment, especially, during and after the HSCT period. Future studies are needed to investigate VTE prevention strategies for this high-risk pt population.

Disclosures:

No relevant conflicts of interest to declare.
Simvastatin Improve ADAMTS13 mRNA and Protein Expression in Podocytes.
Authors: Shen Lei , Lu Guoyuan , Dong Ningzheng , Ma Zhenni , Ruan Changgeng
Published: October 2019

Abstract 2246

Aim:

ADAMTS13 is a specific VWF (von Willebrand factor) -cleaving protease, which severe deficiency is the main cause of thrombotic thrombocytopenic purpura. ADAMTS13 is mainly synthesized and released of the surface of hepatic stellate cells and endothelial cells, but is alsoexpressed in other cells, including kidneypodocytes. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, not only has benefit action on atherosclerosis but also has anti-inflammatory and antithrombotic properties. Recent study indicated that ADAMTS13 can reduce inflammatory plaque formation during early atherosclerosis in mice. In this study, we investigate the effects of simvastatinon inflammatory factors-induced ADAMTS13 expression in podocytes.

Methods:

A conditionally immortalized mouse podocyte cell line was used in the study. Inflammatory factors (TNF-Á, IL-4, IL-6) and simvastatin were added to cell culture medium to exam their effect on ADAMTS13expresion in podocytes. We examedADAMTS13mRNA and protein expressbyquantitative real-time PCR (qRT-PCR) and Western blotting.

Results:

Our results showed that ADAMTS13 mRNA and protein was expressed in podocytes, and its expression levels were significantly decreased in cells treated withdifferent concentrations of IL-4 (1, 5,10 ng/mL) and IL-6(1,10, 100 ng/mL), while TNF-Á almost had no effect on ADAMTS13 expression. When podocytes treated with simvastatin (1 and 10 Ìmol/L), ADAMTS13 mRNA were significantly increased (1.53 ± 0.80 and 3.46 ±1.70, respectively, p<0.01 vs control), and ADAMTS13 protein levels were also increased (2.05 ± 0.18 and 2.22±0.12, respectively, p<0.01 vs control). Simvastatin also can reverse the inhibition effect of IL-6 (100 ng/mL) and IL-4 (10 ng/mL) on ADAMTS13 mRNA and protein expression in podocytes in a dose-independent manner.

Conclusions:

We demonstrate that different inflammatory cytokines had different influence on ADAMTS13's expression in podocytes. Simvastatin can increase the expression of ADAMTS13 in a dose-dependent manner with or without IL-6 and IL-4, which may be a very relevant compartment for the antithrombotic property of simvastatin.

Disclosures:

No relevant conflicts of interest to declare.
Trends in the Incidence of Venous Thromboembolism Adjusted for Body Mass Index (BMI).
Authors: Kevin Cohoon P. , Ashrani Aneel A. , Petterson Tanya M. , Crusan Daniel J. , Bailey Kent R , Heit John A
Published: October 2019

Abstract 2256

Background:

The incidence of venous thromboembolism (VTE) may be increasing due to the obesity epidemic.

Objective:

To estimate the incidence of VTE and describe trends in incidence adjusted for the population's increase in BMI.

Methods:

Using the resources of the Rochester Epidemiology Project, we identified all Olmsted County, MN residents with an incident deep vein thrombosis (DVT) or pulmonary embolism (PE) over the 13-year period, 1988–2000 (n=1,400). Age and BMI at VTE diagnosis and gender were collected for all cases. Age was categorized into 10 year intervals. Using a model for the population distribution of BMI derived from several (n=6) non-VTE Olmsted County cohorts (n=8,011 individuals), we calculated the probability of falling into each of five BMI categories (<18.5, 18.5–24, 25–29, 30–34, 35+) for each age, gender and calendar year. In Olmsted County in the median age group, the prevalence of BMI >30 kg/m2increased from approximately 15% in 1980 to 36% in 2000. We applied these probabilities to the decennial census data for 1980, 1990, 2000 using linearly interpolated census values for intra-census years to obtain Olmsted County age/sex/calendar year/BMI category denominators. Generalized linear modeling assuming a Poisson error structure, and using a log link function, and a log (population) offset was used to assess the relationship of crude incidence rates to gender, year of diagnosis, age and BMI.

Results:

The overall average age- and sex-adjusted annual VTE incidence was 120 per 100,000 person-years (DVT: 66 per 100,000; PE: 54 per 100,000), with higher age-adjusted rates among men than women (129 versus 113 per 100,000, respectively). VTE incidence rates increased exponentially with age for both genders, ranging from 3 to 1,079 per 100,000 for age groups 0–19 to 90–110 years. Age and sex-adjusted VTE incidence increased by increasing BMI category (88, 112, 106, 150, and 195 per 100,000 person-years, respectively, by increasing BMI category). Unadjusted for BMI, VTE incidence was unchanged from 1988 through 2000 (p=0.70). After adjusting for age, gender and calendar year, VTE incidence increased with increasing BMI (p<0.0001). The calendar year incidence slope estimate decreased numerically from 3% per decade to minus 4% per decade after adjusting for increasing BMI, remaining nonsignificant (p=0.57).

Conclusions:

VTE remains a major national health problem, especially among the obese. Despite the dramatically increasing population BMI, and the significant association of BMI category with VTE incidence, the overall incidence of VTE remained unchanged over the timeframe, 1988–2000.

Disclosures:

No relevant conflicts of interest to declare.
Real Life Efficacy and Safety of Rivaroxaban for Extended VTE Treatment – First Results of the Prospective Noac Registry (NCT01588119).
Authors: Koehler Christina , Werth Sebastian , Gelbricht Vera , Haensel Ulrike , Tittl Luise , Beyer-Westendorf Ines , Schreier Thomas , Eulitz Maria , Beyer-Westendorf Jan
Published: October 2019

Abstract 2267

Background:

In the EINSTEIN-EXT trial, rivaroxaban (RX) has been found to be at least as effective and safe as warfarin in extended venous thromboembolism (VTE) treatment, which lead to approval in many countries. However, patients in RCT‘s present a selected population treated under a strict protocol and followed for a short period of time. Consequently, efficacy and safety of new oral anticoagulants (NOAC) need to be confirmed in unselected patients in daily care.

Objectives:

To evaluate the efficacy, safety and management issues of rivaroxaban anticoagulation for extended VTE treatment in daily care.

Patients and methods:

In the district of Saxony, Germany, a network of 200 physicians from private practice and hospitals enrol patients in the prospective NOAC registry. Inclusion criteria are: 1) indication for NOAC anticoagulation >3 month; 2) age > 18 years; 3) written informed consent; 4) availability for follow-up. No Exclusion criteria apply. In the registry, up to 2000 patients will receive prospective follow up (FU) by phone visits at day 30 day and quarterly thereafter to collect efficacy and safety data.

Results:

Until July 31th 2012, 938 patients were registered. Of these, 126 patients received RX for extended VTE treatment (demographic data in table 1). In our registry, the population receiving extended VTE treatment is older than in EINSTEIN-EXT (65.0 vs. 58.2 years). Indication for prolonged treatment is proximal deep vein thrombosis or pulmonary embolism (93.3%). Most patients received 20 mg OD, but a quarter of patients received 15 mg OD due to impaired renal function.

Until July 31th, completed FU cumulate to 44.2 patient years. The results of 1-, 3- and 6-months FU are shown in table 2. Until now, no recurrent VTE or VTE-related death occurred. Two patients experienced major vascular events (1 ACS, 1 TIA). Bleeding events were frequent (24.6%) but only 2 patients (1.6%) experienced major bleeding events, none of which were fatal. Two patients died due to underlying diseases. At 3 and 6 month, 94% resp. 85% of patients were still taking RX.

Conclusion:

In unselected patients in daily care, extended VTE treatment with RX is effective and safe with low rates of events or treatment discontinuation in the first 180 days of treatment. Long-term data will be reported.

Disclosures:

Werth: Bayer Healthcare: Honoraria. Beyer-Westendorf:Bayer Healthcare: Bayer provided a grant to support the NOAC registry in part Other, Honoraria; Boehringer Ingelheim: Boehringer provided a grant to support the NOAC registry in part, Boehringer provided a grant to support the NOAC registry in part Other, Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria.
Deferasirox Inhibit Doxorubicin-Induced Reactive Oxygen Species Generation in Both Acute Myeloid Leukemia and Normal Heart Cell While Maintain the Cytotoxicity of Doxorubicin Only on Acute Myeloid Leukemia
Authors: Yeh Su-Peng , Chang Yu-Chien , Lo Wen-Jyi , Huang Min-Lih , Yang Yang-Sheng , Hsiao Pei Ching , Li Dian-Kun
Published: October 2019

Abstract 3620

Background:

Deferasirox (DFX) was recently found to have anti-leukemia effect both in vitro and in vivo. DFX can also potently inhibit the generation of intracellular reactive oxygen species (ROS). On the other hand, the generation of ROS by Doxorubicin (DOX) is critical for the cytotoxicity on both leukemia and normal heart cells. It is not known whether combining DFX and DOX will have synergistic or antagonizing effect on leukemic cells. Similarly, it is also unknown whether adding DFX to DOX will have protective effect on normal heart cell.

Method:

Cells of human acute myeloid leukemia (AML) cell line THP1, mice AML cell line WEHI3, and rat normal heart cell line H9C2 were treated with Doxorubicin 5microM for various duration in the presence of absence of DFX pretreatment (100microM for 10 minutes). Intracellular ROS generation was measured by the detection of 2,7-dichlorodihydrofluorescein (DCF) fluorescence intensity using flow cytometry. Apoptosis was determined by Annexin V-Propidium Iodide staining using flow cytometry. Cytotoxicity was determined by Trypan blue exclusion assay.

Results:

Although intracellular ROS was reduced, DFX alone induced apoptosis of THP1 (from 3% to 18%) and WEHI3 (from 31% to 49%) AML cells. DOX-induced ROS production was also significantly reduced when THP1, WEHI3, and H9C2 cells were pretreated with DFX (Figure 1a, 1b, 1c respectively). However, the DOX-induced apoptosis of THP1 and WEHI3 AML cells were not antagonized by DFX (Figure 2a). 24 hours after exposure to this physiological dose DOX, all the WEHI3 cells died in both DFX treated or untreated group (figure 2b). More importantly, DFX-pretreated H9C2 heart cells had fewer cell death (3.7%) after exposure to DOX (5microM for 24 hours) compared to non-DFX pretreated cells (8.5%).

Conclusions:

DFX alone induced apoptosis in two different AML cell lines. DFX also markedly reduced the ROS generation due to DOX treatment. However, DFX did not negatively influence the pro-apoptotic and cytotoxic effect of DOX on these AML cell lines. Interestingly, DFX also markedly reduced the DOX-induced ROS generation and DOX-induced cell death in normal rat heart cell, which might have protective effect on DOX-related cardiomyopathy. We are now using Balb/c-WEHI3 AML mice model to test whether DFX can protect cardiomyocytes from DOX-related damage while maintain the cytotoxic effect of DOX on AML cells.

Disclosures:

No relevant conflicts of interest to declare.
A New Simplified Prognostic Index with Age Cut-off of 70 Years for Patients with Diffuse Large B-Cell Lymphoma. A Population-Based Analysis From the Danish Lymphoma Registry, LYFO
Authors: Gang Anne Ortved , Pedersen Michael , d'Amore Francesco , Møller-Pedersen Lars , Pedersen Bo Amdi , Jensen Paw , Pedersen Bjarne Bach , Møller Michael B. , Gadeberg Ole , Ingeberg Steen , mourits-Andersen Torben , Pulczynski Stanislaw , Klausen Tobias Wirenfeldt , De Nully Brown Peter
Published: October 2019

Abstract 3651

Introduction:

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma accounting for 35–40%. Since the 1990s, The International Prognostic Index (IPI) has served as useful tool in daily treatment decision algorithm and in the design of clinical trials.

In the last decade, improvement of overall survival (OS) has been demonstrated with R-CHOP-like regimens. In addition, the median life expectancy has increased substantially since the 1980'ies and more intensive treatment options like autologous BMT are performed successful up to 70 years of age. Thus, prognostic factors, including age, are susceptible to change over time.

Material and methods:

Patients with DLBCL diagnosed in the period 2000–2010 treated with Rituximab and CHOP-like chemotherapy were extracted from the Danish population-based Lymphoma Registry that covers > 97% of Danish lymphoma patients. Clinical and laboratory data at time of diagnosis were analysed, leaving out factors with < 75% completeness. Patients with transformed lymphoma, CNS involvement and HIV+ patients were excluded from the analysis.

Results:

1990 patients (M:F ratio 1.26) were extracted from the LYFO database. The median age was 65 years, median follow-up was 54 months. Overall median survival was 9.7 years, with a 5 year OS of 65%. Analysis of age using 60, 65, 70, 75 years as cut-off, revealed 70 years as the optimal cut-point.

Univariate analysis was performed including age, gender, stage, performance status, extranodal disease, LDH, albumin, immunoglobulin G, bulky disease, lymphocytes and haemoglobin. Only significant factors were included in a Cox proportional hazards model. Results for the entire patient cohort are shown in table I, and for patients <= 70 years in Table II.

Survival analysis of patients with 0–1, 2–3 and 4–6 risk factors showed 5-year survival values of 90%, 71% and 45% respectively (left figure). For patients <= 70 years, the corresponding values were 90%, 81% and 62%, respectively (right figure).

Conclusion:

Two decades after the introduction of the IPI, age, performance status and LDH are still some of the most powerful prognostic factors in DLBCL. Age cut-off at 70 years is meaningful in reflecting clinical practice and, in our analysis, albumin and IgG added significant prognostic importance. In addition, for patients younger than 70 years, male gender is an adverse prognostic factor.

Disclosures:

No relevant conflicts of interest to declare.
Identification of Biologicaly Relevant Enhancers in Human Erythroid Cells
Authors: Su Mack , Steiner Laurie A , Bogardus Hannah , Schulz Vincent P , Hardison Ross C , Gallagher Patrick G.
Published: October 2019

Abstract 368

Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Recent studies utilizing genomic methodologies have shown that enhancers are frequently associated with biologically relevant and disease-associated genetic variants. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulate gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, in primary human erythroid cells. These data were combined with parallel gene expression analyses and candidate enhancers identified. Cell and tissue-type specific enhancers act over distances of tens to hundreds of kilobases, thus bona fide erythroid enhancers are expected to be enriched in the genomic vicinity of genes expressed and functional in erythroid cells. Sites of occupancy were correlated with levels of gene expression. Promoter sites within 2kb of annotated transcriptional start sites (TSS) were excluded. Consistent with their predicted function, there was significant enrichment of p300 peaks within 2–50kb of the TSS of genes highly expressed in erythroid cells c.f. peaks >100kb of a TSS. There was also significant enrichment of combinations of 2, 3, and 4 co-localizing erythroid transcription factor peaks within 2–50kb of the TSS of genes highly expressed in erythroid cells. In contrast, similar to other cell type-specific enhancers, there was no enrichment of p300 or erythroid transcription factor sites within 2–50kb of genes highly expressed in nonerythroid cells. When analyses were performed comparing a set of erythroid-specific genes vs. a random set of genes, there was significant enrichment of combinations of 2, 3, and 4 co-localizing erythroid transcription factor binding sites, but not p300, within 2–50kb of the TSS of erythroid-specific genes. Evolutionary analyses revealed high conservation between man and chimp for p300 and erythroid transcription factors. However, there was a very large falloff between human and mouse, with.

Disclosures:

No relevant conflicts of interest to declare.
A Phase II Trial of Alemtuzumab (Campath) with DA-EPOCH-R in Relapsed or Refractory Diffuse Large B-Cell Lymphoma and Hodgkin Lymphoma
Authors: Grant Cliona , Dunleavy Kieron , Hessler Julieanne , Steinberg Seth M. , Pittaluga Stefania , Jaffe Elaine S. , Wilson Wyndham H.
Published: October 2019

Abstract 3709

Background:

In diffuse large B-cell lymphoma (DLBCL), stromal signatures are predictive of outcome in newly diagnosed patients treated with R-CHOP. These signatures, termed 'stromal 1' and 'stromal 2' are associated with genes expressed by infiltrating mononuclear cells. The stromal 2 signature includes genes associated with angiogenesis and is associated with a poor outcome. Hence, targeting reactive cells in the microenvironment is a rational therapeutic strategy in DLBCL. In Hodgkin lymphoma (HL), the reactive macrophages surrounding Hodgkin Reed Sternberg (HRS) cells are associated with an adverse outcome and may provide survival signals. CD52 is highly expressed in many of these cells in the microenvironment and in most cases of DLBCL.

Methods:

We combined alemtuzumab (a monoclonal antibody against CD52) with DA-EPOCH-R to target reactive cells in the microenvironment and positive tumor cells. Alemtuzumab (30 mgs on day 1) was administered intravenously before DA-EPOCH-R (as previously described) and responding patients received up to 6 cycles of therapy.

Results:

Characteristics of 28 patients accrued: median age (range) 44 (22–72); male sex 15 (54%); stage III or IV disease 20 (71%); median prior regimens (range) 2 (1–6); and prior autologous transplant 7 (25%). Enrolled histologies were HL 12 (43%); DLBCL 10 (36%); and primary mediastinal B-cell lymphoma 6 (21%). Responses by histological subtype are shown in the attached table. The regimen was particularly effective in patients with HL with a CR rate of 73%. At 12 months (with 21 months median potential follow-up), the OS and PFS probabilities for all patients were 68% and 18% respectively. Toxicities included: treatment related mortality (sepsis) in 1 patient; febrile neutropenia in 16%; and grade 4 thrombocytopenia 15% of cycles. CMV reactivation was observed in 52% of patients in the setting of anti-viral prophylaxis.

Conclusions:

Combining alemtuzumab with DA-EPOCH-R is feasible. Patients with a diagnosis of HL had a very high complete response rate. Accrual continues.

Disclosures:

No relevant conflicts of interest to declare.
Cbl Controls the Expression of Axl and Lyn Tyrosine Kinases Mediating Resistance to Nilotinib in Chronic Myeloid Leukemia Cells
Authors: Tregoat Claire , Gioia Romain , Lagarde Valérie , Prouzet-Mauleon Valerie , Lippert Éric , Sirvent Audrey , Roche Serge , Mahon Francois-Xavier , Pasquet Jean-Max
Published: October 2019

Abstract 3734

The second generation of Abl tyrosine kinase inhibitor (TKI) nilotinib has been developed to overcome resistance to imatinib, which was up to now the gold standard treatment of chronic myeloid leukemia (CML). Although nilotinib has been approved recently as the front line treatment of CML, there are still some unresponsiveness nilotinib CML cells. The generation of nilotinib-resistant K562 CML cells led us to characterize the underlying mechanism regulating one of such cell resistance. We have previously shown that overexpression of the Lyn tyrosine kinase (TK) could explain this resistance both in vitro and in vivo (Mahon et al, 2008). Analysis by “Stable isotope labeling with amino acids in cell culture” (SILAC) further showed that this activity of Lyn is associated to overexpression of the TK Axl and the hyper activation of the Spleen TK Syk (Gioia et al, 2011). The inhibition step by step of these TK confirmed the relevance of each TK in the resistance to nilotinib. Similar mechanisms were detected in primary cells from nilotinib-resistant CML patients. Although the mechanism of deregulation of Lyn is currently unknown, is correlated to a transcriptional upregulation. The knockdown of Axl expression in nilotinib-resistant K562 cells (K562-rn shRNA Axl) overcomes resistance that correlates with a decrease of Lyn overexpression and an increase of c-Cbl expression. Interestingly, SILAC analysis also showed a down-regulation of several signaling proteins including the ubiquitin E3-ligase c-Cbl. Since c-Cbl targets several TK for lysosomal recycling or proteosomal degradation, we addressed whether c-Cbl down-regulation plays a role in Lyn and Axl protein accumulation. c-Cbl depletion in K562 cells (shRNA) induced a large increase of Axl and Lyn protein levels. In addition, the depletion of c-Cbl also induced an emerging resistance to nilotinib as assessed in viability and apoptosis assays. Conversely, the overexpression of c-Cbl in nilotinib-resistant K562 cells (K562-rn) dramatically reduced Axl and Lyn levels expression and re-sensitized K562-rn cells to nilotinib. These effects were dependent upon an intact E3-ligase activity of c-Cbl. Looking at c-Cbl mutations in K562 cells and in CML patients who overexpressed Lyn and Axl did not revealed any mutations. Altogether, these results suggest that c-Cbl down-regulation plays a critical role in the increase of Axl and Lyn levels that control cell resistance to nilotinib in CML cells.

Disclosures:

Mahon: Novartis Pharma: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy.
High Risk of Error in Categorizing Treatment Response in Individual CML Patients by Standard Cytogenetic Analysis or PCR Assay with High CV: A Bayesian Analysis
Authors: Laneuville Pierre
Published: October 2019

Abstract 3776

Existing therapeutic guidelines for CML are centered on the establishment of cytogenetic/molecular disease response milestones for assessing prognosis and recommendations for treatment decisions. Categorical risk classifications have been established from the analysis of large cohorts of patients on trial and thousands of cytogenetic and molecular analysis. While such analysis can be regarded as robust, little attention has been given to assessing the reliability of correctly assigning a patient to a given disease response category based on a single bone marrow cytogenetic analysis or quantification of BCR/ABL by PCR at a specific time point. More recently, the achievement of a cytogenetic response of ≤ 35% Ph (MCR) or BCR/ABL ≤ 10%IS at three months on first line treatment with imatinib or second generation TKIs has been shown to predict for significant differences in subsequent MMR, PFS,OS, and its adoption as a new milestone has been proposed. In the DASISION trial, the rate of MCR at 3 months for imatinib and dasatinib was 67% and 81%, and BCR/ABL ≤ 10%IS 64% and 84% respectively. The rate of BCR/ABL ≤ 10%IS at 3 months in the ENESTnd trial was 77% and 91% for imatinib and nilotinib respectively. The posterior probability of a patient having achieved an MCR at three months based on the analysis of 20 metaphases (ie. ≤ 7/20 Ph-positive) based on a Bayesian analysis is shown in Fig. A Calculations were based on the apriori probabilities of achieving an MCR at three months of 67% and 81% for imatinib and dasatinib. Conditional probabilities (probability of achieving ≤ 7/20 Ph+ if in MCR or > 7/20 Ph+ if not in MCR) were derived from the cumulative Poisson distribution. Bayesian analysis of the posterior probability of BCR/ABL ≤ 10%IS is shown in Fig. B using apriori probabilities of 64% and 84% for imatinib and dasatinib, and conditional probabilities based on normal distribution of PCR results with a coefficient of variance (CV) of 40%. The analysis demonstrates that the probability of incorrectly assigning a patient as having failed to reach an MCR is surprisingly high for results of > 7/20 Ph+ metaphases (ie. 20% for 12/20 Ph+ for dasatinib), or BCR/ABL ≤ 10%IS (ie. 20% for BCR/ABL ∼ 35%IS for dasatinib). These results signal the need for caution in assigning the categorization of response and risk in individual patients to those of much larger cohorts and highlight the need to acknowledge current limitations in therapeutic guidelines. Accuracy of cytogenetic testing can only be improved by increasing the number of metaphases analyzed, which is often not feasible. With lower achievable PCR assay CVs the accuracy of PCR can exceed that possible with cytogenetic analysis. Details of the analysis and comparative ROC curves will be presented.

Disclosures:

Laneuville: Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau.