bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact Assessment of Immunogenicity of Romiplostim In Subjects with Immune Thrombocytopenic Purpura (ITP)
Authors: Pamma Rupinderjit , Deshpande Meghana , Hokom Martha , Gupta Shalini , Zhuang Yao , Chirmule Naren , Swanson Steve , Jawa Vibha
Published: October 2019

Abstract 2517

Romiplostim is a peptibody that increases platelet production via binding to the thrombopoietin (TPO) receptor. This molecule has been engineered to have no amino acid sequence homology to endogenous TPO (eTPO); however, a low theoretical risk of developing conformational antibodies that cross-react with eTPO does exist. The current immunogenicity assessment strategy detects antibodies that bind to romiplostim, TPO, or TMP (the active peptide portion of romiplostim) and identifies any antibodies capable of neutralizing the biological effect of the drug or TPO.

In clinical studies, 537 subjects with ITP were dosed with romiplostim; 31 (5.8%) subjects developed binding antibodies to romiplostim and 21 (3.9%) subjects developed binding antibodies to TPO. However, only 2 (0.4%) of these subjects had antibodies that neutralized the biological effect of romiplostim. The binding antibodies detected against romiplostim were not cross-reactive with TPO in the immunoassay and did not neutralize TPO in the biological assay. Pre-existing binding antibodies against romiplostim were detected in 43 (8.0%) subjects and against TPO in 29 (5.4%) subjects. The immunogenicity rates in Japanese ITP subjects (n=46) dosed with romiplostim were comparable to non-Japanese subjects.

As a part of the post-marketing risk management strategy, Amgen performed immunogenicity testing on 37 subjects. Of these subjects, 3 developed binding antibodies against romiplostim that were not cross-reactive with TPO. None of these subjects developed neutralizing antibodies to romiplostim or TPO.

The impact of immunogenicity to romiplostim on safety and efficacy was evaluated utilizing platelet profile as a pharmacodynamic marker. No significant impact of the antibodies against romiplostim and TPO on the development of adverse events was noted. Grade 3 and above hypersensitivity related events were found in subjects that were antibody-positive and antibody-negative in the same proportion, indicating that these events were not related to anti-romiplostim or anti-TPO antibodies. No clinical correlation could be made between the development of antibodies against romiplostim and/or against TPO and the previous drug therapies or concomitant medications. No correlation between binding antibodies to romiplostim or TPO and platelet profiles of these subjects could be established. The subjects that developed a neutralizing antibody response against romiplostim were associated with a trend of lower platelet counts. The reduced platelet counts could be attributed to the presence of binding and neutralizing antibodies to romiplostim. Alternatively, the lowering of the platelet counts could also be attributed to discontinuation of therapy at the end of study.

Disclosures:

No relevant conflicts of interest to declare.
Decreasing Adverse Events From Warfarin In a Community Hospital Setting
Authors: Jubelirer Steven J , Davis Elaine A , Hodges Brian , Morris Lillian D , Miller Karen S
Published: October 2019

Abstract 2554

Background:

Millions of patients across the USA received warfarin to prevent and treat thromboembolism each year. Despite its effectiveness warfarin is associated with a risk of bleeding. The Joint Commission has brought attention to the safety of warfarin and challenged hospitals to “reduce the likelihood of harm associated with the use of anticoagulation therapy” as one of two new national patient safety goals for 2008. At CAMC (Charleston Area Medical Center, Charleston, WV) an anticoagulation safety task force was formed to address this challenge.

Purpose:

This study was initiated to document and reduce harm from warfarin at CAMC.

Methods:

This is an ongoing retrospective review of inpatients that received warfarin and had an INR of ≥4.0. Variables collected included, INR values, warfarin administration (dose and frequency), comorbidities, other current medications, other laboratory values, harm level (as defined by the Institute for Healthcare Improvement), presence of major or minor bleeding, use of blood transfusions, fresh frozen plasma, or vitamin K (and dose).

Results:

During baseline period there was a 3 month average of 15.86 episodes of harm/1000 warfarin doses while post implementation 9.69 episodes of harm were noted. At baseline, of those with harm 83.7% were temporary harm in which patients required an intervention (FFP or vitamin K) and 30.2% of the critical INR's occurred when warfarin was ordered daily instead of after the result was obtained and reviewed by the physician. From baseline April-July 08 compared to Aug 08-Feb 09 the frequency of vitamin K administration for INR < 5 and no bleeding has decreased by 33% (p=0.144). Overall there were 4.15% critical INR's before compared to 0.88% after implementing improvements representing a 79% improvement.

Conclusions:

Baseline data suggested a need for improvement in the safety of warfarin. Implementation of education, order sets (warfarin administration, warfarin reversal, physician pocket cards, and inpatient to outpatient handoff order sets, and warfarin workflow) and drug/drug interaction alerts to reduce adverse events. This study showed a decrease in the episodes of harm and the percent of critical INR's. It is estimated that the annual cost savings (RN time to treat critical INR's, excluding nursing time to administer vitamin K, and length of stay from complications of treatment) was $50,445.

Disclosures:

No relevant conflicts of interest to declare.
Gi-Coupled GPCR Signaling Is Required at Multiple Levels and In Different Lineages In Hematopoiesis.
Authors: Terpager Marie , Kataoka Hiroshi , Cornelissen Ivo , Coughlin Shaun R.
Published: October 2019

Abstract 2635

G protein-coupled receptors (GPCRs) can regulate cell migration, survival, proliferation and differentiation –– key processes in hematopoiesis. The Gi-coupled receptor CXCR4 plays a key role in hematopoiesis, suggesting that related receptors might also contribute. Because all Gi family members except Gz are inhibited by pertussis toxin (PTX), we utilized a ROSA26-CreOnPTX mouse line that expresses PTX in a Cre-dependent manner to broadly probe the role of Gi signaling in hematopoiesis. Mice hemizygous for the hematopoietic lineage-specific Cre transgene Vav-iCre were crossed with ROSA26-CreOnPTX/CreOnPTX mice to generate offspring expressing PTX in hematopoietic lineages (Vav-PTX) and Cre-negative controls in which the PTX allele remained silent. Vav-PTX mice were born at the expected Mendelian rate, and except for a smaller thymus, were grossly normal, but all died with pneumonia between days 2 and 14. Bone marrow in 3 day-old Vav-PTX mice was hypocellular with significant underrepresentation of granulocytic and lymphocytic lineages as well as hematopoietic stem and progenitor cells (lin-, c-kit+, Sca1+). In bone marrow reconstitution studies, cells from Vav-PTX fetal livers (E14.5) showed impaired short-term and no long-term repopulating activity. Additionally, Vav-PTX fetal liver cells were significantly impaired in their ability to form granulocyte/macrophage and erythroid colonies in vitro. Interestingly, when wild-type E14.5 fetal liver cells were grown in vitro in presence of exogenous PTX, only erythroid colony formation was impaired, and flow cytometric analysis of the progenitor populations of Vav-PTX fetal liver revealed a significant decrease in granulocyte-macrophage progenitors (GMPs) as well as in common myeloid progenitors (CMPs) but not in megakaryocyte/erythroid progenitors (MEPs). Thus, reduced progenitor populations may account for reduced granulocyte/macrophage colony-forming activity in fetal liver cell cultures but does not account for reduced erythroid colony-forming activity. Indeed, normal MEP numbers in Vav-PTX livers and the ability of exogenous PTX to inhibit formation of erythroid colonies in wild-type fetal liver cultures suggests that Gi signaling in MEPs or their progeny may contribute to erythropoiesis in fetal liver. Several of the necessary roles of Gi signaling identified above are not accounted for by the function of CXCR4, and, taken together, our data suggest that Gi-coupled GPCRs likely contribute to hematopoiesis at multiple levels and in different lineages. An effort to identify GPCRs that contribute to erythropoiesis is underway.

Disclosures:

No relevant conflicts of interest to declare.
Alterations In HLA-DR Expression In Peripheral Blood Mononuclear Cells Are Associated with An Elevated Tricuspid Regurgitant Jet Velocity and Pulmonary Hypertension of Sickle Cell Disease
Authors: Klings Elizabeth , Dworkis Daniel A , Safaya Surinder , Li Guihua , Reid Lynda , Sebastiani Paola , Baldwin Clinton T. , Steinberg Martin H.
Published: October 2019

Abstract 2640

Sickle cell anemia (SCA) is characterized by a chronic inflammatory vasculopathy. An elevated tricuspid regurgitant jet velocity (TRV) by echocardiography in SCA patients is not only reflective of possible pulmonary hypertension but is also associated with increased mortality risk, relative systemic hypertension, and renal disease, all of which suggest this is a marker of a more diffuse vasculopathy. We hypothesized that differential gene expression in peripheral blood mononuclear cells (PBMCs) occurs in SCA patients with an elevated TRV and might reflect the role of inflammation in sickle vasculopathy. Peripheral blood samples were obtained from 15 steady-state SCA patients (7 of whom had a TRV > 2.5 m/sec). Three cases with increased TRV had right heart catheterization that confirmed the presence of pulmonary hypertension. PBMCs were isolated via a Ficoll separation. RNA was isolated and amplified using the Ambion Illumina Total RNA Prep Kit, and then hybridized to Illumina Human HT-12 V3 Expression Bead Chips and scanned. Expression data was background corrected, log-transformed, and strictly filtered to consider only probes showing expression in each sample and significant expression in at least half of either group of samples. Differential expression was tested for significance using a t-test performed on both native and rescaled datasets, and only probes reaching a significance of p < 0.05 in both datasets were considered further. Of the 50 probes selected as significant in this way, the two of the most interesting were HLA-DRB1 and MYOM2, observed almost exclusively in SCA patients with a normal echocardiogram (p=0.007 and 0.008 respectively). Differential PBMC gene expression across the HLA-DRB1 locus has been associated in other populations with impaired immunity and increased susceptibility to sepsis. Our previous genetic studies have identified a potential link between the HLA loci and leg ulcers, another hemolytic complication associated with an elevated TRV in 219 SCA cases when compared with 1180 controls. In those studies, HLA-B was found to be in linkage disequilibrium with 3 genes found to be significantly associated with leg ulcers. This suggests the possibility that these heritable loci may play a role in disease modulation in SCD. PBMCs in SCA patients might have a pro-inflammatory phenotype and this may play a role in the vasculopathy and increased mortality risk observed in these patients.

Disclosures:

No relevant conflicts of interest to declare.
Improvement In Acute Management of Vaso-Occlusive Pain In Pediatric Sickle Cell Disease with Use of a Clinical Pathway
Authors: Ender Katherine , Freed Jennifer , Babineau John , Tresgallo Mary , Schechter William , Saroyan John , Kharbanda Anupam
Published: October 2019

Abstract 2660

Background:

Sickle cell disease causes considerable morbidity and mortality. Three guidelines exist for the management of vaso-occlusive pain, although studies have shown that these guidelines are not well followed and that there is considerable variation in care. Investigations of clinical pathways for pediatric sickle cell pain have not been published to our knowledge, but research in other pediatric conditions has shown that the implementation of clinical pathways can improve medical care. We initiated an investigation of the effects of a pain management pathway for sickle cell vaso-occlusive crisis in the emergency department (ED) with the hypothesis that the introduction of a clinical pathway would improve the efficiency and efficacy of acute pain management.

Methods:

We conducted a prospective, cohort study from February 2009 to March 2010 in an urban, tertiary care ED. We collected data from patients aged 3–18 years old with sickle cell disease who presented to the ED with vaso-occlusive crisis (VOC) pain. Baseline data was collected for five months prior to the introduction of the clinical pathway, followed by a four-week time interval in which the ED physicians and nurses were in-serviced on the pathway, followed by six months in which data was collected with the clinical pathway in place. Our pathway, which is in checklist format with instructions for triage, monitoring, medication administration, and timing of assessments and interventions, was developed by representatives from the divisions of Pediatric Hematology, Pediatric Emergency Medicine, and Pediatric Pain Medicine, Symptom Management and Palliative Care and based upon current standard-of-care guidelines.

Results:

Over the eleven month study period, 68 patients were enrolled. Random chart audits revealed a 75% capture rate. Significant improvement was demonstrated in time interval to first analgesic from 74 minutes to 42 minutes (p value 0.02), time interval to first opioid from 94 minutes to 46 minutes (p value < 0.01), and time interval to subsequent assessment of pain score from 110 minutes to 72 minutes (p value 0.02). The percentage of patients who received ketorolac also increased from 57% to 82% (p value 0.03). Change in pain scores was not significantly different, nor was admission rate.

Conclusions:

Implementation of a pain management pathway for sickle cell VOC led to an improvement in the time interval to administration of first analgesic and time interval to pain re-assessment, bringing these aspects of patient management closer to accepted guidelines. Whether improvement can be made in admission rates, change in pain ratings, and patient satisfaction will require further study.

Disclosures:

No relevant conflicts of interest to declare.
Integrated Use of Minimal Residual Disease Classification and IKZF1 Alteration Status Accurately Predicts 79% of Relapses In Pediatric Acute Lymphoblastic Leukemia
Authors: Waanders Esmé , van der elden Vincent H.J. , van der Schoot Ellen , van Leeuwen Frank N. , van Reijmersdal Simon V. , de Haas Valerie , Veerman Anjo J. , van Kessel Ad Geurts , Hoogerbrugge Peter M. , Kuiper Roland P. , van Dongen Jacques J.M.
Published: October 2019

Abstract 272

The response to therapy as determined by minimal residual disease (MRD) is currently used for stratification in treatment protocols for pediatric acute lymphoblastic leukemia (ALL). Even though MRD classification clearly identifies patients at low or at high risk for relapse, it also results in a large intermediate group (50 to 60% of patients), which still contains approximately half of all relapse cases. To improve risk stratification, we evaluated the added value of the IKZF1 alteration status, recently identified as a prognostic factor, in precursor-B-ALL patients. In an unbiased cohort of 131 uniformly treated precursor-B-ALL patients, we determined MRD levels at 42 and 84 days after treatment initiation using RQ-PCR analysis of Ig/TCR rearrangements. Based on these levels, patients were divided into three groups: MRD-Low (MRD-L), MRD-Medium (MRD-M) and MRD-High (MRD-H). IKZF1 alterations at diagnosis were determined using multiplex ligation-dependent probe amplification and genomic sequencing. We confirmed the strong prognostic significance of MRD classification, which was independent of IKZF1 status. Importantly, in the large MRD-M group (n=81; 62% of patients) containing 46% of the relapsed patients, IKZF1 alteration status identified 8 out of 11 relapsed patients (72%). The 9 year relapse-free survival (RFS) for IKZF1 mutated patients in this MRD-M group was 27% compared to 96% for patients wild-type for IKZF1 (P<0.001). Based on these results, we defined a new parameter integrating both MRD and IKZF1 status. The favorable risk group included patients classified as MRD-L or MRD-M with IKZF1 wild-type (n=104; 5 relapses), whereas the high risk group consisted of MRD-H patients or MRD-M patients with IKZF1 alterations (n=27; 19 relapses). This parameter showed stronger prognostic value than each of the established risk factors alone (Hazard Ratio[95%CI]: 24.98[8.29-75.31]). Importantly, whereas MRD and IKZF1 status alone identified only 46% and 54% of relapses, respectively, their integrated use allowed prediction of 79% of all relapses with 93% specificity. In conclusion: The use of a new parameter integrating MRD and IKZF1 status results in an unprecedented sensitivity in upfront relapse prediction and has a high potential for future risk stratification, particularly for patients originally classified as non-high-risk, such as the large group of MRD-M patients.

Disclosures:

No relevant conflicts of interest to declare.
Plasma Tyrosine Kinase Activity as A Potential Biomarker In BCR-ABL1-Targeted Therapy
Authors: Yeh Chen-Hsiung , Abdool Adam , Bruey Jean-marie
Published: October 2019

Abstract 2738

Introduction:

In targeted therapy using tyrosine kinase (TK) inhibitors (TKIs), measurement of TK activity could help in diagnosis, identification of potential responders, and monitoring of treatment efficacy. Here we evaluated the feasibility of measuring circulating TK (cTK) activity in plasma in patients with BCR-ABL1-positive leukemia.

Patients and Methods:

Study subjects included 46 patients with newly diagnosed chronic myelogenous leukemia (CML), 24 with multidrug-resistant CML, 24 with BCR-ABL1-positive acute lymphocytic leukemia (ALL), as well as 38 healthy donors. Plasma cTK activity was measured using a chromogenic universal TK assay in which plasma samples and standards were added to wells of a microtiter plate that were coated with synthesized poly(Glu-Tyr) peptide substrates. Specific TK activity was measured by spectrophotometry, with or without addition of TKIs.

Results:

cTK activity was significantly higher in CML (median 801.93 U/mL, range 18.10–3932.30 U/mL) and BCR-ABL1-positive ALL patients (median 659.55 U/mL, range 0–1626.90 U/mL) than in healthy donors (median 82.85 U/mL, range 0.63–852.80 U/mL) (P<0.001). Plasma cTK activity was closely correlated with cellular BCR-ABL1 kinase activation, as indicated by phosphorylation of the downstream signaling proteins CRKL (P<0.001) and STAT-5 (P=0.003). However, cTK activity was independent of BCR-ABL1 transcript level and BCR-ABL1 mutation type. Plasma cTK activity was inhibited by imatinib and dasatinib in ex vivo studies; this inhibition was severely diminished in patients harboring T315I mutations.

Conclusions:

cTK activity is readily detectable in plasma or serum samples without pre-cleaning or enrichment. Ex vivo testing measuring the effect of TKIs on plasma cTK activity thus hold promise as drug sensitivity tests for predicting and monitoring response to specific TKIs.

Disclosures:

Yeh: Quest Diagnostics Inc.: Employment. Abdool:Quest Diagnostics Inc.: Employment. Bruey:Quest Diagnostics Inc.: Employment.
Costimulatory Effects of Interleukin-18 (IL-18) on Human Natural Killer (NK) Cells Activated through Fc Receptors for Immunoglobulin (IgG)
Authors: Srivastava Shivani , Feng Hailin , Yu Menggang , Pelloso David , Robertson Michael
Published: October 2019

Abstract 2780

NK cells play an important role in innate and adaptive immune responses. Most human NK cells express CD16, an Fc receptor for IgG that mediates lysis of antibody-coated target cells and costimulates interferon (IFN)-g production in response to cytokines. IL-18 is an immunostimulatory cytokine with antitumor activity in preclinical animal models. The effects of IL-18 on human NK cell function were examined. Here we show that NK cells stimulated with immobilized IgG in vitro secreted IFN-g; such IFN-g production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-g production by NK cells stimulated with immobilized IgG or CD16 antibodies (Figure 1). NK cell IFN-g production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk, extracellular signal-related kinases (ERK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3-K). Stimulation with IL-18 or immobilized IgG could augment IL-12-induced IFN-g production by STAT4-deficient lymphocytes obtained from lymphoma patients after autologous stem cell transplantation (Figure 2). IL-18 also augmented the in vitro lysis of rituximab-coated Raji cells by human NK cells (Figure 3). These observations that IL-18 can co stimulate IFN-g production and cytolytic activity of NK cells activated through Fc receptors makes it an attractive cytokine to combine with monoclonal antibodies for treatment of cancer.

Disclosure:

No relevant conflicts of interest to declare.
A Recombinant Antibody to EphA3 with Pro-Apoptotic and Enhanced ADCC Activity Against Various Hematologic Malignancies Shows Selective Inhibition of Colony Formation From Long-Term Culture-Initiating Cells (LTC-ICs) In Primary Leukemia Samples
Authors: Palath Varghese , Vekhande Rohini , Baer Mark , Ching Wendy , Tomasevic Nenad , Martinez David , Yi Christina , Bhaskar Swathi , Luehrsen Kenneth , Williams Jason , Leung John , List Alan F. , Zhang Ling , Lackmann Martin , Boyd Andrew , Scott Andrew , Cilloni Daniela , Yarranton Geoffrey , Bebbington Christopher
Published: October 2019

Abstract 2897

EphA3 is a receptor tyrosine kinase expressed at high level on the surface of some solid tumors and certain leukemias. KB004 is a high-affinity non-fucosylated, recombinant antibody developed by KaloBios derived from a mouse monoclonal anti-EphA3 antibody using Antibody Humaneering™ Technology. The antibody binds EphA3 from human and cynomolgus macaque with equivalent affinity (610 pM for human and 570 pM for cynomolgus as determined by surface plasmon resonance (spr) analysis) making the latter a relevant toxicology species. KB004 has a direct pro-apoptotic effect on EphA3-positive cells and a induces potent antibody-dependent cellular cytotoxicity (ADCC) activity mediated by CD16-expressing effector cells. The non-fucosylated antibody has enhanced affinity for both human and cynomolgus CD16a. The affinity of KB004 for cynomolgus CD16a (7.2 nM, determined by spr analysis) was comparable to the affinity for the high-affinity variant of CD16a from humans (6.6 nM), indicating that KB004 has similar potential for ADCC in cynomolgus macaques.

We first demonstrated cell-surface expression of EphA3 by flow cytometry in approximately 50% of primary human leukemia cells. Here we extend the analysis to multiple myeloma (MM) in which 3/5 patient samples show EphA3 expression. EphA3 expression was confirmed in a panel of chronic myeloid leukemia (CML) samples [n=14] and in acute myeloid leukemia (AML) [n=40] in which EphA3+ cells were identified in each French American British (FAB) classification sub-type except M0, for which only two samples were studied. CD123-positive leukemia stem cells (CD34+ CD38− CD123+) also displayed surface expression of EphA3. KB004 induced apoptosis in EphA3+ primary cells from each of the diseases analyzed, including imatinib-sensitive and resistant CML, with no activity in EphA3-negative specimens, including normal CD34+ bone marrow cells. KB004 inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony recovery and also showed dose-dependent inhibition of long-term culture-initiating cells (LTC-IC) from AML stem cells. EphA3 expression was not detected on primary human CD34+ bone marrow cells from healthy individuals.

These data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies. The selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells.

Disclosures:

No relevant conflicts of interest to declare.
Lenalidomide reexposure After Short Interruption In Del(5q) MDS Patients at Relapse of Transfusion Dependence
Authors: Radkowski Robert Marek , Haase Sabine , Schlegelberger Brigitte , Goehring Gudrun , Banisch Stefanie , Heinsch Michael , Aul Carlo , Giagounidis Aristoteles
Published: October 2019

Abstract 2923

Introduction:

Lenalidomide is effective in myelodysplastic syndroms (MDS) in patients with del(5q) chromosomal abnormality (List et al 2006). However, relapse of transfusion dependence occurs consistently and therapeutic measures at that moment are scarce. We report on our experience to achieve a second remission of transfusion dependence using lenalidomide after a variable period of lenalidomide treatment interruption.

Methods:

5 patients with a del(5q) chromosomal abnormality (low- and intermediate-1-risk MDS according to IPSS) were treated within clinical trials, all transfusion-dependent with a median age of 67 years and with female prevalence. Mean MDS duration before start of lenalidomide therapy was 4.2 years. Patients were treated with doses of oral lenalidomide ranging from 5 mg every other day to 10 mg for 28 days of every 28 days cycle. All patients had a response to the initial lenalidomide treatment and achieved transfusion independence. All 5 patients relapsed and became red cell transfusion dependent, again. Median time to relapse was 24.2 months (13-55 months).The therapy was stopped and after a therapy-free interval of 7.2 months (2-13 months) we resumed lenalidomide therapy. The patients were treated with the initial lenalidomide dose.

Results:

3 of 5 patients (60%) became transfusion independent, again. The median increase in hemoglobin from baseline to the maximum hemoglobin achieved was 4.4 g/dl. Patients are followed up and all of them are currently in ongoing transfusion independence with a median time of 16.3 months (11, 15, 23 months, respectively). The other two patients stayed transfusion dependent and lenalidomide was interrupted after 3 and 4 months, respectively. Both later progressed to higher MDS subtypes or acute myeloid leukemia. Responses seemed to be independent on age, time to relapse, interval to retreatment or doses of lenalidomide.

Discussion:

In low/int-1 IPSS del(5q) patients relapsing with red cell transfusion dependence during lenalidomide treatment, current algorithms recommend discontinuation of the drug. Our results suggest efficacy of lenalidomide in a significant portion of the patients when they are rechallenged with a standard dose after a therapy-free interval of 2 to 6 months. Second remissions might be as long lasting as, or exceeding, the initial one. We are unable to define predictive factors for second responses. Lack of further response may be an ominous prognostic sign and, in our experience, heralded progression to higher risk MDS subtypes or acute myeloid leukemia after a short time.

Disclosures:

Giagounidis: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.
Comparison of Stromal Cells of Adipose Tissue From Multiple Myeloma Patients and Healthy Donors
Authors: Hébraud Benjamin , Labat Elodie , Espagnolle Nicolas , Corre Jill , Attal Michel , Bourin Philippe
Published: October 2019

Abstract 2979

Introduction:

Multiple myeloma (MM) is a B-cell neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow (BM) patients causing severe bone disease (with osteolytic lesions, pain, pathological fractures) and cytopenias. These lesions are irreversible, even for patients in complete response to treatment. We have proposed to treat those lesions with mesenchymal stem cells (MSC) of the BM using their ability to differentiate into osteoblasts and to support haematopoiesis. But our previous results demonstrated that MM MSC are abnormal. We thus studied cells from adipose tissue (AT) with similar properties than MSC; the Adipose derived Stromal Cells (ASC). The aim of this study is to demonstrate the normality of ASC in MM context for a potential use in autologous stem cell transplantation. Here we propose the first study comparing ASC issue from MM patients and healthy donors (HD).

Patients and Methods:

We studied ASC from 15 patients with newly diagnosed MM and 15 HD between 18 and 65 years. All gave their informed consent. The stromal vascular fraction was isolated from subcutaneous AT by and centrifugation. The ASC were sorted by adhesion to the plastic flask and expanded for 3 passages. We then performed the following assays to compare ASC from MM and HD:

Results:

The cell culture assay of ASC didn't show any difference between MM and HD for all the studied parameters: expansion capacity (HD 470± 45, MM 208±194; p=0.13), cell population doubling (HD 16.0±0.3, MM 15.0±1.3; p=0.17) and progenitor frequency (HD 3.4%±3, MM 3.7%±5.5; p=0.165). ASC phenotype didn't show any significant difference for all the checked markers and confirm their stromal feature: positive for CD90, CD73 and CD105, and negative for CD14 and CD45. The differentiation assay was evaluated for osteogenic, chondrogenic and adipogenic lineages. We did not underline any difference comparing ASC from MM or HD (Table 1). We previously showed that MM MSC secreted increased amount of IL-6, GDF15 and DKK-1 when compared with HD MSC. Interestingly, no difference was observed between MM ASC and HD ASC (Table 2). In the same way, contrary to MM MSC, MM ASC didn't promote the proliferation of MOLP-6 cell line better than HD ASC (proliferation rate: 1.49±0.34 for HD and 1.66±0.80 for MM; p=0.33).

Conclusion:

To our knowledge, this is the first study to compare ASC from MM patient and HD. These preliminary data suggest that ASC from MM patients are normal and could potentially be used in autologous stem cell transplantation for MM patients. We are currently completing this study by performing haematopoiesis support and microarrays assays.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Multiple Myeloma with Human Ig Light Chain Specific Cytotoxic T Lymphocytes
Authors: Weng Jinsheng , Cha Soung-Chul , Matsueda Satoko , Neelapu Sattva , Kwak Larry W.
Published: October 2019

Abstract 3018

The variable regions of Ig expressed by malignant B cells can serve as a tumor specific antigen. Clinical trials of idiotype (Id) vaccines have demonstrated humoral responses and prolonged remission duration in a recent phase III trial of follicular lymphoma patients in the first remission (Schuster et al, J Clin Oncol 27: 793S, 2009). However, the potentially immunogenic epitopes derived from Ig that stimulate CD8+ T cell immunity have been incompletely characterized. Here, we identified nine out of 14 candidate peptides derived from the Ig L-chain variable region of the human U266 myeloma line, which generated cytotoxic T lymphocytes (CTLs) from 53 HLA A2+ normal donors. These CTLs lines, as well as CTLs line isolated from myeloma patients by stimulation with autologous L-chain Id peptides, specifically produced IFN-γ in response to peptide-pulsed T2 cells and lysed U266 and autologous myeloma cell targets, respectively, but not normal blood B cells. Lysis was HLA class I-dependent, suggesting that primary myeloma cells express Id peptides on the cell surface in combination with HLA molecules. Nine CD8+ Id peptide-specific T-cell clones exhibited the effector memory phenotype and the ability of these T cell clones to eliminate U266 tumor in immune deficient mice is being tested. Finally, sequence analysis revealed shared T-cell epitopes in both framework and CDR regions of the U266 L-chain. CTLs generated against a shared U266 epitope lysed patient-derived myeloma cells expressing the shared sequence, suggesting a strategy to overcome the limitation of patient-specific Id vaccine manufacture. Our data identified novel immunogenic Id L-chain T-cell determinants and suggests that, unlike previously described Ig heavy chains, these sequences harbor common T-cell epitopes that may provide the rationale for shared Id vaccines.

Disclosures:

No relevant conflicts of interest to declare.
Statin Therapy and Levels of Thrombosis Risk Factors In a Healthy Population: the Multi-Ethnic Study of Atherosclerosis
Authors: Adams Nathan B. , Lutsey Pamela , Folsom Aaron , Herrington David , Sibley Christopher T , Zakai Neil , Ades Steven , Burke Gregory , Cushman Mary
Published: October 2019

Abstract 3178

Background:

HMG CoA reductase inhibitors (statins) were recently reported to reduce the rate of venous thromboembolism (VTE) in healthy people. Statins reduce levels of inflammation biomarkers, however the mechanism for a reduction in VTE risk is unknown. We studied cross-sectional associations of statin use with hemostatic factors related to venous thrombosis risk in a large cohort of healthy people.

Methods:

Cross-sectional analyses were performed in the Multi-Ethnic Study of Atherosclerosis (MESA), a cohort study of 6814 healthy men and women age 45–84, free of clinical cardiovascular disease at baseline; 1001 were using statins. Twenty three warfarin users were excluded. Age, race, and sex-adjusted mean hemostatic factor levels were compared between statin users and nonusers, and multivariable linear regression models were used to assess associations of statin use with hemostasis factors, adjusted for age, race/ethnicity, education, income, hormone replacement therapy (in women) and major cardiovascular risk factors.

Results:

The table shows that those using statins had significantly lower levels of D-dimer, C-reactive protein and factor VIII than non-users. Homocysteine and von Willebrand factor did not differ by statin use. Specific adjustment for LDL and triglycerides did not attenuate the observed differences in these factors by statin use.

Conclusions:

Findings of lower D-dimer, factor VIII and C-reactive protein levels with statin use suggest mechanisms whereby statins might lower VTE risk. These associations were not explained by lipid levels on treatment. A prospective study linking these biochemical differences to VTE outcomes is warranted.

Disclosures:

No relevant conflicts of interest to declare.
Decreased Activity of ADAMTS13 In Patients with Deep Vein Thrombosis Associated with Inflammatory Markers
Authors: Mazetto Bruna M , de Andrade Orsi Fernanda Loureiro , Barnabé Aline , Matos Marinez Farana , de Paula Erich , Morelli Vânia Maris , Annichino-Bizzacchi Joyce Maria
Published: October 2019

Abstract 3181

Introduction:

Elevated levels of inflammatory markers and clotting factors have been related to the pathogenesis of DVT. Particularly, the balance between VWF and ADAMTS 13 activity has been previously described in patients with thrombosis. VWF levels are also described to be elevated during inflammatory processes and therefore could play a role linking the activation of inflammatory and coagulation systems in patients with DVT.

Objective:

To evaluate the activity of ADAMTS13 and VWF in patients with DVT and its association with inflammatory markers.

Patients and Methods:

Fifty-five patients with DVT, 6 months to five years after the acute episode, attended at the Hematology Center of UNIFESP were included in this study and 121 normal subjects were selected as controls. The activity of ADAMTS 13 was performed by binding of residual VWF to collagen; VWF antigen, IL-6, IL-8, F1+2 and MCP1 was determined by ELISA and D-dimer and CRP was performed by turbidimetry. Continuous variables were analyzed by Mann- Whitney test, categorical variables by the chi-square test and Spearman rank test was used for correlation analysis.

Results:

Thirty-tree patients had DVT caused by transient risk factor, especially the use of oral contraceptives, and 22 patients had spontaneous DVT. No patient had renal, hepatic or malignant disease. The median ADAMTS 13 activity was not statistically different between patients (median= 65.8%) and controls (median=78.5%, p= 0.3043). The plasma levels of VWF antigen, IL-6 and CRP were higher in patients than in controls (p= 0.008, p= 0.0003 and p= 0.008, respectively). The levels of IL-8 and MCP1 did not differ betwee n patients and controls (p= 0.65 and p= 0.07, respectively). The levels of VWF correlated significantly with CRP (r= 0.28, p= 0.005) and F1+2 (r= 0.3, p= 0,02) in patients. There was an inverse correlation between ADAMTS13 activity and F1+2 concentrations (r= -0,33, p= 0.01).

Conclusion:

This study suggests that, even after the acute episode of DVT, a chronic inflammatory state would contribute to increasing levels of VWF in patients' plasma and hypercoagulability. The downregulation of ADAMTS13 activity may also contribute to the activation of the coagulation. However the role of ADAMTS13 in the interaction between inflammatory and coagulation systems in patients with thrombosis is yet to be determined. These factors together may contribute to recurrent DVT, which affects over 25% of patients within 10 years from the first episode.

Disclosures:

No relevant conflicts of interest to declare.
Pseudoexpression of Glucocorticoid-Induced TNF-Related (GITR) Ligand Upon Coating of Cancer Cells by Platelets Impairs NK Cell Anti-Tumor Reactivity
Authors: Placke Theresa , Kanz Lothar , Salih Helmut R. , Kopp Hans-Georg
Published: October 2019

Abstract 3193

NK cells as part of the innate immune system substantially contribute to cancer immune surveillance. They prevent tumor progression and metastasis due to their ability to mediate cellular cytotoxicity and to produce cytokines like IFN-γ, which, among others, stimulates subsequent adaptive immune responses. NK reactivity results from an integrative response emerging upon recognition of multiple ligands for activating and inhibitory NK cell receptors including various members of the TNFR family. Apart from the direct interaction with their target cells, NK cell activity is further influenced by the reciprocal interplay with various other hematopoietic cells like e.g. dendritic cells. Metastatic tumor spread in experimental animals is dramatically reduced in thrombopenic mice. Additional depletion of NK cells reverses this effect, indicating that platelets may impair NK anti-tumor reactivity. However, the underlying mechanisms have not been fully elucidated, especially in humans. Recently, we demonstrated that NK anti-tumor immunity is impaired by platelet-derived TGF-β, which is released upon interaction of platelets with tumor cells (Kopp et al., Cancer Res. 2009). Here we report that the ligand for the TNFR family member GITR (GITRL) is upregulated on megakaryocytes during maturation resulting in substantial GITRL expression by platelets. Since we recently identified GITR as inhibitory NK receptor involved in tumor immune escape (e.g., Baltz et al., Blood 2008, Baessler et al., Cancer Res. 2009) we investigated how platelet-derived GITRL influences platelet function and NK immune surveillance. Signaling via GITRL into platelets upon interaction with NK-expressed GITR or recombinant GITR-Ig fusion protein did not alter platelet activation as revealed by analysis of the activation marker CD62P and release of TGF-β. Interestingly, we found that GITRL-negative tumor cells rapidly get coated by platelets, which confers a seemingly GITRL-positive phenotype. “GITRL pseudoexpression” on tumor cells caused a substantial reduction of NK cell cytotoxicity and cytokine production. This reduced NK reactivity was not due to induction of apoptosis via GITR and could be restored by addition of a blocking GITR antibody. Thus, coating of tumor cells by platelets inhibits NK reactivity, which is in part mediated by platelet-derived GITRL. Our data provide a functional basis for the previously observed finding that platelets increase metastasis i.e. by enabling evasion of tumor cells from NK-mediated immune surveillance.

Disclosures:

No relevant conflicts of interest to declare.
The Transient Receptor-Potential Channel 6 Plays a Critical Role In Platelet Function
Authors: Murad John P , Ting Harold J , Khasawneh Fadi T.
Published: October 2019

Abstract 3201

In spite of the fact that changes in the intracellular levels of calcium (e.g., calcium entry (CE)) is a central step in platelet activation, the underlying mechanism of this CE is still ill defined. Previous studies have demonstrated that of the transient receptor potential channel (TRPC) family, TRPC1 and, to a higher extent, TRPC6 are expressed in blood platelets, whereas TRPC4 is found in megakaryocytes (at the mRNA level). Furthermore, recent studies have shown that TRPC1 deficient platelets exhibited no defects in platelet function, and unaltered calcium homeostasis. Based on these considerations, we hypothesized that TRPC6 plays an essential role in receptor-operated calcium entry and platelet function. Our preliminary studies reveal that platelets deficient in TRPC6 exhibited a defect in platelet aggregation mediated by the thromboxane receptor (TPR), at least, at low agonist levels (i.e., low levels of receptor activation). Moreover, TRPC6 deficient mice also exhibited a significantly prolonged bleeding time, and were protected against thrombosis development, using a FeCl3 carotid-artery injury model. Together, these findings are consistent with the role of TPRs in hemostasis and the genesis of thrombosis. In future studies, we will investigate the role of TRPC6 in: 1. calcium homeostasis; 2. separate platelet functional responses such as dense granule secretion and shape change; and 3. in platelet activation via separate platelet G-protein coupled receptors such as PAR4 and P2Y1. These studies may define a new therapeutic target for managing multiple thrombosis-based disorders.

Disclosures:

No relevant conflicts of interest to declare.
Mutation In Erythroid Specific Transcription Factor KLF1 Causes Hereditary Spherocytosis In the Nan (Neonatal Anemia) Hemolytic Anemia Mouse Model
Authors: White Robert A , Heruth Daniel P. , Hawkins Troy , Logsdon Derek , Gibson Margaret , Sokolovsky Inna , Nsumu Ndona , Major Stephanie , Fegley Barbara , Woods Gerald , Lewing Karen , Neville Kathleen , Cornetta Kenneth , Peterson Kenneth
Published: October 2019

Abstract 3217

The zinc finger protein Erythroid Krüuppel-like factor (EKLF, KLF1) regulates definitive erythropoiesis and terminal differentiation of red blood cells. KLF1 facilitates transcription through high affinity binding to CACCC elements within its erythroid-specific target genes which include genes encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins lead to the hemolytic anemia Hereditary Spherocytosis (HS). We have identified a new HS gene by studying the hemolytic anemia mouse mutant Nan (Neonatal Anemia). Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in Nan mice. The causative nature of the E339D mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1+/− knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild type KLF1. Competitive inhibition may help explain the apparent disconnect between the finding that Nan/+ heterozygous mice are anemic, whereas Klf1+/− heterozygous mice are normal and haplo-sufficient. This is the first direct association of a KLF1mutation with a disease in adult mammals. After examining a small population of HS patients, we also discovered one HS patient with a KLF1 mutation, which resulted in a significant amino acid substitution (T251I) in the activator/repressor domain, 28 amino acid residues upstream of the first zinc finger domain. This HS subject had no known mutations in the exons or intron/exon boundaries of EMS genes (SPTA1, SPTB, ANK1, SLC4A1) which comprise 85% of HS mutations in humans. The lack of a known genetic mutation in EMS genes leaves this patient's KLF1 mutation as the leading candidate defect. The identification of the gene causing the Nan mutation is significant because the Nan mutant has allowed discovery of a new HS gene which may also cause this disease in humans. In addition, the putative dominant/negative competitive inhibition of the Nan mutation makes the Nan mouse an excellent model system to study the function of KLF1.

Disclosures:

No relevant conflicts of interest to declare.
Effects of Prasugrel In a Rat Model of Myocardial Infarction Induced by Thrombotic Coronary Artery Occlusion
Authors: Sugidachi Atsuhiro , Yamaguchi Shinji , Jakubowski Joseph A , Hashimoto Masami , Niitsu Yoichi
Published: October 2019

Abstract 3317

Purpose:

Prasugrel is the 3rd generation of thienopyridine with an antiplatelet profile distinct from clopidogrel. Its phase 3 study, TRITON-TIMI 38, demonstrated reduced rates of ischemic events relative to clopidogrel. However, effects of prasugrel in models of myocardial infarction (MI) have not yet been reported.

Methods:

Photochemical activation of rose bengal by laser activation at 540 nm is well established method for the induction of endothelial injury resulting in arterial thrombotic occlusion. Hirata et al. applied this approach in the rat to induce MI secondary to photochemically induced thrombosis (Jpn. J. Pharmacol., 67, 51–57, 1995). In the present study, the effects of prasugrel (1, 3 and 10 mg/kg, p.o., 2 hrs before photo-irradiation), were evaluated in this rat model of MI induced by photochemical insult to rat coronary arteries.

Results:

In a vehicle-treated control group of rats the mortality rate was 58.8% during 30-min photo-irradiation. In contrast, mortality rates of prasugrel-treated animals (1-10 mg/kg, p.o.) during the photo-irradiation were 0% for 1 mg/kg, 22.2% for 3 mg/kg and 0% for 10 mg/kg, indicating a protective effect of prasugrel. In surviving rats infarct sizes, the necrosis area/total left ventricular area (NA/TLV), at 24 hrs after irradiation was evaluated. In the control group, NA/TLV was 37.9 ± 6.8% (n=7). At all prasugrel doses tested, infarct size (NA/TLV) was smaller than that in control groups: 14.4 ± 4.0% for 1 mg/kg (p<0.01 vs. control, n=7), 19.8 ± 4.5% for 3 mg/kg (p<0.05 vs. control, n=7), and 14.8 ± 3.6% for 10 mg/kg (p<0.01 vs. control, n=7). We monitored blood pressure (BP), heart rate (HR), and the electrocardiogram in control and 10 mg/kg of prasugrel groups. And found no significant differences between the groups for BP and HR values 24 hrs after photo-irradiation. In contrast, arrhythmia was observed in 5 of 7 animals in the control group at 24 hrs, in contrast no arrhythmia was found in the group treated with prasugrel.

Conclusion:

The present results indicate that in this rat model prasugrel protected against both myocardial infarction and associated arrhythmia.

Disclosures:

Sugidachi: Daiichi Sankyo Co., Ltd.: Employment. Yamaguchi:Daiichi Sankyo Co., Ltd.: Employment. Jakubowski:Eli Lilly and Company: Employment. Hashimoto:DAIICHI SANKYO: Employment. Niitsu:Daiichi Sankyo Co., Ltd.: Employment.