bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Myeloid and Other Stromal Cell Types in Tumor Angiogenesis.
Authors: Ferrara Napoleone 
Published: October 2019

Abstract
Abstract SCI-32
Role of myeloid and other stromal cell types in tumor angiogenesis Napoleone Ferrara Genentech, Inc, 1 DNA Way, South San Francisco, CA, 94080, USA. Vascular endothelial growth factor (VEGF)-A is an important angiogenic factor, involved in both physiological and pathological growth of blood vessels. An anti-VEGF-A monoclonal antibodies and two small molecule VEGF RTK inhibitors have been approved by the FDA for cancer therapy. We have been investigating the mechanisms of refractoriness/resistance to anti-VEGF therapies. Our analysis points to the mobilization and recruitment of CD11b+ Gr1+myeloid cells from the bone marrow in the tumor as key mechanisms for VEGF-independent angiogenesis, at least in mouse models. Tumors that are refractory to anti-VEGF treatment result in increased peripheral mobilization and tumor homing of of CD11b+Gr1+ cells compared to sensitive tumors. Addition of CD11b+Gr1+ cells from resistant tumors was able to sustain the growth of sensitive tumors in the presence of anti-VEGF antibodies. In subsequent studies, we identified the secreted protein Bv8 as a mediator of myeloid cell-dependent angiogenesis. Blocking Bv8 using neutralizing antibodies inhibited the growth of several tumor cell lines transplanted in nude mice and also reduced the angiogenic switch in the RIP-Tag, a transgenic model of insulinoma. The expression of Bv8 by CD11b+ Gr1+myeloid was regulated by hematopoietic growth factor, especially G-CSF. Other studies have implicated tumor-associated fibroblasts as a source of alternative pro-angiogenic factors following VEGF blockade. We identified PDGF-C as one of such mediators. In summary, multiple stromal cell types and signaling pathways may contribute to inherent refractoriness and, potentially, acquired resistance to anti-VEGF therapies in tumors.

Disclosures
Ferrara: Genentech, Inc.: Employment.
Counterpoint: In Some AMLs, Stem Cells are More Mature and Present at High Frequency.
Authors: Cleary Michael L. 
Published: October 2019

Abstract
Abstract SCI-35
Leukemia stem cells (LSCs) are responsible for sustaining and propagating malignant disease and, therefore, are promising targets for therapy. The current paradigm for LSC frequency, maturation and hierarchical organization is primarily based on transplantation studies in xenograft mouse models. To circumvent potential limitations of this experimental approach, investigators have recently employed syngeneic mouse models to study LSCs. In a mouse model of AML initiated by MLL oncogenes, which are associated with the FAB-M4 or M5 subtypes of human AML, LSCs are remarkably frequent, accounting for up to one-quarter of malignant myeloid cells at late-stage disease. Even in this syngeneic setting, however, transplant assays alone markedly underestimate LSC frequency due to poor engraftment efficiency. LSCs are organized in a phenotypic and functional hierarchy, and express myeloid lineage-specific antigens, placing them downstream of the known hematopoietic progenitor compartments. Thus, LSCs in this model are not synonymous with normal upstream progenitors that are targeted for leukemia initiation, but rather constitute myeloid lineage cells that have acquired an aberrant self-renewal program as well as other biologic features of hematopoietic stem cells. Gene expression profiling confirms the downstream myeloid character of LSCs in this model, and further demonstrates the aberrant expression of a stem cell associated transcriptional subprogram. However, LSC maintenance in the self-renewing compartment of AML employs a global transcriptional program more akin to embryonic rather than adult stem cells. Expression of LSC maintenance program genes is enriched in poor prognosis human malignancies, suggesting that the frequency of aberrantly self-renewing progenitor-like cancer stem cells may be linked to prognosis in human cancer. Consistent with this possibility, LSC frequencies in different syngeneic models of Hox-associated AML can vary over three orders of magnitude, depending on the particular initiating oncogene and expression levels of Hox pathway co-regulators, and correlate with leukemia biology. Studies in a human cord blood cell transduction/transplantation model of AML further support the downstream character of MLL LSCs. These findings prompt a revision of the current paradigm that AML leukemia stem cells are always rare and solely located within the most immature bone marrow progenitor compartments. The fact that LSCs can be more analogous to precursors and employ ESC-like genetic programs for their maintenance, may allow for their selective therapeutic targeting that spares HSCs required for hematopoiesis.

Disclosures
No relevant conflicts of interest to declare.
Role of the Glycoprotein (GP)IbαaIb Cytoplasmic Domain in Regulating the Von Willebrand Factor-Binding Functions of the Platelet GPIb-IX-V Complex.
Authors: López José A. , Munday Adam 
Published: October 2019

Abstract
Abstract SCI-39
The platelet glycoprotein (GP) Ib-IX-V complex mediates the initial attachment of platelets to regions of blood vessel damage by binding von Willebrand factor (VWF) exposed in the matrix at the site of injury. This interaction is especially important in the arterial system and microvasculature, given that it is the preferred platelet adhesive event at high shear stress. The interaction is positively and negatively regulated in several ways, including through exposure of the GPIb-binding site on VWF by shear stress or collagen binding, cleavage of VWF by the metalloprotease ADAMTS13, and in the platelet by the targeting of the GPIb-IX-V complex to lipid rafts, cytoskeletal attachments, and binding of the adaptor protein 14-3-3. Association with 14-3-3 has complex roles in regulating accessibility of GPIbα (the ligand-binding subunit of the complex), as several binding sites have been identified within the cytoplasmic domains for this adaptor molecule, and these are regulated by phosphorylation. To address the role of GPIbα phosphorylation in regulating platelet adhesion to VWF, we mutated 5 cytoplasmic Ser and Thr residues that were predicted by bioinformatics programs to be targets for serine/threonine kinases (Thr547, Ser566, Ser587, Ser590, Ser609) and expressed the mutants either as an isolated cytoplasmic domain or in intact GP Ibα as part of a GP Ib-IX complex expressed in CHO cells. We tested whether the following kinases could phosphorylate a recombinant GP Iba cytoplasmic tail: protein kinase A (PKA), protein kinase C (PKC), calmodulin-dependent kinase II (CaMKII) and Akt. All but Akt phosphorylated the region. We then tested polypeptides with various combinations of mutations to these residues for phosphorylation by PKA, and found that this kinase phosphorylated each of the residues except Ser590. We also examined the effect of the mutations on 14-3-3z association and on cell attachment and rolling on VWF. The findings from these studies were complex, with the results of 14-3-3 association suggesting a hierarchy of 14-3-3 binding. This binding did not correlate with the effect of the mutations on attachment on VWF or the rolling velocity profiles. Nevertheless, mutants could be clustered in terms of these effects, the aggregate of the data suggesting a very complex pattern of GPIb-IX-V regulation by the GP Ibα cytoplasmic tail, which will have to be taken into account when considering the association of GP Ibα with 14-3-3 as an antithrombotic target.

Disclosures
No relevant conflicts of interest to declare.
Manipulating Stem Cells for Therapeutic Expansion.
Authors: Sauvageau Guy 
Published: October 2019

Abstract
Abstract SCI-42
“Self-renewal” is a process central to the expansion of normal and cancerous stem cells and its understanding is critical for future advances in transplantation-based therapies and cancer treatment. Even today, many patients are deprived of the benefit of a successful blood stem cell transplant because the number of allogeneic or autologous stem cells available is insufficient, which results in delayed hematopoietic recovery post-transplant, or exclusion of a transplant-based therapeutic option altogether. The molecular machinery controlling self-renewal of hematopoietic stem cells (HSCs) remains poorly defined with the exception of a few genes such as HOX4 and Bmi1. We and others recently demonstrated the capacity of a recombinant HOXB4 protein (TAT-HOXB4 fusion protein) to stimulate mouse and human HSC self-renewal divisions in culture. Technical difficulties inherent to this recombinant protein have postponed the initiation of clinical trials. In part to overcome this hurdle, we have developed a novel in vitro/in vivo gain-of-function screen and identified several nuclear factors which expand hematopoietic stem cell ex vivo. A significant proportion of these factors display HOXB4-like properties and show non-cell autonomous activity. Initial results suggest that some of these new factors are also active with human cord blood derived HSCs. The generation of novel TAT fusion proteins will open new possibilities in the therapeutic expansion of human HSCs.

Disclosures
No relevant conflicts of interest to declare.
Bone Marrow Neutralizes Human Regulatory T-Cells to Permit Graft-Versus-Tumor Immunity In Humanized Mice
Authors: Guichelaar Teun , Rozemuller Henk , Emmelot Maarten , Martini Bianka , Storm Gert , Lokhorst Henk M , Martens Anton , Mutis Tuna 
Published: October 2019

Abstract
Abstract 1017
While application of regulatory T-cells (Tregs) has become a promising tool to prevent Graft-versus-Host Disease (GvHD), Tregs pose the risk of suppressing the curative Graft-versus-Tumor (GvT) effect induced by therapeutically transfused donor lymphocytes. Towards clinical application of Tregs, we explored the in vivo impact of human Tregs on GvT and GvHD in a humanized GvT model. In this model we have previously shown that infusion of human PBMC into tumor bearing Rag2-/-gc-/- mice induces a profound GvT effect but also results in lethal xenogeneic(x) GvHD. We observed that co-infusion of human CD4+CD25+ Tregs suppressed x-GvHD, but allowed effective GvT against human tumors localized within the bone marrow, despite efficient Treg-homing to bone marrow. Remarkably, however, Tregs abrogated the GvT effect against the same tumor when located extramedullary, suggesting that permittance of the GvT effect in the bone marrow was due to the inactivation of Tregs in the bone marrow micro-environment. In detailed exploration of this remarkable phenomenon, we discovered that stromal cells derived from bone marrow neutralize the suppressive phenotype and promotes interleukin-17 in human Tregs through secretion of IL-6/IL-1β. In conclusion, this study for the first time provides a novel in vivo mechanism of how human Tregs can control undesired inflammation while permitting GvT against hematological tumors residing in bone marrow. This mechanism involves the bone marrow stroma, which creates a tumor micro-environment that significantly drives conversion of Tregs into non-suppressor T-cells.

Disclosures:
No relevant conflicts of interest to declare.
Evolution of Human BCR-ABL1 lymphoblastic Leukaemia-Initiating Cells.
Authors: Notta Faiyaz , Mullighan Charles , Wang Jean , Poeppl Armando G , Doulatov Sergei , Phillips Letha A , Ma Jing , Minden Mark D. , Downing James R. , Dick John E. 
Published: October 2019

Abstract
Abstract 1023
Many tumour types are composed of genetically diverse cells, however little is known of how diversity evolves or the impact that diversity has on functional properties. Here, using xenografting and DNA copy number alteration (CNA) profiling of human BCR-ABL1 acute lymphoblastic leukaemia, we demonstrate that genetic diversity occurs in functionally defined leukaemia-initiating cells and that many diagnostic patient samples contain multiple genetically distinct subclones. Reconstruction of the subclonal genetic ancestry of several samples by CNA profiling demonstrated a branching multiclonal evolution model of leukaemogenesis, rather than linear succession. For some patient samples, the predominant diagnostic clone repopulated xenografts, while in others it was outcompeted by minor subclones. Reconstitution with the predominant diagnosis clone was associated with more aggressive growth properties in xenografts, deletion of CDKN2A/CDKN2B, and poor patient outcome. Our findings link clonal diversity with function and underscore the importance of developing therapies that eradicate all subclones.

Disclosures:
No relevant conflicts of interest to declare.
Treatment for Primary Acute Myeloid Leukemia: Results of Two Consecutive Trials From the Spanish CETLAM Group Showing Improvement with the Use of G-CSF Priming and Precise Risk-Adapted Therapy.
Authors: Sierra Jorge , Hoyos Montserrat , Nomdedeu Josep F. , Esteve Jordi , Duarte Rafael F. , Ribera Josep-Maria , Tormo Mar , Guardia Ramon , Llorente Andreu , Bueno Javier , Besalduch Juan , Queipo M. Paz , Marti-Tutusaus Josep M. , Pedro Carmen , Torres Pio , Garcia Antoni , Heras Inmaculada , Bargay Joan , Font Llorenç , Gonzalez Jose , Sampol Antonia , Salamero Olga , Escoda Lourdes , Gallardo David , Calabuig Marisa , Oriol Albert , Arnan Montserrat , Pratcorona Marta , Brunet Salut 
Published: October 2019

Abstract
Abstract 1066
Different approaches have been investigated to improve the prognosis of adult patients with primary acute myeloid leukemia. In two consecutive phase II trials our group has explored the use of intermediate-dose cytarabine in induction associated with idarubicin and etoposide, the addition of G-CSF priming to the previous combination, and risk-adapted postremission therapy.

Objective:
To compare the results of two consecutive trials for primary AML and to analyze the factors influencing the outcome.

Patients and methods:
Adult patients between 17 and 60 years of age with de novo AML, diagnosed between 1999 and 2009, were included in the CETLAM AML-99 and AML-03 trials. Induction chemotherapy (CT) included one or two courses of idarubicin 12 mg/m2 IV days 1,3,5, cytarabine 500 mg/m2/12h over 2h IV days 1,3,5,7 and etoposide 100 mg/m2 IV days 1,2,3. This was followed by one consolidation with mitoxantrone 12 mg/m2 IV from day 4 to 6, and cytarabine 500 mg/m2/12h IV from day 1–6. In the AML 03 trial, patients also received G-CSF priming, 150 mg/m2 subcutaneously (SC) from day 0 to the last day of induction and consolidation CT. Postremission therapy consisted of high-dose cytarabine (CBF AML), autologous or allogeneic hematopoietic transplantation depending on cytogenetics, courses to complete remission (CR), and in the AML-03 protocol also based on molecular abnormalities involving FLT3 or MLL genes and/or the persistence of minimal residual disease after consolidation.

Results:
Overall, 788 patients were included, 353 in the AML-99 trial and 435 in the AML-03. Median age of the patients was 46 years (range 17–60). There were no differences between patients included in the two protocols regarding age, gender, leukocyte counts, cytogenetics and proportion of favourable and unfavourable molecular cases in the group with intermediate-risk karyotype. The main results achieved appear in the table.
Multivariate analysis confirmed the favourable impact of AML-03 protocol on outcome. Other significant factors influencing survival were age, leukocyte counts and cytogenetics.

Conclusion:
G-CSF priming improved the CR rate of adult patients with primary AML and favourable or unfavourable cytogenetics. This fact and a more precise risk-adapted therapy taking into account genetic data and MRD studies translated into improved overall survival.

Disclosures:
No relevant conflicts of interest to declare.
Outcome of Saddle Pulmonary Embolism: A Nested Case-Control Study.
Authors: Bose Gauruv , Gandara Esteban , Carrier Marc , Erkens Petra MG , Rodger Marc , Wells Philip 
Published: October 2019

Abstract
Abstract 1102

Introduction:
The management of saddle pulmonary embolism (PE) is controversial. Evidence about outcomes and management strategies is scarce in the literature due to the small prevalence of saddle PE. Historically it has been recommended that this group of patients should be treated aggressively.

Purpose:
To determine the prevalence and outcomes of patients diagnosed with saddle PE.

Methods:
Retrospective cohort study of consecutive patients with saddle PE diagnosed at the Ottawa Hospital between January 2007 and December 2008. Patients were included if a thrombus was present on computed tomographic pulmonary angiography (CTPA) in the main pulmonary arteries spanning the bifurcation of the main pulmonary trunk. These cases were each matched with two non-saddle controls with proximal PE (thromboemboli in the main pulmonary arteries) based on age, sex, systolic blood pressure greater than or less than 90 mmHg, and the presence or absence of cancer. Demographics, prognostic factors, treatment, and outcomes were collected. Patients were followed over a 30 day period following the diagnosis. RESULTS: A total of 32 (5%) of 724 patients with PE had a saddle event. Baseline characteristics are depicted in Table 1. Differences between the saddle case group and non-saddle control group include the presence of right ventricular dilation (59% of saddle cases vs. 22% of controls, p-value: 0.0007) and in the proportion of patients managed as outpatients (7% of saddle cases vs. 33% of controls, p-value: 0.02). At 30 days no differences were found in patients with saddle PE or proximal PE for all cause mortality (6% vs. 10%; OR: 0.64; 95% CI: 0.08–3.2), PE related mortality (0% vs. 6%; OR: 0.52; 95% CI: 0.01–6.1), major bleeding (3% vs. 5%; OR: 0.65; 95% CI: 0.02–6.4), or recurrent venous thromboembolism (6% vs. 10%; OR: 0.64; 95% CI: 0.08–3.2).

Conclusions:
Patients with saddle PE do not have a worse 30-day prognosis than patients with proximal PE matched by age, sex, systolic blood pressure, and presence of cancer.

Disclosures:
No relevant conflicts of interest to declare.
Prothrombin Complex Concentrate (Octaplex®) In Patients Requiring Immediate Reversal of Vitamin K Antagonist Anticoagulation.
Authors: Song Madeline , Warne Connor , Crowther Mark A. 
Published: October 2019

Abstract
Abstract 1115

Rapid reversal of vitamin K antagonists (VKAs) is required when patients experience acute bleeding or are in need of urgent surgery. Traditionally, in Canada, the only therapeutic option for warfarin reversal was fresh frozen plasma (FFP); recently a four factor prothrombin complex concentrate (PCC) has become available. In this retrospective chart review, we examined the use of Octaplex®, a four factor (FII, FVII, FIX, FX), solvent/detergent (S/D) treated and nanofiltered PCC, in a tertiary care centre over 18 months (November 2008 to May 2010), to determine its efficiency and safety in VKA reversal. The PCC was used 85 times in 82 patients. All patients had received warfarin prior to PCC administration. Patients received a mean dose of 1792 (1000 – 3500) IU of PCC for treatment of bleeding (36), urgent surgery (40) and excessively elevated international normalized ratio (INR) (8). One patient received PCC for an unknown indication and is excluded from the results presented here; a further 6 patients did not have INR tests done immediately before and immediately after PCC administration and are excluded from the laboratory results presented here. The mean INR before administration of the PCC was 5.10 (range 1.2 – 25). The mean INR after administration of the PCC was 1.43 (range 0.9 – 3.3). Forty-seven of seventy-nine [59.5%, 95% CI 47.9 to 70.4%] administrations resulted in an INR of 1.3 or less and 60/79 [75.9%, 65.0 to 84.9%] resulted in an INR of 1.5 or less. Of the 40 patient encounters where PCC was administered due to need for urgent surgery, 39 were able to undergo the procedure. Three patients experienced a thromboembolic event; one had a venous thromboembolism due to a clot extension of a previously diagnosed left leg DVT 8 days after PCC administration, another had a non-occlusive renal vein clot 8 days after PCC administration and the last had a cilioretinal artery occlusion 238 days after receiving PCC. No other adverse events potentially related to PCC were observed. We conclude that the prothrombin complex concentrate Octaplex® provides efficacious, safe and rapid reversal of VKA therapy.

Disclosures:
Crowther: Pfizer: Consultancy, Research Funding; Sanofi Aventis: Consultancy, Research Funding; Leo Laboratories: Consultancy, Research Funding; Artisan: Consultancy. Off Label Use: The product described in this abstract is not available in the US. The described use is “on label” in Canada.
Short Polyphosphates Inhibit the Procoagulant Activities of Long-Chain Polyphosphate: A Novel Role for Platelet-Derived Pyrophosphate?.
Authors: Smith Stephanie A. , Morrissey James H. 
Published: October 2019

Abstract
Abstract 1140

Introduction:
Inorganic polyphosphates are negatively charged, linear phosphate polymers that influence hemostasis via accelerating factor V activation, triggering the contact pathway, and enhancing fibrin polymerization. The latter two effects require long-chain polyphosphates for optimal activity (>1000mers for activating the contact pathway and >250mers for enhancing fibrin polymerization). We explored whether short polyphosphates could inhibit the procoagulant effects of long polyphosphate polymers.

Methods:
Polyphosphate was size-fractionated by gel electrophoresis. “Long-chain polyphosphate” (LCP) was a heterogeneous polyphosphate preparation containing polymers ranging from 400 to several thousand phosphates long. Short-chain polyphosphate preparations were narrow fractions of homogeneous size. We also tested additional phosphate-containing molecules including ADP, ATP, monophosphate, pyrophosphate (PPi), and triphosphate. Clotting assays were performed using pooled normal plasma spiked with 0–20 μM LCP and 0–500 μM small (poly)phosphate; clotting was initiated by factor Xa plus CaCl2, or by CaCl2 alone (contact pathway). Fibrin was formed by clotting 2.6 mg/ml fibrinogen with 10 nM thrombin in the presence of 0–500 μM small (poly)phosphate plus 150 μM LCP and CaCl2.

Results:
Small polyphosphates (size range 23–83mer) reduced the ability of LCP to trigger the contact pathway of blood clotting, while monophosphate, PPi, triphosphate, ADP, and ATP all failed to influence the procoagulant activity of LCP. None of the small (poly)phosphates antagonized the ability of LCP to enhance factor V activation. Interestingly, monophosphate, PPi and triphosphate all inhibited the ability of LCP to enhance fibrin clot structure, with PPi being the most potent. Although ADP and ATP contain di- and tri-phosphates, they did not recapitulate the potent inhibitory activity of PPi. On the other hand, PPi had no measurable effect on the turbidity of fibrin clots formed in the absence of LCP.

Conclusions:
Short-chain polyphosphates inhibit the ability of LCP to initiate the contact pathway of coagulation. Platelet dense granules contain abundant PPi which is secreted in response to platelet agonists, although biological roles for platelet-secreted PPi are unclear. We propose that PPi is a novel modulator of fibrin clot structure, acting to regulate the effects of longer-chain polyphosphates on fibrin fibril formation.

Disclosures:
Smith: University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.