bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterizing the Disease-Initiating Cell Population In a Murine Model of Jak2V617F+ Myeloproliferative Neoplasm
Authors: Mullally Ann , Lane Steven W. , Poveromo Luke , Al-Shahrour Fatima , Kutok Jeffery , Ebert Benjamin L. 
Published: October 2019

Abstract
Abstract 317
The JAK2V617F mutation is the most common somatic mutation in BCR-ABL negative myeloproliferative neoplasms (MPN) and small molecule JAK2 kinase inhibitors are currently being evaluated in clinical trials in MPN patients. We recently reported a Jak2V617F conditional knockin mouse MPN model in which expression of Jak2V617F was under the control of the endogenous Jak2 promoter and the phenotype closely recapitulated the features of human polycythemia vera (PV) (Mullally and Lane et al, Cancer Cell 2010). In this model the MPN phenotype developed as a result of expansion and erythroid skewing of committed myeloid progenitor populations, while the hematopoietic stem cell (HSC) compartment (enriched within LineagelowSca1+cKithigh, LSK cells) was not expanded. In contrast to the expanded progenitor population, only LSK cells had the unique capacity to initiate the MPN. Notably, treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. In light of these findings, we wished to further characterize MPN-initiating LSK cells in this model, with a view to gaining understanding of the differential molecular circuitry of JAK2V617F mutant and normal HSC. The LSK compartment, although enriched for HSC, may be further defined by immunophenotype into long-term HSC (LT-HSC, LSKCD150+CD48-), short-term HSC (ST–HSC, LSKCD150-CD48-), or multipotent progenitor cells (MPP, LSKCD150-CD48+). Gene expression analysis of total LSK cells revealed enrichment for markers of myeloid progenitor differentiation in Jak2V617F LSK cells compared to Jak2 wild-type (WT) controls, although there were no quantitative differences in the LSK sub-populations between the two groups. We therefore sought to evaluate whether the MPN-initiating cell population was contained within the LT-HSC compartment. Employing single cell sorting multi-parameter flow cytometry and limiting dilution bone marrow transplantation assays, we provide further functional characterization of the MPN-initiating population in this model. We demonstrate that Jak2V617F LT-HSCs are highly enriched for MPN-initiating activity in vivo and are able to maintain the MPN through multiple rounds of serial transplantation for periods greater than one year. The MPN phenotype remains stable during this time without evolution to myelofibrosis or leukemia. These findings provide important biological insights into MPN initiation and maintenance and have implications for the development of curative therapeutic strategies for MPN patients.

Disclosures:
No relevant conflicts of interest to declare.
Systematic Review of Antiphospholipid Syndrome and Left Ventricular Thrombus
Authors: Kotlyar David S , Kelesidis Iosif , Pintova Sofya , Bhardwaj Aarti , Chirumamilla Amala , Sommer Per , Menegus Mark A , Ostfeld Robert J 
Published: October 2019

Abstract
Abstract 3182

Background:
Left ventricular thrombus is a potentially devastating complication of anti-phospholipid syndrome (APLS). To date there have been no systematic reviews of this topic.

Methods:
We systematically searched MEDLINE, EMBASE, Cochrane, ISI, and CINAHL for cases of left ventricular thrombi in APLS. We also included a case seen at our institution. Both English and non-English language citations were included. Keywords included “intracardiac” AND “antiphospholipid”, or “antiphospholipid syndrome” AND “cardiac thrombus”, or in MEDLINE with MeSH operator “antiphospholipid syndrome” AND “thrombus.” A total of 669 citations were found. Twenty-six met our criteria.

Result:
There are a total of 28 cases of left ventricular thrombus in APLS (Table 1). The median age was 38 (±15.1 yrs). Fifteen (54%) were women, 12 (43%) were men and 1 was of unknown sex. Five (18%) died, 21 (75%) survived to discharge and 2 (7.1%) had unknown vital status. Nineteen (68%) had a positive cardiolipin antibody, 7 were unknown (25%) and 2 were negative (7.1%). Fourteen (50%) had a positive lupus anticoagulant, and 14 (50%) were unknown. Eight were treated with anticoagulation alone (29%). Of those, 7 recovered (25%) and one was unknown (3.6%). Three (10.7%) were treated with anticoagulation and corticosteroids, and all 3 recovered. In eight (29%), surgical thrombectomy and anticoagulation were utilized. Of the 8 who had surgery, 6 recovered (21%) and 2 died (7.1%).

Discussion:
In this systematic review we have shown that death may be less common than previously reported (33% [Lim, et. al, Intern Med J. 2004 Mar;34(3):135-7] vs. 18% in our study). A high index of suspicion is warranted.

Disclosures:
No relevant conflicts of interest to declare.
Comparative Analysis of Autologous Hematopoietic Cell Transplantation with Radioimmunotherapy (RIT) Based Conditioning Versus Total Body Irradiation (TBI) for High-Risk Diffuse Large Cell Lymphoma (DLCL): Toxicity and Efficacy
Authors: Krishnan Amrita , Palmer Joycelynne , Nademanee Auayporn , Raubitschek Andrew , Yamauchi Dave , Kogut Neil Martin , Federico Laura , Tsai Nicole , Forman Stephen 
Published: October 2019

Abstract
Abstract 32

Background:
RIT based conditioning offers the potential of combining the efficacy of radiation with decreased toxicity over traditional TBI. In pilot trials we demonstrated that combining Yttrium 90 (ibritumomab tiuxetan) with high-dose BEAM (ZBEAM) is feasible and has a toxicity profile similar to high-dose BEAM. Herein we report the results of a comparative analysis designed to evaluate transplant outcomes among DLCL patients who were conditioned with either ZBEAM or a TBI-based conditioning regimen. Patients were matched on age (+/− 5 years), disease status, number of prior regimens, year of diagnosis (+/− 5 years), and year of transplant (+/− 5 years). There was a total of 92 DLCL patients treated from 01/1997-01/2009; 46 patients in each treatment group. The median patient age was 56.5 years (range: 19–78) for the ZBEAM group, and 53 years (range: 21–62) for the TBI group. Both groups had a median of two prior regimens, with 13% (ZBEAM) and 15% (TBI) considered high-risk first remission, 65% beyond 1st CR and 22% induction failures in each cohort. The median length of follow-up for surviving patients was 51–83 months. There was a trend toward improved PFS in the RIT group: 2 year PFS for ZBEAM group 66% (95%CI: 56–74) vs. 50% (95%CI: 43–57) for TBI group (p<0.08). Results to date show that a plateau in PFS appears to have been achieved for both groups (at 2.6 years in the ZBEAM group and 3.7 years for the TBI group), which translates into a 20% improvement in PFS for the ZBEAM patients, >4 years post transplant. Similarly the OS estimate was significantly higher for ZBEAM compared to TBI controls: 84% vs. 59 % (p<0.01). The lower OS rate for the TBI cohort was primarily due to toxicity, with a 2 year non relapse mortality of 0% for ZBEAM vs. 13% for TBI. (p<0.01) The causes of death included: relapse progression n=9 (ZBEAM), n=15 (TBI), infection n=2 TBI, cardiac disease n=2 TBI, pneumonia N= 1 TBI, unknown N=3 TBI.

Conclusions:
RIT based conditioning demonstrated improved survival when compared to traditional radiation based regimens in the treatment of DLCL due to a more favorable toxicity profile, while maintaining potent anti-lymphoma effects.

Disclosures:
No relevant conflicts of interest to declare.
Long-Term Production System of Red Blood Cells Using Human Cord Blood and Stromal Cells
Authors: Kobune Masayoshi , Kikuchi Shohei , Murase Kazuyuki , Iyama Satoshi , Sato Tsutomu , Miyanishi Koji , Sato Yasushi , Takimoto Rishu , Kato Junji 
Published: October 2019

Abstract
Abstract 3211
We have previously shown that primary human stromal cells and hTERT-transduced human stromal cells (hTERT-stromal cells) support cord blood (CB) hematopoietic stem/progenitor cells. However, it is unclear whether human stromal cells maintain the expansion of erythroid progenitor cells without losing erythroid differentiation potential for a long-term ex vivo culture. In an attempt to evaluate the efficacy of human stromal cells, erythroid induction was conducted by SCF, EPO and IGF-1, 2-week after expansion of CB CD34+ cells with or without human stromal cells. The maturation of erythroid cells were evaluated by morphological findings, transferrin receptor (TfR)/glycophorin A (GPA) expression and hemoglobin (Hb) synthesis (MCH, pg/cells). The number of BFU-E upon 2-week coculture with the hTERT-stromal cells was significantly higher than those without hTERT-stromal cells (BFU-E, 639±102 vs. 4078±1935, the initial cell number of BFU-E was 513±10). Hb concentration of erythroblasts that had been derived from coculture with stromal cells, was significantly higher than that derived from stroma-free condition 14 days after erythroid induction (MCH, 0.78±0.11 vs. 2.62±0.12; p<0.05). Moreover, cobblestone area (CA)-forming cells existed beneath stromal layer weekly produced the large number of BFU-E from 4th week to at least 8th week (the total number of BFU-E, 57246±1288)(Figure A). Notably, these BFU-Es derived from CA could simultaneously differentiate into orthophilic erythroblasts with nearly normal Hb synthesis (MHC, 24.5±6.4 pg/cell)(Figure B) and GPA expression. Furthermore, most of these erythroblasts derived from CA underwent enucleation spontaneously after further 7 days culture. Thus, using hTERT-stromal cells, the long-term ex vivo erythroid production could be attained from CB cells. These findings contribute to constructing long-term of ex vivo erythroid production system using human stromal cells.

Disclosures:
No relevant conflicts of interest to declare.
Anti-Leukemia Effect of Rapamycin Alone or Plus Daunorubicin on Acute Lymphoblastic Leukemia Cell Lines
Authors: Yang Xi , Gong Yuping , Niu Ting 
Published: October 2019

Abstract
Abstract 3256
Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine-threonine kinase that integrates signals from multiple inputs, including growth factors, amino acids, and intracellular energy supply, to regulate diverse cellular functions, such as transcription, ribosome biogenesis, translation initiation, and autophagic cell death (autophagy). Aberrant activation of the mTOR signaling pathway has been demonstrated in several tumors, including the majority of acute lymphoblastic leukemia(ALL). The potential anti-leukaemia effect of mTOR inhibitors has received some attention so far in ALL. In this study, we aimed to assess the anti-leukemic activity of Rapamycin (RAPA), an mTOR inhibitor, alone and in combination with daunorubicin (DNR). Here, we demonstrated that RAPA in concentrations of 1–100 nmol/L significantly inhibited the proliferation of Ph+ ALL cell line SUP-B15, whereas exerted poor effect on Ph- ALL cell line CEM. However, RAPA at a dose of 50 nmol/L significantly intensified the inhibition induced by DNR on two ALL cell lines. The synergistic effect was associated with regulation of autophagy and apoptosis, blockage of cell cycle progression in the G1 phase. We also reported that the consequence of DNR-treatment induced the overexpression of the mTOR signaling pathway in two ALL cell lines and primary leukemia cells in vitro, whereas RAPA effectively eliminated this deleterious side effect of the DNR and might enhance DNR ability to kill drug–resistant cancer. Altogether, our results suggested that DNR in combination with RAPA was more effective in the treatment of ALL than DNR alone. Therefore, combination of classical induction chemotherapy with an inhibitor of the mTOR kinase could be a promising strategy in future treatment of ALL.

Disclosures:
No relevant conflicts of interest to declare.
In Vivo Imaging In the Individualized Mouse Model of Acute Lymphoblastic Leukemia Enables Highly Sensitive and Continuous Follow up of Patient-Derived Xenografts
Authors: Terziyska Nadia , Farkasova Katarina , Wagner Ernst , Ogris Manfred , Jeremias Irmela 
Published: October 2019

Abstract
Abstract 3259
In the individualized xenograft mouse model, acute leukemia cells from patients are transplanted into severely immuno-deficient mice to serve as a preclinical animal model. The use of this mouse model for pre-clinical therapy trials is hampered by the low sensitivity of existing readouts disabling the reliable follow up of single animals. Both secretion of leukemic cells into the blood flow as well as clinical signs, if at all, occur at late states of the disease; readouts in further organs by, e.g., immunohistochemistry or flow cytometry analysis, require organ extraction and can thus be performed only once per mouse.
To overcome this obstacle, we established in vivo imaging in the xenograft mouse model of acute lymphoblastic leukemia (ALL). We engrafted pediatric acute leukemia cells from patients at diagnostic bone marrow aspiration of either diagnosis or relapse. In agreement with published data, mice developed leukemia within weeks to months. Engraftment was followed by easy passaging of cells into further generations of mice. We established lentiviral transduction of xenograft cells which enabled expression of transgenes in these cells. Using lentiviral transduction, we stained patient-derived xenograft ALL-cells using luciferase as a reporter. Transgenic xenograft leukemia cells were visualized once per week by bioluminescence in vivo imaging using a charge-coupled device camera.
Our first data show that in vivo imaging enabled the reliable and continuous follow up of single animals over time. The sensitivity of in vivo imaging in measuring leukemic engraftment was significantly higher compared to current readouts like examination of blood cells. In vivo imaging data support that the leukemic pattern of metastases of patient-derived xenograft ALL-cells in mice highly resembles the distribution of the disease in men. Upon intravenous injection, cells first home to the liver, where they stay alive only for a few weeks. Long-term engraftment is seen in the bone marrow of many different bones and constantly increases over time. Engraftment in spleen indicates rather late stages of disease. This kinetic of engraftment remained constant between several transgenic xenograft samples and between different mice engrafted with the same sample.
Taken together, we have established molecular modulation of xenograft cells which enables expression of luciferase and in vivo bioluminescence imaging as a new sensitive and continuous in vivo readout in mice. In vivo imaging will allow realizing precise preclinical trials in the individualized mouse model in the future.

Disclosures:
No relevant conflicts of interest to declare.
Synergism Between Chemotherapy with 6-Mercaptopurine and DNA Vaccination Against Ph+ Acute Lymphoblastic Leukemia In Syngeneic Mice
Authors: Rott Yvonne , Schmidt Manuel , Wittig Burghardt , Herting Egbert , Köchling Joachim 
Published: October 2019

Abstract
Abstract 3265
Long term remission and cure in 70–80 % of children and 40–50% of adults with acute lymphoblastic leukemia (ALL) is achieved with polychemotherapy. The BFM study protocol includes maintenance chemotherapy with 6-mercaptopurine (6-MP) and methotrexate. However, prognosis of patients with Philadelphia chromosome positive (Ph+) ALL remains poor and most patients with Ph+ ALL eventually relapse unless they undergo allogeneic stem cell transplantation. Thus, new relapse prevention strategies are needed for patients with Ph+ ALL.
We compared the efficacy of 6-MP with a DNA-based vaccine in a syngeneic Balb/c mouse model. We also evaluated whether 6-MP inhibits the immunization by the DNA-vaccine or alternatively, acts synergistically in combination therapy. Non-viral minimalistic immunogenically defined gene expression vectors (MIDGE) encoding a BCR-ABLp185 fusion specific peptide, GM-CSF and IL12 were used for in vivo transfection of murine skin. In addition, we used natural DNA-based double stem-loop immunomodulators (dSLIM), exhibiting an unique structure and containing 6 CpG-motifs, as non-specific immune adjuvant. One day after the application of a lethal challenge dose of the syngeneic BCR-ABLp185 expressing leukemia cell line BM185, mice received either daily oral applications of 6-MP (50mg/m2/d) or were immunized twice with the DNA vaccine BCR-ABL/GM-CSF/IL-12/dSLIM intracutaneously or received both 6-MP and the DNA-vaccine.
While chemotherapy with 6-MP lead to a significant longer mean tumor-free and overall survival compared to the control and a survival rate of 10%, immunization with the DNA vaccine BCR-ABL/GM-CSF/IL-12/dSLIM also showed a significant longer mean tumor-free and overall survival compared to the control and a survival rate of 56%. More intriguingly, the combination therapy with 6-MP and DNA vaccination further improved the outcome and lead to a survival rate of 100%. Immunization experiments and CTL assays showed that BCR-ABL specific sequences are essential to prevent Ph+ ALL. In cell depletion studies we show that both CD4+ T cells and natural killer cells and to a lower degree CD8+ cells contribute to the anti-leukemia effect of our DNA-based vaccine. In conclusion, we provide survival and functional data that 6-MP combined with a leukemia-specific DNA-vaccine acts synergistically in the treatment of Ph+ ALL. This approach might provide an option to optimize maintenance therapy in order to prevent a relapse in patients with Ph+ ALL.

Disclosures:
Schmidt: Mologen AG: Employment. Wittig:Mologen AG: Employment, Membership on an entity's Board of Directors or advisory committees.
PAR-3 Activation by Activated Protein C: a Novel Podocyte Protective Signalling Pathway
Authors: Madhusudhan Thati , Wang Hongjie , Müller-Krebs Sandra , Schwenger Vedat , Zeier Martin G. , Nawroth Peter , Esmon Charles , Isermann Berend 
Published: October 2019

Abstract
Abstract 329
Activated protein C (APC) has anti-coagulant and cytoprotective effects. Recently we have shown that APC protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. The cytoprotecive effects of APC in endothelial cells require endothelial protein C receptor (EPCR) and protease activated recetor-1 (PAR-1). However, the signalling mechanism through which APC exerts its cytoprotective effects in podocytes is not known. Here we have used a mouse model of LPS-mediated podocyte injury and show that APC protects against LPS induced podocyte apoptosis and proteinuria. These cytoprotective effects of APC require activation of PAR-3. Supplementary in vitro experiments were performed using immortalized differentiated human and mouse podocytes to delineate the receptor mechanism.
LPS administration (200 μg/mice, i.p.) induced podocyte apoptosis, foot process effacement and albuminuria by 24h. Administration of APC (20 μg/ mice, i.v, 6 h after LPS injection) protected against podocyte apoptosis, foot process effacement and albuminuria. Pre-incubation of APC with HAAPC antibody which blocks the anticoagulant, but not the cytoprotective properties of APC did not abolish the cytoprotective effect, establishing that the protective effect of APC is independent of its anticoagulant property. To demonstrate the direct cytoprotective effect of APC on podocytes, FITC labelled APC was administered i.v. Within 3 min FITC-APC accumulated in the pericapillary space of mouse glomeruli. In addition significantly elevated levels of PC were detected in urine samples of LPS treated mice as well as of diabetic patients or mice. These results suggest that APC transverses the glomerular filtration barrier, thus being able to directly modulate podocyte signalling in vivo. Furthermore experiments with PAR-3-/- mice, EPCR deficient (EPCRd/d) mice or PAR-1 antagonist (pepducin PI pal12S) showed that the cytoprotective effect of APC in podocytes is independent of EPCR but requires PAR-3 and PAR-1. In vitro APC (2 nM) inhibited puromycin aminonucleoside (PAN) induced podocyte apoptosis. These anti-apoptotic effect of APC require limited proteolysis of PAR-3 and cross-activation of either PAR-1 (mouse, rat podocytes) or PAR-2 (human podocytes). In addition, ectopic expression of PAR-3 in mesangial cells which lack PAR-3 is sufficient to render these cells APC sensitive and inhibit staurosporine induced apoptosis.
In conclusion, we demonstrate a novel cyto-protective mechamism of APC in an acute model of nephropathy, which depends on APC mediated limited proteolysis of PAR-3. This novel podocyte protective signalling pathway may lay ground to the delineation of new pathophysiological concepts and therapeutic approaches.

Disclosures:
No relevant conflicts of interest to declare.
the Efficacy and Safety of Prothrombin Complex Concentrate In Patients Requiring Urgent Reversal of Warfarin, A Retrospective Canadian Experience
Authors: Varga Cindy , Papadoukakis Stella , Caplan Stephen N. , Kahn Susan R. , Blostein Mark 
Published: October 2019

Abstract
Abstract 3344

Background:
Major bleeding remains the most frequent adverse effect of oral anticoagulant therapy. A rapid method of reversal is required for patients on warfarin who suffer acute bleeding or require emergency surgery. Octaplex is a prothrombin complex concentrate recently recommended for use by the National Advisory Committee for Blood and Blood Products of Canada (Canadian Blood Services) at a dose of 1000 IU of factor IX activity. It ensures rapid INR correction by providing vitamin K-dependent factors without a large volume load. The main goal of this study was to investigate both the efficacy and safety of Octaplex use in a tertiary care center in Quebec, Canada.

Materials and Methods:
A retrospective review was conducted on the charts of patients who received Octaplex for urgent warfarin reversal between 1 January 2009 and 31 October 2009. Data collected included INRs pre and post Octaplex administration, and documentation of any adverse events.

Results:
We identified 146 occasions when Octaplex was administered for urgent INR reversal. A total of 118 patients were included in the study with a median age of 77. The three most frequent indications for Octaplex were: 1) gastrointestinal bleeding, 2) intracranial hemorrhage, and 3) pre-procedural INR reversal. The majority of patients (90.7%) were treated with Octaplex at a dose of 1000 IU of factor IX activity, with a range of 500 IU to 5000 IU. The average mean pre-and post INRs were 4.00 and 1.65, respectively. Overall, 89.8% (130/146) of patients corrected to an INR < 2.0, of which 51.5% (67/130) corrected to an INR < 1.5. Although 8.2% (12/146) of patients were considered to have a poor response (post INR > 2.1), these patients tended to have a higher mean pre-INR value (5.28 vs. 2.69) and still achieved important absolute drops in INR (range 1.1–9.3, mean 2.91). Only 4 patients (2.7%) did not experience a reversal of their INR. Seventeen patients received second doses of Octaplex, 70.6% (12/17) of whom achieved a post INR < 1.5 and 94.1% (16/17) achieved a post INR < 2.0. There were 8 reported thrombotic events amongst 118 patients: 3 myocardial infarctions, 3 DVTs, 1 ischemic limb and 1 intracardiac clot. All patients with these adverse events had other co-morbities that could have also contributed to these toxicities.

Conclusion:
Octaplex is efficient and appears to be safe in the urgent reversal of INR in patients taking vitamin K antagonists.

Disclosures:
No relevant conflicts of interest to declare.