bluuebunny/crossref_metadata_2025_split huggingface dataset:
Illuminating Clonal Dynamics of a Leukemia Cell Line Using Viral Barcoding and Illumina Deep Sequencing Technology
Authors: Porter Shaina , Highnam Gareth , Mittelman David , Porteus Matthew 
Published: October 2019

Abstract
Abstract 1410
Leukemias are generally clonal in origin, but develop significant heterogeneity as they expand and acquire genetic and epigenetic alterations. Detecting this heterogeneity and tracking the dynamics and evolution of clones within a population are important for our understanding of these cancers and the phenomena of drug resistance and relapse. Attempts to track the contributions of individual cells to the clonality of large populations, however, have been constrained by limitations in sensitivity and complexity. We have created an efficient and high throughput method to overcome these limitations by harnessing the power of viral marking and next generation sequencing technology, allowing us to track the clonal contributions of many thousands of cells with minimal perturbations to the population as a whole. K562s are a common patient-derived CML line featuring rapidly proliferating, highly aneuploid cells. We stably and heritably marked individual cells via the lentiviral-mediated insertion of one of ∼14,000 random, non-coding, 20 basepair DNA “barcodes.” Using Illumina next-generation sequencing we determined the relative frequency of every barcode, thus directly measuring the contribution of each clone to the overall population over 90 population doublings. We anticipated that the the K562 cell line, while very highly aneuploid, would have stable clonal dynamics because it has been grown in culture for years, thus allowing potentially dominant clones to reach equilibrium. We found, however, that K562 cells continue to undergo dramatic changes in clonal representation. As expected, in analysis of our clonally marked population, there was a broad distribution of clones. After culturing under ideal conditions for 90 population doublings, we found that a single clone now represented 9% of the population and that less than 100 clones now represented more than 50% of the population, a massive clonal skewing after just a brief period of time. Cell lines are often assumed to be a homogenous group of identical clones, a seemingly reasonable assumption considering the years of passaging/subculturing. However, our results show that ongoing genomic and/or epigenomic instability leads to proliferative instability, resulting in unexpected clonal dynamics and rapid clonal evolution. These results have profound implications for the experimental use of cell lines, as well as broad applications for clonal analysis of leukemias, other malignancies, and stem cells.

Disclosures:
No relevant conflicts of interest to declare.
Type of Bleeding Among Patients with Nonvalvular Atrial Fibrillation in a Large Managed Care Population
Authors: Deitelzweig Steven , Pinsky Brett , Buysman Erin , Lacey Michael , Makenbaeva Dinara , Wiederkehr Daniel , Graham John 
Published: October 2019

Abstract
Abstract 2073

Background:
Risk of bleeding is an important consideration among patients with nonvalvular atrial fibrillation (NVAF), and different anatomical sites may carry greater risk for bleeding.

Objective:
To describe the incidence of critical anatomical site bleeding events among patients with NVAF in the pre-NOAC (novel oral anticoagulation) era.

Methods:
Administrative claims data were used for this retrospective study. Adults with healthcare claims related to atrial fibrillation (ICD-9-CM 427.31) between Jan 2005 and Jun 2009 but no evidence of valvular disease were included. Patients were followed until the earliest of death, disenrollment from the health plan, or 30 Jun 2010. Bleeding events in the follow-up period were considered major if they were associated with any of the following: inpatient care, blood transfusion, decreased hemoglobin or hematocrit, physician guided medical or surgical treatment, intracranial bleed, or death. Anatomical bleeding sites were identified based on diagnosis codes.

Results:
The mean (SD) age of the study sample (N=48,260) was 67 (13) years and 62.2% of the patients were male. Mean (SD) follow-up duration was 802 (540) days (median 673 days). Over 105,419 patient-years, the critical site with the highest rate of major bleeding events was the gastrointestinal tract, followed by intracranial bleeding (TABLE).

Conclusions:
Gastrointestinal, intracranial, and pericardial bleedings were the most frequently observed major bleedings in the pre-NOAC era. Patients with NVAF have a high rate of major gastrointestinal bleedings.

Disclosures:
Deitelzweig: Bristol-Myers Squibb/OptumInsight: Research Funding, Speakers Bureau. Pinsky:OptumInsight: Employment. Buysman:OptumInsight: Employment. Lacey:OptumInsight: Employment. Makenbaeva:Bristol-Myers Squibb: Employment, Equity Ownership. Wiederkehr:Pfizer: Employment, Equity Ownership. Graham:Bristol-Myers Squibb: Employment, Equity Ownership.
Hedgehog Ligands and Receptors Are Deregulated in Myelodysplastic Syndromes
Authors: Xavier Juliana M. , Dias Amanda I , Latuf-Filho Paulo , Traina Fabiola , Vassallo José , Saad Sara T Olalla 
Published: October 2019

Abstract
Abstract 3817

Background:
The Hedgehog pathway has an important role in self-renew of normal and leukemic stem cells and is upregulated in myeloid leukemias, however there are no studies regarding the Hedgehog pathway in myelodysplastic syndromes (MDS). Hedgehog ligands (Sonic hedgehog [SHh], Indian hedgehog [IHh], and Desert hedgehog [DHh]) are produced by stromal cells and bind to the receptor Patched (PTCH). This binding causes activation of Smoothened (SMO) receptor, resulting in downstream transcription of target genes.

Aim:
To evaluate Hedgehod pathway components in MDS.

Methods:
Bone marrow (BM) samples were collected from 39 MDS (25 low risk, 14 high risk-WHO 2008) patients and 26 healthy donors (HD). Relative expressions of PTCH and SMO were obtained by Real Time PCR. For immunohistochemistry, BM biopsies were collected from 21 MDS (17 low risk and 4 high risk) patients and 7 megaloblastic anemia (MA) were used as control. The BM sections were stained with antibodies for DHh, SHh and c-Kit and the percentage of stained nucleated cells was based on an average of 4 high-powered fields.

Results:
Hedeghog receptors PTCH and SMO were overexpressed in MDS BM cells compared to normal BM as follows; PTCH: [median (min-max)] healthy donors= 2.23 (0.42–9.22); low risk= 6.09 (0.41–25.28); high risk= 3.97 (0.42– 21.01); HD vs low risk and HD vs high risk p= 0.02; SMO: healthy donors= 2.33 (0.00–16.11); low risk= 14.79 (1.89– 41.93); high risk= 34.44 (8.04– 164.28)]; HD vs low risk and HD vs high risk p<0.001. Immunohistochemistry assays showed a higher expression of Hedgehog ligands in MDS bone marrow compared to control. For DHh, the number of stained cells in low risk MDS were higher than in MA [MA = 102(84 –183), low risk= 196(69 – 317) high risk 159(131 – 236)]; MA vs low risk p=0.018 and MA vs high risk p= 0.11. Furthermore, SHh staining was 2 and 2.5 fold higher in low and high risk MDS, respectively, compared to MA [MA= 47(18 – 74), low risk 98.25(53 – 129), high risk 116.25 (93 – 125)] MA vs low risk p= 0.001, MA vs high risk p=0.01. Similar to SHh, c-Kit staining was higher in MDS cases as follows: MA= 95.3(61 – 114), low risk= 106(57–136), high risk= 95.3(61 – 114). Interestingly, a high correlation was found between c-kit and DHh (p=0.002, r=0.63) or c-kit and SHh (p=0.001, r=0.7).

Conclusion:
To our knowledge, this is the first study of the Hedgehog pathway in MDS. We observed overexpression of Hedgehog receptors and ligands. Furthermore, there is a correlation between these ligands and a marker for hematopoietic stem/progenitors cells. According to these data, we propose that deregulation of Hedgehog in MDS may occur in abnormal progenitors found in MDS bone marrow.

Disclosures:
No relevant conflicts of interest to declare.
Thrombin Generation Capacity of Prothrombin Complex Concentrate in an in Vitro Dilutional Model
Authors: Spronk Henri M.H. , Rossaint Rolf , Yvonne Henskens M.C. , van Oerle Rene , Cate Hugo Ten , Grottke Oliver 
Published: October 2019

Abstract
Abstract 4380

Background:
There is a growing use of prothrombin complex concentrates (PCCs) for the treatment of trauma-induced coagulopathy, which is addressed to their propensity to increase thrombin generation. Despite considerable differences in composition of commercially available PCCs, there is lack of data investigating the procoagulant capacity of different PCCs.

Methods:
The vitamin K-dependent coagulation factors, heparin, and antithrombin were assessed in five commercially available PCCs. The procoagulant potential of the PCCs was assessed in plasma and whole blood from 4 healthy donors by means of classical coagulation assays, thrombin generation assay and thromboelastometry. In order to reflect coagulopathy, whole blood was diluted with 20, 40, 60, and 80% Ringer's lactate solution.

Results:
The five different PCCs were characterised by comparable levels of factors II, VII, IX and X (all around 20–30 IU/mL), whereas the heparin (0 to 17.6 IU/mL) and antithrombin (0.06 to 1.29 IU/mL) levels were remarkably different between manufactures. In vitro dilution of blood induced a prolongation of the PT and aPTT, and attenuation of thrombin generation and ExTem induced thromboelastometry. Overall, non- or low-heparin containing PCCs restored the in vitro dilutional coagulopathy, whereas PCCs containing heparin has an anticoagulant effect. The thrombin generation assay showed to be the most sensitive method for assessment of PCC effects.

Conclusions:
This study shows that most available PCCs are not balanced regarding their pro-and anticoagulants. The effect of measured differences in thrombin generation among different PCCs require further investigations to elaborate the clinical meaning in the treatment of trauma induced coagulopathy.

Disclosures:
No relevant conflicts of interest to declare.
An Intact Immunoglobulin IgAλ Multiple Myeloma Patient Exhibiting Light Chain Escape
Authors: Kraj Maria , Kruk Barbara , Warzocha Krzysztof , Szczepinski Andrzej , Endean Kelly , Harding Stephen 
Published: October 2019

Abstract
Abstract 5008
A 48 year old man was referred to the Institute of Hematology and Transfusion Medicine, Warsaw, Poland in April 2008 with anemia (Hemoglobin; 10. 4 g/dl) and mild renal impairment (eGFR; 75. 4 mL/min/1. 73m2). An initial diagnostic monoclonal protein screen (serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE) and serum free light chain (FLC) analysis) revealed an IgAλ monoclonal protein (0. 8g/dL) with monoclonal serum FLC and an abnormal serum FLC κ/λ ratio (0. 0001; RI, 0. 26–1. 65). A bone marrow biopsy at that time confirmed 60% involvement of monoclonal λ - restricted plasma cells; a bone survey did not detect any osteolysis. The patient was diagnosed with multiple myeloma (MM) (ISS stage I, Durie and Salmon stage IA) and was initially treated with 6 cycles of vincristin, doxorubicin and dexamethasone (VAD). The patient responded well to the induction treatment and subsequently underwent a successful autologous stem cell transplantation (ASCT).
The patient was monitored for 3 years subsequent to the ASCT with both serum and urine electrophoresis, serum FLC analysis (Freelite) and heavy chain/light chain (HLC) immunoassays (Hevylite). Sixteen months following the ASCT the dFLC (involved λ FLC– uninvolved κ FLC) concentration began to increase, the FLC κ/λ ratio became abnormal with a trace of λ Bence Jones protein (BJP) detected by urine IFE. However, both SPE and IFE were normal and the HLC ratio (IgAλ/IgAκ) was within the normal range. During the next 9 months the dFLC continued to increase and a λ BJP could now be clearly detected on the urine IFE. 27 months following the ASCT the patient sustained a pathological fracture of the tibiae and was referred to our centre 4 months later. At this point, the dFLC concentration was highly elevated (3168 mg/L) with a λ BJP detectable by both serum and urine IFE. However, there was no detectable monoclonal intact immunoglobulin by serum IFE or HLC analysis, indicating disease relapse by a separate FLC clone; referred to as light chain escape (LCE). A bone marrow biopsy revealed 15% involvement of λ restricted plasma cells; this time a bone survey identified osteolysis. The patient was diagnosed with progression of multiple myeloma and received 6 cycles of bortezomib, cyclophosphamide and dexamethasone (VCD regimen). He responded well to treatment and 3 years following the ASCT achieved a CR as indicated by a normalized κ/λ FLC ratio, negative immunofixation with 1–3% bone marrow plasma cells. The patient is now well and able to continue with normal life.
In this case study the increase in the dFLC levels was the first indication of disease progression and highlights the importance of monitoring intact immunoglobulin MM patients with serum FLC immunoassays for early detection of LCE.

Disclosures:
Endean: The Binding Site Group Ltd: Employment. Harding:Binding Site: Employment.
Thrombelastographic Demonstration of Nitrite Inhibition of Platelet Function
Authors: Park Ji-Won , Piknova Barbora , Nghiem Khanh , Lozier Jay N. , Schechter Alan N. 
Published: October 2019

Abstract
Abstract 5148
We have recently shown that nitrite ions in blood will inhibit platelet aggregation and activation as measured by aggregometry and flow cytometric analysis of P-selectin under partially deoxygenated conditions. We now show that these phenomena can be measured by thrombelastographic analysis, an alternative method which supplies additional parameters of clotting, using whole blood from healthy donors. Nitric oxide (NO) can be generated by endogenous nitric oxide synthase (NOS) as well as by a serial reduction pathway in which nitrate and nitrite are converted to NO via several enzymatic and nonenzymatic mechanisms. We have previously shown that nitrite anions at 0. 1μM inhibit platelet activation and aggregation in the presence of red blood cells through their reduction to NO and this effect was enhanced by deoxygenation. In the present study, we examined the influence of nitrite on the clotting properties of blood to investigate how nitrite may affect overall clotting processes by modulating platelet function. We measured major clotting parameters such as reaction time (R, minute of initial fibrin formation), angle (velocity of clot growth), and maximum amplitude (MA, clot strength) using thrombelastograph hemostasis analyzer (TEG5000®) in whole blood diluted with plasma to yield 20% Hct. Both the NO donor (DEANONOate) and nitrite showed inhibitory effects on clotting parameters resulting in delayed R, decreased angle, and reduced MA in a dose dependent manner. However, the sources of NO did not change any TEG parameters in plasma. These results suggest that this new clinical method may be used to follow NO inhibition of platelet-induced blood clotting and that the physiological effect of factors which determine NO bioavailability, such as endogenous levels of blood and tissue nitrite, could be used as biomarkers for predicting hemostatic potential.

Disclosures:
Schechter: NIH: Employment, co-inventor of NIH patent on use of nitrite in disease, co-inventor of NIH patent on use of nitrite in disease Patents & Royalties.
Novel Method to Study Mouse Bone Marrow Endothelial Cells in Vivo and in Vitro
Authors: Feng Yuxin , Liu Ming , Guo Fukun , Liu Wei , Sampson Leesa , You Li-Ru , Kuan Chia-Yi , Zheng Yi 
Published: October 2019

Abstract
Abstract 617
Self-renewal, differentiation, and proliferation of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) are maintained in a complex microenvironment of the adult bone marrow (BM). BM endothelial cells (ECs) have been proposed to be a key component of HSC and LSC niche. However, in contrast to the well-developed culture system of human ECs, current work of murine BM endothelial cells is hindered by a lack of mouse bone marrow endothelial cell primary culture and suitable assay methods to clearly define murine BMEC functionality in vivo and in vitro, which limits genetic and mechanistic studies by using mouse models. To establish an in vivo approach to study the EC function in adult mice, a strain of Tie2-CreER transgenic mice was generated to allow conditional and inducible manipulation of BMECs by Cre recombinase expression under the Tie2 promoter. In vivo lineage tracing was achieved in a Tie2-CreER/TD-tomato or -EGFP reporter mouse strain. Upon a four day Tamoxifen injection regimen, TD-tomato or EGFP reporter was readily visualized in bone marrow vasculature that colocalizes with CD31+ ECs as determined by immunostaining. FACS analysis of Tie2-CreER/EGFP reporter mice showed that the EGFP+ cells in the BM were exclusively in the CD45- VEGFR2+ and CD31+ cell fraction, with no EGFP+ cells being detectable in the CD45+ hematopoietic lineages or osteoblast/stroma cell fractions, suggesting that the Tie2-driven CreER expression is limited to the endothelial lineage in the adult BM. Next, we developed an in vitro method to culture and assay the mouse BMECs functionally. An in vitro selection process allowed us to establish a primary BM cell culture condition that permitted functional expansion and maintenance of mouse BMECs in long-term tissue culture, yielding homogenous CD45- cells expressing endothelial markers CD31, CD34 and VEGFR2. These cells formed capillary-like structures in 2-demensional and 3-demensional tubes/capillaries, and showed TD-tomato reporter color when derived from the Tamoxifen induced Tie2-CreER/TD-tomato mouse BM. They showed expected adhesion and migration activities and morphology of ECs. Lineage chasing assays using isolated CD45+ and CD45- BM cells from the Tie2-CreER/Td-tomato mice demonstrated that the BMECs in our culture system, bearing the Tie2-promoter driven TD-tomato color and CD31+ marker, were exclusively derived from CD45- non-hematopoietic lineage. Taken together, we have established a faithful assay method for studying murine BM EC functions in vivo and in vitro, allowing the tracking and genetic manipulation of adult BM ECs in mice and in culture. The method can be useful for delineating molecular and cellular mechanisms of BMEC regulation and EC-mediated BM niche function, and may have value in testing anti-angiogenic activities of anticancer drugs in animal models.

Disclosures:
No relevant conflicts of interest to declare.
Clinical Factors Predictive Of Myelofibrotic Evolutions In Patients With Essential Thrombocythemia and Polycythemia Vera
Authors: Tiribelli Mario , De Marchi Federico , Barraco Daniela , Marin Luciana , Codarin Erika , Buttignol Silvia , Damiani Daniela , Fanin Renato 
Published: October 2019

Abstract

Introduction
Evolution to myelofibrosis (MF) represents a relatively rare but always severe event in patients with essential thrombocythemia (ET) and polycythemia vera (PV). Few reports have focused on the clinical and biological features at diagnosis of ET and PV that correlate with progression to MF.

Aims and Methods
We retrospectively studied a series of patients with post-ET and post-PV MF and compared with a group of ET and PV patients with a long follow-up without myelofibrotic evolution, with the aim to identify prognostic factors for MF. Forty-three patients with post-ET (n=29) and post-PV (n=14) MF followed at our institution were compared with 125 ET and 75 PV patients with at least 9 years of follow-up without evolution. Diagnosis of ET and PV was confirmed according to WHO criteria (including JAK2 analysis, performed since 2006 and study), evolution to MF was defined according to IWG-MRT proposed criteria. The following parameters, available for all patients at diagnosis of ET or ET, were taken into consideration to find prognostic risk factors for myelofibrosis: age, platelet (PLT) count, hemoglobin (Hb) and hematocrit (Hct) levels, white blood cell (WBC) count. Statistical analyses were conducted using Student t test.

Results
Median time from diagnosis of ET/PV and progression to MF was 156 months (range: 29-314). Comparing baseline characteristics of patients who evolved to MF and those who did not, we did not found any significant correlation. Mean data at diagnosis for patients with (n = 43) or without (n=200) subsequent evolution to MF were as follow: age 52.1 vs 53.1 years (p=0.79), Hb 15.4 vs 15.7 g/dl (p=0.59), Hct 47.2 vs 47.1% (p=0.67), WBC 9.8 vs 9.1 x 109/l (p=0.11), PLT 713 vs 689 x 109/l (p=0.87). Also when considering only the 29 post-ET MF and the 125 ET patients, there was no clinical feature present at diagnosis that could foresee a future myelofibrotic evolution. Conversely, in the 14 post-PV MF and 75 PV patients, progression to MF was predicted only by higher WBC count (11.4 vs 9.3 x 109/l, p=0.046), while no correlation was found with age, Hb, Hct or PLT [Table 1].

Conclusions
Concordant with some previous reports, our data suggest a possible role of leucocytosis as an adverse risk factor for progression to MF in patients with PV, though not in ET. Other clinical characteristics present at diagnosis, such as advanced age, anemia or polycythemia and thrombocytosis do not seem to be associated with higher risk of fibrotic evolution in patients with myeloproliferative neoplasms.

Disclosures:
No relevant conflicts of interest to declare.