bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dosing of Rviii-Singlechain Based on Clinical Bleeding Phenotypes Results in Low Bleeding Rates and Low Consumption in Patients Treated with Prophylaxis Two or Three Times Weekly
Authors: St Ledger Katie , Blackman Nicole , Veldman Alex , Pabinger Ingrid 
Published: October 2019

Abstract
Introduction: Safety and efficacy of rVIII-SingleChain (AFSTYLA) was investigated in two pivotal and one extension study, comprising the Affinity program. The study design and instructions provided to the investigators allowed the physician to freely choose the dose assignment and regimen, based on the FVIII treatment regimen used prior to enrollment, and the subject's clinical bleeding phenotype. Adjustments of dose and schedule were allowed at any time during the study.
Methods: This analysis investigates initial and final dose assignment and the corresponding total and spontaneous bleeding rates in subjects in the adult and adolescent study assigned to prophylaxis two or three times weekly (>80% of the subjects on prophylaxis therapy) in dosing brackets of 20 to <30 IU/kg, 30 to <40 IU/kg and 40 to ≤50 IU/kg.
Results: In the final dose assignment, 43 subjects were assigned a dose of 20 to <30 IU/kg, 62 subjects were assigned a dose of 30 to <40 IU/kg, and 38 subjects were assigned a dose of 40 to ≤50 IU/kg. No differences were observed in the analysis by dose brackets in the adult and adolescent (12-65) population (complete or stratified by age); neither in the initial nor the final dose assignment analysis. The median annualized bleeding rate for the dosing brackets of 20 to <30 IU/kg, 30 to <40 IU/kg and 40 to ≤50 IU/kg was 1.93, 1.05 and 2.21, respectively. The median annualized spontaneous bleeding rate was 0, 0 and 0.50, respectively (final dose assignment).
Conclusions: This data shows that dosing of rVIII-SingleChain based on clinical bleeding phenotypes results in low bleeding rates and low consumption in patients treated with prophylaxis two or three times weekly, with more than 70% of subjects being maintained at doses of <40 IU/kg.

Disclosures
St Ledger: CSL Behring: Employment. Blackman:CSL Behring: Employment. Veldman:CSL Behring: Employment. Pabinger:CSL Behring: Consultancy.
Superimposed Hypercoagulable and Hypocoagulable States: Elevated Von Willebrand Factor (vWF) Levels Secondary to Polycystic Ovary Syndrome Masking Von Willebrand Disease
Authors: Scheitler Kristen M , Coberly Emily A , Hossain A. Mosharraf , Hammer Richard D 
Published: October 2019

Abstract
Von Willebrand Disease (vWD) is the most common inherited bleeding diathesis and can present with easy bruising, cutaneous bleeding, prolonged bleeding from mucosal surfaces after surgical procedures, and menorrhagia. However, laboratory confirmation of vWD can be challenging, as vWF and Factor VIII are acute-phase reactants, and levels will be elevated in inflammation, stress, physical activity, estrogen therapy, and hyperthyroidism.
Polycystic Ovary Syndrome (PCOS) induces a state of both chronic inflammation and hyperestrogenism. PCOS is further characterized by a prothrombotic state, with elevated circulating levels of vWF, fibrinogen, and PAI-1. The precise origin of PCOS-induced hypercoagulability is yet to be elucidated but is most likely multifactorial, with insulin resistance, hyperestrogenism, hyperandrogenism, LH excess, age, and BMI imparting varying degrees of influence and synergism.
We present a case of a morbidly obese 23 year-old female with PCOS presenting with menorrhagia. She has a history of prolonged bleeding after surgical procedures and was self-referred due to concern for bleeding risk during an upcoming dental extraction. Laboratory evaluation revealed she was blood group A positive with a normal PT and aPTT. Evaluation for vWD was within normal limits, with vWF antigen of 84% (55-200%), vWF activity of 68% (55-200%), Factor VIII activity assay of 75% (55-200%), and normal vWF multimers. Factor IX assay was significantly elevated at 218% (60-150%). Thromboelastography (TEG) showed elevated MA and G, consistent with platelet hypercoagulability and increased clot strength.
Subsequent review of remote external records determined that the patient had a previous evaluation for a bleeding disorder at age three following prolonged bleeding after tonsillectomy. At that time, she had a prolonged aPTT of 36.1 sec (21.0-31.0 sec) that corrected after mixing study to 29.9 sec. vWF antigen and ristocetin cofactor levels were both low. Factors VIII, IX, XI, and XII were within normal limits. The patient was diagnosed with type I von Willebrand disease at that time.
PCOS is a hypercoagulable state associated with hyperestrogenism and elevated vWF levels, which may lead to a false negative workup for vWD. In this case, a patient with PCOS and a remote diagnosis of type I vWD had elevation of vWF levels into the normal range on current laboratory studies. The patient's current laboratory studies are consistent with a hypercoagulable state with an elevated Factor IX level and do not reflect the underlying bleeding diathesis. Management of such patients with coexisting hypercoagulable and hypocoagulable states, particularly perioperatively, may be complex.

Disclosures
No relevant conflicts of interest to declare.
Ginkgo Biloba Extract Blunts the Prothrombotic Effect of Thalidomide on Human Umbilical Endothelial Cells
Authors: Lu Rui-Nan , Wang Ling , Zhou Li , Li Jianyong , Lu Hua 
Published: October 2019

Abstract
Patients with multiple myeloma (MM) are at increased risk of developing venous thromboembolism (VTE) compared to general individuals. With the introduction of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide, substantially elevates the incidence of VTE. It was reported that patients with MM have significantly increased plasminogen activator inhibitor (PAI-1) level resulting in a decrease of fibrinolytic activity. To prevent VTE in MM patients receiving IMiDs, different prophylactic strategies such as low molecular weight heparin, aspirin and warfarin have been recommended. But most of those recommendations are based on limited evidence.
Ginkgo biloba extract (GBE) is one of the most widely used herbal supplements, which has been used in thrombosis prevention. It prompts us to investigate the impact of thalidomide on fibrinolysis and whether GBE has affection on fibrinolysis. In this study, the human umbilical vein endothelial cells (HUVECs) were cultured alone or co-cultured with human multiple myeloma cell line (MMCs) in the medium with or without thalidomide and in the presence or absence of GBE. After 48 hours, culture supernatants were collected and protein concentration of t-PA and PAI-1 were analyzed by enzyme-linked immunosorbent assay.
Results showed that MMCs and thalidomide can both altered the profibrinolytic potential of HUVECs by decreasing t-PA and increasing PAI-1 levels. When co-cultured with MMCs, thalidomide can further elevate PAI-1 levels but not further reduce t-PA. Whether HUVECs alone or co-cultured with MMCs, GBE was able to counteract these effects caused by thalidomide through increasing t-PA and decreasing PAI-1 levels.
Results in this study indicate that GBE blunts the prothrombotic effect of thalidomide on HUVECs. It supports GBE clinic use for the prevention of VTE induced by IMiDs.

Disclosures
No relevant conflicts of interest to declare.
Lef1 Is a Critical Mediator of Wnt/β-Catenin Signaling in T-Cell Acute Lymphoblastic Leukemia (T-ALL)
Authors: Giambra Vincenzo , Gusscott Sam , Gracias Deanne , Song Raymond , Weng Andrew P. 
Published: October 2019

Abstract
T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy characterized by an uncontrolled proliferation of immature T-cells. While current therapies cure ~80% of pediatric patients, adults fare more poorly with ~40% overall survival. Refractory cases and relapses are presumably due to the ineffective targeting of leukemia stem cells (LSC), previously described in human and in mouse models of T-ALL, and thought to be resistant to standard treatments.
Recently, we have reported that active signaling through the Wnt/β-catenin pathway is a defining feature of LSC in T-ALL, and that interruption of this signaling pathway abrogates disease propagation in vivo. Using an integrated, real-time reporter of Wnt/β-catenin signaling (7TGC; composed of 7 Tcf/Lef-binding sites upstream of a minimal promoter and GFP marker), we identified leukemia-initiating cell (LIC) activity to reside asymmetrically within the minor proportion of Wnt-active, GFP+ cells in primary mouse NOTCH1-induced T-cell leukemias. Moreover, inducible deletion of β-catenin in this context eliminated LIC activity.
Here, we report that LIC activity in this Wnt-active subpopulation is dependent on Lef1. Using Lef1loxP/loxP animals, we show that inducible Cre-mediated deletion of Lef1 in established leukemias extinguishes both Wnt/GFP reporter expression and LIC activity. To explore mechanisms underlying asymmetry of LIC activity within the tumor population, we have also investigated the differential expression of various Lef1 protein isoforms in Wnt-active (GFP+) vs. Wnt-inactive (GFP-) leukemic subsets and assessed their function in supporting LIC activity. These results suggest that β-catenin acts via Lef1 to support asymmetric LIC activity within the Wnt-active subset of leukemia cells.

Disclosures
No relevant conflicts of interest to declare.
High-Expressing Cftr Interacts with HDAC2 to Promote the Development of Ph+ Leukemia through HDAC2-Mediated PENT Pathway
Authors: Yan Tianyou , Gong Yuping 
Published: October 2019

Abstract
The cystic fibrosis transembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily and mediates the transportion of Cl- and HCO3-. Recent studies have reported that CFTR is not only an anion channel protein, but also is used as a regulator to regulate the function of other proteins through interaction with other proteins. Our previous study found that CFTR protein expression in Ph+ acute leukemia cells K562, SUP-B15 and Ph+ primary leukemia blasts was significantly higher than that in normal control cells, and CFTRinh-172 (a CFTR specific inhibitor) had the significant anti-proliferative, apoptosis- inducing and cell-cycle-arrest effect on the CFTR-high expression Ph+ leukemia cells, suggesting that high-CFTR expression might be involved in the incidence and development of Ph+ acute leukemia. At the present study, we found with co-immunoprecipitation (co-IP) and LC-MC assay that HDAC2 protein existed in the fragment pulled down by CFTR antibody in K562 cells; Western Blot assay further confirmed that pretreatment with CFTRinh-172 reduced the expression of HDAC2 protein, but not HDAC2 mRNA in the K562 and SUP-B15: Immunfluorescence analysis testified the co-localization of CFTR and HDAC2 in situ in K562 and SUP-B15 cells; What's more, high expression of HDAC2 protein not mRNA were exhibited in the HEK 273 cell with CFTR cDNA transfect, meanwhile, CFTRinh-172 reduced the expression of HDAC2 protein not mRNA in the CFTR-transfect HEK 293 cells. Moreover, MG132, a proteasome inhibitor, recovered partly the expression of HDAC2 reduced by CFTRinh-172. These results demonstrated that CFTR could interact with HDAC2, and the combination of two proteins protected HDAC2 from the degradation via ubiquitin-proteasome pathway and keep HDAC2 high-expression; Next, Chip/qPCR assay were used to check the relation of HDAC2 and PENT transcript in two Ph+ leukemia cells, the results showed that HDAC2 protein bound in the promote region of PENT gene, and the mRNA and protein of PENT decreased when pre-treated by HDAC2 inhibitor as well as CFTRinh-172, in the meantime, PDK1 and mTOR (down-dream signaling of PENT) activity also decreased. At last, MTT test revealed that CFTRinh-172 raised the anti-proliferation of HDAC2 inhibitor. In conclusion The research has provided the strong evidence that high-expression CFTR plays an important role in the development of Ph+ leukemia through HDAC2-mediated PTEN pathway. The work was supported by the Foundation of the Science & Technology Department of Sichuan Province (NO. 2015SZ0234-5)

Disclosures
No relevant conflicts of interest to declare.
Felipe Samaniego
Authors: Jain Neeraj , Sehgal Lalit , Davis R. Eric , Shuttleworth Stephen Joseph , Samaniego Felipe 
Published: October 2019

Abstract
Background: Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of the patients develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. Several reports indicated that the BTK inhibitor ibrutinib successfully blocks B-cell receptor signaling and shows clinical benefit in leukemia and lymphomas, including mantle cell lymphoma [23422267] and DLBCL [26193343], for which ibrutinib is FDA-approved. In phase I/II clinical trials, ibrutinib elicited an overall response rate of 68% in patients with relapsed/refractory MCL. However, in spite of these encouraging results, responses are variable and generally incomplete, acquired resistance is common, and recurrence is anticipated [26430726]. We undertook a study of factors underlying acquired ibrutinib resistance (IR) in initially ibrutinib-sensitive DLBCL cell lines.
Methods: IR DLBCL cell lines were generated by continuous culture of parental (PT) cell lines in increasing concentrations of ibrutinib, up to a maximum concentration of 10µM. Once established, IR cell lines were removed from ibrutinib, expanded, and cultured under the same conditions as the PT cell lines for further experiments. Gene expression profiling (GEP) of IR and PT populations was performed on Agilent 4 x 44K gene microarrays.
Results: Of five ABC DLBCL cell lines tested, two (OCI-LY3, U2932) were initially resistant to ibrutinib (IC50>10µM).Three (TMD8, OCI-LY10, HBL1) were initially sensitive (IC50 < 10 nM), but chronic exposure to ibrutinib generated syngeneic versions with IC50 > 5µM. In comparison to PT versions of these cell lines, IR cells did not form clumps in suspension cultures, displayed irregular cell morphology, elevated colony formation ability in methylcellulose matrix, and had higher proliferation rate. Western blots and GEP data showed increased expression by IR cell lines of IAP family members survivin, cIAP2, and oncogenic BCL2 and BCL6. Reduced B-cell receptor signaling, and enhanced PI3K-Akt activity was identified in IR cell lines. Analysis of PI3K isoforms revealed up-regulation of PI3Kα and PI3Kβ with decreased expression of PI3Kδ and PTEN (PI3K negative regulator). Given the enhanced PI3K isoform expression with IR, we treated cell lines with KA2237, a PI3Kβ/δ isoform targeting drug, and observed reduced metabolic activity (survival) of IR cells compared to PT cell lines.
Conclusion: This study highlights that changes in a regulator (PTEN) and mediator (p110β) of PI3K/AKT signaling have important roles in the development of ibrutinib resistance in DLBCL. Treatment with KA2237 may provide a better outcome for ibrutinib-resistant DLBCL.

Disclosures
Samaniego: Karus Therapuetics: Research Funding.
Erythropoietin Signaling Regulates Heme Biosynthesis
Authors: Chung Jacky , Wittig Johannes G. , Ghamari Alireza , Maeda Manami , Lodish Harvey , Bauer Daniel E. , Orkin Stuart H. , Cantor Alan B. , Maeda Takahiro , Dailey Harry A. , Paw Barry H. 
Published: October 2019

Abstract
Heme plays a fundamental role in a diverse array of cellular processes and is required for the survival of all cells. During erythropoiesis, heme production is drastically upregulated to support the production of oxygen-carrying hemoglobin. This increase in heme production is mediated by transcriptional induction of heme metabolism genes including ferrochelatase (FECH), which is the enzyme that catalyzes the rate-limiting insertion of ferrous iron into protoporphyrin IX in the mitochondria of erythroid cells. However, how heme production is coordinately regulated by extracellular cues is currently unknown.
Here, using complementary biochemical and genetics approaches, we show that erythropoietin (EPO) signaling regulates heme biosynthesis via a protein kinase A (PKA)-dependent mechanism. In its inactive state, PKA is a tetrameric complex consisting of two catalytic subunits (C) that are bound to and inhibited by two regulatory subunits (R). The C subunits become activated to phosphorylate downstream target proteins when they dissociate from the R subunits. We demonstrate that EPO-induced JAK2 (janus kinase 2) activity leads to release of the C subunits from the R subunits. We also find that phosphorylated STAT5 (signal transducer and activator of transcription 5) forms a molecular complex with PKA-C. This suggests that phospho-STAT5 can outcompete PKA-R to release PKA-C to directly phosphorylate FECH at a highly conserved threonine residue located in the catalytic site. We examined the importance of FECH phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. Erythroid cells harboring the homozygous Thr115Ala Fech mutation exhibited a block in hemoglobin production and concomitant intracellular accumulation of upstream protoporphyrin intermediates. Strikingly, this phenotype bears resemblance to erythropoietic protoporphyria (EPP), a human hematologic disorder typically associated with FECHmutations.
Together, our results support a model where EPO signaling during erythroid maturation activates PKA by a previously unrecognized JAK2/STAT5-dependent mechanism. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Furthermore, our data implicates aberrant EPO/JAK2/PKA signaling in the pathogenesis of human EPP.
Figure Figure.

Disclosures
No relevant conflicts of interest to declare.
Aspirin-Platelet Sensitivity in Patients with Polycythemia Vera and Human Platelet Antigen Genotype: Which Are Assays Valid?
Authors: Cacciola Emma , Gentilini Cacciola Elio , Cacciola Rossella Rosari 
Published: October 2019

Abstract
Polycythemia vera (PV) is a myeloid neoplasm characterized by thrombotic risk related to high hematocrit (HCT) and to platelet hyperactivation. According to PVSG, the antithrombotic therapy include low dose aspirin (ASA). However, there are insensitive ASA PV patients. It is debated if enherited thrombophilia is responsible to affect platelet activation and, hence, to cause ASA insensitivity. Therefore, we evaluated gene polymorphism human platelet antigen-1 (HPA-1) as thrombophilic indicator associated with abnormal platelet activity, HCT, platelet count, b-TG and PF4, as markers of platelet activation, the platelet functional activity (PFA) and the maximum clot firmness (MCF), as direct and indirect indicators of ASA sensitivity. We studied 40 patients (28 men, 12 women; mean age 64 years, range 35-85 years) with PV according to WHO criteria. Fifty subjects served as controls. The mean duration of disease was 9 years. All patients were on phlebotomy and ASA (100 mg once daily). Platelets and HCT were measured by automated analyzer. b-TG and PF4 were determined by ELISA. PFA and MCF were measured by Platelet Function Analyzer (PFA-100) and by ROTEM delta. Of 40 patients, 24 were homozygous HPA-1a/a and 16 heterozigous HPA-1a/b. The mean HCT value was 47±3% and platelets were 428±180x109/L. All patients had high b-TG and PF4 (113±47 IU/ml vs 20±11 IU/ml and 45±21 IU/ml vs 6±2 IU/ml, respectively), prolonged C/EPI closure time (CT, unit: s, n.v. 84-160 s) (233±65 s) and normal MCF (MCF, unit: mm, n.v. 50-72 mm) (65±5 mm). These findings suggest that PFA-100 and Thromboelastometric assays may be an useful tool to detect ASA platelet sensitivity in PV patients.

Disclosures
No relevant conflicts of interest to declare.
Antibody Response to Pneumococcal Conjugate Vaccine (PCV13) in Chronic Lymphocytic Leukemia Patients Receiving Ibrutinib
Authors: Andrick Benjamin , Alwhaibi Abulrahman , DeRemer David , Quershi Sameera , Khan Rahil , Shenoy Somanath , Pantin Jeremy M 
Published: October 2019

Abstract
Introduction
Due to immune dysregulation and subsequent increased risk of infection associated with chronic lymphocytic leukemia (CLL), both the CDC and ACIP recommend patients with CLL receive the 13- valent pneumococcal conjugate vaccination (PCV13). The effect of ibrutinib, a selective inhibitor ofBruton'styrosine kinase (BTK) which effects B-antigen receptor (BCR) signaling, on IgG antibody responses to pneumococcal vaccination in patients with CLL has not been evaluated. The aim of this study was to evaluate the immune response to PCV13 in patients receiving in ibrutinib and CLL control patients (no active treatment).
Methods
Study patients were enrolled on an IRB approved protocol at Georgia Cancer Center. Specific inclusion criteria a) histological confirmed CLL as defined by WHO classification b) no current therapy (CLL control) c) ibrutinib 420 mg/day (active) d) ECOG ≤ 2. Exclusion criteria a) Previous vaccination with PCV13 within 2 years b) anti-CD20 therapy within last 6 months c) IVIG therapy within 6 weeks. Serum pneumococcal antibody assessment was performed by microsphere photometry on subjects at Day 0 and Day 30 days following vaccination. Antibody specific serotypes (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23) were determined by ELISA. Pre- and post- vaccination expression of BTK and HACS1 (hematopoietic adapter containing SH3 and SAM protein) of CD19+ lymphocytes was determined by Western Blot analysis. The primary objective was to determine whether concurrent administration of PCV13 in patients receiving ibrutinib generates a ≥ 2 fold increase in ≥ 3 of pneumococcal serotypes as compared to the control group. Secondary objectives were to evaluate expression BTK and HACS in both study groups.
Results
At interim analysis, 9 patients have been enrolled to date, with 8 patients (n=4 ibrutinib, n=4 CLL control) having completed pre and post-vaccination assessments. The median patient age was 69 yo(range: 53-77 yo) with 75% ≥ 65 yo. All CLL control patients (4/4) generated a ≥ 2 fold increase in ≥ 3 of pneumococcal serotypes, whereas (0/4) of ibrutinib patients generated an adequate immune response to PCV13 (p=0.029; Fisher exact). See Table 1 for mean serotype changes in both patient cohorts.
Conclusions
Preliminary data suggests that patients who are receiving ibrutinib group do not generate an effective immune response to PCV13 vaccination compared to untreated CLL controls. This may have an impact on susceptibility and response to infection with pneumococcal infection as well.

Disclosures
No relevant conflicts of interest to declare.
MK2 Is a Therapeutic Target for High-Risk Multiple Myeloma
Authors: Gu Chunyan , Yang Hongbao , Peng Hailin , Xu Zhiye , Chen Ruini , Yang Ye 
Published: October 2019

Abstract
p38 mitogen-activated protein kinase (MAPK) is constitutively activated in human multiple myeloma (MM). However, a major substrate of p38, MAPKAPK2 (MK2) has received little attention in MM. In this study, we disclosed that elevation of MK2 on DNA, mRNA and protein levels in MM cells compared to normal plasma control cells by array-based comparative genomic hybridization (aCGH), Gene expression profiling (GEP) and immunohistochemistry (IHC) respectively. High MK2 expression prognosticated inferior outcome of newly diagnosed myelomas, particularly in patients with high-risk disease based on GEP. Follow-up study demonstrated that enforced overexpressing MK2 promoted MM cell proliferation, drug-resistance and xenograft tumorigenesis in vivo, while knockdown MK2 expression by shRNA abrogated these characteristics. Further mechanism study showed MK2 directly bound and activated AKT signaling in MM, while specific MK2 inhibitor inhibited MM cell growth and clonogenicity through suppressing AKT phosphorylation. Our findings not only indicate that MK2 is a potential therapeutic target in high-risk MM but also suggest the clinical relevance of MK2 inhibition in MM.

Disclosures
No relevant conflicts of interest to declare.