bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effectiveness and Safety of Low Dose Eltrombopag Twice Weekly in ITP Patients That Achieved CR after 50mg Daily Standard Dose
Authors: Iuliano Francesco , Iuliano Eleonora , Perricelli Alessia , Pomillo Angelo , Luci Maria , Abruzzese Elisabetta 
Published: October 2019

Abstract
Introduction. Eltrombopag is an oral, non-peptide thrombopoietin receptor agonist that has been shown efficacy and safety in chronic immune thrombocytopenia (ITP) patients not responding to previous therapy , by raising the platelets count in both continued long-term administration and in a repeated short-term administration. However, when eltrombopag is discontinued, platelet counts usually return to baseline within 2 weeks. As reported in different case series only 15 % of patients achieve a durable response after treatment discontinuation. The aims of this study was to evaluate the efficacy and safety of eltrombopag given at a dose of 25 mg every 3 days as maintenance therapy in responding patients after an initial standard dose of 50 mg orally once daily.
Methods. A total of 7 consecutive adult patients, female (70 %), median age 47 years (range 28 - 79) were enrolled in the study. Patients had ITP for a median of 3.5 years and had failed a median of four prior therapies. Splenectomy was not taken into account because contraindicated or refused. All patients received eltrombopag 50 mg by mouth once a day as induction treatment .A complete blood count was performed at baseline, on day 5, then weekly for 28 days, every 2 weeks during maintenance. Complete Response ( CR) was defined as platelet count ≥ 100 * 10^9/L measured on two occasions > 7 days apart and the absence of bleeding. Relapse Free Survival (RFS) was considered to be from the day of initial response until relapse (Plt <30 × 109/L). Treatment safety profiles and side effects were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0.Maintenance dose of 25 mg every 3 days was initiated in all patients in CR from at least three months.
Results. 7 out of 7 pts achieved CR after eltrombopag induction therapy and were enrolled in the study. 1 out of 7 pts was too early to be evaluated. 6 pts were eligible for the maintenance phase. The median (range) follow-up of this case series was of 13.5 months (4-22), during which all patients maintained a safe platelet count without the need to increase the dose or frequency of administration. No patient relapsed . No adverse events were observed.
Conclusions. Maintenance therapy was manageable and well-tolerated. Maintenance can allow for improved health care resource allocation providing cost savings and potentially improves patients' quality of life by by reducing the need of food restriction/dairy products required when taking eltrombopag.Moreover , maintenance therapy improves adhesion to the treatment and may be an effective alternative for patients who can not discontinue the drug

Disclosures
Abruzzese: novartis, bristol myers squibb, ariad, pfizer, takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees.
The Acquired Von Willebrand Syndrome in Patients with Monoclonal IgA Proteins Is Rare but May be Clinically Severe
Authors: Budde Ulrich , Dittmer Rita , Schneppenheim Sonja , Rausch Tina 
Published: October 2019

Abstract
During the last 10 years our laboratory diagnosed and typed 5585 different patients with inherited von Willebrand disease (VWS) and acquired von Willebrand syndrome (aVWS). Out of these 21% (1217) suffered from aVWS. With 252 patients lymphoproliferative disorders were number three in frequency (21%) after cardiovascular (43%) and myeloproliferative (27%) disorders. Out of the patients with lymphoproliferative disorders patients with a monoclonal IgA protein comprised only 3.6% (9 patients). Much more abundant were monoclonal IgG´s (71%) and IgM´s (26%).
According to our data only one patient was classified as MGUS and the others suffered from myeloma. The most impressive property was the heterogeneity of their laboratory and clinical data with the exception that all patients suffered from severe bleeding complications whenever their hemostatic system was challenged by accidents or invasive procedures (even iliac crest biopsy).
The VWF:Ag ranged from 0.06 – 5.60 IU/ml and only one patient had a VWF:Ag <0.5 IU/dl. The functional test (VWF:CB) ranged from 0.06 and 5.48 IU/ml again with only one patient <0.5 IU/dl. The ratio VWF:CB / VWF:Ag was below 0.8 in three patients. Thus more than 50% of the patients would remain undetected if the VWF multimers were not included in the
diagnostic panel. Two patients displayed the whole set of multimers, while the others showed a mild (5) or severe (2) absence of the large multimers. The hallmark of the multimic pattern from patients with an IgA monoclonal protein was the presence of a lot of smeary material within the electrophoretic lanes diplayed in all patients.
In summary aVWS in the course of lymphoproliferative disorders and monoclonal IgA proteins is clinically severe, although probably not life threatening. It remains undetected with standard VWF tests and might be therefore not as rare as detected in our large patient panel.

Disclosures
No relevant conflicts of interest to declare.
A Structure-Function Study of the Activity of Stem Cell Factor in Stimulating the Expansion of Cord Blood CD34+ Cells
Authors: Shen Bin , Jiang Wenhong , Fan Jie , Dai Wei , Ding Xinxin , Jiang Yongping 
Published: October 2019

Abstract
Stem cell factor is one of the most important growth factors for human hematopoietic stem cells (HSC). Recombinant human stem cell factor (rhSCF) can stimulate HSC expansion and regeneration in vitro, when it is used in combination with other cytokines like Flt-3L and TPO. However, the specific structural region(s) of the rhSCF protein that are critical for its function in HSC expansion are still unknown. Few studies have addressed this problem, to date. We have recently reported the production of a novel monoclonal antibody (named 23C8) against rhSCF, and the demonstration that 23C8 could inhibit the ability of rhSCF to enhance HSC expansion. Here, we report the identification of a short polypeptide from rhSCF that contains the epitope for binding to 23C8, and, like the full-length rhSCF, is able to stimulate the expansion of umbilical cord blood (UCB)-derived CD34+ cells.
Twelve short polypeptides were designed and synthesized, which cover the full length of rhSCF, with 3-5 amino acids overlaps. 23C8 was collected from hybridoma cell culture medium and further purified using protein G affinity chromatography. ELISA was used to identify the polypeptide(s) that positively react with 23C8 among all the synthesized polypeptides. In addition, the effects of the synthetic polypeptides on human HSC expansion capacity were evaluated by supplementing the cell culture medium with 100 ng/ml of a given polypeptide. Total cell number and CD34+ cell number of each group were monitored on day 6. Our novel anti-SCF monoclonal antibody (23C8) partially blocked SCF’s function in human UCB CD34+ cell expansion. Of all the polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 40 to 57, was recognized by 23C8 during ELISA. P0, like the full-length rhSCF, enhanced expansion of CD34+ cells derived from human UCB. P0 addition increased the numbers of total nucleated cells and CD34+ cells by 10.58±0.86 and 4.63±0.43 folds, respectively. For comparison, the extents of increases in cell numbers in the vehicle control group was 3.15±0.99 fold (total nucleated cells) and 1.07±0.11 fold (CD34+ cells), respectively. Residues 40-57 of hrSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34+ cells in vitro. The short P0 peptide is a potential candidate for development as a synthetic substitute for rhSCF in clinic applications.

Disclosures
Jiang: Biopharmagen.corp: Employment. Jiang:Biopharmagen.corp: Employment.
Long Term Follow up of the AML97-Study for Patients with AML Older Than 60 Years: A Study of the East German Hematology and Oncology Study Group (OSHO)
Authors: Kahl Christoph , Krahl Rainer , Al-Ali Haifa Kathrin , Sayer Herbert G. , Schulze Antje , Herold Michael , Hänel Mathias , Scholl Sebastian , Hochhaus Andreas , Uharek Lutz , Maschmeyer Georg , Hähling Detlev , Junghanss Christian , Peter Norma , Kämpfe Dietrich , Kettner Erika , Heinicke Thomas , Fischer Thomas , Kreibich Ute , Wolf Hans-Heinrich , Niederwieser Dietger 
Published: October 2019

Abstract
The incidence of acute myeloid leukemia (AML) is age-dependent with the majority of patients (pts) being older than 60 years at diagnosis. Treatment of these pts needs to be well balanced between sufficient efficacy and tolerable toxicity. Here, we report long term follow-up of the OSHO AML97 study. Pts with AML older than 60 years were registered after informed consent and received age-adapted intensive chemotherapy treatment (curative arm; induction therapy with AraC 2 g/m2 iv day 1, 3, 5, 7 and mitoxantrone 10 mg/m2 iv day 1 to 3 to induce complete remission (CR), followed by 2 consolidation courses with AraC 240 mg/m2 iv day 1 to 5 and mitoxantrone 10 mg/m2 iv day 1 to 2), low dose chemotherapy (palliative arm; idarubicin 10 mg po day 1 and either thioguanine 40 mg po day 1-5, or AraC 80 mg sc day 1-5 or etoposide 100mg po day 1-5) or supportive therapy (best supportive care including transfusions). A total of 618 pts were enrolled (curative arm n=471, palliative treatment n=115 and supportive therapy n=32 pts). In the curative arm, CR was obtained in 66.8% of pts. Treatment related mortality (TRM) was 11.2% after induction and 4.5% after consolidation I, respectively. Median overall survival for all pts in the curative arm was 12 months, event free survival (EFS) at 12 years was 0.11±0.02%. In multivariate analysis, cytogenetics at diagnosis was the most important prognostic factor for CR (p=0.001). With a median follow up of 10 years (range 0.1 - 11.8) probability of overall survival (OS) at 5 years was 0.48±0.11; 0.13±0.03; 0.10±0.04 and 0.08±0.03 for pts with favorable, normal, other and unfavorable cytogenetics. Median survival for pts treated with palliative chemotherapy was 54 days.
In conclusion treatment of older AML pts with an intense dose of AraC in the induction therapy is feasible and able to induce high rates of CR. Nevertheless, despite the high CR rate in this setting OS and EFS are still low. However, importantly, this does not apply for patients with favorable cytogenetics. This result also confirms the need for cytogenetic analysis to be performed in all pts older than 60 years potentially eligible for intensive induction therapy. The treatment results with palliative chemotherapy are disappointing. These results reported here need to be set in relation with the new therapeutic modalities for AML including epigenetic and molecular therapies.

Disclosures
Al-Ali: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Wolf:Bayer: Honoraria; Geo Pharma: Honoraria.
Warfarin Inhibits Vitamin K Epoxide Reductase By Specifically Blocking at a Conformational and Redox State
Authors: Shen Guomin , Zhang Hao , Cui Weidong , Sadler Evan , Gross Michael , Li Weikai 
Published: October 2019

Abstract
Warfarin targets vitamin K epoxide reductase (VKOR) to interfere with blood coagulation in humans. Warfarin is the most widely used oral anticoagulant, but its inhibition mechanism remains largely unknown. Here we use quantitative mass spectrometry to show that warfarin changes the intracellular redox state of human VKOR. Warfarin induces this redox shift at a physiological dosage that correlates well with warfarin-inhibited VKOR activity. The warfarin-induced redox change is prevented by the mutation of two critical cysteines, suggesting that they form a disulfide bond essential for warfarin binding. We also mapped warfarin binding through its interaction with warfarin resistant mutations located at different structural regions of human VKOR. Modeling of these interactions suggests how warfarin binds to a VKOR conformation that is stabilized by the essential disulfide bond. Thus, after sixty years of clinical use of warfarin, we now have a good mechanistic understanding that this drug blocks human VKOR at a specific conformational and redox state.

Disclosures
No relevant conflicts of interest to declare.
Analysis of Highly Self-Renewing GPI-80+ Human Fetal Hematopoietic Stem Cells Identifies Novel Regulators of Stemness
Authors: Calvanese Vincenzo , Prashad Sacha L. , Magnusson Mattias , Mikkola Hanna K. A. 
Published: October 2019

Abstract
Achievements in pluripotent stem cell and reprogramming strategies provide hope for generating hematopoietic stem cells (HSC) in culture and for obtaining unlimited sources of transplantable cells. To reach this goal, deeper understanding of the regulatory mechanisms that distinguish the self-renewing HSC from non-self-renewing progenitors during human development is required. We analyzed the molecular signature of GPI-80 (VNN-2) expressing human second trimester fetal liver HSPC, the only population that harbors the truly self-renewing fetal HSC. Microarray and RNAseq analysis comparing CD34+CD38-CD90+GPI-80+ HSC to CD34+CD38-CD90+GPI80- HPC demonstrated remarkable molecular similarity of these two functionally distinct populations, including comparable expression of many key transcription factors involved in HSC development and maintenance (e.g. SCL, RUNX1, MLL1, HOXA9, BMI1, GFI1, ETV6 etc.). Nevertheless, this analysis identified a subset of transcriptional regulators uniquely up-regulated in GPI80+ HSC, such as MYCT1, HLF, MLLT3 and HIF3A. These factors were down-regulated in human fetal liver HSPC upon expansion on MSC stroma culture, during which they become compromised in in vivo engraftment ability despite maintaining HSC surface immunophenotype. multipotency and expression of most known HSC regulators. Moreover, these factors were absent or expressed at low levels in human ES cell derived HPC, which can acquire HSC surface phenotype but are unable to self-renew. The expression of MYCT1, MLLT3 and HLF was also enriched in undifferentiated HSPC subset as compared to progenitors in the adult bone marrow, while HIF3a expression was low post-natally. Altogether, these analyses implied strong correlation of the expression of these factors with HSC self-renewal properties. Strikingly, knockdown of MYCT1, HLF, MLLT3 and HIF3A in human fetal liver HSPC using lentiviral shRNA impaired maintenance of undifferentiated HSPC in MSC stroma co-culture, while their inducible overexpression augmented HSPC expansion and prevented their premature exhaustion. Uncovering how these novel HSC regulators protect the highly self-renewing fetal HSC will help define the pre-requisites for establishing and maintaining stemness in developing human HSC, and for generating HSC for therapeutic use.

Disclosures
No relevant conflicts of interest to declare.
The Complementary Prognostic Role of Baseline and Interim PET in Predicting Treatment Outcome in Advanced-Stage Hodgkin Lymphoma
Authors: Cimino Giuseppe , Zaucha Jan Maciej , S Cirillo , Saviolo C , Hutchings Martin , El-Galaly TC , Borra Anna , Rago Angela , Gallamini Andrea 
Published: October 2019

Abstract
INTRODUCTION: We retrospectively evaluated the contribution of the positron emission tomography (PET) combined with computed tomography (PET/CT) and contrast enhanced computed tomography (ceCT) in the staging and in the prognostication of untreated advanced Hodgkin’s lymphoma (HL).
METHODS: One-hundred-sixty-two HL patients from Italian, Poland and Danish centers entered this study. Treatment consisted on ABVD with or without radiotherapy. All patients were staged at baseline with ceCT and PET/CT (PET-0) and after 2 ABVD with PET/CT (PET-2). All the ceCT and FDG/PET scans were centrally reviewed. PET-2 was reported according to the 5-point Deauville scale: score 1 to 3 considered negative and score 4-5 positive.
RESULTS: Patients breakdown into (a) stage I-II; (b) III or (c) IV for CeCT and PET/CT-staged patients was as follows: 49.1%, 31.0%, 19.9% and 46.6%, 21.7%, 31.7%, respectively. ceCT and PET-0 were concordant or discordant in 117 (72.2%) and 45 (27.8%) patients, respectively. Combining the PET-0 and ceCT data we demonstrated an extra-nodal sites (ENS) in 57 (35.2%) of the 162 patients. Most (27/57) extra-nodal sites (ENS) were detected by PET-0 alone while ENS were concordantly detected by ceCT and PET-0 in other 25 cases and by ceCT alone in the remaining 5 patients. These latter three patient groups showed similar 2-y EFS rates of 62%, 83% and 56%, respectively.
In patients with PET/CT-documented ENS, a pathological metabolic uptake was documented, respectively, in the BM (18 cases), lung (7 cases) and in two other parenchymal sites (2 cases). Overall, the 2 and 5-year OS and EFS for the entire patient cohort were 97.4%, 97.4% and 86.6% and 82.6%, respectively. PET-2 was positive in 40 cases (25.1%) and negative in 119 (74.84%). At univariate and multivariate analyses ENS disease and a positive PET-2 were the only variables significantly affecting the EFS rates, with a HR of 3.9 (95% CI:1.62-9.36; P=.002) and 2.9 (95% CI:1.82-4.52; P<.00001), respectively. Combining these two variables we stratified the patients in 4 risk groups with significantly different EFS rates (P<.00001). Group 1 (i.e. nodal disease and a negative PET-2) identified patients with a very favorable 3-y EFS (98%), whereas group 4 (i.e. presence of ENS disease and a positive PET-2) had a very dismal prognosis, with a 3-y EFS of 0%. Group 2 (i.e. presence of ENS and a negative PET-2) and group 3 (nodal disease and a positive PET-2) identified patients at intermediate risk.
CONCLUSION: Present findings indicate that (1) PET/CT is mandatory for baseline HL staging while CeCT is of limited value. (2) The combination of ENS and PET-2 in a integrated prognostic model was even more predictive than PET-2 alone on treatment outcome in ABVD-treated HL patients.

Disclosures
No relevant conflicts of interest to declare.
Exceeding MR5.0 Sensitivity in Routine BCR-ABL1 Analysis Using Multiplex Digital PCR
Authors: Andersen Rikke Fredslund , Pallisgaard Niels 
Published: October 2019

Abstract
Objectives and background:
Clinical studies are currently initiated where treatment of CML patients with tyrosine kinase inhibitors is stopped. One of the main criteria for patients to be eligible for these studies is that they must have achieved a molecular response of MR4.0. However, in order to obtain a reliable molecular measurement at the MR4.0 sensitivity level a higher sensitivity is needed in the analysis. We have tested a multiplex droplet digital PCR (ddPCR) approach in order to obtain reliable measurements at the MR5.0 level. When using a standard laboratory protocol with three reference genes and singleplex qPCR, only ¼ of the sample is tested for BCR-ABL1 transcripts. By using multiplex PCR the entire sample can be tested. The advantage of ddPCR is that an absolute concentration is measured and there is no need for calibration or standardisation of the analysis.
Methods:
Blood samples stabilized in Paxgene tubes from 36 CML patients were used for the study. Samples were analysed according to the standard laboratory protocol with qPCR using B2M, BCR and GUSB as reference genes. For the study presented here RNA was purified from 5 million cells using the QiaSymphony RNA kit (Qiagen). 2.5 ug of RNA was used for cDNA synthesis using SuperScript VILO cDNA synthesis kit (LifeTechnologies). Samples were analysed with multiplex dd PCR on the QX100 system (Biorad). The WHO reference genes BCR and GUSB were used as reference. Eight wells were analysed for BCR-ABL1 (FAM labelled probe). Four of the wells were also analysed for BCR and four were analysed for GUSB (HEX labelled probes).
Results:
The median number of GUSB transcripts in the samples was 605,000 copies (range 195,520-934,400). According to the EUTOS “Working definitions of Molecular Response in CML” (ref), MR5.0 sensitivity is obtained when analysing 240,000 GUSB transcripts. All samples except one were above this level. MR5.5 is obtained when analysing 759,000 GUSB transcripts and 20% of the samples (7/36) reached this level. The ddPCR results were compared to the qPCR results and a high concordance was seen in the positive samples (r2=0.94). 21 samples were positive by qPCR, and an additional 4 samples were positive by ddPCR.
Conclusions:
Using the multiplex ddPCR protocol developed 97% of the analysed samples had a sensitivity of MR5.0 or better. Compared to the labs routine qPCR protocol 20% more samples were found positive with ddPCR. Due to the multiplex set-up of the ddPCR the entire cDNA sample can be tested for BCR-ABL1 transcripts. Contrary to qPCR there is no need for calibration of the ddPCR analysis and the method has potential for routine use when a high sensitivity is needed.
References:
“Working definitions of Molecular Response in CML”, EUTOS Molecular Monitoring Steering Group, Nick Cross, Martin Müller, Fabrizio Pane, Andreas Hochhaus, November 2013.

Disclosures
No relevant conflicts of interest to declare.
Glycoprotein Ibα Clustering Induces Macrophage-Mediated Platelet Clearance in the Liver
Authors: Yan Rong , Chen Mengxing , Ma Na , Zhao Lili , Cao Lijuan , Zhang Yiwen , Zhang Jie , Yu Ziqiang , Wang Zhaoyue , Xia Lijun , Ruan Changgeng , Dai Kesheng 
Published: October 2019

Abstract
Objective: Immune thrombocytopenia (ITP) is an autoimmune bleeding disease in which autoantibodies are directed against the patient’s own platelets, leading to platelet destruction and bleeding. Currently, platelet destruction in ITP is thought to be mediated by monocytic and phagocytic cells, and the antibody-coated platelets are removed from circulation by the spleen in an Fc-dependent manner. Conventional treatments for ITP therefore aim at reducing platelet destruction, either by immunosuppression or splenectomy. However, many ITP patients, particularly patients with anti-glycoprotein (GP) Ib-IX autoantibodies, do not respond to conventional treatments such as intravenous immunoglobulin G (IVIG). However, the underlying mechanism remains unclear.
Methods: Anti-GPIb-IX antibodies-induced platelet aggregation was detected by an optical aggregometer. GPIbα clustering was measured by Fluorescence Resonance Energy Transfer (FRET) by flow cytometry. Antibodies-induced platelet depletion and rescue experiments were performed in guinea pigs and cynomolgus macaques. Platelet distribution after antibody-mediated depletion was detected by in vivo Imaging System (IVIS), immunohistochemistry and immunofluorescence microscopy.
Results: We found that anti-GPIbα N-terminus antibody AN51, but not other anti-GPIbα antibodies (AK2, HIP1, VM16d, or WM23), induced integrin αⅡbβ3-dependent platelet aggregation. FRET result showed that AN51 induced obvious GPIbα clustering but not VM16d. After intravenous injection, AN51 dose-dependently induced thrombocytopenia in guinea pigs, and the platelets were mainly removed by macrophages in the liver. N-acetyl-D-glucosamine (GlcNAc), previously shown to inhibited αMβ2-mediated phagocytosis of refrigerated platelets, showed dose-dependent inhibition of AN51-induced platelet destruction. Furthermore, AN51 but not VM16d, induced rapid platelet clearance in the liver of cynomolgus macaques. Five of twenty-two chronic ITP patients had anti-GPIbα autoantibodies, and the autoantibodies from four of the five patients competed with AN51 for binding to platelets.
Conclusion: GPIbα clustering induced by anti-GPIbα N-terminus antibody causes integrin αⅡbβ3-dependent platelet aggregation, phagocytosis, and rapid platelet clearance in the liver. Our findings reveal a novel Fc-independent mechanism underlying the pathogenesis of ITP, and suggest new therapeutic strategies for ITP patients with anti-GPIbα autoantibodies.

Disclosures
No relevant conflicts of interest to declare.