bluuebunny/crossref_metadata_2025 huggingface dataset:
Low- Molecular Weight Heparins (LMWH) Versus Vitamin K Antagonists (VKAs) in the Treatment and Secondary Prevention of Cancer-Associated Venous Thromboembolism (VTE): A Systematic Review and Meta-Analysis of Randomized Controlled Trials (RCTs)
Authors: Thein Kyaw Zin , Tijani Lukman , Oo Thein H.
Published: November 2019

Introduction: Cancer-associated thrombosis accounts for about 20% of all cases of VTE. VTE increases not only mortality but also morbidity in cancer patients. LMWH is currently recommended over VKAs by major consensus guidelines and it is primarily based on a single, large RCT (CLOT trial) and some smaller studies. However, recently published CATCH trial, which enrolled 900 cancer patients with acute VTE, did not detect a statistically significant reduction in recurrent VTE between tinzaparin and warfarin groups. Hereby, we conducted a systematic review and meta-analysis of RCTs to determine the efficacy and safety of LMWH versus VKA in the treatment and secondary prevention of cancer-associated VTE.

Methods: MEDLINE and EMBASE databases were searched through June 30, 2016. All potential studies and their references were evaluated for any additional relevant studies. The RCTs which compare LMWH vs VKA in the treatment of VTE were incorporated in the analysis. Mantel-Haenszel method was used to calculate the estimated pooled risk ratio (RR), and risk difference (RD) with 95% confidence interval (CI). Fixed effects model was applied.

Results: A total of 2196 cancer patients with acute VTE from 6 RCTs and a subgroup of another 2 RCTs were eligible for analysis. Enoxaparin, dalteparin, tinzaparin and nadroparin were used on LMWH group and warfarin or acenocoumarol were used on VKA group. The treatment duration was from 3 to 6 months. The VTE incidence was 77 (6.832%) in LMWH group vs 128 (11.973%) in VKA group with a RR of 0.572 (95% CI: 0.436 to 0.750, P < 0.001). The absolute RD in VTE was -0.051 (95% CI: -0.076 to -0.027, P < 0.001). Major bleeding (MB) events were reported in 49 (4.757%) patients in LMWH group vs 45 (4.473%) in VKA group according to the analysis of 6 RCTs. The pooled relative risk for MB was statistically non-significant at 1.049 (95% CI: 0.705 to 1.562, P = 0.812). Composite clinically relevant non-major (CRNM) and minor bleeding events were noted in 143 (13.883%) patients in LMWH group and in 163 (16.203%) in VKA group according to the analysis of 6 RCTs. The RR for composite CRNM and minor bleeding was statistically significant at 0.773 (95% CI: 0.630 to 0.948, P 0.014), favoring LMWHs over VKAs.

Conclusions: Our meta-analysis confirms that LMWHs significantly decrease recurrent VTE events compared to VKAs without increasing MB in patients with cancer-associated VTE. LMWH therapy appears to have less CRNM and minor bleeding events compared to VKAs.

Figure Figure.

Disclosures

Oo: Daiichi Sankyo: Research Funding.
Intron Retention Mechanisms That Regulate SF3B1 and Mitoferrin Gene Expression during Late Erythropoiesis
Authors: Parra Marilyn , Yeo Gene , Conboy John G.
Published: November 2019

Intron retention (IR) regulates hundreds of erythroid genes in a differentiation stage-specific manner during terminal erythropoiesis. Regulated genes include highly expressed RNA binding proteins (RBPs) such as SF3B1, as well as iron transporters (e.g., mitoferrins 1 and 2), and cytoskeletal proteins (e.g., alpha spectrin). Selected IR transcripts are relatively abundant; 25-50% of the above-mentioned transcripts can be polyadenylated, retained in the nucleus, and efficiently spliced at all but one or two introns, thus limiting the amount of translatable cytoplasmic mRNA. We are studying novel IR regulatory mechanisms involving both splice site strength and deeper intronic elements, using model introns that are differentially regulated in human erythroblasts. SF3B1, a key pre-RNA splicing factor implicated in MDS, exhibits dynamic regulation of intron 4, with low IR in proerythroblasts and high IR in mature orthochromatic erythroblasts. Intron 4 sequences include three ultraconserved elements that encode cryptic exons we term 4a, 4b, and 4c, two of which (4a and 4c) encode premature termination codons (PTCs) expected to induce nonsense-mediated decay (NMD) if spliced into SF3B1 transcripts. We hypothesize that these PTC exons can have an additional function as inefficiently spliced decoy(s), that is, their splice sites may interact with splice sites at the boundaries of intron 4 to form non-productive complexes that do not permit efficient splicing but instead prevent excision of the intron. This concept represents an extension of decoy models previously proposed by others to explain selected exon skipping events, and is supported by several recent findings: eCLIP (enhanced cross-linking and immunoprecipitation) data indicate strong binding of 3' splice site factors U2AF1 and U2AF2 at these exons; low levels of exon 4a and 4c splicing can be seen in NMD-inhibited cells; and deletion of exon 4c from minigene splicing reporters decreases IR. Besides SF3B1, we identified numerous other dynamically-regulated IR events encompassing cryptic PTC exons that bind 3' splice site factors. In contrast to the dynamic regulation of IR in SF3B1, IR in SLC25A37 (mitoferrin-1) and SLC25A28 (mitoferrin-2) is stably maintained at a high level throughout terminal erythropoiesis. Baseline IR levels for stably retained introns correlate with splice site strength, but are also influenced by deeper intronic elements. For example, SLC25A28 intron 2 contains intronic elements that appear to reduce IR, since blocking them with antisense morpholinos leads to substantially increased IR levels. These studies demonstrate that IR in major erythroid genes is regulated by sequences within the retained introns that can either increase or decrease retention, suggesting that multiple IR pathways are employed during terminal erythropoiesis to regulate gene expression.

Disclosures

No relevant conflicts of interest to declare.
Argonaute 2 Silencing of Band 3 in Early Reticulocytes
Authors: Evgenia Bloch M , Branch Donald R.
Published: November 2019

Post-transcriptional gene silencing by RNA-induced silencing complex (RISC) in erythropoiesis is a rapidly evolving field of study. Disruption of microRNA (miRNA) synthesis has been shown to hinder erythropoiesis and has been linked to disease. The presence of miRNA in circulating RBCs has recently been reported; however, clear understanding of their role and effectors has yet to be determined.

In this study, freshly drawn RBC were density separated on discontinuous percoll gradients. The top two fractions, fraction 1 (F1) representative of the top layer containing the youngest, reticulocyte-like cells, and a second fraction with greater density and non-reticulocyte properties (F2), were isolated. Cell populations were analyzed to compare RBC protein levels using western blotting (WB). qRT-PCR was used to evaluate band 3 RNA. Argonaute (AGO2) RNA immunoprecipitation (RIP), was used to ascertain RISC mediated transcriptional repression of band 3 using qRT-PCR.

Band 3 protein expression was elevated in the denser F2 compared to F1, in freshly drawn normal RBCs. RNA levels of band 3 complemented protein levels, as increased band 3 RNA was observed in F2. RIP experiments revealed elevated post-transcriptional control of band 3 through increased association of its RNA with AGO2 in F1 compared to F2 thereby, correlating with greatly reduced, sometimes undetectable protein levels of band 3 in F1.

We confirm the presence of AGO2 in circulating RBCs. We report, for the first time, the post-transcriptional control of band 3, an integral RBC transmembrane protein, in circulating reticulocyte-like cells. These results suggest a possible subpopulation of reticulocytes, a pro-reticulocyte, with ongoing control of band 3 expression following enucleation, and a potential role of miRNA-RISC in band 3 pathology.

Disclosures

No relevant conflicts of interest to declare.
MASL1 Is a Critical Modifier of Hepcidin Expression Via Smad1/5/8 Phosphorylation in the Bone Morphogenetic Protein (BMP) Signaling Pathway
Authors: Kumkhaek Chutima , LaChance Christian , Aerbajinai Wulin , Zhu Jianqiong , Rodgers Griffin P.
Published: November 2019

Iron is essential for hemoglobin synthesis during terminal erythropoiesis. Hepcidin is the main regulator of iron homeostasis and is repressed by erythropoiesis. Although several candidates have been proposed to act as hepcidin inhibitors and erythroid regulators such as growth differentiation factor 15 (GDF15), twisted gastrulation BMP signaling modulator 1 (TWSG1) or erythroferrone (ERFE), their role in hepcidin repression during erythropoiesis is still unclear. We previously demonstrated that malignant fibrous histiocytoma-amplified sequence 1 (MASL1) is important for terminal erythropoiesis. In addition, down-regulation of MASL1 expression in macrophages strongly enhances IL-6 production following LPS or poly IC stimulation. Therefore, we hypothesized that MASL1 could directly (or indirectly) influence hepcidin expression. We found that endogenous MASL1 expression was significantly decreased in CD34+ cells treated with IL-6 compared with EPO-treated CD34+ cells at day 3 (0.35±0.05 fold vs 2.34±0.13 fold, P=0.002) and day 7 (1.03±0.75 fold vs 205.31±10.83 fold, P=0.001) of differentiation. In contrast, endogenous hepcidin expression was markedly increased in CD34+ cells treated with IL-6 compared with EPO-treated CD34+ cells. Interestingly, an increased hepcidin expression was detected in MASL1-knockdown CD34+ cells at day 3 of EPO-induced differentiation when compared with mock (47.47±23.49 fold vs 2.10±2.46 fold, P=0.029) or control lentiviral vector (47.47±23.49 fold vs 2.54±1.29 fold, P=0.029). Of note, ERFE, GDF15 and TWSG1 expression were decreased in MASL1-knockdown CD34+ cells at day 3 of EPO-induced differentiation. In addition, endogenous MASL1 expression is down-regulation after LPS treatment in PMA-induced THP1 cells but IL-6 is enhanced in MASL1-knockdown-PMA-induced THP1 cells after LPS treatment. In human hepatic cells (Huh-7), we found a significant decrease in hepcidin expression in MASL1-overexpressed Huh-7 cells after BMP2 (5.85±0.32 fold vs 9.98±0.97 fold, P=0.028) or BMP6 (10.07±1.35 fold vs 20.3±0.75 fold, P=0.007) stimulation for 24 hrs. Moreover, up-regulation of MASL1 enhanced the phosphorylation of Erk1/2 proteins while inhibited the phosphorylation of Smad1/5/8 proteins in Huh-7 cells after BMP2 or BMP6 stimulation for 1 hr that consequently affect in down-regulation of hepcidin expression. Strikingly, MASL1 overexpressed-Huh7 cells showed moderately decreased nuclear localization of phospho-Smad1/5/8 after BMP2 or BMP6 treatment. Taken together, these data demonstrate that MASL1 is a critical modifier of hepcidin expression potentially via additional mechanisms related to erythropoiesis and body iron homeostasis. Further clarification of these pathways may be the useful in developing novel treatment of anemias or iron disorders.

Disclosures

No relevant conflicts of interest to declare.
An HPFH Mutation in the γ-Globin Gene Promoter of Humanized Cooley's Anemia Mice Rescues Them from Perinatal Lethality and Renders Them Transfusion Independent
Authors: Fontenard Suean Daimia , Huo Yongliang , Liu Shanrun , Lockhart Jonathan , Berlett Michael , Ryan Thomas M
Published: November 2019

Cooley's Anemia (CA), β-thalassemia major, is a genetic disease caused by an impairment in β-globin protein synthesis. The resulting excess in α-globin chains causes the premature destruction of erythroid cells (ineffective erythropoiesis), anemia, and if left untreated, death within the first years of life. Several mutations in the promoters of the fetal γ-globin genes have been identified which impair the silencing of the fetal genes in adulthood, a condition termed hereditary persistence of fetal hemoglobin (HPFH). The amount of fetal hemoglobin (Hb F) expressed and the distribution within the RBC population (pancellular vs heterocellular), varies widely with different HPFH mutations. It has also been observed that thalassemia patients who co-inherit HPFH mutations that express higher levels of Hb F have milder disease symptoms. The purpose of this study is to determine whether the incorporation of a non-deletional HPFH mutation into the promoter of the human g-globin gene in a humanized mouse model of CA can rescue the animals from their perinatal lethality. Heterozygous humanized HPFH -175 mice express pancellular, high-levels of Hb F into adulthood. Homozygous HPFH -175 CA mice are rescued from their perinatal lethality, surviving solely on 100% human Hb F and are transfusion independent for life. This result is significant because it demonstrates that introduction of an HPFH mutation into the γ-globin gene promoter by gene editing may be a viable therapeutic option for CA patients in the future.

Disclosures

No relevant conflicts of interest to declare.
Hemoglobin S Screening Using the « Sickle Scan » - Biomedomics System. the Necker-Enfants Malades Hospital Experience
Authors: MINE Louis , Nguyen-Khoa Thao , Allaf Birch , Ribeil Jean-Antoine , Remus Christelle , Gauthereau Valérie , Enouz Sarah , KIM Jason S. , Yang Xiaoxi , Gluckman Eliane , Munnich Arnold , Cavazzana Marina , Girot Robert
Published: November 2019

Context: The principle of Sickle Scan (Biomedomics, Inc.) is a rapid, point-of-care qualitative lateral flow immuno-assay kit for the identification of sickle cell conditions of Hb A, S and C. Sickle Scan was specifically developed to allow for the identification of sickle cell trait Hb AS, heterozygotes AC, and Hb SS, Hb SC and Sβ° patients. Other sickle cell conditions as SD, SE, SO-Arab, S Lepore,… cannot be identified using Sickle Scan system. The test must be done using venous blood or capillary blood (fresh or dried blood spots).

Patients and methods: Two hundred and fifty patient samples (143 adults and 107 newborns) were analyzed. All tests were performed according the manufacturer's recommendations in one laboratory by 2 observers. The reference tests used for comparison were HPLC (NBS Variant - Bio-Rad) and capillary electrophoresis (Capillarys 3 - Sebia).

Results:

Comments: In adult patients, the 2 observers concordantly detected the presence of Hb A, Hb S and Hb C. There were 4 differences of interpretation between them (no Hb A in a AS patient and no Hb A in 3 SS transfused patients). The percentage of Hb A in these 3 last patients was respectively 13.6%, 17.7% and 18%. There was no false positivity neither in the patient heterozygous AE nor in the patient SD. No false negativity occurred for Hb S and C. In newborns, the accuracy of the test was excellent for the identification of the phenotypes FA, FAS, FAC, FS (SS / Sβ°). The lowest detected values of Hb S and Hb C in FAS and FAC newborns were respectively 2.4 and 3.4 %. We observed an inconstant cross-reactivity of the antibody anti Hb S with the hemoglobins E and D, in respectively 3/25 FAE phenotype and 6/26 in the FAD phenotype. There was no cross reaction with hemoglobin Bart's and O-Arab.

In FAS newborns the mean and extreme values of the percentage of Hb A were m=8.2 (2.6-15.5) and no difficulties occurred for the identification of these low percentages of Hb A. This observation is different from those made in adult patients for which one observer did not find Hb A in transfused patients with highest values of Hb A comprised between 13.6 and 18.

Conclusions: In this series of adult and newborn patients, the Sickle Scan appeared as an accurate method for the identification of AS, AC, SS/Sβ° and SC phenotypes. False positive tests were observed in some patients with hemoglobin E or D but no false negative results were found as regards the identification of Hb S and Hb C.

Table Table.

Disclosures

Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding.
Mathematical Modeling of White Blood Cell Population Dynamics for Diagnosis and Prognosis
Authors: Chaudhury Anwesha , Sohani Aliyah R , Higgins John M.
Published: November 2019

The complete blood count (CBC) is one of the most commonly ordered clinical tests because it provides a snapshot of the current state of the hematologic system in a patient, including an estimate of the number of currently circulating white blood cells (WBC). This simple assessment of patient WBC count is valuable in the screening and monitoring of a very wide range of conditions, from infection to malignancy to autoimmunity. Many routine WBC counts are measured by sophisticated automated hematology analyzers that rely on the principles of flow cytometry and characterize the morphology of thousands of individual WBCs in the process of providing a simple WBC count. These single-WBC measurements collected at high-throughput are usually discarded, but the probability distributions they represent provide a glimpse into the dynamics of the circulating WBC population, potentially enabling clinicians not only to estimate current numbers of circulating WBCs but also to infer whether the numbers of WBC subtypes (neutrophils, lymphocytes, and monocytes) are likely to increase or to decrease in the subsequent days and weeks.

We utilize this otherwise discarded single-WBC information in combination with semi-mechanistic mathematical models of neutrophil, lymphocyte, and monocyte population dynamics to infer patient-specific rates of neutrophil, lymphocyte, and monocyte flux (Figure 1). We characterize differences in average flux for patients with different classes of disease (infection, hematologic malignancy, and cardiac ischemia) and show how the data collected in a simple CBC and differential may be helpful in the early diagnosis and prognosis for a number of important diseases. For instance, we assess the diagnostic efficiency of inferred WBC dynamics to help identify (1) patients with initially negative cardiac troponins who are most likely to have subsequent positive levels, and (2) patients with newly discovered leukocytosis who are more likely to have an underlying malignant hematologic process. In a retrospective study of 73 patients with mild to moderate leukocytosis (11 x103/uL < WBC < 20 x103/uL) of unknown etiology, this approach showed promise in helping to identify patients who were diagnosed with a hematologic malignancy in the subsequent 6 months (Figure 2).

Figure 2: Nuclear maturation distributions of neutrophils (a) and lymphocytes (b) were derived from raw CBC measurements for patients found to have moderately elevated WBC counts (> 11x103/uL and < 20 x103/uL) before an underlying disease process had been identified. A statistically-significant fraction of patients with a subsequent new diagnosis of a hematologic malignancy could be identified based on either their single-neutrophil or single-lymphocyte characteristics at the time of the CBC up to 6 months prior to diagnosis, with multivariate discriminants showing even better performance.

Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Calcium Independent Events Trigger VWF-Dependent Shear-Induced Procoagulant Platelet Formation
Authors: Kholmukhamedov Andaleb , An Ann , Liu Fang , Janecke Rae , Jobe Shawn M
Published: November 2019

Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation.

To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs.

In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibition of SiPP formation in the absence of VWF, in VWF with deletion of the GPIbα-binding A1 domain, and in the presence of antibodies blocking the interaction of GPIb-IX and VWF. Calcium-dependent processes play an essential role in agonist-initiated PSer externalization. However, extracellular Ca2+ is not required for SiPP formation. Inhibition of Ca2+ entry, removal of extracellular Ca2+ and chelation of intracellular Ca2+, all had no effect on SiPP formation. Caspase activation mediates PSer externalization in the apoptotic platelet, but neither pharmacologic inhibition of caspases nor calpain inhibitors prevented SiPP formation. In contrast to the negligible roles of calcium- and caspase mediated events, SiPP formation is abrogated both in the absence of the mPTP potentiator cyclophilin D and in cyclosporine A-treated platelets. Furthermore, SiPP formation was closely correlated with mPTP formation as measured by loss of mitochondrial transmembrane potential. Together, these results identify a new mechanistic pathway of SiPP formation distinct from previously described initiating mechanisms for PSer externalization. Targeting of this distinct pathway may provide a novel pharmacologic strategy for the treatment of high shear hematopathologies, like arterial thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Impaired GPVI Mediated Platelet Functional Responses and Signaling in CD45 Knockout Mice
Authors: Inamdar Vaishali , Kostyak John , Badolia Rachit , Dangelmaier Carol , Kim Soochong , Manne Bhanukanth , Kunapuli Satya P.
Published: November 2019

Background: CD45 is a Receptor Protein Tyrosine Phosphatase C (PTPRC) and regulates Src Family Kinases activation in Lymphocytes. Although it is known to be absent from the platelet surface, proteomics studies prove that the CD45 c-terminal catalytic domain is present in platelets. Thus the aim of this study is to identify presence of CD45 c-terminal domain in platelets and characterize the functional implications of CD45 deficiency in platelets using a global CD45 knockout mouse.

Results: Platelets from CD45-deficient mice displayed a selective impairment of aggregation and dense granule secretion mediated by the collagen receptor Glycoprotein VI. CD45 deficient mice show increased bleeding times, indicating an important role for CD45 in hemostasis. However, there was no difference observed in thrombus generation and thrombus stability using the ferric chloride-induced carotid artery injury model. Signaling downstream of the GPVI receptor, indicated by Src Family Kianse (SFK), Syk and Phospholipase C_2 (PLCg2) tyrosine phosphorylation, was also impaired. In order to establish the presence of CD45 in platelets we used an established primary antibody that recognizes the c-terminal domain of CD45. We observed that this antibody recognized a protein of approximately 65 kDa, which is the expected size of the c-terminal 1 and 2 domains of CD45, in wild type (WT) mice but not in knockout (KO)mice.

Conclusion: Thus we conclude that CD45 is expressed in platelets as a truncated form, possibly generated by proteolytic cleavage, and regulates GPVI signaling, through regulation of Src Family Kinase activation.

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.
Selected Expression and Functional Importance of α4a-Tubulin in Platelet Biogenesis
Authors: Strassel Catherine , Hovasse Agnes , Moog Sylvie , Mageira Magda , Batzenschlager Morgane , Schaeffer-Reiss Christine , Kile Benjamin , Gachet Christian , Janke Carsten , Lanza Francois
Published: November 2019

Platelets are produced from mature megakaryocytes (MK) following a profound cellular reorganization. This includes the assembly of microtubules (MT) into a unique submenbranous coiled structure, the marginal band (MB). This process is thought to depend on a specific αβ-tubulin isotype repertoire. The MK-restricted-β1-tubulin, the predominant isoform of the MB, is already known to be important for platelet biogenesis but the implication of other isotypes is currently unknown.

Our goal was to establish the αβ-tubulin repertoire in platelets and during megakaryopoiesis and to evaluate the implication of selected isotypes in platelet formation.

To establish an exhaustive list of the tubulin isotypes, we used combination of RT PCR and proteomic analyses to quantify the expression of each isotype in human platelets and in human MK differentiated in culture from CD34+ hematopoietic progenitors. Information gained on the hierarchical combination of tubulin isoforms in the course of platelet biogenesis has been extended at the functional level to investigate both their role in marginal band formation and platelet functions

β6-, β5- and α1c-tubulin transcripts were already present in CD34+ cells and decreased during the final stages of megakaryopoiesis. On the other hand, β1-, α4A- and α8-tubulin transcripts were only observed later during MK differentiation and in platelets. Quantitative LC-SRM mass spectrometry confirmed the predominant expression of β1 and α4A-isotypes in platelets. A functional role of the newly identified α4a-tubulin was supported by the thrombocytopenia and enlarged platelets with a decreased number of MT coils (1-3) comprising less-acetylated tubulin in mice carrying a point mutation in tuba4a. Additionally, a tendency to increased responses to several agonists was observed in these platelets.

This study reveals new information on the evolution of the tubulin isotype repertoire in platelet formation pointing to a role of less-widely expressed α-isotypes.

Disclosures

No relevant conflicts of interest to declare.
Effects of RNA Interference on CD70 in Dendritic Cells of Patients with Immune Thrombocytopenia
Authors: Zhou Zeping , Wang Yunlong , Yang Renchi , Li Huiyuan
Published: November 2019

Objective: This study aims to assess the biological properties and functions of CD70 costimulatory molecule on dendritic cells (DCs) from patients with chronic immune thrombocytopenia (ITP).

Methods: DCs were generated from monocytes isolated from peripheral blood, the immunophenotype markers were detected by flow cytometry. Chemically synthesized siRNA was then transferred into the cells by Lipofectamine 2000 to choose the most effective siRNA to block CD70 expression in three candidates. The mRNA expression and protein synthesis were analyzed by real-time RT-PCR and flow cytometry. The CD4+ T cells were co-cultured with DCs to assess the suppression capacity of DCs in the proliferation of CD4+ T cells and the production of IL-10 and IFN-γ by CD4+ T cells. The CD4+ T cells were co-cultured with DCs to assess the ability of DCs in the induction of Regulatory T cells (Tregs).

Results: Dendritic cells were transfected with FITC-labelled scrambled siRNA for 4-6h. The transfection efficiency was average 60%. After transfection (48 h), the results of real-time RT-PCR analysis showed strong and specific downregulation of CD70 mRNA levels in transfected cells (p=0.046, siRNA1\2\3 Inhibition rates (74.5±6.1) %, (78.1±8.0) %, (90.1±2.5) % respectively, but not in negative control or in the absence of siRNA(p=0.157). The CD70-3 siRNA was extraordinarily effective in repressing the expression of CD70. The expression of CD70 on the DCs with siRNA3 was also significantly lower(p=0.043), but showed no difference in negative control (p=0.157). DCs from transfected group could inhibit the proliferation of PHA-activated CD4+ T cells than the negative control in ITP patients(p=0.008), but showed no difference in group of normal control (p=0.08). In chronic ITP patients and normal controls, the DCs from transfected group could inhibit the expression of IFN-γ compared with the group of negative control (p=0.018, p=0.043) and increase the expression of IL-10 (p=0.012, p=0.043). In chronic ITP patients and normal controls, the DCs from transfected group were defective in inducing the CD4+ T cell differentiating to CD4+CD25+CD127low Tregs compared to the negative control (p=0.012, p=0.043).

Conclusions: In our study, siRNA is capable of inducing RNAi in DCs, it can knock down the CD70 gene expression specifically and effectively. The DCs from transfected group could inhibit the CD4+ T cells and induce the CD4+ T cells differentiating to Tregs. This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for autoimmune disease.

Disclosures

No relevant conflicts of interest to declare.
Development of Platforms to Phenotype Variants of Uncertain Significance in VWF
Authors: Yee Andrew , Dai Manhong , Kretz Colin , Meng Fan , Ginsburg David
Published: November 2019

Platelet sequestration at sites of vascular injuries and factor VIII (FVIII) stabilization critically relies on von Willebrand factor (VWF). Specific domains within VWF coordinate these functions and may harbor mutations that result in von Willebrand disease (VWD). To survey all possible mutations that may lead to type 2 VWD, we developed prokaryotic and eukaryotic systems to display VWF fragments that are screened for a specific function and identified by DNA sequencing. We constructed an M13 filamentous phage display library consisting of ~2.8x106 independent clones that express random VWF fragments. Following a single round of selection for platelet binding, bound phage were eluted and analyzed by next generation DNA sequencing, revealing an optimal fragment spanning the A1 domain and a second region of weaker enrichment over the D4 domain. All A1 fragments encompassed C1272-C1458, indicating that intramolecular disulfide bridging of these cysteines optimizes platelet binding. Competitive binding assays between phage displaying a fragment of the D4 domain vs. a control phage resulted in enrichment of the former, suggesting a potential, novel role for the VWF D4 domain. Analogous to our previously reported method for analyzing VWF proteolysis (Kretz CA et al. Proc Natl Acad Sci USA. 2015), we mutagenized and displayed the VWF A1 domain to determine the spectrum of mutations that may alter the interaction between VWF and its platelet receptor, GPIbα. The mutant A1 library comprised ~5x106 independent clones, sufficient to represent all 3,933 single substitutions. Screening this library against platelets in the presence of excess control phage resulted in an enrichment of phage displaying an A1 fragment. DNA sequencing identified mutations located outside the VWF A1/platelet GPIbα interface, indicating allosteric control of this interaction. Screening phage displayed VWF fragments for FVIII binding, however, failed to identify critical residues; thus, we adapted our approach for eukaryotes. When displayed on mammalian cells, the VWF D'D3 domains retained their capacity to bind FVIII. Introduction of the VWD subtype 2N mutation, R816W, into the displayed VWF D'D3 domains abolished FVIII binding, demonstrating feasibility for mutational analyses. Together, these results demonstrate the utility of phage and mammalian display for structure-function analyses and that these platforms provide an efficient method for phenotyping variants of uncertain significance.

Disclosures

No relevant conflicts of interest to declare.
Dabigatran Is Associated with a Significantly Lower Risk of Abnormal Uterine Bleeding Than Warfarin in Female Patients of Childbearing Age with Venous Thromboembolism
Authors: Ferreira Melanie , Klok Frederikus A , Feuring Martin , Fraessdorf Mandy , Kreuzer Jörg , Huisman Menno V
Published: November 2019

Introduction: While Direct Oral Anticoagulants (DOACs) have been shown to have an overall equal efficacy as and better safety profile than warfarin in patients with acute venous thromboembolism (VTE), Xa-inhibitors have been associated with a higher risk of abnormal uterine bleeding (AUB). No data regarding AUB with the direct thrombin inhibitor dabigatran etexilate (dabigatran) have been reported to date.

Aim: To determine the risk of AUB during anticoagulation with dabigatran compared to warfarin in female VTE patients aged 18-50 years.

Methods: We performed a post hoc analysis of the pooled RE-COVER studies, two double-blind randomized trials evaluating dabigatran administered at a dose of 150mg twice daily versus warfarin after an initial treatment with parenteral anticoagulants, and the RE-MEDY trial, a double-blind randomized trial that studied the extended use of dabigatran at the same dose versus warfarin, all in patients with VTE. We compared the incidence of AUB, based on a defined preferred terms search in adverse events, in female patients aged 18-50 years who were treated with dabigatran versus who were treated with warfarin. Patients who rolled-over from RE-COVER into RE-MEDY and stayed on the same treatment were accounted for as "only one patient" taking the whole treatment period into consideration, whereas patients who switched treatment were analyzed as two different study subjects.

Results: Of the 2964 women included in the above mentioned trials, 1280 women were in the relevant age category and included in the current analysis. A total of 643 patients were randomized to treatment with dabigatran and 637 to treatment with warfarin. The overall rate of AUB was 5.9% for the women treated with dabigatran and 9.6% in those treated with warfarin, for an Odds Ratio of 0.59 (95% confidence interval 0.39-0.90).

Conclusion: In female VTE patients of 18-50 years old, dabigatran was associated with a significantly 41% lower risk of AUB than warfarin.

Disclosures

Ferreira: BMS-Pfizer: Consultancy, Other: Grant support; Bayer: Other: Grant support. Klok:Boehringer Ingelheim Pharma GmbH & Co.KG: Research Funding; Bristol-Myers Squibb: Research Funding; Bayer HealthCare: Research Funding; Daiichi-Sankyo: Research Funding. Feuring:Boehringer Ingelheim Pharma GmbH & Co.KG: Employment. Fraessdorf:Boehringer Ingelheim Pharma GmbH & Co.KG: Employment. Kreuzer:Boehringer Ingelheim: Employment. Huisman:GlaxoSmithKline: Other: Grant support; Boehringer Ingelheim Pharma GmbH & Co.KG: Other: Grant support; Bayer HealthCare: Other: Grant support; Pfizer: Other: Grant support; Actelion: Other: Grant support.
So Difficult to Understand : Readability Index Analysis of Online Patient Information on Lymphoma from NCI- Designated Cancer Center.
Authors: Kaur Supreet , Kumar Abhishek , Mehta Dhruv , Maroules Michael
Published: November 2019

Objectives - In current era of information technology, there is abundance of medical information for the patients and families. It is recommended that the online patient information (OPI) should be written no greater than sixth grade as per The National Institute of Health (NIH), American Medical Association, Department of Health & Human Services. We aim is to assess whether OPI on lymphoma from NCI-Designated Cancer Center (NCIDCC) and various cancer associations websites meet the current recommendations on a panel of readability indexes.

Methods - OPI from patient only section of NCIDCC and cancer associations websites were collected. This text was analyzed by 7 commonly used readability tests - Flesch Reading Ease score(FRE), Gunning Fog(GF), Flesch-Kincaid Grade Level(FKGL), The Coleman-Liau Index(CLI), The Simple Measure of Gobbledygook (SMOG) Index, Automated Readability Index(ARI) and Linsear Write Formula(LWF). Text from each article was pasted into Microsoft Word and analyzed using the online software Readability formulas.

Results - The mean FRE score is 55.4 (range 37.4-67.9) which corresponded to difficult level grade. The mean GF score is 12.9 (range 10.8-15.2) that comes between difficult and hard. The FKGL score is 9.9 (range 7.2-11.9) that corresponds to above the level of ninth grader.The mean CLI score is 11.09 (range 9-12) which represented text of twelfth grade. The mean SMOG index is 9.6 (range 8.1-11.1) which corresponded to greater than seventh grade level. While the mean ARI score was 10.5 (range 7.7-12.5) which represents readability suitable for people more than tenth grade. The mean LWF was 11.29 (range 7.8-14.4) that corresponds to tenth grade level of text.

Conclusion - The currently available OPI on lymphoma did not met the set national recommendations level on seven different validated readability indexes. Currently, available literature is difficult to understand and comprehend for average patient and their kins. There is a dire need to revise the currently available material for easy comprehension and understanding by the general patient population.

Disclosures

No relevant conflicts of interest to declare.
the Use of Tailored Feedback to Improve Hematologist Competence and Clinical Decision Making for the Treatment of Patients with Multiple Myeloma: a Novel Web-Based Educational Solution
Authors: Repetto Patricia , Raje Noopur S. , Fagerlie Sara R
Published: November 2019

Introduction/Background:

Recent advances in the understanding and treatment of multiple myeloma (MM) have led to improvements in patient management, including stratifying patients according to disease- and patient-specific risk factors, identifying appropriate patients for autologous stem cell transplantation, selecting treatment and incorporating new therapies into practice, and managing adverse effects.

Materials and Methods: An online educational program using clinical problem-based instruction methodology was developed for hematologists and posted November 30, 2015 (http://www.medscape.org/viewarticle/853712). Each patient case included interactivity in the form of clinical decision questions and knowledge assessmentquestions. Tailored feedback and potential consequences in response to clinical decision questions was provided to each learner. Learners who answered a question incorrectly on the first attempt were provided feedback without revealing the correct answer and given the opportunity to answer the question again (second attempt) [Figure 1]. To determine measurable improvements in competence and clinical decision making, first and second attempt answer choices were evaluated for the clinical decision questions. Overall effect size was calculated using Cohen's d to show the magnitude and strength of the consequence-based feedback learning method. Data were collected through February 17, 2016.

Results: A total of 404 hematologists participated in the activity during the study period, and responses from 129 (those who completed all clinical decision questions) were assessed. Responses to the 6 clinical decision questions show that a range of 28% to 67% of learners answered a question correctly on the first attempt. After receiving feedback specific to each incorrect answer, there was an overall 71% relative improvement in hematologists/oncologists who answered a question correctly on the second attempt [Figure 2]. The consequence-based feedback had a large impact on hematologists' ability to make clinical decisions correctly as demonstrated by the overall effect size of d=.94.

Conclusions: This study demonstrated the success of a web-based CME activity with tailored feedback to learners' responses. Participation in this activity led to large improvements in clinician knowledge and the ability to select appropriate evidence- based practice choices, which may ultimately improve outcomes of patients being treated for multiple myeloma.

Disclosures

No relevant conflicts of interest to declare.
Outcome of Myelodysplastic Syndromes over Time in the US: A National Cancer Data Base Study from 2004-2013
Authors: Al-Kali Aref , Zblewski Darci , Bartley Adam C , Patnaik Mrinal M , Gangat Naseema , Begna Kebede H. , Tefferi Ayalew , Elliott Michelle , Hogan William J , Litzow Mark R. , Go Ronald S.
Published: November 2019

Background: Over 10,000 new cases of myelodysplastic syndrome (MDS) are diagnosed in the US annually (MA X, Cancer 2007). Hypomethylating agents (azacitidine and decitabine), immunomodulating drugs (lenalidomide) and erythropoietin stimulating agents (epoetin α and darbepoetin) have been approved for more than a decade for the treatment of MDS. However, changes in overall outcome after the approval of these treatments are unknown. We used the National Cancer Data Base (NCDB), a nationwide oncology registry covering 70% of the US cancer population to answer these questions.

Methods: We identified patients with MDS diagnosed from 2004-2013 using ICD-O-3 codes 9980-9983 and 9985-99912. Overall survival (OS) was estimated using the Kaplan-Meier method. We compared OS using the log-rank test according to demographic subgroups (sex, age [<40, 41-65, and ≥65 years], race [White, Black, Native American, Asian/Pacific Islander, and other], and ethnicity) across years of diagnosis (Period 1: 2004-2006, Period 2: 2007-2009, and Period 3: 2010-2013).

Results:

i) Characteristics: There were 54,953 MDS patients in our study. The median age at diagnosis was 76 years (78.7% older than 65) and 44% were females. White race (88.9%) was the most common, followed by black (7%). Four percent were Hispanics. A third (30.8%) of all patients were treated in academic centers. Three quarters (75.8%) had either Medicaid or Medicare insurance.

ii) MDS Subtypes: 5q Syndrome was seen in 2008 cases (3.7%) while the most common subtype was MDS-Unclassifiable (U) (55.6%). Therapy related MDS (t-MDS) was seen in 1125 cases (2%), however coding for t-MDS was discontinued in 2009. Refractory anemia (RA), refractory anemia with ring sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), and refractory anemia with excess blasts were seen in 9%, 7.6%, 6%, and15.6%, respectively.

iii) OS: Compared to their counterparts, patients who were male, older, had a diagnosis of RAEB, covered by Medicare insurance, and treated at non-academic centers had the worse OS (all P <0.001). Across Periods 1-3, we did not see improvement in OS among demographic subgroups except for those who were <40 years old (P=0.03).

Conclusions: In the past decade, the overall outcome of MDS did not improve despite the advent of new therapies. More than half of the cases were classified as MDS-U reflecting the difficulty of subtyping MDS based on World Health Organization classification. We found disparity in outcomes by demographics and type of treatment facility. This is the largest study of MDS outcome to-date and should help in understanding the gaps and areas that need future research.

Figure Figure.

Disclosures

Al-Kali: Onconova Therapeutics, Inc.: Research Funding; Celgene: Research Funding.
Male Gender Increases the Risk of Liver Fibrosis in Patients with Thalassemia Major Independent of Iron Overload
Authors: Al-Khabori Murtadha K. , Al Busaifi Said , Al Omairi Al Ghaliya , Hassan Moez , Al Dhuhli Humoud , Al Ajmi Umaima , Al Rahbi Sarah , Daar Shahina
Published: November 2019

Introduction and Objectives: Iron overload in patients with Thalassemia Major (TM) leads to various complications including liver fibrosis. The independent impact of gender on this risk has been previously investigated but not yet confirmed. We, therefore, planned to assess the independent impact of gender in patients with TM on the risk of liver fibrosis.

Methods: We included 96 patients with TM followed and transfused in one academic tertiary hospital. Patients underwent assessment of liver fibrosis using ultrasound elastography (FibroScan device) with a cut off value of 7.8 kPa. The mean ferritin in the 5 years prior to elastography assessment was used to represent iron overload. Association was tested using Chi-squared and the independent impact of gender was confirmed in the multivariable logistic regression with a model that included mean ferritin and gender.

Results: The median age of the 96 included patients was 26 years (Interquartile range [IQR]: 22-30). Males constituted 45% of patients and 33% of patients were splenectomised. The median alanine transaminase, aspartate transaminase, albumin and total bilirubin were 30 U/L (IQR: 18-64), 30 U/L (IQR: 18-46), 46 g/L (IQR: 44-48) and 21 µmol/L (IQR: 14-32) respectively. The median ferritin and liver iron concentration assessed by MRI T2* were 1293 µg/L (IQR: 753-2715) and 6.7 mg/gdw (IQR: 3.5-16.1) respectively. Thirty seven percent of patients had positive serology for HCV while 1% of patients had positive serology for HBV. The proportion of patients with fibrosis as assessed by elastography was 59%. The proportion of male patients with fibrosis was 70% compared to 51% in female patients with a trend towards statistical significance (odds ratio [OR] of 2.2 with a p value of 0.094). In the multivariable logistic regression model, both gender (OR of 3.0, P value of 0.0188) and ferritin (OR of 1.0004, p value of 0.0036) were statistically significant independent predictors of liver fibrosis.

Conclusion: Male gender increases the risk of liver fibrosis independent from iron overload. Our study confirms the previously suspected but unproven association. Follow up and therapy may be tailored to include gender as a decision factor. Larger studies are needed to further confirm these results.

Disclosures

No relevant conflicts of interest to declare.
Quantitation of Suicidal Netosis Using Convolutional Neural Networks in Human Neutrophils
Authors: Elsherif Laila , Metts Carrington A. , Sciaky Noah , Cooper Joshua N. , Holly Stephen P. , Parise Leslie V.
Published: November 2019

Suicidal NETosis (Neutrophil Extracellular Traps) is a mode of cell death involving de-condensation of chromatin and fusion with cytoplasmic granule proteins to create a net-like structure for entrapping pathogenic elements. Despite its name, NETosis does not occur exclusively in neutrophils but rather in all myeloid cells of the immune system except dendritic cells. A growing list of diseases and conditions implicate NETs in their etiology and the progression of symptoms. These conditions include sepsis, cystic fibrosis, lupus nephritis, acute respiratory distress syndrome (ARDS) and deep vein thrombosis. There are several different quantitative methods to assess NETosis making comparative analysis across the field of NETosis research extremely challenging. To further define NETosis and facilitate new discoveries in this field, we have developed a novel assay that combines high-content microscopy with Convolutional Neural Networks (CNN). CNN is the revolutionary image recognition technology used in popular social media facial features recognition systems to 1) quantify the percentage of NETotic and non-NETotic nuclei in a cell population, 2) classify NETotic, necrotic and normal nuclei in a mixed population of neutrophils and 3) differentiate between two different NETotic nuclei generated by two of the most commonly used agonists in NETosis research, the PKC activator PMA and A23187, a Ca+2 ionophore. Furthermore we have optimized neutrophil isolation and culture conditions to yield reproducible results with low inter-and intra-assay variability. Image acquisition was performed on adherent neutrophils using the automated BD Pathway Bio-imaging Systems with autofocusing function that allows imaging of approximately 10% of a well in a 96-well plate. An algorithm was created for image segmentation and recognition of NETotic nuclei based on size, DNA staining intensity and pattern. Data generated by this semi-automated analysis system were used to train a CNN to count NETotic and non-NETotic nuclei. These methods allowed us to calculate the EC50 for PMA (2.2 nM) and A23187 (2.5 uM) and to differentiate between their distinct mechanisms of NETosis. CNNs are able to classify NETotic phenotypes, without the use of expensive antibodies or reagents. Our assay represents a crucial first step for standardizing and quantitating NETosis, which will allow better data interpretation across different labs and provide a potential screening tool for known and as yet undiscovered NETosis agonists.

Disclosures

No relevant conflicts of interest to declare.