bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use Of Novel Oral Anticoagulants For Treatment Of Venous Thromboembolism (VTE) In Patients With Malignancy: Meta-Analysis Of Randomized Controlled Trials
Authors: Jiang Bo , Malik Qasim , Crevecoeur Louis Romel 
Published: October 2019

Abstract
Use of Novel oral anticoagulants for treatment of venous thromboembolism (VTE) in patients with malignancy: meta-analysis of randomized controlled trials

Background
Venous thromboembolism (VTE) is a frequent complication of cancers. Patients with cancer have at least a 6 fold increased risk for VTE compared to those without cancer, and the diagnosis of VTE in cancer patient is associated with a 2-4 fold decreased survival during the first year. The mainstays of anticoagulant treatment in cancer patients remain unfractionated heparin, LMWH, and the Vitamin K antagonists (VKAs), with current guideline favoring the use of LMWH. Novel oral anticoagulants (NOAs) that directly inhibit factor Xa and thrombin, including dabigatran, rivaroxaban, and apixaban, represents a milestone in the prevention and treatment of VTE. However, there have been no clinical trials to test the efficacy of NOAs in cancer patients, therefore, use of these agents in cancer population is an extrapolation of published results with general population.

Objectives
Determine the efficacy of novel oral anticoagulants (thrombin inhibitor; dabigatran, and direct factor Xa inhibitors; rivaroxaban and apixaban) in VTE treatment in patients with cancer.

Data source
A systematic review was conducted using MEDLINE and EMBASE up to July 2013. Key words used included venous thromboembolism, pulmonary embolism, deep venous thrombosis, cancer, dabigatran, rivaroxaban, and apixaban. Due to the fact that no randomized controlled trials (RCTs) in cancer population, we combined data that were extracted from major RCTs that include cancer patients and performed a sub-group analysis.

Results
Three randomized controlled trials (RCTs) were reviewed, and a total of 550 patients with cancers were analyzed. NOAs are not inferior to the current standard anticoagulant therapy with enoxaparin followed by an adjusted-dose vitamin K antagonist, to prevent symptomatic recurrent venous thromboembolism. Odd ratio (OR) is 0.988 (95% CI, 0.51-1.94). There is no significant heterogeneity. The major bleeding events were not analyzed due to lack of sub-group data in the trials.

Conclusion
Cancer patients have different hemodynamics and unique features that make the treatment of VTE challenging. These includes: tumor-associated pro-coagulant, venous stasis and endothelial damage from chemotherapy and catheters, an increased risk of anticoagulant-related bleeding, and the complexity of anticoagulant control because of the occurrence of urgent procedures. The development of NOAs provided new modalities to treat VTEs in cancer patients, as it showed that NOAs is non-inferior to the current standard anticoagulant therapy. However, large scale randomized controlled trials specifically targeted cancer patients are needed.

Disclosures:
No relevant conflicts of interest to declare.
Longevity Promoting Interventions Inhibit Molecular and Functional Changes In Aging Hematopoietic Stem Cells
Authors: Yalcin Safak , Hu Wenhuo , Miller Richard A , Shin Yusup , Park Christopher Y. 
Published: October 2019

Abstract
Recent studies strongly suggest that aging results in the decreased regenerative capacity of adult somatic stem cells, but this process is poorly understood. Hematopoietic stem cell (HSC) aging is associated with myeloid-biased differentiation, reduced capacity for self-renewal, and reduced production of lymphoid and red blood cells. These functional changes lead to alterations in peripheral blood cell counts and reduction in adaptive immunity, as well as an increased incidence of myeloid malignancies. Previous studies have demonstrated that cell-intrinsic changes underlie HSC aging and persist through transplantation. MicroRNAs (miRNAs) are small non-coding RNAs that regulate HSC self-renewal and lineage specificity by suppressing the expression of hundreds of genes. Since rapamycin treatment has previously been shown to inhibit age-associated phenotypes in old HSCs, we hypothesized that investigating the expression and function of miRNAs in HSCs purified from mouse models of longevity may yield significant insights into the molecular mechanisms that underlie the phenotypic changes observed in the aging hematopoietic system.
Thus, we have characterized the cellular and functional alterations in aged HSCs after anti-aging interventions [rapamycin treatment and calorie restriction (CR)] in old mice versus age-matched non-treated controls. We found that anti-aging interventions significantly reduce the expansion of HSC numbers, restore erythroid and B-lymphocyte numbers, reverse age-related thrombocytosis in C57BL/6 mice, and reduce reactive oxygen species (ROS) levels specifically in the HSC compartment. Moreover, the diminished function of aged HSCs in vivo was restored by anti-aging interventions. To identify the transcriptional regulatory networks involved in age-associated functional HSC alterations, we performed miRNA expression profiling studies using highly purified HSCs from young, age-matched non-treated, and treated old mice (12 weeks old or 27 months old, n=5 per group). Analysis of age-associated miRNA expression changes revealed differential expression (p<0.05 compared with internal controls) of 30 in 768 (3.9%) mouse miRNAs tested. Compellingly, one third of these age-associated miRNA expression changes were inhibited by CR and rapamycin treatment. Thus, our findings provide evidence that anti-aging interventions also inhibit HSC aging, and that these effects are evident at the level of miRNA expression. Overall, these data provide a molecular view of HSC aging and identify numerous candidate genes that may regulate HSC aging.

Disclosures:
No relevant conflicts of interest to declare.
The Role Of tet2 DNA Methylcytosine Dioxygenase In Zebrafish Early Hematopoiesis
Authors: Lin Huan-Chau , Lim Ken-Hong , Chiang Yi-Hao , Wang Wei-Ting , Lin Ching-Sung , Chang Yu-Cheng , Hsieh Ruey-Kuen , Kuo Yuan-Yeh , Chou Wen-Chien 
Published: October 2019

Abstract
Loss-of-function mutations in Ten-Eleven-Translocation 2 (TET2) gene have been identified in various human myeloid and lymphoid malignancies. Recently, the TET gene family (TET1, TET2, and TET3) was found to function as DNA methylcytosine dioxygenase that is able to oxidize 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). In Tet2-deficient mouse models, Tet2 has been shown to play an important role in regulating self-renewal and differentiation of hematopoietic stem cells. These Tet2-deficient mice would gradually develop a chronic myeloid neoplasm resembling human chronic myelomonocytic leukemia suggesting that TET2 may function as a tumor suppressor. In the present study, we investigated the role of tet2 in zebrafish early hematopoiesis. During zebrafish early development, the expression of tet1, tet2, and tet3 by qRT-PCR can be detected mainly after the segmentation stage (26-somite), with fluctuated expression levels thereafter. Whole-mount in situ hybridization revealed that tet2 expression was strong over aorta-gonad-mesonephros region at 48 hours post-fertilization (hpf). Morpholino oligonucleotide (MO) knock-down of tet2 increased the expression of tet1, tet3, dnmt3aa, gata-1, alpha-Hb and fli1a (48 hpf) as well as rag2 and lck (4 days post-fertilization), and the expression of spi1b and mpo decreased (48 hpf). The expression of primitive hematopoietic stem cell markers scl and lmo2, as well as dnmt3ab, beta-Hb, l-plastin, and rag1 were unaffected. The levels of 5-mC and 5-hmC measured by ELISA were also decreased after MO knock-down of tet2. The number of gata-1 expressing red blood cells increased after tet2 MO knock-down as evaluated by flow-cytometry indicating that tet2 deficiency increased erythropoiesis. These preliminary results suggest that tet2 might play a role in the epigenetic regulation of zebrafish early hematopoiesis including erythropoiesis. Recently, transcription activator-like effector nuclease (TALEN) has been shown to generate targeted genomic editing in zebrafish. To validate our observation, we therefore utilized customized TALENs pair to generate tet2 knock-out zebrafish animal model. We designed a pair of TALENs targeting first exon of tet2 and our tet2 TALENs were able to generate insertion and/or deletion in targeted region of tet2 exon 1 in 25% to 44% zebrafish embryos. We obtained a total of fifteen different tet2 mutation genotypes F1 fish, and seven of them were predicted to cause early termination of transcription. The in-cross of these F1 genotypes matched the Mendelian inheritance. The tet2-/- knock-out F2 zebrafish is not embryonic lethal and can grow to sexually mature adult fish. The detailed analysis of tet2-/- knock-out zebrafish early hematopoiesis will be presented at the meeting.

Disclosures:
No relevant conflicts of interest to declare.
Role Of Long Pentraxin 3 (PTX3) In Wound Closure Induced By Bone Marrow-Derived Mesenchymal Stromal Cells
Authors: Cappuzzello Claudia , Doni Andrea , Dander Erica , Pasqualini Fabio , Nebuloni Manuela , Bottazzi Barbara , Mantovani Alberto , Biondi Andrea , Garlanda Cecilia , D'Amico Giovanna 
Published: October 2019

Abstract
Although several studies have shown the capacity of mesenchymal stromal cells (MSCs) to repair and regenerate different tissues, the mechanisms underlying these processes are not understood. Long Pentraxin 3 (PTX3) is a multifunctional protein produced by MSCs and other cell subsets upon activation with inflammatory cytokines. PTX3 is involved in innate immunity, inflammation and extracellular matrix deposition. In the present study we analyzed the potential role of PTX3 in wound repair process induced by MSCs.
PTX3 knockout MSCs (PTX3-/-MSCs) were collected from bone marrow of PTX3-/- mice. After 3-5 culture passages the expression of surface markers was analyzed by flow cytometry and their osteogenic and adipogenic differentiation capacity was detected by alizarin red O and oil red S staining, respectively. The ability of PTX3-/-MSCs to abrogate T cell proliferation was evaluated by co-culturing MSCs and PBMCs previously activated with Phytohaemagglutinin. Finally, equal number of both PTX3-/-MSCs and wild type (WT) MSCs were implanted into excisional wounds created by a biopsy punch on the back of allogenic WT and PTX3-/- mice. Wound area was measured up to 14 day and calculated using an image analysis program. The wound specimens were collected at 2, 7 and 14 days and processed for histological analysis.
We demonstrated that PTX3-/-MSCs, similarly to WT MSCs, displayed typical fibroblastoid morphology, they expressed common MSC markers and were able to differentiate into adipocytes and osteoblasts. In addition, they drastically decreased the mitogen-induced proliferation of lymphocyte. Importantly, in a mouse model of wound healing, PTX3-/- MSCs showed a highly significant defect in wound closure compared to WT MSCs at each time point. Histological evaluation of skin samples treated with PTX3-/- MSCs showed a reduction of the granulation tissue and a significant increase of neutrophils (GR-1+) in the wound bed. Moreover, wounds treated with PTX3-/- MSCs were characterized by an excessive accumulation of fibrin at the 2nd day after injury. Accordingly, PTX3-/- MSCs showed a defective ability to degrade the fibrin matrix in vitro. Finally, PTX3-/- MSCs failed to close the ulcers in PTX3-/- mice.
In conclusion, we demonstrated that PTX3 deficiency does not alter the phenotype and the capacity of MSCs to differentiate into mesengetic lineages; however, the production of PTX3 represents an essential requirement for MSC ability of enhancing tissue repair.

Disclosures:
No relevant conflicts of interest to declare.
Defective Nucleotide Metabolism Contributes To p53 Activation In Diamond-Blackfan Anemia
Authors: Danilova Nadia , Elena Bibikova , Covey Todd , Nathanson David , Dimitrova Elizabeth , Anne Lindgren , Glader Bertil , Radu Caius , Sakamoto Kathleen , Lin Shuo 
Published: October 2019

Abstract
Diamond-Blackfan Anemia (DBA) is a rare childhood bone marrow failure disorder, characterized by the presence of red cell aplasia, congenital abnormalities, and increased levels of adenosine deaminase (ADA). Haploinsufficiency of ribosomal proteins (RPs) due to mutations in RPS19 and RPL11 occurs approximately 25% and 5% of DBA patients, respectively. The pathogenesis of DBA has been associated with activation of p53, but the mechanism of how this leads to the erythroid defect in DBA patients is not well understood.
To understand the molecular pathways leading to DBA, we used previously published zebrafish models of RPS19 and RPL11 deficiency, in addition to primary human fetal liver CD34+ cells transduced with RPS19 shRNA to evaluate signaling pathways upstream of p53. One of the earliest responses to RP deficiency was upregulation of rrm1, a subunit of ribonucleotide reductase (RNR), responsible for de novosynthesis of dNTPs. Since ADA expression is increased in DBA and ADA is involved in nucleotide catabolism, we hypothesized that RP deficient zebrafish and primary human fetal liver CD34+ cells have defects in nucleotide metabolism, which result in replication stress and activation of p53 through the ATR/ATM/Chk1/2 pathways.
Several genes involved in de novo nucleotide synthesis, nucleotide catabolism, and purine salvage are upregulated in RPL11 and RPS19 deficient zebrafish, including rrm1 (2-7 fold), ada (2.8 fold), xdh (2.3 fold), and hprt1 (3-fold). In contrast, genes involved in pyrimidine salvage, such as tk1 and dck were downregulated in RP deficient zebrafish (0.4 and 0.8 fold, respectively). RP deficient zebrafish also had imbalanced dNTP pools, with increased dTTP and decreased dCTP levels compared to controls. To test whether these alterations in nucleotide metabolism lead to replication stress, we evaluated DNA damage and stress-induced proteins in primary human fetal liver CD34+ cells transduced with RPS19 shRNA and found increased phosphorylation of p53 (Ser15 and Ser37), ATM (Ser1981), 53BP1 (Ser1778), Chk1 (Ser345), and Chk2 (Thr68), indicating activation of the ATR/ATM/Chk1/2/p53 pathway in RP deficient cells. To rescue the RP-deficient zebrafish, we treated them with a mixture of exogenous nucleosides. Nucleoside treatment decreased p53 expression, restored rrm1, ada, tk1, and dck to normal levels, decreased number of apoptotic cells, and increased the number of Gata1-expressing cells in RP deficient zebrafish. Our data suggest that defective nucleotide metabolism contributes to p53 upregulation in DBA, and that nucleoside supplements alleviate replication stress and may prove beneficial for patients with DBA.

Disclosures:
No relevant conflicts of interest to declare.
Paroxysmal Nocturnal Hemoglobinuria and Thrombosis Before and After Eculizumab
Authors: Muñoz-Linares Cristina , Pastrana Miguel , Ojeda Emilio , Fores Rafael , Cabero Martin , Morillo Daniel , Bautista Guiomar , Navarro Belen , Krsnik Isabel , Gil Santiago , Regidor Carmen , de LaIglesia Almudena , Bueno Jose Luis , Garcia-Marco Jose A , Cabrera Jose Rafael 
Published: October 2019

Abstract
Thrombosis, mainly venous but also arterial, is the leading threat in Paroxysmal Nocturnal Hemoglobinuria (PNH) patients, caused by the continuous hemolysis. It constitutes the first cause of death in all reported series. Since 1964, a total of 56 patients with PNH clone were evaluated in our Hematology Unit. According with PNH Parker´s Classification, most of the patients were Classical type (28 patients), and the remaining included in the other subsets: 21 in the setting of another bone marrow failure syndrome (BMFS) and 7 subclinical.
Since November 2007, Eculizumab (an anti-C5 antibody) is employed in the disease to abrogate the hemolysis in our patients. In the last years, sixteen patients have been treated with this drug in our series. PNH patients previously anticoagulated with warfarin because thrombosis, continued on therapy after the addition of Eculizumab. Also, patients with more than 50 % PNH clone (established by Cytometry with FLAER on granulocytes) and platelets >50 x109/L received oral anticoagulation.
The incidence and localization of thrombotic events in the patients without Eculizumab was as follows:Parker’s ClassificationClassicalBMFSSubclinical28216Thrombosis cases (%)14 (50)4 (20)2 (33)Thrombotic episodes:2675Deep calf642Multiple cerebral ischemic infarcts212 (1 death)Large Cerebrovascular311Budd-Chiari11 (1 death)Portal2 (1 death)Retinal2Cava1Pulmonary thromboembolism2 (1 death)Myocardial infarction3 (1 death)Arterial ischemia2 (1 amputation)Skin ischemic2 (vasculitis, livedo reticularis)
After introduction of Eculizumab, sixteen patients have been treated with this drug and active thrombosis resolved in all cases, as was the case of a patient with a large persistent thrombosis in the inferior cava vein despite the isolated anticoagulation therapy. Only one patient on Eculizumab therapy experienced a thrombotic event and suffered a transient ischemic attack with aphasia after a prolonged catheter ablation procedure for an atrial fibrillation. This patient had previous signs of small vessel disease in MR imaging techniques. The episode occurs despite heparin anticoagulation and anticipated additional Eculizumab dose and resolves thereafter.
Eculizumab had a clear favourable impact in preventing thrombosis complications in our series of PNH patients. Careful monitoring of LDH levels and shortening the Eculizumab interval doses it is indicated in any chirurgical or invasive procedures in these patients.

Disclosures:
Pastrana: Alexion Pharmaceuticals: Speakers Bureau. Ojeda:Alexion Pharmaceuticals: Consultancy, Speakers Bureau.
Loss Of Tet2 Collaborates With AML1-ETO In Inducing Leukemogenesis
Authors: Rasmussen Kasper D , Jia Guangshuai , Johansen Jens V. , Bernard Olivier A. , Helin Kristian 
Published: October 2019

Abstract

Disruption of the epigenome can contribute to both cancer initiation and tumor progression. The methylcytosine dioxygenase TET2, which is involved in the regulation DNA methylation patterns, is frequently mutated in Acute Myeloid Leukemia (AML) patients and plays a central role in normal hematopoiesis. However, Tet2–deficient mouse models do not readily develop acute leukemia suggesting that additional oncogenic events are necessary for full-blown transformation.
The translocation t(8;21)(q22;q22) results in expression of the AML1-ETO (AE) fusion protein and is present in ∼5-10% of AML patients. Interestingly, the combination of t(8;21)(q22;q22) and TET2 mutations, although rare, have been observed in several studies in patients groups covering both pediatric and adult de novo AML. To confirm a direct role of TET2 mutations in the development of t(8;21)(q22;q22)-rearranged leukemias we expressed the AE fusion protein in hematopoietic stem and progenitor cells (HSPC) isolated from WT or Tet2-deficient animals. Serial replating assays in semisolid media as well as growth in liquid culture suggested a cooperative effect of AE and Tet2 deficiency as indicated by a significant increase in proliferation and colony formation efficiency. We then used a bone marrow transplantation model to assess the in vivo leukemic potential of AE;Tet2-/- HSPCs. The majority of recipient mice transplanted with AE;Tet2-/- (n=5/6) HSPCs succumbed to an aggressive and transplantable AML-like condition with a median latency of 5-6 months, whereas mice receiving AE HSPCs remained healthy over a 12 month observation period (n=6/6). The morbid mice showed many signs of disease including loss of weight, anemia, splenomegaly and large number of leukemic blasts (CD45neg) in circulation. Further analysis of the bone marrow and spleen revealed a massive expansion of a linneg (CD3e, Gr-1, Mac-1, B220, Ter119), c-Kitlow-high, CD16/32pos cell population indicating a block of differentiation and expansion of an immature myeloid progenitor population from AE:Tet2-/- HSPCs. To investigate how Tet2-deficiency contributes to transformation of t(8;21)(q22;q22)-rearranged cells we generated and cultured pre-leukemic AE-transduced cells in vitro from Tet2fl/fl HPSCs harboring the inducible Rosa26-CreERT2 allele. Genome-wide analysis of methylation patterns and gene expression showed that induction of Tet2-deficiency in pre-leukemic cells leads to progressive hypermethylation of gene-regulatory elements and altered expression of several genes implicated in tumorigenesis. Thus, this study illustrates how aberrant DNA methylation patterns can contribute to disease and confirm the role of Tet2 as a tumor suppressor preventing leukemogenesis.

Disclosures:
Helin: EpiTherapeutics : Consultancy, Equity Ownership.
A Genetic Screen In Zebrafish Identified Dlst As a Potential Therapeutic Target For Human Acute T-Lymphoblastic Leukemia
Authors: Anderson Nicole M , Peng Hongling , Helman Daniel , Harrold Itrat , Sanda Takaomi , Grant Trevor , Nagykery Nicholas , Samaha Yasmina , Le Meng , Amsterdam Adam , Neuberg Donna , Mansour Marc R. , Denton Travis , Kanki John , Singh Anurag , Look A. Thomas , Feng Hui 
Published: October 2019

Abstract
Despite improvements in treatment for T-Acute Lymphoblastic Leukemia (T-ALL), only 70-80% of children and 40% of adults achieve long-term remission. To discover novel targets for the development of molecular therapeutics, we conducted a dominant genetic modifier screen to identify genes whose inactivation delays the onset of leukemia. We have generated a Myc-driven zebrafish model of T-ALL that recapitulates its human counterpart genetically and pathologically. Utilizing this zebrafish model of T-ALL, we conducted a genetic screen that identified a gene encoding dihydrolipoamide S-succinyltransferase (DLST), whose heterozygous loss significantly delays the onset of leukemia in zebrafish. DLST is the E2 component of α-ketogluterate dehydrogenase complex (KGDHC), a key enzyme in the TCA cycle that converts α-ketogluterate (α-KG) to Succinyl CoA. Western blot analysis revealed that DLST was upregulated in zebrafish tumor cells, T-ALL cell lines and primary human T-ALL samples. Zebrafish T-ALL cells with heterozygous inactivation of dlst have decreased cell size and slowed cell cycle progression, compared to T-ALL cells without dlst inactivation. To demonstrate that DLST is a relevant target in human T-ALL, we used two structural analogs of α-KG (α-keto-n-valeric acid sodium salt (KV) and α-keto-β-methyl-n-valeric acid (KMV)) to inhibit the KGDHC activity. Inhibition of KGDHC by both KV and KMV decreased cell viability and promoted apoptotic cell death in a panel of human T-ALL cell lines. Genetic inactivation of DLST, via shRNA knockdown, also decreased cell viability in MOLT16 human T-ALL cell line, confirming the therapeutic potential of DLST in human T-ALL treatment. In vivo treatment of our zebrafish T-ALL model with KV promoted tumor regression within seven days of treatment. Taken together our findings indicate that DLST is an important contributor to T-ALL pathologenesis, and suggest that DLST is a potential target for further therapeutic development to treat human T-ALL.

Disclosures:
No relevant conflicts of interest to declare.