bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Clinical Characteristics and Efficacy of Chronic Myeloid Leukemia Patients with T315I Mutation
Authors: Chen Chen , Xu Na , WanEr Wu , Liang Liu , Zhou Xuan , JiXian Huang , Changxin Yin , Cao Rui , Liu Qifa , Xiaoli Liu 
Published: October 2019

Abstract
Background: The application of tyrosine kinase inhibitor (TKIs) has greatly improved the overall survival (OS) and quality of life of chronic myeloid leukemia (CML) patients.However, in the TKIs era, 10% to 25% of CML patients still develop TKIs resistance, and ABL kinase point mutations are the most common reason.Most of the ABL kinase region mutations resistant to imatinib could be alleviated by second generation TKIs, but the T315I mutation resistance to the first and second generation TKIs. Ponatinib is a multi-target tyrosine kinase inhibitor, which belongs to the third generation of TKI inhibitors,and is sensitive for CML or Ph postitive ALL patients with T315I mutation. But,how to apply ponatinib bridging graft or whether ponatinib preventive therapy is needed after transplantation is uncertainty.
Methods: 18 CML patients with T315I mutation detected by ABL1 kinase region mutation in Southern Hospital from March 2013 to April 2018 were retrospectively analyzed.G-banding method was used for chromosome analysis and real-time quantitative PCR method was used to detect mutations in ABL1 kinase region by BCR-ABL1 fusion gene Sanger sequencer.
Result:18 CML patients with T315I mutation :13 cases chronic phase (CP) ,2 cases in accelerated phase,3 cases in blastcrisis phase(BP); 9 cases in high risk group, 6 cases in middle risk group and 3 cases in low risk grou by Sokal score score system.15 patients by imatinib ,3 patients first-line treatment with dasatinib .In imatinib group, 13 cases conversed to dasatinib because of drug resistance or intolerance, and 5 cases (5 / 13) were converted to ponatinib because of T315I mutation.One case in dasatinib group converted to ponatinib because of T 315 I mutation.A total of 6 patients (6 / 18) were treated with ponatinib. 6 patients (6 / 18) treated by allogeneic hematopoietic stem cell transplantation (Allo-SCT).The median stage of T315I mutation was 12.5 m from the beginning of treatment to the detection of T 315I mutation in 18 patients.At the end of the follow-up, 8 cases died of recurrence and 10 survived: (CMR 2 cases, CHR 1 cases, PR 3 cases, NR 3 cases, 1 cases not regularly followed up, unable to evaluate the disease state), including 6 patients with PO treatment.
Conclusion:The point of T315I mutation was detected in patients with CML resistance after long-term sequential therapy frequently. The recurrence rate was still high even if these patients experience allogeneic hematopoietic stem cell transplantation.However,these patients treatment with ponatinib before and after transplantation maybe reduce the recurrence rate and improve prognosis.
Key words:Chronic myeloid leukemia;BCR-ABL;T315I;ponatinib.

Disclosures
No relevant conflicts of interest to declare.
CHD1 - a Novel Epigenetic Regulator in Myeloid Malignancies with a Role in DNA Repair
Authors: Sinha Arpan , De La Garza Adriana , Verma Amit , Frazer J. Kimble , Bowman Teresa V. 
Published: October 2019

Abstract
Myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) are clonal hematopoietic disorders resulting from genetic alterations in hematopoietic stem cells. These myeloid disorders are clinically heterogeneous and biologically complex. Despite major advances in understanding the genetic and molecular landscape of MDS/AML, along with the introduction of newer and targeted therapies, the cure rates in AML are still only about 60% in children, and much lower in adults. Exploiting the genetic tractability of the zebrafish (Danio rerio) vertebrate model, we are investigating the role of a novel epigenetic regulator, Chromodomain helicase DNA binding protein-1 (CHD1) in hematopoiesis and its misregulation leading to MDS and AML. CHD1 is located at chromosome 5q21, which lies within the most frequent breakpoints seen with the deletion of the long arm of chromosome 5 [del (5q)] in patients with MDS and AML. In addition to del (5q), we found that CHD1 levels are significantly lower in bone marrow cells of patients with other forms of MDS, relative to normal controls. Using CRISPR/Cas9-mediated targeted mutagenesis in zebrafish, we created chd1 homozygous mutant fish. We confirmed a marked decrease in chd1 gene expression in these mutant fish. Chd1 homozygous mutants are viable and fertile as adults, with no significant developmental or hematopoietic phenotypes observed during embryogenesis. As CHD1 can act as a tumor suppressor and is linked to the DNA damage response, we hypothesized that chd1 mutant zebrafish would be more sensitive to DNA damaging agents. Indeed, we found that chd1 mutants have increased sensitivity to ionizing radiation as evidenced by elevated brain cell death measured by whole mount imaging of live embryos and immunofluorescence for activated Caspase 3, a marker of apoptosis. We also generated chd1het; tp53het zebrafish to test whether chd1 haploinsufficiency could accelerate tumor rates in tp53 mutant fish. Single heterozygotes chd1het or tp53het usually do not form tumors at one year of age, but chd1het; tp53het double heterozygous zebrafish showed substantial tumor growth by one year of age. Taken together, our data suggest that CHD1 may play a key role in protecting genomic integrity, explaining why diminished CHD1 levels could contribute to the pathogenesis of MDS and AML. This genetic interaction may be especially crucial in patients with combined del (5q) and TP53 alterations, and could contribute to the increased severity seen in this group. Our findings suggest these and other CHD1-deficient patients may be resistant to standard therapies due to attenuated DNA damage responses, allowing their AML to survive DNA damage caused by conventional anti-cancer treatments.

Disclosures
No relevant conflicts of interest to declare.
A Restructured Approach to Diagnosis of Heparin Induced Thrombocytopenia in a Large Tertiary Hospital
Authors: Swarup Sriman , Ball Somedeb , Adhikari Nimesh , Welch Courtney Alice , Fackrell Jaden , Thein Kyaw Zin , Sultan Anita , Swarup Khatrina , Awasthi Sanjay , Tijani Lukman 
Published: October 2019

Abstract
Background
The standard practice for diagnosis of heparin-induced thrombocytopenia (HIT) involves a combination of 4T score and laboratory tests, such as enzyme immunoassay for detection of antibodies. We noted a lack of widespread use of 4T score in our practice setting. We also found that our laboratory utilized Particle Immunofiltration Assay (PIFA) for HIT screening, which has been shown to have questionable diagnostic utility in HIT diagnosis (Warkentin et al., 2007). The study aims to improve the rate of 4T score usage in conjunction with an improved laboratory diagnostic test for patients with suspected HIT in a cost-efficient manner.
Method
We initiated a quality improvement project involving the review of all patients with laboratory orders for PIFA testing between March 2017 to March 2018, explicitly assessing for documented 4T scores before the ordering of PIFA. Three of the investigators also calculated 4T scores for these patients at the time of laboratory testing and noted the results of the serotonin release assay (SRA), if ordered. We further collected data on any alternative anti-coagulation used in such patients for a cost-efficacy analysis later.
Results
A total of 170 PIFA tests were ordered during the period of investigation. Only five (0.02%) of these patients had a documented 4T score at the time of testing. One hundred thirteen patients (66.4%) had a low 4T score per investigator-calculation. Forty-seven patients (27.6%) were noted to have intermediate 4T scores. Lastly, ten patients (0.05%) were observed to have high 4T scores. A total of 32 SRAs were ordered; five of which were positive (four with an intermediate 4T score and one with high 4T score). PIFA was false-negative in two confirmed cases of positive SRA and false-positive in 13 instances of negative SRA. Thus, in this study, the sensitivity of PIFA was noted to be 60%, and specificity was observed to be 50%. Nineteen patients also received alternative parenteral anti-coagulation (fondaparinux or argatroban); seven of these were with low, eight with intermediate, and four with high 4T scores.
Conclusion
The study highlights the need for improving 4T score usage rates in our hospital as well as a need for switching to an alternative HIT screening test to promote patient safety and cost efficacy. Hence, we have begun the integration of 4T score with laboratory testing into the electronic medical record, alongside a shift in our HIT screening test from PIFA to the recently FDA-approved automated latex immunoturbidimetric assay. We will be continuing analysis of patients with suspected HIT for another six months to assess the effects of the above interventions.
Figure. Figure.

Disclosures
No relevant conflicts of interest to declare.
Adenosine Regulation of cAMP through Phosphodiesterases
Authors: Teng Bunyan , Darlington Daniel N , Cap Andrew P 
Published: October 2019

Abstract
Introduction: Adenosine, an autacoid and metabolite of ATP, has been known to have anti-platelet properties. Of the 4 adenosine receptors (ARs), only A2A AR have been implicated in adenosines anti-platelet properties in human. A2A AR is a G-Protein Coupled Receptors associated with a stimulatory G-Protein (Gs) that can activate adenylyl cyclase (AC) and increase intracellular cAMP. An elevation of cAMP has been shown to inhibit platelet aggregation to natural stimuli. Regulation of intracellular cAMP is balanced between synthesis by adenylate cyclase and degradation by phosphdiesterases (PDE). There are 3 PDE subtypes found in platelets: PDE2, PDE3, and PDE5. However, it is not know which subtype(s) is (are) responsible for regulating cAMP level in human platelets after adenosine stimulation.
Materials and Methods: Platelet-rich plasma (PRP) was isolated from whole blood of human volunteers, and centrifuged at 200g for 10min. Light transmission aggregometry was performed after stimulation of platelets with 100uM ADP, with or without NECA (non-specific AR agonist), DPCPX (A1 AR antagonist), and Sch 58261 (A2A AR antagonist). PRP treated with NECA, DPCPX, Sch 58261, and PDE inhibitors (EHNA, E in figures, for PDE2, Trequinsin, T in figures, for PDE3, and 4-{[3'4'-(methylenedioxy) benzyl]amino}-6-methoxyqunazolin, 4 in figures, for PDE 5). Cyclic AMP was measured in platelets after treatment by liquid chromatography/ Tandem Mass Spectroscopy (Quantiva, ThrermoFisher) after treated with these drugs.
Results: ADP-induced platelet aggregation was inhibited in a dose dependent manner by the non-specific adenosine agonist, NECA (Figure 1) and the effect was blocked by A2A specific antagonist Sch 58261, not by the A1 AR antagonist, DPCPX (Figure 2). NECA inhibition of platelet aggregation was likely due to an elevation of intracellular cAMP (1 uM, 5min incubation, Figure 3). Inhibition of PDE3 alone, significantly increased intracellular cAMP, suggesting that basal PDE3 activity is present. PDE 3 inhibition combined with NECA elevated cAMP even higher than PDE inhibition or NECA alone (Figure 3), suggesting that NECA (A2A stimulation) effects PDE activity. Inhibition of PDE2 or 5 had no effect on basal or NECA stimulated cAMP (Figure 3). Inhibition of all 3 PDE (2,3,5) combined with NECA elevated cAMP to levels higher then NECA+ PDE3 inhibition, again suggesting that NECA maybe effecting the activity of the PDEs (Figure 3). The potentiation of cAMP by PDE3 inhibition + NECA was block by A2A, but not A1 antagonist (Figure 4) suggesting that the nonspecific adenosine agonist is elevating cAMP through A2A.
Conclusion: 1. In human platelets, NECA stimulates cAMP through A2A receptors and this elevation is likely due to an elevation in adenylate cyclase via Gs coupled to A2A. PDE3 is basally active and likely regulated by adenosine receptors.

Disclosures
No relevant conflicts of interest to declare.
Protective Effect of Thrombopoietin on CoCl2-Induced Apoptosis of HUVEC Cells through PI3-k/Akt Pathways
Authors: Ye Jieyu , Wang Jun-yan , Liang En-yu , Yang Mo 
Published: October 2019

Abstract
Endothelial damage is a serious syndrome following by stem cell transplantation. However, a useful treatment for attenuating the periods of this disorder was still under explored. Our previous studies have demonstrated that in patients with acute inflammation states, serum thrombopoietin (TPO) levels were significant higher compared with healthy subjects (181.11 ± 35.38 vs 96.13 ± 9.7 pg/ml, p<0.001), which may act as an acute response protein to protect the body. TPO is now recognized as a potent proliferative factor for outer hematopoietic system, such as cardiomyocytes and neurocytes. This study is aim to investigate the protective effect of TPO on human endothelial cells, and to explore its potential mechanism. Four experimental groups were set up using human umbilical vein endothelial (HUVEC) cells, which are: Normal control, CoCl2 alone (800 μmol/L), TPO(100 μg/L) + CoCl2 and TPO alone groups. CoCl2 alone decreased the viability of HUVEC cells in a dose dependent manner (r=-0.997), while addition of TPO significantly rescued this effect (n=5, p<0.01). We further tested the anti-apoptotic effect of TPO on HUVEC cells. CoCl2 alone was able to induced apoptosis as demonstrated by Annexin V (n=4, p<0.001), active-caspase-3 (n=4, p<0.01) and Mitochondrial Membrane potential (JC-1) assays (n=4, p<0.01). Consistently, TPO plus CoCl2 showed a significant anti-apoptotic effect compared with CoCl2 alone group in Annexin V (n=4, p<0.01), active-caspase-3 (n=4, p=0.039) and Mitochondrial Membrane potential (JC-1) assays (n=4, p=0.038). In order to find out the underlying mechanism, we further tested the expression of phospho-Akt (p-Akt). Our results demonstrated that CoCl2 alone suppressed the PI-3k/Akt pathway. TPO plus CoCl2 was shown to activate the expression of p-Akt by western blot. In summary, our findings showed that TPO had a protective effect on human endothelial cells. This effect was likely mediated by activation of PI-3k/Akt pathway, which leads to anti-apoptosis on these cells. This may provide us a new clue to identify novel strategy in treating endothelial syndromes.

Disclosures
No relevant conflicts of interest to declare.
Bendamustine with Total Body Irradiation Limits Murine Graft-Versus-Host Disease in Part Via Myeloid-Derived Suppressor Cells
Authors: Hoffman Emely , Stokes Jessica , Molina Megan Stanley , Katsanis Emmanuel 
Published: October 2019

Abstract
Background
GvHD remains a significant challenge in allogeneic hematopoietic cell transplantation (HCT). An under-investigated strategy to reduce GvHD is the modification of the preparative conditioning regimen. Cyclophosphamide (CY) and total body irradiation (TBI) is a commonly utilized myeloablative conditioning regimen, but is associated with GvHD. Bendamustine (BEN) is an alkylating agent that has been applied recently in nonmyeloablative chemotherapy-based conditioning regimens, demonstrating reduced myelosuppression, transplant related mortality, and acute graft versus host disease (GvHD). We have recently published that BEN can effectively replace CY when given following murine haploidentical bone marrow transplantation. We therefore hypothesized that BEN can also have advantages over CY when given in conjunction with TBI as a myeloablative preparative regimen.
Methods
We used a major histocompatibility mismatched murine BMT model to evaluate GvHD following BEN-TBI and CY-TBI conditioning. BALB/c (H-2d) mice received equivalent doses of BEN or CY (based on maximum tolerated dose), followed by TBI (400 cGy) and infusion of bone marrow (BM) cells with splenocytes (SC) or purified T-cells from C57BL/6 (H-2b) mice. GvHD morbidity and mortality and engraftment were monitored and MDSC frequencies were assessed by flow cytometry and immunofluorescence.
Results
We demonstrate that BEN-TBI conditioning, while facilitating complete donor chimerism, results in significantly less GvHD and improved survival compared to CY-TBI. In BEN-TBI conditioned mice, suppressive CD11b+Gr-1high myeloid cells are increased in the blood, bone marrow (BM), and spleen, with no evident differences in suppressive function. The ratio of Gr-1+ cells to T-cells is significantly increased in BEN-TBI mice in the intestines. When Gr-1high cells are depleted prior to transplant, the beneficial effects of BEN-TBI are partially lost. Alternatively, administration of G-CSF, which promotes CD11b+Gr-1+ myeloid cell expansion, trends towards an increase in survival in BEN-TBI mice.
Conclusions
These findings indicate a potential role of granulocytic myeloid-derived suppressor cells (MDSCs) in the mechanism by which BEN allows engraftment with reduced GvHD. BEN-TBI conditioning reduces GvHD morbidity and mortality compared to CY-TBI and may present a safer alternative to CY-TBI as a myeloablative conditioning regimen in allogeneic HCT.

Disclosures
No relevant conflicts of interest to declare.
Molecular and Clinical Aspects of Aα Dysfibrinogenemia in 25 Patients from the Czech Republic
Authors: Ceznerova Eliska , Stikarova Jana , Roselova Pavla , Kaufmanova Jirina , Suttnar Jiri , Louzil Jan , Kotlin Roman , Salaj Peter , Dyr Jan E. 
Published: October 2019

Abstract
Fibrinogen is a 340 kDA glycoprotein that plays a vital role in the hemostasis. The three pairs of polypeptide chains (Aα, Bβ and γ) form a heterodimer structure of fibrinogen, each chain is encoded by a distinct gene (FGA, FGB and FGG). Congenital dysfibrinogenemia is a rare qualitative disorder characterized by structural alterations in fibrinogen molecule resulting in its defective function. The clinical manifestation of majority cases is asymptomatic (50%); about 30% are associated with bleeding tendencies and only some cases are thrombotic (20%).
In this study, we evaluated 25 patients with heterozygous missense mutations in exon 2 of FGA gene from 13 unrelated families, all with low levels of functional fibrinogen and prolonged clotting time. All mutations were located around thrombin cleavage site at N-terminus of a fibrinogen Aα chain. Mutation Aα G13E was found in 6 families (13 patients), Aα R16H in 4 families (6 patients), Aα R16C in 1 family (4 patients), and Aα R19G in 1 family (2 patients). Correlations between the genotype and phenotype were studied by fibrin polymerization, fibrinolysis, kinetics of fibrinopeptides release, and electron and confocal microscopy. The viscoelastic properties of fibrin clots were measured. All mutations caused a delay or absence of fibrinopeptide A release and prolonged fibrin polymerization. Clinical phenotypes of mutations were, unexpectedly and importantly, highly heterogeneous. Out of 25 patients 17 were asymptomatic, 7 had the bleeding tendencies and 1 patient had thrombosis. One woman with Aα G13E had 2 spontaneous abortions and 3 pregnant women with Aα G13E, Aα R16C and Aα R19G mutations suffered for hemorrhages or postpartum bleeding. Eventually, all had successful delivery under provided care with a hematologist. Small surgeries were performed to patients with Aα R16H supplied with fibrinogen concentrates and antifibrinolytics. Our observations show that the patients should be carefully treated based both on the results of the laboratory analyzes and personal and family history.
This work was supported by the project of the Ministry of Health, Czech Republic, for conceptual development of research organization 00023736, by Grant from the Academy of Sciences, Czech Republic nr. P205/12/G118, and by ERDF OPPK CZ.2.16/3.1.00/24001.

Disclosures
No relevant conflicts of interest to declare.
Therapeutic Potential of AO-176, a Next Generation Humanized CD47 Antibody, for Hematologic Malignancies
Authors: Wilson W. Casey , Bouchlaka Myriam N , Capoccia Ben J , Hiebsch Ronald R , Donio Michael J , Puro Robyn J , Carter Alun J , Chakraborty Prabir , Manning Pamela T , Karr Robert W , Sung Vicki , Pereira Daniel S 
Published: October 2019

Abstract
Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and over-expressed on several tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPalpha) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models.
We have developed a next generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPalpha interaction and induces phagocytosis of hematologic and solid tumor cells, but also exhibits several unique functional properties. The first property is the ability of AO-176 to induce direct tumor cytotoxic cell death in hematologic (ex. Jurkat, Raji and Molt-4) as well as solid human tumor cell lines by a cell autonomous mechanism (not ADCC). Secondly, AO-176 exhibits preferential binding to tumor versus normal cells, including red blood cells (RBCs), T cells, endothelial cells, skeletal muscle cells and epithelial cells. A0-176 also does not affect the function of any of these primary cells when assayed ex vivo. The negligible binding of AO-176 to RBCs versus hematologic (ex. Jurkat, Raji or Molt-4) or solid tumor cells is particularly profound and different from other reported anti-CD47 antibodies. AO-176 also does not induce hemagglutination of RBCs. These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. Consistent with this attribute, AO-176 was well tolerated in cynomolgus monkeys with no adverse effects in general nor with respect to RBCs which was consistent with ex vivo results. A third novel property of AO-176 is its enhanced binding to tumor cells at acidic pH. Because the microenvironment of leukemic bone marrow and solid tumors has an acidic pH, this enhanced binding of AO-176 at low pH has the potential added advantage of tumor-specific targeting. Lastly, we show that AO-176 demonstrates dose-dependent anti-tumor activity in hematologic and solid tumor xenograft models.
Taken together, the unique properties and anti-tumor activity of our next generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPalpha axis targeting agents as it progresses to clinical development.

Disclosures
No relevant conflicts of interest to declare.