bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calr Mutational Status in Egyptian Patients with Myeloproliferative Neoplams
Authors: Soliman Eman A. , El-Ghlban Samah I. , Abdelaleem Abdelaleem H. , Abdel-Aziz Sherin , Shamaa Sameh , Abdel-ghaffar Hasan A. 
Published: November 2019

It has been known that the insertion/deletion mutation of CALR gene is the second deriver mutation in myeloproliferative neoplasm (MPN) of essential thrompocythemia (ET) and primary myelofibrosis (PMF). As the molecular workup has been incorporated for the prospective screening and diagnosis of MPN in our Oncology Center. An Egyptian 87 cohort of patients with non-mutated JAK2 (58 ET and 29 MF) were investigated using polymerase chain reaction (PCR) as a pilot study. We found that 37 out of 87 patients (42%) were carrying CALR mutations (30 out of ET (52%) and 7 out of MF (24%)). Sanger sequencing was used to determine the type of CALR mutations in all positive patients and we found that 13 out of 37 (35%) had type 1/type 1-like and 36 out of 37 (97%) with type 2/type 2-like. This CALR mutation profile in Egyptian patients appear different from the western status as type2/type 2-like is the highest in our patients (97%) versus 35-45% and type1/type 1-like was 35% versus 55-65% compared to western results. Meanwhile, the clinical course and phenotype of our cohort of patients is not similar to that in western as there is no significant difference of overall survival between type1/type1-like and type2/type2-like. This finding might be due to the different environmental and genetic backgrounds of Egyptian population. A part of it might be related to the HCV infection as 12 out of 37 (32%) had HCV infection. Further study is in progress on a large number of patients to correlate that with the clinicopathological status, response to therapy and the mechanistic pathway of oncogenic transformation.

Disclosures
No relevant conflicts of interest to declare.
Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4
Authors: Renna Stephanie , Edelstein Leonard C. , McKenzie Steven Edward 
Published: November 2019

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017).
We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets.
Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets.
In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates.
Figure

Disclosures
No relevant conflicts of interest to declare.
Fludarabine and Cyclophosphamide Based Conditioning Is Associated with Good Outcomes in Patients Undergoing Matched Sibling Donor Transplants for Aplastic Anaemia
Authors: George Biju , Abraham Aby , Korula Anu , Devasia Anup , Lakshmi Kavitha M , Kulkarni Uday Prakash , Abu Backer Fouzia N , Srivastava Alok , Mathews Vikram 
Published: November 2019

Background: Fludarabine based conditioning protocols are increasingly being used for transplant (SCT) in patients with aplastic anaemia (AA) especially in those who have received multiple transfusions.
Patients and Methods: Between January 2004 and June 2019, 229 patients with AA and HLA identical sibling donors underwent SCT at CMC Vellore India using Fludarabine [180 mg/m2 over 6 days] and Cyclophosphamide [120 mg/kg over 2 days]. Few patients received low dose ATG [ATGAM 40 mg/kg]. Data on HSCT and outcomes were collected from the institutional database and individual medical records.
Results: The median age was 23.9 years (range: 1.5 - 58) including 151 males and 78 females including 78 children (33.4%). Donors were matched sibling (n = 215) or family donors (n = 14). The graft source was Bone marrow in 11 and peripheral blood stem cells in 218. GVHD prophylaxis consisted mainly of cyclosporine and methotrexate mainly with few receiving post-transplant cyclophosphamide.
Engraftment occurred in 90% with graft failure in 2.6% and early death in 7.4%. Regimen related toxicity (RRT) was seen in 4.7% and included veno-occlusive disease of liver and hemorrhagic cystitis. Acute GVHD (grade 2-4) occurred in 26.4% while chronic GVHD was seen in 40.3%. The 5 year overall survival (OS) for the entire group is 75.9 + 4.9%. The 5 yr OS was 81.4 + 3.9% for ages 0 - 20, 76.3 + 4.4% for ages 21-40 and 55.3 + 9.3% for ages > 40 years. Age > 40 years (p = 0.000), presence of fever requiring hospital admission within 4 weeks prior to SCT (p = 0.001) and acute GVHD (p = 0.051) were identified as risk factors associated with a poor outcome on a univariate analysis while on a multivariate analysis, older age and fever continued to remain significant.
Conclusion: This is the largest series of patients with AA undergoing SCT using a fludarabine based conditioning and is associated with improved survival following sibling donor HSCT for AA. Presence of fever requiring a hospital admission immediately prior to SCT is associated with poor outcomes.

Disclosures
No relevant conflicts of interest to declare.
Targeting Polo-like Kinase 4 for Therapeutic Benefit in the Patients with Diffuse Large B-Cell Lymphoma
Authors: Zhao Yi , Han Yang , Yang Juan , Hu Shunfeng , Wang Xin 
Published: November 2019

Abstract:
Polo-like kinase 4 (PLK4) is a serine/threonine kinase that plays a critical role in regulating centriole duplication. PLK4 overexpression causes centriole amplification and genomic instability, which is the hallmark of cancer. Thus, PLK4 has been suggested as a tumor promoter role in solid malignancies. CFI-400945 is a highly selective small-molecule inhibitor of PLK4. CFI-400945 treatment results in centriole duplication defects, which leads to mitotic catastrophe and cell death. The antineoplastic effects of CFI-400945 have been reported in many cancers and this inhibitor has entered Phase I clinical trials for solid tumors. However, the role of PLK4 and cellular effects of CFI-40045 in hematological malignancies remains elusive.
In the present study, we found that PLK4 expression is up-regulated at both transcriptional and translational levels in diffuse large B-cell lymphoma (DLBCL) cell lines (Fig. 1a, Fig. 1b). DLBCL patients displayed high protein levels of PLK4 as visualized by immunohistochemistry (Fig. 1c). In vitro, treatment of DLBCL cell lines with CFI-400945 induced a decrease in cell proliferation and an increase in cell apoptosis and a block at the G2-M transition in a dose-dependent manner (Fig. 1d, Fig. 1e, Fig. 1f). Furthermore, we found that the antineoplastic effects of CFI-400945 in DLBCL cell lines were due to the increased DNA damage, as observed by increased accumulation of γH2AX through phosphorylation of CHK1/CHK2 (Fig. 1g). Since PLK4 inhibition affects DNA damage, we next aimed to explore the effects of PLK4 expression on sensitivity of DNA damage-inducing drugs (e.g., doxorubicin). We found that silencing of PLK4 via shRNA improve the anti-tumor effects of doxorubicin in DLBCL cell lines (Fig. 1h, Fig. 1i). PLK4 inhibition in combination with doxorubicin significantly increased phosphorylation of H2AX compared to cells treated with doxorubicin alone (Fig. 1j).
In summary, our data indicates that PLK4 is aberrantly expressed in DLBCL cell lines and tissues. Targeting PLK4 with the selective inhibitor CFI-400945 suppresses cell proliferation and induces apoptotic deaths and causes DNA damage in vitro. Our findings established PLK4 as a potential therapeutic target in DLBCL. Silencing of PLK4 expression induces accumulation of DNA damage and enhances the cytotoxic effects of doxorubicin. Thus, the levels of PLK4 may serve as a determinant of doxorubicin sensitivity and a predictive biomarker of the patients with DLBCL treated with R-CHOP based therapy.
Figure 1

Disclosures
No relevant conflicts of interest to declare.
Anemia Is Negatively Correlated with Cardiac Index in Hereditary Hemorrhagic Telangiectasia(HHT) Patients with Liver Arteriovascular Malformations (AVMs)
Authors: Farooq Hafsa , Lee Alfred I , Young Lawrence , Henderson Katharine 
Published: November 2019

OBJECTIVE:Patients with hereditary hemorrhagic telangiectasia (HHT) and liver arteriovascular malformations (AVMs) may develop high output cardiac failure. Iron deficiency anemia is common in HHT patients and is believed to adversely impact cardiac index (CI) but has not been systemically studied in this population. We performed a retrospective study to assess the relationships among hemoglobin, iron, and CI.
METHOD:Chart records were reviewed of HHT patients with liver AVMs who were seen at Yale HHT Center from 2003-2019 and who had undergone cardiac catheterization with calculation of CI. For each patient, clinical history, laboratory studies, and CI were recorded. All variables were categorized according to median of interquartile range and analyzed using Spearman's correlation coefficients.
RESULT:Seventeen patients with HHT and liver AVMs were identified who had undergone cardiac catheterization. Median age was 68 years (range, 36-85); 15 were female and 2 males. Median values of hemoglobin, ferritin, and CI were 10.6 g/ld., 35 ng/mL, and 4.77L/m/m2, respectively. A statistically significant, inverse correlation between hemoglobin and CI was observed (R = -0.45, P = 0.03). Serum iron and CI were also negatively correlated although the association was not significant (R = -0.3, P = 0.17). No significant correlation of serum ferritin with CI was seen (R = 0.05, P = 0.8).
CONCLUSION:Anemia is negatively correlated with CI in HHT patients with liver AVMs. The impact of iron indices on CI is uncertain and partly limited by our small sample size. Larger studies incorporating multivariable analyses are warranted.
Figure

Disclosures
No relevant conflicts of interest to declare.
Cytokine Release Syndrome Is an Independent Risk Factor Associated with Platelet Transfusion Refractoriness after CAR-T Treatment for Relapsed/Refractory Acute Lymphoblast Leukemia
Authors: Liu Yadan , Hu Yongxian , Huang He 
Published: November 2019

Platelet transfusion refractoriness (PTR) has been widely observed in hematopoietic stem cell transplantation (HSCT) and chemotherapy in some researches. However, PTR not only has unclear mechanism but no recognized standard treatment guidelines, even has a life-threatening effect on hematological patients. Our study is aim to evaluate the rate of PTR and related factors of PTR in patients with refractory/relapsed acute lymphocytic leukemia (R/R ALL) who received chimeric antigen receptor T cell therapy (CAR-T). We retrospectively collected the data of 49 R/R ALL patients between July 2011 and June 2019 in our single center, and divided them into chemotherapy group and CAR-T group, including 26 patients only receiving chemotherapy and 23 undergoing CAR-T therapy. All R/R ALL patients underwent multiple first lines or second lines of chemotherapies. As for CAR-T group, the fludarabine and cyclophosphamide were administrated three days to lower the tumor burden before CAR-T cell infusion. Age, sex, height, weight, the platelet count before and after transfusion, transfusion unit are all included in our data. In terms of the CAR-T group ,we also collect the peak body temperature , the peak c-reactive protein ,CRS grade , IL6 , IL10 , IFN-γ. The pre-transfusion and post-transfusion platelet count were obtained after each transfusion, the 12h CCI<5000 (corrected count increment) is used to evaluate if the patient has PTR. By chi-square test, we found 13 patients became PTR in all patients, of 2 patients (15.4%) is in chemotherapy group while 11 (84.6%) is in CAR-T group. Moreover, 7.7% (2/26) PTR in chemotherapy group is markedly lower than 47.8% (11/23) in CAR-T group (P=0.001). The associated factors with PTR in this study is cytokine release syndrome (CRS) grade. Then we found the significant elevated cytokine levels in CAR-T group. By Mann-Whitney U test, the mean levels of IL-6 in PTR group versus non-PTR group were 6.58 vs 17.91 (p<0.001). Serum mean levels of IL-10 in PTR group versus non-PTR group were 7.58 vs 16.82 respectively (p=0.001) while serum levels of IFN-γ were 6.58 versus 17.91 respectively (P<0.001). With the decline of cytokines, the 12h CCI of partial patients with PTR will be increased than before, some patients even recover to normal platelet transfusion increment. The rate of PTR in patients undergoing CAR-T therapy was much higher than those who received chemotherapy only. CRS was the only independent risk factor associated with PTR after CAR-T treatment. High serum levels of cytokines (IL-6, IL-10 and IFN-γ) during CRS might contribute to is PTR.

Disclosures
No relevant conflicts of interest to declare.
Safety and Utilization of Midline Catheters in the Management of Acute Myeloid Leukemia: A Single Center Experience
Authors: Torregrosa Diaz Jose Miguel , Gardeney Helene , Gallego Hernanz Maria Pilar , Desmier Deborah , Leleu Xavier , Rammaert Blandine 
Published: November 2019

BACKGROUND: Tunneled central venous catheters (TCVCs) are used in cancer patients for irritant or vesicant infusion such as chemotherapy or antibiotics. However, they can be associated which significant complications. Midline catheters (MCs) are peripheral intravenous access devices that ends in a large peripheral vein, easier to install and which may reduce the need for TCVCs. Therefore, the aim of this study is to assess the rate of adverse outcomes with both kind of catheter, in hospitalized patients for the treatment of acute myeloid leukemia.
METHODS: We retrospectively compared the use and outcomes of TCVCs and MCs at our institution between December 2017 and December 2018. Data were collected using electronic medical records. Both devices were used indistinctly among our hospitalized patients for chemotherapy, antibiotics and parenteral nutrition. The following complications were recorded: catheter related bloodstream infection (defined as documented bacteriemia and differential time to positivity pointing to TCVC as main source), local signs of infection (such as erythema or inflammation) at insertion site, thrombosis which required an anticoagulant treatment, extravasation and local bleeding at insertion site.
RESULTS: Through this period, 39 TCVCs and 50 MCs were used among 56 different patients. As expected duration of the device was shorter for MCs (mean 24days) as they were ablated on discharge, compared to TCVCs (mean 32.5 days). Median aplasia duration (defined as polymorphonuclear neutrofils count in peripheral blood <500/mm3) of each chemotherapic episode was 13 days [0 - 55 days] for patients harboring a TCVCs and 17 [0 - 46] for patients with MCs, with no statistically-significant difference. Main complications were infection related, TCVCs being more prone to have positive bloodstream cultures [20 (52.6%) vs 15 (29.4%), p= 0.027] and local signs of infection [7 (18.4%) vs 1 (2%); p=0.019] than MCs (and probably related to length of stay).
No extravasations were found in any group. As expected local bleeding was more frequent inTCVCs [6 (15.8%) vs 1 (2%); p=0.039].
Whole complications (infection, thrombosis and bleeding) were more frequent among TCVCs (n=35) than among MCs (n=18) even when reported to the length of the catheter (1.08 complications/day in TCVCs and 0.75 complications/day in MCs).
SUMMARY/CONCLUSION: MCs insertion -quick and easy- makes them user-friendly for hematologists and can be safely used among acute leukemia patients.

Disclosures
Leleu: Oncopeptide: Honoraria; Carsgen: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Sanofi: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; BMS: Honoraria; Merck: Honoraria; Karyopharm: Honoraria.
A Hydrogel Based 3D Culture System for Hematopoietic Differentiation of Induced Pluripotent Stem Cells
Authors: Sidhu Ishnoor , Barwe Sonali , Kolb E. Anders , Kiick Kristi Lynn , Gopalakrishnapillai Anilkumar 
Published: November 2019

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent cells (iPSCs) holds a great potential in development of cell therapies, modeling hematological malignancies, and testing new drugs. Hematopoiesis is regulated by bone marrow (BM) microenvironmental factors such as BM architecture, and cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs rely on cell-cell interaction (embryoid body formation and feeder cell co-cultures) or cell-matrix interaction (ECM coated dishes). Generation of a 3-dimensional (3D) culture environment attempts to capture both cell-cell and cell matrix interactions in studying the differentiation of the iPSCs. This study reports development of a 3D culture system for hematopoietic differentiation of iPSCs.
iPSC colonies were encapsulated in 3D Matrigel or polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) and cultured in feeder-free cell culture and maintenance medium (mTeSR1™, Stem Cell Technology). There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig 1A). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG compared to Matrigel, whereas colonies in 2D were more diffused. Viability of the encapsulated colonies was evaluated in situ using cytotoxicity kit. The 3D encapsulation did not have an adverse effect on the viability and growth of iPSCs (Fig 1B). The encapsulated iPSCs maintained the pluripotent phenotype in 3D as assessed by TRA-1-60 staining (Fig 1C).
To test the efficiency of these 3D model systems to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff™ Hematopoietic Kit (Fig 1D). Following differentiation, the single cells were collected and immunophenotype analysis was performed by flow cytometry. Above 50% population stained positive for HSPC surface marker CD34 both in PEG hydrogel and Matrigel (Fig 1E).
To test the robustness of the 3D system in inducing hematopoietic differentiation, we encapsulated the isogenic trisomic 21 (Tri21) iPSCs and subjected to hematopoietic differentiation and flow cytometry analysis as described above. As reported previously in 2D system (Banno et al., 2016, Cell Reports), Tri21 iPSCs showed higher percentage of hematopoietic commitment into erythroid and megakaryocytic lineages compared to disomic 21 (Di21), as characterized by the presence of respective surface markers (Fig 1F).
In conclusion, 3D culture system was suitable for inducing hematopoietic differentiation of iPSCs in isogenic disomic and trisomic lines. The 3D model can be used to generate patient-specific HSPCs.
Figure 1

Disclosures
No relevant conflicts of interest to declare.
Thrombopoietin Has Protective Effect in Neonatal Hypoxia-Ischemia Rat Model
Authors: Li Liang , Yang Liuming , Xin Hongwu , Chong Beng H , Yang Mo 
Published: November 2019

Thrombopoietin (TPO) is a growth factor for the megakaryocytic lineage. The expression of TPO and TPO receptor (c-mpl) in the central nervous system (CNS) and the role of TPO in neural cells and brain damage models were investigated. Our results showed the expression of TPO in human cerebral hemisphere, cerebellum, cerebrospinal fluid and blood plasma. We found that TPO had a protective effect in hypoxic-ischemic rat model, as indicated by the increased ipsilateral brain weight and neuron density in a neonatal rat model of hypoxic-ischemic brain damage. Recoveries of sensorimotor functions and histopathology were observed in these animals that received TPO. In addition, TPO could promote C17.2 cells proliferation by activating PI3K/Akt signaling pathway, and the proliferation could be reduced to nearly basal level by the pre-treatment with LY 294002. The phosphorylation of AKT, which is a hallmark of activation of each molecule was significantly enhanced after the treatment with TPO in the cells, peaking at 30 min after stimulation with TPO. TPO was also found to have an anti-apoptotic effect which mediated via Bcl-2/BAX and suppressing the mitochondrial membrane potential. Results showed the increased level of Bcl-2 and decreased level of BAX were in the time-dependence manner (0, 5, 15, 30 and 60 mins) in these cells. In addition, the mitochondrial membrane potential was significantly decreased by adding 100 ng/ml TPO. Our results indicated that TPO have neural protective effects.

Disclosures
No relevant conflicts of interest to declare.