bluuebunny/crossref_metadata_2025 huggingface dataset:
PNH Clone Size By Flow Cytometry and Its Correlation with PIG Gene Mutation
Authors: Jeong Dajeong , Park Hee Sue , Choi Seongmin , Seo Heewon , Kim Sung-Min , Im Kyongok , Yun Jiwon , Kim Jung-Ah , Hwang Sang Mee , Kim Kwangsoo , Kim Ju Han , Lee Dong Soon
Published: October 2019

Background: Routine diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is based on flow cytometric measurement of PNH clone in RBC and granulocyte with sensitivity of 1%, and recent introduction of high-sensitive flow cytometry (FCM) makes it possible to detect 0.01% clone. However, the clinical significance of small PNH clone has not been elucidated, so even the necessity to treat or not is controversial. We investigated whether quantitative results of PNH clone size measured by FCM correlate with mutant burden of PIG gene.

Methods: A total of 44 specimens from 20 patients whose PNH clone size was >1% in either RBC or granulocyte by routine FCM were enrolled (classical PNH n=10, AA/PNH n=8, MDS/PNH n=2). To detect small quantity of cells with PIG gene mutation, we performed ultra-deep sequencing (average depth 3000X) for PIGA, PIGM, PIGT, and PIGX genes on these 44 consecutive specimens.

Results: Sixteen patients (80.0%) were found to harbor PIG gene mutations: 15 patients had PIGA mutation and 1 patient had PIGM mutation. Granulocyte PNH clone size and variant allele frequency (VAF) of PIG gene mutation showed higher correlation (Spearman's r=0.73, p=0.0002) than that of RBC (Spearman's r=0.61, p=0.0073). All patients harboring PIG gene mutation showed more than 10% PNH clone by FCM. In contrast, 4 patients who did not have PIG gene mutation showed less than 10% PNH clone by FCM.

Conclusion: Considering the mechanism of PNH development, the presence of PIG gene mutation is a definitive evidence supporting PNH diagnosis. PNH granulocyte clone size more than 10% by FCM seems to be clinically significant in relevance to PIG gene mutation. We suggest that neither high sensitivity FCM nor the detection of PIG gene mutation is a requisite for routine clinical diagnosis of PNH.

Disclosures

No relevant conflicts of interest to declare.
Perioperative Management of Hemophilia a Using Recombinant Factor VIII in Patients Undergoing Major or Minor Surgery
Authors: Okamoto Atsushi , Yamamoto Kenta , Eguchi Go , Kanai Yoshitaka , Yamaguchi Terufumi , Maeda Yasuhiro
Published: October 2019

[Introduction]

It has been known that Hemophilia needs careful management from the bleeding tendency in the perioperative period. In this study, we performed endoscopic nasal pituitary adenomatectomy for growth hormone-producing pituitary adenoma in patients with hemophilia A. There were no reports using efraloctocog alpha (ELOCTATE®, Bioverativ, Cambridge, MA, USA, rFVIIIFc) for perioperative management in major surgery, intracranial surgery, for a hemophilia A patient.

[Case]

A 28-year-old man admitted to our Hospital because of endoscopic nasal pituitary adenomasectomy for growth hormone-producing pituitary adenoma. rFVIIIFc was used as a management of hemophilia A at the perioperative period. And we created a regimen for administration of rFVIIIFc regarding the guidelines for hemostasis treatment for hemophilia patients without inhibitors (revised 2013) published by the Japan Thrombohemorrhagic Society. In our hospital, the results of factor VIII activity can not be obtained as an emergent examination, so we used APTT as an indicator.

[Result]

The surgery was terminated with 150 ml of intraoperative bleeding volume that was almost the same amount as expected bleeding volume. Despite a risk of nasal bleeding after surgery was expected, only a small amount of nasal bleeding was occurred twice. The course of factor VIII activity was also good with APTT.

[Conclusion]

We concluded that rFVIIIFc may be available reagent in perioperative management of intracranial surgery with hemophilia A without inhibitors. The advantage of perioperative management by the bolus administration method (=BI method, This involves repeated administration of a bolus injections.) using half-life extended drugs is that these drugs need to be administered intravenous injection only once a day, and such a treatment protocol is easy to perform at a hospital. Furthermore, the BI method is also economical as it reduces the amount and thus the cost of the drug, as compared with the continuous administration method (=CI method, A syringe pump continuously administers coagulation factors after an initial bolus administration.) using the existing coagulation factor preparations. For perioperative management using extended half-life drugs, we consider that further case studies are necessary to prepare dosing regimens. However, such drugs have the potential to impact not only periodic replacement therapy, but also perioperative management in hemophilia patients. As mentioned above, we feel that the extended half-life drugs have the potential to significantly impact hemophilia treatment.

Disclosures

No relevant conflicts of interest to declare.
Hematopoietic Stem and Progenitor Cell Proliferation and Differentiation Requires the Trithorax Protein Ash2l In Vivo
Authors: Chatain Nicolas , Lüscher-Firzlaff Juliane , Kuo Chao-Chung , Bochyńska Agnieszka , Ullius Andrea , Denecke Bernd , Costa Ivan , Koschmieder Steffen , Lüscher Bernhard
Published: October 2019

Gene transcription is controlled by post-translational modifications of core histones. Methylation of lysine 4 of histone H3 (H3K4), together with acetylation of H3K27, is closely associated with open chromatin and gene transcription. H3K4 methylation is catalyzed by KMT2 lysine methyltransferases that include the mixed-lineage leukemia 1-4 (MLL1-4) and SET1A and B enzymes. For efficient catalysis all six require a core complex of four proteins, WDR5, RBBP5, ASH2L, and DPY30.

In a newly-generated pIC-inducible Mx1-Cre mouse model, we report that targeted disruption of Ash2l in the hematopoietic system results in death of the mice due to a rapid loss of mature hematopoietic cells in the bone marrow and the spleen, associated with a significant reduction of the RBC, WBC and platelets. However, Lin-Sca1+Kit+ (LSK) cells, which are highly enriched in hematopoietic stem and multi-potent progenitor cells, were significantly increased in the bone marrow (18-fold increase). This increase was associated with a 15-fold loss of the myeloid progenitor (MP) population (p < 0.001). In detail, total cell number of granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythrocyte progenitors (MEPs) and common-myeloid progenitors (CMPs) was significantly reduced, while the relative proportion remained essentially unchanged. In LSK cells, Ash2l loss interfered with H3K4 methylation and resulted in broadly deregulated gene expression, including down-regulation of mitosis-associated genes. As a consequence, LSK cells accumulated in the G2/M-phase of the cell cycle and were unable to proliferate and form colonies in semi-solid medium. Lentiviral complementation with human ASH2L in the lineage-negative population partly but not entirely restored colony forming capacity, suggesting that ASH2L-mediated shaping of hematopoietic differentiation is a very early process. In addition, we demonstrated that those genes that harbor H3K4me3 and H3K27ac marks at their promoters were preferentially transcriptionally down-regulated. In contrast, up-regulated genes were characterized by low H3K4me3 and H3K27ac. Thus, these findings suggest that H3K4me3 and H3K27ac are not essential for transcription of all genes and that other, so far unidentified chromatin marks, allow recruitment of the RNA polymerase complex and transcription. Together, our data identify Ash2l as a novel essential factor for balanced gene expression and for hematopoietic stem and multi-potent progenitor cell physiology. These data may have important implications for future therapies of hematopoietic malignancies, including epigenetic targeting of malignant stem cells.

Disclosures

No relevant conflicts of interest to declare.
Inhibition of DOT1L and PRMT5 Promotes Anti-Tumor Activity in a Human MLL Leukemia Model Induced By CRISPR/Cas9
Authors: Secker Kathy-Ann , Keppeler Hildegard , Duerr-Stoerzer Silke , Schmid Hannes , Schneidawind Dominik , Hentrich Thomas , Schulze-Hentrich Julia , Mankel Barbara , Fend Falko , Kanz Lothar , Schneidawind Corina
Published: October 2019

MLL rearrangements play a crucial role in leukemogenesis. They are associated with a poor prognosis and new treatment strategies are urgently needed. Here, we used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4/-AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient leukemic cells faithfully mimicking the nature of the disease. Thus, it serves as ideal fundamental basis for pharmacological studies.

The catalytic activity of disruptor of telomeric silencing 1-like histone methyltransferase (DOT1L) is critical for the transforming potential of MLL oncogenes. Therefore, inhibition of DOT1L deals as one specific therapeutic approach for MLL-rearranged leukemias currently tested in clinical trials. However, the previous success was limited by restricted response warranting further investigation of DOT1L combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines in vitro and in MLL mouse models. Here, we used DOT1L (EPZ004777) and PRMT5 (EPZ015666) inhibitors alone or in combination in our human MLL-rearranged model demonstrating a dose-dependent reduced proliferation, impairment of cell cycle, upregulation of differentiation marker CD14 and apoptosis measured by flow cytometry and downregulation of known MLL target genes determined by qPCR. Strikingly, the combination of both compounds led to complementary anti-tumor effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias.

Disclosures

No relevant conflicts of interest to declare.
Incidence of Silent Thrombosis in Myeloproliferative Neoplasm Patients below 60 Years : Single Center Egyptian Study
Authors: Mattar Mervat , Nassef Sahar , El Husseiny Noha M , Morad Mohamed Abdel Kader , Salah Marwa , El Masry Mohamed Roshdy , Abdul Gawad Ahmed
Published: October 2019

Background : Identification of JAK-2 mutation even in absence of myeloproliferative disorders was found to be related to venous thromboembolism occurrence. The aim of this work is to screen myeloproliferative neoplasm ( MPN) patients for venous thrombosis and study its correlation with both JAK 2 allele burden and with symptoms the patients presented with. Methods: We enrolled 73 cases with JAK2 positive MPN in the period between August 2015 till Feb 2017. All patients were screened for thrombosis in venous system in neck, upper and lower limbs, superior and inferior Venae Cavae and portal and mesenteric venous systems system using color Doppler Ultrasound. Results: 53 patients (72.6%) were below 60 years. Forty even (64.4%) were females and 26(35%) were males. Twenty two (30%) of cases were Essential Thrombocytosis (ET), 35(248%) were Polycythemia Vera (PV) and 16 (22%) were Myelofibrosis (MF). Twenty seven venous thrombotic attacks were reported in twenty two patients (30.1%). Seventeen patients (23%) had mesenteric and portal vein thrombosis,six patients had iliofemoral (8%) and 4 (5%) had combined lower limb and portal thrombosis. Eight patients (10.8%) had active thrombosis at screening. Only three (4%) patient were symptomatising with pain during screening. Sixteen patients with thrombosis were below 60 (30% of those below 60 years) and 6 were above sixty years (also 30% of those above sixty years). Correlation analysis between JAK2 allele burden and thrombosis was not statistically significant (r=0.3 ,p value=0.5). However, JAK 2 allele burden was statistically higher in those above sixty years in both thrombosed and non-thrombosed cases in comparison to those below sixty years (p= 0.03, 0.017 respectively). The incidence of pruritis (p =0.02) and of abdominal pain (p=0.039) was significantly different between thrombosed and non-thrombosed cases. Comparison of 8 cases with active thrombosis to old thrombosis revealed no statistical difference in the MPN10 score (p>0.05). Conclusion: We recommend routine screen for venous thrombosis in any case of MPNs once diagnosed and screening for MPNs in any case with venous thrombosis . Further research in MPN group age below 60 years of age is highly recommended.

Disclosures

No relevant conflicts of interest to declare.
Long-Term Outcomes of De Novo Acute Myeloid Leukemia (AML) Patients (pts) with Life Threating Complications
Authors: Bazhenov Aleksei , Parovichnikova Elena N. , Galstyan Gennadiy M. , Troitskaya Vera V. , Gribanova Elena O. , Savchenko Valery G.
Published: October 2019

Background. Induction of remission in pts with AML is often associated with the life-threating complication and ICU admission. The long-term outcomes of these AML pts discharged from ICU are unknown. The aim was to estimate the long-term outcomes of de novo AML pts required ICU admission due to life-threating complications during induction of remission.

Methods. All de novo AML pts, younger than 60 y.o, median age 34 (17-60 yo), from 2013 to 2016 were enrolled in the study. All pts were divided into 2 groups: pts who required ICU admission during induction chemotherapy due to life-threating complications and pts who didn't require ICU admission during induction chemotherapy. Overall survival (OS) and disease free survival (DSF) were assessed by the Kaplan - Meier method, log rank value p<0.05 consider as significant. Univariate analysis was performed with χ 2 tests or Fisher's exact tests for categorical variables to find an independent ICU mortality predictor.

Results In total, 73 AML pts included in study, 26 (36%) of them were admitted in ICU during induction of remission (tab. 1).

ICU pts had more advanced AML. Reasons for ICU admissions were acute respiratory failure (ARF) (62%); neurological events (16%); septic shock (SS) (16%); cardiac arrhythmia (7%). ICU survival rate was 69%. 8 pts died in ICU (5 SS and 3 ARF). Needs for mechanical ventilation, vasopressors and ≥2 organ dysfunctions were independent predictors (p<0.05) of ICU mortality. 18 of 26 (69%) pts received chemotherapy during their stay in ICU. Modifications of chemotherapy were required in 46% of ICU pts and in 36% in non-ICU pts (p>0.05). There were differences in 30-day OS between ICU and non-ICU pts (69% vs. 0%, p=0.001). However, a landmark analysis for long term OS for patients who survived the first 30 days of treatment were the similar in ICU and non-ICU groups (p=0.946)( picture1, A). DSF also were the similar in ICU and non-ICU groups (p=0.946) (picture 1,B)

Conclusion. During remission induction 36% of AML pts needed ICU admission due to life-threating complications. Mortality rate in ICU is high (31%). However, after discharging from ICU AML patents had the similar long-term outcomes as well as non-ICU AML pts.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Platelet (hem)ITAM Signaling Mediated Cancer-Associated Venous Thrombosis
Authors: Wang Xia , Wu Jingchang , Gu Haidi , Qian Jun , Ruan Changgeng , Zhao Yiming
Published: October 2019

Background-Patients with cancer are at a high risk of venous thromboembolism (VTE) and underlying mechanisms are unclear. Recent studies have shown that podoplanin (PDPN) expressing highly in primary brain tumors is associated with increasingly risk of VTE by activate platelets. The C-type lectin-like receptor 2(CLEC-2) is a type II transmembrane protein expressed on the surface of platelets and PDPN is the only known endogenous ligand for it. CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif ((hem)ITAM).

Objective: We tested the hypothesis that the platelet (hem)ITAM signaling is involved in the development of VTE in patients with malignant tumors, and targeted inhibitors may reduce thrombosis and improve prognosis in patients with cancer.

Materials and Methods-We selected two human malignant cell lines (NCI-H226 and C8161) expressing high levels of PDPN as models to study the role of the platelet (hem)ITAM signaling in cancer-associated venous thrombosis in vitro and in vivo.

Results and Conclusions-Our results showed that both types of cell lines expressing PDPN triggered platelet activation via CLEC-2 in vitro, which could be abrogated by an anti-PDPN antibody SZ-168 or syk inhibitor R406. Further, in vivo study showed that injection of NCI-H226 or C8161 in two mouse models of venous thrombosis activated platelets, increased platelet counts and enhanced thrombosis. These results suggested that platelet activation induced by PDPN was correlated with hypercoagulability, contributed to thrombosis in cancer patients. Importantly, PDPN enhanced thrombosis was reduced in mice treated with SZ168 or R406. Immunohistochemical staining using anti-PDPN antibody was also performed in pulmonary squamous cell carcinoma specimens of 166 patients. During up to 4-year-follow-up, 20 (12.05%) patients developed VTE. 105 tumor specimens stained positive for PDPN (27 high expression, 43 medium expression, 35 low expression). High PDPN expression was associated with an increased risk of VTE and poor prognosis, independently of age, sex and tumor type. In this study, we found that PDPN expression in tumors induced platelet aggregation and was associated with a high risk of VTE via platelet (hem)ITAM signaling. Our data also suggested that SZ168 inhibited PDPN-induced platelet aggregation in vitro and decreased the incidence of VTE in mice. In conclusion, our research demonstrated novel insights into the pathogenesis of cancer-associated venous thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Novel MEIS1-FOXO1 Fusion Gene in a Case of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia
Authors: Jiang Chuang , Qian Jiabi , Hao Wenge , LIU Wei , Shen Shuhong , Jiang Hua , Zhang Hui
Published: October 2019

Background: Thanks to the total therapy and systemic basic-translation research, the overall survival rate in children with acute lymphoblastic leukemia (ALL) has dramatically improved to almost 90% over these past few decades. FOXO1 gene belongs to the forkhead family of transcription factors, which play roles in myogenic growth and differentiation. Translocation of FOXO1 with PAX3 has been reported in pediatric alveolar rhabdomyosarcoma. In B-cell precursor ALL, two cases with FOXO1 fusions have been identified already, while its function on ALL remains unknown. Here, we report a novel MEIS1-FOXO1 fusion gene in a case with B-ALL. Methods: Flowcytometery, karyotype, RT-PCR and fluorescence in were employed, MEIS1-FOXO1 was identified as novel fusion gene in a case of pediatric BCP-ALL. Using IL-3 dependent BaF3 cells as study model to test the leukemia transformation potential of MEIS1-FOXO1. Results: A novel MEIS1-FOXO1 fusion was identified in one cease of pediatric B-ALL. Panel next generation sequencing (NGS) showed that the leukemia clone had concurrent NRASG12D, TP53R273H, WHSC1E1099K, ABCC1R1166X, PHGR1H37P, HOXA3P219L and DSTP4606L somatic mutation. This patient was enrolled in CCCG-ALL2015 clinical trial (ChiCTR-IPR-14005706) and achieved completed remission and low minimal residual disease (MRD) level (MRD<0.01%) at day 19 from induction therapy. Functional study showed that MEIS1-FOXO1 fusion gene can potentiate BaF3 cells growth independent of IL3 supplement, as compared to those without MEIS1-FOXO1 fusion transduction. In the meanwhile, we have found that MEIS1-FOXO1 fusion gene can drive cells into S-phase with concurrent decreased G0/G1 phase, which might be its oncogenic role in leukemogenesis. Using qPCR methods, we have found that MEIS1-FOXO1 fusion gene altered the cell cycle related genes expression. Conclusions: Integrating the FOXO1-fusion reports, our data have added more evidence to underline the role of FOXO1 deregulation in the pathogenesis of acute lymphoblastic leukemia. Novel fusion of MEIS1-FOXO1 can potentiate B-ALL via cell cycle entry. Detailed mechanisms involved into the MEIS1-FOXO1 should be further investigated.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Platelets Promote Activation of the Complement System in Ovarian Cancer
Authors: Gonzalez Pagan Omayra , Cho Min Soon , Afshar-Kharghan Vahid
Published: October 2019

Platelets promote metastasis and growth of ovarian cancer. We have shown that platelets extravasate into the tumor microenvironment (TME) and increase proliferation and epithelial-mesenchymal transition (EMT) in ovarian cancer cells. We have also shown that activation of the complement system in TME of ovarian cancer enhances tumor growth. Ovarian cancer cells secrete complement proteins that upon activation in the TME increase proliferation of cancer cells and promote EMT via an autocrine pathway. The activators of the complement system in the TME have not been identified. We have demonstrated that upon activation platelets activate the complement system on their surface. In the current study, we examined whether extravasated platelets inside tumors contribute to the complement activation in the TME.

1) We examined the effect of antiplatelet reagents on platelet extravasation into TME, using murine models of ovarian cancer. Tumors induced by injection of ovarian cancer cells into the peritoneum of Nu/Nu mice were resected after 6-8 weeks and the number of extravasated platelets was determined by immunostaining tumor sections and counting the number CD42 (GPIb) positive cells that were outside the blood vessels (CD31 positive). We found that platelet extravasation is an active process and platelet inhibition by aspirin or ticagrelor reduces the number of extravasated platelets. Furthermore, P2Y12 deficient platelets extravasate less than normal platelets. In all of these experiments, the number of extravasated red blood cells were significantly less than extravasated platelets and was not affected by the inhibition of platelets.

2) We examined the effect of platelet inhibition on the activation of the complement system in the TME. We immunostained resected tumors from aspirin- or ticagrelor-treated tumor-bearingmice and from P2Y12-deficient tumor-bearingmice for the endproductof complement activation (C5b-9 or membrane attack complex). Inhibition of platelet function by aspirin or ticagrelor,or the presence of hypoactive platelets in P2Y12 deficient mice reduced the amount of C5b-9 deposited in the tumors induced in murine models of ovarian cancer.

Our result showed that complement activation in the TME is at least partially dependent onextravasated platelets. We propose that platelets in addition to directly increasing proliferation of ovarian cancer cells, also enhance tumor growth by activating the complement system in the vicinity of cancer cells. Our study links platelets, complement activation, and ovarian cancer growth; and raises the possibility of using antiplatelet reagents and complement inhibitors as novel synergisticanti-tumor reagents in ovarian cancer.

Disclosures

No relevant conflicts of interest to declare.
Functional Characterization of Epigenetic Modifiers of Demethylating Therapy in AML
Authors: Redhaber Desiree Melanie , Phely Laurent , Ehrenfeld Sophia , Veratti Pia , Schäfer Silvia , Mitschke Jan , Beckert Jessica , Shoumariyeh Khalid , Duyster Justus , Miething Cornelius
Published: October 2019

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults, and remains a difficult to treat disease, especially in elderly patients. AML arises in hematopoietic stem and progenitor cells and frequently carries alterations in genes involved in epigenetic regulation. Furthermore, azacitidine (AZA) and decitabine (DAC), two nucleoside analogs inhibiting DNA methylation (DNMTi), have shown clinical efficacy in the therapy of myelodysplastic syndrome (MDS) and AML, highlighting the importance of epigenetic regulation in AML. Up to now, the mechanism of action of AZA and DAC in AML has not been fully elucidated, and reliable biomarkers of therapy response to these drugs do not yet exist.

To identify epigenetic response modifiers towards AZA and DAC treatment, we conducted a pooled shRNA-based screen in three human leukemia cell lines using an inducible custom shRNA library targeting more than 660 different genes involved in epigenetic pathways. After selection of retrovirally infected cells, shRNA expression was induced and a fraction of cells were treated with either AZA, DAC or left untreated. 10 days after shRNA induction, genomic DNA was extracted and deep-sequenced to identify genes conferring a selective advantage/disadvantage under these conditions. By comparing shRNA representation changes between the differentially treated groups, we identified multiple candidate genes sensitizing or protecting leukemic cells to/from AZA and DAC treatment. Notably, the DNMT1 gene itself expectedly emerged as an important sensitizer to DNMTi, as did other CXXC domain-containing genes binding to (un-)methylated CpG domains. Another prominent set of genes in the pooled analysis belonged to the DNA damage pathway, including genes such as PARP1 and BRCA1. The identified candidate genes were validated and confirmed in more than 80% when tested in single shRNAs competition assays. Their impact on DNA methylation and gene expression is currently being assessed, with ongoing experiments focusing on the mechanistic role of some of the validated target, as well as the clinical potential for combinatorial therapies. Thus, using an unbiased functional genetic approach, we identified multiple genes including CpG binding proteins and members of the DNA damage pathway as important modulators of DNMTi response. Our results will increase our understanding of the molecular mechanisms driving DNMTi efficacy, and may help to identify new biomarkers and novel targets for combinational therapy in AML.

Disclosures

No relevant conflicts of interest to declare.
Endothelial Cell-Derived Extracellular Vesicles Mitigate Radiation-Induced Hematopoietic Injury
Authors: Piryani Sadhna O. , Jiao Yiqun , Kam Angel Y.F. , Liu Yang , Vo-Dinh Tuan , Chen Benny J. , Chao Nelson J. , Doan Phuong L.
Published: October 2019

Hematopoietic stem cells (HSCs) reside in specialized microenvironments and adjacent to endothelial cells (ECs), from which they receive regulatory cues. Following injury from radiation or chemotherapy, transplanted ECs can facilitate hematopoietic regeneration. Since these ECs do not engraft in the marrow, we hypothesize that ECs facilitate hematopoietic regeneration through secretion of extracellular vesicles (EVs). EVs are becoming recognized as a mode of cell-to-cell communication through transfer of their cargo of nucleic acids and proteins. Using ultracentrifugation, we visualized these EVs by transmission electron microscopy and measured their size to be a mean size of 160 nm (range 121-217 nm, n=18). Using flow cytometric analysis with Syto13, we demonstrate that these EVs contain nucleic acids in 30% of the particles. Quantitative real-time PCR show that these EVs express CD31 and VECadherin, and not Fibroblast Specific Marker 1 (FSP1), indicating EVs bear EC markers. At day 7 following 300 cGy, C57BL/6 cKit+Sca-1+Lineage- (KSL) cells cultured with EVs demonstrated total cell expansion and colony-forming unit cells (CFCs) compared to cultures with cytokines alone (*p< 0.0001 and 0.01, respectively, n= 3-6/group). Following myelosuppressive, sub-lethal radiation dose of 500 cGy, C57BL/6 mice treated with EVs on days 1-4 display increased marrow cellularity (*p= 0.003, n= 2-3/group), preserved vascular endothelial cells by mouse endothelial cell antigen immunohistochemistry (*p=0.03, n=2-4/group), CD150+CD48-KSL cells (*p= 0.03, n= 5/group), and increased CFCs (*p= 0.03, n= 6/group) compared to saline-treated mice. No differences were detected in these parameters when EV-treated mice were compared to mice treated with primary, cultured C57BL/6 marrow ECs. Following lethal-dose irradiation of 800 cGy, mice that received EVs on day 1-4 display prolonged survival compared to saline-treated mice (*p= 0.008 by Log-rank Analysis, n= 10/group). At day 30 following irradiation, 5 of 10 mice were alive in the EV-treated group compared to 0 of 10 in the saline-treated group. No differences in survival were observed between EV-treated and EC-treated mice (p= 0.4). Treatment of irradiated C57BL/6 hematopoietic stem/progenitor cells (HSPCs) with EVs generated from a genetically distinct strain (BALB/c mice) showed similar levels of cell expansion and CFCs compared to treatment of HSPCs from syngeneic EVs. Our findings show that syngeneic or allogenic EVs could be cell-derived therapy to deliver physiologic doses of nucleic acids and growth factors to hematopoietic cells to accelerate hematopoietic regeneration.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Exon Skipping of AIMP2 and Lymphomagenesis
Authors: Kim Doyeun , Song Seulki , Kim Dae-Yoon , Kim Dongchan , Kwon Nam Hoon , Kim Byung-Su , Lee Hyun Jung , Kim Hyo Jung , Lee Jung Lim , Yoon Sung-Soo , Jeon Yoon Kyung , Kim Sunghoon , Koh Youngil
Published: October 2019

ARS-interacting multifunctional protein 2 (AIMP2), the component of aminoacyl-tRNA synthase complex, acts as a potent tumor suppressor in conjunction with p53. The second exon deficient AIMP2 generated by alternative splicing mechanism (AIMP2-DX2) induces loses its important role as a signaling modulator. Previously, it predisposes mouse to increase lung cancer, but it did not induce tumorigenesis by itself.

To further validate the oncogenic role of AIMP2-DX2 in mouse model, we generated inducible transgenic mouse which express DX2 in doxycycline dependent manner. Unexpectedly, we report the late onset spontaneous lymphoma development instead of lung cancer in this model. Polyclonal expansion of pre-B cells and infiltration to multi-organ, especially kidney, was major events. More importantly, switching off the transgene by doxycycline removal reduced pre-B cell hyperplasia by inducing apoptotic cell death. Destabilization of p53 and dysregulation of cell cycle gene by DX2 overexpression underlined the increased lymphoid cell proliferation. Coincident with the results, in International Cancer Genome Consortium (ICGC) data analysis, AIMP2-DX2 expression was the highest in Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) among various cancer subtypes. When analysis was performed using RNA sequencing data of various B-cell lymphoid malignancies, germinal B-cell origin (GCB) DLBCL, B-acute lymphoid leukemia and BL showed completely different gene expression pattern according to the AIMP2-DX2 expression status suggesting a distinct disease entity. AIMP2-DX2 expression in DLBCL cell line and tissue samples was validated and regulation of DX2 in lymphoma cell line affected cell viability as well as p53 stability. Lastly, we confirmed expression of AIMP2-DX2 in FFPE tissue of 35 lymphoma patients using RNA in situ hybridization (ISH) with specifically designed probes that capture the exon 1-3 junction of AIMP2 transcript.

In summary, our data demonstrate the causality of AIMP2-DX2 in lymphoma oncogenesis and provide evidence that AIMP2-DX2 is a potential therapeutic target especially in DLBCL and BL.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
LSD1 Inhibitors Induce Fetal Hemoglobin in Primary Human Erythroid Cells
Authors: LE Cuong , Myers Greggory , Habara Alawi , Chui David H.K. , Steinberg Martin H. , Engel James Douglas , Cui Shuaiying
Published: October 2019

Significant amount of clinical literatures have supported that high level of fetal hemoglobin (HbF) improves the disease pathophysiology of β-globinopathies [sickle cell disease (SCD) and β-thalassemia]. Thus, we certainly can treat β-globinopathies by increasing the HbF level in adult erythroid cells.

We originally reported that the lysine-specific histone demethylase 1 (LSD1) plays an important role in the regulation of the fetal γ-globin genes. Inhibition of LSD1 by using RNAi and monoamine oxidase inhibitor tranylcypromine (TCP) in primary human erythroid progenitor cells induces HbF to therapeutic levels. Furthermore, LSD1 inhibitor RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with SCD.

Most recently, we examined thein vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 (including GSK-LSD1, LSD1-C12, LSD1-C76, OG-L002, and S2101) on HbF synthesis and erythroid physiology in SCD mice. There was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment with GSK-LSD1 or OG-L002 in SCD mice. Here, we report the effects of these two inhibitors in primary human erythroid cell derived from peripheral blood CD34+ cells. We isolated CD34+ cells using MACS column and cultured them using the two-phase-culture system. After seven days in expansion phase (phase 1) and three days in differentiation phase (phase 2), cells were treated with different doses of GSK-LSD1 and OG-L002 LSD1 inhibitors along with controls (DMSO, hydroxyurea and TCP) for 3 or 5 days in phase 2 culture. Flow cytometric assays showed that the percentage of HbF positive cells were significantly high when CD34+ cells treated with OG-L002 LSD1 (~50% at 0.1 µM) or GSK-LSD1 (~30% at 0.1 µM) as compared to control DMSO (~20%) after 5 days. These results suggest that GSK-LSD1 and OG-L002 could be two new promising HbF inducers based on LSD1 inhibition. These findings provide additional evidence to support that LSD1 comprises a useful molecular target for possible therapeutic intervention in treating SCD. Further study will be necessary to address the potential therapeutic effects of the compounds in SCD patients.

Disclosures

No relevant conflicts of interest to declare.
"Part of the Ritual": a Qualitative Study of Patient and Physician Perspectives on the Use of Anticoagulation for Improving Pregnancy Outcomes in Obstetrical Antiphospholipid Syndrome
Authors: Skeith Leslie , Rodger Marc A. , Bates Shannon M. , Gonsalves Carol , Taylor Taryn S.
Published: October 2019

INTRODUCTION: Antiphospholipid syndrome is an acquired thrombophilia associated with recurrent pregnancy loss. Treatment with low-molecular-weight heparin (LMWH) and/or aspirin (ASA) during pregnancy to prevent future loss is based on limited trial data with mixed results. Given the clinical equipoise, we sought to understand how patients and physicians navigate the decision-making process for use of antepartum LMWH and/or ASA. This research is needed to inform the feasibility of future research that seeks to resolve the question of treatment efficacy in this patient population.

METHODS: Following constructivist grounded theory (CGT) methodology, we interviewed 10 patients and 10 thrombosis physicians in Ottawa, Canada from January 2017 to March 2018. Patients were drawn from women included in the TIPPS (Thrombophilia in Pregnancy Prophylaxis Study) screening logs and from consecutive women attending a specialty Thrombosis Clinic who met the revised Sapporo/Sydney laboratory criteria and had at least 1 prior late loss or 2 early losses (<10 weeks gestation). Data collection and analysis occurred iteratively, in keeping with CGT methodology.

RESULTS: Our analysis generated three predominant themes, present across both patient and physician interviews, which captured a patient-led decision-making experience: (1) Accepting Uncertainty, (2) Focusing on Safety and (3) Managing High Stakes. Many patients accepted the uncertainty of their situation; they felt hopeful but were also reluctant to expect a better pregnancy outcome. This was echoed in the physician interviews, which emphasized the uncertainty of LMWH/ASA in improving outcomes when counselling patients on their options. Both groups focused on the low-risk nature and safety of these medications. Patients focused on safety to the fetus, with little concern for maternal risk. Finally, patients and physicians acknowledged the high emotional burden and what was at stake: avoiding further pregnancy loss. Patients responded to their situation by taking action (i.e. using LMWH injections became a "ritual") whereas physicians reacted by removing themselves from the final decision and "[leaving] it up to the patient".

CONCLUSION: In this setting of clinical equipoise and in the absence of serious safety concerns, decision-making around antepartum LMWH/ASA was largely directed by informed patient choice. These findings should be considered when designing future research on the role for LMWH/ASA in this population, as it suggests that the perceived benefits of treatment go beyond improving pregnancy rates. Rather, patients seem to derive psychological benefit from the process of taking action, even in the absence of a guaranteed good outcome.

Disclosures

Skeith: Leo Pharma: Honoraria; CSL Behring: Research Funding. Rodger:Biomerieux: Research Funding.
Association of 30 Day Readmission between Psychosocial, Behavioral and Epidemiological Factors in Adult SCD Patients
Authors: Saha Aparna , Poojary Priti , Pandya Dishita , Kumar Abhishek , Nadkarni Girish , Kaur Supreet
Published: October 2019

Background: Sickle-cell disease (SCD) has been identified as a major public health problem by the World Health Organization (WHO) and is one of the most common hematological genetic diseases. In United States, there are approximately 100,000 admissions per year attributed to SCD. Our objective is to provide nationally representative rates for unplanned 30-day readmissions in SCD patients as well as identify reasons for, characteristics and predictors of these readmissions.

Methods: We performed retrospective analysis using the 2013 Nationwide Readmissions Database (NRD) of the Healthcare Cost and Utilization Project (HCUP) from AHRQ. We used chi-square tests, t tests, and Wilcoxon rank-sum tests for descriptive analyses and survey logistic regression to calculate adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for associations with readmissions adjusting for confounders.

Results : Of the 46,842 hospitalizations with SCD as the primary diagnosis for the index admission, 11,787(25.2%) had at least one 30-day unplanned readmission and 30% of these readmissions were within the first week after discharge. Among 11,787 readmissions, 83% of the readmissions were attributed to SCD, including 81.5% due to acute painful crisis. Patients who got readmitted were likely to be younger, with more comorbidity burden including chronic kidney disease, asthma, drug abuse and hypertension. Predictors of readmissions included: age 18-29 years (aOR 1.5, 95% CI 1.3 - 1.6; P<0.01), chronic kidney disease (aOR 1.3, 95% CI 1.1 - 1.6, P <0.01), depression (aOR 1.2, 95% CI 1.1 - 1.4, P<0.01), drug abuse (aOR 1.2, 95% CI 1.1 - 1.4, P 0.04), and discharge against medical advice (AMA) (aOR 1.8, 95% CI 1.5-2.1, P<0.01. AMA discharges and depression were responsible, respectively, for 76% and 23% increases in the odds of 30-day readmission.

Conclusion: About a quarter of patients admitted with SCD get re-admitted within a month of discharge. This calls for identification of modifiable risk factors and timely intervention with safe hospital discharge which is critically important for patient care and national health expenditure.

Disclosures

No relevant conflicts of interest to declare.
VWF Mediates Platelet Activation and Secretion to Promote Osteosarcoma Metastasis
Authors: Zhao Yiming , Wang Qi , Jiang Miao , Ruan Changgeng
Published: October 2019

[Background] As a multimer protein that mediates the adhesion of platelets to vascular surface, VWF has been found to participate in tumor metastasis, and may also be involved in the interaction between platelets and tumor cells. We observed in our clinical studies that the expression of VWF in lung metastatic osteosarcoma tumor is significantly higher than that of in primary osteosarcoma. Based on these observations, we speculate that VWF and platelets may contribute to promoting osteosarcoma metastasis. Thus, this study is to explore the effects of VWF-mediated platelet activation and secretion on the migration and invasion ability of SAOS2 cells with VWF-expressing osteosarcoma cell line SAOS2 as a model, as well as the inhibition of the interaction between VWF and platelets by specific antibodies.

[Methods] SAOS2 cells were stimulated with inflammatory factor PMA and resulted in increased expression of vWF. Then the adhesion of labeled platelets to SAOS2 cells was evaluated under static and flow conditions, respectively. The above processes were intervened with SZ-2(anti-GPIb), SZ-123(anti-VWF) and AVW3 (anti-VWF) mAbs which specifically blocked the vWF-GPIbα pathway. Transwell assay was used to explore the effect of vWF-mediated migration and invasion ability of SAOS2 cells. PDGF-BB, TGF-β and VEGF that could be secreted by platelets to promote tumor cells growth in the co-culture system of platelets and SAOS2 cells were detected by ELISA.

[Results] Under static and flow conditions, SAOS2 cells treated with PMA exhibited significantly higher platelets-adhesion capacity compared to the cells untreated with PMA, and the adhesion could be partially inhibited by SZ-2 and AVW3 mAbs. Furthermore, platelets significantly promoted the migration and invasion ability of SAOS2 cells, and PDGF-BB and TGF-β secreted by platelets were also increased in these processes. Both of SZ-2 and AVW3 mAbs partially decreased the migration and invasion ability of SAOS2 cells.

[Conclusions] vWF were expressed highly in some kind of non-endothelial origin cancer cells, such as SAOS2, which mediates platelet activation and secreting factors such as PDGF-BB and TGF-β to promote tumor cells growth. Activated platelets have a significant promoted effect on the migration and invasion ability of SAOS2 cells and this process can be inhibited by antibodies that specifically block the pathway of VWF-GPIbα.

Disclosures

No relevant conflicts of interest to declare.
Home Care of Acute Leukemia Patients: From Active Therapy to End-of-Life and Palliative Care. a Three-Year Experience of a Single Centre
Authors: Capodanno Isabella , Alfieri Pierluigi , Tamagnini Enrica , Codeluppi Katia , Luminari Stefano , Merli Francesco
Published: October 2019

Purpose:

Since July 2012, the Haematological Home Care (HHC) program was activated in our Institution to improve quality of assistance of patients with haematological malignancies, both in the terminal phase of disease and during active chemotherapy.

Methods:

We conducted a retrospective study to describe the 3-year experience of our HHC program; its efficacy was measured for acute leukaemia patients only analysing number of hospitalizations, number of accesses to the Emergency department and place of death. Our study population was also compared with historical data from a small group of 17 patients with acute leukaemia assisted at home, by their general practitioner, in the previous 3 years, when the HHC program was not available.

Results:

The study group consisted of 44 patients, 36 of whom (82%) needed a palliative treatment, while 8 (18%) had an ongoing active chemotherapy. The mean number of hospitalizations was 0.64 (range 0-7) per patient and the number of emergency department visits was 0.82 (range 0-4) per patient. Place of death was home environment in 51.4% and hospital in 40.5% of patients.

When compared to data of the historical group, HCC program significantly reduced the number of hospitalizations (0.64 vs 2.53 per patient, range 0-9; P 0.001), significantly increased the number of patients who died at home (51.5% vs 6%; P=0.003) and reduced those who died in hospital (40.5% vs 65%; P 0.003).

Conclusions:

Home care for patients with acute leukaemia is feasible, decreases the number of hospitalizations, and increases the number of patients who die in their own home.

Figure. Figure.

Disclosures

Luminari: Servier: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Roche: Consultancy; Sandoz: Consultancy.
ELN Genetic Risk Stratification Not Affect the Mid-Term Survival in Childhood AML: Report from Southern China Childhood AML Group
Authors: Feng Xiaoqin , Li Jian , He Xiangling , Xu Honggui , Zheng Mincui , Wen Hong , He Yingyi , Liu Sixi , Ruan Yongsheng , Li Chunfu
Published: October 2019

OBJECTIVE: To evaluate the survival of children with acute myeloid leukemia (AML) in the ELN genetic risk stratification. To explore the precise treatment and efficacy in childhood AML.

Methods: The information of genetics of 151 newly diagnosed AML children enrolled in the Southern China Childhood AML Collaboration Group from December 2014 to June 2017 were retrospectively analyzed. These patients was divided into low-risk, intermediate-risk and high-risk groups according to the ELN genetic risk stratification. All cases were treated with the Southern China Childhood AML Collaborative Group protocol. The induction of remission was not randomized using FLAG-IDA and DAE regimens for 2 courses of treatment. The consolidation therapy was performed using HAE and MidAC regimens. 20 cases underwent hematopoietic stem cell transplantation. The mediate follow-up time was 18 months. Survival analysis and Log-Rank test were investigated by Graphpad Prism software.

RESULTS: Of 151 children with AML, AML1/ETO positive in 46(30.5%) cases, CBFβ/MYH11 positive in 12(7.9%) cases, MLL gene in 18(11.9%) cases (including MLLT3-KMT2A in 7 cases). There were mutation gene FTL3/ITD in 24(15.9%) cases, ASXL1 mutation In 10(6.6%) cases, NPM1 mutation in 3(2.0%) cases and CEBPA double mutation in 4(2.6%) cases. Chromosome -7 or 7q- was found in 5(3.3%) cases. According to the ELN genetic risk stratification, 60/151(39.7%), 52/151(34.4%) and 39/151(25.8%) of patients were classified as low-risk, intermediate-risk and high-risk respectively. In the cohort of DAE induction, the CR+CRi rate was 95%, 69% and 80% in the low-risk, intermediate-risk and high-risk group. In the cohort of FLAG-IDA induction, the CR+CRi rate was 100%, 95% and 100% in the low-risk, intermediate-risk and high-risk group. The significance of CR+CRi rate only were be seen in the intermediate-risk group( P=0.02). The 2 year overall OS and DFS were 82.56% and 75.74% respectively. The 2-year DFS of the FLAG-IDA and DAE groups were 85.38% and 53.72% respectively (P=0.0004). The 2-year DFS of the LR, IR, and HR groups in the FLAG-IDA group were 89.23%, 92.29%, and 91.67% respectively, and no statistical difference was found among the groups (P=0.909). The 2-year DFS of the chemotherapy group and the hematopoietic stem cell transplantation group were 82.24% and 63.38%. There were no statistical differences between the groups.

Discussion: The FLAG-IDA induction group significantly improved DFS compared with the DAE group. Hematopoietic stem cell transplantation failed to improve the survival of children with AML in the short period in our multicentre study. Our mid-term results show that the ELN genetic risk stratification had not a significant impact on the prognosis of newly diagnosed childhood AML with high-intensity therapy.

Disclosures

No relevant conflicts of interest to declare.