bluuebunny/crossref_metadata_2025 huggingface dataset:
Incidence and Rate of Venous Thrombosis in Medical Inpatients: the Medical Inpatients and Thrombosis (MITH) Study
Authors: Zakai Neil , Callas Peter , Repp Allen , Cushman Mary
Published: October 2019

Abstract 1239

Introduction:

While various government agencies mandate monitoring for in-hospital VT and use of VT prevention strategies, the incidence and risk factors for symptomatic venous thrombosis (VT) in medical inpatients has not been established in general medical populations with confirmation of the VT outcomes. In order to design effective preventive strategies, the scope of the problem must be understood in a real-world setting.

Methods:

Between January 2002 and June 2009 all cases of VT complicating medical admissions were identified using ICD-9 codes and confirmed by medical record review at a 500 bed teaching hospital in the United States. 601 control charts without VT ICD-9 codes were also reviewed. A case of VT was defined as VT occurring as a complication of medical admission (not on admission or a reason for admission) and required definitive imaging or autopsy evidence of VT. The incidence of VT was calculated using the number of admissions and the patient days in the hospital over the same time period cases were ascertained.

Results:

299 cases of VT complicated 64,034 admissions and 871 patient-years of observation. No cases of VT complicated admissions among controls. The occurrence of VT (per 1000 admissions and per 1000 patient-years, 95% CI) is presented in the Table. The overall VT incidence was 4.7 per 1000 admissions or 0.34 per patient-year. The rate was highest on the oncology service (0.65 per patient-year), intermediate on the medical service (0.38 per patient-year) and lowest on the cardiology service (0.13 per patient-year). Upper extremity DVT was common, at 91/180 (51%) of all deep venous thrombosis (DVT). There were 11/91 (12%) PEs among patients with upper extremity DVT, 22/86 (26%) PEs among patients with lower extremity DVT and 20% of patients with distal and proximal DVT had PE. VT occurred on median hospital day 5 (interquartile range 3–10).

Conclusion:

We report the incidence and rates of symptomatic VT in a general medical inpatient population. Prior studies relied on administrative databases and did not confirm all VT cases by record review. In our medical population, no systematic screening for VT occurred and diagnosis required clinical suspicion for VT; thus, rates under-estimate the burden of VT. While oncology patients did not have an increased incidence per admission, their rate of VT was higher when accounting for the time spent in the hospital. For the first time, we report the high incidence of upper extremity DVTs in medical inpatients, which is likely due to increasing use of vascular access devices. The occurrence of VT on median day 5 suggests that strategies to encourage VT risk assessment and provide VT prophylaxis on admission are important for VT prevention.

Disclosures:

No relevant conflicts of interest to declare.
Impact of Interim Hospitalizations on Risk of Venous Thromboembolism (VTE) Recurrence: A Nested Case-Cohort Study
Authors: Ashrani Aneel A. , Farmer Sara A. , Petterson Tanya M. , Bailey Kent R. , Leibson Cynthia L. , Heit John A.
Published: October 2019

Abstract 1241

Background:

Hospitalization (with or without surgery) is a major risk factor for incident venous thromboembolism (VTE); however, the contribution of interim hospitalization to risk of recurrent VTE is unknown.

Objective:

To estimate risk of recurrent VTE related to interim hospitalization by conducting a population-based longitudinal review of provider-linked detailed medical records.

Methods:

We performed a nested case-cohort study. The cohort consisted of all Olmsted County residents with incident VTE 1988–2000 and ≥1 day follow-up. Cases were cohort members with recurrent VTE. Subjects were followed for all interim hospitalizations and warfarin use from incident VTE until earliest of emigration, death, recurrent VTE, or 12/31/2005. Data were analyzed using Cox proportional hazards and time dependent covariates to test for the effects of interim hospitalization and prophylaxis on VTE recurrence, adjusting for gender and age at incident VTE. Analyses were limited to subjects who survived free of death and recurrent VTE for ≥ 6 months.

Results:

Of 1262 incident VTE events (cohort), there were 309 VTE recurrences (cases). We randomly sampled 272 subjects from the cohort and 163 cases. Of the random samples, 210 incident events and 83 cases survived ≥ 6 months free of death and recurrent VTE and form our analysis population. The rate of secondary (interim) prophylaxis was approximately 50% for both incident events and cases, and was not predictive of recurrence (p=0.73). Male gender and interim hospitalization were associated with increased VTE recurrence even after adjusting for age at incident VTE and use of secondary warfarin prophylaxis. The hazard of recurrent VTE was nearly 10-fold higher for subjects with interim hospitalization versus those with none (HR: 9.6; 95% CI: 6.6, 13.8); men had a 1.5-fold increased recurrence rate compared with women (HR: 1.5; 95% CI: 1.1, 2.1).

Conclusions:

Our results, for the first time, show the importance of interim hospitalization as a predictor of VTE recurrence.

Disclosures:

No relevant conflicts of interest to declare.
Association of Gene-Gene Interactions with Venous Thromboembolism (VTE): A Merged/Imputed Genome-Wide Scan/Candidate-Gene Case-Control Study
Authors: Heit John A. , Armasu Sebastian M. , Matsumoto Martha E. , Petterson Tanya M. , Rider David N. , Cunningham Julie M. , Asmann Yan W. , de Andrade Mariza
Published: October 2019

Abstract 1242

Background:

While bivariate interactions between Factor V Leiden, prothrombin G20201A and hereditary deficiency of antithrombin, protein C and protein S compound VTE risk, whether other gene-gene interactions are associated with VTE is largely unknown.

Objective:

To test novel gene-gene interactions for an association with VTE.

Methods:

Genome-wide scan (Illumina 660Q [557,112 SNPs]) and candidate gene (12,551 SNPs in 764 genes within the anticoagulant, procoagulant, fibrinolytic and innate immunity pathways) genotypes from 1270 non-Hispanic adults of European ancestry with objectively-diagnosed VTE (cases; no cancer, venous catheter or antiphospholipid antibodies) and 1302 controls (frequency-matched on case age, gender, race, MI/stroke status) were merged and imputed to about 2.5 million SNPs with MACH using HapMap Phase 2 (60 CEU). We tested all pairwise SNP-SNP interactions using a 2-stage procedure (fast epistasis in the first stage and age, gender, MI/stroke status and state of residence adjusted analysis using logistic regression in the second stage).

Results:

The mean ± standard deviation case and control ages were 54.4 ± 16.2 and 55.6 ± 15.8 years, respectively and 51% were female. Among 2.299×10^12 pairwise interactions tested, 900 million reached a p-value<E-06 and were be tested in the second stage. Of these, 2417, 278 and 22 SNP-SNP interactions reached p-values ≤1E-9, 1E–10, and1E-11 respectively; 121 SNP-SNP interactions reached the Bonferroni statistical significance threshold (≤5.555659E-11). The gene-gene pairwise interactions with p-values≤1E-11 were: (1) chromosome 3 open reading frame 70 (C3orf70) – short-chain dehydrogenase/reductase (SDR family) member 4 (DHRS4), (2) 1-acylglycerol-3-phosphate O-acyltransferase 4 (AGPAT4) - NIMA (never in mitosis gene a)-related kinase 6 (NEK6), and (3) TatD DNase domain containing 2 (TATDN2) – WAS protein family, member 3 (WASF3). No gene function data are available for C3orf70. DHRS4 encodes for a 3-ketosteroid reductase important for active steroid hormone regulation, AGPAT4 is important for signal transduction and lipid biosynthesis, NEK6 encodes for Nek6 which downregulates p53-induced cancer cell senescence, TATDN2 encodes a deoxyribonuclease, WASF3 encodes for a member of the Wiskott-Aldrich syndrome protein family which are key regulators of signal transduction and cytoskeletal reorganization in hematopoietic cells.

Conclusion:

Pairwise gene-gene interaction analyses suggest potential associations between VTE and novel genes for future testing in replication studies.

Disclosures:

Heit: Daiichi Sankyo: Consultancy, Honoraria.
Amount of Platelet Inhibition Produced When Initiating Aspirin Therapy: Comparison of Two Doses 81 Vs. 325
Authors: Schwartz Kenneth A. , Chivu Simona , Davis John
Published: October 2019

Abstract 1250

Introduction:

Patients with occlusive arterial disease are commonly treated with different daily doses of aspirin usually either 81 or 325mg. This treatment practice continues despite studies showing equal protection from future vascular events with daily aspirin doses that exceed 81mg. We evaluated the hypothesis that the amount of platelet inhibition observed 96 hours after initiating aspirin therapy with 3 daily doses of 81mg would be equivalent to that observed using a dose of 325mg.

Methods:

The amount of platelet inhibition produced when initiating daily aspirin therapy was evaluated using the slope of the platelet prostaglandin agonist (PPA) stimulated light aggregometry curve at 4 time points: time 0 before the first dose, 2 hours after the first dose, 24 hours after the first dose (just before the second dose) and 96 hours after the first dose (24 hours after the 3rd dose). After a 7 day wash out period, during which subjects were instructed not to take aspirin or other NSAIDs, the subjects repeated the study using a 325mg aspirin dose. Because of the large variation in reactivity of platelets among people, each normal subject served as their own control and the results were evaluated using a paired “t” test. We plan to have studied a minimum of 12 normal subjects by the time of the ASH meeting.

Results:

To date 6 normal subjects were studied. 96 hours after initiating aspirin therapy, 24 hours after the 3rd aspirin dose, there was no difference between the PPA slopes observed for the 81mg=36 and the 325mg=28 (p=.28). Immediate platelet inhibition observed in the 24 hours after initiating aspirin therapy was far greater with the 325mg than the 81mg dose. Two hours after the 81mg dose the PPA slope was 77 compared to the 325mg slope of 12 (p=.000). Similarly, the slopes 24 hours after initiating aspirin were 68 for the 81mg dose and 35 for the 325mg dose (p=.006). The time 0 slopes were also statistically different 81mg=91 and 325=78 (p=.024).

Conclusions:

1. Initiation of aspirin therapy with a dose of 325mg dose produces a more immediate inhibition of platelet function with maximal platelet inhibition observed 2 hours after the first dose. 2. Platelet inhibition with an aspirin dose of 81mg occurs more gradually with maximal platelet inhibition observed 24 hours after the 3rd dose at 96 hours. 3. Whether daily aspirin therapy is initiated with a daily dose of 81mg or 325mg, the amount of platelet inhibition observed 96 hours after starting aspirin therapy, 24 hours after the 3rd dose, is equivalent.

Comment:

This data supports the clinical observations that protection from future vascular events is equivalent with either 81 or 325mg daily dosing and suggests that the current practice of increasing the dose of aspirin in patients who experience a subsequent event may not be beneficial.

Disclosures:

No relevant conflicts of interest to declare.
Replacement Therapy for Minor Surgery and Invasive Procedures in Factor VII Deficiency: The STER Experience
Authors: Napolitano Mariasanta , Dolce Alberto , Ingerslev Jorgen , Giansily-Blaizot Muriel , Di Minno Matteo ND , Auerswald Guenter K.H. , de Saez Arlette Ruiz , Tagliaferri Annarita , Batorova Angelika , Caimi Teresa Maria , Chuansumrit Ampaiwan , Fernandez Maria Fernanda Lopez , Bertrand Marie-Anne , Verdy Elisabeth , Zanon Ezio , Abecassis Lien , Aronis Sophie , Siragusa Sergio , Orecchioni Assunta , Mariani Guglielmo
Published: October 2019

Abstract 1262

Introduction:

Surgical bleeding is among the most frequent symptoms in Factor VII (FVII) deficient patients: in an analysis by our group, post-surgical bleeding was reported in 24% of cases (Mariani G. et al. Thromb Haemost 2005;93:481). A recent retrospective study showed a similar prevalence of surgical bleeding (15%) with a significant relation between this type of bleeding, deep hematomas and a FVII coagulant activity (FVIIc) of less than 7% (Benlakal F et al. J Thromb Haemost 2011;9:1149). Little is known about prevention of bleeding in ‘minor surgical interventions’, important tools in modern medicine, bearing not a negligible risk of bleeding, as local hemostasis may not always be punctually achieved.

Aim:

Analysis of Replacement Therapy (RT) for minor surgeries in patients with FVII deficiency, prospectively reported in the Seven treatment Evaluation Registry (STER). Clinicaltrials.gov identifier: NCT01269138.

Methods:

Analysis of RTs used in 38 minor surgical procedures (34 patients; FVIIc:<1–20%). Minor surgical procedures were defined as suggested by Kitchens (Surgery and hemostasis. Textbook of Consultative Hemostasis and Thrombosis. 2007).

Results:

Reported interventions included: oral surgery (n=15), endoscopic biopsies (n=7), catheter insertions (n=3), ear-nose-throat and head-neck (n=5), mixed type (n=8). RT schedules were based on recombinant-activated FVII (rFVIIa; n=29), plasma-derived FVII (pdFVII; n=8), or Fresh-Frozen-Plasma (n=1). One-day RT schedule was employed in 27 procedures, 2–10 days in 11 procedures. Total doses ranged from 7.2–510 μg/kg (rFVIIa) and 9–300 IU/kg (pdFVII). FFP was given at a total dose of 50 ml/kg (1-day treatment, split into 4 doses of 12.5 ml). Antifibrinolytics were administered together with RT in 16 procedures (11 dental) for a minimum of 1 day to a maximum of 7 days. No bleeding nor thrombotic events occurred; one patient developed an inhibitor (Table 1).

Conclusion:

For most of the uncomplicated minor surgery procedures (single dental extractions, catheter insertions, endoscopic biopsies), one-day RT is sufficient with low to medium doses of rFVIIa (median 25 μg/Kg/bw) or pdFVII (median 18.5 UI/Kg/bw), possibly with more than one administration. In complicated and more elaborate interventions, longer-lasting RT schedules and higher doses may be needed.

Disclosures:

No relevant conflicts of interest to declare.
Genome-Wide shRNA Screen for DNA Damage Response Regulators in Human Hematopoietic Stem and Progenitor Cells
Authors: Gan Olga I , Milyavsky Michael , van Delft Mark F , Buzina Alla , Kalatskaya Irina , Ketela Troy , Stein Lincoln , Moffat Jason , Dick John E.
Published: October 2019

Abstract 1289

Hematopoietic stem cells due to their life-long function should protect genome integrity to avoid accumulation of genetic aberrations leading to malignant transformation or bone marrow failure. We reported recently that human HSC of cord blood origin are exquisitely sensitive to DNA damage-induced apoptosis. Thus, following 3Gy of ionizing radiation HSC show evidence of persistent DNA damage response and greater p53-dependent apoptosis in comparison with commited myeloid progenitors. To elucidate the molecular basis of these observations we carried out a genome-wide loss-of-function genetic screen using a library of 80 000 shRNA vectors targeting more than 16 000 human genes. A screen was performed on immortalized (by TLS-ERG infection) cord blood cells (TEX), which radio-sensitivity is similar to early hematopoietic cells. To find out the radio-protective hits we exposed infected cells to four rounds of 4Gy irradiation in three independent experiments. TEX cells exhibited steady increase of their proliferative potential after each irradiation exposure. Cells were gathered after each irradiation round and their DNA was subjected to sequencing to determine protective hits. Upon the analysis we recognized known regulators of DNA damage response (e.g. p53) and identified many genes that previously were not connected to genotoxic stress response. Thus, the knockdown of these genes was at least as effective as p53 knockdown in protection of TEX cells against gamma-irradiation. The validation of the chosen hits on TEX cells showed that about half of them indeed mediate the protection against irradiation. Further validation included real-time PCR of infected cells to ensure the absence of off-target effect along with Western blot analysis of affected protein. We followed up the investigation of chosen hits on primary human lineage-negative cord blood cells and observed that only few candidates were mediating the same effect on HSC/progenitors cells as on TEX cells. To further validate the effect of the most prominently effective hits we employed several shRNA vectors for the same target gene. Following this step of validation, we choose to investigate the knockdown of CHEK2, the gene with reported role in DNA damage response in several murine tissues. Our preliminary studies in long-term cytokine-supplemented cultures demonstrated that CHEK2 is involved in DNA damage response of human HSC and progenitors. The further investigation of the effect of CHEK2 knockdown on repopulating human HSC is currently underway. An integrated analysis of our observations revealed several putative CHEK2-centered molecular networks, which connect DNA damage response to HSC function.

Disclosures:

No relevant conflicts of interest to declare.
Characterization of Hematopoietic Stem Cell Function and Sensitivity in the Homologous Recombination Deficient Exonuclease1mut Mouse Model
Authors: Desai Amar , Qing Yulan , Gerson Stanton L.
Published: October 2019

Abstract 1293

Hematopoietic stem cell (HSC) maintenance is essential for sustained longevity and tissue function. The HSC population has lifelong self-renewing capabilities and gives rise to subsets of multipotent progenitor cells, and in turn a progeny of terminally differentiated mature cells consisting of all subtypes of the myeloid and lymphoid lineages. Long term reconstituting HSCs are necessary to replace these differentiated cells after losses caused by normal degradation or damage accumulation, with failure to replenish these stores being linked to a variety of human pathogeneses as well as aging phenotypes.

HSC populations require functional DNA repair pathways in order to maintain their reconstitution capabilities but little is known about the pathways involved or the mechanism of regulation. While the majority of HSCs are quiescent at steady state, endogenous or exogenous stress can force these cells into proliferation, and previous evidence has suggested that the HSC reliance on DNA repair changes with this mobilization. Quiescent HSCs are believed to depend on non-homologous end joining (NHEJ) for repair but prior literature has shown that once forced into cycle, the DNA repair dependency shifts and is shared between homologous recombination (HR) and NHEJ.

We use Exo1 deficiency as a model for homologous recombination loss in mice and demonstrate in vivo that HR is dispensable in quiescent HSCs. This is in contrast to loss of the complementary double strand break repair pathway NHEJ which has been shown to result in severe defects in HSC function. However when we force mobilize HSCs into cycle in vivo using the anti metabolite 5-fluorouracil we are able to demonstrate that the HR defects become detrimental to the animal as shown by increased cellular IR sensitivity and subsequent animal death. Additionally we use competitive repopulation studies to show that indeed the Exo1mut HSC population is more radiation sensitive after forced mobilization. This work begins to elucidate the consequences of the loss of homologous recombination in hematopoietic stem cells as well as the interplay between cell cycle status and DNA repair dependency.

Disclosures:

No relevant conflicts of interest to declare.
P/E Selectins and Their Ligand PSGL-1 Are Crucial for Macrophage-Mediated Multiple Myeloma Chemoresistance
Authors: Zheng Yuhuan , Yang Jing , Qian Jianfei , Hanabuchi Shino , Liu Zhiqiang , Lu Yong , He Jin , Li Haiyan , Shah Jatin J , Lan Yongsheng , Yi Qing
Published: October 2019

Abstract 130

Multiple myeloma (MM) bone marrow (BM) provides a homing microenvironment, which is important for tumor development, growth, proliferation, migration and chemoresistance. Our previous research has shown that macrophages (MΦ) are associated with malignant BM and confer MM cell chemoresistance in vitro. Based on these findings, we designed mechanistic studies to elucidate MΦ-mediated MM chemoresistance. MΦ protected MM cells from different chemotherapeutics, such as bortezomib, doxorubicin, dexamethasone, melphalan, or their combination-induced apoptosis. Cell-to-cell contact was crucial for such protection. Under a coculture condition, MΦ protected MM cells with wide ranging MM/MΦ ratio. Inhibition of phagocytosis did not affect the protection. An experiment examining MM patients' BM showed that MΦ was a dominant accessory cell population in patients' BM. Patients PBMC-derived MΦ or primary MΦ isolated from patient BM had protective activity. Further mechanistic study showed that surface molecules, such as P/E selectins (on MΦ) and their ligand PSGL-1 (on MM cells), were important for MΦ-mediated MM chemoresistance. Blockade antibodies against either P/E selectins or PSGL-1 significantly repressed MΦ-mediated drug resistance. MΦ had limited protection on PSGL-1-knocked down MM cells. Next, we examined intracellular signal transduction in MM cells, upon interaction with MΦs. Src family kinase and Erk1/2 kinase were activated in MM cells, after coculture with MΦs. Coculture also stimulated PSGL-1 and c-myc overexpression in MM cells. Inhibition of Src family kinase, Erk1/2 kinase, or c-myc by small molecule inhibitors impaired MΦ-mediated MM chemoresistance. The overexpression of PSGL-1, under coculture condition, could be repressed by IFN-α neutralization antibody. More importantly, coculture with MΦ could not stimulate Erk1/2 activation or c-myc overexpression in PSGL-1-knocked down MM cells, suggesting that Erk1/2 and c-myc were downstream of PSGL-1-initiated signal transduction. Finally, we established a NOD-SCID mouse model to test MΦ-mediated MM chemoresistance in vivo. MM tumors with MΦ infiltration grew significantly faster than MM, only, tumors and were more resistant to melphalan treatment. Overall, our data demonstrated a mechanism of MΦ-mediated chemoresistance, both in vitro and in vivo. Based on our findings, MΦ-targeted therapy in MM treatment may be beneficial to improve the effectiveness of MM chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Runx1-Cbfb Interacts with CHD7, a Chromatin Modifying Enzyme with a Potential Role in Hematopoiesis
Authors: Hsu Jingmei , Lee Chung-Tsai , Gerber Scott , Yu Shuqian , Speck Nancy A.
Published: October 2019

Abstract 1300

Mutations in RUNX1 and CBFB are among the most common genetic alterations in hematologic malignancies, including acute myeloid and lymphoid leukemia (AML, ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome and myeloproliferative neoplasms. Loss of Runx1-CBFb causes a failure of hematopoietic stem cell emergence during embryogenesis. Critical roles for Runx1-CBFb in adult hematopoiesis include hematopoietic stem and progenitor homeostasis, and lymphoid and megakaryocytic differentiation. We took an unbiased co-immunoprecipitation and mass spectrometry approach to identify Runx1-CBFb co-regulators in T cells, and identified chromodomain helicase binding protein 7 (CHD7) as a potential interacting partner. CHD7 is an ATP-dependent chromatin remodeling protein that primarily occupies enhancer and promoter regions. Autosomal dominant mutations in CHD7 cause CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). It was shown that CHD7 interacts with Sox2, and its occupancy correlates with H3K4me1/2 modifications and P300 binding at enhancer regions, and H3K4me3 marks at promoters. We confirmed the interaction of endogenous Runx1 and CHD7 in T cells. We demonstrate that the Runx1 transactivation domain, which is critical at all stages of hematopoiesis, is required for the CHD7 interaction. To elucidate an in vivo function for CHD7 in hematopoiesis, we generated a conditional pan-hematopoietic Chd7 deletion in mice using a floxed Chd7 allele and Vav1-Cre. Deletion of Chd7 in hematopoietic cells appears to cause no lineage specific defects. However, CHD7 deficient bone marrow cells had a competitive advantage in T cell reconstitution as compared to wild type cells, suggesting a role for CHD7 in restraining T cell numbers in the adult. Determining how CHD7 exerts its functions should shed light on underlying mechanisms in hematopoietic stem cell formation, T cell development, and hematopoietic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
RUNX1-Induced Silencing of Non-Muscle Myosin Iib (MYH10) Is Required for Megakaryocyte Polyploidization
Authors: Bluteau Dominique , Lordier Larissa , Antony-Debré Iléana , Jalil Abdelali , Legrand Céline , Rameau Philippe , Bluteau Olivier , Jouni Dima , Mercher Thomas , Leon Catherine , Gachet Christian , Debili Najet , Vainchenker William , Chang Yunhua , Raslova Hana
Published: October 2019

Abstract 1308

Megakaryocytes (MK) are unique mammalian cells that undergo polyploidization during differentiation, which leads to an increase in cell size and protein production that precedes platelet production. The molecular basis of MK polyploidization, denoted endomitosis, which is strongly altered in hematological malignancies and various other disease states, remain poorly understood. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis, associated with a contractile ring defect. Here, we demonstrate that the myosin non-muscle IIB heavy chain (MYH10) is expressed in immature human and mouse megakaryocytes and its expression is repressed during differentiation. In immature MK, MYH10 is specifically localized in the contractile ring while MYH9 is mainly present in the cytoplasm suggesting that they occupy two different functions during MK differentiation. Importantly, MYH10 down-modulation by shRNA or by addition of a chemical inhibitor blebbistatin, increases polyploidization by inhibiting the return of 4N cells to 2N. Conversely, re-expression of MYH10 in MKs prevents polyploidization and the transition of 2N cells to 4N cells. Furthermore, we demonstrated that RUNX1 directly repress the transcription of MYH10. In vitro and in vivo RUNX1 invalidation inhibits MK polyploidization and increases expression of MYH10. Accordingly, in patients with a germline mutation of RUNX1 (FPD/AML), MYH10 is still expressed in platelets. Altogether ours results demonstrated that the RUNX1-mediated silencing of MYH10 is needed for the switch from mitosis to endomitosis linking thus polyploidization with MK differentiation.

Disclosures:

No relevant conflicts of interest to declare.
Stepwise Signaling and Low Oxygen Promote Hematopoietic Progenitor Emergence From Human Pluripotent Stem Cells
Authors: Endo Hiroshi , Takayama Naoya , Koike Tomo , Nakauchi Hiromitsu , Eto Koji
Published: October 2019

Abstract 1317

Hematopoietic systems in mouse models have been well characterized based on the defined cell surface markers present on fetal and adult hematopoietic stem cells (HSCs) and their blood derivatives. In humans, by contrast, hematopoietic ontogeny and the subsequent hierarchy have not been determined. Human pluripotent stem cells (ES cells, ESCs and iPS cells, iPSCs) are embryo-type cells and a promising cell source for studying the ontogeny of blood cells within a differentiation system.

We previously established an in vitro co-culture method using C3H10T1/2 mesenchymal stromal cells, whereby vascular endothelial growth factor (VEGF) promotes the appearance of CD34+ hematopoietic progenitor cells (HPCs) from human ESCs or iPSCs (Takayama et al., Blood, 2008; Takayama et al., J Exp Med, 2010). Here we demonstrate that the use of a combination of low oxygen and growth factors is suitable for cells at specific developmental stages that include CD56+CD326- mesodermal progenitors during the early phase (days 0–4), CD34+CD56+CD90+CD105+CD43-KDR- hemangioblasts during the mid-phase (days 5–7), and CD43+ hematopoietic and KDR+ endothelial cells during the late phase (days 8–10).

During mid-phase, application of basic fibroblast growth factor (bFGF, 10 ng/ml) under 1% O2 significantly increased numbers of CD43+ cells by 5-fold, as compared to cells without bFGF under 21% O2. Administration of a MYC inhibitor (50 μM) to cells during the early-phase, and tumor growth factor beta (TGF-beta) receptor inhibitor (SB431542, 10 μM) during mid-phase, also stimulated generation of CD34+CD43+ HPCs (1.5-fold and 3-fold in increase respectively). Notably, although this protocol resulted in a prominent yield of mesodermal progenitors during the early phase and of HPCs during the late phase (which were 50% and 10% of all derivatives from human iPSCs, respectively), this signaling manipulation had the opposite effect at other stages. For example, bFGF or TGFbeta receptor inhibition significantly depressed HPCs during the early-phase.

The signaling modulation and low oxygen conditions in our co-culture system did not require factors known to affect ex vivo human CD34+ HSC / HPC expansion from cord blood cells, which contain stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, erythropoietin, interleukin (IL)-3 and IL-6.

Our novel culture protocol implicates new players in the stepwise development from a pluripotent state to blood cell generation. These players appear to be governed by circumstances resembling a developmental niche with lower oxygen conditions.

Disclosures:

No relevant conflicts of interest to declare.
CD56(+)CD146(+) Cells in Normal Human Bone Marrow-Stroma Proliferated to Form Tumors in severe Combined Immunodeficiency Mice
Authors: Shirasaki Ryosuke , Tashiro Haruko , Oka Yoko , Yamamoto Tadashi , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki
Published: October 2019

Abstract 1328

Background and Aims:

We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and in vivo. Recently, we also reported that myelogenous leukemia-derived myofibroblasts formed blastomas in non-obese diabetes severe combined immunodeficiency (NOD/SCID) mice, in which the CD56-positive cell-fraction was selectively proliferated (16th EHA). To ascertain whether normal human bone marrow-stroma-derived cells also behave like leukemia-derived cells, stromal cells were injected to NOD/SCID mice, and tumor-formation was observed.

Materials and Methods:

Bone marrow cells were collected from informed normal individuals, whose adherent cells were separated and were cultured for one month to eliminate monocyte/macrophage, vascular cell, and pericyte to prepare stromal myofibroblast-rich fraction. Cells were injected to NOD/SCID mice intra-venously, peritoneally, and subcutaneously (3 × 106/mice). When the tumor formation was observed or mice were dead, they were sacrificed and the engrafted cells were analyzed.

Results and Discussion:

Between at day 70 and 90 after injection mice were dead. Autopsy findings revealed tumor formation at subcutaneous injection sites, and cells were also infiltrated to the liver and ascitic fluid. These cells expressed CD56, CD146, and Nestin strongly, but not other lineage-specific markers including CD133; however, the ratio of CD56(+)CD146(+) fraction in the injected stromal cells was below 0.1%. When the tumor cells were cultured in vitro, they exhibited spindle-shaped appearance, and doubling time was 12 to 16 hours. They expressed c-Myc, Klf4, Nanog, Sox2, and other molecules that are expressed in an immature somatic stem cell. These observations indicate that CD56(+)CD146(+) cell-fraction in bone marrow-stroma is very immature and highly proliferative. We are now analyzing biological characteristics of this specific cell-fraction, and determining the contribution to normal hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Regulatory Potential of miRNAs on CXCL12 Expression in Human Mesenchymal Stem Cells (MSC)
Authors: Illmer Thomas , Heder Carolin , Stölzel Friedrich , Fierro Fernando , Ehninger Gerhard
Published: October 2019

Abstract 1332

CXCL12 is the major CXC chemokine produced by various cell types of the hematopoietic niche. It is well known for it‘s regulatory potential in physiological processes like stem cell mobilization and homing. In addition it participates in regulation of proliferation and survival of cells with expression of it‘s receptor CXCR4. Thus, variable CXCL12 levels can influence a multitude of mechanisms associated with stem cell migration, homing and proliferation, e.g. during stem cell mobilisation CXCL12 levels are down regulated in MSC and osteoblasts thereby enabling conversion of CD34 positive cells into the circulation. The regulatory mechanisms of this process are however incompletely understood. In a systematic approach we investigated whether miRNAs are able to mediate CXCL12 regulation in human MSC. We transfected the immortalized human MSC cell line SCP-1 with a library consisting of 702 different pre-miRNAs. Subsequently, CXCL12 level determination by ELISA revealed a total of 27 miRNAs downregulating CXCL12 and 34 miRNAs which upregulated CXCL-12. Since the most important effect of miRNAs is the interaction with the 3 ‘UTR of target genes we further validated our candidate miRNAs with a luciferase assay using the 3 ‘UTR of CXCL12 transcript variant 2. Here, 8 miRNAs (miR-23, 130b, 135a, 200b, 200 c, 216, 222 and 602) were shown to interact significantly with CXCL12. Next, expression of candidate miRNAs was determined in 46 human resting primary MSC samples which proved strongest expression of miR-23 in MSC, miR-130b and 222 are expressed at lower but detectable levels, whereas the other candidate miRNAs showed no expression in human MSC. Subsequently, transfection of pre-miR-23 molecules in primary human MSC reduced both CXCL12 protein values and the attraction of migrating cells in a transwell assay.

In conclusion, using a large scale library transfection approach we identified miR-23 as an important regulator of CXCL-12 In human MSC.

Disclosures:

No relevant conflicts of interest to declare.
Ionizing Radiation Induces Hematopoietic Stem Cell Senescence and Long-Term Bone Marrow Suppression in a p16Ink4a/Arf-Independent manner
Authors: Shao Lijian , Feng Wei , Li Hongliang , Wang Yong , Sharpless Norman , Zhou Daohong
Published: October 2019

Abstract 1345

Many patients receiving chemotherapy and/or ionizing radiation (IR) develop residual (or long-term) bone marrow (BM) injury that can not only limit the success of cancer treatment but also adversely affect their quality of life. Although residual BM injury has been largely attributed to the induction of hematopoietic stem cell (HSC) senescence, neither the molecular mechanisms by which chemotherapy and/or IR induce HSC senescence have been clearly defined, nor has an effective treatment been developed to ameliorate the injury. The Ink4a-Arf locus encodes two important tumor suppressors, p16Ink4a (p16) and Arf. Both of them have been implicated in mediating the induction of cellular senscence in a variety of cells including HSCs. Therefore, we examined the role of p16 and/or Arf in IR-induced HSC senescence and long-term BM suppression using a total body irradiation (TBI) mouse model. The results from our studies show that exposure of wild-type (WT) mice to a sublethal dose (6 Gy) of TBI induces HSC senescence and long-term BM suppression. The induction of HSC senescence is not associated with a reduction in telemore length in HSCs and their progeny, but is associated with significant increases in the production of reactive oxygen species (ROS), the expression of p16 and Arf mRNA, and the activity of senescence-associated β-galacotosidase (SA-β-gal) in HSCs. However, genetical deletion of Ink4a and/or Arf has no effect on TBI-induced HSC senescence, as HSCs from the Ink4a and/or Arf knockout mice after exposure to TBI exhibit similar changes as those seen in the cells from irradiated WT mice in comparison with the cells from un-irradiated mice with correspondent genotypes. In addition, TBI-induced long-term BM suppression is also not attenuated by the deletion of the Ink4a and/or Arf genes. These findings suggest that IR induces HSC senescence and long-term BM suppression in a p16Ink4a/Arf-independent manner.

Disclosures:

No relevant conflicts of interest to declare.
Runx1 Is Required for Cbfb-MYH11 Activity During Primitive Hematopoiesis
Authors: Hyde R. Katherine , Zhao Ling , Alemu Lemlem , Liu Pu Paul
Published: October 2019

Abstract 1357

Inversion of chromosome 16 (Inv(16)) is found in nearly all patients with acute myeloid leukemia (AML) subtype M4 with eosinophilia. Inv(16) results in the fusion of the transcription factor gene CBFB, and the MYH11 gene, which encodes Smooth Muscle Myosin Heavy Chain (SMMHC). It has been proposed that the Inv(16) fusion protein, CBFβ-SMMHC, initiates leukemogenesis through repression of the transcription factor RUNX1. However, we recently found that CBFβ-SMMHC also has activities that are independent of Runx1 repression. Mice expressing a knockin allele of Cbfb-MYH11 (Cbfb+/MYH11) show a severe differentiation defect during primitive hematopoiesis. Interestingly, this severe defect is not seen in mice homozygous for a null allele of Runx1 (Runx1−/−). We also found genes deregulated uniquely in Cbfb+/MYH11 embryos were also expressed in leukemic cells from mice and Inv(16) patients. These findings imply that CBFβ-SMMHC's Runx1 repression independent activities play a role during leukemogenesis.

There are two potential models to describe these observations: a Runx1-independent model and a Runx1-dependent model. In the first model, Cbfb-MYH11 initiates leukemia through a novel mechanism that does not involve Runx1. In the second model, Cbfb-MYH11 cooperates with Runx1, but rather than repressing Runx1 activity, Cbfb-MYH11 alters it. To test these two models, we generated mice that express a conditional allele of Cbfb-MYH11 on a Runx1−/− background. Surprisingly, we found a complete rescue of the Cbfb-MYH11 induced primitive hematopoietic defect: the primitive blood from Runx1−/−; Cbfb+/MYH11 embryos was indistinguishable from Runx1−/−; Cbfb+/+ embryos. Preliminary data showed that Cbfb-MYH11 expression in the embryos was not affected by the loss of Runx1, indicating that Runx1 is not regulating Cbfb-MYH11 expression, but is required for its activity. Interestingly, another allele of Runx1 in which Runx1 is fused to the β-galactosidase gene, lacZ (Runx1lzd) and that has been shown to have dominant negative activities (Nancy Speck, personal communication) also rescued the primitive blood defect in the Cbfb+/MYH11 embryos. This demonstrates the Cbfb-MYH11 requires Runx1 activity for its primitive hematopoietic differentiation defect. Based on this finding, we hypothesize that Cbfb-MYH11 may have a similar requirement for Runx1 activity during leukemogenesis. Consistent with this idea, we found that adult mice expressing Cbfb-MYH11 and the Runx1-lzd allele showed a significant delay in the development of leukemia as compared to their Cbfb+/MYH11; Runx1+/+ littermates.

Disclosures:

No relevant conflicts of interest to declare.
Anthracycline Treatment Induces Extracellular Calreticulin in Acute Myeloid Leukemia
Authors: Mans Sarah , Schaubitzer Kerstin , Shan Deborah , Mueller Beatrice U , Pabst Thomas
Published: October 2019

Abstract 1376

The myeloid key transcription factor CEBPA (CCAAT-enhancer binding protein alpha) is blocked by the mRNA binding protein calreticulin (CRT) thereby affecting myeloid differentiation. We previously reported that the unfolded protein response (UPR) is activated in 24.5% of AML patients, involving the up-regulation of endoplasmatic reticulum (ER) stressors, such as CRT. In addition, anthracycline treatment was shown to trigger the UPR and to specifically induce CRT cell membrane surface expression in some solid tumor types. This ecto-CRT is specifically targeting dendritic cells and initiates immunogenic cell death of cancer cells by acting as an “eat-me” signal. However, UPR induced translocation of CRT in leukemogenesis has hardly been investigated so far.

This study aimed at analyzing anthracycline induced CRT exposure and extracellular release of CRT in leukemic cells as well as at elucidating the candidate pathways involved such as the UPR transducers PERK, ATF6 and IRE1.

In leukemic cell lines, we observed that anthracycline treatment was inducing CRT expression on the cell membrane surface within 2 hours. This effect was caused by intracellular translocation, but not de novo synthesis. Exposure of CRT was decreased by Brefeldin A, which blocks the vesicle transport from the ER to the Golgi apparatus within 2 hours of treatment, suggesting that exocytosis is critical for CRT delocalization. By specific inhibition of the PERK pathway of the UPR using Salubrinal, we observed reduced CRT expression on the cell membrane. In contrast, blocking the ATF6 or the IRE1 pathway did not affect CRT exposure.

Mean CRT levels on the cell membrane of blasts from 53 AML patients at diagnosis were significantly (21-fold; p<0.0005) increased compared to CRT levels of myeloid cells from healthy volunteers (n=75). Using an ELISA assay investigating serum from 104 AML patients at diagnosis and 86 healthy volunteers, we found that extracellular CRT was significantly (2.2-fold; p<0.0001) increased in AML patients compared to healthy controls. We observed a more favorable outcome in AML patients with increased CRT expression on the cell membrane, as well as in patients with higher levels of extracellular CRT in the serum. Finally, we identified a strong correlation between CRT serum levels and CRT expression on the cell surface of leukemic cells. Ongoing studies aim at elucidating the mechanisms of CRT secretion from the cell membrane to the serum.

In conclusion, CRT translocation to the cell membrane and increasing levels of secreted CRT in the serum represent early events of anthracycline treatment in leukemic cells, and both are associated with a more favorable outcome in AML patients.

Disclosures:

No relevant conflicts of interest to declare.
Mechanisms of Drug Resistance Against JAK2 Inhibitors
Authors: Azam Mohammad
Published: October 2019

Abstract 1426

The discovery of activating somatic mutations of JAK2 kinase as a contributing factor in the pathogenesis of myeloproliferative neoplasms (MPNs) —polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF)—prompted the development of JAK2-targeted therapies for these diseases1–3. Therapy with JAK2 inhibitors (INCB018424 and TG101348) induces rapid and marked reductions in spleen size and can lead to remarkable improvements in constitutional symptoms and overall quality of life suggesting that therapeutic targeting of JAK2 holds great promise for the treatment of JAK2 mediated disorders. It is likely that these two clinical agents will soon be approved by the FDA for the treatment of these diseases. Given our experience in development of resistance in tyrosine kinase inhibitor therapy (TKI) especially in the context of BCR/ABL targeting by Gleevec, we anticipate that resistance to JAK2 inhibitors will develop as well. To understand the mechanism of resistance against JAK2 targeted therapies, we performed mutagenesis studies and in vitro screening for resistance as previously reported for selection of resistance mutation in BCR/ABL (Azam et. al. (2003), Cell and Azam et. al. (2006), PNAS.).

We sequenced the resistant clones isolated from INCB018424 resistant screening and identified 125 different amino substitutions spanning across FERM, SH2, pseudokinase and kinase domains. Frequency of resistant mutations demonstrated several major hot-spots clustered in the FERM, pseudokinase and kinase domain. We observed sixty different mutations in the kinase domain that clustered into eight major hot-spots with several minor-spots in regulatory regions including the hinge region, catalytic-loop, activation-loop and in the helical region of c-lobe. Structural modeling of these mutations suggests that the JAK2 kinase, like ABL kinase, is subject to auto-inhibited regulatory mechanisms and selects for drug resistant mutations from allosteric domains (FERM, SH2 and Pseudokinase domain). We anticipate that these results will be valuable in diagnosis of clinical resistance and in the identification and design of next generation small molecule JAK2 inhibitors

Disclosures:

No relevant conflicts of interest to declare.
Heterogeneity in Infants with Acute Myeloid Leukemia: Retrospective Analysis of a Japanese Nationwide Survey
Authors: Shimada Akira , Tomizawa Daisuke , Kinoshita Akitoshi , Hamamoto Kazuko , Tsukimoto Ichiro , Ogawa Atsushi , Taga Takashi , Imamura Toshihiko , Tawa Akio , Horibe Keizo , Taki Tomohiko , Hayashi Yasuhide , Adachi Souichi
Published: October 2019

Abstract 1477

Introduction:

When compared to older patients, infants with acute leukemia exhibit distinct cytogenetic features, such as higher prevalence of MLL gene rearrangement (MLL-R), and are known to have higher vulnerability to intensive cytotoxic therapy, such as hematopoietic stem cell transplantation. In contrast to acute lymphoblastic leukemia (ALL), there have been few reports on acute myeloid leukemia (AML) in infants. To develop more appropriate therapeutic strategies for infants with AML, it is necessary to elucidate the distinct clinical features of this subgroup. We therefore performed a retrospective analysis on infant AML in Japan. Patients: Infants with AML, aged less than 1 year at diagnosis, registered in any of the 6 Japanese AML clinical trials between 1991 and 2010 (TCCSG M91-13, TCCSG M96-14, AML99, CCLSG9805, CCLSG9805RE, and JPLSG AML-05) were included in this study. Patients with Down syndrome were excluded. Results: A total of 122 infant AML patients were included in the present analysis, which comprised approximately 10% of all pediatric AML patients. The most frequent FAB classification type was M5 (28.7%), followed by M7 (22.9%) and M4 (10.8%). About 30% of patients had 11q23 abnormalities/MLL -R, but there was no impact on prognosis. Several cases with normal karyotype were revealed to be MLL -R on FISH analysis or on MLL -fusion chimeric transcript analysis by RT-PCR. t(8;21), inv(16) and t(15;17) cases were very rare among the infant cohorts. Furthermore, 7.8% had t(1;22)(p13;q13), and 2.5% had t(7;12)(q36;p13). Genetic mutation results could be obtained in 11 cases in the AML99 study; only one case each was confirmed to have NRAS, KRAS or KIT gene mutation. No cases with FLT3-ITD were detected among the 11 cases in the AML99 or the 44 cases in the AML-05 study. Survival rate varied based on treatment received; 5-year OS rate was 58.3% to 71.4%, and 5-year EFS rate was 49.4% to 64.2%. Discussion: Survival rate in infant AML was identical to that in older pediatric AML. However, there was a possible underestimation of MLL -R patients based on sole chromosome analysis; the prevalence of MLL -R was less than 50% in infant AML patients, without any prognostic impact. Other well-known genetic alterations in pediatric AML also had no effect on outcome of infant AML. Infant AML is a heterogeneous subgroup of pediatric AML, and further studies, as well as novel biomarkers, will be necessary to fully understand its biology.

Disclosures:

No relevant conflicts of interest to declare.