bluuebunny/crossref_metadata_2025_split huggingface dataset:
Shortened Erythrocyte Life Span and Increased Oxidative Stress In Erythroid Precursors Are Consistent with Normocytic Anemia In Mice with Chronic Inflammation
Authors: Layman Andrew J. , Langdon Jacqueline M. , Yu Qilu , Prince Olivier D. , Walston Jeremy D. , Civin Curt I. , Roy Cindy N. 
Published: October 2019

Abstract
Abstract 3205
The anemia of inflammation is a common co-morbid condition associated with various infections, autoimmune disorders, and other chronic disease states. In general, hemoglobin levels are mildly to moderately decreased. In vitro studies have demonstrated that pro-inflammatory cytokines can inhibit erythropoiesis at various stages of development, but many of these studies are limited to the analysis of early developmental stages and primarily assess proliferation as opposed to maturation or survival. Sufficient data from in vivo studies and whole animal models are lacking. We characterized the anemia in 8–10 week old C57BL/6 mice with turpentine oil-induced sterile abscess. We found that following 3 weeks of sterile abscess, the mice had a normocytic, normochromic anemia.
Using in vivo biotin-labeling of peripheral blood, we found the coefficient describing erythrocyte life span significantly decreased from -0.044 ± 0.002 SE in control mice to -0.048 ± 0.002 SE in mice with sterile abscess (p = 0.04). In further support of the mechanism of increased erythrocyte turnover, we found that splenic macrophages isolated from mice with abscess significantly increased expression of Heme-regulated gene 1 (Hrg-1) 2.5 fold, (p < .001) and significantly increased expression of Ferroportin (Fpn) 2.76 fold (p = 0.003).
Oxidative stress can lead to increased erythrocyte turnover and can inhibit erythroid maturation. To determine whether erythrocytes from mice with sterile abscess had impaired capacity for survival, we assessed staining of peripheral blood and erythroid precursors with chloromethyl dichlorodihydrofluorescein diacetate, acetyl ester (DCF). DCF fluoresces upon oxidation. We found that mean DCF fluorescence intensity (MFI) in peripheral blood increased from 3.2 ± 1.0 in control mice to 5.5 ± 0.6 in mice with abscess (p=0.004). Similarly, we found a greater percentage of bone marrow derived erythroid precursors from mice with abscess had high DCF staining (16.2 ± 3.9%) when compared to control mice (6.7 ± 1.9%, p=0.003). In conclusion, these data support the hypothesis that increased oxidative stress in erythroid precursors of mice with sterile abscess results in increased erythrocyte turnover and normocytic, normochromic anemia. Future studies will investigate whether pro-inflammatory cytokines inhibit the expression of key erythrocyte anti-oxidant enzymes or whether reactive oxygen species from neighboring granulocytes damage erythroid precursors in the bone marrow.

Disclosures:
No relevant conflicts of interest to declare.
Phase I Trial of the Oral Poly (ADP-ribose) Polymerase (PARP) Inhibitor Veliparib (ABT-888, V) Combined Wtih Topoecan (T) and Carboplatin (C) for Adults with Relapsed and Refractory Acute Leukemias
Authors: Karp Judith E. , Pratz Keith W , Litzow Mark R. , Ji Jiuping , Chen Alice , Rudek Michelle , Kaufmann Scott H. 
Published: October 2019

Abstract
Abstract 3276
PARP is activated in response to DNA single strand (SS) breaks and is pivotal to the base excisional repair pathway for chemotherapy-damaged DNA. The orally bioavailable PARP inhibitor V delays DNA repair and potentiates the cytotoxicity of multiple classes of chemotherapy drugs including topoisomerase I inhibitors and platinating agents in leukemia cell lines. A previous clinical trial of T+C yielded promising results in adults with refractory acute leukemia at the maximum tolerated dose (MTD) of T 1.6 mg/m2/d + C 150 mg/m2/d by intravenous continuous infusion (IVCI) × 120 hrs. We are conducting a Phase I dose-escalation trial of V given orally twice daily Days 1–8 with T+C given by 120 hr IVCI Days 3–7.
Acute Myelogenous Leukemia (AML, 33 patients) Acute Lymphoblastic Leukemia (ALL, 4 patients) Median Age (range) 570 (40-75) 55 (42-62) Relapsed 8 No. Prior CRs 2 (1-3) Prior Stem Cell Transplant 2 0 Refractory 25 4 No. Primary Refractory 10 1 No. Prior Regimens 3 (1-4) 3 (1-4) Prior Stem Cell Tranpslant 3 1 Secondary AML 12 Anecedent Hematologic Dx 6 Treatment-Related 6 Cytogenetics Intermediate 10 2 Adverse: Single/Complex 7/16 2 Ph+ (1 with complex) BRCA-1/BRCA-2 1 (mutation)/ 1 (13q14 deletion) 0
Deaths due to myocardial infarct and splenic infarcts with ascites occurred in one pt each when C 150 mg/m2 was added to V 10 mg BID and T 1.3 mg/m2, resulting in adjustment of the dose escalation schema for C and T. Of 27 pts treated at doses of V 10–20 mg orally BID Days1-8 and T 1.0–1.2 mg/m2/d + C 120–150 mg/m2/d for 120 hrs IVCI Days 3–7, 8 (30%) have experienced > grade 3 non-hematologic toxicity, 4 (15%) have had grade 4 toxicity and 1 (4%) died from sepsis with multi-organ failure. Overall response rate is 22% (2 CR, 1 CRi, 3 PR), with 4 in relapsed AML, 1 each in refractory AML and refractory ALL. Pharmacokinetic (PK) studies in pt plasma, marrow supernatant, and marrow blasts demonstrated that V does not accumulate with multiple dose administrations in plasma but does in marrow, is not altered by T or C administration, and is detectable in marrow supernatant and cells. Nuclear staining for phosphorylated histone H2AX (γH2AX) demonstrated that V 10 mg BID increased DNA damage by > 2-fold in 11/22 (50%) on Day 1 and 13/18 (72 %) on Day 4 after 24 hrs of T+C. A PAR ELISA demonstrated that V suppresses PAR levels to <50% of pretreatment values in 21/29 (72%) blood and 9/13 (69%) marrow samples. The MTD has not been reached and dose escalation is ongoing.

Disclosures:
No relevant conflicts of interest to declare.
Differential Dependence On Wnt Signaling Allows Chemical Mediated Eradication of Human Acute Leukemia without Affecting Normal Blood Stem Cells
Authors: Risueño Ruth M. , Szabo Eva , Dang Uyen Maria , Levadoux-Martin Marilyne , Chung Jae-Uk , Xenocostas Anargyros , Leber Brian , Bhatia Mickie 
Published: October 2019

Abstract
Abstract 3278
Specific targets of signaling pathways that control self-renewal and survival of acute myeloid leukemic stem cells (LSCs) vs. normal hematopoietic stem cells (HSCs) are largely unknown. Here, using a small molecule (CWP232228) derived from a parent compound that inhibits Wnt/TCF targets, we demonstrate reduction of primary human AML-blast growth and clonogenic capacity ex vivo, without effects on normal hematopoietic progenitors. Upon establishment of AML or normal hematopoiesis in immune-deficient recipients, in vivo administration of CWP232228 reduced leukemic disease and abolished LSC self-renewal, with no effect on normal HSC function. In vivo gene profiling and ex vivo molecular studies revealed that CWP232228 induces apoptosis and differentiation of AML-blasts via inhibition of Wnt/b-catenin signaling and activation of non-canonical Wnt signaling which phenocopies the effects of this small molecule. Our study reveals an in vivo differential dependence of AML on canonical vs. non-canonical Wnt signaling that allows therapeutic targeting of LSCs whilst sparing normal HSCs.

Disclosures:
Chung: Choongwae Pharma Corporation: Employment.
New Fluorescent Assay for the Direct Quantification of Heparin and Low-Molecular-Weight Heparins In Clinical Samples
Authors: Szelke Helga , Giese Christina , Krämer Roland , Harenberg Job 
Published: October 2019

Abstract
Abstract 3325
Heparin, a linear polydisperse polysaccharide, is used to prevent blood clotting in various diseases. It consists predominantly of a trisulfated disaccharide repeating unit and has a high negative charge density. Currently accepted methods to control blood levels of heparin or of its more commonly used low-molecular-weight (LMW) fractions include chromogenic substrate assays that analyze the biological inhibition of blood coagulation factor Xa (FXa). There are no established methods to directly determine the concentration of heparins in clinical samples.
A modified positively charged perylene diimide (Heparin Red) was developed that binds specifically to heparin and LMW-heparin (Szelke H et al., Chem. Commun. 2010;46:1667). A red fluorescence signal is dose dependently quenched in the presence of heparin and LMW-heparins. LMW-Heparin was added to human serum, plasma or urine. The chromogenic S2222 substrate assay was used to determine the anti-FXa activity of the anticoagulants. Fluorescence was recorded with a portable fluorimeter, equipped with a 550 nm excitation filter and a 610 nm emission filter.
For plasma samples spiked with 0.6–1.0 IU/mL LMW-heparin, the intraday (n=5) and interday (n=5) coefficient of variations (CVs) of the fluorimetric Heparin Red assay were in the range of 2.9–6.1% and 4.3–13.8%, respectively. The CVs of the chromogenic S2222 assay for same plasma samples were the range of 3.3–18.8% (intraday) and 6.2–13.7 (interday).
In serum and plasma samples of patients treated with LMW-heparin, correlation between the Heparin Red assay and the chromogenic anti-FXa test (S-2222) was r = 0.66. The correlation between the Heparin Red assay and the anti-FXa test in urine samples was r = 0.83. This is probably due to its lower protein concentration in urine compared to plasma and serum samples.
The fluorescent perylene diimide Heparin Red allows sensitive quantification of heparin and LMWHs in plasma, serum and urine samples. Ease of application combined with matrix-independent response make this heparin probe attractive for both routine laboratory and fast point-of-care testing as well as high-throughput pharmacokinetic studies, ideally without the need for patient-specific calibration.

Disclosures:
No relevant conflicts of interest to declare.
A Role for Splicing Factors and R-Loop Formation in the Pathophysiology of Fanconi Anemia.
Authors: Green Allison M , Collins Natalie , Longerich Simonne , Sung Patrick , Kupfer Gary M 
Published: October 2019

Abstract
Abstract 3371

Introduction:
Fanconi anemia(FA) is an autosomal and X-linked recessive genetic disorder characterized by congenital defects, aplastic anemia, and a predisposition to cancer. At the cellular level, patients with FA display hypersensitivity to DNA crosslinking agents and increased levels of chromosomal instability. Because of these cellular phenotypes, the FA pathway has been thought to function in DNA maintenance and repair. Our data suggest that RNA and R-loop formation may account for at least part of the genomic instability seen in FA cells.

Methods:
Nuclear extracts were incubated with ribohomopolymers to determine whether FANCD2 is able to bind RNA. In vitro binding assays were performed to determine whether FANCD2 is able to bind R-loop nucleic acid structures. siRNA transfections were used to reduce protein levels of splicing factors. Survival assays were performed to determine cell sensitivity to the interstrand crosslinking drug mitomycin C (MMC) and the topoisomerase I inhibiting drug camptothecin (CPT).

Result:
In vitro binding studies with ribohomopolymers revealed that the non-ubiquitylated form of FANCD2 is able to bind polyG RNA preferentially. Because G-rich RNA is associated with R-loop formation at IgG switch regions (Yu et al, Nat. Immunology, 2003), we wanted to determine whether FANCD2 is also able to bind R-loops. A human recombinant protein complex containing FANCD2, FANCI, and FANCE was shown to bind R-loop structures in vitro. Since depletion of the splicing factor ASF/SF2 has been demonstrated to increase cellular genomic instability through the formation of increased levels of R-loop structures (Li et al, Cell, 2005), we next wanted to determine what effect depletion of ASF/SF2 would have on the FA pathway. siRNA mediated knock down of ASF/SF2 protein levels resulted in decreased levels of FANCD2 ubiquitylation following treatment with MMC. As FANCD2 ubiquitylation is often considered a marker of activation of the FA pathway, this result suggests that depletion of ASF/SF2 inhibits activation of the FA pathway. siRNA mediated knock down of ASF/SF2 also increased cellular sensitivity to MMC and CPT. siRNA mediated knock down of the splicing factor SC35 yielded a similar result, inducing an increase in sensitivity to MMC and CPT and reducing levels of FANCD2 ubiquitylation. However, siRNA mediated knock down of a third splicing factor, SRp55, had no effect on FANCD2 ubiquitylation or cellular sensitivity to MMC and CPT.

Conclusion:
These results suggest that R-loop formation may be a source of some of the genomic instability present in FA cells. We also demonstrate that splicing factors may play a role in activation of the FA pathway. These findings may potentially provide new targets for therapeutic treatments for FA.

Disclosures:
No relevant conflicts of interest to declare.
Genome-Wide Single-Nucleotide Polymorphism-Array Based Karyotyping Detects Clonal Aberrations, and Predicts the Risk of Imatinib Failure In Chronic Myeloid Leukemia.
Authors: Han Boram , Huh Jungwon , Yi Jun Ho , Lee Ha Yeon , Kim Jong-Won , Kim Hee-Jin , Kim Yeo-Kyeoung , Kim Hyeoung Joon , Moon Joon Ho , Sohn Sang Kyun , Jung Chul Won , Kim Dong Hwan (Dennis) 
Published: October 2019

Abstract
Abstract 3387

Background:
The current study investigated the diagnostic and clinical role of genome-wide, single nucleotide polymorphism arrays (SNP-A) which can detect microscopic copy number changes in chronic myeloid leukemia (CML).

Methods and Materials:
Genome-Wide Human SNP 6.0 Array (Affymetrix, CA, USA) was used for SNP-A karyotyping analysis in 132 CML patients. All patients were treated with imatinib and treatment outcomes were compared according to the presence of clonal aberrations (CAs) detected by SNP-A or additional cytogenetic abnormalities (ACAs) detected by metaphase cytogenetics (MCs).

Result:
Forty four clonal aberrations were identified in addition to t(9;22) (40 losses, 2 gains, 2 loss of heterozygosity [LOH]) that were not detected by MCs. The 9q34 deletions were found in 11% of cases, while 22q11.2 deletions were observed in 14% of cases. Nine cases harbored both 5'-ABL and 3'-BCR deletions adjacent to the t(9;22) breakpoint (7%). Copy number gains were identified at 8p and 9p, and losses at 2q, 7q, 8q, 11q, 13q, and 16p. Two cases with variant translocation showed additional deletions on the 3rd chromosome with involvement in variant translocations, which were missed by MC. We defined 3 commonly deleted regions (CDRs) on 9q34 and 22q11.2: CDR1 on 9q34 spanned approximately 162 kb between 9q34.11 and 9q34.12; CDR2 on 22q11.23 spanned 138 kb; CDR3 on 22q11.23 encompassed 102 kb.
When we compared treatment outcomes according to the combination of the presence of CAs by SNP-A and/or ACAs, the patients having both CAs and ACAs showed a higher risk of failure following imatinib therapy than those with either CAs/ACAs or none (p=0.015).

Conclusion:
The present study suggests that SNP-A analysis is a useful tool for detection of clonal aberrations in the CML genome, and could improve the prediction of prognosis in CML patients. In addition, our data suggest that SNP-A analysis is a useful tool to detect cryptic aberrations in CML genome, could improve the prediction of prognosis in CML patients and enables us to delineate CDRs on 9q34 and 22q11.2 which might be associated with CML leukemogenesis.

Disclosures:
No relevant conflicts of interest to declare.
Hammersmith Score Is Able to Identify Chronic Myeloid Leukemia Patients with Poor Prognosis Before Treatment with Second-Generation TKIs.
Authors: Breccia Massimo , Latagliata Roberto , Stagno Fabio , Gozzini Antonella , Abruzzese Elisabetta , Rossi Antonella Russo , Santopietro Michelina , Cannella Laura , Sorà Federica , Vigneri Paolo , Trawinska Malgorzata , Sica Simona , Specchia Giorgina , Santini Valeria , Alimena Giuliana 
Published: October 2019

Abstract
Abstract 3409
A score aiming at early identification of CML patients showing sensitivity to second generation TKIs was proposed by the Hammersmith group. The score was created by analizing 80 patients and was based on 3 prognostic factors: previous cytogenetic response to imatinib, Sokal risk and recurrent neutropenia during imatinib. Subsequently, the score was validated in a small series of 28 patients. Aim of our study was to confirm the validity of this score and to establish its strength on a large group of CML patients resistant to imatinib and treated with second generation TKIs. One hundred twenty-seven patients were collected from 6 different Italian hematologic centers. There were 66 males and 61 females, median age 54 years (range 25–80). Twenty-seven patients received interferon before imatinib. Thirty patients had primary resistance, whereas 97 patients received second-generation TKI after acquired resistance to imatinib. The application of Hammersmith score was possible in 118 patients with available data: 52 patients were identified as good risk, 27 patients as intermediate risk and 38 patients as poor risk. The 1-year cumulative incidence of complete cytogenetic response (CCR) was 73% in good risk patients, 40% in intermediate risk patients and 23% in poor risk patients (p=0.0001). Similarly, the cumulative incidence of major molecular response (MMR) was 52% in good risk, 28% in intermediate risk and 13% in poor risk category (p=0.001). In the evaluation of event-free survival (EFS), events were considered loss of hematologic or cytogenetic response, disease progression, death for any cause, toxicity: the estimated 2-year event-free survival (EFS) was 89% in good risk, 70% in intermediate risk and 55% in poor risk group (p=0.0001). Progression-free survival (PFS) was defined as survival without evidence of accelerated or blastic phase: the estimated 2-year PFS was 97% in good risk, 93% in intermediate risk and 87% in poor risk category (p=0.05). Kaplan Meier estimated 2-year overall survival (OS) was 100% in the good risk, 93% in the intermediate risk and 82% in the poor risk category (p=0.001). In conclusion, as suggested by Milojkovic et al, some prognostic factors before starting second generation TKIs might predict cytogenetic response and outcome. As far as we known, the so-called Hammersmith score was not yet validated in large series of patients: we demonstrated that this score was able to discriminate patients at high risk of failure and consequent progression before treatment with second generation TKIs.

Disclosures:
No relevant conflicts of interest to declare.
Large Granular Lymphocyte Expansion Following Allogeneic Hematopoietic Stem Cell Transplantation Is Associated with the Presence of CMV Reactivation and Probably with Acute GvHD and Shows An Indolent Outcome.
Authors: Nann Sabine , Tzankov Alexander , Cantoni Nathan , Halter Jörg , Heim Dominik , Tsakiris Dimitrios , Arber Caroline , Buser Andreas , Gratwohl Alois , Tichelli Andre , Rovo Alicia 
Published: October 2019

Abstract
Abstract 3461
Expansion of CD3+ large granular lymphocytes (T-LGL) can be observed in situations such as viral infection, autoimmune disease, malignant neoplasm, and following allogeneic hematopoietic stem cell transplantation (HSCT). We sought to evaluate in patients treated with allogeneic HSCT incidence, characteristics, and clinical significance of persistent post-transplant T-LGL expansion. In this single center retrospective cohort study, we included all patients seen between January/08 and December/09 in our out-patient clinic for their regular follow-up. In patients with persistent lymphocytosis (>3 G/l for >3months) and an abnormal CD4/CD8 ratio, an extensive immunophenotyping was assessed; in case of an abnormal expansion of T-LGL cells a TCR gene rearrangement was performed. In 14 (7%) out of 215 evaluated patients a T-LGL expansion was diagnosed. Patients' characteristics with and without T-LGL expansion are summarized in Table 1. The median time between HSCT and diagnosis of lymphocytosis was 12 months (1-58). The median lymphocyte count was 4.24 G/l (3.0-26.5). The median duration of lymphocytosis was 29 (4-176) months. In 13/14 cases there was a CD3+/CD8+ immunophenotype, in 1 case was CD3+CD4+. In 5/14 patients a clonal TCR-gene rearrangement was observed. None of the patients presented neutropenia. Mild anemia was observed in 8/14 patients (57%), and thrombocytopenia in 2/14 patients; both changes were most probably not related with the T-LGL expansion. None of the patients had typical clinical signs of a T-LGL leukemia. In the univariate analysis acute GvHD and CMV reactivation were the only variables associated with T-LGL expansion, In the multivariate the relative risk (RR) of CMV reactivation was 5.063 (95%CI: 1.586–16.160; p=0.006) and the RR of acute GvHD grade 2–4 was 2.831 (95%CI: 0.831–9.648; p=0.096).

Conclusion:
we detected a T-LGL expansion in 7% of patients after HSCT. No symptoms or clinical signs related to T-LGL leukemia were observed. The T-LGL expansions, even when monoclonal, showed a chronic but indolent course. They have to be considered rather as an expression a chronic stimulation, triggered by causes such CMV reactivation or acute GvHD rather than as a malignant transformation. The question whether a T-LGL expansion plays a GvL role could not be answered in this study due to the small number of patients and the study design.

Disclosures:
No relevant conflicts of interest to declare.
Treatment of FLT3-ITD Positive AML Relapsing After Allogeneic Hematopoietic Stem Cell Transplant (HSCT) with Sorafenib.
Authors: Sharma Manish R , Ravandi Farhad , Chiattone Alexandre , Kebriaei Partow , Popat Uday , Giralt Sergio , Bashir Qaiser , Qazilbash Muzaffar H. , Nieto Yago , Yuen Carrie Hai-Lai , Parmar Simrit , Kantarjian Hagop M. , Cortes Jorge , Kadia Tapan , Borthakur Gautam , Champlin Richard E. , Lima Marcos De 
Published: October 2019

Abstract
Abstract 3471
AML with the internal tandem duplication of the Fms-Like Tyrosine kinase-3 gene (FLT3-ITD) has a poor prognosis, with high relapse rates. Sorafenib is an oral multikinase inhibitor of tyrosine kinases including Flt-3 with activity in AML. We used this drug to treat patients relapsing after allogeneic HSCT and herein present the results.

Patients and Methods.
We retrospectively evaluated 16 patients with FLT3-ITD AML that received sorafenib for at least a 7-day course either alone (n=8; 50%) or in combination (n=8; 50%) to treat AML relapsing at a median of 3 months (range, 1–7) after HSCT. Median age was 34 years (range, 20–603). Cytogenetics was diploid in 69%, high-risk in 19% and unknown in 12%. Treatment history prior to HSCT included sorafenib in 38% of the cases, and 30% were status post a 2nd HSCT. Donors were related (50%) or unrelated (50%). Seven patients (44%) received and failed other salvage chemotherapy prior to starting sorafenib (median=2 treatments; range, 1–5). At start of sorafenib salvage treatment, median WBC was 22.5 K/mm3 (range, 0.6–119), and median bone marrow blast was 58% (range, 12–88%), while 75% of the patients had circulating blasts. Sorafenib doses were as follows: 400 mg (n=3) or 800 mg daily (n=4) in combination to idarubicin/Ara-C, or 800 mg daily with 5-azacitidine (n=1); single agent: 800 mg (n=6) or 1200 mg (n=2) daily. Median duration of sorafenib single agent therapy was 39.5 days (range, 10–100), and in combination it was 7 days (range, 7–32). Complete remission (CR) rate was zero; 2 patients achieved a PR. Median reduction in bone marrow (n=9) and peripheral blood (n=12) blasts was 0% (range, 0–46), and 50% (range, 0–88), respectively. Two patients were ‘bridged’ to a 2nd HSCT, which led to CR durations of 53 and 106 days. Median survival after sorafenib initiation was 81 days and it was similar with single agent versus combination therapy (Figure 1 and 2). All patients have died.

Conclusions.
Treatment of overt AML relapse with sorafenib was unsuccessful as described here. It is possible that ‘pre-emptive’ treatment in the presence of minimal residual disease will be more effective, but this remains to be proven.

Disclosures:
No relevant conflicts of interest to declare.