bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Throughput Screening Reveals Unique Sensitivity of CD34+CD38- AML Cells to Isotretinoin and Tretinoin
Authors: Xie Wen , Hassane Duane C , Jordan Craig T. , Roboz Gail J. , Guzman Monica L. 
Published: October 2019

Abstract
Abstract 1430
Acute myeloid leukemia (AML) is a heterogeneous disease, originating from leukemic stem cells (LSCs). This relatively rare sub-population shows mostly a CD34+CD38- immunophenotype and has been proposed to be responsible for disease relapse and chemotherapy resistance. In order to identify effective drug(s) for clinical use that can eliminate LSCs, we have screened a library of 2000 compounds from the SPECTRUM collection (MicroSource Discovery Systems, Inc.) using a multi-parameter flow cytometry high-throughput screening. The drugs compiled in the library are 40% US FDA approved drugs, 30% natural products, 10% approved drugs for use in Europe and Japan, and the remainder consistent of bioactive compounds.
The initial screen aimed to identify potential compounds that can decrease viability or increase differentiation of AML cells at 24 and 72 hours of treatment. Compounds demonstrating autofluorescence were eliminated as false positive hits.
We found 137 compounds that decreased viability in all populations. Furthermore, 37 drugs demonstrated ability to increase cell death (more than 50%) in phenotypically described LSCs, progenitor and blasts populations, but with viability over 50% for normal residual lymphocytes within the same AML specimen. Interestingly, only 4 drugs displayed higher specificity to LSCs when compared to total blast populations. Surprisingly, two of these drugs are different forms of retinoic acid (isotretinoin and tretinoin).
The selectivity of isotretinoin and tretinoin was confirmed in different primary AML samples. We observed that phenotypically described stem cell subpopulations were affected when exposed to retinoic acid in 4 out of 5 AML samples. LSC populations display increased apoptosis and differentiation assessed by flow cytometry. Characterization of what confers sensitivity to these types of drugs is under investigation. Together, a multiparameter flow cytometry screen revealed specific anti-LSC activity for retinoid acid, which was not detectable when analyzing total populations and would have been missed using traditional screening. These studies underscore the utility of LSC-based assays for defining novel therapies and suggest that retinoid-based therapies merit further investigation in AML.

Disclosures:
No relevant conflicts of interest to declare.
High Incidence of Fanconi Anemia in Patients with a Morphological Picture Consistent with Refractory Cytopenia of Childhood
Authors: Yoshimi Ayami , Niemeyer Charlotte M. , Baumann Irith , Schwarz-Furlan Stephan , Schindler Detlev , Ebell Wolfram , Strahm Brigitte 
Published: October 2019

Abstract
Abstract 2780

Introduction:
Refractory cytopenia in childhood (RCC) is the most common subtype of myelodysplastic syndrome (MDS) in children. Differential diagnosis from inherited bone marrow failure (IBMF) such as Fanconi anemia (FA) remains an intriguing challenge, because most patients with RCC have a hypocellular bone marrow (BM) and dysplastic features in haematopoiesis are observed in both RCC and IBMF. Moreover the spectrum of phenotypic findings in FA is extremely wide. Some FA patients have a mild phenotype without malformation. The purpose of this study is to estimate the incidence of FA in an RCC cohort without a full clinical feature of FA, but subsequently diagnosed by chromosome breaking test.
Patients and Methods: Between 01/2007 and 12/2010 reference pathologists of the European Working Group of MDS in Childhood (EWOG-MDS) provided a morphological report consistent with RCC in 137 children studied in Germany. Seventeen patients with hypercellular BM or abnormal karyotype, 2 patients, in whom dyskeratosis congenital was diagnosed after initial inclusion and one patient, in whom chromosome breaking test was not performed, were excluded.

Results:
Seven of remaining 117 patients had facial and/or skeletal anomalies typically noted in FA and one patient had a brother with FA. In these 8 patients, FA had been suspected by their local physicians (group FA-1). Nine patients (8.3%) without these typical anomalies were subsequently diagnosed of FA by chromosome breakage test (group FA-2). The diagnosis of RCC was finally made in the remaining 100 patients with negative chromosomal breakage test (group RCC). The clinical features of patients in each group are summarized in the Table. The mean corpuscular volume of red cells (MCV) was elevated (> +2SD) for ages in all patients with FA, but only 42 % in patient with RCC. In some children of group FA-2 additional non-haematological abnormalities were also observed. However, they were not evident and or typical to prompt the treating physicians to suspect FA. A few patients in the group RCC also had some physical anomalies, not specific for any of the known IBMF disorders. Possibly that other known or not yet described IBMF disorders remain uncovered in children with “de novo” RCC.

Conclusion:
Our results illustrate that the same haematological features and congenital anomalies can be noted in FA and RCC. More importantly, they indicate that the exclusion of FA by a chromosomal breakage test or other methods is mandatory in all patients prior to diagnosis RCC. Chromosomal breakage analysis may identify patients with FA in 8% of patients with a morphological description of RCC without a full clinical picture of FA.

Disclosures:
No relevant conflicts of interest to declare.
Identification and Characterization of Human Leukemia Stem Cell Functional Regulators
Authors: Eppert Kolja , Hermans Karin G , Dick John E. 
Published: October 2019

Abstract
Abstract 2955
Emerging evidence shows that many cancers are organized as cellular hierarchies sustained by a subpopulation of cancer stem cells (CSC). It is thought that CSC properties influence therapy response, disease relapse, and overall survival. However, little is currently known about the molecular pathways that control stem cell behaviour. Our goal is to identify and characterize the molecular regulatory networks that govern the self-renewal and developmental program of human leukemia stem cells (LSC).
To expand our understanding of LSC function, we selected genes from our previously established LSC transcriptional program as novel candidate regulators of stem cell function. The LSC transcriptional program was identified by fractionation of 16 primary human acute myeloid leukemia (AML) samples into four populations that were subjected to sensitive in vivo LSC assays. mRNA expression analysis was performed on each fraction and a global LSC-specific signature was determined from functionally defined LSC fractions. Using similar methodology, a hematopoietic stem cell (HSC) enriched gene signature was established from human cord blood. Bioinformatic analysis identified a core transcriptional program that LSCs and HSCs share, revealing the molecular machinery that underlies stemness properties. The LSC and HSC signatures have prognostic significance independent of other factors when validated on a large cohort of cytogenetically normal AML patients. We have begun experiments to determine the role of 17 candidate stem cell regulators using functional in vivo and in vitro assays.
We first examined the effect of candidate gene overexpression in normal HSC using xenograft assays. Lineage negative cord blood cells were transduced with lentiviral overexpression vectors, injected into immune-deficient mice and engraftment of human cells was measured after 12 weeks. In the preliminary round of in vivo analysis, over-expression of GPR56 increased the engraftment capability of HSCs in immune-deficient recipients compared to control (p=0.0019). GPR56 is involved in cell adhesion and differentiation and evidence is accumulating for a role in various cancers such as melanoma, esophageal cancer, and glioma. By qPCR analysis we observed that GPR56 mRNA is more highly expressed in both LSC-enriched and primitive normal hematopoietic cell populations, including highly purified HSC, compared to more mature cell populations such as AML blasts and normal lineage committed progenitor cells.
In conclusion, our LSC and HSC data established that determinants of stemness influence clinical outcome of AML patients. We have identified multiple novel candidate stem cell-related genes and provided evidence for a role for GPR56 in hematopoietic stem cell regulation.

Disclosures:
No relevant conflicts of interest to declare.
Deficiency of CD73/Ecto-5'-Nucleotidase Enhances Acute Graft-Versus-Host Disease
Authors: Chernogorova Petya , Tsukamoto Hiroki , Ayata Korcan , Gerlach Ulrike V. , Rughani Ankur , Ganesan Jayanthi , Follo Marie , Thompson Linda F. , Idzko Marco , Zeiser Robert 
Published: October 2019

Abstract
Abstract 312
Acute graft-versus-host disease (GvHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT). We have recently shown that extracellular adenosine triphosphate (ATP) enhances GvHD by activation of the purinergic receptor P2X7R. The abundance of extracellular ATP is regulated by ecto-nucleotidases such as CD39, which dephosphorylate ATP to ADP and AMP. Ecto-5'-nucleotidase (CD73) degrades AMP to adenosine, which can itself exert tolerogenic functions via activation of A2A and/or A2B adenosine receptors (AR). Extracellular ATP and adenosine have immunoregulatory roles under different inflammatory conditions. Deficiency of CD73, the enzyme that metabolizes AMP to adenosine, could therefore have pro- or antiinflammatory activity by reducing adenosine levels in the microenvironment. Currently, the roles of CD73 and adenosine in GvHD are unclear.
By using cd73−/− donor or recipient mice and a specific CD73/ecto-5'-nucleotidase inhibitor we observed enhanced GvHD severity when CD73 was genetically deleted or pharmacologically blocked. CD73 was upregulated on CD4+ and CD11c+ cells after irradiation, suggesting an inducible rescue mechanism along with CD39 to counteract ATP accumulation resulting from tissue damage due to the preconditioning treatment. CD73 deficiency led to enhanced T cell expansion and IFN-γ and IL-6 production in GvHD mice. Migratory capacity of cd73−/− T cells was increased, compatible with the ability of adenosine to inhibit transendothelial migration. A2A receptor deficiency led to increased numbers of proliferating allogeneic T cells in allo-HCT recipient mice, suggesting that signaling through the A2A receptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GvHD. Furthermore, pharmacological blockade of CD73 improved graft-versus-tumor activity in an in vivo B cell leukemia model.
In conclusion, we demonstrate that CD73 plays a protective role in GvHD with enhanced disease severity in its absence, while blocking CD73 led to increased graft-versus-tumor effect. These data have potential clinical implications as the alloimmune response could be enhanced by CD73 blocking in patients with residual tumor burden after transplantation. Conversely, immune modulation by addition of CD73 enzyme or an adenosine receptor agonist could improve the outcome of GvHD patients.

Disclosures:
No relevant conflicts of interest to declare.
Persistent Endothelial Activation Is Associated with An Increased Mortality During Therapy of Children with ALL – a Pilot Study
Authors: Niedzielska Ewa , Doroszko Adrian , Porwolik Julita , Krupska Ewa Szahidewicz- , Chybicka Alicja , Szuba Andrzej 
Published: October 2019

Abstract
Abstract 4232

Background:
Activation of endothelial cells is associated with a worse prognosis in numerous disorders treated at intensive care units. The goal of this study was to analyze if an activation of vascular endothelium despite of the treatment with the ALLIC protocol in children with ALL might have a predictive value for the survival rate.

Methods:
N=18 children at age of 4–18 years with ALL treated with the ALLIC protocol were investigated. Plasma levels of markers of endothelial activation (VCAM-1, ICAM-1, VEGF, E-selectin, P-selectin and PAI-1) were analyzed at baseline, during the 33rd and 78th day of therapy (beginning of the protocol M).

Results:
Higher baseline levels of ICAM-1 (467.1±72.8 vs. 206.3±21.7 ng/ml, p<0.05) and E-selectin (56.1±10.9 vs. 28.1±10.0 ng/ml, p<0.05) were observed in children treated unsuccessfully. Moreover, an increase in E-selectin levels and a non-decreasing trend in ICAM-1 levels within the first three days of the ALLIC protocol as well as significant increase in the PAI-1 levels were positive predictors for death in children with ALL.

Conclusions:
In this pilot study, we have demonstrated for the first time that persistent pro-inflammatory and pro-aggreagatory activation of endothelium might be a positive predictor for death in children with ALL.

Disclosures:
No relevant conflicts of interest to declare.
Combination of Mitoxantrone,Ara-C and Homoharringtonine In Treatment of Refractory and Relapsed Acute Myeloid Leukemia
Authors: Hu Jianda , Liu Tingbo , Cai Chunxia , Chen Xinji , Chen Buyuan 
Published: October 2019

Abstract
Abstract 4294
Advances in effective chemotherapy have improved clinical outcomes in acute leukemia in recent years. 5-year survival rate approaches 50–60% for acute myeloid leukemia (AML). However, the prognosis remains poor for patients who are relapsed or refractory to first-line therapy. Drug resistance and early disease recurrence are major contributing factors in the limited survival of patients with AML. The strategy for treating these patients is through reinduction chemotherapy followed by allogeneic stem cell transplantation. New combinations of different agents were employed in refractory patients to overcome drug resistance. The current study is to evaluate the efficacy of a MAH regimen comprising Mitoxantrone,Ara-C and Homoharringtonine in refractory or relapsed AML. 37 patients aged 14–65 years with refractory or relapsed AML (15 refractory AML patients, 22 relapsed AML patients) were treated with the MAH regimen(Mitoxantrone 10mg qd, iv.gtt, for 2□‘3 days;Ara-C 100mg bid, iv.gtt, for 5□‘7 days; Homoharringtonine 4mg qd iv.gtt, for 5□‘7 days). Chemotherapy duration lasted for 5 or 7days depended on bone marrow cellurarity. 15 (40.5%)and 1 (2.7%) patients achieved complete remission (CR) and partial remission (PR) respectively. The overall response rate was 43.2%. There was no relation between remission duration and previous chemotherapy. All patients who achieved CR received a consolidation and intensification therapy. The median overall survival (OS) for all patients was 97 days (range 18–487 d). For the patients who were in CR or PR,the median relapse-free survival(RFS) was 147 days(range 4 to 341 d). All patients experienced profound myelosuppression. The most common observed side effect of the regimen was infection because of grade ‡W neutropenia, which could be observed in 33 patients(89.1%). 4 patients died in aplasia due to severe infection and brain hemorrhage. In patients achieving remission, the median time to reach absolute neutrophil count (ANC) more than 0.5×109/l was (16.0±6.4)d. Platelet levels of more than 20×109/l were achieved in a median time of (12.7±6.2)d. Nonhematological side effects, consisting mainly of gastrointestinal toxicity(21/37,56.8%) and transient liver ALT and AST increase (4/37), were generally mild to moderate and tolerated. To a conclusion, MAH regimen can be employed in treatment of the refractory or relapsed AML patients who were not responded to other regimen. It is effective and is good tolerant.It could provide some refractory patients the chance to receive hematopoietic stem cell transplantation.

Disclosures:
No relevant conflicts of interest to declare.
Findings of Feasibility Assessment From Multicenter Clinical Trials of Antithrombotic Therapy in Pediatric Venous Thromboembolism
Authors: Wallace Amy E , Young Guy , Manco-Johnson Marilyn J. , Goldenberg Neil A. 
Published: October 2019

Abstract
Abstract 4323
BACKGROUND:Two major multicenter pediatric randomized controlled clinical trials (RCTs) in pediatric venous thromboembolism (VTE) have been conducted in the last decade, and each was closed early due to poor accrual. With the recent trend in reduced federal grant support for clinical trials, an emphasis on demonstration of feasibility is critical.OBJECTIVE: In order to inform the design and execution of future RCTs in pediatric VTE, we sought to report findings of feasibility assessment from two ongoing and one proposed clinical trials in the field. METHODS: In the UNBLOCK study (ongoing phase 1 trial of bivalirudin in acute DVT; clinicaltrials.gov registration #NCT00812370), feasibility was assessed informally in 2008/9 during grant development teleconferences and early investigator meetings. In the Kids-DOTT trial (ongoing phase 3 RCT of shortened vs. conventional duration of therapy in acute VTE associated with a transient/reversible clinical risk factor; NCT00687882), feasibility was assessed in 2005 and updated in 2011 using standardized questionnaires, with response data summarized via descriptive statistics. The latter approached was also used in 2011 for the PHLO trial (proposed phase II/III RCT of catheter-directed thrombolysis followed by therapeutic anticoagulation vs. anticoagulation alone in completely veno-occlusive acute DVT of the proximal leg). RESULTS: Response rate (Kids-DOTT and PHLO questionnaires) was 100%. Number of investigative sites/principal investigators assessed and geographical distribution, by trial, were as follows: UNBLOCK: n=6 (all USA); Kids-DOTT: n=26 (25 USA, 1 Canada); PHLO: n=7 (all USA). Of the 26 site PIs in Kids-DOTT, 84% reported past experience with 5 or more trials, with a median experience of 20 per PI. Across all 3 trial feasibility assessments, all but one site used electronic health records in clinical care, and all but one investigative team had trial experience in using electronic data capture systems. With regard to size of the screening population for pediatric VTE trials, Figure 1 shows the reported average annual volume of new pediatric VTE cases managed by each Kids-DOTT investigator/site, using data from the prior 2 years. All sites reported use of inpatient/consult lists in screening efforts. CONCLUSIONS: These findings indicate appropriate site experience and capacity to execute multicenter clinical trials in pediatric VTE. Therefore, feasibility of ongoing and future trials in the field is more likely related to such issues as: suitability of funding and number of sites to targeted enrollment; experience/capabilities of the clinical coordinating and data coordinating centers; and pragmatic protocol design, particularly eligibility criteria, enrollment/randomization windows, and frequency, timing, and invasiveness of follow-up and endpoint determinations.

Disclosures:
No relevant conflicts of interest to declare.
Use of Emergency-Release, Uncrossmatched O Negative Red Blood Cells (RBCs) in a Tertiary Care Children’s Hospital
Authors: Andrews Jennifer C. , Viele Maurene , Goodnough Lawrence T 
Published: October 2019

Abstract
Abstract 4341

Background:
Transfusion services must offer means of issuing blood products quickly for emergent transfusions. At Lucile Packard Children’s Hospital (LPCH), off-site refrigerators were installed in the Neonatal Intensive Care Unit (NICU), the LPCH operating room (OR) and the Cardiovascular Intensive Care Unit (CVICU) inventoried with uncrossmatched O negative RBCs for immediate emergency use. Uncrossmatched O negative RBCs are also provided to children undergoing cardiac catheterization at the discretion of the Pediatric Cardiologist, since these patients commonly do not have type and screen samples drawn until large vessel venous cannulation via insertion of the catheter. Uncrossmatched blood products are also provided for children per our massive transfusion protocol (MTP). The purpose of this study was to assess the utilization of these uncrossmatched blood products in children and its impact on transfusion service (TS) inventory of O negative RBCs.

Methods:
Orders received for emergency-release uncrossmatched RBCs for patients ages 0 days to 18 years including MTPs from January 1 2011 to March 31 2011 were evaluated retrospectively. Variables collected include: patient demographic information and diagnosis; blood products ordered, released and transfused; location of the patient and location from where blood was dispensed (off-site refrigerator versus [vs] TS).

Results:
Median patient age was 3.46 years (range 0 days to 15.62 years), and 82% of the patients had congenital heart disease. Sixty four RBCs were issued to 33 patients during the 3-month study period. Of those, 32 RBCs were transfused, 8 RBCs were wasted because temperature parameters were exceeded before return to the TS, and 24 RBCs (38%) were not transfused and returned to TS inventory. Nineteen of the 32 RBCs were transfused, representing 2% of the total 964 O negative RBC units transfused at our institution for that time period. Nineteen (58%) units were for children in the cardiac catheterization suite. Seven patients were in the CVICU, five children were in the NICU, and two were in the Pediatric Intensive Care Unit. Two units of O negative RBCs were dispensed from the emergency off-site refrigerator in the CVICU.

Discussion:
The majority of children (79%) who received emergency-release uncrossmatched O negative RBCs at LPCH were those with congenital heart disease undergoing cardiac catheterization or being cared for post-operatively in the CVICU. There were 2 instances of children requiring RBCs from off-site refrigerators for an emergent transfusion. Emergency-release, uncrossmatched O negative RBCs at LPCH either from the TS or from three off-site refrigerators caused no undue strain on our supply and inventory of donor O negative RBCs.

Disclosures:
No relevant conflicts of interest to declare.