bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow Cytometry-Based Maturity Score As a Novel Prognostic Parameter in AML
Authors: Flörcken Anne , Tanja Schneider , Singh Anju , Türkmen Seval , Thomas Burmeister , Anagnostopoulos Joannis , Pezzutto Antonio , Dörken Bernd , Westermann Jörg 
Published: October 2019

Abstract
Background: Based on cytogenetic and molecular phenotypes, the European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). Single leukemic blast cell antigens were also shown to have prognostic value, but the influence of AML blast maturity within the different ELN risk groups is largely unknown.
Methods: Our study includes 300 AML patients, who were diagnosed at our institution between 01/2003 and 04/2012 and in whom morphologic, immunophenotypic, cytogenetic and molecular data from primary diagnosis were available. A flow cytometric maturity score was developed in order to distinguish mature AML (AML-ma) from immature AML (AML-im) by means of a quantitative analysis of the expression of early progenitor cell antigens (CD34, CD117, and TdT). Clinical outcome analysis was performed both in the whole cohort and in the different ELN risk groups using Chi Square and Fisher's Exact Test (CR/CRi) and Logrank Test (RFS, OS).
Results: A mature AML blast phenotype (AML-ma) was associated with a significantly longer RFS (7.5 vs. 4.3 years) and OS (5.3 vs. 2.7 years) than an immature phenotype (AML-im) (p < 0.001). Low-risk phenotypes according to ELN (NPM1, PML-RARA) significantly accumulated in the AML-ma group, whereas high-risk aberrations (monosomal and complex aberrant karyotypes, -5 or del(5q), -7) had a higher frequency in AML-im.
Interestingly, the differences in RFS and OS were maintained in a subgroup analysis within the different ELN risk groups. Statistical significance was obtained in the intermediate risk group with AML-ma being superior to AML-im (RFS: 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS: 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). In contrast, there was no correlation between AML blast maturity and CR/CRi rates.
Conclusions: Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome, even within the different ELN risk groups. Particularly for intermediate risk patients according to ELN, the score may be useful in post remission therapy decision-making. It remains to be clarified whether these results reflect differences in genetic instability and/or a different frequency of leukemia stem cells in AML-ma and AML-im.
Figure 1 Figure 1. Figure 2 Figure 2.
RFS (A) and OS (B) of AML patients with mature (AML-ma) and immature (AML-im) blast phenotype within the ELN intermediate risk group. AML-ma show a significantly longer RFS and OS compared to AML-im.

Disclosures
No relevant conflicts of interest to declare.
Single Cell Whole Exome Sequencing in NPM1/Flt3 Positive Pediatric Acute Myeloid Leukemia
Authors: Walter Christiane , Hofmann Winfried , Reinhardt Katarina , Reinhardt Dirk , von Neuhoff Nils 
Published: October 2019

Abstract
Introduction:
Acute myeloid leukemia (AML) is one of the most frequent forms of leukemia in children younger than 15 years. The detection of several mutations in a blast population of pediatric AML (pAML) is supposed to be caused by a clonal evolution from a leukemic stem cell (LSC) to leukemic blasts. LSC are believed to be more resistant to chemotherapy, to be able to survive during treatment and to be responsible for the emergence of a relapse due to the persistence in the bone marrow (BM) niche. Since LSC and potential leukemic subclones are only present in small subpopulations, it has been a major technical challenge to particular analyse only the specific population. To acquire a better understanding of the underlying mechanisms of mutagenesis, clonal evolution and leukemogenesis, the aim of this study was to establish methods that allow the analysis and detection of mutations in single cells of a subpopulation known to contain HSC as well as LSC (CD34+CD38-). We especially focused on a pAML subgroup with mutations in Nucleophosmin (NPM1) and/or fms related tyrosine kinase 3 (Flt3).
Methods and Results:
We established methods to perform single cell sorting, whole genome amplification (WGA) using multiple displacement amplification (MDA) technology (Qiagen) and subsequent whole exome sequencing. The sorting efficiency was checked as Hoechst stained cells were sorted onto glas slides with 48 defined spots and the presence of single cells was checked under an inverse fluorescent microscope. Subsequently, single CD34+CD38- patient derived cells were sorted into 0,5ml low binding tubes containing 4µl PBS followed by WGA and whole exome sequencing. The mutational status of the sorted single cells from three patients suffering from pAML was analysed and compared to mutations detected at initial diagnosis in DNA from a bulk of BM cells. WGA from single CD34+CD38-PI- cells resulted in an amount of 29 to 31.7µg DNA from each of five single cells. The quality of the amplified DNA was sufficient for whole exome sequencing. A 4bp insertion in exon 12 of NPM1 reflecting a common NPM1 mutation (MutA) initially detected from a bulk of cells was identified in amplified DNA from single cells using whole exome sequencing in 2/3 patients. Internal tandem duplications in Flt3 indicated by mismatches in the alignment could be detected in amplified DNA from single cells of two patients. The detected ITD resemble those initially detected in DNA from a bulk of BM cells.
Discussion and Conclusion:
Single cell sequencing provides a useful tool to amend the detection of genetic aberrations from a bulk of cells and to confirm the presence of specific mutations in single cells from small subpopulations. It therefore helps to get further insights into the clonal evolution in pAML.

Disclosures
No relevant conflicts of interest to declare.
Identification of Cytogenetically Normal Human CD34+CD38+ Hematopoietic Stem/Progenitor Cells from Inv(16)+ Leukemic Bone Marrow
Authors: LeGrand Jason N. , Heidemann Stephanie C. , Swindle C. Scott , Klug Christopher A. 
Published: October 2019

Abstract
For many subtypes of AML including cases with the inv(16), mutations that give rise to the leukemic phenotype occur, at least in part, in the hematopoietic stem/progenitor (HSPC) cell subset, as suggested by studies showing that primitive CD34+ CD38- bone marrow cells can function as leukemia-initiating cells (LIC) when transferred into immunodeficient mice. A significant challenge has been that LIC share many of the same cell-surface markers as their normal HSPC counterparts, thus making it difficult to purify and functionally characterize either subset from the bulk bone marrow of leukemia patients. Here we report the FACS analysis of several previously reported human LIC markers on bone marrow samples from inv(16) AML patients and show that a combination of TIM3, CLL1, and CD33 can significantly enrich for a rare population of CD34+ CD38- cells that lack the inv(16) fusion mRNA when tested by nested RT-PCR. Heterogeneous expression of these markers among different patient samples often causes incomplete elimination of the fusion mRNA when FACS-sorting the CD34+ CD38- population as single TIM3-, CLL1-, or CD33- subsets. The combination of TIM3 with CLL1 and/or CD33 leads to a more consistent elimination of the fusion mRNA from the FACS-sorted CD34+ CD38- subsets. Results from methylcellulose assays showed that the TIM3- CLL1- CD33- subset of CD34+CD38- cells could form multiple colony types, including CFU-GEMM, that were all negative for the fusion mRNA by RT-PCR. In contrast, colonies derived from bulk bone marrow were all positive for the fusion mRNA. The TIM3- CLL1- CD33- subset of CD34+CD38- cells displayed greater than 600-fold enrichment for progenitor activity compared to bulk bone marrow but did not form additional colonies upon serial re-plating. These results have important implications for the therapeutic targeting of inv(16)+ hematopoietic stem/progenitor cells in patients with relapsed and refractory disease and for purification of normal HSPC from leukemic bone marrow samples.

Disclosures
No relevant conflicts of interest to declare.
Infused Autograft Lymphocyte to Monocyte Ratio and Survival in Classical Hodgkin Lymphoma
Authors: Porrata Luis F. , Inwards David J. , Ansell Stephen M. , Micallef Ivana N. , Johnston Patrick B. , Hogan William , Markovic Svetomir N. 
Published: October 2019

Abstract
Infused autograft absolute lymphocyte count (A-ALC) is a prognostic factor for survival post-autologous peripheral hematopoietic stem cell transplantation (APHSCT) for lymphoma. Myeloid-derived suppressor cells (MDSCs) affect tumor growth by suppression of host anti-tumor immunity. Thus, we set out to investigate if the infused autograft lymphocyte /monocyte ratio (A-LMR), as a biomarker of host immunity (i.e., lymphocytes) and MDSCs (i.e., monocytes), affects survival post-APHSCT in patients with classical Hodgkin lymphoma (cHL). Only patients that collected their stem cells through the peripheral blood were included in the study. From 1994 to 2012, 183 cHL patients qualified for the study. The 183 patients were randomly divided into a training set (n = 122) and a validation set (n = 61). Receiver operating characteristic and area under the curve (AUC) identified an A-LMR ≥ 1 as the best cut-off value (AUC = 0.8, p < 0.01) and validated by the k-fold cross-validation (AUC = 0.8) in the training set. Multivariate analysis showed A-LMR to be an independent prognostic factor for survival in the training set. Patients with an A-LMR ≥ 1.0 experienced a superior overall survival (OS) versus patients with an A-LMR < 1.0 (median OS was not reached vs 40.4 months, 5-year OS rates of 86% (95 CI, 72%-93%) vs 43% (95 CI, 28-58 %), p < 0.0001, respectively) in the training set. In the validation set, an A-LMR ≥ 1 showed a median OS of not reached vs 41.8 months for an A-LMR < 1, 5 year OS rates of 90% (95 CI, 73%-97%) vs 48% (95 CI, 29-68%), p < 0.0001, respectively. A-LMR provides a platform to engineer an immunocompetent autograft to improve clinical outcomes in cHL patients undergoing APHSCT.

Disclosures
No relevant conflicts of interest to declare.
Using the Rothman Index to Predict Discharge and Readmission Rates in an Inpatient Hematology Unit
Authors: Kim Roger Y. , Yao Xiaopan , Longley Peter , Rimar Joan , Kournioti Chryssanthi , Lee Alfred Ian 
Published: October 2019

Abstract
BACKGROUND: Limited data exists regarding predictors of discharge (DC) disposition and readmission (RA) rates for hematology-oncology patients who are hospitalized. The Rothman Index (RI) is a single numerical score ranging from -91 to 100 derived from 26 data elements including vital signs, laboratory results, and nursing assessments. It is automatically calculated and displayed in real-time in the electronic medical record by a commercial software system. A previous analysis at our institution revealed a significant association between RI and DC disposition, and between RI and RA rates, for patients with solid tumors who were hospitalized in a dedicated inpatient oncology unit. We sought to determine the effectiveness of RI in predicting DC disposition and RA rates for hospitalized patients with hematologic malignancies.
METHODS: Medical records of patients with hematologic malignancies admitted to Yale Cancer Center between 3/1/13 and 2/28/14 were reviewed. Admissions for elective chemotherapy were excluded. DC disposition was categorized as home (Hm), nursing home (NH), hospice (Hp), or death (D). RI at DC was examined as a predictor of DC disposition, overall RA rate, and RA rate of patients discharged to Hm. As an individual patient might have several admissions, generalized estimating equation models were used to assess the effect of RI at DC on DC disposition and the possibility of RA by controlling the within subject correlation.
RESULTS: A total of 281 patients comprising 520 admissions were included in the final analysis. The median age was 59 (range: 19-93; age 70 or older: 21%), and 53% of patients were male. The most common hematologic malignancies were non-Hodgkin lymphoma (33%), acute myeloid leukemia (25%), and multiple myeloma (19%). Among all admissions, DC disposition was as follows: Hm, 439 (84%); NH, 53 (10%); Hp, 18 (4%); and D, 10 (2%). RI at DC was a significant predictor of DC disposition (p = 0.0001), with median RI at DC as follows: Hm, 82.1; NH, 61.4; Hp, 38.7; and D, -1.3. Patients with lower RI at DC were significantly more likely to be readmitted (p = 0.0006), even when the analysis was restricted to patients who had been discharged to Hm (p = 0.0131). Among Hm patients, RA rate was 61% for those whose RI at DC was < 60 and 46% for RI at DC > 60.
CONCLUSIONS: The RI at DC significantly correlated with patient DC disposition and RA rates and may assist health care providers in DC planning with the goal of reducing hospital RA rates and ultimately health care costs.

Disclosures
Lee: Pfizer: Consultancy.
Hyperexpression of Inflammatory Genes in Placental Tissue in Patients with Hemoglobin SC Disease
Authors: Baptista Letí­cia Carvalho , Fertrin Kleber Yotsumoto , Ferreira Regiane , Lanaro Carolina , Albuquerque Dulcinéia Martins , Costa Fernando Ferreira , Costa Maria Laura , Melo Monica Barbosa 
Published: October 2019

Abstract
Introduction: Pregnant women with sickle cell disease (SCD) are at higher risk of obstetrical complications, such as preeclampsia and preterm delivery. The placental circulation is susceptible to vaso-occlusion, which may contribute to fibrosis, necrosis and infarction of the placental villi. Restricted uteroplacental circulation is associated with low birth weight and fetal growth restriction. The mechanisms by which SCD affects placental physiology are largely unknown, and the chronic inflammatory state may be involved in this pathological process. The aim of this study was to evaluate the expression of mediators of inflammatory response in the placenta of pregnant women with SCD.
Methods: This is an ongoing case control study that has recruited 4 pregnant patients with hemoglobin SC disease and 4 healthy control pregnant women paired for gestational age and route of delivery. Placental tissue was collected for total RNA extraction and synthesis of cDNA. Samples were run on a qPCR Array (Inflammatory Response and Autoimmunity RT2 Profiler, Qiagen), which includes 84 genes. Gene expression levels were normalized to housekeeping genes (ACTB, B2M, and RPLP0), and will be further validated by RT-qPCR.
Results: Two patients underwent cesarean delivery and one patient delivered preterm. The analysis of placental gene expression showed that 16 genes were upregulated. The top 5 most upregulated genes were CCL24 (18.1 fold), TLR9 (8.6 fold), inducible nitric oxide synthase NOS2 (6.8 fold), IL17A (5.4 fold), and CCL16 (4.8 fold). Upregulated genes also included other chemokines and their receptors (CCL21, CCL22, CXCL3, and CCR7), interleukins (IL1RAP, IL5, and IL9), another toll-like receptor (TLR3), transcription factor BCL6, CD40 ligand (CD40LG), and kininogen 1 (KNG1). Interestingly, the two genes found to be downregulated were CRP (C reactive protein) and IFNG (interferon gamma).
Conclusion: This is the first study of differential gene expression in placenta of patients with SCD. Despite patients with hemoglobin SC disease being clinically less severe than homozygous sickle cell anemia, we have found that placental tissue in this subset of patients displays increased expression of several genes involved in inflammatory response. Our data support local inflammatory changes in the placenta of these patients that might explain the development of complications in SCD pregnancies. Although the association between obstetric complications and SCD has been long described, our data should contribute to better define the mechanisms affecting the placental physiology that lead to complications, such as preeclampsia.

Disclosures
No relevant conflicts of interest to declare.
Evidence for Transient Acute Liver Injury in Mouse Models of Sickle Cell Disease during Steady State Health
Authors: Wandersee Nancy J. , Holzhauer Sandra L. , Retherford Dawn M. , Foster Thomas D. , Hillery Cheryl A. 
Published: October 2019

Abstract
Individuals with sickle cell disease experience episodes of acute vaso-occlusive complications interspersed by periods of feeling well and apparent health during “steady state”. It is likely that episodic and subclinical sickling and vaso-occlusion occur on an ongoing basis even during periods of steady state. In order to determine whether mice with sickle disease have acute vaso-occlusion sufficient for organ injury at apparent steady state health, we chose to study 2 mouse models of severe sickle cell disease with serial blood draws over time. Since we have shown in previous studies that the liver is highly susceptible to acute sickling/vaso-occlusion induced by hypoxia, we first examined serial ALT measurements in a total of 13 Berkeley sickle cell (BERK-SS) mice, initially from week-to-week and month-to-month, and then day-to-day time frames. Individual BERK-SS mice showed week-to-week and month-to-month variations of up to 5-fold over baseline ALT levels, followed by return to baseline levels in most mice. This suggests that BERK-SS mice, during steady state health, experience acute, yet transient, liver injury. Surprisingly, we also found day-to-day variations of up to 3-fold in ALT levels in BERK-SS mice. To determine if these changes in ALT levels were mirrored by changes in other biomarkers, we measured serum amyloid P (SAP) as a marker of acute inflammation (similar to CRP in humans) in the same plasma samples. We found up to 1.6-fold variation in SAP over baseline plasma levels over time. However, there was less overall variation in SAP levels in individual mice compared to the striking changes in ALT. In addition, we found that variations in SAP did not consistently precede, follow, or coincide with variations in ALT levels, suggesting that SAP is a marker of sickle cell-induced inflammation or vaso-occlusion independent of liver injury. We next sought to confirm our findings with daily serial ALT and SAP measurements from Townes sickle cell (TOWNES-SS) mice (n=8), a second commonly used mouse model of severe sickle cell disease. Our data showed that baseline ALT levels are higher and SAP levels are lower in TOWNES-SS as compared to BERK-SS mice, likely reflecting differences in strain background between the two models. However, we again found daily fluctuations in ALT and SAP levels in individual TOWNES-SS mice, similar to those seen in individual BERK-SS mice. We propose that these temporal variations indicate that both the Berkeley and Townes mouse models of sickle cell disease exhibit fluctuations in steady state health, similar to those seen in human individuals with sickle cell disease. In addition, the results suggest that studies in these mouse models should be designed and interpreted with these temporal fluctuations in mind.

Disclosures
No relevant conflicts of interest to declare.
Chd1 Regulates Primitive State of Bone Marrow Mesenchymal Stromal Cells without Affecting Hematopoietic Support; An Insight on the Dissociation Between Primitive State and Niche Function
Authors: Oh Il-Hoan , Choi Hyun-Kyung 
Published: October 2019

Abstract
Mesenchymal stromal cells (MSCs) are characterized by heterogeneity in the proliferation/self-renewal potentials and hematopoietic supporting activity among subpopulations. Numerous studies have suggested that a primitive state of MSC subpopulation are correlated to its niche function to support hematopoietic stem cells (HSCs), but the mechanisms regulating primitive state of MSCs remains poorly understood.
In the present study, we examined the role of a chromatin remodeling enzyme, chd1 in the maintenance of open chromatin and undifferentiated state of MSCs. We analyzed for expression in MSCs, the expression level of chd1 progressively decreased during in-vitro subculture (from 7 to 18 passages) in a manner proportional to the passage numbers. Moreover, chd1 expression was down regulated in the MSCs during their differentiation into adipogenic or osteogenic lineages, compared to proliferative state, indicating the correlations between MSC proliferation potentials and expression level of chd1. Next, we transduced human bone marrow-derived MSCs with shRNAs against chd1 and found that chd1 knock down MSCs (chd1-KD) exhibit significant loss of colony forming activity (CFU-F), decrease of cell proliferation and loss of multi-lineage differentiation towards osteogenic or adipogenic lineages. Moreover, chd1-KD MSCs exhibited lower level expression of pluripotency-related genes, oct-4, sox-2 and nanog, with concomitant increase of H3K9me3 on the promoters and decreased chromatin accessibility in the oct-4 promoter, suggesting that chd1 regulate open chromatin and multi-lineage potential of MSCs.
However, KD of chd1 in MSCs did not affect the HSC-supporting activity of MSCs; human cord blood-derived CD34+ cells co-cultured on chd1-KD MSCs exhibited rather higher maintenance of primitive phenotype (CD34+90+) and higher repopulating activity in NOD/SCID-ɤC KO mice compared to those co-cultured on control group MSCs. Together, these results show that, while primitive state of MSCs are regulated by chromatin remodeling complex,chd1, the hematopoietic niche activity of MSCs is not directly influenced by the primitive state of MSCs, raising a questions on the prevailing notion that undifferentiated MSCs can better support hematopoietic function.

Disclosures
No relevant conflicts of interest to declare.
Desoxyribonucleoprotein Thromboembolism Following Total Body Irradiation: Case Report
Authors: HRUBAN ZDENEK , PIERCE MILA I. , NAIR SUMATI , WARNER NANCY E. 
Published: October 2019

Abstract
A case of sudden death shortly after total body radiation is reported in a patient with acute stem cell leukemia. The sudden death was associated with capillary DNA thromboembolism.
OBSERVATIONS ON THE EFFECT OF IRRADIATION IN CHRONIC ACQUIRED HEMOLYTIC ANEMIA EXHIBITING HEMOLYTIC ACTIVITY FOR TRANSFUSED ERYTHROCYTES
Authors: EVANS ROBERT S. , DUANE ROSE T. 
Published: October 2019

Abstract
The history and course of a middle-aged woman with a chronic acquired hemolytic anemia have been presented. Splenectomy resulted in a complete remission of the disease for 18 months before a relapse occurred which led eventually to a fatal termination.
The original cause of the abnormal hemolytic process is not known, but the onset of the relapse was closely associated with the resumption of gold therapy.
The acceleration of the hemolytic process was associated with an increasing tendency toward spherocytosis and susceptibility of the erythrocytes to hemolysis in hypotonic solution.
It is demonstrated that transfused normal erythrocytes shared in the hemolytic process and were eliminated from the circulation at an abnormal rate although no atypical isohemolysin was demonstrated by in vitro tests.
An attempt was made to alter the rate of hemolysis in several ways. The injection of thorium dioxide was followed by signs of transitory slowing of red cell destruction. Injections of Congo red were apparently without effect. Irradiation of the mediastinal and periaortic nodes was begun to ascertain if the production of a hemolytic antibody in lymphatic tissue could be altered. Irradiation was followed by a fall in leukocyte and lymphocyte counts in the peripheral blood. Transfusions were then followed by partial remissions in the hemolytic process for varying periods of time in two instances. Irradiation of a second patient with a chronic hemolytic anemia of similar character produced a fall in leukocyte count and lymphocyte count, but in the absence of transfusions no slowing of the hemolytic process occurred.
No definite conclusions can be drawn from these fragmentary observations on the effect of irradiation, but further investigation as to the character of the hemolytic substance and possible methods of modifying its production are indicated when the opportunity is afforded.