bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Functional Characterization of Human Erythroid Progenitors: BFU-E and CFU-E
Authors: Bhagia Pooja , Mohandas Narla , An Xiuli
Published: October 2019

Abstract 1028

The two committed erythroid progenitor populations that have been functionally defined by colony assays are burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E). While significant progress has been made in defining these two progenitor populations in the murine system, their characterization in the human system is incomplete. To address this issue, we have characterized the dynamic changes in surface expression levels of number of proteins including CD34, c-kit, IL-3R, CD36, CD71, GPA and CD45 during proliferation of purified human CD34+ cells from cord blood during the first phase of the two-phase in vitro erythroid culture system. In the presence of stem cell factor, IL-3 and erythropoietin during this phase, CD34+ cells differentiate first into BFU-E and then into CFU-E during 7 days of culture with peak levels of BFU-E at day 4 and of CFU-E at day 6. During this period of time, the expression levels of CD34 and IL-3R decreased, while that that of CD36 and CD71 increased. CD45 was expressed during the entire 7 day culture period while there was no expression of GPA. Based on these findings, we sorted pure populations of CD34+CD36−IL3-R+ and CD34− CD36+IL-3R− cells and characterized their behavior in colony forming assays. The sorted CD34+CD36−IL3-R+ population gave rise to BFU-E colonies while CD34− CD36+IL-3R− population gave rise CFU-E colonies, both at a purity of over 80%. The identity of the sorted BFU-E and CFU-E cells was further supported by their differential responsiveness to dexamethasone and lenalidomide (Narla A et al Blood 2011), with increased proliferation BFU-E population by dexamethasone and increased proliferation of CFU-E by lenalidomide. These findings were further validated by isolation of pure populations of BFU-E and CFU-E from primary human bone marrow based on the identified markers. The ability to isolate pure populations of human BFU-E and CFU-E progenitors should enable detailed molecular and cellular characterization of these distinct erythroid progenitor populations. Furthermore, enumeration of the number of these progenitor populations in human bone marrow may help in delineating mechanisms of disordered erythropoiesis in various disorders such as bone marrow failure syndromes.

Disclosures:

No relevant conflicts of interest to declare.
Sphingomyelinase-Induced Remodeling of the Erythrocyte Membrane
Authors: Dinkla Sip , Wessels Katharina , Verdurmen Wouter P.R. , Joosten Irma , Brock Roland E. , Bosman Giel J.C.G.M.
Published: October 2019

Abstract 1032

Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, a major lipid component of cell membranes, generating phosphocholine and ceramide. Inflammation arising from various diseases including sepsis is known to enhance SMase secretion in the circulation. Also, chronic inflammation typically induces anemia through reduced production and enhanced clearance of red cells, which is often associated with poor disease outcome. Incubation of red cells with plasma of septic patients with enhanced SMase activity has been described to result in ceramide formation and exposure of phosphatidylserine (PS). Therefore, we studied the effect of SMase on various pathophysiologically relevant parameters of red cell homeostasis in fresh and stored erythrocytes.

Using time-lapse confocal microscopy we observed a typical sequence of events during SMase treatment of erythrocytes. The cells first lost their discoid shape, PS became externalized, and ultimately there was a sudden loss of cytoplasmic content. Additional confocal microscopy, flow cytometry and immunoblot analyses showed that SMase treatment resulted in ceramide-induced alterations in red cell membrane-cytoskeleton interaction and organization, including lipid raft formation. These changes probably underlie the increase in osmotic fragility, membrane vesiculation and invagination, as well as large hemoglobin aggregates that we also observed. The membrane lipid scrambling, altered morphology, enhanced fragility and presence of rigid membrane patches may all contribute to enhanced removal of sphingomyelinase-exposed red cells.

Generation of very low ceramide levels in the membrane by SMase, as detected by Positive-Ion MALDI-TOF MS, already affected red cell shape, membrane rearrangement and osmotic responsiveness. Combined with the observation that 24 hour incubation of red cells with pathological SMase levels induced shape change and PS exposure, these data suggest that red cell homeostasis could well be disturbed by SMase during chronic inflammation. Of note, red cell storage greatly increased the sensitivity to SMase-induced changes. This may well have implications for red cell transfusion in critically ill patients.

Disclosures:

No relevant conflicts of interest to declare.
The Lymphoid Chemokine CCL21 Enhances the Migration- and CTL-Inducing Functions of Dendritic Cells
Authors: Hong Cheol Yi , Kalinski Pawel , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Abstract 1127

The migration of dendritic cells (DCs) to secondary lymphoid organs is very important to elicit an adaptive immune response in cancer immunotherapy. Here, we show the effect of lymphoid cytokine on the ability of maturing DCs to migrate in response to the lymph node-associated chemokines. The secondary-lymphoid organ chemokine (SLC/CCL21) during DC maturation dramatically enhanced DC migratory capacity responding to CCL21 and CCL19, and, moreover, produced strongly enhanced cytotoxic T cells, although it did not affect the expression of cell surface markers such as CD80, CD83, CD86, and CCR7 and the production of cytokines such as IL-12p70, IL-10, and IL-23. Mature DCs (mDCs) exposed by chemokine produced higher levels of CXCL10 (IP-10) that is one of the chemokines involved in Th1 attraction, but did not affect the production of Th2-attracting cytokine CCL22, compared with unstimulated mDCs. CCL21-exposed DCs induced strongly enhanced numbers of the interferon-g (IFN-g)-expressing antigen-specific CD8+ T cells against tumor-specific antigens in an CXCL10-dependent manner. Cytotoxic CD8+ T cells stimulated with CCL21-exposed DCs expressed higher level of IFN-g than those stimulated with control mDCs. Interestingly, generation of cytotoxic T cells (CTLs) stimulated by TNFa/IL-1b/IL-6/PGE2-treated DCs (sDCs) supplemented with IP-10 produced strong cytotoxic T cells expressing higher level of IFN-g. Tetramer assay showed that CCL21-treated DCs enhanced generation of antigen-specific CTLs. Taken together, our data suggest that mDCs pre-stimulated by chemokine CCL21 enhanced migratory capacity to secondary lymphoid organs and produced strong cytotoxic T cells via IP-10 signaling pathway.

Disclosures:

No relevant conflicts of interest to declare.
Waveform Analysis Extends the Capabilities of aPTT and PT Clotting Assays for Detecting Factor Deficiency and Monitoring Procoagulant Drug Therapy
Authors: Leong Lilley , Jiang Xiaoqiao , Cifrese Jill , Murphy John E. , Haaning Jesper , Myles Timothy
Published: October 2019

Abstract 1205

Coagulation assays, such as activated partial thromboplastin time (aPTT) and prothrombin time (PT), are important for the diagnosis and treatment of bleeding disorders. Waveform analysis (WA) of the optical data generated in performing coagulation assays may provide additional information that will allow further understanding of coagulation disorders and their treatment (Hemophilia. 2006;12(suppl 3):76). Since WA employs assays currently in use in the clinical laboratory, it has the potential to be easily adapted to the clinical setting. To assess the utility of WA in procoagulant drug discovery for hemophilia, we determined its ability to: 1) discriminate between different factor deficiencies by factor-specific coagulation assays and by standard aPTT; and 2) discriminate between hemophilic plasma (with and without inhibitors) spiked with therapeutic proteins used to treat hemophilia. The ultimate goal of the study was to determine the feasibility of WA in monitoring hemophilia therapy in the clinic.

WA of plasma from factor (F)-deficient (FVIII, von Willebrand factor [type III vWD], FIX, and FVII) donors showed both absorbance and first-derivative waveform differences. Differences in first-derivative curves for severe hemophilia A plasma relative to vWD type III plasma were observed. The hemophilic plasma had a longer lag time (113 vs 93 seconds, respectively) and lower peak amplitude (29.7 vs 54.7 AU) than the vWD type III plasma. To determine whether WA might be useful for assessing the procoagulant activity of FVIII and FVIII bypass therapeutic agents, we compared waveforms of plasma-derived FVIII (WHO 8th) with pegylated FVIII (BAY 94–9027) in hemophilia A plasma or of rFVIIa variant and BAY 86–6150 in hemophilia A plasma containing 112 BU inhibitor. The waveforms obtained with WHO 8th and BAY 94–9027 were almost identical by ellagic acid aPTT with the first derivative giving lag times of approximately 31 seconds for both 5% WHO 8th and 5% BAY 94–9027. WA was able to detect rFVIIa and BAY 86–6150–mediated coagulation at 1.4 nM by first-derivative waveform in silica aPTT. Comparison of rFVIIa with BAY 86–6150 at this concentration indicated that BAY 86–6150 was more procoagulant than rFVIIa, with a shorter lag time of 75.9 seconds vs 97.9 seconds (measured by taking the first derivative of the waveform). Therefore, in addition to clot time or activity levels currently available by standard aPTT/PT, WA allows further insight into the kinetics of coagulation. In conclusion, WA holds promise as a global hemostasis assay that will extend the current utility of optical coagulation analyzers used in the clinic.

Disclosures:

Leong: Bayer HealthCare: Employment. Jiang:Bayer HealthCare: Employment. Cifrese:Bayer HealthCare: Employment. Murphy:Bayer HealthCare: Employment. Haaning:Bayer HealthCare: Employment. Myles:Bayer HealthCare: Employment.
Pharmacodynamic Differences in the Two Generic Brands of Enoxaparin
Authors: Hoppensteadt Debra , Jeske Walter , Gray Angel , Walenga Jeanine M. , Wahi Rakesh , Thethi Indermohan , Fareed Jawed
Published: October 2019

Abstract 1251

Several generic versions of enoxaparin have recently become available. While these generic versions of enoxaparin exhibit similar molecular profiles and comparable anti-Xa activities; product specific differences in global anticoagulant (APTT, Heptest and thrombin generation inhibition) have been reported. The purpose of this study was to compare a generic version of enoxaparin Sandoz from Argentina (Fibrinox lot 002) and from the US (enoxaparin lot 914786) in various in vitro whole blood and plasma based clotting tests. Despite comparable molecular profile and anti-Xa potency, product specific differences were noted between the products and the US generic enoxaparin showed a cumulatively stronger activity in most of the assays. To further test the pharmacodynamic profile of these products, individual groups of monkeys (n=4–8) were administered with each product at a 1 mg/kg SC and blood samples were collected for up to 28 hours. Clot based assays such as the APTT, Heptest, thrombin time, amidolytic anti-Xa and anti-IIa activities were carried out. In addition, tissue factor pathway inhibitor (TFPI) antigen, thrombin activatable fibrinolysis inhibitor (TAFI) activity and thrombin generation assays were also performed. Variable differences were noted in the clot based and amidolytic assays. Interestingly, the US generic product exhibited a lower release in the TFPI antigen whereas in the thrombin generation assays it produced a stronger inhibition of thrombin in terms of the AUC. TAFI activity profile also showed wide variations. These differences were more prevalent during the 1–4 hour time period. No differences were noted at >6 hours. The hysterisis PK/PD plots revealed marked differences between the two products. These results indicate that the products for the same generic suppliers may exhibit variations according to market places. Moreover, these observations underscore the need for a more stringent pharmacodynamic profile to demonstrate product equivalence.

Disclosures:

No relevant conflicts of interest to declare.
A Subset of MicroRNAs and Genes Involved in AML Has a Pivotal Role in the in Vitro differentiation of Hematopoietic Stem Cell Precursors
Authors: Pulecio Julian , Laricchia-Robbio Leopoldo , Izpisua Juan Carlos , Barragan Montserrat , Vitaloni Marianna , Martinez Ignacio Sancho , Nivet Emmanuel
Published: October 2019

Abstract 1290

After the finding of a set of transcription factors capable of reprogram any somatic cell into an embryonic stem-like cell by Yamanaka's group a lot of effort has been put to differentiate and produce in-vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. Nonetheless, so far there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally in vivo blood-depleted mice. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells capable of distinguish into sub lineages in in vitro assays, while another group obtained blood precursors by transdifferentiation of fibroblasts; however a complete recovery of the hematopoietic lineages in vivo was not seen. Our hypothesis is that the gap missing in the current protocols to obtain repopulating blood stem cells can be filled by the microRNA profiling of Cord Blood (CB) progenitors, in order to find the key players in the maintenance of blood stemness. In particular, it has been shown that population with the highest capacity to be engrafted in mice is the CD34+/CD90+ from CB. Our preliminary results depict a set of miRNAs that are specifically overexpressed in the CD34+/CD90+ population from CB cells when compared against a less specific CD34+ population. These miRNAs are currently being tested as a tool to improve the efficiency of iPS differentiation and fibroblasts conversion towards blood progenitors by means of lentiviral infection of the miRNA precursors. Interestingly, we have found that these miRNAs have been previously reported to have a main role in the occurrence of Acute Myeloid Leukemia in humans and mice. These results led us to look for genes that are highly expressed in blood progenitors but also have been shown to be correlated with AML.As a safety study, we are currently evaluating the effect of overexpressing AML related factors (miRNAs and genes) when added to the established protocols to obtain blood progenitors from iPS and fibroblasts. Surprisingly, our initial results show that the overxpression of the above mentioned genes and miRNAs have an intrinsic potential to induce in vitro differentiation or conversion from iPS and fibroblasts towards blood progenitors.

Disclosures:

No relevant conflicts of interest to declare.
Enhanced Generation of Human Hematopoietic Stem/Progenitor Cells From ES and Patient Derived IPS Cells Using a Novel Compound Screen
Authors: Rönn Roger Emanuel , Moraghebi Roksana , Guibentif Carolina , Woods Niels-Bjarne
Published: October 2019

Abstract 1294

The ability to generate hematopoietic stem and progenitor cells from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to identify novel pathways involved in hematopoietic stem and progenitor cell generation and expansion from human ES and iPS cells. By using a small molecule compound library with our optimized iPS-2-blood lineage differentiation protocol, we have identified two novel chemical compounds that specifically enhance the generation of phenotypic adult hematopoietic stem cells. Compound A and compound B both enable increases of CD45/43+CD34+CD38-CD90+CD45RA- cells by 183+/−53%, n=5 and 275%, n=1, respectively. This is in comparison to DMSO carrier control wells, where the percentage of blood cells (CD45/43+) produced per total cells for these experiments was 73+/−2.9%, n=5. The increase in hematopoietic cell output using compound A was highly significant for the adult phenotypical hematopoietic stem cell fraction with a P-value of 0.03, n=5 (see Figure). Hematopoietic progenitor, CD45/43+ CD34+, counts were also slightly increased for compound A and B at 129+/−32%, n=5 and at 211%, n=1, respectively. Interestingly, no statistically significant increase in the number of total blood CD45/43+ cells was detected at the time of harvest, with either compound; at 108% +/− 8.4%, n=5, for compound A, and 164%, n=1, for compound B. In addition to the improvements, as measured by FACS, compounds A and B both increased the numbers of clonogenic progenitors as measured by CFU-assay, allowing for a 241+/−67%, n=4, and 443%, n=1, increase in total hematopoietic colony counts, respectively. These results identify 2 novel compounds with the ability to expand the more primitive fractions of hematopoietic cells with preferential expansion of the most primitive fraction of hematopoietic cells derived from human ES and iPS lines. We are currently performing transplantation experiments with cells generated using these compounds to assess their repopulating potential. Further studies are being performed to investigate the molecular mechanisms of these compounds for hematopoietic stem and progenitor cell generation from ES and iPS cells.

Disclosures:

No relevant conflicts of interest to declare.
Effects of Enhanced Physical Activity on Hematopoietic Stem Cell Aging in PEPCK-Cmus Transgenic Mice
Authors: Qu William W. , Hakimi Parvin , Berger Nathan A. , Hanson Richard W.
Published: October 2019

Abstract 1297

Hematopoietic stem cells (HSCs) are self-renewing multipotent precursor cells that give rise to all blood cell lineages. As HSCs age, expansion of the pool of HSCs and abnormal production of blood cells occur, resulting in a loss of immune function and an increased incidence of myeloid leukemia. Exercise has been associated with an extended lifespan, yet the connection between HSC fitness and enhanced physical exercise is currently unclear. Transgenic mice that over-express the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) in their skeletal muscle (PEPCK-Cmus mice) have remarkable endurance, can run five to six kilometers at a speed of 20 meters per minute without stopping, and have a markedly longer lifespan. We thus determined the effects of enhanced physical activity on HSC aging by measuring the pool size and functional ability of HSCs in both aged PEPCK-Cmus transgenic and control mice. Cell surface immunostaining and flow cytometric analysis were performed to determine the total quantity of HSCs within the bone marrow cell population of both groups of animals. Preliminary studies showed that age-associated expansion of the HSC pool was absent in PEPCK-Cmus mice. However, the efficiency of HSCs to produce B lymphoid progenitors in PEPCK-Cmus mice was surprisingly 11-fold higher than that in control mice while the age-associated skewness of HSC differentiation toward the myeloid lineage was diminished in PEPCK-Cmus mice. Furthermore, reactive oxygen species (ROS), known to be associated with aging when at high levels, were lower in the HSCs of PEPCK-Cmus mice than was noted in the HSCs of control animals. Together, these preliminary data imply that the functional capacity of HSCs is maintained in old PEPCK-Cmus mice when compared to that of the HSCs in control animals of the same age. This suggests that enhanced physical activity may slow HSC aging, possibly attributed in part to decreased ROS levels.

Disclosures:

No relevant conflicts of interest to declare.
The Midostaurin (PKC412) Metabolite CGP52421 Shows Little Growth-Inhibitory Activity Against Against Neoplastic Mast Cells but Retains Inhibitory Effects on IgE-Dependent Activation and Histamine Release
Authors: Peter Barbara , Blatt Katharina , Stefanzl Gabriele , Hadzijusufovic Emir , Dutreix Catherine , Gross Stefan H , Roesel Johannes L , Valent Peter
Published: October 2019

Abstract 1417

The multikinase inhibitor midostaurin (PKC412) is currently tested in clinical trials in patients with advanced systemic mastocytosis (SM). Although clinical symptoms improve in many patients and sometimes the proliferation of neoplastic mast cells (MC) can be kept under control for some time, most patients progress after a variable latency period, even if their mediator-related symptoms are still completely suppressed. In vivo, midostaurin is metabolized to two major and active metabolites, namely CGP62221 and CGP52421. Whereas the in vitro effects of midostaurin on growth and activation of MC are well documented, only little data on effects of midostaurin-metabolites are available. We examined the effects of midostaurin and its pharmacologically relevant metabolites CGP52421 and CGP62221, on IgE-dependent mediator secretion in basophils as well as growth of primary neoplastic MC and the human MC leukemia cell line HMC-1. All three compounds were found to inhibit IgE-dependent secretion of histamine in blood basophils, with comparable IC50 values (<1 μM). Midostaurin and CGP62221 were also found to inhibit the proliferation in HMC-1.1 cells and HMC-1.2 cells with IC50 values ranging between 50 and 250 nM. However, the second metabolite of PKC412, CGP52421, did not produce comparable anti-proliferative effects, even when applied at concentrations up to 1 μM. Corresponding results were obtained when analyzing drug-effects on primary neoplastic MC obtained from two patients, one with indolent SM and one with aggressive SM. Furthermore, whereas midostaurin and CGP62221 induced apoptosis in HMC-1.1 and HMC-1.2 cells as evidenced by light microscopy, caspase 3 staining and TUNEL assay, CGP52421 did not induce apoptosis in neoplastic MC. Finally, midostaurin and CGP62221 were found to induce dephosphorylation of KIT V560G and KIT D816V in HMC-1 cells, whereas no effects of CGP52421 on KIT activation were seen. In drug competition experiments, the metabolite CGP52421 did not interfere with midostaurin- or CGP62221-induced growth inhibition in HMC-1 cells. In conclusion, the pharmacologically relevant midostaurin metabolite CGP52421 inhibits IgE-mediated histamine release but does not efficiently block KIT-dependent proliferation of neoplastic MC. This observation may help to explain clinical responses including improvement of mediator-related symptoms seen in midostaurin-treated patients with advanced SM, and would favor the development of new treatment concepts employing midostaurin as an inhibitor of both MC activation and MC growth in patients with SM.

Disclosures:

Dutreix: Novartis: Employment. Gross:Novartis: Employment. Roesel:Novartis Pharma AG: Employment. Valent:Novartis: Consultancy, Honoraria, Research Funding.
Mir-222-221 Induce Dexamethasone Resistance by Targeting BBC3/PUMA in Human Multiple Myeloma
Authors: Zhao Jian-Jun , Chu Zhangbo , Lin Jianhong , Tai Yu-Tzu , Munshi Nikhil C. , Anderson Kenneth C. , Carrasco Ruben
Published: October 2019

Abstract 1419

Dexamethasone is a conventional drug that effectively induces multiple myeloma (MM) cell death at presentation and in relapsed patients and continues to be used against MM alone or in combination with the new therapeutic agents. However, still remain unclear the mechanism of Dexamethasone resistance in clinic. We now report new mechanisms of Dexamethasone resistance implicating microRNAs (miR). MicroRNAs are noncoding small RNAs (18–25nt) that play an important role in the regulation of gene expression. Recently our profiling study in MM1S and MM1R cells has shown that miR-222-221 are highly expressed in MM1R cells as compared with MM1S cells. Our further investigation showed that enforced expression of miR-222-221 could render cells resistant to Dexamethasone and that the resistance to this drug could be partially restored by ectopic expression of PUMA without 3'UTR region. The list of microRNAs regulators for PUMA (BBC3) 3'UTR region in targetscan website includes miR-221 and miR-222. We also found a negative relationship between miR-222-221 levels and therapeutic response in clinical samples by using Q-RT-PCR, IHC and ISH methods. So we propose that miR-222-221 could target PUMA to induce Dexamethasone resistance in MM. Ectopic expression of miR-222-221 in MM1S cells resulted in decreased expression of PUMA protein and mRNA, whereas knockdown expression of miR-222-221 could restore PUMA expression in MM1S cells. Notably, miR-222-221-transfected MM1S cells became resistance to Dexamethasone as compared to vector-treated cells. Further, the knockdown of miR-222-221 sensitizes the MM1R cells to Dexamethasone-induced cell growth arrest and apoptosis. These findings indicate that the miR-222-221 play a significant role in regulation of PUMA expression level and could be potential targets for restoring PUMA expression and response to Dexamethasone therapy in a subset of MM patients. We are currently investigating the role of miR-222-221 in Dexamethasone resistant in vivo using well established mouse xenograft models of MM.

Disclosures:

No relevant conflicts of interest to declare.
Germline Mitochondrial DNA Mutations As a Novel First Event in Childhood Myelodysplastic Syndrome
Authors: de Vries Andrica , Zwaan Christian M. , Beverloo H. Berna , Wagner Anja , Lankester Arjan , te Boekhorst Peter , de Coo Rene , Schoonderwoerd Kees , Smeets Bert , Polychronopoulou Sophia , Pieters Rob , van den Heuvel Marry M.
Published: October 2019

Abstract 1711

Introduction:

Mutations in the mitochondrial DNA (mtDNA) have been found in 50–60% of adult MDS patients, with an increasing frequency with rising age and ahigher incidence in more advanced MDS. In general, cells contain different amounts of mitochondria which can simultaneously harbour wildtype and mutated mtDNA. Co-existence of normal and mutant mtDNA is referred to as heteroplasmy, whereas the existence of only mutated mtDNA is called homoplasmy. As yet no information is available on the role of mtDNA mutations in pediatric MDS. We recently identified a family with (germline) mtDNA mutations and childhood MDS. We hypothesized that mtDNA mutations, catalyzed by ATP deficiency, reflect the genetic instability of the stem cells that facilitates that the development of MDS clone initiation and subsequent clonal evolution to acute myeloid leukemia triggered by type I and II events.

Methods:

Based on our findings in the above mentioned cases we analyzed the role of mtDNA mutations in the index family, in combination with studying oxidative phosphorylation, as well as in an extended cohort of 19 childhood MDS patients, including sporadic primary, therapy-related and familial MDS, using Mito-Chip (Affymetrix) and direct sequencing validation approach. To investigate whether the mutations were germline or somatic, in a subset of patients, germline mtDNA mutation analysis was performed.

Results:

In 14/19 of the pediatric MDS patients non-recurrent mtDNA mutations were found. Mt-mutational status was not correlated with the different WHO subgroups of childhood MDS. Heteroplasmic mutations were only found as somatic events, whereas. germline mutated cases were solely homoplasmic.

Conclusion:

We describe the first family in which germline mtDNA mutations trigger the devlopment of MDS, and show for the first time, that also in sporadic MDS cases, germline homoplasmic mutations may genetically predispose for developping childhood proliferative myeloid disease.

Disclosures:

No relevant conflicts of interest to declare.
Analysis of Efficacy and Costs of Antifungal Prophylaxis in Patients Undergoing Chemotherapy for Acute Leukemia: “Real Life” Evaluation
Authors: Bertz Hartmut , Drognitz Kathrin , Proskurin Tatjana , Lubbert Michael
Published: October 2019

Abstract 2081

Background:

Antifungal prophylaxis (AP) and therapy (AT) are increasing the costs during treatment for acute myeloid leukemia (AML). Efficacy of AP, empirical (EMP) and diagnostic tool led pre-emptive (PRE) AT still need to be evaluated and improved.

Methods:

In 2010 we performed a prospective study analysing all consecutive patients (pts) treated for AML in terms of AP/AT, choice and costs of the applied diagnostic procedures and EMP or PRE AT. All pts received regular lung CT scans, Galactomannan (Gal) and ß D-glucan (ß-D) tests. AP switched every three months between posaconazole (P) and fluconazole (F) to evaluate P-prophylaxis. Planned EMP consisted mainly of liposomal Amphotericin (LAMB) 1 mg/kg and planned PRE of LAMB 3 mg/kg.

Results:

In 37 pts. 86 cycles (cs) of induction (IND; n=51)/consolidation (CONS; n=35) chemotherapy have been documented. F 200mg/d as AP was given in 46 and P 600mg/d in 40 cs. EMP or PRE was initiated in 9+9 /46 cs (IND n=14; CONS n=4) of F (39%) and in 8+9 / 40 cs of P (42.5%; p=n.s.) (IND n=14; CONS n=3); EMP/PRE AT consisted mainly of LAMB 1 (n=12) or LAMB 3 (n=17) with no further antifungal drug escalation in 26/29 cs (89.6%); in 4 cs toxicity led to modification after 10 days in median of LAMB. 23/121 CT scans (19%; 119€ each), 31/277 ß-D (11%; 55€ each) and 13 / 440 GAL tests (3 %; 9€ each) were positive resulting in mean diagnostic costs of 372 €/c. In only 5/86 cs (6%) all 3 and in 7/86 cs (8%) 2 procedures showed positive results simultaneously. No proven, 10 probable (P and F AP: n=5 each) and 7 possible fungal infections (P n=5; F n=2) were detected. No patient died due to IFI, 3/37 pts. due to AML and none experienced > grade 3 toxicity by antifungal therapy.

Conclusions:

Recommended posaconazole AP showed no advantage compared to F but strikingly higher expenses (mean cost 2438 € vs. 24 €/c). LAMB as primary AT showed very convincing clinical results. These results underline that clinical signs/judgement and CT scan are still the leading, inexpensive decision tools for starting AT. Our consequences are stop of P as AP and of regular GAL/ß-D testing. This project is supported with a grant by GILEAD.

Disclosures:

Bertz: MSD: Membership on an entity's Board of Directors or advisory committees; GILEAD: Research Funding.
FLT3-ITD-Associated Accumulation of Dendritic Cell Precursors, Depletion of Mature Dendritic Cells and Abnormal Cytokine Production in Acute Myeloid Leukemia: Analyses At Diagnosis, Remission and Relapse
Authors: Stripecke Renata , Rickmann Mareike , Sundarasetty Bala Sai , Ganser Arnold
Published: October 2019

Abstract 2525

FLT3-ITD is a frequent (30%) mutation in AML, conferring proliferative advantage, self-renewability and blocking of myeloid differentiation. We had previously shown that ITD+ AML blasts show an accumulation of cells with characteristics of mixed-lineage dendritic cell (DC) precursors, but the effects of FLT3-ITD on the mature dendritic cell compartment and cytokine profile had not been previously described.

The frequency of different DC types in peripheral blood has been established for healthy donors (HD) such as Pan myeloid DCs (Lin−/DR+/CD11c+: 0.5 ± 0.3% frequency in HD), Pan plasmacytoid DCs (Lin−/DR+/CD123+: 0.15 ± 0.08% in HD), mature myeloid DCs type-1 (CD19−/CD14−/BDCA-1+: 0.35 ± 0.17% in HD), mature myeloid DCs type-2 (CD19−/CD14−/BDCA-3+: 0.04 ± 0.02% in HD), and mature plasmacytoid DCs (CD19−/CD14−/BDCA-2: 0.23 ± 0.2% in HD). In this study, we analyzed the frequency of DCs in diagnostic samples of 12 ITD+ compared with 22 ITD- AML patients, and in sub-cohorts of ITD+ patients in remission and relapse.

We demonstrate that ITD+ patients at diagnosis showed a significantly higher frequency of mixed-lineage DC precursors (Lin−/DR+/CD11c+/CD123+: 13 ± 12%) than ITD- patients (5 ± 4.8%) whereas the frequency of all types of mature DCs was lower for ITD+ patients compared with ITD- patients. Analyses of remission samples (collected at 3–15 months after first diagnosis) revealed a regression of the mixed-lineage DC precursors, but the frequency of mature DCs in ITD+ patients remained consistently lower than for the ITD- or HD cohorts.

Remarkably, for one ITD+ patients who eventually relapsed (at 15 months), a gradual reduction in the frequency of BDCA-1 and BDCA-2 positive DCs was clearly noticeable at 9 months after diagnosis, at a time point when the ITD mutation was not yet detectable by RT-Q-PCR, whereas relapse was correlated with the resurgence of the mixed lineage CD11c+/CD123+ DC population (17% of PBMCs) and reduction in the frequency of BDCA-1 (0.07%) and BDCA-2 positive DCs (0.1%).

Luminex analyses of several cytokines secreted by the PBMCs from 6 ITD+ patients showed that samples collected at diagnosis expressed lower levels of inflammatory and homeostatic cytokines than in remission, such as TNF-alpha (36.67 vs. 101.22 pg/ml, p-value: 0.22), IL-6 (35.27 vs. 119.15 pg/ml, p-value: 0.19), IL-7 (2.84 vs. 16.27 pg/ml, p-value: 0.07) and IL-8 (5682.59 vs. 10108.98 pg/ml, p-value: 0.07).

This is the first clinical evidence that FLT3-ITD undetectable minimal residual disease may affect the differentiation of DCs, potentially subverting the normal inflammatory and homeostatic immune reconstitution and thus hampering the anti-leukemia immune surveillance.

Disclosures:

No relevant conflicts of interest to declare.
EBV Copy Number Variation in Twins Discordant for Young Adult Hodgkin Lymphoma
Authors: Cozen Wendy , Hwang Amie , Marshall Vickie , Mack Thomas M , Whitby Denise
Published: October 2019

Abstract 2631

Background:

Epstein-Barr Virus (EBV) is a risk factor for some types of Hodgkin lymphoma (HL). Host response to the virus may play a role in pathogenesis and is in part determined by genetic factors. Therefore, using only traditional unrelated controls may not be appropriate.

Methods:

We compared EBV DNA copy number in peripheral blood mononuclear cells (PBMCs) collected from 63 young adult HL patients in remission and their unaffected co-twins, and 43 spouse controls. Viral load was measured with TaqMan quantitative PCR assays, tested in triplicate and reported as viral copies per million cells. Samples positive in all 3 replicates were classified as positive, and those positive in 1–2 samples were classified as qualitative positive (QP). EBV copy number was log-transformed for some analyses. A binomial proportion test was used to compare EBV copy number in cases and their unaffected co-twins (n=50 like-sex pairs). Conditional (case vs. unaffected co-twin) and unconditional logistic regression (case and unaffected co-twin vs. spouse controls) were used to estimate the effect of EBV copy number on risk as a categorical (positive, QP, or negative) or continuous variable (SAS 9.1). Unconditional logistic regression analyses were adjusted for sex and age.

Results:

26% of case, 18% of unaffected co-twins and 9% of spouse controls had positive EBV copy number measurements. Like-sex young adult HL cases had higher EBV copy numbers compared to their unaffected co-twins (P = 0.04). When EBV copy number was considered as a categorical variable, both cases and monozygotic unaffected co-twins were more likely to be positive compared to controls (cases v. controls: Odds Ratio [OR] = 3.2, 95% confidence intervals [CI] = 0.9, 11.1; unaffected co-twins v. controls: OR= 3.6, 95% CI= 0.9, 14.6). However, there was little difference between dizygotic unaffected co-twins and controls (OR= 0.7, 95% CI = 0.5, 5.5).

Conclusions:

That young adult HL cases had higher EBV DNA copies than their unaffected co-twins implies that an acquired pre-disease factor or disease affect altered the host response to EBV. However, that both cases and unaffected co-twins had higher EBV DNA copies compared to controls suggests that there is also a heritable component to the host response to EBV.

HL cases > co-twins (acquired or disease effect)

HL cases and co-twins > controls (heritable effect)

Disclosures:

No relevant conflicts of interest to declare.
Immunoregulation of Human Myeloid Dendritic Cells and Monocytes by Vascular Endothelial Growth Factor,
Authors: Sanchez Washington Yamandu , Wang Ran , Wilkinson Andrew , Khalil Dalia , Hart Derek NJ , Catley Laurence , Vuckovic Slavica
Published: October 2019

Abstract 3212

Vascular endothelial growth factor (VEGF), produced by many tumours including multiple myeloma (MM), has been reported to regulate the function of myeloid cells inducing myeloid-derived suppressor cells that suppress T cell responses. Little is known about the effect of VEGF on human myeloid cells including myeloid dendritic cells (MDC) and monocytes during their lifespan in bone marrow (BM) and peripheral blood (PB). We analysed the capacity of rhVEGF165 to regulate. MDC (CD11c+CD16+MDC and CD11c+CD16−MDC) and CD14+ monocytes that reside in the BM of humanised (hu)NOD/SCID mice or in peripheral blood (PB) of adult healthy subjects.

The CD11c+CD16−MDC and monocytes located in the femur, tibia and pelvic bones had elevated VEGFR-1 and VEGFR-2 surface expression compared to those located in the vertebrae and skull. Surface expression of VEGFR-1 and VEGFR-2 was negligible on CD11c+CD16+MDC, CD11c+CD16−MDC and monocytes in PB, however, VEGFR-1 mRNA was detected in CD11c+CD16+MDC and monocytes, and VEGFR-1 and VEGFR-2 mRNA in CD11c+CD16−DC. Treatment with rhVEGF165 had no effect on maturation status of MDC and monocytes residing in BM and PB, as measured by MHC-class II, CD40, CD86, CD83 expression. However, rhVEGF165 treatment regulated the capacity of MDC and monocytes to stimulate T cell responses by inducing several disparate outcomes, enhancing the capacity of CD11c+CD16−MDC, diminishing the capacity of monocytes and no effect on CD11c+CD16+MDC.

Our data suggests that the paradigm of VEGF-induced myeloid-derived suppressor cells is not as simple as originally predicted and it is likely that VEGF regulates myeloid cells to varying degrees depending on their developmental status.

Disclosures:

No relevant conflicts of interest to declare.
Positron Emission Tomography Status and Tandem High-Dose Therapy Favorably Influence Outcome of Patients with Relapsed and/or Refractory Hodgkin Lymphoma
Authors: Devillier Raynier , Coso Diane , Castagna Luca , Brenot-Rossi Isabelle , Anastasia Antonella , Chitit Arturo , Schiano Jean Marc , Santoro Armando , Chabannon Christian , Balzarotti Monica , Blaise Didier , Bouabdallah Reda
Published: October 2019

Abstract 335

The cure of relapsed or refractory Hodgkin Lymphoma (HL) still remains a challenge. High dose chemotherapy followed by autologous stem cell transplantation (ASCT) is the standard of care but almost half of patients relapse after ASCT and have poor outcome. Predictive factors including an interval from end of first line therapy to relapse shorter than 12 months, an Ann-Arbor stage III or IV at relapse, and relapse in previously irradiated field are currently used to identify patients with poor outcome. Development of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) assessment improves evaluation of response both in first line and salvage treatments. The aim of our study is first to confirm the predictive value of PET status before ASCT and then to compare ASCT strategy (single versus tandem) in patients with relapsed and/or refractory HL. We here report a series of 111 consecutive patients with relapsed and/or refractory HL who achieved at least partial remission (PR) at PET evaluation after one line of salvage chemotherapy and who underwent single or tandem ASCT. PET response assessment showed 85 (77%) patients in CR (PET- group) and 26 (23%) in PR (PET+ group). Five-year overall (OS) and progression free survival (PFS) were 81% and 64% respectively. There were significant differences in 5-year PFS (79% versus 23%, p<0.001) and 5-year OS (90% versus 55%, p=0.001) between PET- and PET+ groups respectively. This predictive value remained significant in both favorable/intermediate and unfavorable subgroups. In PET+ subgroup analysis, tandem ASCT dramatically improved 5-years PFS, from 0% to 43% (p=0.034) compared to single ASCT. Multivariate analysis showed that PET status (HR: 5.26 [2.57–10.73]) and tandem ASCT (HR: 0.39 [0.19–0.78]) but not relapse risk (HR: 1.77 [0.80–3.92]) significantly influenced PFS, while only PET status significantly influenced OS (HR: 4.03 [1.38–11.75]). Our results suggest that I) PET status before ASCT must be considered as a strong and significant prognostic factor influencing outcome and identifying high risk patients for more intensive strategy II) Tandem ASCT improved outcome compared to single ASCT, especially for PET+ patients.

Disclosures:

No relevant conflicts of interest to declare.
Plerixafor Abrogates the Supportive Function of MSC for Self-Renewal of Human Hematopoietic Stem Cells,
Authors: Ludwig Annette , Saffrich Rainer , Eckstein Volker , Lenze Angela , Diehlmann Anke , Ho Anthony D. , Wuchter Patrick
Published: October 2019

Abstract 3408

The CXCR4-SDF1α axis plays an important role in maintaining the stemness of human hematopoietic stem cells (HSC). In the present study we established a surrogate model for the bone marrow niche by culturing HSC on a feeder-layer of human mesenchymal stromal cells (MSC) and investigated the proliferation and differentiation behaviour upon perturbation by Plerixafor.

HSC (CD34+ cells) were isolated from umbilical cord blood by fluorescent activated cell sorting (FACS). MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were stained with carboxyfluorescein succinimidyl ester (CFSE) and cultured on MSC feeder-layer for 6 days. For evaluating the influence of the culture medium on MSC cultures, three different media conditions (M1-M3) were used. Medium 1 (M1) contained 2% fetal calf serum (FCS), medium 2 (M2) contained 10% FCS and medium 3 (M3) contained GMP-grade human platelet lysate (hPL). Proliferation of HSC was calculated by analyzing the distribution of the CFSE dye (measured at day 1 and day 6). Plerixafor was added in concentrations of 0.1, 1.0 and 10.0 μM. On day 6, HSCs were harvested and analyzed by flow cytometry for CD34, CD38 and CXCR4 expressions in relationship to the cell division rate.

When co-cultured with MSC, the division kinetics of HSC was increased, while the proportion of CD34+ cells remained significantly higher compared to HSC without MSC. Accordingly, more CD38− HSC were found after 6 days upon co-culture with MSC. All three MSC preparations supported self-renewing proliferation of HSC, whereas MSC M1 induced the strongest effect. This underlines that co-culture with MSC has a significant supportive function for hematopoiesis. The additional exposure to Plerixafor in the co-culture system partially reversed this effect in a dose-dependent manner: division rate of HSC and the proportion of CD34+ and CD38− cells were reduced with higher concentrations of Plerixafor. The reduction of self-renewing proliferation by Plerixafor was not observed in controls consisting of HSC without MSC. Plerixafor also rendered the CXCR4 receptors undetectable on the surface of CD34+ cells for up to 6 days, most probably due to a persisting blockade of the antibody-binding site.

Human HSC co-cultured with MSC showed an increased cell division rate and produced a higher proportion of CD34+/CD38− cells. Different MSC culture media were systematically analysed in this setting and subtle differences in the supportive function could be observed. The addition of Plerixafor neutralized the effects of MSC, leading to an earlier loss of “stemness” and to lineage-commitment of HSC, thus providing evidence for the role of the CXCR4/SDF1α axis in terms of supportive function of MSC for self-renewal of HSC.

Disclosures:

Ho: Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
Dysregulated Bone Wound Repair and Marrow Functions in Senescence Accelerated Mice (SAMP6),
Authors: Epperly Michael W. , O'Sullivan Regina P. , Cao Shaonan , Dixon Tracy M. , Goff Julie P. , Glowacki Julie , Greenberger Joel S.
Published: October 2019

Abstract 3415

Introduction:

The Senescence Accelerated-Prone mouse (SAMP6) shows normal growth followed by rapid aging, development of osteopenia and shortened lifespan, compared with control R1 mice. The bone defect has been attributed to reduced osteoblast potential of marrow stromal cells. We compared in vivo repair of tibial bone wounds. We compared in vitro hematopoiesis, irradiation sensitivity, stromal cell biology, and osteoblastogenesis with SamP6 and R1 marrow. We tested the hypothesis that SAMP6 mice have poor healing of osseous wounds and that in vitro properties of SAMP6 marrow are abnormal.

Methods:

As a model for spontaneous bone repair, a 2-mm unicortical wound was made in each proximal tibia. At intervals, wound diameter was measured on radiographs. Long term bone marrow cultures (LTBMCs) were established from other SAMP6 and R1 mice to measure hematopoiesis and to establish marrow stromal cell lines (MSCLs) for analysis. MSCL gene expression and sensitivity to irradiation were measured; osteoblastogenic potential was determined by culture in osteoblastogenic medium (1% FBS, 10 nM dexamathasone, 5 mM b-glycerophosphate, 50 mg/mL ascorbate-2-phosphate) and gene expression analysis for osteoblast markers alkaline phosphatase (ALP), PTH-receptor-1 (PTHR1), and Osteocalcin (OC).

Results:

There was a faster rate of closure of the tibial wounds in R1 than in SAMP6 mice; at day 21, wounds in R1 mice were 47% the size of those in SAMP6 mice (p = 0.032). For the first 24 weeks, SAMP6 LTBMCs had significantly greater hematopoiesis than R1, shown by more cobblestone islands (sites of stem cell activity) and by more multilineage colonies from nonadherent cells transferred to semisolid medium. After 24 weeks, however, hematopoiesis ceased in SAMP6 cultures but continued in the SAMR1 cultures for 40 weeks. There was constitutive upregulation of TGF-b, an inhibitor of hematopoiesis, in SAMP6 MSCLs, compared with R1 cells. SAMP6 MSCLs were more sensitive to radiation (Do of 1.5 ± 0.1 Gy), compared with R1 (Do of 4.0 ± 0.4 Gy; p = 0.0080). There was unexpected constitutive expression of ALP, PTHR1, and OC in SAMP6 MSCLs, compared with R1. In osteoblastogenic medium, there was greater expression of osteoblast marker genes in the SAMP6 line compared to R1.

Conclusion:

SAMP6 showed delayed bone wound repair. Marrow from SAMP6 mice showed shortened in vitro hematopoiesis and greater radiation sensitivity, indicative of greater oxidative stress, but the unexpected finding of constitutive expression of osteoblast genes suggest that in vivo factors are more important than innate cellular defects in marrow to account for impaired bone healing.

Acknowledgments:

This project was supported by NIAID U191A168021-06 and NIA R21AG034254.

Disclosures:

No relevant conflicts of interest to declare.