bluuebunny/crossref_metadata_2025 huggingface dataset:
Risk Score at Diagnosis and the Dynamics of Response to TKI Therapy In Chronic Myeloid Leukemia.
Authors: Lenearts Tom , Castagnetti Fausto , Traulsen Arne , Pacheco Jorge M , Rosti Gianantonio , Dingli David
Published: October 2019

Abstract 1236

Background:

Although most patients with early chronic phase CML (ECP-CML) respond to TKI therapy, the depth and speed of response can be different. While it is known that both the Sokal and Hasford scores have an impact on the speed and depth of response, no mechanisms explaining these differences have been identified.

Objective:

To provide explanations for any potential differences in CML response as a function of the Sokal and Hasford score using a computational model.

Methods:

We utilize a computational model of hematopoiesis and CML, together with serial quantitative data of disease burden under nilotinib therapy to determine the fraction of CML cells responding to therapy (z) and the impact of TKI on the self-renewal probability of CML cells (e) under therapy. Patients were stratified at diagnosis on both the Sokal and Hasford scoring system. A non-linear least squares method was used to separately fit the model to serial Q-RT-PCR data for BCR-ABL in response to therapy in each cohort.

Results:

A total of 73 patients were studied. The number of patients with low, intermediate and high risk disease based on the Sokal score was 34, 29 and 10 respectively while the respective distribution of patients on the Hasford score was 29, 43 and 1. Although the impact of nilotinib on the self-renewal probability of CML progenitor cells was similar across all risk groups (there were substantial differences in the fraction of cells responding to therapy: For the Sokal groups, the fraction of cells (z) responding to therapy decreased from 0.09 to 0.086 and 0.069 respectively for low, intermediate and high risk disease. In the case of the Hasford score, the difference in z between low and intermediate categories becomes more pronounced, i.e. z is 0.093 for low and 0.08 for intermediate risk disease.

Conclusions:

The risk score at diagnosis of CML has a direct impact on the dynamics of response to TKI therapy. Patients with a lower risk score respond faster to the same therapy when compared to high risk patients. The impact of TKI on the self renewal of CML cells appears to be the same regardless of the risk score but the fraction of cells responding decreases as the risk score increases. This suggests that subclones that may be less sensitive to TKI therapy may be emerging. Strategies that increase the fraction of cells responding to therapy in patients with higher risk disease may be indicated.

Disclosures:

Rosti: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau.
5-Azacytidine and G-CSF Derepressed Chromatin Structure of PU.1 and Its Targets Cebpa and Cbfb In Myelodysplastic Syndrome (MDS)
Authors: Stopka Tomas , Burda Pavel , Basova Petra , Vargova Karin , Curik Nikola , Jonasova Anna , Trneny Marek
Published: October 2019

Abstract 124

The myelodysplastic syndrome (MDS) represents a heterogeneous disorder characterized by ineffective hematopoiesis and evolution to acute myelogenous leukemia that is strikingly refractory to current therapeutic approaches. Novel epigenetic drugs including DNA-methyltransferase inhibitor 5-Azacitidine (5-AZA, Vidaza) are currently considered to improve clinical response in patients with MDS. MDS is characterized by abnormal differentiation and blocked maturation responsive to 5-AZA, therefore we studied major regulator of hematopoietic differentiation, transcription factor PU.1 as a candidate target of the epigenetic therapy. Transcription factor PU.1 represents very important myelo-lymphoid regulator of differentiation. PU.1 expression is regulated by Upstream Regulatory Element (URE) and its deletion in mouse caused downregulation of PU.1 leading to acute leukemia (Rosenbauer 2004). Our laboratory recently demonstrated that PU.1 in murine acute leukemic cells binds and promotes derepression of CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) (Burda 2009) that encode two key hematopoietic transcription factors involved in myeloid differentiation. Furthermore, transcriptional regulation through PU.1 binding sites of Cebpa and Cbfb loci involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3 lysine K9. Others reported that Cebpa expression is augmented by G-CSF (Dahl 2003). To determine if 5-AZA regulates PU.1 and its targets we determined their expression and chromatin structure following the 5-AZA treatment in MDS patient-derived blasts and in cell lines derived from MDS (MOLM-13, OCI-M2, SKM-1) and AML (K562). Our data provide evidence that in the chosen cell lines and in so far limited number of patients-derived cells (N=4) the gene expression of PU.1 and its direct targets Cebpa and Cbfb is stimulated by 5-AZA and this effect is further enhanced by G-CSF. Furthermore, marks of activated chromatin structure including histone H3K9 hyperacetylation and H3K4 hypermethylation are increased at the URE of the PU.1 gene again documenting its transcriptional activation. Conversely, levels of H3K9 methylation at URE are significantly reduced upon 5-AZA treatment documenting 5-AZA stimulates loss of repressive chromatin structure near PU.1 gene. These observations are currently compared with responsiveness of the patients to 5-AZA in vivo and expanded to larger set of patients. Our data collectively supports importance of the chromatin structure upstream of PU.1 gene and of its direct targets Cebpa and Cbfb in patients with MDS that may add to better understanding of effectiveness of epigenetic therapy in MDS. (Grants # IGA 10310-3, MSMT 2B06077, SVV-2010-254260507, MPO FR-TI2/509, GAUK 251135 82210).

Disclosures:

No relevant conflicts of interest to declare.
Zygomycosis After Allogeneic Hematopoietic Stem Cell Transplantation: A French Multicenter Cohort Study (2003-2008)
Authors: Xhaard Alienor , Fanternier Fanny , Dannaoui Eric , Bergeron-Lafaurie Anne , Lacroix Claire , Herbrecht Raoul , Legrand Faezeh , Mohty Mohamad , Michallet Mauricette , Cordonnier-Carlet Catherine , Malak Sandra , Guyotat Denis , Couderc Louis Jean , Socie Gerard , Lortholary Olivier , Ribaud Patricia
Published: October 2019

Abstract 1263

Background:

To evaluate the clinical characteristics and outcome of zygomycosis (ZG) in allogeneic hematopoietic stem cell transplant (HSCT) recipients.

Methods:

Retrospective cohort study involving HSCT patients with ZG diagnosed between 2003 and 2008, from 19 centres. During this period, 4138 allogeneic HSCT were performed in these centers.

Results:

ZG incidence was 0.56% (n=23). Twenty patients (87%) were male. Median age was 44 years (range: 3 to 63 years). Donors were non HLA-identical in 14 cases and conditioning regimens were reduced intensity in 8. ZG occurred a median of 217 days post transplant (range: 0 to 2693 days). Fifteen patients had active graft-versus-host disease (GVHD). Twenty patients were receiving steroids at a median dose of 1 mg/kg/day (range: 0.1 to 2 mg/kg/day) at ZG diagnosis. Ten patients (43%) had diabetes mellitus. Nine patients (39%) had experienced a previous proven or probable invasive filamentous fungal infection (Aspergillus: 8, Trichoderma:1) a median of 115 days (range: 8 to 392 days) before ZG diagnosis. ZG was a breakthrough infection in 20 cases. Before ZG diagnosis, 13 patients were on voriconazole therapy for a median duration of 61 days (range 3 to 394 days), 4 patients were on posaconazole (PCZ) for a median of 61 days (range 7 to 88 days). Other patients were receiving caspofungin, itraconazole or voriconazole. Lungs were the most common site of infection (65%); two patients had a disseminated infection. Histology was performed in 52% of the patients, and contributed to diagnosis for 83% of biopsies. Culture was positive in 83% of cases. The main causal species were Lichtheimia spp. and Rhizopus spp. Median time between the first clinical symptom and diagnosis was 8 days (range: 2 to 34 days). Twenty patients received an antifungal. All but 2 patients received a lipid formulation of amphotericin B as first-line treatment. Eleven patients received PCZ, two as first-line treatment. Surgery was performed in eight patients. Median treatment duration was 47 days (range: 2 to 730 days). Nine patients responded to treatment. Nineteen patients (83%) died a median of 46 days after ZG diagnosis (range: 0 to 1449 days). ZG attributable mortality was 79% (15/19 patients). Four patients are alive a median of 706 days after ZG diagnosis (range: 705 to 1955 days).

Conclusion:

ZG is a late event with a poor prognosis after allogeneic HSCT. Because ZG diagnosis is difficult, special attention must be paid to patients on long-term antifungal treatment for a previous mold infection, and with protracted GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Pre-Engraftment Syndrome Following Umbilical Cord Blood Transplantation Is a Strong Predictor of Acute Graft-Versus-Host Disease
Authors: Wang Xingbing , Sun Zimin , Liu Huilan , Geng Liangquan , Ding Kaiyang , Zhu Weibo , Wang Zuyi
Published: October 2019

Abstract 1267

Pre-engraftment syndrome (PES) has been described in patients receiving umbilical cord blood transplantation (CBT). However, PES remains poorly characterized, and the prognosis and appropriate management are unclear. Therefore, we retrospectively analyzed the incidence, risk factors, manifestations, and clinical outcome of PES in CBT recipients treated for hematological malignancies at our transplantation center.

A total of 60 patients (median 20 years, range 3–48) received either myeloablative (n=52) or reduced-intensity (n=8) conditioning. 28 patients received double unit grafts to augment engraftment. Cyclosporine-A and mycophenolate mofetil were used as GVHD prophylaxis, and all patients received post-transplant granulocyte colony-stimulating factor.

In this study, PES was defined as noninfectious fever (>38.3°C) and/or unexplained rash occurring before neutrophil engraftment. 38 patients (63.3%) fulfilled PES criteria: 33 with fever and 36 with rash. The median onset was 8 days (range 3–15) post-transplant (a median of 12 days before neutrophil recovery). 34 patients received IV methylprednisolone (MP) (0.5-2 mg/kg/d) with 27/34 (79.4%) having fever and/or rash resolution in <48 hours. Univariate analysis identified myeloablative conditioning and younger age as significant risk factors for developing PES. Use of TBI or ATG in conditioning regimen, the infused total nucleated cell dose, gender, weight, blood type mismatch, transplantation of UCB units and degree of donor-recipient HLA disparity were not significantly associated with the development of PES in our series. The cumulative incidence of sustained donor engraftment was 83.3%. Patients with PES had a higher incidence of developing grade II -IV acute GVHD, but PES was not associated with sustained donor engraftment, the day to neutrophil recovery and chronic GVHD. Overall survival did not significantly differ between patients with and without PES.

We conclude that PES is common following CB transplant (CBT), predicts for aGVHD, and responds promptly to corticosteroids.

Disclosures:

Wang: Fund of the Key Medical Project of Anhui Provincial healthy department (2010A005): Research Funding; Clinical Technology foundation of Anhui Provincial healthy department (2008A011): Research Funding; Fund of Anhui Provincial “115” Industrial Innovation Program: Research Funding; Anhui Provincial Outstanding Young Investigator Program (08040106810): Research Funding.
The ZZ Domain of Sequestosome-1/p62 Plays An Important Role In Stromal Cell Support of Myeloma Cell Growth and Osteoclast Formation
Authors: Teramachi Jumpei , Windle Jolene J. , Roodman David , Kurihara Noriyoshi
Published: October 2019

Abstract 128

The marrow microenvironment provides a critical supportive role in myeloma (MM) and enhances both tumor growth and bone destruction through activation of multiple signaling pathways in stromal cells. We reported that sequestosome 1 (p62) plays a key role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with MM. These results suggest that p62 is a potential therapeutic target for blocking the supportive effects of the marrow microenvironment in MM. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling, as a means to develop inhibitory peptides/molecules as potential therapeutic agents for MM. To pursue this objective, we generated deletion constructs of p62 that lacked specific p62 domains: ΔSH2, ΔPB1, ΔZZ, Δp38, ΔTBS and ΔUBA domains. We then transfected these constructs into a p62-knockout (KO) stromal cell-line we established from p62-KO mice and examined their RANKL, IL-6, and VCAM-1 expression induced by TNF-a. GFP-labeled MM1.S myeloma cells or normal CFU-GM, a source of OCL precursors, were then co-cultured with the p62-KO cells transduced with the different p62 deletion constructs and compared to wild type (WT) stromal cells. IL-6 production and VCAM-1 expression induced by TNF-a was 50% lower in non-transduced p62-KO stromal cells compared to WT stromal cells. Further, in contrast to WT cells, RANKL was not induced by TNF-a in the p62-KO stromal cell-line, and OCL formation in co-cultures of p62-KO stromal cells with CFU-GM was very low. Transduction of p62-KO stromal cells with the ΔSH2, ΔPB1, Δp38 and ΔUBA constructs restored stromal cell support of MM growth, VCAM-1 and IL-6 production. However, p62-KO stromal cells transduced with the ΔZZ construct did not increase MM cell growth, or increase IL-6 and VCAM-1 expression, or fully restore the capacity of the p62-KO stromal cells to support OCL formation. These results demonstrate that the ZZ domain of p62 is required for stromal cell support of MM cell growth, increased IL-6 and VCAM-1 expression, and OCL formation. These results suggest that dominant negative constructs or small molecules that target the ZZ domain of p62 should block p62 function and inhibit support of MM cells and OCL formation by the marrow microenvironment.

Disclosures:

Roodman: Amgen, Celgene, Acceleron & Millennium: Consultancy.
Chronic Lymphocytic Leukemia (CLL) Signaling Profiling Identifies CLL-Specific Signaling Impairment That Discriminates CLL B-Cells From Normal Circulating B Cells
Authors: Palomba M. Lia , Piersanti Kelly , Zelenetz Andrew D , Brink Marcel R.M. van den , Altan-Bonnet Gregoire
Published: October 2019

Abstract 1374

CLL is the most common leukemia in the Western world. It is characterized by the clonal expansion of CD5+/CD19+ B cells, expressing surface immunoglobulins (sIg), most often of the IgM subset. Clinically, it can either manifest as an indolent disease with little impact on the lifespan of a subset of patients or as an aggressive, highly drug-resistant disease with lethal outcome but all possible scenarios in between these extremes are routinely observed. The degree of somatic mutations of the sIg has been shown to affect survival; patients expressing highly mutated sIg have a better prognosis than patients whose sIg are less than 2% different from germline sequences. Therefore, it is intuitive that signaling through the sIg, or B-cell receptor (BCR), must have a role in determining the fate of the CLL cells, veering the intracellular machinery towards proliferation or apoptosis.

We have systematically investigated the downstream events caused by BCR stimulation via crosslinking of the sIg in a series of about 60 heterogeneous CLL patients and 15 normal donor peripheral blood mononuclear cells (PBMCs). Using multiplexed phosphoflow cytometry we were able to assess the intensity of multiple signaling events at specific stages of the BCR signaling pathway. By combining phosphospecific staining and classic immunophenotyping we were also able to evaluate signaling intensity at the at the single cell level as well as correlating the signal intensity of CLL-specific surface markers to that of the phosphoproteins analyzed. We used principal component analysis to combine phosphospecific staining within individual cells and generate a variable that discriminates efficiently between CLL and PBMCs B-cells.

We found that signaling properties of CLL B-cells were impaired when compared to normal B cells from healthy donors, both at the level of upstream members of the BCR signaling pathway (pBLNK, pSYK, pZap70, pBTK, pPLCg2) and downstream components such as pp38 and pERK. However, within the cohort of CLL samples we identified a small subpopulation in which signaling proceeded as well or more efficiently than in normal B cells. We also used our principal component variable to quantify the heterogeneity of signaling responses within populations of CLL B-cells and correlate them with clinical parameters that are known prognostic indicators in CLL.

In conclusion, multiplexed phosphoflow cytometry of individual CLL cells provides a novel and highly specific method to determine the use of signaling pathways (such as BCR-mediated) in comparison to normal B cells. Our studies indicate a significant decrease in BCR signaling of CLL B cells, which involves both upstream and downstream components.

Disclosures:

van den Brink: Cytheris: Research Funding.
Toxico-Kinetics of Recombinant VWF In Non-Human Primates and Rabbits
Authors: Muchitsch Eva-Maria , Dietrich Barbara , Rottensteiner Hanspeter , Gritsch Herbert , Ehrlich Hartmut J. , Turecek Peter , Schwarz Hans Peter
Published: October 2019

Abstract 1414

Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. We showed previously by in vitro and ex vivo studies that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine and rat ADAMTS13, whereas ADAMTS13 from rabbits and cynomolgus monkeys is able to cleave human rVWF.

Here we tested the pharmacological behavior of human rVWF in rabbits and cynomolgus monkeys. The animals were infused once with different doses of human rVWF. VWF antigen rose sharply in a dose-dependent manner (∼25 IU/ml VWF:Ag for the highest dose, 15 min after injection) and then declined gradually (∼7 IU/ml VWF:Ag for the highest dose, 18 hours after injection). By contrast, the ADAMTS13 activity did not show relevant changes throughout the entire test period in the rabbit or in the monkey samples, indicating that an excess of intravenously administered rVWF as the substrate does not exhaust its enzyme ADAMTS13. Most importantly, neither rabbits nor cynomolgus monkeys showed any exaggerated pharmacological or toxic effects upon rVWF administration. Even the administration of 14 daily doses of rVWF to cynomolgus monkeys did not lead to any toxicological effect.

Our data indicate that rabbits and cynomolgus monkeys, two species with a sufficient rVWF cleavage capacity by endogenous ADAMTS13, are appropriate animal models for a meaningful preclinical evaluation of the rVWF product, which allows the therapeutic safety margins for human patients to be determined.

Disclosures:

Muchitsch: Baxter Innovations GmbH: Employment. Dietrich:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment.
Cleavage of Recombinant and Plasma-Derived VWF by Human ADAMTS13
Authors: Rottensteiner Hanspeter , Varadi Katalin , Vejda Susanne , Ehrlich Hartmut J. , Scheiflinger Friedrich , Schwarz Hans Peter , Turecek Peter
Published: October 2019

Abstract 1415

A recombinant human CHO-expressed von Willebrand factor (rVWF) consisting of ultra-high molecular weight (UHMW) multimers resembles the VWF stored in Weibel-Palade bodies of endothelial cells. Once secreted into plasma, UHMW multimers are rapidly cleaved by ADAMTS13 and are usually missing in plasma-derived VWF (pdVWF).

Here we analyzed in vitro whether the kinetics of cleavage of rVWF by ADAMTS13 is similar to that of pdVWF. The kinetics of ADAMTS13-mediated proteolysis of rVWF were explored under denaturing conditions (1.5 M urea) or under shear stress so as to expose the ADAMTS13 cleavage site of VWF.

Multiple assays showed that rVWF was efficiently cleaved by ADAMTS13. UHMW multimers disappeared within seconds at physiological concentrations of ADAMTS13. Using lower concentrations of ADAMTS13 (10-30 mU/ml, equivalent to 1–3% of normal human plasma), UHMW were cleaved within 30 minutes. The typical satellite bands appeared very early in an ADAMTS13 dose-dependent manner. Virtually the same results were obtained when human plasma was used as a source for ADAMTS13. Although pdVWF differs from rVWF in its multimeric structure, the decrease in activity was similar for rVWF and pdVWF. Finally, the extent of ADAMTS13 cleavage was similar for rVWF and pdVWF when exposed to shear stress using a cone-plate viscometer.

Our data clearly indicate that rVWF is a good substrate for ADAMTS13. Ongoing phase 1 studies demonstrated that rVWF is indeed processed by the protease when administered in humans with severe VWD.

Disclosures:

Rottensteiner: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Vejda:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.
Identification of a VPS33B-Binding Protein That Facilitates Alpha Granule Formation In Human Megakaryocytes and Platelets.
Authors: Urban Denisa , Li Ling , Wasmuth James , Christensen Hilary , Parkinson John , Kahr Walter H.
Published: October 2019

Abstract 1444

Human platelets contain α-granules, dense (δ-) granules and lysosomes that release their contents upon platelet activation. Platelet granule release is important for hemostasis, since patients with inherited granule defects have bleeding problems. α-granules are absent in the gray platelet and ARC syndromes, while deficient δ-granules are observed in isolation, in combination with α-granule deficiency, or as part of a syndrome in the Hermansky-Pudlak, Chediak-Higashi and Griscelli syndromes. The biogenesis of α-granules is poorly understood. Our laboratory has identified VPS33B as a central player in the formation of platelet α-granules. VPS33B has yet to be characterized in detail, however, its homolog VPS33A is known to be part of a multiprotein complex involved intracellular vesicle trafficking. Studies in our laboratory suggest that VPS33B is also part of a multiprotein complex. We performed a yeast two-hybrid library screen with VPS33B as bait and found another member of the complex: the unidentified gene product of chromosome 14 open reading frame 133 (C14orf133). Sequence analysis indicated this to be human VPS16B. Our studies show that VPS16B specifically binds to VPS33B but not its homologue, VPS33A. Furthermore, we show that VPS33B forms a distinct complex from that of its homologue VPS33A. VPS16B was found to co-localize with trans-Golgi, late endosome and α-granule markers in megakaryocytic Dami cells. Ongoing studies suggest that knockdown of VPS16B affects α-granule formation. We conclude that VPS16B, much like its binding partner VPS33B, plays a crucial role in megakaryocyte and platelet α-granule biogenesis.

Disclosures:

No relevant conflicts of interest to declare.
The Risk of Low Bone Mineral Density with Long-Term G-CSF Therapy for Severe Chronic Neutropenia.
Authors: DiMeglio L. A. , Bolyard Audrey Anna , Marrero Tracy M. , Alter Blanche P , Bonilla Mary Ann , Boxer Laurence A. , Link Dan , Newburger Peter E. , Rosenberg Philip S. , Shimamura Akiko , Dale David C.
Published: October 2019

Abstract 1484

Low bone mineral density (BMD) is a known risk factor for fractures. Low BMD has been reported in individuals with severe chronic neutropenia (SCN), and attributed both to the diseases causing neutropenia and to G-CSF therapy. However, given the rarity of SCN, few data exist regarding associations of BMD z-scores with disease characteristics such as type of neutropenia and duration of G-CSF therapy. We present data here obtained from BMD reports collected through the Severe Chronic Neutropenia International Registry (SCNIR).

We reviewed BMDs on 128 subjects [40 children (< 21 years of age), 87 females] having sufficient information about lumbar spine BMD by dual-xray absorptiometry (DXA) for evaluation. For subjects with multiple BMD measurements available, the most recent one was used for analysis. Mean age was 32.0 years (range 0.6–85 years).

57 subjects had idiopathic SCN (mean age 40 years), 40 had congenital (mean age 15 years), 28 were cyclic (mean age 41 years) and 3 were autoimmune (mean age 18 years). 122 subjects had received G-CSF at the time of the BMD assessment (mean 8.8 years, range 0.1–19.9 years). 11 of the adults were on bisphosphonate therapy for low BMD at the time of the BMD assessment; no children were on anti-resorptive therapy.

BMDs in these subjects were, on average, low. For the children, the BMD z-score (age matched mean ±1 standard deviation) was -1.0 ± 1.1, with 17.5% of children having BMDs that were low for age (Z-score < -2.0). For the adults the BMD t-score was -1.1 ± 1.4, with 46% of adults meeting t-score criteria for osteopenia (≤ -1.0) and 9% meeting criteria for osteoporosis (< -2.5). BMDs were lowest in those with congenital neutropenia, followed by those with cyclic neutropenia.

For children, BMDs were lower in those who had received longer G-CSF therapy (r= -0.506, p=0.002). This association was not seen in adults (r= 0.074, p= 0.5).

The low BMDs and the correlation of lower BMD with longer G-CSF treatment in children suggests there is an association of bone loss with the childhood diseases causing SCN. The data also suggest that regular assessments of bone health should be made in SCN patients, particularly those on long-term G-CSF therapy.

Disclosures:

Boxer: Amgen, Inc.: Equity Ownership. Dale:Amgen: Consultancy, Research Funding.
Leukocyte Integrin αXβ2 Is the Principal Receptor for Candida Albicans on Macrophages and Is Essential for Antifungal Host Defense.
Authors: Jawhara Samir , Soloviev Dmitry
Published: October 2019

Abstract 1493

The induction of cell-mediated immunity to pathogens is the prime task of cells of the innate immune system. Integrin αMβ2 (Mac-1, CR3, CD11b/CD18) is expressed on polymorphonuclear neutrophils (PMNs), monocytes, macrophages (MFC) and NK cells and has been implicated in diverse responses of these cells to infection, including phagocytosis, cell-mediated killing, chemotaxis and cellular activation. In PMNs integrin αMβ2 serves as the principal receptor involved in recognition of the common opportunistic fungal pathogen Candida albicans, and this integrin recognizes the fungal mannoprotein pH-regulated Antigen 1 (Pra1). Professional antigen-presenting cells of myeloid lineage MFC and dendritic cells express integrin αXβ2 (p150,95, CD11c/CD18) which is highly homologous to Integrin αMβ2, but the role of αXβ2 in the immune response to bacterial and fungal infections remains unclear. Employing αM - and αX-KO mice and mutated strains of C. albicans in a model of murine systemic candidiasis, we first demonstrate that mice deficient in αXβ2 exhibit significantly increased susceptibility to the invasive fungal infection. C. albicans induced lethality in αXβ2-KO mice more rapidly (2- to 3-fold faster) than the same inoculum in wild-type mice. MFC derived from mice deficient in α×β2 have a reduced ability to kill or phagocyte C. albicans. When the fungus was injected into mice, 6 hrs after peritoneal of thioglycollate, neutrophil recruitment to C. albicans showed a 20%-30% clearance of the fungus in the αMβ2-KO murine intraperitoneal lavages and a 50–60% rise in the αXβ2-KO lavages, which is consistent with our prior data showing αMβ2-dependency for PMNs-mediated antifungal defense activity. In contrast, when the fungus was injected into the peritoneal cavity 16 hrs after thioglycollate, when most recruited leukocytes are MFC, the C. albicans injected into αXβ2-KO mice showed a 23%-25% clearance of the fungus from the lavages while fungal clearance in αMβ2-KO lavages increased by only 30–40%. Thus, MFC antifungal activity is αXβ2-dependent. These results were further confirmed by in vitro fungicide assays employing isolated murine peritoneal PMNs and MFC. Fungal survival was greatest with PMN derived from αMβ2-KO mice and MFC derived from the αXβ2-KO mice-derived MFC. Disruption of the PRA1 gene of C. albicans, which is the primary ligand for αMβ2, had no effect on MFC fungicide activity, which suggests that αXβ2 recognizes a C. albicans ligand different from Pra1. Taken together, these results suggest that αXβ2 is a principal receptor for C. albicans on macrophages and plays critical role in host antifungal defense in a Pra1-independent mechanism.

Disclosures:

No relevant conflicts of interest to declare.
Overall Survival of Lymphoma Patients In Randomized Controlled Trials Comparing Primary G-CSF Chemotherapy Support to Control.
Authors: Lyman Gary H. , Crawford Jeffrey , Culakova Eva , Poniewierski Marek S. , Huang Mingyan , Kuderer Nicole M. , Dale David C.
Published: October 2019

Abstract 1502

Background

Randomized controlled trials (RCTs) have shown that granulocyte colony-stimulating factor (G-CSF) can reduce the risk of neutropenic complications as well as early mortality (Kuderer et al. JCO 2007). The impact of G-CSF support of chemotherapy on overall survival in patients with lymphoma is assessed here in a systematic review of RCTs of chemotherapy with versus without G-CSF in studies with at least 2 years of follow up.

Methods

A systematic review of RCTs of patients with malignant lymphoma receiving chemotherapy with versus without primary G-CSF support and reporting overall mortality by study arm identified 16 eligible studies. Data extraction was conducted by two independent reviewers. Heterogeneity was evaluated using Cochran's Q-statistic and the Inconsistency Index (I2). Weighted summary estimates of relative risk (RR) and absolute risk (AR) were based on the method of Mantel and Haenszel. Chemotherapy relative dose intensity (RDI) with G-CSF support compared to control was meta-regressed on the logarithm of RR for mortality. Egger's regression intercept was utilized to assess publication bias.

Results

Sixteen (16) eligible RCTs of patients receiving chemotherapy with (n=2,731) versus without (n=2,757) primary G-CSF support were identified. Deaths were reported in 927 patients randomized to receive primary G-CSF and in 1,064 controls. No significant heterogeneity was observed (Q=12.50, P=.641; I2=0%). Overall RR for mortality with versus without G-CSF support of chemotherapy was 0.876 [95% CI: 0.822 – 0.935; P<.001]. The AR reduction for mortality in G-CSF-supported patients versus control was 4.8% [95% CI: 2.4% - 7.15; P<.001]. An inverse trend was observed between the difference in RDI between treatment arms and RR for mortality [Slope = -0.39; 95% CI: -0.78 – 0.01; P=.057]. The greatest reduction in mortality was observed in the 5 RCTs of dose dense chemotherapy [RR=0.830; 95% CI:0.742-0.930; P=.001]. Significant treatment effect was observed in 7 studies limited to older patients [RR=0.910;95% CI: 0.847–0.978;P=.010] and 9 permitting all adult age groups [RR=0.813;95% CI: 0.715–0.926;P=.002]. No difference in treatment effect was observed based on study funding source and no evidence for publication bias was observed.

Conclusions

A highly significant reduction in overall mortality was associated with G-CSF support compared to control in 16 RCTs of lymphoma patients receiving chemotherapy. Patients receiving greater RDI with G-CSF support, especially those receiving dose dense regimens, experienced the greatest reduction in mortality.

Disclosures:

Lyman: Amgen: Research Funding. Crawford:Amgen: Consultancy. Dale:Amgen: Consultancy, Research Funding.
Isolation and Characterization of Renal Erythropoietin-Producing Cells: Contribution of HIF3α In Vivo.
Authors: Pan Xiaoqing , Suzuki Norio , Hirano Ikuo , Yamazaki Shun , Yamashita Toshiharu , Ohneda Osamu , Minegishi Naoko , Yamamoto Masayuki
Published: October 2019

Abstract 1541

Understanding the nature of renal erythropoietin-producing cells (REPs) remains a central challenge for understanding the mechanisms involved in hypoxia/anemia-induced erythropoietin (Epo) production in adult mammals. Previous studies have shown that REPs are renal peritubular cells, but further details are lacking. Here, we describe our approach used to isolate and characterize REPs. We bred mice bearing Epo/GFP combined with Epo-3’-enhancer deletion. These mice exhibit perinatal anemia (average Hct = 18% at P4–6). This enabled identification and purification of REPs based on GFP expression in the kidney. Light and confocal microscopy revealed that GFP immunostaining was confined to fibroblasts residing in the peritubular interstitium, which is consistent with our previous observation from the 180-Kb Epo/GFP reporter assay. Flow cytometry revealed that the GFP fraction constituted approximately 0.2% of the whole kidney cells; with most (63%) cells co-expressing CD73, a well-known marker for Epo-expressing cells. qRT-PCR confirmed that Epo expression was increased by approximately 100-fold in the purified REP population compared with that of the unsorted cells or CD73 fraction. Gene expression profiling showed Hif2α and Hif3α enrichment in the purified GFP/REP population. Moreover, in mouse hypoxia experiments, Epo expression was approximately 2.5-fold higher in Hif3α–/– kidney cells than in wild-type cells. The molecular strategy described here provides a means to isolate a pure population of REPs, allowing the analysis of gene expression profile of a defined population of cells essential for Epo production in the kidney. Our findings have led to a novel hypothesis that positive regulation by HIF2α and negative regulation by HIF3α might be necessary for correct renal Epo induction.

Disclosures:

Yamamoto: Chugai Pharmaceutical, Co., Ltd.: Research Funding.
A Screen In Primary Erythroblasts Reveals a MicroRNA-Centered Homeostatic Mechanism for Regulating Cyclin E Activity.
Authors: Xu Yanfei , Sengupta Tanushri , Minella Alexander C.
Published: October 2019

Abstract 1543

A growing body of evidence highlights the importance of microRNAs in regulating the expression of mediators of cell cycle progression. A theme emerging from these studies is that microRNAs participate in feedback or feed-forward circuits to provide bistability for key transition points in the cell cycle. We previously have shown that proper regulation of cyclin E activity is required for normal erythroid cell maturation in vivo, using cyclin ET74AüT393A knock-in mice, which have markedly dysregulated cyclin E due to its failure to interact with the Fbw7 ubiquitin ligase complex. We hypothesized that we could identify novel, microRNA-based molecular circuitry for maintaining appropriate levels of cyclin E activity by screening cyclin E knock-in erythroblasts for alterations in microRNA expression. We analyzed data we obtained from multiplex real-time PCR arrays comparing the expression of over 500 microRNAs in cyclin ET74A T393A knock-in versus wild-type erythroblasts (Ter119+/CD71+) and found down-regulated expression of a number of microRNAs targeting CDK inhibitors. We also identified down-regulated expression of potential microRNA regulators of Fbw7 expression. We found that overexpression of miR-223, in particular, significantly reduces Fbw7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the Fbw7 3’ untranslated region. We then found that reduced miR-223 expression leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression in K562 cells is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway and that dysregulated Fbw7 expression alters the erythroid differentiation capacity of these cells. Mir-223 plays an important role in myeloid and erythroid differentiation by regulating multiple substrates involved in these maturation programs. Here, we identify Fbw7 as a novel target of miR-223. Our data also indicate that miR-223 modulates Fbw7 expression as part of a homeostatic mechanism to regulate cyclin E activity and provide the first evidence that activity of the SCFFbw7 ubiquitin ligase can be controlled by the microRNA pathway.

Disclosures:

No relevant conflicts of interest to declare.
Small Rab GTPase Rab7b Promotes Megakaryocytic Differentiation by Enhancing IL-6 Production and STAT3-GATA-1 Association.
Authors: He Donghua , Chen Taoyong , Yang Mingjing , Zhu Xuhui , Cao Xuetao , Cai Zhen
Published: October 2019

Abstract 1549

Interleukin 6 (IL-6) is a pleiotropic cytokine acting on a variety of cell types, and plays important roles in hematopoiesis. GATA-1 (GATA binding protein 1) is an important transcription factor involved in either megakaryocytic or erythrocytic differentiation. However, the mechanisms for IL-6-induced megakaryocytic differentiation and regulation of GATA-1 have not been fully elucidated. By using phorbol-12-myristate-13-acetate (PMA)-induced megakaryocytic differentiation of K562 cells as a model, we investigated the roles of Rab7b, a late endosome/lysosome-localized myeloid small GTPase, in megakaryocytic differentiation. We find that Rab7b can promote PMA-induced megakaryocytic differentiation, as evidenced by typical morphological alterations, increased fibronectin-specific adhesion, increased polyploidy formation, and increased expression of CD41a. The GTP-bound status and lysosomal localization of Rab7b are required for promotion of K562 megakaryocytic differentiation, which can be blocked by inhibitor of nuclear factor κB (NF-κB) and neutralizing antibodies for IL-6 and gp130. In Rab7b-silenced K562 cells, PMA-induced activation of NF-κB, IL-6 production and megakaryocytic differentiation are impaired. Furthermore, we demonstrate that IL-6-induced activation of signal transducer and activator of transcription 3 (STAT3) and the subsequent association of STAT3 with GATA-1 may contribute to PMA-induced and Rab7b-mediated transcriptional upregulation of megakaryocytic differentiation markers. Therefore, our data suggest that Rab7b may play important roles in megakaryopoiesis by activating NF-κB and promoting IL-6 production. Our study also indicates that the IL-6-induced association of STAT3 with GATA-1 may favor megakaryopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Klotho Polymorphisms and Priapism In Sickle Cell Disease.
Authors: Souza Claudia R Lustosa , Shimmoto Marily M Azevedo , Vicari Perla , Arruda Martha Mariana A S , Roizenblatt Marina , Pedro Ana C Cabanas , Hayashida Debora Y , Mecabo Grazielle , Figueiredo Maria Stella
Published: October 2019

Abstract 1653

Background:

Sickle cell disease (SCD) is a monogenic disorder with phenotypic heterogeneity, possibly determined by polymorphisms (SNPs) in genes whose products modify the pathophysiology of the disease. Priapism is one of the most common vaso-occlusive complications of SCD, and it occurred in more than 30% of males. The Klotho (KL) gene appears to be associated with vascular function and nitric oxide biology and the presence of SNPs could affect its function. Association between KL and priapism in SCD patients was suggested by Nolan et al. in 2004. However, other authors could not confirm this finding (Elliot et al., 2007).

Objective:

We decided to evaluate the relevance of SNPs rs2249358, rs211234 and rs9536314 to the occurrence of priapism in patients with SCD followed at Outpatient clinic at Escola Paulista de Medicina/UNIFESP.

Methods:

Forty male patients with SCD were enrolled, 39 (97.5%) with sickle cell anemia (SS) and one (2.5%) SC hemoglobinopathy. The manifestation of priapism was identified through analyses of medical records. The SNP rs2249358 was identified by PCR followed by restriction with XbaI. The other SNPs, rs211234 and rs9536314, were analyzed by allele specific PCR. Statistical analysis: t test, Chi2 or Fisher. This study was approved by Ethical Committee, and all patients agreed in participate.

Results:

The median age of the patient was 28.5 years-old (20-68 y.o.). Fourteen out of 40 patients had priapism (35%), each one with SS disease. The group of patients with priapism were older (32.5 y.o., 25–68 y.o.) than the group without this manifestation (27.5 y.o., 20–56 y.o.) (p=0.03). There was no statistical difference in the distribution of the SNPs rs211234 and rs9536314 between the two groups of patients (p=0.51 and p= 0.09, respectively). Regarding the distribution of SNP rs2249358, the group with priapism presented 8 individuals (57.1%) with GG genotype, 5 (35.7%) with AA and 1 (7.17%) with AG, whereas in the group without priapism, the distribution was different: 5 (19.2%) with GG, 7 (26.9%) with AA and 14 (53.8%) with AG genotype (p=0.0212). When we compare the presence of at least one A allele (AA or A-) with the G allele in homozygosis (GG), we observed that the A allele has a protector effect (OR: 0.1786; CI: 0.04232–0.7535) (p=0.031).

Conclusions:

In a relatively small group of patients with SCD, it was observed a significant proportion of individuals with priapism, which reinforces the importance of this manifestation. We also observed correlation between SNP rs2249358 of KL gene and priapism, as suggested previously.

Disclosures:

No relevant conflicts of interest to declare.
DIGE-Based Proteomic Analysis Identifies NPM/B23 and Nucleolin C23 as Overexpressed Proteins In Relapsed /Refractory Acute Leukemia
Authors: Hu Jian Da , Lin MinHui , Liu TingBo , Li Jing , Chen XinJi , Chen BuYuan
Published: October 2019

Abstract 1711

Resistance to chemotherapy is a challenge in treatment of acute leukemia. Although the classic multidrug resistance (MDR) phenotype is often characterized by expression of drug efflux pump P-glycoprotein or by multidrug resistance-associated proteins, precise molecular mechanisms are largely unknown. To investigate novel protein changes involved in resistance mechanism, protein expression profiles between human myeloid leukemia HL-60 cell lines and adriamycin- resistant HL-60 cell lines (HL-60/ADR) was compared, which was based on a differential proteomic approach — 2 dimensional difference in gel Electrophoresis(2D-DIGE) followed by mass spectrometry (MALDI-TOF-MS) and complemented by western blot validation. 16 protein spots were identified as being differentially expressed (> 1.2 fold change and p≤ 0.05) between above two cell lines, among which 13 protein spots were identified as up-regulated and 3 as down-regulated in the HL-60/ ADR cell line. Proteins found to have higher abundance levels in the resistant HL-60/ADR cells included enzymes, proteins and oncogenes related to signal transduction, protein synthesis, cell growth regulation and metabolism. 3 lower abundance proteins are related to transcription. From 16 proteins, 2 proteins, nucleophosmin B23 (NPM B23)and nucleolin C23, were selected and verified in leukemia cell lines and primary leukemia samples by western blot. Compared to healthy control samples, which showed no expressions of these 2 proteins, leukemic cell lines revealed an obvious up-regulation of B23 and C23. Moreover, significantly higher expressions of B23 and C23 were found in 3 resistant leukemic cell lines, HL-60/ADR, K562/ADR and KG01 cells, compared to the parent HL-60 and K562 cells, and other leukemic cell lines. In de novo leukemia samples, 43.8%(35/80) expressed B23 and C23 proteins, 37.9% (22/58) AML and 59.1% (13/22) ALL respectively. Meanwhile, concomitant expression of B23 and C23, both positive or negative, was noted in 97%(79/80)patients. Over-expressions of B23 and C23 were observed in 68.8% relapased/refractory leukemia patients. With regard to treatment outcome,among those patients who achieved ongoing CR, fewer patients expressed 2 proteins, only 13.35% (7/52) AML and 46%(7/15) ALL respectively. It implicated that B23 and C23 may be involved in drug resistance and be useful in assessing treatment outcome and prognosis of leukemia. To a conclusion, these results provide a novel clue for the molecular mechanism of MDR and suggest that B23 and C23 are prognostic indicators for leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Single 4.5mg Dose of Rasburicase for Tumor Lysis Syndrome In Adults
Authors: Yim Barbara , Navaleza Anne , Haidau Augustin , Sreenivasappa Shylendra , Kassem Mohammed A. , Ciobanu Bety C. , Braik Tareq , Gaddh Manila , Rubinstein Paul G. , Shah Moushmi , Telfer Margaret , Catchatourian Rose
Published: October 2019

Abstract 1779

Introduction:

Tumor Lysis Syndrome (TLS) is an oncologic emergency that leads to a host of metabolic disturbances which can ultimately result in acute renal failure and death.

Currently rasburicase, Elitek™, is FDA approved for the treatment of TLS in patients with leukemia, lymphoma, and solid tumor malignancies which have risk factors for TLS. The approved adult dose is 0.2mg/kg/day over 30 minutes for up to 5 days. Chemotherapy should be initiated within 24 hours of rasburicase administration. Since rasburicase's introduction to the market, various studies have examined single dose use (3mg and 6mg) in the adult population, trying to resolve the dilemma of determining a dosing regimen for adults that is as effective as the FDA approved dosing regimen, while minimizing cost.

This study was aimed at determining if a single 4.5mg dose of rasburicase would be adequate in reducing uric acid levels (UAL) as compared to the FDA approved conventional weight based approach; it also sought to determine the cost-effectiveness of this approach.

Method:

This is a retrospective study of the John H. Stroger Jr. Hospital of Cook County (JHS) patients who were administered a single dose of 4.5 mg rasburicase for TLS from 12/2007 to 06/2010. We included patients ≥ 18 years old, with a hematologic malignancy and on chemotherapy or about to start receiving chemotherapy within 24 hours of rasburicase administration. Patients with low risk for TLS or an indication other than prevention and management of TLS were excluded.

Result:

Demographics:

Characteristics:

Responders are defined as patients that achieved more than 50% reduction in the UAL at 24 hours, 48 hours or 96 hours or a decrease of UAL to normal levels. Two patients required a second dose to achieve response. The non-responders did not receive any additional doses for unclear reasons. No adverse events were noted.

Conclusion:

This retrospective study provides evidence that a single 4.5mg dose of rasburicase effectively reduced plasma UAL to within normal limits. In addition, decrease in plasma UAL was observed within 24 hours after administration. No effect on electrolytes and serum creatinine was noted.

The cost-effectiveness of a single 4.5mg dose of rasburicase was evaluated based on dose and drug cost. The potential cost saving was substantial when compared with the FDA approved dose.

In our opinion, this study validates the use of a single dose of 4.5mg rasburicase in the treatment and prophylaxis of TLS. This dose can be considered as a possible alternative to the FDA approved adult dosing regimen.

Disclosures:

No relevant conflicts of interest to declare.