bluuebunny/crossref_metadata_2025 huggingface dataset:
Derivation and Drug Screening of Growth Factors- and Stroma-Dependent Human Multiple Myeloma Cells
Authors: Shieh Jae-Hung , Su Tsann-Long , Shieh Jason , Moore Malcolm A.S.
Published: October 2019

Abstract 5020

Multiple myeloma (MM) is the second most common hematological malignancy in the U. S. MM is treatable but not curable with a median survival of 5–7 years with advanced treatment. There are several established human MM cell lines that grow in the absence of any cytokine dependence or stromal interaction. However, most primary MM patient specimens are difficult to maintain or to expand in vitro for pathophysiology investigation and drug screening. To address this issue, we systemically evaluated effects of various culture parameters on the growth of primary MM cells. A serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system containing cytokine cocktail is capable of expanding purified CD138+ cells from primary MM specimens in vitro. Optimal expansion of the MM CD138+ cells requires a combination of stromal cells, human interleukin (IL-6) and human hepatocyte growth factors (hHGF). These MM cells can be maintained and repeatedly recovered following cryopreservation. The in vitro effect of five novel alkylating agents (BO-1055, −1090, 1099, −1393 and −1509) was evaluated for an inhibition of proliferation of the MM cells, and on human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 Cobblestones area forming cells (CAFC; an in vitro surrogate assay for hematopoietic stem cells) assay with CB CD34+ cells. BO-1055 shows a promising therapeutic window between the MM cells and other normal tissues. For the MM cells, IC50 was ∼1. 02 μM. In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC was >10, >25, 8, and >5 μM, respectively. Therefore, our stromal culture system supports human primary MM cells and closely recapitulates the growth of MM cells in their niche in vivo. We also identified a novel BO-1055 molecule that inhibits primary MM cells but is well-tolerated by other normal tissues. This in vitro stromal co-culture system provides a powerful tool to dissect the pathophysiology of human MM, and to screen new drugs for MM therapy.

Disclosures:

No relevant conflicts of interest to declare.
Modulation of Proinflammatory Cytokines and Insulinlike Growth Factor-1 (IGF-1) in Human Immunodeficiency Virus-1 Smoldering Multiple Myeloma (HIVSMM) Patients
Authors: McPherson Eugene , Tassy Philippe
Published: October 2019

Abstract 5043

Multiple myeloma is a B-cell malignancy involving germinal center B cells characterized by blood and urinary monoclonal proteins, osteolytic bone lesions and infiltration of bone marrow with plasma cells wit pathology involving aberrant chromosomal translocation with increased glucose uptake (Warburg Phenomena). This glucose transporter (GLUT) is activated and over expressed along with proinflammatory cytokines produced by malignant plasma cells both smoldering and multiple myeloma (MM). These cytokines may control growth, progression and dissemination. Interleukin-6 (IL-6) is a major cytokinetic growth factor in MM but IGF-1 is a more prominent growth factor that binds to IGF-1 receptor (IGF-1R) and is a strong indicator of prognosis which can augment anti-apoptotic effects of IL-6. Modulation of IGF-1 and proinflammatory cytokines in HIVSMM patients with ritonavir-based highly active antiretroviral therapy (RTV-based HAART) may suppress nuclear factor kappa b (NF Kb) and inhibit the overexpression of GLUT-1–4 required for HIVSMM growth and viability. A selenium adjuvant RTV-based HAART abrogates GLUT activity with specificity for GLUT-4 (prominent growth factor in MM cells. We present a 44 YOF with an with HIV disease, anemia, CD4 cell count of 74/cumm, viral load > 1 million copies/ml, SMM with IgG kappa/lambda 3. 68 grams/L, a hypocellular bone marrow biopsy with > 60 % plasma cell (polytypic) and with an increase in immature forms. Her serum free light chains (SFLC) on diagnosis was 324 mg/dl/121 mg/dl kappa/lambda ratio of 2. 68, bone scan with no evidence of lytic lesions and beta-2-microglobulin (B2MG) of 14. 40 mg/L: soluble interleukin-2 receptor (sIL2R) of 10, 833. 47 pg/ml; IGF-1 was 346 ng/ml (Nl = 94–250 ng/ml); selenium level was 137 mg/dl; C-reactive protein (CRP) of 38 mg/dl. After three years of selenium-RTV-based-HAART therapy her proinflammatory markers trended downward. CD4 cell count now is 348/cumm; viral load of 25. 7 copies/ml; SMM IgG 0. 972 grams/L; SFLC of 35. 9mg/dl/35mg/dl kappa/lambda ratio of 1. 03; B2MG of 2. 24 mg/L; sIL2R of 6546. 14 pg/ml; IGF-1 of 128 ng/ml; selenium of 150mg/dl; and CRP of 0. 26 mg/dl.

Conclusions:

Modulation of myeloma proinflammatory cytokines with protease inhibitor RTV, selenium adjuvant based HAART in HIVSMM patients may suppress the GLUT OVEREXPRESSION activity and prevent development TUMORGENESIS OF MM.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Value of Interim PET/CT in Patients with Poor Risk Diffuse Large B-Cell Lymphoma (DLBCL). Experience of the Spanish Geltamo Group
Authors: Coronado Mónica , Pardal Emilia , Couto Rosa , de la Cruz Fatima , Panizo Carlos , Grande Carlos , Arranz Reyes , Ramirez María José , Hernández Roberto , Martin Alejandro , Caballero Dolores
Published: October 2019

Abstract 5076

Background:

Interim FDG-PET appears to be useful to guide risk stratification of patients with DLBCL, but remains controversial because the absence of consensus criteria for assessment. The reduction of maximum Standardized Uptake Value (SUVmax) between baseline and interim PET improves the accuracy and reproductibility of this method (Casasnovas, Blood 2011).

Patients and Methods:

A prospective non randomized phase II trial (EudraCT:2006-005254-68) was undertaken in young patients (pts) newly diagnosed of poor risk DLBCL. Therapy was changed after 3 cycles of R- MegaCHOP based on PET (using local assesment and visual scale); pts with positive PET received early salvage therapy. Primary end points were Progression free survival (PFS) and Overalll survival (OS). Retrospectively, central review was done by three experts using visual assessment (Deauville criteria) and semiquantitative asessment. Baseline and interim SUVmax, and ΔSUVmax were evaluated (cutt off ΔSUVmax: 66%). Significance of PET parameters in OS was analyzed.

Results:

71 pts were enrolled and central review was possible in 50 pts, from which 80% have complete follow up (mean 37, 6 months, 3. 4–56. 4). OS was significantly influenced by interim PET result, using visual (p=0. 046) but mainly by semiquantitative analysis (p=0. 0008). ΔSUVmax had impact on the overall survival (p=0. 007) whereas basal SUVmax did not.

Conclusions:

Our preliminary results show that outcome of DLBCL pts with a positive interim PET is worse despite change of therapy. Semiquantitative PET evaluation seems to be necessary, being ΔSUVmax a good prognostic parameter.

Disclosures:

No relevant conflicts of interest to declare.
HIF-1α Induces a Chemoresistance Phenotype in a Non-Hodgkin‘s Lymphoma Cell Line Via up-Regulation of Bcl-xL
Authors: Hernandez-Luna Marco A , Rocha-Zavaleta Leticia , Vega Mario I , Huerta-Yepez Sara
Published: October 2019

Abstract 5091

Non-Hodgkin's Lymphoma (NHL) is one of the most common cancers in childhood. The development of chemoresistance in tumor cells is one of the principal causes of treatment failure. This resistance has been associated to different mechanisms, being the overexpression of anti-apoptotic proteins such as Bcl-xL. It has been shown that this protein is regulated by the transcription factor HIF-1α, which is overexpressed in several tumors, including NHL, and the overexpression of both proteins may result in resistance to chemotherapy. We investigated the role of HIF-1α in resistance to chemotherapy via the induction of the Bcl-xL expression in NHL cell lines, using a pharmacological modulation of HIF-1α. Our data showed that treating the Ramos cell with ethyl 3, 4-dihydroxybenzoate, the inhibitor of prolylhydroxylase, induces accumulation of HIF-1α and this correlates with an increase of Bcl-xL as well as resistance to apoptosis after the exposition to chemotherapeutics drugs. In contrast, the treatment of Ramos cells with 2-methoxyestradiol, the inhibitor of HIF-1α activity, induced downregulation of the Bcl-xL expression, and this correlates with the sensitization of tumor cells to chemotherapeutic drugs. This data demonstrated that the up-regulation of the anti-apoptotic protein Bcl-xL in NHL cells correlates with HIF-1α expression and activity, through which induces a phenotype of chemoresistance.

Disclosures:

No relevant conflicts of interest to declare.
Anemia in Lymphoma: The Role of Cytokines
Authors: Shao Zonghong , Wang Ting , Tu Meifeng , Zhu Jun , Zheng Wen
Published: October 2019

Abstract 5103

Purpose

This study was to investigate the role that cytokines play in the pathogenesis of lymphoma associated anemia.

Methods

IFN-γ, IL-6, TNF-α, IL-1β and EPO in plasma samples from 45 lymphoma patients and 12 controls were analyzed using enzyme-linked immunosorbent assays and EPOR on their bone marrow cells were detected by flow cytometry. Their bone marrow CFU-E were cultured in vitro also.

Results

Of 45 initial lymphoma patients, 25 (55. 6%) had anemia before diagnosis, 13(28. 9%) had anemia during therapy, 7(15. 5%)never had anemia. Plasma IFN-γ and TNF-α levels were significantly higher in severe and moderate anemia patients than those in mild anemia patients, no anemia patients and controls. Patient's plasma EPO, IL-6 and IFN-γlevels showed an inverse correlation to their Hb. Lymphoma without anemia patients had significantly high bone marrow EPOR level than that of lymphoma associated anemia patients and controls. Patients bone marrow CFU-E showed positive correlation to their Hb and EPOR.

Conclusion

Increased plasma IFN-γ, TNF-α and IL-6 might contribute to anemia in lymphoma, EPO and EPOR levels were elevated to balance such negetive effects and maintain hemotopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Incidental Venous Thromboembolism in Kidney Cancer Patients: A Retrospective Case-Control Study
Authors: Yokom Daniel W , Ihaddadene Ryma , Le Gal Gregoire , Carrier Marc
Published: October 2019

Abstract 5134

Introduction:

The incidence of venous thromboembolism (VTE) in kidney cancer patients has been reported at 1. 2–3. 2% with an estimated hazard ratio for death of 1. 3–3. 2. Computed Tomography (CT) scans are used frequently to stage cancer, and as the resolution of the scans increases more incidentally discovered asymptomatic VTE are being diagnosed. Currently, it is unclear if incidentally found VTE have the same effect on survival as symptomatic VTE in kidney cancer patients.

Methods:

We undertook a retrospective case-control analysis of all kidney cancer patients treated at our institution between January 1, 2005 and July 1, 2012. Cases with VTE were matched in a ratio of 1:2 to controls by gender, age and stage at cancer diagnosis. All charts were reviewed for comorbidities at cancer diagnosis, cancer type, stage, and treatment plan. We reviewed imaging studies to identify the location of the VTE and whether it was unsuspected – which was defined as a VTE found on a CT which was indicated for cancer staging.

Results:

From a cohort of 1166 primary kidney cancer patients we identified 68 (5. 8%) patients who developed a VTE. There were 30 cases of pulmonary embolism (PE), 24 lower extremity deep vein thrombosis (DVT), 5 DVT of the upper extremity and neck and 9 intra-abdominal VTE. Of these, 35% were discovered incidentally and 65% were symptomatic.

Compared to matched controls, all patients with VTE did not have worsened survival (hazard ratio [HR] = 0. 87; 95% CI: 0. 54–1. 42). As demonstrated in Figure 1, symptomatic and incidentally discovered VTE were at no increased risk of death compared to controls (HR = 0. 72; 95% CI: 0. 40 – 1. 31 and HR = 1. 12; 95% CI: 0. 61 – 2. 27 respectively). Also, when looking specifically at symptomatic and incidentally discovered pulmonary embolisms, no difference in survival was detected (HR = 1. 44; 95% CI: 0. 68 – 3. 06 and HR = 0. 78; 95% CI: 0. 31 – 1. 97 respectively).

Conclusions:

In this analysis of kidney cancer patients we found a high rate of VTE (5. 8%) of which 35% were found incidentally on CT staging. The results from this study show that kidney cancer patients with VTE do not have worse survival outcomes than those without VTE. Moreover, the difference in survival between patients with any symptomatic VTE or PE does not differ from incidentally found VTE or PE.

Disclosures:

No relevant conflicts of interest to declare.
Treatment of Serum Ferritin Deficiency Regardless Haemoglobin Level
Authors: Al-Jafar Hassan A.
Published: October 2019

Abstract 5166

Introduction

The concentration of serum ferritin is positively correlated with the size of the total body iron stores in the absence of inflammation. [1] It is mainly concentrated in the liver, spleen and bone marrow in humans. The serum ferritin is present in all cells of the body. [ 2] Normal values are generally 12 – 300 ng/mL for men and 12 – 150 ng/mL for women. Some patients have clinical symptoms of iron deficiency anaemia as fatigue, general weakness, hair loss, depressed mode although their haemoglobin and serum ferritin are in the lower normal values [3].

Method

Patients sample are 1- females 2- aged from 15–70 years 3-who have problems with oral iron either not getting benefit from it or can't tolerated it or not compliant with oral iron treatment. On presentation patients subjected for the following tests and then repeated after one month of intravenous iron infusion 1- complete blood count, 2- serum ferritin, 3- serum vitamin B12 and folate, 4- HPLC, 5-G6PD, 6- Coagulation profile (INR, APTT), 7 – LFT and RFT.

Results

Group 1: responded laboratory and clinically to treatment.

Group 2: responded laboratory and clinically to treatment.

Group 3: responded laboratory and clinically to treatment.

Discussion

Three group of patients noticed:

Group 1 Low Hb and low s. ferritin where symptoms of iron deficiency are exist in this group of patients.

Group 2 Hb is normal and s. ferretin where symptoms of iron deficiency are exist in this group of patients.

Group 3 both Hb and s. ferretin where symptoms of iron deficiency are exist in this group of patients.

Group 4 control group was just observed and reassured.

Iron is essential element in all cells of human [4], so when it is low it means dysfunctions in other cells and organs like hair, muscles and brain.

Conclusion

Lower normal level of Serum Ferritin is enough for normal Hb level but it is may be not enough for normal functions in other organs where the iron is needed. So that why it might be need to treat S. ferritin deficiency and shouldn't wait to reach to the level of IDA. According to that it might be indicated to: 1- Treat patients when s. ferritin is low even if Hb is normal. 2 - Lower normal value has to be increased to at least to 50mg to ensure that not only Hb will become normal but also other organ function.

Putting in mind that the range of Hb could reach to 305ng/ml without reaching to the toxic level of iron overload.

Reference

Aim

Is to assess the benefit of raise up Serum Ferritin level to a new higher reference lower value as 50 ng/mL instead of the present lower values, because the present lower normal value 11 ng/mL, may be enough to get haemoglobin to the normal level and function, but it seems not enough for normal functions of other different organs as the hair, muscles, brain, which could be the reason of hair lose, general weakness, fatigue, depressed mode, . ect although haemoglobin is normal and s. ferritin is low or in lower normal value.

Disclosures:

No relevant conflicts of interest to declare.
Thalassemia – Increasingly Frequent Cause of Microcytic Anemias in Children in Poland
Authors: Albrecht Katarzyna , Adamowicz-Salach Anna , Burzynska Beata , Pawelec Katarzyna , Matysiak Michal
Published: October 2019

Abstract 5184

Thalassemias are a group of genetically determined hemolytic anemias related to disturbed synthesis of globin chains, and are classified as quantitative hemoglobinopathies.

We would like to present our experience in diagnosis of thalassemia in children as an increasingly frequent cause of microcytic anemias as well as to point out that it is the cause of microcytic anemia in Poland.

Between 2005–2012 we established the diagnosis of thalassemia minor in 94 children of Polish origin, referred to the Department of Pediatrics, Hematology and Oncology, Medical University of Warsaw. At admission all patients presented microcytosis, erythrocytosis, hypochromia and normal or elevated iron status. Most of them had long-lasting uneffective iron therapy in anamnesis. Their age ranged from 3 to 17 years. The group consisted of 40 girls and 54 boys.

Routinely employed diagnostic methods, including blood morphology, determination of iron and ferritin, electrophoretic separation of hemoglobins, determination of HgbA2 and HgbF, as applied in all cases, often turn out to be insufficient to diagnose thalassemia, substantiating the need to use modern molecular biological techniques.

Thalassemia alfa was diagnosed in 9 children in whom we detected deletion α 3, 7 using molecular biological techniques.

In 2 children coexistence of both thalassemia alfa and beta was established. Both children presented macrocytosis and normal range of haemoglobin and red blood cells count. The molecular biological techniques used in both cases identified deletion α 3, 7 characteristic for alfa thalassemia, and Hb Koln (Codon GTC>ATG) and Del 4, 2 (Taq1) characteristic for beta thalassemia.

In the remaining 83 patients we made the diagnosis of thalassemia beta. The diagnosis was possible after the evaluation of Hgb A2 and F levels. In 24 cases we managed to performed molecular biological techniques, identifying mutation IVS I-6 (T>C) as the most frequent.

Generally associated with the Mediterranean Basin, thalassemia is, however, more and more often diagnosed in Poland. This is due to the international human traffic. Growing availability of diagnostic tests, like molecular biological techniques, especially useful in equivocal cases, is also important.

The amplification of diagnostic possibilities by genetic tests in the future may be useful in detection of genetic mutations responsible for thalassemia and characteristic for Polish population.

Disclosures:

No relevant conflicts of interest to declare.
The Role of STAT5 in FLT3-Mediated Leukemogenesis
Authors: Müller Tony Andreas , Dechow Rebekka , Istvanffy Rouzanna , Hennighausen Lothar , Peschel Christian , Duyster Justus
Published: October 2019

Abstract 771

Introduction:

Activating mutations of FLT3 are among the most frequent alterations in AML. Two distinct types of mutations have been described: Duplication of the juxtamembraneous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). Although both lead to constitutive FLT3 signaling, FLT3-ITD signals through the STAT5 pathway whereas FLT3-TKD and FLT3 WT activate STAT5 to a much lesser degree. In a murine bone marrow transplantation model, FLT3-ITD leads to a myeloproliferative neoplasm; FLT3-TKD induces a lymphoid disease with significantly longer latency. Interaction of SRC with FLT3-ITD was recently shown to be necessary for STAT5 activation.

Methods:

Fetal liver cells from constitutive STAT5 knockout mice were infected with FLT3 oncogenes and cultured on methylcellulose. Conditional STAT5 knockout mice were analyzed for phenotype and latency of hematologic disorders after transplantation with FLT3-ITD or FLT3-TKD-infected bone marrow. Expression of STAT5 target genes was determined using Realtime-PCR. Phospho-STAT5 levels were quantified by intracellular staining and flow cytometry.

Results:

Depletion of STAT5 leads to a dramatic decrease in the ability of fetal liver cells to form colonies in Methylcellulose when infected with FLT3-ITD, but not when infected with FLT3-TKD. In-vivo studies using a murine conditional knockout model proved that STAT5 is necessary for the induction of a myeloproliferative disease by FLT3-ITD, while FLT3-TKD is not dependent on STAT5. Mice with deletion of the STAT5 allele displayed expansion of mainly lymphoid cells with an increased latency similar to the FLT3-TKD disease. In additional studies differential activation of STAT5 in myeloid and lymphoid progenitors was found. The STAT5 target gene Oncostatin M is exclusively expressed in myeloid progenitor cells infected with FLT3-ITD and in mice with intact STAT5 that were transplanted with FLT3-ITD. Furthermore, elevated SRC expression levels could be detected in proliferating myeloid progenitor cells compared to lymphoid progenitors.

Conclusion:

STAT5 is critical for the induction of a myeloproliferative disease by FLT3-ITD in a murine transplantation model. Interaction of SRC and FLT3-ITD leads to the activation of STAT5 in myeloid progenitors, but not in lymphoid progenitors expressing low levels of SRC. Oncostatin M upregulation is likely to be a downstream process contributing to the phenotype and latency of the disease.

Disclosures:

No relevant conflicts of interest to declare.
Impaired Neutrophil Function in 24p3 Null Mice Contributes to Enhanced Susceptibility to Bacterial Infections
Authors: Devireddy L.R. , Liu Zhuoming
Published: October 2019

Abstract 833

Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores are chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins of secondary granules. Neutrophils from SGD patients lack normal functions and are prone to bacterial infections, but the importance of 24p3 for neutrophil dysfunction in SGD is not known. Here we show that neutrophils of 24p3−/− mice are defective for many neutrophil functions. Specifically, neutrophils in 24p3−/− mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3−/− neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3−/− neutrophils. Further, 24p3−/− neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3−/− mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3−/− mice to these pathogens is not due to sequestration of siderophores to limit the iron availability but as a consequence of impaired neutrophil functions.

Disclosures:

No relevant conflicts of interest to declare.
p21-Activated Kinases Regulate Directional Migration and Cytoskeletal Organization in Human Neutrophils
Authors: Itakura Asako , Aslan Joseph E. , Kusanto Branden T. , Phillips Kevin G. , Insall Robert H. , Chernoff Jonathan , McCarty Owen J.T.
Published: October 2019

Abstract 834

Neutrophil chemotaxis is controlled by coordinated processes of directional sensing, polarization and motility. This study was designed to characterize the role of p21-activated kinases (PAKs) during the chemotaxis of human primary neutrophils. PAKs are known as effectors of the Rho GTPases Rac and Cdc42. It has been shown that PAK1 and PAK2 are strongly activated downstream of the f-Met-Leu-Phe (fMLP) receptor via Rac (Huang et al., MCB 1998). PAK1 is known to localize in lamellipodia at the leading edge of human neutrophils (Dharmawardhane et al., JLB 1999) and mediate persistent directional migration via Cdc42 in a neutrophil-like cell line (Li et al., Cell 2003). However, little is known about the specific role of PAK isoforms in spatial/temporal regulation of cytoskeletal dynamics in human neutrophils.

Our data show that human neutrophils express PAK1, 2 and 4. Under an fMLP gradient, human neutrophils developed morphological polarity with a distinct leading edge and rear, and migrated up the fMLP gradient at the speed of 7.5 ± 0.56 μm/min. Inhibition of Rac or PI3K impaired directionality but did not significantly affect migration speed of chemotaxing neutrophils (6.3 ± 0.56 μm/min or 6.2 ± 0.85 μm/min, respectively). In contrast, neutrophils treated with the PAK inhibitor, PF3758309 (PF), displayed random migration, less polarization and reduced motility (3.1 ± 0.21 μm/min). These results suggest that PAK regulates neutrophil chemotaxis independently of the Rac-PI3K axis. The presence of PF did not abrogate intracellular Ca2+mobilization in fMLP-driven chemotactic condition. Instead, the decreased migratory ability by PAK inhibition was associated with multiple Ca2+ spikes. Immunofluorescence imaging shows that PAK2 but not PAK1, was phosphorylated and translocated from cytosol to actin-rich leading edge in the proximity to GTP-bound Rac within 3 min of fMLP stimulation. Notably, PF treatment resulted in partial neutrophil spreading and actin/myosin II translocation in the absence of extracellular stimuli, suggesting that basal level of PAK phosphorylation may be required for cytoskeletal integrity of resting neutrophils. Neutrophils pretreated with PF displayed less activation and translocation of PAK2 and Rac.

In summary, our data demonstrate for the first time the distinct roles of PAK isoforms in human neutrophil morphological polarity and directional migration and suggest that PAK2 is activated downstream of fMLP receptor through Rho-family small GTPases.

Disclosures:

No relevant conflicts of interest to declare.
The Red Cell: How Erythropoiesis Modulates Hepcidin Expression
Authors: Ginzburg Yelena
Published: October 2019

Abstract SCI-24

Erythroid precursors in the bone marrow require transferrin-bound iron for hemoglobin synthesis. Therefore, it is not surprising that the regulation of erythropoiesis and iron metabolism is interlinked. Iron demand for erythropoiesis is communicated to the iron-regulatory machinery through incompletely understood mechanisms. At the core of systemic iron homeostasis is the peptide hormone hepcidin, restricting cellular iron export to plasma by inducing the endocytosis and proteolysis of ferroportin. Hepcidin, a liver-synthesized peptide hormone, is released in response to increased iron load, and there is early evidence that circulating hepcidin concentrations affect the distribution of iron between the macrophage storage compartment (favored by higher hepcidin concentrations) and parenchymal cells, including cardiac myocytes and hepatocytes (favored by low hepcidin). Furthermore, ferroportin has recently been identified on erythroid precursors. Its purpose in this cell type and its function in the interface between erythropoiesis and iron metabolism are unclear. Additionally, in response to bleeding or the administration of erythropoietin, expansion of erythroid precursors suppresses hepcidin, most likely through one or more mediators released by the bone marrow and acting on hepatocytes. Iron-loading anemias with ineffective erythropoiesis, in particular β-thalassemia, demonstrate the effects of pathological “erythroid regulators” of hepcidin. Although erythrocyte transfusions are the main cause of iron loading in patients who receive them (β-thalassemia major), lethal iron overload is seen also in patients who are rarely or never transfused (β-thalassemia intermedia). Here, iron hyperabsorption is the cause of iron overload and, as in hereditary hemochromatosis, is caused by low hepcidin. Decreased hepcidin expression in β-thalassemia, with concurrent ineffective erythropoiesis and iron overload, indicates that the “erythroid regulator” may play an even more substantial role in iron metabolism than the “stores regulator.” Two members of the bone morphogenetic protein (BMP) family, growth differentiation factor (GDF) 15 and Twisted Gastrulation (TWSG1), have been implicated as candidate bone marrow-derived hepcidin suppressors in β-thalassemia. Neither factor is responsible for the physiologic hepcidin suppression in response to hemorrhage-induced stress erythropoiesis, and the physiologic suppressor is not known. We focus here on the current state of knowledge regarding the regulation of iron metabolism and attempt to elucidate the interface between iron regulation and erythropoiesis using evidence in part derived from animal models of β-thalassemia. A more complete understanding of the coregulation of erythropoiesis and iron metabolism may lay the foundation for improving diagnosis, increasing treatment options, and ultimately impacting the well-being of patients afflicted with different anemias and/or iron overload related-disorders.

Disclosures:

No relevant conflicts of interest to declare.
Mechanisms of Transcriptional Regulation and Transformation by HOXA9
Authors: Hess Jay L. , Collins Cailin , Bronstein Joel , Sun Yuqing , Nagaraja Surya
Published: October 2019

Abstract SCI-30

HOXA9 plays important roles in both development and hematopoiesis and is overexpressed in more than 50 percent of acute myeloid leukemias (AML). Nearly all cases of AML with mixed lineage leukemia (MLL) translocations show increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. In most cases, upregulation of HOXA9 is accompanied by upregulation of its homeodomain-containing cofactor MEIS1, which directly interacts with HOXA9. While HOXA9 alone is sufficient for transformation of hematopoietic stem cells in culture, the addition of MEIS1 increases the transformation efficiency and results in rapidly fatal leukemias in transplanted animals. Despite the crucial role that HOXA9 plays in development, hematopoiesis, and leukemia, its transcriptional targets and mechanisms of action are poorly understood. We have used ChIP-seq to identify Hoxa9 and Meis1 binding sites on a genome-wide level in myeloblastic cells, profiled their associated epigenetic modifications, identified the target genes regulated by HOXA9 and identified HOXA9 interacting proteins. HOXA9 and MEIS1 cobind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/P300 binding. These include many proleukemogenic gene loci, such as Erg, Flt3, Myb, Lmo2, and Sox4. In addition, HOXA9 binding sites overlap a subset of enhancers previously implicated in myeloid differentiation and inflammation. HOXA9 binding at enhancers stabilizes association of MEIS1 and lineage-restricted transcription factors, including C/EBPα, PU.1, and STAT5A/B thereby promoting CBP/p300 recruitment, histone acetylation, and transcriptional activation. Current efforts are focused on using both biochemical and genetic approaches to assess the role of HOXA9 “enhanceosome” components C/EBPα, PU.1, and STAT5A/B in transcriptional regulation and leukemogenesis. Studies to date suggest that C/EBPα and PU.1 binding can occur in the absence of HOXA9/MEIS1, supporting a model in which these proteins act as pioneer transcription factors for establishment of poised, but not activated, HOXA9-regulated enhancers. Work is under way to assess the impact of high-level HOXA9 and MEIS1 on enhanceosome assembly and the role of recruitment of transcriptional coactivators involved in target gene up- or downregulation, including histone acetyltransferases and chromatin remodeling complexes. Collectively, our findings suggest that HOXA9-regulated enhancers are a fundamental mechanism of HOX-mediated transcription in normal development that is deregulated in leukemia.

Disclosures:

No relevant conflicts of interest to declare.
In Vitro Production of Erythrocytes
Authors: Douay Luc
Published: October 2019

Abstract SCI-39

The generation of red blood cells (RBCs) in vitro using biotechnologies could represent an interesting alternative to classical transfusion products, in that it would combine adequate supplies with the specific production of blood products of a particular phenotype and the reduction of infection risks. This presentation will review how it is now possible to obtain in vitro complete maturation of the erythroid line to the stage of enucleation, starting from hematopoietic stem cells (HSCs) from peripheral blood, bone marrow or umbilical cord blood, or embryonic stem cells or adult pluripotent stem cells (induced pluripotent stem cells, iPSCs). This presentation will discuss how the functionality of cultured human RBCs (cRBCs) is settled in terms of deformability, hemoglobin maturation, oxygen carrying capacity, enzyme content, and terminal maturation from the reticulocyte stage to mature RBC after infusion into the NOD/SCID mouse model. The clinical feasibility of this concept has recently been demonstrated by reporting that cRBCs generated in vitro from peripheral HSCs under GMP conditions encounter in vivo the conditions required for their maturation and that they persist in the circulation for several weeks in humans. These data have established the proof of principle for transfusion of in vitro-generated RBCs and the pathway toward new developments in transfusion medicine. The most proliferative source of stem cells for generating cRBCs is cord blood, but it is limited in terms of HSCs and is dependent on donations. Pluripotent stem cell technology represents a potentially unlimited source of RBCs and opens the door to the development of a new generation of allogeneic transfusion products. Because iPSCs can be selected for a phenotype of interest, they are obviously the best candidate for organizing complementary sources of RBCs for transfusion. It is established that only three human iPSC clones would have been sufficient to match more than 99 percent of the patients in need of RBC transfusions. As a whole, a very limited number of RBC clones would provide for the needs of most alloimmunized patients and those with a rare blood group. Generating cRBCs from iPSCs has been done but needs to be optimized to lead to a clinical application in blood transfusion. Several crucial points remain to be resolved, notably, the choice of the initial cell type, the method of reprogramming (i.e., to ensure the safety of the iPSCs and to ensure their clinical grade), the optimization of the erythrocyte differentiation, and the definition of GMP conditions for industrial production. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, this presentation will highlight the potential applications for alloimmunized patients and those with a rare blood group.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Bromodomains in Myeloma
Authors: Bradner James E.
Published: October 2019

Abstract SCI-6

In cancer, epigenetic proteins are among the most promising and intently pursued targets in drug discovery. This relates to the growing appreciation that cancer growth pathways are centrally influenced by proteins that modify chromatin. Already, inhibitors of DNA methyltransferases and histone deacetylases have demonstrated substantial clinical efficacy, leading to regulatory approval for use in hematologic malignancies. These events have triggered intense competition to develop inhibitors of the chromatin-modifying enzymes known as epigenetic “writers” and “erasers.” Perhaps owing to perceptions regarding the feasibility of targeting protein-protein interactions, small-molecule inhibitors of chromatin-binding modules or epigenetic “readers” have received considerably less attention. Recently, we reported the first potent small-molecule inhibitor of human bromodomains, JQ1, which exhibits selectivity for the BET family of bromodomain-containing transcriptional coactivators. Of broad relevance to cancer, we demonstrated that c-Myc expression is critically dependent on BRD4 function and localization to discrete upstream regulatory regions. Exposure of cancer cells to JQ1 prompts immediate downregulation of c-Myc expression, leading to suppression of a transcriptional program associated with proliferation, survival, and metabolic adaptation. In translational models of MYC-dependent hematologic malignancies, in particular multiple myeloma, the efficacy of JQ1 treatment establishes a mechanistic rationale for the leveraged clinical development of drug-like JQ1 derivatives. Toward this objective, we have completed chemical optimization of novel, drug-like inhibitors of BET bromodomains as reagents capable of supporting human clinical investigation. This research has established the feasibility of inhibiting epigenetic reader proteins with efficient, cell-permeable small molecules. In addition, we have developed a high-throughput platform for modeling mechanism-based, drug-drug combinations to achieve synergy in translational model systems of multiple myeloma. In support of an open-innovation model of chemical biology, we have created a chemical probe allowing the broad study of chromatin biology, which has already been provided to more than 350 academic, governmental, and industrial laboratories worldwide. Together, we have rapidly identified mechanism-based opportunities for clinical translation in cancer.

Disclosures:

Bradner: SHAPE Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tensha Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Exome Sequencing Reveals The Genetic Landscape Of Rare Atypical e6a2 CML
Authors: Rao Arati V. , Fedoriw Yuri D. , Richards Kristy L. , Sun Zhen , Love Cassandra L , Dave Sandeep
Published: October 2019

Background

Over 90% of Ph-positive chronic myelogenous leukemia (“typical CML”) patients have breakpoints in the M-bcr, which typically result in b2a2 (e13a2) and/or b3a2 (e14a2) fusion mRNAs, both of which are translated into the p210 BCR-ABL protein. CML patients with the p190 BCR-ABL (m-bcr) or p230 BCR-ABL (μ-bcr) fusion genes have been reported. Atypical BCR breakpoints outside these cluster regions are extremely rare. For instance, only 8 cases have been described of e6a2 fusion CML. Very little is known about the clinical or biological characteristics of this subtype of CML, including the role of collaborating gene mutations in the development of disease. In this study, we defined the gene mutations that occurred in a rare e6a2 CML case and compared the observed gene mutations to those in “typical” chronic phase (CP)-CML cases. To our knowledge, this is the first comparison of the genetic mutations occurring in typical CML and in this rare atypical form of CML.

Methodology

We identified the index e6a2 CML patient, and eight additional typical CML patients for whom we had bone marrow aspirate, peripheral blood and paired normal tissue. We performed whole-exome sequencing for all of these samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 3 GB of sequencing data using high throughput sequencing on the Illumina platform.

Results

We identified 15 candidate cancer genes that were somatically mutated in our e6a2 CML patient. Commonly implicated biological processes comprising these genes included transcription (STAT5A, TET2, GTF2F1), cellular differentiation (TP73), and signal transduction (GPR116). Interestingly, the majority of these mutations also occurred in typical CML, albeit at lower frequency. Thus, genes mutated common to our atypical case and typical CMLs included STAT5A, TET2, GTF2F1, ABL1 and CYP2A6. Thus, while atypical e6a2 BCR-ABL fusion CML cases are extremely rare, they appear to share many aspects of the biology with typical CMLs.

Conclusion

This study represents an in-depth analysis of a rare e6a2 CML in combination with one of the first analyses of gene mutations that occur in typical CML. Our data provide a significant first step to identifying genes that play a role in the pathogenesis along with BCR-ABL that perhaps contribute to drug resistance, and ultimately impact overall survival.

Disclosures:

No relevant conflicts of interest to declare.
Peripheral Arterial Occlusive Disease (PAOD) In Chronic Phase Chronic Myeloid Leukemia Patients Treated With Nilotinib
Authors: Jeon Young-Woo , Lee Sung-Eun , Choi Soo-Young , Kim Soo-Hyun , Park Jin-Eok , Jeon Hye-Rim , Jang Eun-Jung , Kim Dong-Wook
Published: October 2019

Introduction

ENESTnd trial, nilotinib showed superior efficacy and good tolerability compared with standard dose of imatinib in chronic phase chronic myeloid leukemia (CP CML). However, serious vascular adverse events such as ischemic heart disease (IHD) and peripheral arterial occlusive disease (PAOD) have been reported. Therefore, we have prospectively evaluated for emergence of PAOD in all (CML patients treated with nilotinib.

Materials and methods

Between May 2005 and April 2013, a total of 98 CP CML patients on nilotinib treatment were tested with laboratory studies, electrocardiography and ankle brachial index (ABI) + pulse wave velocity (PWV). Abnormal ABI index was defined with below 1.0 and abnormal PWV as over 1200∼2000 cm/sec adjusted by age. and patients who had abnormal ABI or PWV were performed with computed tomography angiography.

Results

So far, 88 of total 98 patients (57 male) were evaluated with screening test. 17 patients of 88 showed abnormal ABI or PWV. Finally, 3 patients (3.4%) of 17 were diagnosed with overt PAOD by CT angiography (2 upper extremity, 1 lower extremity). The median age was 46 year old (range, 19∼72). The median duration of disease was 49 months (range,1.2 ∼ 65) and 51.3 months (range,36 ∼ 115) in the patients treated with first-line and second-line nilotinib, respectively. The median mean daily dosage of nilotinib was 710mg (range, 656∼800mg) and 737 mg (range, 500∼800mg) in each group. The duration of nilotinib treatment was 46.3 months (range, 0.7∼64.2) and 25.0 months (range, 2.8∼55.8) in each group. Comparing first and second-line nilotinib treated patients, an abnormal ABI or PWV (1 versus 16 patients) and overt PAOD (0 versus 3 patients) was more frequent in patients with second-line nilotinib (p=0.042, p< 0.001, p<0.001).

Conclusion

Our preliminary data suggest an importance of PAOD evaluation in patients with long-term nilotinib treatment (p=0.042) and a routine assessment of PAOD including ischemic heart disease (IHD) is strongly recommended on regular basis.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation Of hOCT1expression In Patients With Chronic Myeloid Leukemia (CML) Treated With Imatinib In First Line
Authors: Couto Mascarenhas Cintia Do , Almeida Maria Helena , Miranda Eliana C M , Virgilio Bruna , Delamain Marcia Torresan , Duarte Gislaine O. , Duarte Vagner O , De Souza Carmino A , Pagnano Katia B.
Published: October 2019

Introduction

The majority of chronic myeloid leukemia (CML) patients (pts) in chronic phase (CP), present satisfactory response to imatinib treatment. However, 25-30% of these pts exhibit suboptimal response or treatment failure. The probability of achieving optimal response may be related with several factors. The human organic cation transporter 1 (hOCT1, SLC22A1), an influx transporter, is responsible for the uptake of imatinib into chronic myeloid leukemia (CML) cells The aim of this study was to analyze hOCT-1 levels at diagnosis of CML patients and correlate with cytogenetics and molecular responses.

Methods

hOCT-1 expression was evaluated in 58 newly diagnosed CML pts. Pts were treated with imatinib 400-600mg in first line. Samples were collected from peripheral blood at diagnosis and RNA was obtained from total leucocytes. For cDNA synthesis, 3 ug of RNA was used. hOCT-1 expression was evaluated by real-time PCR with TaqMan probe SLC22A1 (Applied Biosystems) and endogenous GAPDH control. The results were analyzed using 2-ΔΔCT. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter if CCR was achieved. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RT-PCR (RQ-PCR). Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1%.

Results

58 CML pts, 60% male, median age of 46 years (19-87) were evaluated, 71% in chronic phase (CP), 21% in accelerated phase (AP) and 5% in blast crisis (BC). The mean and median of hOCT-1 transcript levels in the total group was 2.03 and 0.961 respectively (0.008–19.039) and CP pts was 1.86 and 1.00 (0.008-10.34).The median duration of imatinib treatment was 27 months (1-109) and 96.6% achieved complete hematological response, 79.3% complete cytogenetic response and 69% major or complete molecular response. The regression analysis showed correlation between higher transcript levels of hOCT-1 and BCR-ABL transcripts<10%) at 3 months analysis (p<0.0001). Albeit, there was no influence of the hOCT-1 transcript levels at diagnosis in the achievement of cytogenetic and molecular response at 24 months of treatment.

Conclusions

In this report, we found that high hOCT-1 expression was predictive of BCR-ABL transcripts<10% at 3 months, although we did not find correlation between hOCT-1 levels at diagnosis and the achievement of molecular response at 24 months, studies show that there is correlation between BCR-ABL log reduction in the first months of treatment and the achievement of molecular response.

Disclosures:

No relevant conflicts of interest to declare.