bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Clonal Pre-B Cell Progenitors of Plasma Cells In Multiple Myeloma
Authors: Perez Lia , Boucher Kelly , Parquet Nancy , Alsina Melissa , Baz Rachid , Anasetti Claudio , Dalton William S.
Published: October 2019

Abstract 1921

The lack of specific molecules to define the multiple myeloma (MM) malignant progenitor cells responsible for the development of the disease has hampered the evaluation of minimal residual disease (MRD) in MM. Syndecan (CD138) expression is limited to terminally differentiated plasma cells (PC) and studies of myeloma cell biology using CD138+ selected cells are limited in scope since earlier PC progenitors involved in the disease process are likely CD138 negative. We have identified a bone marrow (BM) CD138- subset that co-express CD19 with identical kappa or lambda light chain restriction as the abnormal plasma cells, as previously shown by others. Further characterization of CD138-/CD19+ cells (23% ± 18% of total BM cells) has shown that this subset co-expresses Notch-1 (90 ± 5%), c-Kit (20 ± 5%), CD20 (5 ± 2%), CD27 (20 ± 13%), CD34 (21 ± 15%) and lack CD56. CD138-/CD19+ cells represent two distinctive populations being CD34+/CD20- or CD34-/CD20+. A small percentage (1.1 ± 0.4%) of CD138-/CD19+ cells showed aldehyde dehydrogenase (ALDH) activity. To further study MM BM progenitor we developed a multicolor flow cytometry assay to flow sort MM BM cell subsets allowing > 95% purity using a FACS Aria. Isolated BM populations were grown in methylcellulose supplemented with 5% PHA-leukocyte conditioned medium to detect progenitor cells committed to differentiate into mature PC. CD138+ cells did not form colonies, whereas CD138-/CD38+/CD19+/light chain+ regardless of CD34 expression grew colonies with a low efficiency of 1 in 25,000. However, only CD138-/CD38+/CD19+, but not CD34+ (HSC), differentiated into a mature syndecan (CD138+) expressing PC. Isolated CD138-/CD38+/CD19+ cells were relatively bortezomib and melphalan resistant when compared to CD138+ plasma cells. We hypothesize that the CD138-/CD38+/CD19+/CD34+ population contains earlier progenitor B cells that differentiate into the malignant PC. Surrogate assays for stem cell activity and xenotransplant models should determine cancer stem cell activity of these cells. Ongoing experiments will test whether Notch-1, CD34 or c-Kit expression is required for malignant PC progenitor function. Research studies of these CD138- MM putative progenitor cells will allow developing novel treatments to eradicate the MM minimal residual disease reservoir.

Disclosures:

No relevant conflicts of interest to declare.
Mean Corpuscular Volume as An Early Predictor of Malignant Evolution of Monoclonal Gammopathy of Undetermined Significance
Authors: Khalife Mustapha A. , Kuriakose Philip , Hammoud Marwa
Published: October 2019

Abstract 1925

Introduction:

Monoclonal gammopathy of undetermined significance (MGUS) is an asymptomatic premalignant clonal plasma cell proliferative disorder. It occurs in over 3 percent of the general population over the age of 50. MGUS can progress to multiple myeloma (MM) or related cancers at a rate of about 1.0 to 1.5 percent per year. There are no findings at diagnosis of MGUS that reliably distinguish patients who will remain stable from those who will progress to a malignant disease. Size and type of serum monoclonal protein, as well as serum free light chain ratio have been suggested in some studies as independent risk factors for disease progression. In this study, we tried to analyze the relationship between Mean Corpuscular Volume (MCV) and the risk of progression of MGUS into a malignant lymphoproliferative condition.

Methods:

A total of 1744 patients, from a single institution, with a first diagnosis of MGUS from April 18th, 1995 till June 24th, 2010 were included in this study. Patients analyzed were divided into two groups. Group 1 consisted of 345 patients with an elevated MCV (>100fL/red cell) and group 2 consisted of 1399 patients with a normal MCV (between 80fL/red cell and 100fL/red cell). Patients with short follow up (<3 months), low B12/folate levels, hypothyroidism, liver disease or a serum monoclonal protein >1.5g/dL were excluded. The Chi-square test was used to compare the proportion of patients who progressed in group 1 relative to group 2.

Results:

After a median follow up of 51 and 58 months for the high MCV and normal MCV respectively, there were 78 progressions (22.6%) versus 267 non-progressions (77.4%) among the high MCV patients (Group1) and 110 progressions (7.9%) versus 1289 non-progressions (92.1%) among the normal MCV patients (Group 2). The difference in progression between the two groups was statistically significant (Chi-square test p-value < 0.001). The odds ratio for the progression comparison of the high versus normal MCV groups was 3.4 with a 95% confidence interval of 2.5 to 4.7.

Conclusion:

In our single institution, retrospective study, there seems to be a higher incidence of malignant transformation in the subset of patients with MGUS having an elevated MCV. MCV is a cost effective method that might help identify, at presentation, patients with benign monoclonal gammopathies requiring stricter monitoring.

Disclosures:

No relevant conflicts of interest to declare.
TNFα Mediated IL-6 Secretion Was Regulated by JAK/STAT Pathway but Not by MEK Phosphorylation and AKT Phopshorylaton In U266 Multiple Myeloma Cells
Authors: Oh Jeong-In , Park Juwon , Lee Chansu , Jung Woojune , Ryu Jiyeon , Kim Byoung Kook , Ahn Kwang-Sung , Yoon Sung-Soo
Published: October 2019

Abstract 1930

Serum levels of IL-6 and TNF-a were significantly increased in patients with active multiple myeloma (MM) when compared with normal controls. Furthermore, MM patients with advanced aggressive disease had significantly higher levels of IL-6 and TNFa than those with MM in plateau phase. However, the detailed mechanisms involving TNFa-mediated cell signaling pathways resulting in increased IL-6 secretion from MM cells are largely unknown. In our study, we found that MEK and AKT phosphorylation was induced by TNFa treatments. TNFa increased MM-derived Interleukin-6 (IL-6) production. TNF-a-stimulated IL-6 production was abolished by inhibition of JAK2 and IKKa, but not of MEK, p38, PI-3K, or by small interfering RNA (siRNA) targeting TNF receptor (TNFR). Also, TNFa increased phosphorylation of STAT-3, -5 including c-Myc and cyclinD1. Three different types of JAK inhibitors decreased the activation of the above pathways. In conclusion, blockage of JAK/STAT mediated NF-kB activation was highly effective in controlling the growth of MM cells and consequently, a new inhibitor of TNFa mediated IL-6 secretion may be a potential new therapeutic agent for MM.

Disclosures:

No relevant conflicts of interest to declare.
Bone Marrow Stroma-Mediated Paracrine Inhibition of Ruxolitinib (INCB018424) Induced Apoptosis of JAK2V617F-Mutated Cells
Authors: Manshouri Taghi , Quintás-Cardama Alfonso , Estrov Zeev , Knez Liza , Zhang Ying , Kantarjian Hagop , Verstovsek Srdan
Published: October 2019

Abstract 1976

Janus kinases (JAK) comprise a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events via signal transducer and activator of transcription (STAT) proteins. The activating somatic mutation JAK2V617F is highly prevalent among patients with myeloproliferative neoplasias (MPNs), which prompted the development of JAK2 inhibitors for patients with these malignancies.

We have reported ruxolitinib (INCB018424) to be a potent orally bioavailable inhibitor of JAK1 and JAK2. Ruxolitinib inhibits IL-6 signaling and the proliferation of JAK2V617F Ba/F3 cells (IC50 127nM). We have streamlined a co-culture experimental platform designed to interrogate the interplay between JAK2V617F–positive cells and the stromal cells. Ruxolitinib caused marked apoptosis of human JAK2V617F mutant SET2 (IC50 56nM) and human UKE-1 (IC50 329nM) cells without any effect on the proliferation of the wild-type JAK2 human mast cell lines MHC1.1 or MHC1.2 which carry mutant KIT. The proapoptotic effect was markedly attenuated when JAK2V617F mutant cells were co-cultured on either immortalized or primary human marrow stromal cells (MSC: HS5, NK.tert or TM-R1). Co-culturing with each human-MSCs monolayer also hampered the ability of the JAK1/2 inhibitor to dephosphorylate STAT3 and STAT5, and prevented cytochrome c release and the cleavage of PARP protein. This protective effect was maintained in non-contact co-culture assays (cell lines separated by 0.4 μm micropore membranes), suggesting a paracrine effect. Cytokine profiling of supernatants from co-cultures of JAK2V617F–positive cells and human MSC monolayers are being analyzed to determine paracrine signals evoked upon ruxolitinib treatment. Neutralizing antibodies against over-secreted cytokines and downregulation of mRNAs encoding such cytokines with shRNAs will further validate the putative protective effects of such cytokines.

Our preliminary results suggest that marrow stroma secreted cytokines play a critical role in protecting JAK2V617F–positive clones from JAK2 inhibitor therapy, thus highlighting the possible significant role of the microenvironment not only in the pathogenesis of MPNs but also in the resistance to JAK2–directed therapies of bone marrow JAK2V617 F-positive cells. Complete results on experiments using the clinically relevant JAK1/2 inhibitor ruxolitinib will be presented.

Disclosures:

Estrov: Celgene Corporation: Consultancy. Verstovsek:Incyte Corporation: Research Funding.
MicroRNA-101 Is Down-Regulated In PV and ET Granulocytes and Its Decrease Is Associated with Over-Expression of Histone Methyltransferase In EZH2 In MPN Patients
Authors: Swierczek Sabina , Yoon Donghoon , Hickman Kimberly , Prchal Josef T.
Published: October 2019

Abstract 1989

Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF) are hematological malignancies due to clonal proliferation arising from a multilineage stem cell and have been classified as myeloproliferative neoplasms (MPNs). A somatic point JAK2 V617F mutation occurs in the majority (>95%) of PV patients and about 50–60% of ET and MF patients. The nonspecificity of this mutation, presence of JAK2V617F-negative PV patients, and evidence of both JAK2V617F-negative and positive PV clones demonstrate that the JAK2V617F mutation is not the initial or sole somatic event in the pathogenesis of PV. However, JAK2V617F plays an important role in the dysregulation of hematopoiesis in MPNs.

MicroRNAs (miRs) have been shown to be important regulators of hematopoiesis. We searched for miRs that directly regulate JAK2 expression by in silico analysis. Two of these miRs, namely miR101-1 and miR101-2, had JAK2 as a putative target. Interestingly, miR101-2 is located on chromosome 9p adjacent to the JAK2 locus. miR101 also negatively regulates the enhancer of zeste homolog 2 (EZH2), a member of the polycomb group of proteins involved in controlling and maintaining stem cell pluripotency and cell proliferation. Loss of miR101 in the cancer genome leads to overexpression of EZH2, contributing to cancer progression.

We analyzed the expression of miR101 in 35 PV, 15 ET and 3 MF patients and 20 normal controls by quantitative RT-PCR and found it was decreased in PV, ET and MF patients compared to normal controls (PV; P<0.03 and ET; P<0.01). We next investigated whether miR101 expression inversely correlates with EZH2 mRNA levels in MPN patients, as had been previously reported in human tumors. We examined the expression level of EZH2 for the same cohort of PV, ET and MF patients by quantitative RT-PCR and found a significant increase of EZH2 expression compared with normal controls (PV; P<0.02 and ET; P<0.05).

Analysis of the molecular bases of these observations is in progress. We will test the hypothesis that the dysregulation of these miRs further increases the expression of EZH2 and contributes to the severity and pathophysiology of the MPNs.

Disclosures:

No relevant conflicts of interest to declare.
Increased Risk of Second Malignancies In Young Patients with Mycosis Fungoides Diagnosed Before 30 Years of Age: A Population-Based Study
Authors: Ai Weiyun , Keegan Theresa , Fish Kari , Laura Pincus , Soon Christopher , Kim Youn H. , Chang Ellen
Published: October 2019

Abstract 1994

Background:

It has been reported that adults with mycosis fungoides have an increased risk of developing second malignancies. Although children and young adults with mycosis fungoides appear to have a higher proportion of early-stage disease and a more favorable outcome than adults, the risk of second malignancies in this population is not known.

Patients and Method:

A total of 178 patients diagnosed with mycosis fungoides or Sézary syndrome (MF/SS) before 30 years of age were identified from nine Surveillance, Epidemiology, and End Results (SEER) cancer registries in the U.S. from 1973 to 2007. The risk of second malignancies was assessed by calculating the standard incidence ratio (SIR), which compares the observed cancer incidence in the MF/SS cohort with the expected incidence in the age-, sex-, race-matched general population. Overall survival was calculated among 166 patients diagnosed with MF/SS before age 30 years, identified from the California Cancer Registry between 1988 and 2007.

Results:

In the MF/SS cohort from 1973 to 2007, 8 second cancers at all sites were reported in 7 patients, among which 5 cases were Hodgkin's and Non-Hodgkin's lymphomas. The SIR for a secondary malignancy at any site was 3.60 (95% confidence interval [CI]=1.55-7.10). Significantly increased risks of lymphoma and melanoma were observed, with SIR=24.23 (95% CI=7.87-56.55) and SIR=10.61 (95% CI=1.29-38.33), respectively. The median time to secondary malignancy was 2.9 years (range = 1.4 to 15.8 years). Among MF/SS patients diagnosed in California from 1988 to 2007, the 5- and 10-year overall survival was 96% (95% CI=91.4-98.5) and 92% (95% CI=86.1-96.5), respectively.

Conclusion:

Patients with MF/SS diagnosed before 30 years of age have favorable overall survival. However, these patients appear to have an increased risk of developing secondary cancers, similar to what we have seen in the adult MF/SS patients. Additional multi-center studies on the long term outcome of this young patient population are warranted.

Disclosures:

No relevant conflicts of interest to declare.
Prediction of Survival In Diffuse Large B-Cell Lymphoma Based On the Expression of Two Genes Reflecting Tumor and Microenvironment
Authors: Alizadeh Ash A. , Gentles Andrew J. , Alencar Alvaro J. , Kohrt Holbrook E , Houot Roch , Goldstein Matthew J , Zhao Shuchun , Natkunam Yasodha , Advani Ranjana , Gascoyne Randy D. , Briones Javier , Tibshirani Robert J. , Myklebust June Helen , Plevritis Sylvia K. , Lossos Izidore S , Levy Ronald
Published: October 2019

Abstract 2006

Background:

Several gene expression signatures predict survival in diffuse large B cell lymphoma (DLBCL), but the lack of practical methods for genome scale analysis has limited translation to clinical practice.

Methods:

We examined the power of individual genes to predict survival across different therapeutic eras. In studying 787 patients with DLBCL, we built and validated a simple model employing one gene expressed by tumor cells and another expressed by host immune cells, assessing added prognostic value to the clinical International Prognostic Index (IPI). We validated models in an independent cohort using diagnostic formalin-fixed specimens.

Results:

We verified expression of LMO2 as an independent predictor of survival and ‘Germinal Center B-cell’ subtype. We identified expression of TNFRSF9 from the tumor microenvironment, as the best in bivariate combination with LMO2. We studied distribution of TNFRSF9 tissue expression in 95 patients. A model integrating these two genes (TGS) was independent of ‘cell of origin’ classification, ‘stromal signatures’, IPI, and added to the predictive power of the IPI. This bivariate model and a composite score integrating the IPI (TGS-IPI) performed well in three independent cohorts of 545 previously described patients. Both models robustly stratified outcomes in a simple assay of routine specimens from 147 newly diagnosed patients as depicted.

Conclusion:

Measurement of a single gene expressed by tumor cells (LMO2) and a single gene expressed by the immune microenvironment (TNFRSF9) powerfully predicts overall survival in patients with DLBCL. A simple test integrating these two genes with the IPI can readily be used to select patients of different risk groups for clinical trials.

Disclosures:

No relevant conflicts of interest to declare.
Microarray Analysis of the Peripheral Monocytes From Waldenstrom's Macroglobulinemia Patients Reveals a Distinct Gene Expression Profile
Authors: Jiang Jingrui , Yang Guang , Liu Xia , Hunter Zachary , Manning Robert , Xu Lian , Patterson Christopher , Treon Steven P.
Published: October 2019

Abstract 2010

Background:

Waldenstrom's macroglobulinemia (WM) is a B-cell malignancy characterized by the accumulation, predominantly in the bone marrow, of clonally related IgM-secreting lymphoplasmacytic cells. Macrophage derived inflammatory factors are elevated in WM suggesting a possible contribution by monocytes to the growth and survival of WM cells.

Patients and Method:

Monocytes (CD14+) from the peripheral blood of 8 untreated WM patients, and 6 healthy donors (HDs) were isolated by immunomagnetic bead sorting. Gene expression profiling was then performed using Human Genome U133 Plus 2.0 chips, and data obtained from microarray was analyzed by dChip software with fold change >=2 and p<=0.05 as cut off points. Validation was performed by real time PCR.

Results:

Using Panther classification, 284 transcripts were identified as significantly different in monocytes derived from WM patients versus HDs. The resulted transcripts are involved in many critical signaling pathways, such as Toll-like immune receptor pathway (i.e. TLR1, TLR4, TLR8, TICAM); inflammatory response (i.e. CD40, PTAFR, FPR2), integrin binding (i.e. RAC2, ILK), chemokinesis (i.e. CCR2, CX3CR1), apoptosis (i.e. TNFSF10), p53 signaling (i.e. GADD45A, 1433F) and G-protein coupled receptors (i.e. CX3CR1). Fifteen of 21 genes were validated by real-time PCR, and were over-expressed in peripheral blood monocytes from WM patients in comparison to healthy age matched donors:

Conclusion:

Peripheral monocytes from WM patients demonstrate a distinct gene expression profile characterized by up-regulation of genes affecting Toll-like innate immunity, inflammation, and apoptosis. These studies define a distinct microenvironmental signature, and provide a framework for the exploration of novel targets for prognosis and therapy in WM.

Disclosures:

No relevant conflicts of interest to declare.
C-Cbl-PI3K Interaction Is Important for Platelet Outside-In Signaling
Authors: Buitrago Claudia Lorena , Kunapuli Satya P. , Sanjay Archana
Published: October 2019

Abstract 2014

Platelet activation by outside-in signaling is initiated by the binding of fibrinogen to alphaIIbbeta3, an integrin only expressed in platelets and megakaryocytes. Signals transduced by alphaIIbbeta3 regulate actin cytoskeleton resulting in filopodia and lamellipodia formation, cell spreading and retraction.

c-Cbl protein is abundantly expressed in platelets and functions as E3 ubiquitin ligase and scaffolding protein to mediate protein-protein interactions. Importantly, c-Cbl tyrosine 731 has been shown to interact with p85 subunit of phosphotidylinositol 3-kinase (PI3K) modulating the actin cytoskeleton. Although previous reports showed c-Cbl activation downstream of alphaIIbbeta3, the mechanisms and implications of this activation or the downstream targets remain to be elucidated. We have studied the role of c-Cbl in platelet outside-in signaling: Using human platelets we have demonstrated that c-Cbl Y700, Y731 and Y774 residues undergoes tyrosine phosphorylation upon platelet adhesion to immobilized fibrinogen. These phosphorylation events are completely inhibited in the presence of the pan Src Family Kinases (SFKs) inhibitor (PP2) suggesting that c-Cbl is phosphorylated downstream of SFKs. Spleen tyrosine kinase (Syk) is also involved in this signaling pathway since its inhibition significantly reduce c-Cbl phosphorylation at residues Y774 and Y700; interestingly, tyrosine 731 phosphorylation, which allows the interaction with the p85-subunit of PI3K, is not affected by Syk inhibition. The physiological role of c-Cbl in platelet outside-in signaling was studied using c-Cbl knock-out mice. We found that in contrast to WT platelets, c-Cbl KO platelets had a significantly reduced spreading over a fibrinogen-coated surface. Furthermore, clot retraction analysis demonstrated that c-Cbl KO platelets retraction time was delayed when compared to WT platelets, suggesting a retraction defect. To further elucidate the physiological role of c-Cbl-PI3K interaction we used a knock-in mouse in which the c-Cbl residue Y 731 was substituted with phenylalanine (Y731F) thereby abolishing the PI3K binding site on c-Cbl. Importantly, platelets from Y731F mice showed spreading and clot retraction defect that were comparable with the c-Cbl KO. These result indicates that in large part, the role of c-Cbl in platelets outside-in signaling is determined by its interaction with PI3K.

In conclusion, we have demonstrated that c-Cbl plays an important role in platelet outside-in signaling, and its interaction with PI3K through tyrosine 731 is of pivotal importance in platelet spreading and retraction.

Disclosures:

No relevant conflicts of interest to declare.
Agonist-Induced Binding of the Regulatory Scaffold Protein Spinophilin to Protein Phosphatase-1 In Platelets
Authors: Sinnamon Andrew , Ma Peisong , Brass Lawrence F.
Published: October 2019

Abstract 2029

Platelet regulation plays a critical role in hemostasis. Underactivation can result in failure to stop bleeding, whereas inappropriate platelet activation can cause thrombus formation. The 130-kDa scaffold protein spinophilin (SPL) has recently been shown to play a role in preventing platelet overactivation by forming a complex with the proteins RGS10, RGS18, and the tyrosine phosphatase SHP-1. This complex dissociates when platelet are activated by thrombin or thromboxane A2 and evidence from spinophilin knockout mice suggests that this regulates platelet activation in vitro and in vivo. Spinophilin was originally isolated as a binding partner for the serine/threonine phosphatase, PP-1, in neurons. Here we asked whether PP-1 forms a complex with spinophilin in human platelets and, if so, whether the complex is affected by platelet activation. The approaches that we used to answer this question included Western blotting with antibodies to PP-1 and spinophilin, and co-precipitation studies looking for an association between spinophilin and PP-1. The results of the Western blots confirm the presence of PP-1 in platelets. The initial co-precipitation studies show that little, if any, PP-1 is associated with spinophilin in resting platelets, but there is a time-dependent increase in the SPL/PP-1 complex when platelets are activated with the PAR1 (thrombin receptor) activating peptide, SFLLRN. Thus it appears that within approximately the same time frame that the SPL/RGS/SHP-1 complex is decaying in activated platelets, the SPL/PP-1 complex is forming. Targets for PP1 have not been fully identified in platelets, but it is known that spinophilin localizes to the plasma membrane upon platelet activation. Since spinophilin is thought to direct PP1 targeting in neurons, it is reasonable to propose that it may be directing PP1 to targets in platelets in a similar manner. The studies described in this abstract were supported in part by a 2010 ASH Trainee Research Award to Andrew Sinnamon, who is a first year medical student at the University of Pennsylvania.

Disclosures:

No relevant conflicts of interest to declare.
Release of Alkaline Phosphatase Caused by PIGV Mutations In Patients with Hyperphosphatasia-Mental Retardation Syndrome (HPMR), a Recently Found Second Inherited GPI Anchor Deficiency.
Authors: Murakami Yoshiko , Krawitz Peter M , Robinson Peter N , Mundlos Stefan , Maeda Yusuke , Kinoshita Taroh
Published: October 2019

Abstract 2031

Recent whole exome sequencing of three sibs of non-consanguineous parents demonstrated PIGV mutations in Hyperphosphatasia-Mental Retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels (Eur J Med Genet, vol.53, issue2, p85, 2010). PIGV is the second mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K and H385P. We have shown that these mutant PIGV proteins are unstable and the mutant cDNAs restored only subnormal GPI biosynthetic activity after transfection into PIGV deficient CHO cells. The backbone of GPI-anchor is synthesized in the endoplasmic reticulum (ER) and is transferred to the proteins which have GPI attachment signal at the C-terminal. It is known that GPI-anchored proteins are not expressed on the surface of GPI deficient cells due to degradation within the cells or secretion. ALP, a GPI anchored protein, was efficiently secreted into medium from PIGV deficient CHO cells, in which incomplete GPI bearing one mannose was accumulated. In contrast, ALP was degraded in PIGL, DPM2 or PIGX deficient CHO cells, in which GPIs lacking mannose were accumulated. Secretion of ALP required GPI transamidase that cleaves the C-terminal GPI attachment signal peptide and replaces it with GPI. It seems that GPI transamidase is activated by GPI bearing at least one mannose, cleaving the hydrophobic signal peptide and resulting in secretion of soluble ALP. It is well known that hypophosphatasia is caused by a deficiency of liver-, bone-, kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. This inherited disorder is characterized by defective bone mineralization and some patients develop epilepsy caused by the disturbance of pyridoxal-5-phosphate (PLP) dependent metabolism of neurotransmitters due to the defect in ALPL which converts PLP to pyridoxal (PL) thereby facilitating passive uptake of this freely diffusible form at the cell surface. Some of the HPMR patients have seizures and the patients with previously reported inherited GPI deficiency caused by defective PIGM encoding the first mannosyltransferase (Nature Med. 12, p846 2006) also have seizures. This common symptom may be caused by the loss of membrane bound ALPL. On the other hand, patients with HPMR do not have the portal vein thrombosis seen in the patients with PIGM deficiency. The affected cell types and the degree of deficiencies in various GPI-anchored proteins might be the cause of the variability of the clinical features among GPI deficiencies.

Disclosures:

No relevant conflicts of interest to declare.
Organ Siderosis In a Murine Graft-Versus-Host-Model After Allogeneic Stem Cell Transplantations Unrelated to Red Cell Transfusion
Authors: Nogai Axel , Ziebig Reinhard , Muckenthaler Martina U. , Thiel Eckhard , Uharek Lutz , Notter Michael
Published: October 2019

Abstract 2048

Introduction:

Organ siderosis (OS) occurs frequently after allogeneic stem cell transplantations and is associated with worse survival. Although commonly attributed to transfusions of erythrocyte sediments, we present the occurrence of OS in mice suffering from Graft-versus-Host-disease (GvHD) independent of transfusions.

Methods:

Prior to transplantation (day 0), C57BL/10-wildtype (wt) mice received treosulfan 2000mg/kg on day -3 to -1 and cyclophosphamide 200 mg/kg on day-1. Mice were transplanted with 5×106 bone marrow cells and 3×106 splenocytes of Balb/c (allogeneic) or C57BL/10 (syngeneic) origin. Mice were sacrificed on day+9. Liver iron content was determined by atomabsorption spectroscopy in graphit tubes. In addition, a semi-quantitative analysis of liver, kidney and spleen sections was performed in kinetic studies. Macroscopic and histological GvHD-Scores were assessed according to standard scores.

Results:

Treatment of mice without additional transplantation or transplantation of syngeneic cells did not increase the iron content of the liver compared to untreated mice (naïve: 6.7 +/− 1.3 μmol/g; chemotherapy only: 7.2 +/− 1.9; syngeneic: 7.2 +/− 2.1). In contrast, allogeneic transplantated mice suffering from GvHD developed a siderosis of the liver of 13.6 +/− 3.1 (p=0.01, Kruskal-Wallis test). Furthermore, the iron deposition was correlated with the macroscopic and histologic GvHD score.

Conclusion:

These results show the occurrence of OS in wildtype animals after allogeneic stem cell transplantation. Since the detection of iron deposition occurs early after transplantation, OS is unlikely due to increased intestinal iron absorption, but appears to be a consequence of blocked iron export out of hepatic epithelial cells and macrophages and potentially by redistribution of iron as a consequence of liver cell destruction. Further studies are needed to elucidate the role of regulatory proteins such as hepcidin and ferroportin for OS in the context of GvHD following stem cell transplantation. In view of GvHD-associated OS in transplanted patients occuring frequently independent of transfusions, the effect appears clinically relevant with treatment options remaining to be defined.

Disclosures:

No relevant conflicts of interest to declare.
CBL Mutations In Therapy-Related Leukemia and Infant Leukemia
Authors: Shiba Norio , Taki Tomohiko , Park Myoung-ja , Nagasawa Masayuki , Takita Jyunko , Kato Motohiro , Kanazawa Takashi , Sotomatsu Manabu , Arakawa Hirokazu , Hayashi Yasuhide
Published: October 2019

Abstract 2149

Recent reports of somatic mutations of the CBL proto-oncogene in myeloid neoplasms are intriguing, because these CBL mutations were shown to results in aberrant tyrosine kinase signaling, which would lead also to activation of RAS signaling pathways. We and others reported that CBL mutations occurred in a variety of myeloid neoplasms, including de novo acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and MDS/myeloproliferative neoplasm, especially in chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML). The importance of CBL mutations concerning leukemogenesis is substantially increased. We investigated CBL mutations in 20 therapy-related leukemia/MDS (t-Leuk/MDS) cases and 26 infant leukemia cases. Homozygous mutation of theCBL gene (P417R), which was located in the RING finger domain, was identified in one out of 20 (5%) t-Leuk/MDS cases. This patient was a 5 year-old boy, whose biopsied specimen of the buccal lymph node showed malignant lymphoma (diffuse large T cell type, MT1(+), MB1(-), UCHL1(+)). No pathogenic nucleotide changes were identified in the CBL gene in the initial sample. Subsequently, the patient was treated with chemotherapy including VP-16 (200 mg/m2) given twice weekly. Nineteen months after initial diagnosis, he was diagnosed as having therapy-related leukemia with t(5;21) and MLL gene rearrangement due to VP-16. Furthermore, CBL gene mutations were found in 3 of 26 (12%) infant leukemia cases with 11q23 translocation/MLL gene rearrangement. CBL gene mutations were located in splice site in intron 8 and the RING finger domain (Y371H, in 2 cases), SNP array analysis (Affymetrix, GeneChip) of these cases with mutated CBL gene disclosed 11q-acquired uniparental disomy in all cases, but not in cases with wild-type CBL. CBL mutation has not been reported in acute leukemia with 11q23 translocation/MLL gene rearrangement. To our knowledge, these are the first t-Leuk/MDS case and infant cases with 11q23 translocation/MLL rearrangement, suggesting that CBL is mutated in a unique subset of t-Leuk/MDS and infant leukemia which is considered to play a pathogenic role in the development of t-Leuk/MDS and infant leukemia. Further accumulation of cases with t-Leuk/MDS and infant leukemia having the CBL mutation is needed.

Disclosures:

No relevant conflicts of interest to declare.
Frequency of Leukemia Stem Cell Candidates Predicts Refractoriness to Conventional Chemotherapy and Adverse Clinical Outcome
Authors: Ran Dan , Taubert Isabel , Eckstein Volker , Bellos Frauke , Wuchter Patrick , Saffrich Rainer , Schubert Mario , Ho Anthony D.
Published: October 2019

Abstract 2160

We have shown that leukemia stem cells candidates (LSCC) can be prospectively identified by high activity of aldehyde dehydrogenase (ALDHbr) and expression of CD34 among the leukemia blasts from the marrow of patients with AML. In this study we have examined the relationship between the frequency of LSCC at diagnosis with persistence of leukemia blasts after induction chemotherapy as well as with long-term clinical outcome. Using single cell sorting, we have further separated subsets among the LSCC and correlated their individual functional properties with the respective marker constellation. The percentage of LSCC in 101 patients ranged from 0.01% to 12.90% with a median of 0.51%. Frequencies of LSCC among the leukemia

blasts at diagnosis correlated significantly with the persistence of leukemia after the first induction chemotherapy (n=79, Spearman R=0.7797, P<0.0001). During the observation period of 24 months, 21 of 60 patients with high levels of LSCC died as compared to 7 of 41 patients with low levels of LSCC (p=0.029). The overall survival (OS) probability for the patients with high levels of LSCC was significantly worse (p=0.05) than in those with low LSCC. Characterization of these LSCC at a single cell level showed that a varying proportion, i.e. 15% to 78% of their progeny cells demonstrated the same chromosomal aberrations as the original leukemia population, indicating the presence of residual normal HSC. Thus high frequencies of LSCC at the time of diagnosis predict persistence of leukemia blasts, failure to achieve CR within the first cycle and poor overall clinical outcome. Our prospective separation of LSCC permits precise characterization of the biological properties of the subsets of stem cell candidates in human AML.

Disclosures:

Ho: Genzyme: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees.
Bone Marrow Hypoplasia Induced by Conditional Knockout of the RNase III Domain of Dicer-1
Authors: Huang Tai-Chung , Yang Yi , Brayton Cory , Chou Wen-Chien , Pandey Akhilesh
Published: October 2019

Abstract 2226

Background:

MicroRNAs (miRs) play important roles in normal hematopoiesis as well as in hematopoietic malignancies. In generating mature miRs, Dicer-1, an RNase III in the cytosol, excises the hairpin-loop of precursor miRs and assembles mature miRs onto an RNA-Induced Silencing Complex. Among 27 exons of Dicer1, exons 21 and 22 comprise the RNase IIIa domain. Knockout of this domain was shown to be lethal in mouse embryos. In this study, we used tamoxifen to induce Cre-mediated exon deletion and our goal was to investigate the impact of Dicer1 knockout on hematopoiesis in adult mice.

Material and Methods:

Two C57BL/6 transgenic mice were crossed: ROSA26-CreERT2 and floxed Dicer1 exons 21 & 22. At the age of 8 week, three female mice were treated with tamoxifen (1 mg/day, Day 1 to 5) and two with placebo. On Day 8, mice were sacrificed for examination. DNA genotyping and qRT-PCR were used to confirm Dicer1 genomic deletion and to quantify the transcription copy number of Dicer1. Cardiocentesis was done for complete peripheral blood count. Femoral and tibial bone marrow, spleen and inguinal lymph nodes were evaluated by flow cytometry whereas formalin-perfused sternum and thymus were subjected to histopathological examination.

Results:

Two tamoxifen-treated mice presented with unkempt appearance, hunched posture and diarrhea from Day 7. Vehicle-treated control mice remained clinically normal. qRT-PCR showed lower copy number of Dicer1 exons 21 & 22 in tamoxifen-treated group than in control (0.09 vs. 1.00). In tamoxifen-treated group, the spleen weights were lower, white blood cell and platelet counts were lower and bone marrow histopathology revealed hypocellularity, with markedly reduced and immature myeloid elements, consistent with arrested or abrogated development of myelomonocytic and megakaryocytic elements.

Conclusions:

The RNase III domain of Dicer-1 is crucial for the normal differentiation of marrow myeloid series in adult mice, probably at the stage of common myeloid progenitor.

Disclosures:

No relevant conflicts of interest to declare.
A Microrna Feed-Forward Loop Amplifies GFI1N382S Transcriptional Reprogramming In Severe Congenital Neutropenia
Authors: Velu Chinavenmeni Subramani , Kazanjian Avedis , Colmenares Clemencia , Grimes H. Leighton
Published: October 2019

Abstract 2239

The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell self renewal and myeloid differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 that generate dominant negative acting proteins. Moreover, GFI1-mutant SCN patients and Gfi1-/- mice display a unique accumulation of myeloid progenitors. Recently we showed that Gfi1 regulation of HoxA9, Pbx1 and Meis1 underlies these phenomena, in that the Gfi1-Hox transcriptional circuit controls the accumulation of myeloid progenitors in vivo. We have also shown that Gfi1 regulates miR-21 during myelopoiesis, and that miR-21 is deregulated by Gfi1N382S expression. Our new data link these concepts by demonstrating that forced expression of miR-21 in bone marrow cells results in the accumulation of myeloid progenitors in transplant recipients. Moreover, miR-21 directly targets the Ski oncoprotein, and Ski-/- bone marrow cells show an accumulation of myeloid progenitors. Thus, Gfi1-/-, miR-21 overexpressing-, and Ski-/- myeloid progenitors accumulate in the marrow. Strikingly, Ski is dramatically reduced in miR-21 overexpressing Lin- bone marrow cells. Nearly undetectable Ski expression in Gfi1-/- bone marrow cells can be completely rescued by antagonizing miR-21 activity. Since Ski is a corepressor and Gfi1 is a transcriptional repressor, we next tested whether the two proteins physically interact. Indeed, endogenous Ski and Gfi1 can be coimmunoprecipitated. Synthetic Ski and Gfi1 proteins reveal that the interaction is mediated through Ski carboxy-terminal and Gfi1 zinc-finger domains. Chromatin immunoprecipitation reveals Ski and Gfi1 co-occupy several Gfi1 target genes (including HoxA9), which are derepressed upon Gfi1 or Ski knockdown. However, while Gfi1 binds and regulates the miR-21 gene, Ski is not bound to the miR-21 gene, and Ski knockdown has no effect upon miR-21 levels. Thus, the data point to a novel feed-forward transcriptional circuit. Gfi1N382S deregulation of miR-21 amplifies the dominant-negative effect of Gfi1N382S through miR-21 targeting of Ski, leading to further derepression of Gfi1-Ski target genes.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Stem Cell Transplantation for Patients with Chronic Myeloid Leukemia After Prior Treatment with Nilotinib or Dasatinib
Authors: Schleuning Michael , Scholten Marijke , Biezen Anja van , Nagler Arnon , Apperley Jane F. , Michallet Mauricette , Grigg Andrew , Silver Richard T. , Leitner Armin , Gratwohl Alois , Ekblom Marja , Khoury H. Jean , de Witte Theo , Olavarría Eduardo
Published: October 2019

Abstract 2348

Stem cell transplantation (SCT) will continue to be a treatment option for patients with chronic myeloid leukemia, despite the introduction of tyrosine kinase inhibitors (TKI). However, many patients will have received prior therapy with TKI, including Nilotinib or Dasatinib at the time of allogeneic SCT. While the use of Imatinib prior to SCT seems to have no adverse impact on the outcome of allogeneic SCT little is known on the impact of prior use of second generation TKI. Therefore we conducted a retrospective registry study and identified 56 patients with CML who received an allotransplant after having been treated with Nilotinib and/or Dasatinib. Best responses to second generation TKI were major molecular response in 11%, complete cytogenetic response in 7%, partial cytogenetic response in 18%, complete hematologic remission in 25% and no response in 34%, respectively. At SCT, 37% of the patients were in accelerated or in blast phase, 36% in CP2 or higher and 27% in first chronic phase. Graft failure occurred in two patients. The median follow-up for surviving patients is 19 months. At 24 months the estimated non-relapse mortality was 33% and the relapse incidence 15%. Probability of survival is more than 85% at 2 years in patients transplanted in CP1. In univariate analysis there was a non significant trend in favor for pretreatment with Nilotinib as compared to the other groups. However, in multivariate analysis only stage of the disease was a predictor for survival. With respect to overall survival no significant differences could be identified for the following variables: patient age, donor type, stem cell source, intensity of the conditioning, time diagnosis to transplant, in or ex vivo T-cell depletion, response to treatment with second generation TKI. Patients transplanted in blast crisis had a significant higher risk of non relapse mortality. In summary, despite the shortcomings of a retrospective study, the data reported clearly show the feasibility and efficacy of allo SCT in patients pretreated with second generation TKI and it should be emphasized that the timing of allogeneic stem cell transplantation remains crucial to avoid unacceptable high treatment related mortality.

Disclosures:

Ekblom: Bristol-Myers Squibb: Honoraria.
Identification of Somatically Acquired Gene Mutations In the Kineome and Exome In CLL
Authors: Zhang Xiaosong , Li Meng , Ouillette Peter , Diaz Luis , Reis Marcelo , Velculescu Victor , Kinzler Kenneth , Malek Sami , Vogelstein Bert
Published: October 2019

Abstract 2410

The pathogenesis of CLL remains incompletely understood. While acquired chromosomal aberrations have been demonstrated to influence CLL biology and clinical behavior, it remains unclear what single gene defects other than p53 or ATM mutations contribute to or cause the CLL phenotype. In particular, recurrent gene mutations that are increasingly found in other hematological malignancies have not yet been identified in CLL. Further, the discovery of stereotypical immunoglobulin receptor genes in CLL has been advanced in support of the involvement of autoantigenes in the pathogenesis of CLL and thus a pro-proliferative or anti-apoptotic gene mutation may possibly not exist in CLL.

Given that kinase genes are often the targets for recurrent gene mutations and given the prospect for identification of drugable targets, we have analyzed 515 kinase genes in a discovery panel of 23 well characterized CLL samples (in DNA from CD19+ sorted cells) and tested their somatic nature in CD3+ cell-derived DNA from the same patients. A total of 8308 different exon sequences were analyzed. We identified six individual kinase gene mutations (WEE1, NEK1, BRAF, KDR, MAP4K3 and TRPM6), each occurring once in a separate CLL case as well as >1450 non-synonymous polymorphisms but no recurrent kinase gene mutations were identified. Interestingly, mutations in all these genes but NEK1 had previously been identified in cancer genome projects of cancers of the breast, colorectum, pancreas and glioblastome multiforme and WEE1, BRAF and KDR have established roles in cancer biology.

Next we used solution exon capture of genomic DNA followed by Solexa-based next gen exome sequencing on 8 paired CLL derived DNA samples (FACS-sorted CD19+ versus CD3+ paired samples), including 2 samples with known p53 mutations. Using stringent criteria for mutation calling, we identified 69 mutated genes including the 2 cases of p53 mutations. None of the mutated genes were recurrent. Work is in progress to estimate the frequency of mutations in these genes in a wider panel of CLL cases.

In summary, our data provide practical information about the mutations state of the CLL kineome and exome with implications for CLL biology/pathogenesis. This data should motivate additional exome sequence analyses of more CLL samples to fully capture and describe the mutation load in CLL.

Disclosures:

No relevant conflicts of interest to declare.
Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells
Authors: Duque-Afonso Jesus , Essig Aitomi , Solari Leticia M , Berg Tobias , Pahl Heike L. , Lübbert Michael
Published: October 2019

Abstract 2474

Background:

The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells.

Methods:

To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively.

Results:

The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2.

Conclusions:

In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype.

Disclosures:

No relevant conflicts of interest to declare.