bluuebunny/crossref_metadata_2025_split 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
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
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
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
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
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
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
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
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
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
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.