bluuebunny/crossref_metadata_2025 huggingface dataset:
A Newly Identified Platelet and Megakaryocyte Lysyl Oxidase-Adhesion to Collagen Axis in Human Primary Myelofibrosis
Authors: Balduini Alessandra , Abbonante Vittorio , Matsuura Shinobu , Rosti Vittorio , Ravid Katya
Published: October 2019

Controlling platelet function is central to management of various pathologies, including Primary Myelofibrosis (PMF), which is associated with increased incidence of thrombosis and cardiovascular disease. In recent studies we showed that the matrix cross-linking enzyme, Lysyl Oxidase (LOX) is elevated in platelets and megakartocytes of myelofibrotic mice, and transgenic upregulation of LOX increases platelet and megakaryocyte adhesion to monomeric type I collagen (preferred by alpha2β1 collagen receptors), and augments propensity for in vivo thrombosis. Here, we examined the relevance of these findings to human disease, by first determining platelet LOX level, as well as platelet and megakaryocyte adhesion to collagen using samples derived from PMF patients and matching controls. In analyzing 10 PMF platelet samples (5 males and 5 females; 6 JAK2V617F; 4 CALR mutations; age range 30-55; PMF grade 1-3), we found a nearly 20 fold upregulation of LOX expression compared to matching healthy controls (p<0.001). Intriguingly, there was a significant increase in adhesion (plt/mm2) and spreading (pixel2) of PMF platelets relative to control on monomeric, pepsinated acid soluble collagen (PSCI) (p<0.05), while no differences were observed between the samples on native triple helical acid soluble collagen type I collagen (ASCI). To examine the role of LOX in this phenotype, we treated control and PMF-derived human megakaryocytes, differentiated from peripheral blood CD34+ cells, grown in presence or not of LOX inhibitor, β-aminopropionitrile (BAPN) from day 2 of culture. Our preliminary data, based on a cohort of 2 controls and 5 PMF samples, demonstrated that although on ASCI megakaryocyte adhesion is not altered by BAPN treatment both in CTRL and PMF derived megakaryocytes, on PSCI the adhesion of PMF derived megakaryocytes was reduced by about a 50% by BAPN treatment, while the adhesion of CTRL derived MKs was not significantly affected. Taken together, we identified LOX level to be upregulated in human PMF platelets and megakaryocytes, and LOX activity to be important for PMF cells adhesion to collagen. These newly identified properties are highly relevant to megakaryocyte adhesion to the niche, and to platelet activation in PMF.

Disclosures

No relevant conflicts of interest to declare.
Immunohematologic Complications after Nonmyeloablative Hematopoietic Progenitor Cell Transplantation in Patients with Sickle Cell Disease
Authors: Allen Elizabeth S. , Hsieh Matthew M. , Fitzhugh Courtney D. , Klein Harvey G. , Tisdale John F. , Flegel Willy A.
Published: October 2019

Abstract

Background: Hematopoietic progenitor cell (HPC) transplantation can cure sickle cell disease (SCD). Anonmyeloablative conditioning regimen has lower morbidity and mortality, and typically results in donor-derived erythrocytes and stable mixedchimerism of recipient- and donor-derived leukocytes. There is a risk ofimmunohematologic complications due to red cell antibodies induced by transfusions during theperi-transplantation period or exposure to donor antigens from the HPC graft. We described the incidence ofimmunohematologic complications in a cohort of patients with SCD undergoing HPC transplantation.

Study design and methods: All patients with SCD (42 with HLA-matched and 19 withhaploidentical donors) enrolled in 3 clinical trials before March 31, 2015, were retrospectively evaluated for the formation of new red cell antibodies after transplantation or any red cell incompatibility between donor and recipient.

Results: Of the 61 patients, 9 experienced immunohematologic complications. Before HPC transplantation, 3 patients had antibodies that were incompatible with their donors. After HPC transplantation, new antibodies were observed in 6 patients (11 allo-, 2 auto-), 3 of whom developed antibodies that were incompatible with donor or recipient red cells, while 3 developed antibodies that were compatible. The occurrence of new alloantibodies was not significantly associated with allo- or autoantibodies at enrollment, number of pre-enrollment transfusions, recipient sex, or ABO blood group. On average, the 3 patients with antibodies at enrollment that were incompatible with donor red cells received more red cell transfusions and depended on transfusion for longer time periods than comparison groups (51 vs. 13 units, p=0.015; 419 vs. 38 days, p=0.009). Among the 9 patients withimmunohematologic complications, the clinical course was highly variable: some had no significant effects attributable to the antibodies, while others experienced prolongedreticulocytopenia, severe anemia, or became almostuntransfusable. In the 47 patients who maintained their grafts long-term,immunohematologic complications did not negatively impact hemoglobin concentration or hemoglobin S expression after transfusion independence. There was no significant correlation betweenimmunohematologic complications and graft failure, rejection or death.

Conclusions:Immunohematologic complications occurred in 15% of patients with SCD undergoingnonmyeloablative HPC transplantation. Clinical effects ranged from seemingly insignificant to potentially fatal. The formation of new antibodies was not predictable. In individuals with SCD, careful evaluation of donor and recipient phenotypes using red cell genotyping aids in preventing and managingimmunohematologic complications.

Disclosures

No relevant conflicts of interest to declare.
Excellent Clinical Outcome for Relapsed and Refractory Lymphoma Patients with Haplo-Cord Allogeneic Stem Cell Transplantation
Authors: Hsu Jingmei , Artz Andrew , Mayer Sebastian A. , Bishop Michael R. , Phillips Adrienne A. , Smith Sonali M. , Gergis Usama , Kline Justin , Shore Tsiporah B. , van Besien Koen
Published: October 2019

Background:

Umbilical cord blood transplantation has been used for hematologic malignancy patients lacking matching donors but slow engraftment has hindered its wide usage. We have previously described combining CD34 selected -haploidentical grafts with umbilical cord cells to accelerate neutrophil and platelet engraftment Here, we examine the outcome of patients with lymphoid malignancies undergoing haplo-cord transplantation at the University of Chicago and Weill Cornell Medical College from May 2008 to February 2016.

Results:

Median age was 45 years (23-69). 9 (23%) pts had Hodgkin's lymphoma, 5 (13%) patients had CLL and 25 (64%) patients had non-Hodgkin lymphoma including 6 T-NHL. Five (13%) and 1(2%) patient had prior autologous and allogeneic stem cell transplant respectively. Three (7%), 18 (46%) and 18 (46%) patients has low, intermediate and high/very high CIBMTR disease risk index prior to transplant. All patients received fludarabine/melphalan/ATG conditioning regimen and post-transplant GVHD prophylaxis with tacrolimus and MMF. The median time to neutrophil engraftment is 11 days (9-60) and platelet engraftment is 19 days (11-88).

Cumulative Incidence of NRM is 10% at 100 days and 16 % at one year. Cumulative incidence of relapse is 15% at 100days and 18% at one year. With a median follow up for survivors of 18 months, one year overall survival (OS) is 73% (95% CI 59-87). OS at one year is 58% for NHL patients, 80 % for HL patients and 100 % for patients with CLL. None of the survivors have chronic GVHD.

Conclusion:

HaploCord Transplantation offers an excellent treatment alternative for patients with recurrent and refractory lymphoid malignancies who lack matching donors. Count recovery is rapid, non relapse mortality is limited, excellent disease control can be achieved and the incidence of chronic GVHD is limited.

Figure 1 Figure 1. Cumulative incidence of RM and NRM and overall survival of relapsed/refractory lymphoma patients undergoing haplo-cord stem cell transplantation at one year

Disclosures

Smith: Celgene: Consultancy; Juno: Consultancy; TGTX: Consultancy; Portola: Consultancy; Amgen: Other: Educational lecture to sales force; AbbVie: Consultancy; Gilead: Consultancy; Pharmacyclics: Consultancy; Genentech: Consultancy, Other: on a DSMB for two trials . Kline:Merck: Honoraria, Research Funding; Vasculox: Research Funding.
A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Erythrocytes Cultured from CD34+ Cells with Sickle Cell Disease
Authors: Zhu Jianqiong , Chin Kyung , Aerbajinai Wulin , Kumkhaek Chutima , Li Hongzhen , Hsieh Matthew M. , Fitzhugh Courtney D. , Rodgers Griffin P.
Published: October 2019

Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in CD34+ cells from healthy and sickle trait donor's blood. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from CD34+ cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 2.0-fold, δ-globin gene expression 4.3-fold, and γ-globin gene expression 2.6-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.3-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2+HbF was increased from 8.1 % in vector-transduced cells to 19.7% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 14.4% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 79.8% in medium-form Eklf-GATA-transduced cells as compared with 89.8% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. Our results shown that long form Eklf-GATA1 fusion protein has major effects on d- and g-globin induction than β-globin; the medium form Eklf-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Our results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation.

Disclosures

No relevant conflicts of interest to declare.
Long-Term Expression of Von Willebrand Factor Via Piggybac-Mediated Gene Transfer
Authors: Johnson Blake A , Chauhan Anil K. , Staber Janice M
Published: October 2019

von Willebrand disease (vWD), caused by a deficiency in von Willebrand factor (vWF), is the most common inherited bleeding disorder, affecting up to 1% of the population; those affected have a varied bleeding phenotype. von Willebrand factor plays two important roles in hemostasis including platelet adhesion and stability of factor VIII (FVIII). As such, patients with von Willebrand disease type 3 have no measurable vWF protein and demonstrate low levels of FVIII. To date, persistent long-term expression of full-length vWF using gene transfer strategies has not been demonstrated, largely due to the immense size of vWF cDNA (8.4 kb). Non-viral vector systems, such as piggyBac, are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a "cut and paste" mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize PB-mediated vWF gene transfer will confer long-term expression and improve the bleeding phenotype in an animal model of vWD. We engineered PB transposon to carry a human von Willebrand factor cDNA (PB vWF). When transfected with PB-vWF and a hyperactive transposase, iPB7, the hepatocarcinoma cell line, HepG2, demonstrated secretion of vWF into supernatants compared to mock transfected controls (p < 0.01). Next, we evaluated the in vivo gene transfer efficiency in von Willebrand deficient mice by hydrodynamic tail-vein injection using PB-vWF driven by the murine albumin enhancer/human alpha anti-trypsin promoter. vWF null mice received 25 or 50 micrograms each of the PB-vWF transposon and iPB7 to determine long-term expression and phenotypic correction. vWF levels were measured prior to injection and then every 4 weeks for up to 24 weeks. Results revealed therapeutic levels (on average 50% normal mice) of vWF post gene transfer with stable expression for 24 weeks in most mice. These data indicate the PB transposon vector system may provide a long-term gene transfer strategy for vWD. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. PB-vWF gene transfer resulted in at least partial bleeding phenotype correction via tail clip. Additionally, treated mice demonstrated a rescue of the FVIII activity. These data show that the PB vector can be used to deliver this large transgene expression cassette to the liver and achieve long-term expression and phenotypic correction in vivo.

Disclosures

Staber: Emergent BioSolutions: Honoraria; Baxalta: Honoraria.
Structural and Functional Characterization of a Codon Optimized Coagulation Factor VIII
Authors: Shestopal Svetlana A , Kurasawa James H , Hao Jian-Jiang , Karnaukhova Elena , Liang Yideng , Lin Min , Ovanesov Mikhail V , Lee Timothy K , McVey John H , Sarafanov Andrey G
Published: October 2019

Background. Production of recombinant factor VIII (FVIII) is challenging due to its low expression. Previously, it was shown that codon optimization of a B domain-deleted (BDD) FVIII resulted in its increased expression (Ward et al, Blood 2011; 117: 798-807). However, in some cases, synonymous mutations are known to affect the protein's post-translation modifications, conformation, fidelity of amino acid sequence, and functions. Thus, for each particular codon optimization of a given protein, confirmation of its biochemical characteristics is necessary. Recently, we established conditions for expression and purification of a codon optimized BDD-FVIII (CO), in parallel, testing the BDD-FVIII (WT) expressed from the wild-type cDNA sequence (Shestopal et al, ISTH-2015 meeting, Abstract PO196-WED). In present work, we verified if the characteristics of the CO remain unchanged upon modification of its coding sequence.

Objective. To characterize structural and functional properties of the BDD-FVIII encoded by either a codon-optimized or wild-type cDNA sequence (CO and WT, respectively).

Experimental Approach. Several preparations of each WT and CO, purified from independent CHO cells clonal lines, were analyzed by: polyacrylamide gel electrophoresis (PAGE), before and after thrombin treatment; Western-blot analysis; ELISA; mass-spectrometry upon specific protein fragmentation; circular dichroism; chromogenic, clotting and thrombin generation assays to test the FVIII activity; and by surface plasmon resonance to test the binding to von Willebrand factor and a fragment of the low-density lipoprotein receptor-related protein 1 (LRP).

Results. The average purification yield of CO was approximately 7-fold higher than that of WT. The proteins were identical in the amino acid sequences, covered by 99% by mass-spectrometry, and were very similar in: i) patterns of the molecular fragments, including those produced upon thrombin cleavage by PAGE, ii) recognition by anti-FVIII antibodies by both Western-blot and ELISA, iii) glycosylation and tyrosine sulfation by mass spectrometry, iv) secondary structures by circular dichroism and v) binding to von Willebrand factor, and vi) to a fragment of the low-density lipoprotein receptor-related protein 1 by surface plasmon resonance. By chromogenic, clotting and thrombin generation assays, the CO had about 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT.

Conclusions. The higher specific activity of CO was attributed to better preservation of its structure due to consistently higher concentrations than WT at all steps of the production. Thus, we concluded that the codon optimization of the BDD-FVIII resulted in a significant increase of its expression, while did not affect the protein's properties.

Disclosures

No relevant conflicts of interest to declare.
The Pan-PIM Kinase Inhibitor LGB321 Affects Apoptotic Pathways and Microenvironmental Interactions in CLL
Authors: Decker Sarah , Kissel Sandra , Aumann Konrad , Zenz Thorsten , Zirlik Katja , Claus Rainer , Duyster Justus , Dierks Christine
Published: October 2019

Introduction

The emergence of kinase inhibitors like Ibrutinib has drastically altered treatment strategies and improved outcomes in CLL patients, but lack of cure and resistance to therapy still remain serious problems. The three PIM kinases are involved in various important disease mechanisms in CLL, with PIM1 regulating CXCR4 surface expression impacting its interaction with the microenvironment, and PIM2/3 affecting the apoptotic machinery by regulating BAD. The Pan-PIM kinase inhibitor LGB321 (Novartis) targets all three PIM kinases and therefore affects both, CLL apoposis and its interaction with the microenvironment.

Results

In the study presented here, we investigated the effect of the Pan-PIM kinase inhibitor LGB321 on CLL in vitro and in vivo. LGB321 was highly effective in inducing apoptosis in primary human CLL cells, independent of risk factors or the mutation status. Apoptosis induction correlated with reduced pBAD and BAD levels. LGB321 was also effective in the presence of protective stromal cells and could completely overcome the stroma protective effects. Furthermore, we found that Pan-PIM inhibitor treatment blocked the CXCR4/CXCL12 axis by dephosphorylating the CXCR4 receptor on Ser339, by reducing total CXCR4 protein levels, and by blocking the externalization of the CXCR4 receptor. Concordantly, LGB321 blocked CXCR4 functions like migration towards a CXCL12 gradient (P<.0001), and reduced homing of LGB321-pretreated primary CLL cells towards the bone marrow (P=.0001) of NOG mice. In vivo experiments comfirmed the efficacy of LGB321 in 4 different CLL xenograft studies. Transplantation of primary human CLL cells into NOG mice and treatment with LGB321 for 2 weeks strongly reduced WBC counts, spleen size and spleen infiltration with human CLL cells (P=.0295) in all four CLL cases, and blocked BAD as well as CXCR4 phosphorylation also in vivo.

Conclusion

Our results demonstrate, that the Pan-PIM kinase inhibitor LGB321 might be an effective treatment option for CLL patients by impairing PIM2/3 mediated CLL-cell survival, and by blocking the PIM1/CXCR4-mediated interaction of CLL cells with their protective microenvironment in vitro and in vivo. Future clinical trials should be performed to validate its efficacy in human CLL.

Disclosures

Claus: Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel Funding.
Early Enhancement of IgM Expression and Signaling Capacity during Ibrutinib Therapy in CLL Patients
Authors: Drennan Samantha , Chiodin Giorgia , D'Avola Annalisa , Johnson Peter W , Trentin Livio , Packham Graham , Steele Andrew J , Stevenson Freda K , Forconi Francesco
Published: October 2019

In chronic lymphocytic leukemia (CLL), B-cell receptor (BCR) signaling is key to survival and proliferation, and can be effectively targeted by the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. BCR surface IgM (sIgM) levels and signaling capacity are low but variable in CLL. The variability has clinical consequences and relatively high sIgM in circulating cells is associated with aggressive disease, a higher proliferative component and higher levels of adhesion and migration receptors (D'Avola, Drennan et al, Blood 2016).

The aim of this study was to investigate the expression and function of sIgM and other surface markers on the circulating tumor cells of CLL patients following one week of ibrutinib therapy. Expression of markers associated with the BCR complex (IgM, IgD, CD19), and with adhesion or migration (CD38, CD49d, CXCR4) was assessed by flow cytometry in 13 CLL patients prior to and following one week of therapy. Signaling capacity was measured by immunoblotting following sIgM or CXCR4 stimulation. N-glycosylation status of the µ-heavy chains was measured by immunoblotting after biotinylation of cell surface proteins to determine IgM maturation (Krysov et al, Blood 2010).

Following one week of treatment basal activity of BTK was significantly reduced compared with pre-treatment (P=0.002), and anti-IgM induced BTK auto-phosphorylation was completely suppressed, confirming full inhibition of BTK in vivo. Expression of sIgM, but not of sIgD, increased dramatically (P=0.005), with little change in the other receptors. The increase was similar in the tissue-egressed CXCR4dimCD5hi cells and in the CXCR4brightCD5low long-resting cells of the circulation. Increased sIgM expression associated with the fully N-glycosylated form of sIgM, characteristic of the absence of antigen engagement. Also, it correlated with increased capacity to phosphorylate SYK following anti-IgM stimulation (r=0.91, P<0.0001). Conversely, CXCL12-induced CXCR4 signaling, measured by ERK phosphorylation, was fully suppressed.

Whilst ibrutinib induces a rapid and persistent redistribution of CLL cells into the blood, early suspension is typically associated with reduction of the lymphocytosis, nodal progression and short survival. These data reveal a dangerous early phenomenon during ibrutinib in which IgM expression has increased in the circulating cells, resulting in enhanced IgM-mediated signaling capacity through SYK. CXCR4 inhibition may prevent migration into tissue during ibrutinib and avoid (super)antigen encounter, but cells could be at risk of increased IgM mediated proliferation and disease progression if ibrutinib is withdrawn at this stage.

Disclosures

Packham: Aquinox Pharmaceuticals: Research Funding; Karus Therapeutics: Other: Share Holder & Founder. Steele:Portola Pharmaceuticals: Honoraria.
3' Untranslated Region (UTR) Alterations Are Frequently Targeted By MM-Related Mirnas and Affects the Clinical Outcome
Authors: Samur Mehmet K. , Singh Irtisha , Shih-Han Lee , Sperling Adam Samuel , Fulciniti Mariateresa , Tai Yu-Tzu , Parmigiani Giovanni , Leslie Christina S. , Mayr Christine , Munshi Nikhil C
Published: October 2019

More than half of human genes use alternative cleavage and polyadenylation to generate alternative 3' untranslated region (3'UTR) isoforms that play a role in gene expression regulation. The 3' untranslated region (3'UTR) of mRNA contains elements that play regulatory roles in polyadenylation, localization, translation efficiency, and mRNA stability. Although relative contributions of different regulatory mechanisms remain poorly understood, several recent studies showed that alterations in 3'UTRs might affect protein localization as well as their interactions with other proteins.

Here, we obtained global measurements of 3' UTR sequencing and RNA-seq in MM and normal plasma cells to study the effects of 3'UTR alterations in MM. Whole transcriptome sequencing (RNAseq) data from 420 uniformly treated newly diagnosed MM samples were compared with 18 normal plasma cell by using dynamic analyses of alternative PolyAdenylation and results were compared with alternative 3'UTR isoforms measured using 3'-seq, an established quantitative 3' end sequencing method from 15 samples pilot study.

We observed ~10000 different isoforms with a median frequency of 297 (range 1-2495) altered 3'UTRs per sample. We observed 563 isoforms that have distal alternative poly A (APA) site and 449 isoforms that have proximal APA site compared to normal plasma cells in at least 10% of our patients. Enrichment analysis showed that short UTR genes are significantly targeted by miR-506, miR-133, miR-130, miR-27, miR-181 and miR-200 (FDR q-value < 0.05). We also observed that expression of 90 genes significantly increased between those samples with shorter UTR vs. regular UTR. Longer UTR genes are enriched in mir-124, mir-186, mir-200, mir-302, mir-495 and expression of 123 genes were significantly downregulated between long UTR and others. There were no tumor suppressor genes and 8 oncogenes in short UTR genes and 4 tumor suppressor and 14 oncogenes in longer UTR genes.

Our results were confirmed using the 3'seq study which showed that 170 genes have longer UTR in MM compared to normal plasma cells and 187 genes have shorter UTR. ~60% of longer and ~65% of shorter UTR genes reported by 3'seq were also observed with RNAseq. Furthermore, we focused on common genes reported by both high-throughput sequencing methods and analyzed any connection between outcome and UTR shortening or lengthening. We observed 26 genes with altered UTR regions have impact on PFS or OS.

In conclusion, we report significant alternate UTR usage, including intronic UTRs in MM affecting the disease biology and clinical outcome. Our data suggests the need to further investigate the molecular impact of alternate UTR usage and its relationship with miR in myeloma.

Disclosures

No relevant conflicts of interest to declare.
Mesenchymal Stem Cells (MSC) from Patients with Multiple Myeloma Promote Myeloid Cells to Become Granulocytic-Myeloid-Derived Suppressor Cells (G-MDSC) with Immunosuppressive, Bone Resorption and Pro-Angiogenic Activity
Authors: Giallongo Cesarina , Tibullo Daniele , Parrinello Nunziatina Laura , La Cava Piera , Camiolo Giuseppina , Caporarello Nunzia , Anfuso Carmelina Daniela , Puglisi Fabrizio , Conticello Concetta , Brundo Maria Violetta , Romano Alessandra , Di Raimondo Francesco
Published: October 2019

Purpose

A well-recognized feature of MM is the intimate relationship between plasma cells and bone marrow microenvironment, which is mainly composed of MSC, endothelial cells, immune cells and extracellular matrix. G-MDSC accumulate in the tumor microenvironment during tumor development promoting tumor growth and immunosuppression.

Aim

Analyzing MSC from MGUS, Smoldering myeloma (SMM) and MM patients in promoting G-MDSC generation.

Methods

Human peripheral blood mononucleated cells (PBMC) isolated from healthy subjects (HS) were cultured alone and with HS- (n=10), MGUS- (n=10), SMM- (n=4) or MM-MSC (n=14)(1:100). After 6 days, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype(CD11b+CD33+CD14-HLADR-) was confirmed by cytofluorimetric analysis.

Results

Only G-MDSC educated by SMM- and MM-MSC co-cultures (MSCed-G-MDSC) were able to suppress T cell proliferation when cultured with normal lymphocytes (p<0.001) compared to G-MDSC control (isolated from PBMC cultured in medium alone). SMM- and MM-MSCed-G-MDSC significantly up-regulated Arg1, NOS2, TNFα and CEBPA, a transcription factor promoting suppressive phenotype. Since also the angiogenic factor BV8 was significantly up-regulated, we next investigated the pro-angiogenic effect in vitro co-culturing MSCed-G-MDSC with Human Brain Microvascular Endothelial Cells (HBMEC) (1:2). After 5 h, we observed that MM-MSCed-G-MDSC were able to increase both tube length and number of branch points compared to G-MDSC control (p<0.05). Moreover, MM-MSCed-G-MDSC were able to digest bone matrix in vitro (p<0.01).

Adding Bortezomib (5 nM), Lenalidomide (10 μM) or Pomalidomide (1 nM) during co-culture with MM-MSC, isolated G-MDSC showed a significant reduction of pro-angiogenic and bone resorption activity (p<0.05) but did not lose immunosuppressive ability.

Conclusion

MSC play a key role promoting tumor microenvironment transformation in SMM and MM patients.Indeed, only SMM- and MM-MSC and not MGUS-MSC are able to activate myeloid cells in G-MDSC with immunosuppressive, pro-angiogenic, and bone resorptionactivity.

Disclosures

No relevant conflicts of interest to declare.
Global Genomic Analysis of Newly Diagnosed t(4 ;14) Multiple Myeloma Reveals a Specific Mutational Spectrum and Identifies PKD2 As a Potential Therapeutic Target
Authors: Song Xiu Ly , Szalat Raphaël , Talbot Alexis , Nguyen HaiVu , Samur Mehmet K. , Mosen-Ansorena David , Munshi Nikhil C. , Minvielle Stephane , Avet Loiseau Herve , Cuccuini Wendy , Fermand Jean-Paul , Arnulf Bertrand , Bories Jean-Christophe
Published: October 2019

In Multiple Myeloma (MM), the t(4;14) translocation is associated with a poor outcome. However, beside this translocation, the genetic events which determine the adverse evolution of the disease and the resistance to treatments remain elusive.

In this study we performed whole exome or RNA sequencing analysis of samples from 65 newly diagnosed t(4;14) MM. We found that NRAS, KRAS, MAPK and FGFR3 are frequently mutated (12%, 9%, 13.8%, and 20% respectively). Overall, the FGFR3/RAS/BRAF/MAPK genes were mutated in 36 cases (54%). There was a negative correlation between mutations in FGFR3 and those occurring in NRAS, KRAS and BRAF as expected from the mutually exclusive occurrence of mutations in these genes. In addition to alterations in TP53 and DIS3, we found marked elevated frequency of mutations in PRKD2 (10.7%), ATM/ATR (10.7%) and MYCBP2 (7.6%), reduced frequency in FAM46C (1.5%) and no mutation in TRAF3 and CCND1. Mutations in ATM/ATR were strongly associated with the MB4-2 breakpoint (Bp) (p = 1.62 10-4) and significantly correlated with mutations affecting genes coding for members of the MAPK family. We observed a positive correlation between non-silent mutations in PRKD2 and the MB4-1 or MB4-3 Bp (p = 1.3 10-2). Of note, PRKD2 mutations are exclusively found in 3 t(4;14) MM cell lines and among the 84 MM sequenced by Bolli et al. (1), none of the non t(4;14) patient were mutated in PRKD2, indicating that this genetic lesion is associated with t(4;14) MM.

In the NCI-H929 t(4;14) MM cell line, which is mutated for PRKD2, encoding the PKD2 serine/threonine kinase, we observed elevated levels of phosphorylated PKD2. Furthermore, inhibition of PKD, decreased PKD2 phosphorylation and triggered reduced proliferation and apoptosis of MM cell lines and fresh plasma cells from patients in vitro. These results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients.

Altogether, these results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients.

Reference

1. Bolli, N., Avet-Loiseau, H., Wedge, D.C., Van Loo, P., Alexandrov, L.B., Martincorena, I., Dawson, K.J., Iorio, F., Nik-Zainal, S., Bignell, G.R., et al. (2014). Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat Commun 5, 2997.

Disclosures

Munshi: Janssen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties.
Prospective, Multicenter Clinical Trial of Everolimus As Primary Therapy in Waldenstrom Macroglobulinemia (WMCTG 09-214)
Authors: Treon Steven P , Meid Kirsten , Tripsas Christina K. , Heffner Leonard T. , Eradat Herbert , Badros Ashraf Z , Xu Lian , Hunter Zachary , Yang Guang , Patterson Christopher J , Gustine Joshua , Castillo Jorge J. , Matous Jeffrey V. , Ghobrial Irene M.
Published: October 2019

Purpose: Everolimus inhibits mTOR, a component of PI3K/AKT pro-survival signaling triggered by MYD88 and CXCR4 activating mutations in Waldenstrom's Macroglobulinemia (WM).

Experimental design: We evaluated everolimus in a prospective, multicenter study of 33 symptomatic, previously untreated WM patients. Intended therapy consisted of everolimus (10 mg/day) until progression or unacceptable toxicity. Dose de-escalation was permitted. The study was registered at www.clinicaltrials.gov(NCT01470196).

Results: At best response, median serum IgM levels declined from 4,440 to 1,360 mg/dL (p<0.0001); median hemoglobin rose from 10.8 to 12 g/dL (p=0.001); and median bone marrow disease burden declined from 75% to 52.5% in serially biopsied patients. The ORR and major response rates were 72.7% and 60.6%, respectively. Among genotyped patients, non-responders associated with wild-type MYD88 and mutated CXCR4 status. Median time to response was 4 weeks. Discordance between serum IgM levels and BM disease burden was common. With a median follow-up of 13.1 (range 1.6-64.6 months), median time to progression was 21 months, and 33 months for major responders. Discontinuation of everolimus led to rapid serum IgM rebound in 7 patients, and symptomatic hyperviscosity in two patients. Toxicity led to treatment discontinuation in 27% of patients, including 18% for pneumonitis.

Conclusions: Everolimus is active as primary therapy in WM. IgM discordance is common, and treatment cessation can often lead to rapid serum IgM rebound. Pneumonitis also appears more pronounced in untreated versus previously treated WM patients. The risks and benefits of everolimus should carefully be weighed against other primary WM therapy options.

Disclosures

Treon: Janssen: Consultancy; Pharmacyclics: Consultancy, Research Funding. Heffner:Celgene: Research Funding; Millennium: Research Funding; AbbVie: Research Funding; Pharmacyclics: Research Funding. Eradat:AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria. Castillo:Millennium: Research Funding; Janssen: Honoraria; Otsuka: Consultancy; Pharmacyclics: Honoraria; Biogen: Consultancy; Abbvie: Research Funding. Ghobrial:Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Noxxon: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; BMS: Honoraria.
Two Doses of Antithymocyteglobulin As Graft-Versus-Host Disease Prophylaxis in Haploidentical Allogeneic Stem Cell Transplantation: A Multicenter Prospective Study
Authors: Lin Ren , Wang Yu , Huang Fen , Fan Zhiping , Zhang Shen , Yang Ting , Xu Yajing , Liang Xinquan , Ji Yu , Xu Na , Xuan Li , Ye Jieyu , Sun Jing , Huang Xiaojun , Liu Qifa
Published: October 2019

Background

Graft-versus-host disease (GVHD) is an important complication after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Antithymocyteglobulin (ATG) has been widely used for GVHD prophylaxis. However, the delay of T-cell reconstitution causes by ATG may increase the risk of opportunistic infections. In this multicenter prospective study, we compared the outcomes of the patients undergoing haplo-HSCT who received 7.5 mg/kg ATG or 10 mg/kg ATG as GVHD prophylaxis.

Methods

A total of 368 consecutive patients undergoing haplo-HSCT were randomized in 5 hospitals between May 2013 and March 2016. ATG with a total dosage of 7.5 mg/kg were administered in 184 patients and 10 mg/kg ATG were administered in 181 for GVHD prophylaxis. Three patients did not received allocated intervention and transplantation due to leukemia relapse before transplant or toxicity of conditioning regimens. The primary endpoint was the incidence of acute GVHD grade II to IV on day 100. Noninferiority of 7.5mg/kg ATG against 10mg/kg ATG was established if the difference of the 95% confidence interval (CI) in acute GVHD grade II to IV between the two groups was within 15%.

Results

The incidences of acute GVHD grade II to IV on day 100 were 31.5% and 28.2% in 7.5 mg/kg and 10 mg/kg ATG (P=0.485), respectively. The difference between these two groups was 3.3% with 95% CI of -6.1% to 12.7%, which indicated that 7.5 mg/kg ATG was not inferior to 10 mg/kg ATG. The incidences of acute GVHD grade III to IV were similar in the two groups (8.2% in 7.5 mg/kg group vs. 5.0% in 10 mg/kg group, P=0.220). No difference in chronic GVHD was found in the two groups (21.2% in 7.5 mg/kg arm vs. 19.8% in 10 mg/kg arm, P=0.748). More patients developed EBV viremia in 10 mg/kg group (37.5±3.8%) than those in 7.5 mg/kg group (25.2±3.3%, P=0.018). The 2-years overall survival were 69.5±4.3% and 66.7±4.0% for 7.5 mg/kg and 10 mg/kg group (P=0.356). And the 2-year disease-free survival were also comparative in the two groups (66.3±5.7% in 7.5 mg/kg arm vs. 66.6±3.8% in 10 mg/kg arm, P=0.288).

Conclusion

Compared with 10 mg/kg ATG, the 7.5mg/kg ATG applied in haplo-HSCT appears not to increase GVHD and might reduce the risk of EBV infection after transplantation.

Disclosures

Lin: National Natural Science Foundation of China 81400141: Research Funding; National Natural Science Foundation of China 81270647: Research Funding; Science and technology planning project of Guangdong Province 2014B020226004: Research Funding; The project of health collaborative innovation of Guangzhou City 201400000003-4: Research Funding.
Bortezomib Based Induction Is Superior to Thalidomide Based Induction in Reducing Early Relapses Following Upfront HDM: an Analysis By the Asian Myeloma Working Group
Authors: Gopalakrishnan Sathish Kumar , Ong Shin-Yeu , Kim Seok Jin , Kim Hae Su , Lee Ji Yun , Lim Sung Hee , Chen Yunxin , De Mel Sanjay , Ooi Melissa G. , Surendran Shilpa , Lin Adeline , Tan Daryl , Durie Brian G M , Chng Wee Joo , Kim Kihyun
Published: October 2019

Background: Multiple myeloma is a heterogeneous disease with certain genetic features associated with worse outcomes. The benefit of Bortezomib for high risk genetic patient subgroups remains unclear in an Asian population. We performed a retrospective analysis to compare bortezomib-based induction versus thalidomide-based induction in Asian patients who were treated with upfront autologous transplant.

Methods: We retrospectively analyzed outcomes of 240 patients who received autologous transplant in two centers in Singapore (n=167) and one center in Korea (n=73) after bortezomib-based (n=120) versus thalidomide-based induction (n=120) between 2006 and 2014. Patients were staged according to International Staging System (ISS), and responses were defined according to International Myeloma Working Group criteria. Early relapse was defined as relapse within 12 months post-transplant. High risk cytogenetics included the presence of 17p13 deletion, t(14;16), or t(4;14) by FISH.

Results: Baseline characteristics (age, gender, ISS, cytogenetics) were not significantly different between groups receiving bortezomib versus thalidomide induction. Bortezomib induction was associated with improved rates of complete response (CR) post-induction (40% vs 25%, p=0.013) and post-transplant (52% vs 48%, p=0.038). Median overall survival (OS) was prolonged with bortezomib (53 months vs 27 months, p=0.027). Multivariate analysis adjusted for baseline characteristics showed bortezomib reduced the risk of early relapse (HR 0.23, 95% CI 0.09-0.57, p=0.001), and improved overall survival (OS) (HR 0.2, 95% CI 041-0.93, p=0.021). Factors independently associated with poorer OS were high risk cytogenetics (HR 1.77, p=0.011), failure to achieve very good partial response (VGPR) or better post-induction (HR 1.70, p=0.005), early relapse (HR 6.20, p<0.001), but not advanced ISS stage or hypodiploidy karyotype. In subgroup analysis, patients with high-risk cytogenetics (n=46) who received bortezomib had improved OS (HR 0.39, CI 0.17 to 0.93, p=0.033), but patients with standard risk cytogenetics did not have significant OS benefit with bortezomib (HR 0.71, p=0.139).

Conclusion: Our results suggest that despite upfront autologous stem cell transplantation, patients benefit from bortezomib versus thalidomide induction. Furthermore, bortezomib may be beneficial in overcoming the adverse prognostic effect of high-risk cytogenetics.

Disclosures

Durie: Janssen: Consultancy; Takeda: Consultancy; Amgen: Consultancy.
A District General Hospital Experience of Patients Receiving Apixaban, Dabigatran, Rivaroxaban or Warfarin for Anticoagulation
Authors: Raiman Lewis , Antbring Richard H , Abu-Sitta Abed , Al-Refaie Faris N
Published: October 2019

Introduction

Novel oral anticoagulants (NOACs) are becoming increasingly more prevalent as a preventative measure for venous thromboembolism and stroke prevention in atrial fibrillation (AF)1. NOACs are considered non inferior to Vitamin K antagonists (VKAs) in terms of efficacy and clinical trial evidence has suggested lower rates of complications with the use of NOACs2. There is limited published literature on the complication rates of NOAC use in the district general hospital setting. In this retrospective study, the bleeding rates amongst patients on oral anticoagulants in a district general hospital in the United Kingdom were established.

Methods

All patients prescribed a NOAC (Rivaroxaban, dabigatran or apixaban) or a VKA at the Princess Alexandra Hospital, a district general hospital in Harlow, United Kingdom, between 01/01/2015 and 31/12/2015 were identified from hospital pharmacy records. Electronic patient hospital records of these individuals were reviewed to identify indication for anticoagulation and any subsequent bleeding complications after initiating therapy as classified according to ICD-10 up until 31st of June 2016.

Results

A total of 521 patients were prescribed a NOAC. 283 patients were prescribed Rivaroxaban, 217 patients Apixaban and 21 patients were prescribed Dabigatran. 969 patients were prescribed a VKA. From 1st of January 2015 to June 31st 2016, 5.9% of NOAC patients suffered a bleed requiring a hospital visit during this time, versus 7.1% patients on a VKA. Amongst patients prescribed a NOAC, common causes of bleeding included GI haemorrhage (34.5%), epistaxis (38.0%), haemoptysis (13.8%). Amongst patients prescribed a VKA, common causes of bleeding included gastrointestinal bleeding (57.0%), epistaxis (25.2%), haemoptysis (11.9%). Rates of cerebral haemorrhage were low in both groups (0.77% of patients prescribed a NOAC and 0.61% of patients prescribed a VKA).

Conclusion

This data illustrates real-world experience of bleeding rates requiring hospital admission of patients on NOACs and VKA. The data suggests a lower bleeding complication rate amongst a district general hospital patient population taking NOAC compared to VKA, in keeping with previously published data.

References

1. Beyer-Westendorf J, Förster K, Pannach S, Ebertz F, Gelbricht V, Thieme C, Michalski F, Köhler C, Werth S, Sahin K, Tittl L. Rates, management, and outcome of rivaroxaban bleeding in daily care: results from the Dresden NOAC registry. Blood. 2014 Aug 7;124(6):955-62.

2. Chai-Adisaksopha C, Crowther M, Isayama T, Lim W. The impact of bleeding complications in patients receiving target-specific oral anticoagulants: a systematic review and meta-analysis. Blood. 2014 Oct 9;124(15):2450-8.

Disclosures

No relevant conflicts of interest to declare.
Risk of Venous Thromboembolism in Hospitalized Patients with Sickle Cell Disease
Authors: Taylor Joshua , Anghel Alexandra , Corsi Daniel J , Carrier Marc , Tinmouth Alan , Saidenberg Elianna
Published: October 2019

Background: Patients with sickle cell disease (SCD) are at an increased risk of developing venous thromboembolism (VTE). However, the underlying risk of VTE complication during hospitalization is unclear in this patient population. We sought to report the incidence of VTE and its associated risk factors in hospitalized SCD patients.

Patients/Methods: A retrospective cohort study of SCD patients requiring hospitalization was undertaken at a tertiary care center. Incidence ratios of VTE per hospitalization for different risk factors (Thromboprophylaxis use, central venous catheter (CVC), past history of VTE, surgery during hospitalization) were assessed. Univariate, age adjusted and multivariate Poisson models were estimated accounting for the repeated hospitalizations per patients.

Results: A total of 101 patients with at least one hospitalization were included in the study. The mean of number of admissions per patients was 8.9. Overall, 17 out of 896 (1.9%) admissions were complicated by VTE. The incidence of VTE varied by risk factors, from 0.8% in patient without CVC to 6.7% among patients admitted with previous history of VTE. Age adjusted and multivariate Poisson models for incidence rate ratios of VTE per hospitalization among patients with SCD for different risk factors are depicted in Table 1.

Conclusion: The risk of VTE seems low in hospitalized SCD. A prior history of VTE and a hospitalization for surgery might be associated with higher risk of VTE complication. Future studies assessing these risk factors to tailor thromboprophylaxis regimens are needed.

Disclosures

No relevant conflicts of interest to declare.
Are Bleeding Scores Predicting Severity and Outcome in Hemophilia and Rare Bleeding Disorders?
Authors: Shahriari Mahdi , Karimi Mehran
Published: October 2019

Background: Hemophilia is an inherited severe bleeding disorder, Factors VIII and IX deficiencies have X linked inheritance. Most rare bleeding disorders (RBD) have autosomal recessive inheritance. The ISTH-BAT is a valuable research tool that is applicable to clinical practice and scores patients' bleeding symptoms from 0 to 4. ISTH-BAT has the potential to avoid unwanted laboratory testing, predict the risk of bleeding, describe symptom severity and inform treatment.

Aims: To assess the utility of ISTH-BAT in our bleeding disorder population, compare the bleeding score (BS) in adult and pediatric groups and investigate its association with plasma factor levels.Von Wilebrand disease and thrombasthenia was excluded from this study. One hundred cases of epistaxis whose coagulation workups were within normal limits were enrolled in the control group.

Methods: This is an observational analytical study. The ISTH-BAT was used to calculate bleeding scores (BS) in a group of hemophilic patients and healthy controls. Ethics approval and informed consents were secured prior to the study.

Results: A total of 100 patients, (38 hemophilia A; FVIII deficiency,22hemophilia B; FIX deficiency, 10 hemophilia C; factor XI deficiency, 10 factor VII deficiency, 8 factor X deficiency, 6 factor V deficiency, 5 Afibrinogenemia, Factor I deficiency) and 100 controls were analyzed using the ISTH BAT. Mean BAT score in hemophilia A, B and C were 12.8±8.2; 11.6±8.6; and 7.5 ±5.52respectively.While in RBD were and 7.97±5.56. BAT score in 30 controls was 5.35±4.48. Bleeding Scores were significantly higher in hemophilia A and B patients as compared to controls (P < 0.05) but it was not significant when BAT Scores of hemophilia C patients compared with controls (P=0.35). ANOVA test revealed BAT Score were significantly different among the mild, moderate and severe hemophilia A and B in both adult and pediatric patients but there was no difference in Hemophilia C patients. BAT scores were linked to hematomas; minor wound bleeding in hemophilia A, B patients in the pediatric group while more linked with epistaxis bleeding after dental extraction and surgical interventions in the adult group. Mean BAT Scores of rare bleeding disorders was significantly higher than control group (10.97± 5.65; compared with 5.35±4.48; P<0.05). BAT Score of 10 cases of factor VII deficiency and 5 cases of Afibrinogenemia were higher than other case of RBD, and control group (14.35 ± 7.87; and 13.54±5.56 compared with 5.35±4.48; P< 0.001 and P<0.05respectively).

Conclusions: Our data revealed that the ISTH-BAT can help diagnose the bleeding condition in hemophilia and RBD patients and can be considered a predictor for the bleeding risk or severity. This will decrease need of subspecialty coagulation tests; it ultimately improvesdecision making and the clinical management of patients.

Disclosures

No relevant conflicts of interest to declare.