bluuebunny/crossref_metadata_2025 huggingface dataset:
A Rhesus Macaque Model to Optimize Adoptive NK Cell Therapy
Authors: Lopez Rebecca , Lundqvist Andreas , Sellers Stephanie , Berg Maria , Ramanathan Muthalagu , Vasu Sumithira , Smith Aleah , Dunbar Cynthia E. , Childs Richard W.
Published: October 2019

NK cell based immunotherapy represents a promising treatment approach for patients with cancer. Although preliminary clinical trials in humans suggest NK cell infusions can mediate anti-tumor effects, animal models are needed to provide insight into methods to enhance both the function and in vivo longevity of adoptively infused NK cells. Research conducted in our laboratory has shown that ex vivo expanded human NK cells are highly activated, up-regulating NKG2D, Granzyme B, TRAIL and Fas-ligand expression making them much more cytotoxic to tumor cells compared to freshly isolated NK cells. However, important questions remain regarding whether in vitro expansion alters the capacity of these cells to replicate, and traffic to tissues in vivo following their adoptive infusion into recipients. Differences in the genotype and phenotype of mouse NK cells compared to human NK cells limit the value of murine animal models to address these questions. In contrast to mice, Rhesus macaques have orthologues to most of the human MHC class I and II genes and possess NK cells expressing KIRs that are phenotypically and functionally similar to human NK cells, thus providing an excellent model system for evaluating questions related to adoptive NK cell therapy. We developed an in vitro method to expand macaque NK cells to characterize their in vivo longevity and tissue trafficking following adoptive infusion. Macaque NK cells were enriched from peripheral blood mononuclear cells by depleting CD3+ cells using immunomagnetic beads and were then expanded in vitro with autologous plasma and a human EBV-LCL feeder cell line using culture conditions identical to those used to expand NK cells from humans. NK cell cultures expanded 50- to 100-fold over 7 to 20 days, were greater than 99% CD3 negative, and had a similar phenotype to human NK cells including a large proportion of CD16/CD56 double positive cells, and ubiquitous expression of NKG2D, KIR2D, LFA-1, granzyme B, and CXCR3. In contrast to mice but analogous to human NK cells, macaque expanded NK cells upregulated surface expression of TRAIL and were highly cytotoxic to K562 cells and other human tumor lines (Figure). CFSE labelling of expanded NK cells did not alter their phenotype or tumor cytotoxic function. Data characterizing the longevity, proliferative capacity, and tissue trafficking patterns in the blood, bone marrow and lymph node of in vitro expanded and adoptively infused CFSE labeled NK cells (up to 1 × 108 NK Cells/kg i.v.) in macaque recipients will be presented from this analysis.

Figure Figure
Oxidative Modification of Von Willebrand Factor Inhibits Its Cleavage by ADAMTS13
Authors: Chen Junmei , Fu Xiaoyun , Ling Minhua , McMullen Brad , Kulman John , Chung Dominic , Lopez Jose A
Published: October 2019

During inflammation, activated neutrophils go through the oxidative burst, releasing various oxidants, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). Activated neutrophils also release myeloperoxidase (MPO), which generates HOCl from hydrogen peroxide and chloride ions. HOCl preferentially oxidizes cysteine and methionine residues to cysteine sulfenic acid and methionine sulfoxide, respectively, at rates ~100 times faster than it oxidizes tyrosine, another commonly oxidized amino acid. HOCl can also oxidize tyrosine to chlorotyrosine. Of great interest in this regard is the fact that the ADAMTS13 cleavage site in VWF, the Tyr1605–Met1606 peptide bond, contains two residues that are potential targets for myeloperoxidase-mediated oxidation. Given previous studies from our laboratory that VWF cleavage by ADAMTS13 is inhibited by oxidants, we hypothesized that neutrophil oxidants might oxidize either or both of these two amino acid residues and thereby potentially inhibit ADAMTS13-mediated cleavage. We tested our hypothesis using a peptide substrate for ADAMTS13 based on the VWF A2 sequence Leu1591–Arg1668. We incubated the VWF A2 peptide either without HOCl or with 25 or 75 μM HOCl, followed by quenching the oxidant with free methionine. The peptides were then incubated with purified recombinant ADAMTS13 and the reaction sampled every 15 min for one hour. We analyzed the cleavage reaction in two ways:

by electrophoretic separation on a Tricine gel and densitometric quantification of the cleavage product, and by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) to determine the location and extent of oxidative modification and quantity of the cleavage product.

We found that, after exposure to 75 μM HOCl, the A2 peptide contained methionine sulfoxide at position 1606 in 99% of the molecules in the sample, whereas only 0.3% contained both chlorotyrosine at position 1605 and methionine sulfoxide at 1606. The rate of substrate cleavage by ADAMTS13 was markedly reduced with oxidation, as measured by both assays, with the rate for the peptide treated with 75 μM HOCl being only 20% of that of the non-oxidized peptide. Taken together, these findings suggest that oxidants released by activated neutrophils during inflammation have a prothrombotic effect, mediated at least in part by inhibition of VWF cleavage by ADAMTS13.
Target TMPRSS6 for the Treatment of Hereditary Hemochromatosis
Authors: Booten Sheri , Knox Daniel , Alvarado Luis , Guo Shuling , Monia Brett P.
Published: October 2019

Abstract 1047

Hereditary hemochromatosis (HH) is a genetic disorder in which hyperabsorption of dietary iron leads to accumulation of iron in multiple tissues including liver and heart. A common clinical manifestation in HH patients is cirrhosis and hepatocellular carcinoma as a result of iron-mediated injury in liver. The most prevalent genetic defect for HH is the failure to up-regulate hepcidin, a peptide hormone that inhibits the absorption of iron in duodenum and the release of iron from intracellular iron storage such as macrophages. Mutations in a number of genes have been identified as the cause for HH, including hepcidin itself. However, the most common mutation is C282Y mutation in HFE, which is a positive regulator for hepcidin expression. C282Y mutation represents about 85% of the HH population. HFE C282Y HH is an autosomal recessive disease with a ∼50% penetrance. Currently, the only treatment available for iron overload is phlebotomy which will continue throughout the patient's life.

Hepcidin is mainly expressed and secreted by the liver and its expression is regulated predominantly at the transcription level. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor for hepcidin expression. It's been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of TMPRSS6 in Hfe−/− mice could ameliorate the iron overload phenotype (Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg KE et al., Blood, 2011).

Using second generation antisense technology, we identified antisense oligonucleotides (ASOs) targeting mouse TMPRSS6 for the treatment of HH. These compounds were first identified through in vitro screens in mouse primary hepatocytes. After 4 weeks of treatment in C57BL/6 mice on normal chow, we observed an 80% to 90% reduction of liver TMPRSS6 mRNA with a subsequent 2–3 fold induction of liver hepcidin mRNA. Serum iron and transferrin saturation levels were reduced by ∼50%. These ASOs are currently being evaluated in a diet-induced iron overload model and an Hfe−/− iron overload model. Our preliminary results demonstrate that targeting TMPRSS6 is a viable approach for the treatment of hereditary hemochromatosis and possibly other iron-loading diseases associated with suppressed hepcidin levels.

Disclosures:

Booten: Isis Pharmaceuticals: Employment. Knox:Isis Pharmaceuticals: Summer Intern. Alvarado:Isis Pharmaceuticals: Employment. Guo:Isis Pharmaceuticals: Employment. Monia:Isis Pharmaceuticals: Employment.
BCL11A Polymorphism and HbF Response to Hydroxyurea in Adult Patients with Sickle Cell Disease
Authors: Sobash Philip , Kutlar Ferdane , Brown Harrison C. , Clair Betsy , Kutlar Abdullah
Published: October 2019

Abstract 1051

Several genetic polymorphisms are known to influence baseline hemoglobin F (HbF) levels in patients with sickle cell disease (SCD). These include XmnI polymorphisms in the Gγ globin promoter, polymorphisms of HBS1L-MYB and more recently BCL11A. Over the past several years, many studies have shown that polymorphisms in the BCL11A gene are strongly associated with baseline HbF levels in various populations with SCD. The strongest association has been found with a SNP (G→A) in the intron2 of the BCL11A gene (rs4671393). SCD patients on hydroxyurea (HU) therapy display a wide range of HbF responses. The factors underlying this heterogeneity are not clearly delineated. We undertook a study of the BCL11A rs4671393 polymorphism in a population of adult SCD patients who have been on HU therapy for an extended period. Fifty-nine African-American patients with SCD (25 male, mean age 33.9 and 34 female, mean age 34.5) were studied for BCL11A rs4671393 polymorphism and its correlation to HbF response (percent change in HbF from baseline to maximal response). The minor allele frequency (MAF) of A was 0.288. The distribution of HbF response (δHbF) was plotted against the genotype and is shown in the figure. As can be seen, patients with an A allele had a higher δHbF (12.1%) as opposed to those with the wild type (G/G), which was at 10.6%. Those homozygous for the minor allele (A/A) showed a 10.8% increase in HbF in response to HU therapy. These results are suggestive of a higher HbF response to HU when an A allele is present at this position. The number of homozygous for the minor allele are too small to reach a definite conclusion.

Disclosures:

No relevant conflicts of interest to declare.
Glia Maturation Factor-Gamma Negatively Modulates Fatty Acid-Induced Inflammation in Macrophages Via PI3K/AKT Pathway
Authors: Aerbajinai Wulin , Chin Kyung , Zhu Jianqiong , Rodgers Griffin P
Published: October 2019

Abstract 1096

The macrophage-mediated inflammatory response plays a critical role in the development of obesity-related tissue inflammation and insulin resistance. Free-fatty acids (FFAs) are well-characterized factors causing production of inflammatory factors and insulin resistance. Glia maturation factor-gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in the macrophages immune response remains unclear. In this study, we investigated whether GMFG may affect FFAs-induced inflammatory reaction in macrophages. We show here that silencing of GMFG (80% inhibition of the endogenous expression levels) by transfected with GMFG siRNA significantly enhanced FFAs-induced production of proinflammatory cytokines and chemokines, including TNF-α, IL-6 and IL-8 compared to transfected non-targeting silencing siRNA in human peripheral blood monocytes-derived macrophage (1.2 × 104 ± 224.2 vs. 4.6 × 103 ± 98.4 molecules/ng, P < 0.001; 1.2 × 103 ± 56.1 vs. 2.3 × 102 ± 8.4 molecules/ng, P =0.001; 1.3 × 107 ± 5.8 × 105 vs. 4.9 × 106 ± 2.8 × 105 molecules/ng, P < 0.001) as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay (TNF-α: 9.4 × 102 ± 38.6 vs. 5.6 × 102 ± 31.1 ng/mL, P < 0.01; IL-6: 4.5 × 102 ± 31.3 vs. 2.2 × 102 ± 22.7 ng/mL, P < 0.01; IL-8: 2.2 × 103 ± 205.5 vs. 1.3 × 103 ± 113.9 ng/mL, P < 0.01). These increased inflammatory cytokines resulted from an increased activation of NF-κB and the ERK1/2 MAPK signaling pathway (based on increased NF-κB p65 phosphorylation, IκBα loss and enhanced phosphorylation of ERK1/2 in Western blot analysis) and a reduced phosphorylation of the PI3K/Akt/GSK3-β pathway following FFAs stimulation. Overexpression of GFP-GMFG in GMFG-silenced cells abrogated enhanced phosphorylation of NF-κB p65, ERK1/2 MAPK and reduced phosphorylation of Akt and GSK3-β. Furthermore, in cells in which GMFG was knockdown, FFAs induced an increase in the expression of SHIP1, a phosphatase that negatively regulates the PI3K signaling pathway, suggesting increased SHIP1 expression may be responsible for the augmentation of inflammatory cytokines following FFAs stimulation. We also show that silencing of GMFG enhances the oxidized low density lipoprotein (oxLDL) induced expression of TNF-α and IL-6 (2.9 × 104 ± 204.8 vs. 1.3 × 104 ± 153.9 molecules/ng, P < 0.01; 2.5 × 103 ± 31.3 vs. 9.2 × 102 ± 22.7 ng/mL, P < 0.01) in MDM. Taking together with the data that GMFG is constitutively expressed in macrophages and its expression is down-regulated by FFAs stimulation, GMFG might function as a novel negative regulator through participating in the PI3K/Akt signaling pathway, suggesting that macrophage-specific modulation of GMFG may be beneficial in the treatment of FFAs induced inflammation.

Disclosures:

No relevant conflicts of interest to declare.
LDL and HDL Counteract Hematopoietic Stem/Progenitor Cells in Regulation of Inflammation and Atherosclerosis
Authors: Feng Yingmei
Published: October 2019

Abstract 1288

Low-density lipoprotein (LDL) is an independent risk factor in cardiovascular disease. Hypercholesterolemia-associated monocytosis and transformation of monocytes into macrophages are the main features underlying the proatherogenic property of LDL. Different from LDL, high-density lipoprotein (HDL) and its major apolipoprotein, apoA-I, counteract atherosclerosis by reversing cholesterol transport, inhibiting inflammatory cell adhesion to plaques, maintaining endothelial integrity, inhibiting oxidation, and as has recently been shown, suppressing hematopoietic stem cell (HSC) proliferation. As inflammatory cells in atherosclerotic plaques are exclusively derived from hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM), we hypothesized that differential effects of LDL and HDL on HSPC proliferation and differentiation may play a role in atherosclerotic plaque development. We explored the effect of HDL and LDL on HSPC number and function using LDL receptor knockout (LDLr ko) mice, fed with high fat diet, and C57BL/6 mice infused with purified apoA-I and or reconstituted (r)HDL. Compared to mice on normal diet, the number of lineage−/cKit+Sca-1+ (KLS) cells was 5-fold higher in LDLr ko mice on high-fat diet. By contrast, infusion of either 8 mg/kg apoA-I or 8, 12 and 16 mg/kg rHDL decreased the KLS cell frequency in BM. Using BrdU incorporation, we demonstrated that LDL induced but HDL inhibited KLS cell proliferation. When lineage negative cells, exposed to LDL or rHDL were used in competitive repopulation studies, HDL-treated cells supported greater chimerism than untreated controls, whereas LDL-treated cells competed less well. In addition, for HDL-treated Lin− cells, skewing of reconstitution to the B-lymphoid lineage at the expense of the granulocyte/monocyte lineage was seen. In vitro, LDL promoted KLS cell differentiation towards the monocytic lineage, which was abrogated by addition of HDL. In conclusion, LDL and HDL have opposing effects on HSPC number and function. These differential effects may contribute to the opposing effects of HDL and LDL on atherosclerosis development.

Disclosures:

No relevant conflicts of interest to declare.
Pivotal Role for Gsk3 in Hematopoietic Stem Cell Homeostasis
Authors: Huang Jian , Klein Peter S.
Published: October 2019

Abstract 1292

Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our recent study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a μ-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by μ-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. These findings identify unexpected functions for GSK3 in HSC homeostasis, suggest a therapeutic approach to expand HSCs in vivo using currently available medications that target GSK3 and mTOR, and provide a compelling explanation for the clinically prevalent hematopoietic effects of lithium, a widely prescribed GSK3 inhibitor. In the following study, we found that the combination of Gsk3 inhibitor and mTOR inhibitor can expand phenotypic HSCs in vivo and maintain functional HSC in ex vivo culture. This study will provide the basis for a new clinical approach to improve the efficiency of bone marrow transplantation.

Disclosures:

Klein: Follica: Consultancy.
The Cell Cycle Regulator CDC25A Is a Target for JAK2V617F Oncogene
Authors: Gautier Emilie-Fleur , Recher Christian , Laurent Camille , Marty Caroline , Villeval Jean-Luc , Demur Cécile , Delhommeau Francois , Hexner Elizabeth , Giraudier Stephane , Bonnevialle Nicolas , Ducommun Bernard , Laurent Guy , Manenti Stéphane , Mansat- De Mas Véronique
Published: October 2019

Abstract 1385

The JAK2V617F mutation is present in the majority of polycythemia vera (PV) patients and half of those with essential thrombocythemia and primary myelofibrosis. JAK2V617F is a gain-of-function mutation resulting in constitutive JAK2 signalling involved in the pathogenesis of these diseases. JAK2V617F has been shown to promote S phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell cycle transition, is constitutively over-expressed in JAK2V617F -positive cell lines, JAK2-mutated patient CD36+ progenitors and in vitro differentiated proerythroblasts. Accordingly, CDC25A is also overexpressed in bone marrow and spleen of Jak2V617F knock-in mice compared to wild-type (WT) littermates. Using murine FDC-P1-EPOR and human HEL and SET-2 cell lines, we found that JAK2V617F-induced CDC25A up-regulation was not caused by increased transcription or CDC25A stability or the involvement of its known upstream regulators AKT and MAPK. Instead, our results suggest that CDC25A is originaly regulated at the translational level through the implication of the transcription factor STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2V617F-expressing erythroid progenitors, while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2V617F patients, making of this protein an attracting potential therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.
MicroRNA-146a Deficiency Leads to Increased Myeloid Cell Proliferation and Activation
Authors: Zhao Jimmy L. , Rao Dinesh S. , Boldin Mark , Taganov Konstantin , Baltimore David
Published: October 2019

Abstract 2815

NF-κB is a key regulator of inflammation and the immune activation against pathogens. On the other hand, constitutive and dysregulated NF-κB activation has been shown to lead to hematologic malignancies. Thus, NF-κB activity has to be properly regulated to achieve this delicate balance. Recent studies on microRNA-146a (miR-146a) suggest it to be an important negative regulator of NF-κB activation by targeting TRAF6 and IRAK1. To characterize the function of miR-146a in the context of NF-κB activation in the hematopoietic system, we have created a knockout (KO) mouse with germline deletion of miR-146a gene. We have shown that over time miR-146a KO mice develop a myeloproliferative condition in spleen and bone marrow that progresses to splenic myeloid sarcoma and bone marrow fibrosis. The myeloproliferative disease is driven by dysregulated NF-κB activation as the KO spleen and bone marrow cells show increased NF-κB activity and reduction in NF-κB level effectively rescues the myeloproliferative disease. In addition, the KO mice exhibit a chronic inflammatory state with increased cytokine production and macrophages from the KO are hyper-activated upon primary LPS stimulation and secondary re-stimulation. In this study, we have provided genetic evidence that miR-146a functions as a tumor suppressor in the hematopoietic system and highlighted the important role of miR-146a as a negative regulator of NF-κB activation and inflammation.

Disclosures:

Taganov: Regulus Therapeutics Inc: Employment.
A Novel Mutation in MPL (Y252H) Results in Increased Thrombopoietin Sensitivity and Moderate Thrombocytosis in a Pediatric Patient with Essential Thrombocytosis
Authors: Lambert Michele , Jiang Jing , Tong Wei
Published: October 2019

Abstract 2817

Myeproliferative neoplams (MPNs) constitute a group of hematopoietic malignancies that feature enhanced proliferation and/or survival of one or more myeloid lineage cells, including Essential Thrombocythemia (ET). MPN development is rare in children with an estimated annual incidence of ET of 0.09/106 children. The WHO defines ET as persistent platelet count >600 k/mcL in the absence of known cause of reactive/secondary thrombocytosis. The JAK2 V617F mutation is most commonly reported in both children and adults with ET although the reported frequency varies in pediatric populations from 0 to 36% of patients. Mutations in the thrombopoietin receptor (TPO receptor or MPL) intracellular domain, specifically W515K/L, have also been reported in both adult and pediatric ET patients. Here we report a novel mutation in the MPL extracellular domain, Y252H, causing mild thrombocytosis. The patient presented at 2 years of age with a platelet count of 765 k/mcL. During the 3-year follow-up period, she possessed platelet counts between 600–700k/mcL, without any obvious indication of reactive/secondary thrombocytosis. Because of the persistently increased platelet count, her bone marrow was evaluated and it demonstrated increased numbers of megakaryocytes with focal clustering. JAK2 mutation analysis was negative and cytogenetics did not show any clonal abnormalities. Sequencing of the MPL gene showed a missense variant at c.754 T>C resulting in a tyr252his amino acid substitution. To investigate if this Y252H mutation in MPL dysregulates TPO/MPL- mediated cell growth, we introduced it into cytokine-dependent BaF3 cells. Cells stably expressing the mutant MPL allele showed increased proliferation to TPO, in particular at low concentration, in comparison to cells expressing wildtype (WT) MPL. Upon cytokine withdrawal, BaF3 cells expressing the MPL Y252H mutant survived better than that of WT MPL. Primary bone marrow cells from this patient along with the healthy control were subjected to colony forming unit -megakaryocyte (CFU-meg) assays in response to a serial dose of TPO. The Y252H MPL bone marrow showed significantly increased megakaryocyte colonies at low dose of TPO when compared to control bone marrow (17.5 ± 2.5 colonies versus 4.75 ± 1.1 colonies at 15 ng/mL TPO, p<0.001). These results are consistent with the clinically mild thrombocytosis. In summary, our results suggest a novel MPL mutation, Y252H, results in pediatric ET. Further evaluation of the mechanisms of increased TPO sensitivity imparted by this mutation should contribute to our understanding of the molecular pathogenesis of ET.

Disclosures:

Lambert: Cangene: Honoraria.
Immune Thrombocytopenia in Patients with Chronic Lymphocytic Leukemia Is Associated with Stereotyped B-Cell Receptors
Authors: Maura Francesco , Visco Carlo , Novella Elisabetta , Giaretta Ilaria , Tuana Giacomo , Agnelli Luca , Lionetti Marta , Fabris Sonia , Guidotti Francesca , Reda Gianluigi , Gritti Giuseppe , Barcellini Wilma , Baldini Luca , Neri Antonino , Rodeghiero Francesco , Cortelezzi Agostino
Published: October 2019

Abstract 2847

Chronic lymphocytic leukemia (CLL) is frequently complicated by autoimmune disorders, primarily autoimmune hemolytic anemia and immune thrombocytopenia (ITP). In order to investigate biological features related to ITP development, we retrospectively analyzed 463 CLL patients characterized for immunoglobulin heavy-chain variable (IGHV) gene mutational status, cytogenetic features and B-cell receptor (BCR) configuration (HCDR3) at the time of diagnosis. Thirty-six patients (7.7%) had developed ITP, according to our previously reported criteria (Visco et al, Blood 2008): i) an otherwise unexplained rapid (< 2 weeks) and severe fall (at least half of the initial level and below 100*109/L) of the platelet count; ii) normal or augmented number of megakaryocytes in the bone marrow; iii) no or limited (not palpable) splenomegaly and iv) no cytotoxic treatment in the last month. Stereotyped BCR configuration was defined by means of pair-wise alignment with known stereotyped sequences available from different public databases (Stamatopoulos et al, Blood 2007; Bomben et al, Br J Hematol 2009; Murray et al, Blood 2008), specifically excluding pairs of sequences whose length differed more than 3 amino acids and sharing more than 60% identity on alignments showing less than 3 gaps, as described by others and by our group (Stamatopoulos et al, Blood 2007; Maura et al, Plos One, in press). Our results indicated that the occurrence of ITP was strongly associated with unmutated (UM) IGHV (p<0.0001), unfavorable cytogenetic lesions (P =0.005), and interestingly, the occurrence of stereotyped HCDR3 (P =0.006). Additionally, the UM IGHV mutational status, unfavorable cytogenetic lesions, or stereotyped BCR were significantly associated with shorter time to ITP development (P <0.0001, P =0.02 and P =0.005 respectively) compared to other patients. As regards to the most represented HCDR3 subsets, we observed that subsets #1 (IGHV1-5-7/IGHD6-19/IGHJ4; 16/16 UM) and #7 (IGHV1-69 or IGHV3-30/IGHD3-3/IGHJ6; 13/13 UM) were significantly associated with ITP development (P =0.003 and P =0.001, respectively). Moreover, restricting the analysis to UM patients, subset #7 retained a significant association with the occurrence of ITP (P =0.02). Time to ITP development was also significantly shorter in subsets #1 and #7 patients than in all others patients (P =0.0076 and P <0.0001, respectively). Overall, our data suggest that patients with CLL and peculiar BCR conformations are at higher risk of developing secondary ITP, and that stereotyped BCR may be involved in the pathogenesis of this complication.

Disclosures:

No relevant conflicts of interest to declare.
Truncation of NF-KB2 Is Associated with Poor Response to Bortezomib Treatment in Multiple Myeloma
Authors: Ramachandiran Sampath , Guo Xiangxue , Garderet Laurent , Ikhlef Souhila , El-cheikh Jean , Weil Michael R , Young Andrew N , Bagirov Mahir , Lonial Sagar , Chen Zhengjia , Kowalsky Jeanne , Du Yuhong , Khoury Hanna J. , Sunay Susan , Kaufman Jonathan L. , Boise Lawrence H. , Rossi Michael , Bernal-Mizrachi Leon
Published: October 2019

Abstract 2891

The proteasome inhibitor, bortezomib, is a powerful agent for the treatment of Multiple Myeloma (MM). However, many patients fail to achieve an optimal response. To identify biomarkers that could predict an optimal response to bortezomib, we assessed the role of the noncanonical NF-kB pathway in the response to bortezomib. Our in vitro studies identified that NF-kB2/p100 protein levels correlated directly with bortezomib sensitivity. Based on this finding, we measured at diagnosis the mRNA levels of different NF-kB2 regions in 64 patients schedule to receive bortezomib-based regimens. While no difference in expression of the 5'end regions was found between the CD138 (+) and CD138 (-) cells in these patients, half of them demonstrated low or absent 3'end expression levels in the CD138 (+) cells. Among forty-seven patients treated with four cycles of Bortezomib and dexamethasone, we found that low plasma cell NF-kB2 3'end levels were associated with a lower overall response rate (low: 47.8% vs. high: 96%, see table). In contrast, low plasma cell NF-kB2 3'end levels failed predict bortezomib response when lenalidomide or thalidome was added to the therapeutic regimen. To further understand the cause for NF-kB2 truncation, we are currently analyzing RNA sequencing in 3 non-responder patients and whole genome sequencing of 38 MM patients to determine the potential causative mechanisms. Preliminary results suggest that NF-kB2 truncation result from multiple genetic alterations, including: premature stop codons produced by ALU insertions, inversions and splicing variants. Overall, this study substantiates the basis of a more precise therapeutic algorithm according to NF-kB2 3'end plasma cell levels, which include the use of bortezomib and dexamethasone in patients with high NF-kB2 3'end plasma cell levels and immunomodulators or DNA damaging agents in patients with low NF-kB2 3'end plasma cell levels.BD+BTD or BRD++NF-kB2 3' end mRNA (CD 138(+)/CD138(−)LowHighLowHigh> Very Good Partial Response5 (21.7)9 (37.5)1 (11)4 (50)Partial Response6 (26.1)14 (58.3)7 (78)4 (50)Stable Disease4 (17.4)1 (4.2)0 (0)0 (0)Progressive disease8 (34.8)0 (0)1 (12)0 (0)Overall Response11 (47.8)23 (95.8)8 (88)8 (100)*Bortezomib + Dexamethasone**Bortezomib+Lenalidomide/Thalidomide+Dexamethasone

Disclosures:

Kaufman: Millenium, Onxy, Novartis, Keryx: Consultancy; Merck, Celgene: Research Funding.
Survival Outcome of Young Multiple Myeloma (MM) Patients in the Era of Novel Therapies
Authors: Kapoor Prashant , Gertz Morie A , Rajkumar S. Vincent , Lacy Martha Q , Dingli David , Hayman Suzanne R , Russell Stephen J. , Lust John A , Buadi Francis K , Zeldenrust Steven R , Greipp Philip R. , Dispenzieri Angela , Kyle Robert A , Kumar Shaji
Published: October 2019

Abstract 2950

Background:

The median age of patients diagnosed with MM is approximately 70 years, and it is a relatively uncommon disease in the younger population. Both conventional chemotherapy and stem cell transplantation (SCT) have favorably impacted the overall survival (OS) of young MM patients. The median survival of patients < 40 years of age, diagnosed prior to 1993 was reportedly 54 months at our institution. However, the impact of novel agents in this patient population is not well known. We studied the outcome of young patients presenting to our institution over the last decade.

Methods:

Records of 1538 patients presenting between 01/99 to12/08 were reviewed. The demographics, features and therapeutic interventions in patients up to 45 years of age at diagnosis were analyzed using descriptive statistics. The survival outcome of this group was compared with the remainder of the cohort using the Kaplan-Meier method.

Results:

One-hundred (6.5%) out of 1538 newly diagnosed MM patients were ≤ 45 years of age at diagnosis. The median follow-up of this group of young patients was 86 months. The disease and patient characteristics are outlined in table 1. A median of 5 regimens (range 1–14) was administered, and 85% patients had received high-dose therapy. 15 patients underwent allogeneic SCT after at least one prior autologous SCT. Novel agents were used both in the front-line and relapsed setting (lenalidomide 60%, thalidomide 56%, pomalidomide 9% and bortezomib 49%), and 45% of initial regimens were novel agent based. The 5 and 7-year OS from diagnosis was 69% and 59%, respectively and the median OS was 93 months. In contrast, the median OS of patients >45 years diagnosed during this period was 53 months (P=0.001, figure 1).

Conclusion:

A marked improvement in OS of young patients with MM diagnosed in the novel agent era is noted in comparison to the historical control of similar age group. In addition, the age associated survival benefit in the younger patient population prevails in the era of novel therapies.

Disclosures:

Gertz: Celgene: Honoraria. Lacy:Celgene: Research Funding. Dispenzieri:Binding site: Honoraria; Celgene: Research Funding. Kyle:Millennium: Consultancy; Binding Site: Honoraria. Kumar:Millennium Pharmaceuticals, Inc.: Research Funding; Celgene: Consultancy, Research Funding; Genzyme: Research Funding; Novartis: Research Funding; Merck: Consultancy, Honoraria.
Impact of ATG on New HLA Groups for Unrelated Donor Allogeneic Stem Cell Transplantation
Authors: Moon Joon Ho , Lee Yoo Jin , Lee Soo Jung , Do Young-Rok , Suh Jang Soo , Sohn Sang Kyun
Published: October 2019

Abstract 3017

Background:

The outcomes of unrelated donor transplantation have improved with refinements in HLA testing. Recently, grouping of HLA matching for unrelated donor was suggested, defining by well-matched, partially-matched, and mismatched. In the current study, the role of ATG for each group was evaluated.

Methods:

A total of 92 patients diagnosed as hematologic diseases and received allogeneic stem cell transplantation (SCT) from unrelated donor were retrospectively analyzed.

Results:

Nineteen patients were classified as well-matched, 42 as partially-matched, and 31 as mismatched. Among them, 57 patients received anti-thymocyte globulin (ATG) as graft-versus-host disease (GVHD) prophylaxis. The overall survival (OS) rate was higher for well-matched group (83%) compared to partially-matched (54%) and mismatched (34%, p=0.076). For partially-matched group, the OS was significantly improved with ATG (83.3% vs. 38.6%, p=0.018). But, the OS was not different between groups with or without ATG for well-matched (87.5% vs. 66.7%, p=0.487) and mismatched (32.4% vs. 41.7%, p=0.215). ATG decreased the cumulative incidence of grade 3–4 acute GVHD (10% vs. 40.2%, p=0.068) and severe chronic GVHD (21.2% vs. 52.2%, p=0.037). The use of ATG (HR=0.248, p=0.029) was related with favorable OS for partially-matched group. However, the favorable effect of ATG was not observed in well-matched and mismatched groups.

Conclusion:

ATG effectively improved survival rate for partially-matched group in unrelated donor transplantation. However, the positive effect of ATG was not observed in well-matched and mismatched group.

Disclosures:

No relevant conflicts of interest to declare.
Association Between Genetic Variants in Immune Response Genes and Outcomes After Hematopoietic Cell Transplantation
Authors: Thyagarajan Bharat , Jackson Scott , Basu Saonli , Jacobson Pamala A. , Gross Myron D , Weisdorf Daniel J. , Arora Mukta
Published: October 2019

Abstract 3049

Background:

Despite rapid advances in allogeneic hematopoietic cell transplant (HCT) techniques, the procedure continues to be associated with a high morbidity and mortality with transplant related mortality (TRM) ranging from 15% to as high as 50%. Acute graft versus host disease (GvHD) remains a major cause of morbidity and an important cause of early TRM in patients undergoing allogeneic HCT. Though variations in major histocompatibiliy (MHC) genes are well known determinants of acute GvHD, there is increasing evidence that genetic variation in immune/cytokine response pathways also contributes significantly to the pathogenesis of acute GvHD.

Methods:

We evaluated the association of single nucleotide polymorphisms (SNPs) in non-MHC dependent immune/cytokine response pathways (n= 77 SNPs in recipients and donors) with acute GvHD and TRM at one year in a cohort of 425 recipient-donor pairs who underwent allogeneic HCT for treatment of hematologic malignancies at the University of Minnesota between 1998 and 2007. We used a Fine and Gray proportional hazards model adjusted for competing risk to evaluate the association between the genetic variants and time to acute GvHD and time to TRM.

Results:

After adjustment for recipient age at transplant, race, diagnosis, disease status at transplant, gender mismatch, CMV serostatus of recipient and donor, recipient and donor sex, donor type, conditioning regimen (myeloablative or reduced intensity) and year of transplant, one recipient SNP, rs646005 in the TIRAP gene and one donor SNP, rs2232596 in the LBP gene were marginally associated with increased risk for grade II-IV acute GvHD (Hazard ratio (HR) = 1.33 (95% Confidence interval (CI): 1.07 – 1.62) and 1.32 (1.07 – 1.66) respectively; p =0.01). Similar analyses for TRM showed recipient SNP rs5498 in the ICAM1 gene was associated with a decreased risk of TRM (HR: 0.67(95% CI: 0.50 – 0.89); p=0.0006) while recipient SNPs rs1739654 in the LBP gene and rs3804100 in the TLR2 gene were associated with an increased TRM risk (HR: 1.86 (95% CI: 1.16 – 2.97) and 1.66 (95% CI: 1.11 – 2.48) respectively; p =0.01).

Conclusions:

These findings are consistent with previous reports that support a role for genetic variants in the innate immune response pathways among both recipients and donors can influence the risk of acute GvHD and TRM among patients undergoing allogeneic HCT. These findings indicate that a systematic search for genetic variants in the innate immune response pathways may identify novel biomarkers that can be used to identify patients at high risk for acute GvHD and TRM.

Disclosures:

Weisdorf: Genzyme: Consultancy, Research Funding.
Implications of Serial Magnetic Resonance Imaging and Positron Emission Tomography Scanning for Survival of Untreated Myeloma Patients Treated with Total Therapy 3
Authors: Usmani Saad , Mitchell Alan , Sexton Rachael , Panozzo Susan , Nair Bijay , Waheed Sarah , Alsayed Yazan , Crowley John , Barlogie Bart
Published: October 2019

Abstract 3082

Introduction:

We have previously reported on the prognostic implications of state-of-the-art imaging with magnetic resonance imaging (MRI) in Total Therapy 2 and with fluoro-deoxy-glucose positron emission tomography (PET) in Total Therapy 3 (TT3). With both techniques, baseline focal bone lesion number and reduction and resolution of focal lesions (FL) imparted favorable clinical outcomes. Here we are updating both MRI and PET data as they pertain to survival of patients enrolled in TT3A protocol employing VTD (bortezomib, thalidomide, dexamethasone), now with longer follow-up of 6.2 yr compared to 3.6 yr at the time of original publication. The details of the protocol and clinical outcomes have been reported previously. Methods: Analyses were restricted to 239 truly untreated patients in TT3A, as even one cycle of prior therapy pertaining to 64 patients had been shown to alter PET results. Cox regression modeling was employed for overall survival (OS) and progression free survival (PFS). Results: OS and PFS were adversely affected by the presence of FL number at baseline, whether defined by MBS (p<0.0001 and p=0.0009, respectively), MRI (p=0.03 and p=0.03, respectively), or PET (p<0.0001 and p<0.0001, respectively); in addition, extra-medullary disease (EMD) detected by PET especially affected OS (78% vs 43%, p=0.003) and PFS (73% vs 29%, p=0.003) unfavorably. In the context of GEP-defined 70-gene risk model (GEP70), FL number distinguished significantly poorer outcomes in low-risk myeloma whereas, for high-risk disease, only a trend was observed (Figure 1A).Complete FDG suppression prior to first transplant affected subsequent OS and PFS favorably (Figure 1B), especially in the GEP-70-defined high-risk subset so that such patients fared almost as well as the low-risk group (Figure 1C). Earlier FDG suppression by day 7 of induction therapy also favorably affected the OS (84% vs 78%, p=0.05). In multivariate analysis, >3 PET lesions featured with poorer OS (HR=1.98 95%CI, p=0.011) and PFS (HR=1.78, 95%CI, p=0.021). Conclusions: The data presented herein, confirm and extend the previously reported results on the role and utility of MRI and PET imaging in the diagnosis and management of newly diagnosed multiple myeloma. Presence of MM lesions on MRI & PET, presence of EMD and failure of FDG suppression with induction chemotherapy and transplantation carry adverse outcome and can serve as prognostic indicators of long-term survival. FDG suppression after first transplant was especially significant in further distinguishing the poor responders within the GEP70-defined high-risk myeloma.

Disclosures:

Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties.
Intermediate Thrombin Activation Is Associated with Reduced Cardiovascular Death Independently of Markers of Lipid Metabolism and Inflammation
Authors: Isermann Berend H. , Schneider Jochen , Kleber Marcus E , Wang Hongjie , Boehm Bernhard O , Grammer Tanja B , Prüller Florian , Nawroth Peter P , März Winfried
Published: October 2019

Abstract 31

Clinical trials showed an increased frequency of cardiovascular death (CVD) in patients receiving direct thrombin inhibitors. Considering the detrimental effect of local coagulation activation during an acute cardiovascular event these observations are deemed unexpected. These observations raise the question whether enhanced thrombin generation may convey protective effects during non-acute disease stages of cardiovascular disease.

To address the question whether increased endogenous thrombin generation may convey beneficial effects in regard to cardiovascular disease we evaluated the association of the ETP and prothrombin fragment 1+2 (F1+2) with cardiovascular death (CVD) in a large prospective study (3156 individuals, median follow up ten years, Ludwigshafen RIsk and Cardiovascular Health (LURIC)-study). Furthermore, we explored the association of ETP with markers of inflammation (CRP, SAA), endothelial dysfunction (sICAM-1, sVCAM-1), and inflammatory cell activation (PAFAH).

An inverse association between ETP and CVD was observed, with the lowest hazard ratio (HR) in the 3rd ETP quartile. This effect remained significant after adjusting for age, gender, DM, BMI, smoking, hypertension, hsCRP, LDL, HDL and triglycerides and after excluding patients receiving anticoagulants (HR for MOR 0.675, CI 0.531–0.858, P=0.001, and HR for CVD 0.667, CI 0.493–0.903, P=0.009). The nadir of sICAM-1 or sVCAM-1 was observed in the 3rd, for PAFAH in the 4th ETP quartile. When stratifying patients according to F1+2 we observed a similar U-shaped association with CVD (lowest HR in the 2nd quartile: 0.718, CI 0,524–0.983, P=0.039). In contrast, the HR for CVD according to fibrinogen strata increased continuously across the quartiles (highest HR in the 4th quartile: 1.828, CI 1.318–2.536, P<0.001).

These results establish that intermediate levels of ETP or PTF1+2 are associated with a reduced HR for CVD and with lower plasma levels of markers of endothelial dysfunction or inflammatory cell activation. Based on these data we propose that low but sustained levels of thrombin generation mediate vasculo-protective effects translating long term into a reduced risk of CVD. These data may provide a rationale for the observed increased frequency of myocardial infarction in clinical studies evaluating direct thrombin inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Population Level Survival for Patients with Multiple Myeloma: Variation and Growing Disparity for Patients of Minority Race or Ethnicity
Authors: Pulte Dianne , Redaniel Maria Theresa , Brenner Hermann , Jansen Lina , Jeffreys Mona
Published: October 2019

Abstract 3126

Background

Survival for patients with multiple myeloma has increased during the first decade of the 21st century, especially in people diagnosed at a younger age. It is currently unknown whether the improvements in survival have extended equally to people of minority ethnic and racial groups. A recent publication found higher incidence and lower survival for AA versus whites. However, the above publication did not include detailed data on changes in survival in different time periods and little data is currently available for the Hispanic or Asian population. Here, we examine changes in survival in patients with myeloma by racial and ethnic group.

Methods

Using cancer registry data from the Surveillance, Epidemiology and End Results Program (SEER9), we assessed trends in 5-year relative survival for patients with multiple myeloma by racial and ethnic group, including non-Hispanic whites (nHw), African-Americans (AA), Hispanics, and Asian-Americans (Asian) for the periods 1993–97 and 2003–07. Model-based period analysis was used to obtain the most up-to-date measurements of survival. Age adjusted analysis was performed using age-standardized survival with age adjustment according to the International Cancer Survival Standard.

Results

Overall, five-year relative survival improved for the time periods studied. Ethnic/racial analyses showed that this was the case for nHw and AA patients. Hispanic and Asian patients had the highest relative survival in the earlier time period, but smaller improvement in survival was seen in these compared to other groups (see table). Age at diagnosis varied significantly between racial and ethnic groups. Age adjusted 5-year relative survival rates showed an increase of 11.7 percentage units for nHw and smaller improvements for AA, Hispanic and Asian patients (see table.)

Conclusions

Although survival improved greatly for nHw with myeloma between 1993–97 and 2003–07, smaller improvements were observed for people of other racial or ethnic groups. Possible reasons for this disparity include lower rates of transplant among patients of minority ethnic or racial background, less frequent use of newer treatment options, later diagnosis, and differences in response rates to newer medications. Further explanatory studies are required to investigate these possibilities.

Disclosures:

No relevant conflicts of interest to declare.