bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemochromatosis in Italy in the Last 30 Years. Role of Genetic and Acquired Factors.
Authors: Fracanzani Anna Ludovica , Piperno Alberto , Valenti Luca , Fraquelli Mirella , Coletti Sabina , Maraschi Alessandra , Consonni Dario , Coviello Enzo , Conte Dario , Fargion Silvia
Published: October 2019

Abstract 2003

Poster Board I-1025

Background & Aims Clinical presentation of hereditary hemochromatosis markedly changed in the recent years. The aim of the study was to analyze a large series of consecutive Italian patients with hemochromatosis diagnosed between 1976 and 2007 to define whether the genetic background and the presence of acquired risk factors influenced the severity of iron overload and the natural history of the disease across the years. Methods: A cohort of 452 Italian patients with iron overload, of whom 338 HFE-related (C282Y homozygotes or compound C82Y/H63D heterozygotes, and 114 non-HFE-related, prospectively followed for a median of 112 months. Results: Alcohol intake, smoking habits and iron removed to depletion were similar in patients with and without HFE-related iron overload. HBV (4% and 10% p=0.03) and HCV (9% and 17% p=0.02) infections were more frequent in patients with non-HFE-related iron overload. Seventy-three percent and 61% of the patients with HFE and non-HFE-related disease had no acquired risk factor. Cirrhosis was significantly more frequent in non-HFE patients, independently of the presence of acquired risk factors (p=0.02). Gender, alcohol intake, prevalence of smokers, HCV infection, glucose, lipids, iron-related parameters and prevalence of C282Y/H63D significantly differed across the years. At enrolment cirrhosis was present in 145 cases, being significantly more frequent in the first decade (80%, 47% and 13%, p=0.001). Survival did not differ across the decades in cirrhotic patients, HCC occurring similarly in HFE and non-HFE patients. Conclusion: Patients with HFE and non-HFE related iron overload have comparable iron overload and similar clinical history. Patients, unless cirrhotics at enrolment, diagnosed during the last 10 years have less severe disease and lower prevalence of acquired risk factors, independently of genetic background.

Disclosures:

No relevant conflicts of interest to declare.
Severe Thalassemia Intermedia Resulting From Coinheritance of IVSI-110 G>A Beta Gene Mutation and Duplication of Alpha-Globin Gene Cluster in Homozygosis.
Authors: Refaldi Chiara , Barcellini Wilma , Cassinerio Elena , Graziadei Giovanna , Cappellini Maria Domenica
Published: October 2019

Abstract 2011

Poster Board I-1033

Introduction

The clinical severity of thalassemia intermedia depends on the degree of a/non-a-chains iimbalance. Among the molecular mechanisms responsible for thalassemia intermedia is the coinheritance of excessive a-globin gene production with a defective beta-globin gene.

Materials and Methods:

we describe an Italian family where thalassemia intermedia apparently segregates as a dominant form but it tourned out to be due to the coinheritance of a beta-globin mutation and a duplication of the alpha-globin gene cluster. The father (aged 51yrs) showed a well tolerated severe chronic hemolytic anemia (Hb 7.5-8.5 g/dL) not transfusion dependent, jaundice, splenomegaly and leg ulcers:The mother (aged 46yrs) has a completely normal hematological and hemoglobin pattern. Two sons (19 and 14 yrs) showed more severe clinical manifestations than the father. They underwent splenectomy at 12 and 13 years respectively without any benefit and afterwards they become transfusion dependent.

Results:

The hemoglobin analysis revealed that the father and the sons were heterozygotes for the beta mutation IVSI-110 G>A. MLPA analysis of the alpha-globin gene cluster disclosed a full duplication of the alpha-globin locus, spanning a 175 kb from the telomere to the 3'HVR downstream of the alpha-globin gene and including the upstream regulatory element HS-40. This rearrangement increases the number of the active alpha-globin genes in cis from 2 to 4.Surprisengly it was found in heterozygosis in both parents and in homozygosis in both sons. The hematological and molecular data of the family are reported in the table.

In the father the 6 alpha-globin genes led to increased synthesis of alpha-chains; the coinheritance with a beta-thalassemia mutation causes a moderate/severe thalassemia intermedia phenotype. The presence of 8 alpha-globin genes in the sons raises further the degree of globin-chains imbalance and exacerbates the clinical phenotype. It is important to note that splenectomy worsened the clinical course.in the 2 homozygotes for the alpha duplication.

Conclusions:

Based also on previous experience we suggest that splenectomy in patients with a real excess of alphaa chain production is unconvenient since a large amount of circulating red cells with precipitated alpha chains may be responsible for increased hemolysis as well as increased risk of thrombosis This family moreover raises concerns regarding genetic counselling, suggesting that whenever one of the partner is affected by TI it is advisable a complete molecular screening of the couple in order to exclude any possible alpha gene defects interaction

Disclosures:

No relevant conflicts of interest to declare.
Generation of Autologous Pre-B Acute Lymphocytic Leukemia (ALL) Reactive T Cell Lines Using ALL Blasts as Antigen Presenting Cells.
Authors: Zhong Rui-kun , Lane Thomas A. , Ball Edward D
Published: October 2019

Abstract 2035

Poster Board II-12

Although hematologic remissions can be achieved in the majority of patients with adult acute lymphocytic leukemia (ALL) by chemotherapy, long term survival is only 30–40%. The inability of the immune system to recognize and eliminate residual malignant leukemia cells may be an important mechanism contributing to relapse. In this study, we investigated the possibility of using pre-B-ALL cells as antigen presenting cells in an in vitro culture to induce autologous ALL reactive T cells. After 7 day culture of Pre-B-ALL peripheral blood mononuclear cells (CD19+ 93±4%) in 96 well culture plates in culture medium supplemented with a cytokine combination of IL-2/IL-3/IL-4/IL-7/GM-CSF, CD80, CD86, CD83, CD54, HLA-Dr and CD40 expression was analyzed. Significant enhancement of CD80, CD86, CD83 and CD40 on ALL cells was observed (n=8, P≦0.001-0.02). Addition of lipopolysaccharide (LPS) to the cytokine combination further increased CD80, CD86 or CD40 expression by 3 of 8 ALL samples above the baseline enhancement by cytokines (Figure), while CD40 ligand (CD40L) enhanced expression of the co-stimulatory molecules in 4 of 8 cases. Autologous T cells remaining in the culture after day 7 were then expanded with high dose IL-2 and autologous ALL reactive T cell lines were identified by IFN-g release by T cells in response to autologous ALL cells. Autologous ALL reactive T cells were generated from 5 of 8 pre-B ALL samples studied. The data from 3 experiments demonstrated that although the cytokine combination plus LPS or CD40L could effectively induce ALL cell expression of co-stimulatory molecules, the presence of CD40L and LPS in the culture induced significantly greater activation of autologous ALL-reactive T cells than did the cytokine combination plus LPS alone, as assessed by average IFN-g release of 24-48 culture wells (P≦0.004). ALL-reactive T cell lines selected by high IFN-g release response showed effective elimination of autologous ALL cells in a 2 day co-culture assay with an E:T ratio of 1:1 by flow cytometry analysis. Average residual CD19+ cells was 2.0±1.4% for highly reactive T cell lines (n=17) vs 16.5±25.9% for the less reactive control T cell lines (n=9) (P<0.02). Conclusion: Autologous ALL-reactive T cells can be induced from most ALL patients in an in vitro culture with a cytokine combination. Adoptive transfer of these anti-ALL CTL to patients is a possible therapeutic approach for further study.

Disclosures:

No relevant conflicts of interest to declare.
Gender-Based Differences in Hemostatic Responses. Implications in Effective Anticoagulant Management.
Authors: Iqbal Omer , Sadeghi Nasir , Bakhos Fadi , Hoppensteadt Debra , Fareed Jawed
Published: October 2019

Abstract 2103

Poster Board II-80

Abstract: Recent reports from the National Heart, Lung and Blood Institute indicate that as many as 3 million women (particularly young) in the United States suffer from a form of heart disease fundamentally different from that in men, characterized by more even plaque development inside major and smaller blood vessels, posing diagnostic and treatment challenges leading to increased morbidity and mortality. It is hypothesized that women have variable but attenuated hemostatic responses to anticoagulant drugs when compared to men. In order to validate this hypothesis the hemostatic responses in healthy males (n=10) and females (n=10) were evaluated by performing the global clotting assays, fibrinokinetic assays and thrombin generation assays in the presence of Rivaroxaban, an oral Factor Xa inhibitor likely to replace warfarin, Enoxaparin, a low molecular weight heparin and saline as a control. Blood (20 ml) was drawn from healthy volunteers, males (n=10) and females (n=10) and placed into citrated tubes with one part of 3.2% sodium citrate to 9 parts of blood. The citrated whole blood was supplemented with Rivaroxaban (FC=0.3mg/ml), Enoxaparin (FC=5mg/ml) and saline as a control. The samples were analyzed to determine the whole blood APTT and Heptest clotting assays. The remaining citrated blood was centrifuged at 3000 rpm to obtain platelet poor plasma that was aliquoted and kept frozen at -70°C until further analysis. The plasma was then thawed and supplemented with saline, rivaroxaban (FC=0.3mg/ml) and Enoxaparin (FC=5mg/ml). A statistically significant difference between males and females was noted in APTT (p=0.0442)) and Heptest (p=0.0345) assays in the saline control values. However, the anticoagulant response to supplementation of the plasma samples with rivaroxaban at a final concentration of 0.3ug/ml and Enoxaparin at 5 ug/ml showed a statistically significant difference between males and females in the Heptest (P=0.0423) while the APTT assay felt a little short of statistical significance (P=0.0511).

Fibrinokinetics was performed and absorbance recorded (405 nm) at every minute for the next 30 minutes. There are gender-based differences in fibrinokinetic responses to anticoagulant drugs with females showing faster fibrin formation than males. The attenuated hemostatic responses observed in women compared to men may interfere in achieving adequate and effective anticoagulation leading to thrombotic complications.

Time (min) 0 30 Gender Male ODs Female ODs Male - ODs Female - ODs Male - ODs Female - ODs Saline control 0.857±0.31 0.611±0.22 1.367±0.28 1.214±0.28 1.377±0.26 1.24±0.28 Rivaroxaban (0.3ug/ml) 0.853±0.32 0.602±0.21 1.353±0.27 1.221±0.18 1.363±0.27 1.23±0.18 Enoxaparin (5ug/ml) 0.713±0.35 0.507±0.24 0.794±0.33 0.621±0.23 0.802±0.33 0.629±0.23

Disclosures:

No relevant conflicts of interest to declare.
Platelet Senescence and Phosphatidylserine Exposure.
Authors: Dasgupta Swapan Kumar , Abdel-Monem Hanan , Prakasam Anthony , Thiagarajan Perumal
Published: October 2019

Abstract 2117

Poster Board II-94

The transbilayer movement of phosphatidylserine from the inner to the outer leaflet of membrane bilayer occurs during platelet activation and is essential for platelet procoagulant activity. Expression of phosphatidylserine also occurs in platelets stored more than 5 days in the blood bank. We have examined the role of mitochondrial pathways in activation-induced platelet procoagulant activity and during in vitro senescence. Collagen and thrombin-induced exposure of phosphatidylserine is associated with a decrease in mitochondrial membrane potential (MMP). Cyclosporin A, a known inhibitor of cyclophilin D, inhibited phosphatidylserine exposure and the loss of MMP. Consistent with this, platelets from CypD deficient mice had decreased loss of MMP and impaired phosphatidylserine exposure when treated with a combination of thrombin and collagen. However, there is no release of cytochrome c into the cytosol. Also there is no activation of caspase-3 or Rho associated kinase I (ROCK1). In sharp contrast to this, in platelets stored for more than 5 days, phosphatidylserine exposure is found to be associated with caspase-3 and ROCK1 activation. To delineate the mechanism we used ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis via Bcl-2 family of proteins. ABT737-induced phosphatidylserine exposure was also associated with cytochrome c release, caspase-3 and ROCKI activation and it is unaffected in platelets isolated from CypD knock out mice or in platelets treated with cyclosporine A prior to activation. Caspase-3 inhibitor (z-DVED-fmk and ROCK1 inhibitor Y-27632 reduced ABT-737 induced phosphatidylserine expression.

Our results also show that activation-induced phosphatidylserine exposure is dependent on mitochondrial membrane depolarization and CypD but is independent of cytochrome c release or caspase-3 or ROCK1 activation. In contrast, the phosphatidylserine exposure in stored platelets occurs via mitochondrial cytochrome c release, caspase-3 activation and ROCKI activation and does not depend on mitochondrial membrane depolarization or CypD.

Disclosures:

No relevant conflicts of interest to declare.
Effect of B-Cell Depletion On Inhibitor Antibody Formation and Immune Tolerance Induction to Human Factor VIII in Hemophilia A Mice.
Authors: Zhang Ai-Hong Allan , Skupsky Jonathan , Scott David W.
Published: October 2019

Abstract 2137

Poster Board II-114

B-cell depletion using anti-human CD20 monoclonal antibodies has been reported to be effective in autoimmunity and in temporarily eliminating inhibitory antibodies in hemophilia A patients. In the current study, we examined the effect of anti-murine CD20 (αCD20) depletion on the immune response to factor VIII (FVIII) and its influence on an immune tolerance induction (ITI) protocol. Previous studies have shown that IgG subclasses of anti-murine CD20 monoclonal antibody (αCD20) have differential effects on B-cell depletion in the mouse. Thus, IgG1 αCD20 selectively depletes follicular B cells, while sparing marginal zone (MZ) B cells. Combined with evidence that MZ B cells may be tolerogenic antigen-presenting cells, we tested the hypothesis that follicular B-cell depletion using αCD20 IgG1 might favor tolerance induction to human FVIII. Hemophilic (FVIII knockout) mice were primed with physiological doses of recombinant human FVIII by weekly IV injection, followed by αCD20 IgG1 or control IgG1 treatment. Ten days after the αCD20 treatment, the mice were treated with daily high dose (2μg) FVIII IV injections to model ITI in hemophilia A patients. After 4 weekly injections, 70% of the mice developed titers of anti-FVIII IgG as high as 1:12,800. Unlike whole B-cell depletion, subsequent follicular B-cell depletion did not significantly decrease the anti-FVIII IgG titer, compared with mice receiving control IgG1. Repeated high dose FVIII injections to mimic ITI significantly increased the anti-FVIII IgG titer in both groups. However, in the mice that received αCD20 IgG1 treatment, the increase of anti-FVIII IgG levels were significantly lower than that in control IgG1 treated mice. In conclusion, we found that follicular B-cell depletion by αCD20 IgG1 antibody in hemophilia A mice did not switch the immune response to tolerance, but it diminished the immunogenicity of human FVIII in vivo in hemophilic mice. (Supported by NIH R01 HL061883)

Disclosures:

No relevant conflicts of interest to declare.
Ex Vivo Expansion of Highly Purified Human NK Cells..
Authors: Gao Chunji , Li Xiaohong , Ma Jian , Wu Xiaoxiong , Wang Feifei , Li Meng , Yu Li , Da Wanming
Published: October 2019

Abstract 2157

Poster Board II-134

Object

To optimize the expansion of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after Ex vivo expansion.

Methods

NK cells were isolated from PBMNC by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II(Miltenyi Biotec, Germany), then they were cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-γ secretion at the end of the culture period.

Results

In group IL2+IL15 and IL2+IL15+IL12, cells were expanded 50.46±4.31 and 52.35±6.72 fold, respectively, much more higher than others(P<0.01), but no significant difference between them (P>0.05). And the purity of CD3−CD56+NK cells was over 94% in all groups except the control. The expressions of perforin and granzyme B mRNA of expanded NK cells cultured with cytokines was significantly higher than the starting population(P<0.01), although IL2+IL15+IL12 group was slightly higher than that of IL2+IL15 group, without significant difference (P>0.05). There was great increase in IFN-γ levels in the supernatants of NK cells culture in the presence of cytokines; IL2+IL15+IL12 group and IL2+IL12 group was significantly higher than others(P<0.01).

Conclusion

High purity NK cells could be efficiently expanded in culture with IL2+IL15, and its biological functions were enhanced in this condition.

Disclosures:

No relevant conflicts of interest to declare.
Splice Variant JAK2 Transcript Deleting Exon 14 in Patients with Chronic Myeloproliferative Neoplasms.
Authors: Ma Wanlong , Kantarjian Hagop , Zhang XI , Wang Xiuqiang , Zhang Zhong , Yeh Chen-Hsiung , Sferruzza Anthony , O'Brien Susan , Albitar Maher
Published: October 2019

Abstract 2161

Poster Board II-138

The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs). While other point mutations and small deletions and insertions in exons 12, 13, and 14 have been reported in the JAK2 JH2 domain, deletion of the entire exon 14 is rarely detected in patients with MPNs. In a series of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by direct sequencing of mRNA, we detected a complete exon 14 (88 bp) deletion mutation in <1% of those with JAK2 mutation. This appears to be an alternative splicing mutation, not detectable with DNA-based testing, leading to a frameshift deletion in the JAK2 JH2 domain and the expression of a truncated protein (S593I fsX8). Although this mutation was present at detectable levels (>15% of total JAK2 transcript) in a small proportion of MPN cases, we decided to investigate the possibility it may be expressed at low levels (<15% of JAK2 transcript) in patients with MPNs. Using a sensitive reverse transcription-PCR—based fluorescent fragment analysis method to quantify the percentage of truncated (minus exon 14) JAK2 mRNA as compared to wild-type, we tested 61 patients with confirmed MPN; 183 patients with suspected MPNs (93 V617F positive, 90 V617F negative); and 46 healthy normal control subjects. The wild-type PCR product was 273 bp, while the exon 14 splice mutant displayed a 185-bp peak. All samples with a splicing mutant expression to wild-type ratio >15% were confirmed by direct sequencing. The JAK2 exon 14 splice mutation was detected at low levels in 9 of the 61 MPN patients (15%), accounting for 3.96% to 33.85% (mean=12.04%) of JAK2 transcript in these individuals. Among the 183 suspected MPN patients, the exon 14 splice variant was detected in 20 of the 93 (21.5%) with V617F (mean expression level relative to wild type=5.41%, range=2.13%-26.22%) and 31 of the 90 (34.4%) without V617F (mean expression=3.88%; range=2.08%-12.22%). All 46 normal individuals were considered negative for the alternatively spliced transcript. In conclusion, the expression of an alternatively spliced JAK2 mRNA with deletion of exon 14, leading to a truncated JAK2 protein, is a common abnormality in patients with MPNs. This alternatively spliced transcript is more common in MPN patients without V617F mutation and might contribute to the leukemogenesis in these patients. Low levels of JAK2 exon 14 splice mutation may also contribute to the effects of the V617F mutation in patients with MPNs. However, this mutation is missed if DNA and not the RNA is used for testing for JAK2 mutations.

Disclosures:

No relevant conflicts of interest to declare.
Tyrosine Kinase Proteins profiling of Nilotinib Resistant Chronic Myelogenous Leukemia Cells Unravels a Tyrosine Kinase-Mediated Bypass.
Authors: Pasquet Jean-Max , Gioia Romain , Drullion Claire , Lagarde Valerie , Leroy Cedric , Roche Serge , Cardinaud Bruno , Mahon Francois-Xavier
Published: October 2019

Abstract 2175

Poster Board II-152

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or –intolerant disease. We have shown that one of the mechanisms of resistance to nilotinib is an increasing expression of the p53/56 Lyn kinase, both at mRNA and protein level in cell lines. This result was confirmed in vivo in nilotinib-resistant CML patients (Mahon et al. Cancer Res., 2008, 68(23):9809-16.). To elucidate Lyn mediated-nilotinib resistance, a phosphoproteomic study was performed by Stable Isotope Labelling with Amino acid in Cell culture (SILAC) which highlights the potential role of downstream tyrosine kinases. Among different candidate proteinsThe Spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl were the most relevant in the nilotinib resistant cell line as compared to the sensitive counterpart. Syk hyperphosphorylation was confirmed in the nilotinib resistant cell line using western blot at least on tyrosine residues Y323 and Y525/526, two critical tyrosine residues respectively involved in Lyn-mediated Syk phosphorylation and autophosphorylation-associated Syk activation. Lyn interacts with Syk as detected in Syk immunoprecipitates in nilotinib resistant cells. Furthermore, Syk-Lyn interaction is inhibited by dasatinib suggesting the requirement of Lyn kinase activity and Syk phosphorylation. Targeting Syk expression in nilotinib resistant cells by siRNA or tyrosine kinase activity by pharmacological inhibitors leads respectively to a partial (35%) or to a full restoration of nilotinib sensitivity. Moreover, the identification of Axl by SILAC is correlated to a 9 fold increase of its level of expression in the resistant cell line and the inhibition of Axl tyrosine kinase activity decreases proliferation of both nilotinib sensitive and resistant CML cells. All together these results disclose a new pathway for tyrosine kinase inhibitors resistance in CML involving at least the two Lyn downstream tyrosine kinases Syk and Axl.

Disclosures:

Mahon: Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.
Small Interfering RNA against Bcr-Abl Transcripts Sensitized Mutated T315I Cells to Nilotinib.
Authors: Koldehoff Michael , Elmaagacli Ahmet H
Published: October 2019

Abstract 2188

Poster Board II-165

Background:

Selective inhibition of the BCR-ABL tyrosine kinase by RNA interference has been demonstrated in leukemic cells. Therefore, we evaluated the specific bcr-abl small interfering RNA (siRNA) silencing in BCR-ABL positive cell lines, including those resistant to imatinib (IM) and particularly those with the T315I mutation.

Design and Methods:

The factor-independent 32Dp210 bcr-abl oligoclonal cell lines and in human IM-resistant bcr-abl positive cells from different patients with leukemia disorders were investigated. The effects of bcr-abl siRNA or the combination of bcr-abl siRNA with both IM and nilotinib were compared with those of the ABL inhibitors IM and nilotinib.

Results:

Coadministration of bcr-abl siRNA with IM or nilotinib dramatically reduced the .{/MAIN;133}BCR-ABL expression in wild-type (wt) and mutated bcr-abl cells and increased the lethal capacity. The bcr-abl siRNA significantly induced apoptosis and inhibited proliferation in wt (p<0.0001) and mutated cells (H396P, T315I, p<0.0001) versus controls. Cotreatment of bcr-abl siRNA with IM or nilotinib resulted in an increased inhibition of proliferation and induction of apoptosis as compared to IM or nilotinib (p<0.0001) in T315I cells. Furthermore, the combination of bcr-abl siRNA with IM or nilotinib significantly (p<0.01) reversed multidrug resistance gene 1-dependent resistance of mutated cells. In T315I cells bcr-abl siRNA with nilotinib has shown powerful effect on the cell-cycle distribution.

Conclusions:

Our data suggest that silencing by bcr-abl siRNA with IM or nilotinib may be associated with an additive antileukemic activity against tyrosine kinase inhibitor-sensitive and –resistant BCR-ABL cells, and might be an alternative approaches to overcome BCR-ABL mutations.

Disclosures:

No relevant conflicts of interest to declare.
Chronic Myelogenous Leukemia Cells Contribute to a Bone Marrow-Stroma in In Vivo NOD/SCID Mouse System.
Authors: Shirasaki Ryosuke , Tashiro Haruko , Oka Yoko , Sugao Toshihiko , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki
Published: October 2019

Abstract 2191

Poster Board II-168

Aims: The stroma-forming cells in a bone marrow are derived from hematopoietic stem cells. We reported previously that non-adherent leukemia blast cells converted into myofibroblasts to create a microenvironment for proliferation of leukemia blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system chronic myelogenous leukemia (CML) cells are also differentiated into myofibroblasts to contribute to a bone marrow-stroma in vivo. Materials and Methods: Bone marrow cells were collected from informed CML patients, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, non-adherent mononuclear cells were injected to the priory 2.5 Gray-irradiated non-obese diabetes (NOD)/SCID mice intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Bcr-Abl transcript, and mice were sacrificed after chimeric mRNA was demonstrated. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select CML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and Discussion: When non-adherent CML cells were transplanted to NOD/SCID mice, CML cells were engrafted after 2 months. In the murine bone marrow human stromal cells were identified, in which BCR and ABL gene was fused with FISH analysis. When the parental CML cells were cultured on the CML-derived myofibroblasts, CML cells grew extensively in a vascular endothelial growth factor-A-dependent fashion. These results indicate that CML cells can create their own microenvironment for proliferation in vivo.

Disclosures:

No relevant conflicts of interest to declare.
Sirolimus Yields High Response Rates in Hematopoietic Allograft Recipients with Steroid-Refractory Acute Graft-Versus-Host Disease.
Authors: Pidala Joseph A. , Hoda Daanish , Salgado-Vila Norma , Kim Jongphil , Perkins Janelle , Bookout Ryan , Field Teresa , Perez Lia , Ayala Ernesto , Ochoa-Bayona J. Leonel , Raychaudhuri Jyotishankar , Alsina Melissa , Greene John N , Janssen William E. , Fernandez Hugo F. , Anasetti Claudio , Kharfan-Dabaja Mohamed A
Published: October 2019

Abstract 2235

Poster Board II-212

Acute graft-versus-host disease (aGVHD) remains the major cause of morbidity and mortality in allogeneic hematopoietic cell transplant recipients. There is no consensus in regards to the best therapy for patients who fail to respond to, or do not tolerate, systemic glucocorticoids. We evaluate the efficacy of sirolimus in 34 pts, median age of 49 (23-67) years, with steroid-refractory (N=31) or steroid-intolerant (N=3) aGVHD. The diagnosis of aGVHD was established at a median of 34 (7-1042) days after transplantation, and confirmed by biopsy in all cases. Initial treatment of aGVHD consisted of prednisone up to 2 mg/kg. Sirolimus was initiated at a median of 9 (1-255) days after glucocorticoid initiation. A loading dose of sirolimus was administered to 19 (56%) of 34 pts, median 6 (3-8) mg, followed by maintenance therapy of 1-2 mg per day with doses adjusted to target therapeutic trough levels between 4 and 12 ng/ml; therapeutic levels were achieved in all cases. The overall response rate was 76%. Fifteen (44%) of the 34 pts achieved a CR after sirolimus initiation and without requiring additional immune suppressive agents. CR was achieved in 11/31 (42%) steroid-refractory patients, and in 2/3 (67%) steroid-intolerant patients. The median overall survival (OS) after initiation of sirolimus was 5.6 months, and one year OS was 44% (95% C.I. 27%-60%). Maximum decrease in dose of steroids over 12 weeks following sirolimus was a median of 50% (range 0-100%). Fourteen (42%) of 33 pts reached a dose < 20 mg of prednisone at a median of 4 months (95% CI: 3.5-13.5) after initiation of sirolimus. Seven (21%) of 34 pts were free from steroids after a median of 20 months (95% CI: 9.2-20.0) after initiation of sirolimus. Two pts were shown to be successfully liberated from all immunosuppressive agents without recurrent GVHD at 2.7 and 7.1 months after initiation of sirolimus, respectively.

These data indicate the sirolimus is effective in controlling steroid-refractory aGVHD. Further studies are needed to determine the most appropriate timing for sirolimus after transplantation, whether prophylaxis, primary or secondary GVHD therapy.

Disclosures:

Off Label Use: Sirolimus for graft-versus-host disease.
Long-Term Results of Donor Lymphocyte Transfusions for the Treatment of Relapse After Allogeneic Transplantation for Chronic Myelogenous Leukemia - Is There a Role for Immunotherapy in CML?.
Authors: Kolb Hans-Jochem , Bund Dagmar , Schmetzer Helga , Schmid Christoph , Buhmann Raymund , Tischer Johanna , Ledderose Georg
Published: October 2019

Abstract 2258

Poster Board II-235

The long-term outcome of donor lymphocyte infusions (DLI) as first line treatment of relapse of chronic myeloid leukemia (CML) after allogeneic stem cell transplantation was studied and compared to other treatment modalities. Forty five patients were treated with DLI and 36 without DLI. Patients given DLI first did not differ significantly from those given other treatments in gender, age, donor, gender and histocompatibility of the donor, source of stem cells (blood vs marrow), depletion of T cells, stage of the disease, time from diagnosis and year at the time of transplant, acute and chronic graft-versus-host disease (GVHD) and remission duration. The survival of the DLI group was 66.4 percent at 10 and 15 years after transplantation, it was 42.2 and 23.8 percent respectively in the non-DLI group (p=0.019, log rank). Excluding patients with early relapse in the first 6 months the survival of the DLI group was 78 percent at 15 and 20 years, 70 and 26 percent respectively of the non-DLI group (p=0.009). Recurrent leukemia was the predominant cause of death in both groups (11 of 14 patients of the DLI-, 21 of 23 patients of the non-DLI group). Three patients in the DLI group died of recurrent infections, bronchiolitis obliterans (BO) and heart failure respectively, and 2 patients of BO in the non-DLI group. The proportion of surviving patients with positive PCR for bcr/abl was not different in both groups (2 of 13 patients of the non-DLI and 7 of 30 patients in the DLI group). The better survival of the DLI group indicates a better control of residual leukemia by a persistent immune effect. The response to DLI was improved by simultaneous treatment with low doses of interferon-a and GM-CSF. The combination of these cytokines without DLI and transplantation remains to be defined in patients that do not tolerate or respond incompletely to Imatinib. The role of immunotherapy for induction and maintenance of remission is well established in transplant patients.

Disclosures:

No relevant conflicts of interest to declare.
Potential Eradication of Myeloma Stem Cells by Combining ATRA with WNT and HH Signaling Pathways Inhibitors.
Authors: Tricot Guido J. , Wang Siqing , Shi Lei , Xu Hongwei , Xu Chunjiao , Zangari Maurizio , Zhan Fenghuang
Published: October 2019

Abstract 2839

Poster Board II-815

Multiple myeloma (MM) remains largely incurable. The existence of the putative myeloma stem cell population may account for drug-resistance and relapse. Previous investigation showed that the CD138-negative (CD138-) MM cell fraction contains the putative MM stem cells. However, little is known about the molecular characteristics of MM stem cells, which makes it difficult to specifically target such cells. In this study, by comparing gene expression profiles (GEP) of CD138+ and CD138- cells isolated from 10 MM cell lines, we discovered that RARα is the top one over-expressed gene in CD138- MM stem cells. RARα has two major isoforms, RARα1 and RARalpha2. Real-time PCR detected significantly higher expression of RARalpha2 but not of RARalpha1 in CD138- MM cells compared with CD138+ cells. We recently reported that increased RARalpha2 expression at diagnosis resulted in a significantly shorter overall-survival (P = 0.003); importantly, ATRA selectively killed RARalpha2-positive myeloma cells, but not RARalpha2-negative cells. These results suggested that ATRA could be used to eradicate specifically RARalpha2-over-expressing MM stem cells. Indeed, we found that ATRA selectively killed CD138- MM stem cells (P < 0.01) from ARP-1 and OCI-MY5 human myeloma cell lines and 5T33 murine myeloma cells but spared the CD138+ tumor cells from these cell lines. WNT and Hedgehog (HH) signaling pathways were activated in CD138- stem cells. To our surprise, ATRA treatment (10-6M) further increased WNT and HH signaling pathways in CD138- myeloma cells, based on increased protein levels of β-catenin and cleaved Shh/Ihh in ATRA-treated cells. Stimulation of these signaling pathways by LiCl (5 mM) and/or SHH(20 μg/mL) partially abrogated ATRA-induced cytotoxities on CD138- MM cells, demonstrating that stimulation of WNT and HH signaling induced partial ATRA-resistance in CD138- cells. A combinational treatment of ATRA (1 μM), a COX-2 inhibitor celecoxib (shown to inhibit WNT signaling, 50 μM) and a HH signaling inhibitor cyclopamine (10 μM) induced synergistic effects on cell death and growth inhibition of CD138- and RARalpha2-over-expressing MM cells in-vitro. In the 5T33 murine myeloma model, the combination of ATRA (20mg/Kg), celecoxib (10mg/Kg) and cyclopamine (20mg/Kg) induced synergistic inhibition of tumor growth in-vivo. Thus, our study provides novel approaches to specifically target MM stem cells.

Disclosures:

No relevant conflicts of interest to declare.
Gene Expression Signature of Host Immune Response Is Predictive of Follicular Lymphoma Patient Survival in Independent Cohorts, and Correlates with Transformation to Diffuse Large B-Cell Lymphoma.
Authors: Alizadeh Ash A , Gentles Andrew J , Plevritis Sylvia K , Levy Ronald
Published: October 2019

Abstract 2951

Poster Board II-927

Background:

Expression signatures of infiltrating immune cells [1] have been shown to predict survival in follicular lymphoma (FL), but have not been cross-validated in independent patient cohorts [2,3]. These signatures may relate biologically to the frequency of infiltrating including T-cells and macrophages, or to specific transcription programs within tumor cells and/or the tumor microenvironment. We sought to evaluate the validity of this model in an independent cohort of patients with FL, assessing its relationship to outcomes including histological transformation and death.

Methods:

The immune response (IR) predictor score proposed by Dave et al. [1] was applied to gene expression data from an independent cohort of 88 FL patients [4] with known survival outcomes and history of transformation to diffuse large B-cell lymphoma (DLBCL). Genes (n=66) corresponding to IR1 and IR2 signatures were mapped from Affymetrix microarrays [1] to a custom cDNA array [4] via Entrez Gene ID, and the composite IR score was calculated per the scheme proposed by Dave et al.

Results:

The IR score was predictive of patient outcome in the 88 patient test set as a continuous variable (p=0.001, HR=2.01, 95% CI 0.50-1.30). Partitioning of patients into high and low risk groups based on the median IR score across the cohort robustly separated survival curves (Figure A). The IR score was significantly higher in FL patients known to undergo transformation to DLBCL (Figure B: mean IR score of -0.6 in non-transforming FL vs. -0.2 in transforming FL; p∼10-11, t-test).

Conclusions:

The IR score of Dave et al. was highly significant as a predictor of survival in the independent patient cohort [4]. Moreover, the score was significantly associated with propensity of FL to transform to DLBCL. To our knowledge, immune cell infiltration has not previously been implicated in transformation.

1. Dave SS et al. (2004) Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N Engl J Med 351(21): 2159-2169.

2. Tibshirani R (2005) Immune signatures in follicular lymphoma. N Engl J Med 352: 1496-1497.

3. Chu G Hong WJ, Warnke R, Chu G (2005). Immune Signatures in Follicular Lymphoma (Corres). N Engl J Med. 352: 1496-1497.

4. Glas AM et al. (2005) Gene expression profiling in follicular lymphoma to assess clinical aggressiveness and to guide the choice of treatment. Blood 105(1): 301-307.

Disclosures:

No relevant conflicts of interest to declare.
Bcr/Abl Increases Bcatenin Protein and Activity in An ICSBP-Dependent Manner in Myeloid Cells.
Authors: Huang Weiqi , Horvath Elizabeth , Eklund Elizabeth A.
Published: October 2019

Abstract 2958

Poster Board II-934

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor (also referred to as IRF8). Similar to other IRF proteins, ICSBP regulates transcription of genes involved in the inflammatory response. However, ICSBP also functions as a myeloid leukemia tumor suppressor. Specifically, decreased ICSBP expression in myeloid progenitor cells results in cytokine hypersensitivity and resistance to apoptosis in response to Fas-activation or IL3 withdrawal. Consistent with function as a tumor suppressor, decreased ICSBP-expression is found in the bone marrow of human subjects with chronic myeloid leukemia (CML). Expression of ICSBP increases during remission and decreasing ICSBP expression is associated with progression to CML blast crisis (BC). In murine models, ICSBP-deficiency induces a myeloproliferative disorder (MPD) which resembles CML. And, ICSBP overexpression blocks MPD in mice transplanted with Bcr/abl expressing bone marrow. Increased Bcatenin activity is also associated with CML-BC. However, the impact of ICSBP on Bcatenin expression or activity has not been previously investigated. In these studies, we hypothesized that ICSBP-deficiency in CML-BC increases Bcatenin-expression. In support of this hypothesis, we found that Bcatenin protein and activity were increased in myeloid cell lines with ICSBP-knock-down and decreased in cells with ICSBP-overexpression. Bcatenin protein and activity were also increased in primary myeloid progenitors from ICSBP-/- mice in comparison to wild type or ICSBP+/- mice, in an ICSBP-dose dependent manner. Expression of Bcr/abl decreased ICSBP-expression in myeloid cells and increased Bcatenin protein and activity. Also supporting our hypothesis, the effect of Bcr/abl on Bcatenin was abolished by re-expression of ICSBP in Bcr/abl expressing myeloid cell lines or primary murine myeloid progenitor cells. In each of these situations, the increase in Bcatenin protein was not due to increased expression of Bcatenin mRNA. These results suggest ICSBP influences expression of target genes involved in Bcatenin protein stability. Therefore, these studies identify a pathway by which Bcr/abl activity impairs ICSBP expression in immature myeloid cells, thereby increasing stability of Bcatenin protein and Bcatenin activity. Poor prognosis and BC are associated with Fas resistance and increased Bcatenin activity in CML leukemia stem cells (LSC). Bcr/abl activity is also associated with decreased expression of ICSBP. Our prior studies implicated ICSBP-deficiency in Fas-resistance in CML via the ICSBP target gene PTPN13. Our current studies implicate ICSBP-deficiency in increased Bcatenin expression in CML. These results suggest that decreased ICSBP expression drives multiple aspects of the poor prognosis LSC phenotype in CML.

Disclosures:

No relevant conflicts of interest to declare.
Role of a Conserved Helicase Motif in the Transformation of Primary Human CD34+ Cells by the NUP98-DDX10 Oncogene.
Authors: Yassin Enas R , Abdul-Nabi Anmaar M , Takeda Akiko , Yaseen Nabeel R.
Published: October 2019

Abstract 2966

Poster Board II-942

NUP98 gene rearrangements occur in acute myeloid leukemia and other hematopoietic malignancies, and result in the expression of fusion proteins. One of the most frequent NUP98 fusions is NUP98-DDX10 that consists of an N-terminal portion of NUP98 and a C-terminal segment of DDX10, a putative DEAD-box RNA helicase. Here we express NUP98-DDX10 in primary human CD34+ hematopoietic cells and show that it localizes within the nucleus in a punctate distribution. It dramatically increases the proliferation and self-renewal of primary human CD34+ cells and disrupts their erythroid and myeloid differentiation. Expression gene profiling shows dysregulation of many genes by NUP98-DDX10 in primary human CD34+ cells starting within 6 h. Comparison of the dysregulome of NUP98-DDX10 to that of another leukemogenic NUP98 fusion, NUP98-HOXA9, reveals a number of genes dysregulated by both oncoproteins, including HOX genes, COX-2, MYCN, angiopoietin-1, renin, HEY1, SOX4, and others, that may account for the induction of AML by these and other NUP98 fusion oncogenes. The HRAGRTAR sequence in the DDX10 portion of NUP98-DDX10 corresponds to a major motif shared by DEAD-box RNA helicases that is required for their ATP binding/hydrolysis, RNA-binding, and helicase functions. Mutating this motif diminished the transforming ability of NUP98-DDX10, indicating that it plays a role in leukemogenesis. These data demonstrate for the first time the transforming ability of NUP98-DDX10 and show that it is partially dependent on one of the consensus helicase motifs of DDX10. They also point to common pathways that may underlie leukemogenesis by different NUP98 fusions.

Disclosures:

No relevant conflicts of interest to declare.
Organization and Physiologically Mediated Differential Release of Angiogenic Regulatory Proteins From Distinct Platelet Alpha Granules.
Authors: Battinelli Elisabeth , Italiano Joseph
Published: October 2019

Abstract 3045

Poster Board II-1021

In addition to their primary roles in hemostasis and thrombosis, platelets have been reported to participate in other physiological and pathological processes, including but not limited to inflammation, wound healing, and tumor metastasis. Although platelets are presumed to contribute to new blood vessel growth by providing numerous pro-and anti-angiogenic factors, the cellular and molecular basis by which platelets regulate angiogenesis is poorly understood. Previously we have shown that platelets differentially package angiogenic regulatory proteins in their alpha granules. More recently, we have further defined the organization of angiogenic proteins within platelet alpha granules using immunofluorescence and immunogold labeling experiments and have uncovered at least three distinct types of alpha granules. These granules were found to contain selective members of pro- and anti-angiogenic regulatory proteins, respectively. In addition, we have analyzed platelets from nine patients with metastatic renal cell carcinoma using immunofluorescence labeling for VEGF and endostatin. In comparison to volunteer control platelets without known malignancy, there is a significant increase in granules containing VEGF in patients with metastatic disease, strongly suggesting a shift of the angiogenic balance towards a more pro-angiogenic environment in the platelets from patients with malignancy. Previously we have shown that differentially packaged alpha granules are susceptible to selective release in response to proteinase-activated receptors. To explore whether selective release also occurred in physiologic conditions, we activated human platelets with known platelet agonists and looked for evidence of selective release. We have demonstrated that differential release of VEGF can be physiologically regulated by activation with ADP. Human platelets exposed to 50uM ADP result in release of VEGF and retention of endostatin, both by immunofluorecence labeling and by ELISA assay. This study provides mechanistic evidence to support an interplay between tumor cells and platelets, by illustrating a key role for platelets in tumor angiogenesis through the delivery of specific angiogenic factors. This work has implications beyond basic platelet biology as it provides a model for the platelet as a delivery system, with important clinical implications.

Disclosures

No relevant conflicts of interest to declare.
Novel SIRT1 Agonists, SRT501 and SRT2183, Induce Expression of DNA Repair Genes and Apoptosis of Ph− Acute Lymphoblastic Leukemia Cells Alone and in Combination with the HDAC Inhibitor LBH589..
Authors: Scuto Anna , Kirschbaum Mark , Cermak Jennifer M , Atadja Peter , Jove Richard
Published: October 2019

Abstract 3083

Poster Board III-20

Histone Deacetylase Inhibitors (HDACi) such as LBH589, which inhibit the zinc containing catalytic domain of HDAC of classes I, II, and IV, demonstrate activity against various malignancies, particularly lymphoid malignancies. SIRT1 is an NAD+ dependent class III histone deacetylase, which deacetylates histones as well as non-histone proteins and is not affected directly by HDACi such as LBH589. It remains controversial whether inhibition of SIRT1 or its activation is more efficacious in anticancer therapy. We have studied the activity of two novel SIRT1 activators, SRT501 and SRT2183, in Philadelphia chromosome negative acute lymphoblastic leukemia (ALL) cell lines. Both pre B (NALM-6, Reh) and T cell (MOLT-4) ALL lines were treated with either SRT501 or SRT2183, as well as in combination with LBH589 and evaluated for biological and gene expression responses. SRT501 induced growth arrest and apoptosis at doses ranging from 10-100 uM, with even the lowest doses inhibiting growth at 72 hours. SRT2183 is much more potent, with growth arrest and apoptosis induced at doses ranging from 1-20 uM. PCR array analysis revealed that SRT2183 treatment leads to increased mRNA levels of pro-apoptosis, growth arrest, and DNA damage response genes. We have previously demonstrated that the activity of LBH589 is mediated in part through upregulation or acetylation of proteins involved in the DNA damage response pathways. Quantitative real-time PCR confirms that the combination of LBH589 with SRT2183 leads to significantly higher expression of GADD45A and GADD45G than either agent alone. The combination of LBH589 plus SRT2183 showed enhanced inhibition of c-Myc protein levels, phosphorylation of H2A.X, and interestingly, increased acetylation of p53 (acetylation of p53 was not seen with SRT2183 alone). In summary, the novel SIRT1 activators SRT501 and SRT2183 show growth inhibitory and pro-apoptotic activity in Ph- ALL alone and enhanced activity in combination with LBH589. Clinical studies of these agents, particularly in combination with HDACi are warranted.

Disclosures

Kirschbaum: Novartis: Consultancy. Cermak:Sirtris: Employment. Atadja:Novartis: Employment.