bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel, Potent and Selective JAK2 Inhibitors.
Authors: Radimerski Thomas , Baffert Fabienne , Regnier Catherine H. , De Pover Alain , Pissot Carole , Gerspacher Marc , Brueggen Josef , Tavares Gisele , Blasco Francesca , Ledieu David , Nolan Lynda , Ruetz Stephan , Chene Patrick , Erdmann Dirk , Drueckes Peter , Furet Pascal , Lang Marc , Trappe Joerg , Vangrevelinghe Eric , Wartmann Markus , Hofmann Francesco
Published: October 2019

Abstract 3777

Poster Board III-713

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and from primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative disorders and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2V617F and JAK2 wild type enzymes by novel small molecule inhibitors, which act in an ATP-competitive manner. The profile of a lead compound from this class will be presented that displays more than twenty-fold selectivity towards JAK2 within the JAK family, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, the inhibitor exhibited good oral bioavailability and is efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2V617F cell-driven mouse mechanistic model as well as polycythemia and extramedullary erythropoiesis in mouse and rat models.

Disclosures:

Radimerski: Novartis: Employment, Equity Ownership. Baffert:Novartis: Employment. Regnier:Novartis: Employment. De Pover:Novartis: Employment. Pissot:Novartis: Employment. Gerspacher:Novartis: Employment. Brueggen:Novartis: Employment. Tavares:Novartis: Employment. Blasco:Novartis: Employment. Ledieu:Novartis: Employment. Nolan:Novartis: Employment. Ruetz:Novartis: Employment. Chene:Novartis: Employment. Erdmann:Novartis: Employment. Drueckes:Novartis: Employment. Furet:Novartis: Employment. Lang:Novartis: Employment. Trappe:Novartis: Employment. Vangrevelinghe:Novartis: Employment. Wartmann:Novartis: Employment. Hofmann:Novartis: Employment.
Thrombin Generation in Pediatric Patients Treated with L-asparaginase.
Authors: Tousovska Katerina , Zapletal Ondrej , Vytiskova Sona , Slanska Martina , Ruzickova Petra , Sterba Jaroslav
Published: October 2019

Abstract 3983

Poster Board III-919

Introduction

Treatment with L-asparaginase is associated with an disturbance of coagulation cascade. Increased thrombin generation is the most frequently cited coagulation anomaly associated with L-asparaginase treatment. Until now, almost all studies aimed on estimation of thrombin generation, have used circulating procoagulant markers such are thrombin/antithrombin complexes(TAT),D dimers(DD),prothrombin fragments (F1,F2).Calibrated automated thrombogram (CAT) allows the precise estimation of the whole amount of thrombin generated in vivo at present time. We show the first data on thrombin generation measured by CAT among pediatric patients with acute lymphoblastic leukemia treated with L-asparaginase.

Patients and methods

Thrombin generation was measured in platelet poor plasma of 18 patients (age 7-17years) by means of CAT. Samples were obtained at pre-defined time points during the induction and reinduction phase of protocol ALL-IC BFM 2000. We simultaneously checked the APTT,PT,AT III,fibrinogen and DD from each blood sample. Detailed patientsx data were collected prospectively.

Results

We collected 22 series of CAT measurements from 18 patients. The average endogenous thrombin potential (ETP) value was 1237,64±334,49nM/min during protocol I(PI) and 892,31±405,03 during protocol II resp.III(PII/III) (p .000013). Maximum ETP was reached on day 24 in PI (1310,15±256,3nM/min) and on day 15 in PII/III (978,20±262,30 nM/ min). The average ETP value in 20 healthy controls was 1218,12±129,00 nM/min. Three patients developed deep venous thrombosis (DVT) during the study period, all of them on PI. Their average ETP value on the last measurement before thrombosis was 1510,54±78,6 nM/min. Four patient had an infection gr. III/IV, according WHO, during the study period. Their average ETP value after the infection was 1711,43±106,0 nM/min. Two of those four patient developed DVT within 5 resp.8 days after the first sign of infection. Fibrinogen level <1g/l was detected in 19 samples out of 104. From those 19 samples with fibrinogen < 1,0,the ETP value from the same sample was normal in 14 cases and low in 5 cases. There were no bleeding episodes in that time.

Conclusions

Thrombin generation is preserved in majority of patients treated with L-asparaginase in accordance with ALL-IC BFM 2000 protocol, despite the associated hypofibrinogenemia. Patients on induction treatment generate significantly more thrombin than patients on reinduction treatment. Our results show the sharp increase in ETP right after the infection with risk of DVT. Our data should be proved on larger group of patients.

Disclosures:

No relevant conflicts of interest to declare.
Subsegmental Pulmonary Embolism Diagnosed by Computed Tomography: Incidence and Clinical Implications. A Systematic Review and Meta-Analysis of the Management Outcome Studies.
Authors: Carrier Marc , Righini Marc , Wells Phil , Perrier Arnaud , Anderson David , Rodger Marc , Le Gal Gregoire
Published: October 2019

Abstract 4002

Poster Board III-938

Background

Multiple-detectors CTPA appears to have a higher sensitivity for PE as compared to single-detector CTPA. In particular, multiple-detectors CTPA allows better visualization of segmental and subsegmental pulmonary arteries, hence the proportion of patients with suspected PE in whom isolated subsegmental thrombus are reported might be higher using multiple-detectors CTPA.

The clinical significance of subsegmental PE is unknown. In the PIOPED study, PE limited to subsegmental pulmonary arteries were most prevalent among patients with low-probability ventilation/perfusion (V/Q) scans. Patients with non diagnostic (low or intermediate probability) V/Q scans can be safely managed without anticoagulation. Nonetheless, patients with isolated subsegmental PE detected on CTPA are more commonly receiving anticoagulation than not.

Purpose

To determine whether multiple-detectors CTPA increases the proportion of PE diagnosis limited to subsegmental arteries and to assess the safety of diagnostic strategies based on CTPA.

Data Source: A systematic literature search strategy was conducted using MEDLINE, EMBASE, the Cochrane Register of Controlled Trials and all EBM Reviews.

Study Selection

Twenty four articles met all the inclusions criteria (21 prospective cohort studies; 3 randomized controlled trials).

Data extraction

Two reviewers independently extracted data onto standardized forms.

Data Synthesis

A total of 2674 patients with suspected PE were included in the analyses. Of these, 1140 and 1534 patients underwent a single and multiple-detectors CTPA respectively.

Conclusion

The use of multiple-detectors CTPA in diagnostic strategies for PE appears to increase the proportion of patients diagnosed with subsegmental PE with comparable outcomes in patients with negative tests. This suggests that patients with subsegmental PE appear to not require anticoagulation.

Disclosures:

Rodger: Biomerieux: Research Funding; Boehringer Ingelheim: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Leo Pharma: Research Funding; Bayer: Research Funding; GTC Therapeutics: Research Funding.
Minimal Risidual Disease monitoring in Childhood Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia: Preliminary Results of JPLSG Ph+ALL04.
Authors: Kawasaki Hirohide , Adachi Souichi , Ohara Akira , Kikuta Atsushi , Sato Atsushi , Takahashi Hiroyuki , Chin Motoaki , Tsuchida Masahiro , Hamamoto Kazuko , Matsumoto Kouichi , Yagi Keiko , Yabe Hiromasa , Yoshida Makoto , Watanabe Tsutomu , Manabe Atsushi
Published: October 2019

Abstract 4116

Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) is very rare and poor prognostic condition in children. Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) conducted first nationwide clinical trial for Ph+ALL in children (Ph+ALL04). Ph+ALL04 consists of five therapy phases; Induction phase (five drugs induction), Intensification phase (high-dose CA and BFM Ib), Re-induction phase (four drugs re-induction), Imatinib mono-therapy phase, and hematopoietic stem cell transplantation phase (VP+CY+TBI condioning). Before and after each phase, BCR-ABL minimal residual disease (MRD) was monitored by the real-time PCR method. During the period 2004-08, 44 patients were enrolled in the Ph+ALL04 study. Out of 44 patients, five were resistant to Induction Phase and did not achieve CR. Their levels of BCR-ABL transcript at diagnosis were significantly higher than that of patients achieved CR after Induction phase. After Induction phase, 39 patients achieved CR, and seven of 39 patients also achieved MRD negative. After Intensification phase, two patients relapsed. Thirty-four patients were still in CR, and 10 patients also achieved MRD negative after Intensification phase. One patient relapsed after Re-induction phase and three patients relapsed after Imatinib mono-therapy phase. Thirteen patients were MRD negative before Imatinib mono-therapy phase, and 14 patients were MRD negative after Imatinib mono-therapy phase. Before Hematopoietic stem cell transplantation phase, 14 patients were MRD negative. Twenty-six patients received hematopoietic stem cell transplantation. Out of 26 patients, three relapsed after hematopoietic cell transplantation. Twenty-three patients continued to be in CR and MRD negative for median 2 years after hematopoietic stem cell transplantation. In conclusion, amount of BCR-ABL transcript is a possible predictor for achieving remission after induction therapy in Ph+ALL children. MRD detection during therapy is confirmed the important prognostic indicator in the context of the intensive chemotherapy and hematopoietic stem cell transplantation in Ph+ALL children.

Disclosures:

No relevant conflicts of interest to declare.
Decitabine Can Induce Complete Cytogenetic Responses When Used as Initial Therapy in Older AML Patients.
Authors: Cashen Amanda F
Published: October 2019

Abstract 4123

Background

The incidence of acute myeloid leukemia (AML) increases with advancing age. At present, there are limited treatment options available for older patients because of lack of efficacy and increased toxicity. In a previously reported Phase II AML trial (n = 55), older patients not previously treated for AML experienced an overall response rate of 25% with decitabine (Cashen et al. Blood 2008). Response rate was similar in patients with poor or intermediate cytogenetic risk at baseline. In addition, 5 (15%) of the 34 patients who had cytogenetic abnormalities at baseline achieved a complete cytogenetic response in this trial. As older AML patients are more likely to present with unfavorable cytogenetic profiles, it was of interest to review the patient characteristics and describe the responses of the 5 patients who had a complete cytogenetic response.

Methods

The patients for this analysis were from a multicenter, Phase II study. In this study, patients over the age of 60 with AML (>20% bone marrow blasts) and no prior therapy for AML were treated with decitabine, 20 mg/m2 IV for 5 consecutive days of a 28-day cycle until disease progression or unacceptable adverse event. Cytogenetic profile was assessed at screening and after every cycle, beginning with cycle 2.

Results

The 5 patients (2 males, 3 females; 5 white; 2 M1, 1 each for M2, M6, and M7) with a complete cytogenetic response had baseline characteristics that are associated with high risk. Three were >70 yrs of age, and all 5 had AML secondary to prior therapy (n=2) or MDS (n=3). Four patients had poor-risk cytogenetics: 2 pts with -7, -5q, and ≥ 3 abnormalities and 2 pts with deletions of chromosome 5 and ≥ 3 abnormalities. One patient had intermediate-risk cytogenetics. The median baseline bone marrow myeloblast count was 33% (range 21 to 60). These patients were treated with a median 10 cycles (range 6 to 19) of decitabine. The median time to a complete cytogenetic response was 5 cycles (range 2.3 to 6). The median time to relapse from a complete cytogenetic response was 8.1 cycles (range 3.4 to 19.8). The overall median survival was 21.6 months (95% CI: 9.5, 24.7), which compares favorably to the median overall survival of the entire study cohort (21.6 months vs. 7.7 months, Cashen et al. Blood 2008).

Conclusions

Treatment with decitabine can induce complete and durable cytogenetic responses, even in older patients with high risk disease characteristics and poor risk cytogenetics. Patients who achieve a cytogenetic response may have a survival benefit. A presently ongoing Phase III survival trial with decitabine will help further characterize the effect of decitabine on cytogenetic responses in older AML patients.

Disclosures:

Cashen: Eisai, Inc.: Consultancy.
Analysis of the Coagulation Factor XII 46 C/T Polymorphism in a Pedigree and Related Literature Review.
Authors: Chang Da-Yu , Ouyang Jian , Zhou Rong-Fu , Shao Xiao-Yan
Published: October 2019

Abstract 4210

To detect the gene mutation of a pedigree with coagulation factor XII (FXII) deficiency.

Methods

The peripheral blood samples were collected from the proband and his parents, and the plasma FXII:C were determined. All the exons and exon-intron boundries of F12 gene were amplified with PCR and sequenced directly.

Results

The activity of FXII were 52.5%, 78.6% and 89% for the proband, his father and mother, respectively. Direct sequencing suggested that the proband was 46 T/T genotype in exon1, while both his parents were 46 C/T genotype in the same alle. No other base change was detected in F12 gene.

Conclusion

The 46 T/T polymorphism of F12 gene might be the cause of FXII:C deficiency for the propositus.

Disclosures:

No relevant conflicts of interest to declare.
CD200 Is Preferentially Expressed in CD138 dim Multiple Myeloma Cells: Implications for a Myeloma Stem Cell Subpopulation with Potential Immune-Resistance.
Authors: Chen Xiaochuan , Stein Rhona , Goldenberg David M.
Published: October 2019

Abstract 4877

Introduction

The CD138negCD20+ cell population has been suggested as the putative multiple myeloma (MM) cancer stem cells, both in MM cell lines and in patient specimens. CD200 is an immunosuppressive membrane glycoprotein reported to be co-expressed with other stem-cell markers in prostate, breast, brain, and colon cancers. Also, CD200 is a negative prognostic factor for MM. It remains unknown whether CD200 is expressed in the CD138negCD20+ MM cell population, the putative MM cancer stem cells. Here we addressed this question by assessing CD200 expression in different subpopulations of MM cells according to their CD138 expression levels.

Methods

Two MM cell lines (RPMI8226 and NCI-H929) were co-stained with FITC-labeled anti-CD200, PE-labeled anti-CD20, and APC-labeled anti-CD138 antibodies for multicolor flow cytometry analysis. The cells were gated into 4 subpopulations (h1>h2>h3>h4 corresponding to CD138 expression levels: high>dim>low>negative). In each gated cell population, CD200 expression was assessed, and the CD200+ and CD200neg cell subpopulations were further gated for analysis of their CD20 expression levels.

Results

In the RPMI 8226 cell line, the distribution of gated h1, h2, h3, and h4 populations was 87.51%, 5.86%, 5.26%, and 1.29%, respectively. In the h2 (CD138dim) population, 22.88% of the cells were CD200+. In contrast, CD200 was expressed at much lower levels in the other three populations, ranging from 7.28% (h1), 6.65% (h3), to 0.48% (h4). In CD200+ cells from the gated h1, h2, h3, and h4 population, CD20+ cells were 19.50%, 23.03%, 27.67%, and 18.75%, respectively, while in the CD200neg cells, CD20+ cells were 20.79%, 22.50%, 25.08%, and 28.02%, respectively. In the NCI-H929 cell line, 18.59% of the cells in the h2 population were CD200+, whereas only 1.61%, 1.69%, and 0.47% of the cells in the h1, h3, and h4 populations were CD200+. In each population, there were more CD20+ cells in CD200+ cells than in CD200neg cells, which were 24.52% vs 0.04% (h1), 41.94% vs 11.34% (h2), 11.11% vs 3.06% (h3), and 50.00% vs 2.25% (h4), respectively.

Conclusions

These results demonstrate that the immunosuppressive molecule CD200 is preferentially expressed in a CD138dim subpopulation of multiple myeloma cells. Depending on cell line, the putative myeloma stem cell marker CD20 is either preferentially, or not preferentially, expressed in the CD200+ cells, suggesting that an immune-resistant subpopulation of MM stem cells might exist, which may have important implications for designing immunotherapeutic approaches to treat this disease.

Disclosures

Goldenberg: Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Oncogenic RAS Enables DNA Damage- and p53-Dependent Differentiation of Acute Myeloid Leukemia Cells in Response to Chemotherapy.
Authors: Meyer Mona , Rübsamen Daniela , Slany Robert K. , Illmer Thomas , Stabla Kathleen , Roth Petra , Stiewe Thorsten , Eilers Martin , Neubauer Andreas
Published: October 2019

Abstract 5040

Introduction

Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and patients with RAS mutations benefit most from high-dose cytarabine as postremission therapy (Neubauer et al., J Clin Oncol 2008). The molecular reason for this phenomenon is not well understood.

Methods

We used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. In addition, we used primary human inversion 16 positive AML samples with or without oncogenic RAS mutations to corroborate our findings.

Results

There was no difference in cell cycle kinetics, apoptosis or cellular senescence in MLL-ENL cell expressing control vector vs. MLL-ENL cells expressing oncogenic RAS in response to cytarabine treatment. However, we observed an increased activation of DNA damage checkpoints in MLL-ENL-cells expressing oncogenic RAS after incubation with chemotherapeutic agents. This resulted in an Atm/r as well as p53-dependent genetic program causing dramatically reduction of clonogenicity in MLL-ENL cells expressing oncogenic RAS due to induction of myeloid differentiation. Co-expression of dominant – negative p53 completely abolished this differentiation resulting in the rescue of the clonogic potential. Activation of p53 as a result of inhibition of Mdm2-mediated degradation of p53 further enhances this myeloid differentiation. Of note, in primary AML cases, oncogenic RAS also was associated with a more differentiated phenotype.

Conclusions

The data can explain the beneficial effects observed in AML patients with oncogenic RAS mutations treated with high dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy since stronger acitvation of p53 by oncogenic RAS and chemotherapeutic agents cause the differentiation of hematopoietic stem cells to more differentiated progenitors. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.

Disclosures

No relevant conflicts of interest to declare.
Antiphosplipid Antibodies and Thrombosis in Cancer Patients.
Authors: Jajeh Ahmad
Published: October 2019

Abstract 5071

Antiphospholipid antibodies,Abs are common in hospitalized patients,particularly with cancer and acquired human virus immunodeficieny HIV/AIDS. The role of antiphospholipid antibodies in deep vein thrombosis DVT, pulmonary embolism PE and thrombocytopenia is underestimated in clinical practice. The matter is also complicated by many other confounding factors affecting the interpretation of the result for lupus anticoagulant LAC; a coagulation/qualitative test and ELISA test for anticardiolipin antibodies(quantitative). Anticoagulation therapy, presence of antithrombin antibodies, coagulation factors deficiency, the lag time between collection of blood and testing, reagent used and the dilution are all contributing to the accuracy of results. In this study all patients with cancer and thrombosis, thrombocytopenia with coagulopathy not related to coumadin or disseminated intravascular coagulopathy or coagulation factors deficiency were tested for antiphospholipid antibodies. A total of three hundred patients over a year period were randomly selected. All patients were consulted for hematological evaluation. Fifty patients had thrombotic events. this include DVT with or without PE. Eighty percent had cancer. Twenty percent had other problems including HIV infection or AIDS. Female:male ratio is 1:1. Fifty percent were LAC positive with moderately to high titers for phospholipid antibodies. Thirty percent were LAC borderline with discordant results for ELISA anticardiolipin antibodies IgG and IgM. All patients had negative workup for other hypercoagulability panel. This study was done at a single institution with limited internal bias regarding ordering physician. In conclusion: This study indicates the impact of antiphospholipid antibodies in the pathophysiology of thrombosis. Larger prospective multicenter trials needed to confirm this fact.

Disclosures

No relevant conflicts of interest to declare.
Mitogen Stimulated Direct Antiglobulin Test Compared with Traditional Methods for the Detection of Anti-RBC Autoimmunity.
Authors: Barcellini Wilma , Revelli Nicoletta , Imperiali Francesca Guia , Villa Maria Antonietta , Manera Maria Cristina , Paccapelo Cinzia , Zaninoni Anna , Zanella Alberto
Published: October 2019

Abstract 5091

Background

The diagnosis of autoimmune hemolytic anemia (AIHA) is based on a positive direct antiglobulin test (DAT), which is performed by various methods with different sensitivity. Recently mitogen stimulated (MS)-DAT was suggested able to disclose a latent anti-erythrocyte autoimmunity Aims: To compare different traditional (tube, microcolumn and solid phase) and new (mitogen stimulated, MS)-DAT methods in different diagnostic conditions: 54 consecutive cases of hemolytic anemia of suspected autoimmune origin, 28 idiopathic AIHA in clinical remission, and 12 difficult cases of DAT-negative AIHA, and 2) to correlate results with hematologic and hemolytic parameters.

Methods

DAT tube, microcolumn, solid phase, and eluates were performed by standard techniques. MS-DAT was performed by stimulating whole blood with PHA, PMA and PWM and antibodies were detected by competitive solid phase ELISA.

Results

Forty out of 54 consecutive cases of suspected AIHA were positive by one or more tests namely 14 DAT-tube, 19 microcolumn, 19 solid phase and 35 MS-DAT. Among the 14 DAT-tube positive cases, 11 were confirmed by all test, and 3 by one or more (1 microcolumn, 1 solid phase, and 1 MS-DAT), eluates were positive in 11, and the majority of patients (10/14) showed hemolytic anemia. As regards the 26 DAT-tube negative cases, 7 were positive in microcolumn and solid-phase (eluates positive in 2/8, panreactive), and 16 in MS-DAT, although in both groups anemia and hemolytic signs were less clear. Mitogen stimulation increased the amount of RBC-bound IgG in all groups, suggesting that MS-DAT could disclose a latent autoimmunity. Tube-negative/other methods positive cases included patients with B-CLL, myelodysplasia/aplasia, and thalassemia intermedia, in which autoimmune phenomena are more frequently observed than overt clinical autoimmune diseases. MS-DAT failed to detect anti-erythrocyte antibodies in half cases AIHA in clinical remission which still were tube-positive. Finally, MS-DAT was the only positive test in 10 cases of AIHA of difficult diagnosis, and mitogen stimulation allowed the identification of autoantibody specificity in culture supernatants of 2 cases which gave weak positive results in microcolumn/solid phase only.

Conclusion

We concluded that a battery of tests rather than a single test is recommended for the diagnosis of AIHA, depending on the specific clinical context: tube is still a good choice for first screening, microcolumn and solid-phase, which are the automated routine, should be confirmed by a positive eluate to diagnose AIHA, although positivity without a clinical equivalent may be taken into account as part of an autoimmune habitus. MS-DAT could be considered as an additional test for selected cases of difficult diagnosis.

Disclosures

No relevant conflicts of interest to declare.
Genome Variation and Donor-Recipient Compatibility in Graft-Versus-Host Disease
Authors: McCarroll Steven A
Published: October 2019

Abstract SCI-12

Graft-versus-host disease (GVHD) after hematopoetic stem cell transplantion (HSCT) arises from differences between the genomes of donor and recipient. The fact that GVHD is common when HSCT involves HLA-identical siblings but almost never occurs when HSCT involves identical twins has long indicated that the human genome harbors histocompatibility loci outside the HLA locus. An important, unresolved question has involved what specific genomic loci might have potent effects on transplantation outcomes. Although GVHD is a disease of genome compatibility, it was until recently impossible to know all the genomic differences between any specific donor and recipient, or among the individuals in a population. 1. An ongoing revolution in genome analysis methods is making it possible to characterize genome variation almost completely. I will first describe what the complete sequencing of large numbers of human genomes in the 1000 Genomes Project is revealing about how antigen repertoire varies among the individuals in a population, or between any particular candidate donor and recipient. These results have surprising implications for research strategies to identify the genetic basis of histocompatibility barriers in human transplantation. 2. In this and earlier work, we have found a surprising number of genome polymorphisms that remove entire protein-coding genes from people's genomes. Many of these gene deletion alleles are sufficiently common that individuals inherit them from both parents and therefore lack completely an entire protein that is expressed in other individuals. The immune system of such an individual may tend to recognize epitopes encoded by that gene as alloantigens. Colleagues from the Dana Farber Cancer Institute, the Helsinki University Central Hospital and Finnish Red Cross, the University of Michigan, the Fred Hutchinson Cancer Research Center and the Broad Institute recently came together to evaluate the relationship of six of these gene deletions to GVHD in clinical cohorts. We found that donor-recipient mismatch for one of these gene deletions – deletion of the UGT2B17 gene – associated in multiple clinical cohorts with the occurrence of acute GVHD after HSCT. This same gene has been found to give rise to histocompatibility antigens in individual HSCT patients, offering a molecular and cellular mechanism for its association with GVHD. 3. Finally, I will describe a strategy for identifying the genetic bases of histocompatibility barriers among individuals, based on our emerging understanding of how the human genome varies in populations.

Disclosures:

No relevant conflicts of interest to declare.
New Strategies to Define Regulators of Fetal Hemoglobin
Authors: Orkin Stuart H.
Published: October 2019

Abstract SCI-17

The beneficial effect of hemoglobin F (HbF, α2γ2) expression on the phenotypes of sickle cell disease and the β-thalassemias has been recognized for many years. Therefore, retardation of the fetal-to-adult (HbF to HbA, α2b2) switch or reactivation of g-globin expression in erythroid cells of adults has been a long sought goal. Despite molecular analysis of the human β-globin gene cluster and intense study of transgenic mice harboring human globin genes, the nuclear factors mediating hemoglobin switching and maintaining γ-globin gene silencing have remained unknown. The situation has changed recently with the success of new genetic tools, including genome-wide association study (GWAS), in the identification of candidate gene regions in which genetic variation correlates with HbF levels. Three genomic regions have been fingered: the β-globin cluster itself, the BCL11A gene on chromosome 2, and the HB1SL-Myb region on chromosome 6. Taken together, these regions account for ∼40% of the variation in HbF levels. Functional studies have shown that BCL11A encodes a dose-sensitive, zinc-finger repressor that is required for maintenance of silencing of γ-globin in adult erythroid cells. BCL11A also controls the developmental switches from embryonic to adult β-like globins in the mouse and fetal to adult globin in human transgenes harbored in the mouse. BCL11A binds downstream of the γ-globin gene and in the locus control region, but not at the γ-promoters. Mechanistic studies reveal that BCL11A cooperates with another factor, SOX6, that binds within the γ-promoters. The identification of genetically validated regulators has therapeutic implications for directed reactivation of HbF in adults. For stage-specific repressors, such as BCL11A, inhibition of their expression, or interference with their function or interactions with other regulators, could constitute new therapeutic strategies. While transcription factors have been generally considered “undruggable”, advances in high-throughput screening of complex libraries of small molecules and in the use of RNA interference in vivo may provide new platforms for discovery and refinement of HbF inducers. The presentation will illustrate some of these potential approaches.

Disclosures:

No relevant conflicts of interest to declare.
Safety and Efficacy of 90Y Ibritumomab Tiuxetan (Zevalin®) for Untreated FollicularNon-Hodgkin's Lymphoma (FL) Patients, An Italian Cooperative Study
Authors: Pica G. , Nati S. , Vitolo Umberto , Galimberti Sara , Zinzani Pier Luigi , Cascavilla Nicola , Petrini Mario , Carella A. M.
Published: October 2019

Abstract 100

90Y ibritumomab tiuxetan (Zevalin®) combines the targeting advantage of monoclonal antibody with the radiosensitivity of FL. Previous studies showed that Zevalin resulted safe and highly effective in relapsed/refractory indolent NHL, irrespective to prior treatment with rituximab.

Based on these results, we designed a multicenter trial to evaluate the safety and the efficacy of “upfront” single-agent Zevalin in FL. The primary endpoint was the incidence of responses in terms of overall remission rate (ORR) and complete remissions (CR). The secondary endpoints were the treatment safety by monitoring hematology and biochemistry parameters as well as adverse events.

Fifty patients, with a median age of 59 years (range, 35–81), were treated. Forty-eight percent had bone marrow involvement (<25%) and 14% an elevated LDH. Thirty-four percent of patients had high risk FLIPI. Forty-six patients were also assessed by qualitative and quantitative PCR for Bcl2/IgH or IgH clonal rearrangement, for total 30 cases PCR-positive (65.2%).

Results:

The ORR was 93% (45/48) with a CR rate of 82% (41/48). Twenty-six patients, who were PCR-positive at diagnosis, were assessed at week 14. Twenty out of 26 (77%) became PCR-negative. After a median follow up of 24 months, the 2-year EFS for all patients was 85%; moreover, 15 patients (55%), who were PCR-positive at diagnosis, maintain PCR negativity.

As expected, the main toxicity was moderate myelosuppression, with 30% and 26% of patients developing Grade 3/4 neutropenia and thrombocytopenia, respectively. Very few patients required platelets transfusion (4%) or growth factor use (6%). None of the patients experienced grade 3/4 non hematologic toxicity.

In conclusion, Zevalin is highly effective and safe treatment for newly diagnosed FL patients. In the next future, the role of radioimmunotherapy - i.e. including optimal sequencing with chemotherapy - should be established in randomized studies.

Disclosures:

No relevant conflicts of interest to declare.
Antioxidant Vitamins C and E Supplementation Does Not Improve the Hemolytic Profile of Sickle Cell Anemia Patients: A Randomized, Double-Blind, Placebo-Controlled Trial
Authors: Arruda Martha Mariana A S , Mecabo Grazielle , Rodrigues Celso Arrais , Matsuda Sandra S , Rabelo Iara Baldim , Figueiredo Maria Stella
Published: October 2019

Abstract 1063

Background:

Erythrocytes of sickle cell anemia (SCA) patients continuously produce larger amounts of pro-oxidants than normal cells, and oxidative stress seems to play a significant role in the pathophysiology of this disease. In erythrocytes, oxidative stress primarily affects the membrane and results in hemolysis. The use of antioxidants in vitro reduces the generation of pro-oxidants, which could prevent adhesion and phagocytosis of oxidized erythrocytes.

Aims:

To evaluate the impact of antioxidant vitamins C (vitC) and E (vitE) supplementation in the hemolytic profile in SCA patients.

Patients and methods:

Homozygous SCA or S-β-thalassemia patients, over 18 years, were randomly assigned to receive VitC 1,400 mg + VitE 800 mg per day or placebo, administered orally for 180 days. Pregnant women and patients with iron overload out of chelation therapy were excluded. Patients were evaluated clinically and blood samples were collected at days 0 and 180 for complete blood count, automated reticulocyte count, indirect bilirubin, lactate dehydrogenase (LDH), haptoglobin, uric acid, and serum levels of VitC and VitE.

Results:

Overall, 83 patients were enrolled (44 vitamins, 39 placebo). The median (range) age was 27 (18–68) years, and 53 (64%) were female. There were no significant differences between the two groups as regards clinical complications of SCA or baseline laboratorial tests and serum vitC and vitE. Sixty percent of the patients were VitC deficient (30% with severe deficiency), 70% were VitE deficient (33% with severe deficiency) and 44% were deficient in both vitamins. Vitamin supplementation increased VitC from 27.2 to 62.6 μMol/L (p<0.0001) and VitE from 13.9 to 20.2 μMol/L (p<0.0001). No changes in vitC or vitE were observed in patients receiving placebo. No significant changes in hemoglobin levels, hematocrit, mean corpuscular volume were observed in either group. However, patients receiving vitamin supplementation presented a significant increase in the median reticulocyte count (from 152 to 195 ×106/μL, p=0.01), LDH (from 396 to 425 U/L, p=0.018), indirect bilirubin (from 1.45 to 1.73 mg/dL, p<0.0001), and uric acid (from 4.75 to 5.15 mg/dL, p=0.02), and a decrease in the haptoglobin levels (from 3.95 to 3.45 mg/dL, p=0.06).

Conclusion:

Supplementation with vitamins C and E did not improve anemia and, surprisingly, increased markers of hemolysis in patients with SCA and S-β-thalassemia. The exact mechanisms to explain our findings and their clinical significance remain to be determined.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Microparticles Increase Pro-Angiogenic Properties of Prostate Cancer Cell Lines
Authors: Dashevsky Olga , Shai Ela , Varon David
Published: October 2019

Abstract 1147

Platelets are key players in hemostasis, but are also involved in fundamental processes of vascular biology as angiogenesis, tissue regeneration and tumor metastasis. Platelet Microparticles (PMP) are vesicular membrane fragments shed from activated platelets, containing the platelet cytoplasmic content with a typical platelet membrane.

Prostate cancer is the most frequently diagnosed noncutaneous cancer in men in western countries. A major factor of the life-threatening course of this disease is the high rate of metastasis. Circulating tumor cells encounter platelets and may activate them, resulting in a production of microparticles.

We have previously reported a pro angiogenic effect of PMP, both in vitro and in vivo. We also demonstrated that prostate cancer cells that were pre-incubated with PMP acquired invasive properties. This was shown by rapid and transient increase of MMP-2 mRNA level and increased secretion of metalloproteinase-2 (MMP-2) as demonstrated by gelatin zymography.

In the present study, in vivo experiments of injection of prostate cancer cells pre-incubated with PMP to the mouse tail vain showed more rapid localization of cancer cells to the lungs, compared to untreated cancer cells. Microarray assay of prostate cancer cells pre-incubated with PMP has revealed significant differences in transcriptional regulation of genes related to cell growth, survival, tumorigenicity, invasiveness, and angiogenesis that suggested a more aggressive feature of the cells. We then investigated effect of PMP on release of the pro-angiogenic cytokine IL-8 from prostate cancer cells. RT-PCR demonstrated that protein release was accompanied by increase of IL-8mRNA and changes in regulation of related transcription factors. We have shown the involvement of the signaling molecules PI3, p38 and ERK in synthesis and expression of IL-8 from prostate cancer cells and revealed the contribution of the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) and angiopoietin 1 (Ang-1) to the process.

Better understanding of the role of PMP in cancer development and identification of the mechanisms responsible for prostate cancer progression and aggression by the tumor microenvironment may lead to the development of novel effective therapies for metastatic disease.

Disclosures:

No relevant conflicts of interest to declare.
Strong Correlation of High Endogenous Plasma Levels of Tissue Factor Pathway Inhibitor (TFPI) with Poor Blood Clotting and Corrective Action of BAX 513 Under FVIII-Inhibited Conditions
Authors: Dockal Michael , Gorczyca Monika , Knappe Sabine , Palige Michael , Ehrlich Hartmut J. , Scheiflinger Friedrich , Jilma Bernd
Published: October 2019

Abstract 1201

TFPI is the major physiological inhibitor of the extrinsic pathway of blood coagulation. Thus, blocking TFPI activity is a possible approach to treat bleeding disorders such as hemophilia A and B. BAX 513, a fucoidan derived from the brown seaweed L. japonica, and other non-anticoagulant sulfated polysaccharides (NASPs) improve coagulation in hemophilic blood and plasma (Liu et al. Thromb Haemost 2006;95:68). This procoagulant activity of NASPs can at least partially be attributed to the inhibition of TFPI. For this study, we predicted that endogenous TFPI levels are associated with decreased hemostatic capacity under hemophilic conditions. We correlated clotting parameters measured by global hemostatic assays with the total and full-length (FL) TFPI levels in human plasma samples in the absence or presence of BAX 513.

Tissue factor-triggered coagulation of normal and FVIII-inhibited plasma or blood from 30 healthy subjects was monitored by calibrated automated thrombograph (CAT) assay and rotation thromboelastometry (ROTEM). The experiments were performed with or without factor VIII blockade with a goat anti-human Factor VIII antibody to simulate acquired hemophilia. The total and FL-TFPI plasma levels were quantified by ELISA. We observed longer clotting and clot formation times by ROTEM and decreased thrombin generation in the presence of high TFPI levels in normal plasma (p<0.05). These correlations became substantially stronger when FVIII was inhibited (reaching up to r2= 0.64; p<0.001). In the presence of BAX 513 (0.1–1.2 μg/mL), most plasma samples showed a pro-coagulant response with an at least 50% increase in thrombin peak and a decrease in time to peak. The BAX 513-mediated increase in thrombin generation correlated strongly with the individual FL-TFPI plasma level. The effect was even more apparent when FVIII was inhibited, where the extrinsic pathway is the only driver of thrombin formation.

These data indicate that high endogenous TFPI levels critically compromise hemostasis in acquired factor VIII deficiency and that TFPI is a threshold factor for the intrinsic amplification of thrombin formation. As low TFPI levels are associated with increased blood clotting, TFPI inhibition is a rationale treatment approach in patients with hemophilia. Importantly, our findings validate the inhibition of TFPI activity as a mode of action for BAX 513 and related pro-coagulant compounds.

Disclosures:

Dockal: Baxter Innovations GmbH: Employment. Gorczyca:Medical University of Vienna: Employment. Knappe:Baxter Innovations GmbH: Employment. Palige:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.
Kinase Domain Mutations in JAK2V617F Confer Resistance to the Novel JAK2 Inhibitor Ruxolitinib
Authors: Deshpande Anagha , Reddy Mamatha M. , Schade Georg , Ray Arghya , Chowdary Tirumala K. , Griffin James D. , Sattler Martin
Published: October 2019

Abstract 125

The JAK2V617F kinase mutation is frequently associated with myeloproliferative neoplasms (MPNs) and thought to be instrumental for the overproduction of myeloid lineage cells. Ruxolitinib (INCB018424) and several additional small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would cause resistance to ruxolitinib. Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would lower sensitivity towards ruxolitinib, using a cell-based assay. We found 5 different non-synonymous point mutations that conferred drug resistance, including Y931C, G935R, R938L, I960V and E985K. Cells containing mutations had a 9 to 33-fold higher EC50 for ruxolitinib. Results were analyzed by evaluating the three-dimensional structure of ruxolitinib bound to the ATP binding pocket of the monomer JAK2 kinase domain, using a docking algorithm. The inhibitor is held in the pocket by polar contacts and extensive hydrophobic interactions with several residues that line the binding pocket. A880, L855, V863 and M929 interact with the inhibitor on one side, and V911 and L983 provide hydrophobic interactions on the other side. The pyrazol ring of ruxolitinib appears to have a π-π interaction with the Y931 ring. Most mutations that were identified in our screen are either residues that interact with ruxolitinib or are in proximity to the binding pocket and hence are likely to indirectly alter inhibitor binding. The structural analysis indicates that there are additional residues in the kinase domain that may be critical for optimal binding of ruxolitinib and point mutations at these sites may also alter the affinity of the drug. Our analysis further suggests that ruxolitinib and other JAK2 inhibitors may bind in a similar way to the kinase domain. Consequently, these identified mutations conferred cross-resistance to all JAK2 kinase inhibitors tested in cell growth assays, including AZD1480 (5.6 to >7-fold higher EC50), TG101348 (2.2 to 2.8-fold increase in EC50), lestaurtinib (CEP-701) (2.6 to 3.3-fold increase in EC50) and CYT-387 (5.1 to 7.4-fold increase in EC50). Surprisingly, introduction of the ‘gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (4-fold increase in EC50). In addition, the E985K and Y931C mutations reduced the sensitivity to ruxolitinib and offered a growth advantage to cells during treatment, compared to native JAK2V617F expressing cells. These results suggest that the development of point mutations in JAK2 could result in clinical resistance to a wide variety of JAK2 inhibitors currently in clinical trials in patients with myeloproliferative neoplasms.

Disclosures:

No relevant conflicts of interest to declare.
A T Cell Receptor Signaling Mutant Reveals Two Distinct Populations of IL-17 Producing CD4+ T Cells
Authors: Kim Jiyeon S , Smith-Garvin Jennifer E , Jordan Martha S , Koretzky Gary A
Published: October 2019

Abstract 186

Interleukin-17 (IL-17) producing CD4+ T cells (Th17 cells) are essential for immune responses in mucosal and epithelial sites which are the first line of host defense. Th17 cells play a critical role in the pathogenesis of many inflammatory and autoimmune diseases, and the role of IL-17 and Th17 cells in cancer has recently become the focus of extensive investigation. Most studies to date have focused on elucidating the cell extrinsic requirements for differentiation of Th17 cells from naïve CD4+ T cells in peripheral effector sites. Here we report an unconventional population of Th17 cells, “natural Th17 cells” (nTh17), that acquire effector function during development in the thymus, thereby distinguishing them from conventional Th17 cells which require antigen encounter and differentiation in the periphery. We show that these nTh17 cells are present and indeed develop in the thymus using fetal thymic organ culture. nTh17 cells express surface markers consistent with an innate and/or activated phenotype and their development is dependent on selection by MHC class II in the thymus. Yet unlike conventional CD4+ T cells, MHC class II expression on thymic cortical epithelium is not sufficient for their development, rather expression on medullary epithelium is necessary. In addition, T cell receptor (TCR) repertoire analysis of nTh17 cells revealed unique characteristics in TCR gene usage compared to conventional Th17 cells. A mouse model with a mutation in the TCR signaling protein SLP76 (SLP76 Y145F mice) further highlights the difference between the two distinct Th17 populations. SLP76 Y145F mice have increased numbers of nTh17 cells in the thymus compared to wild-type mice. However, peripheral naïve CD4+ T cells from these mice showed severely defective IL-17 production when cultured in vitro under conditions promoting Th17 cell differentiation. This defect was reflected in vivo as CD4+ T cells in the small intestinal lamina propria of SLP76 Y145F mice fail to produce IL-17. Using mixed radiation bone marrow chimeras, we found that the aberrant Th17 phenotype in the thymus and periphery of SLP76 Y145F mice is cell-intrinsic. Finally, adoptive transfer of purified nTh17 cells into RAG-deficient host mice revealed preferential homing of nTh17 cells to thymus and lung compared to other comparison/competitive populations that were co-transferred. Taken together, our data suggest a distinct population of Th17 cells that have characteristics of innate lymphocytes that function at the interface between innate and adaptive immunity. Understanding the biology of nTh17 cells will provide insight into the recently identified Th17 cells in human thymi and umbilical cord blood.

Disclosures:

No relevant conflicts of interest to declare.