bluuebunny/crossref_metadata_2025 huggingface dataset:
Knockdown of MicroRNA-10a in Acute Myeloid Leukaemia Cells Bearing the Nucleophosmin1 Mutation Causes Cell Death and Reduced Clonogenicity
Authors: Bryant Adam J , Palma Catalina A , Lutherborrow Mark , Jayaswal Vivek , Yang Yee Hwa , Ma David
Published: October 2019

Abstract 1367

Acute Myeloid Leukaemia (AML) with a mutation in the Nucleophosmin1 gene (NPM1c+) accounts for one of the largest subtypes of AML, with an unknown etiology. MicroRNA dysregulation has now been implicated in the oncogenesis of many cancers including AML. We sought to investigate the role of microRNAs in the initiation and development of AML with the NPM1c+ mutation.

MicroRNA profiling of bone marrow samples from 28 AML patients and confirmation by qRT-PCR demonstrated a unique microRNA signature in AML-NPM1c+ samples dominated by miR-10a over-expression of 19.6-fold compared to Nucleophosmin1 wild type (NPM1) samples. Functional assessments were performed in the human OCI-AML3 cell line, which is the only cell line to harbour NPM1c+. miR-10a repression was induced by transfection with miRCURY LNA microRNA knockdown probes (Exiqon). Cell growth (MTS) assay demonstrated a significant decrease of 19% in miR-10a knockdown cells compared to the Scrambled control. AnnexinV and Caspase 3 assays assessed the effect of miR-10a knockdown on apoptosis. miR-10a knockdown increased the proportion of AnnexinV positive events when compared to control treated cells by 34.9% and 39.3% at 24 and 48 hours respectively, but had no effect on Caspase 3 expression. Proliferation (BrdU uptake) assays did not show a change, however, clonogenic assays demonstrated a 26.1% decrease in colony number in miR-10a knockdown cells compared to the control. Potential mechanisms were elucidated by determining miR-10a mRNA targets in silico and confirmed by luciferase reporter assays. These included ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C.

In this study, we have demonstrated that miR-10a was highly differentially expressed between AML-NPM1c+ cells compared to leukaemic cells bearing wild type NPM1. Knockdown of miR-10a in OCI-AML3 cells resulted in increased cell death as detected by AnnexinV binding (but not Caspase 3, indicating an effect independent of the classical apoptotic pathways) and reduced clonogenic capacity. These effects are thought to occur through miR-10a mediated modulation of ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C, all of which are associated with neoplastic transformation. Taken together, our results suggest that aberrant miR-10a over-expression in AML-NPM1c+ patients promotes cell survival.

Disclosures:

No relevant conflicts of interest to declare.
Role of CD44 in Endogenous Kras G12D-mediated Myeloproliferative Neoplasm
Authors: Du Juan , Liu Yangang , Tan Lixuan , Lo Juinn Cherng , Wang Jinyong , Ryu Myung-Joem , Zhang Jingfang , Zhang Jing
Published: October 2019

Abstract 1398

Oncogenic KRAS mutations are identified in virtually all the cancer types, making KRAS one of the most frequently mutated genes in human cancers. Our lab established a mouse model of myeloproliferative neoplasm (MPN) initiated by oncogenic Kras mutation (Kras G12D) expressed from its endogenous locus. Because CD44, a cell adhesion molecule and a cell signaling regulator, was found to involve in regulating leukemic stem cells in chronic myeloid leukemia (Krause, Lazarides et al. 2006) and acute myeloid leukemia (Jin, Hope et al. 2006), we have generated compound mice expressing oncogenic Kras but deficient for CD44 (Kras G12D; CD44−/−) to examine the role of CD44 in endogenous oncogenic Kras- mediated MPN.

Our preliminary data show that loss of CD44 prolonged survival of compound Kras G12D; CD44−/− mice and greatly downregulated GM-CSF signaling in hematopoietic stem/progenitor cells. However, CD44 deficiency did not significantly affect the development of MPN, suggesting that GM-CSF signaling is not essential for Kras G12D-initiated MPN and CD44 deficiency might not affect other cytokine signaling. We are currently investigating this issue.

Because Kras G12D hematopoietic stem cells (HSCs) are required to initiate MPN phenotypes, we then examined whether loss of CD44 would affect the behavior of Kras G12D HSCs. Our preliminary results demonstrate that CD44 deficiency did not significantly alter HSC frequencies in bone marrow and spleen, nor their cell cycle profile and apoptosis status. When Kras G12D; CD44−/− bone marrow cells were transplanted into lethally irradiated mice by retro-orbital injection, ∼19% of recipient mice developed MPN, similarly to recipient mice transplanted with same number of Kras G12D cells (∼14%). These MPN mice simultaneously developed acute T-cell lymphoblastic leukemia/lymphoma. Our results suggest that CD44 is dispensable for the homing and engraftment of Kras G12D HSCs. We are currently investigating whether CD44 function is essential for the homing and engraftment of leukemic initiating cells of MPN.

Disclosures:

No relevant conflicts of interest to declare.
Investigational NEDD8-Activating Enzyme (NAE) Inhibitor, MLN4924, Demonstrates Activity Against Primary AML Blast, Progenitor and Stem Cell Populations
Authors: Sen Siddhartha , De Leon Jeanne P. , Smith Peter G , Roboz Gail J. , Guzman Monica L.
Published: October 2019

Abstract 1414

Acute myelogenous leukemia (AML) has a poor prognosis. Most patients die of either primary refractory or relapsed disease. It has been proposed that relapse may result from ineffective ablation of leukemia stem cells (LSCs) by chemotherapy. Therefore, in order to improve therapy, it is imperative to identify agents that eliminate leukemic progenitor and stem cell populations in addition to blasts. We have previously demonstrated that proteasome inhibition can result in selective ablation of LSCs, without harming normal hematopoietic stem cells (HSCs). Thus, we sought to investigate the activity of a more specific inhibitor of the ubiquitin-proteasome pathway, an investigational NEDD8-Activating Enzyme (NAE) inhibitor MLN4924. Treatment of primary human AML and blast crisis CML samples with MLN4924 resulted in a dose-dependent decrease in the total viability of the blast-cell populations, with an average LD50 of 0.367 μM (0.203–0.530 95% CI; N=17). The average LD50 for phenotypically described LSCs was 0.645μM (0.304–0.986; 95%CI; N=12). Interestingly, when performing progenitor/stem functional assays, we observed a 64% and 86% decrease in colony formation after treatment with 0.25μM and 1 μM of MLN4924 (p< 0.001; N=5; p< 0.001 N=8 respectively). Furthermore, MLN4924 had little to no effect on colony formation of normal hematopoietic cells. Thus, MLN4924 may impair the activity of AML stem/progenitor cells, but not normal cells, in vitro. Importantly, primary AML samples that were resistant to standard chemotherapeutics such as cytarabine in vitro were sensitive to MLN4924. Treatment of primary AML samples with MLN4924 showed increased γH2AX foci, indicating activation of stress responses. Together, these data suggest that MLN4924 can eliminate AML blasts, progenitor and stem cell populations in vitro.

Disclosures:

Smith: Millennium Pharmaceuticals: Employment.
Comparison Between the International Standardised Approach and a Single Tube Ten Colour Flow Cytometric Assay for Detection of Minimal Residual Disease in CLL
Authors: Sartor Mary M , Bradstock Kenneth Francis , Gottlieb David
Published: October 2019

Abstract 1460

The eradication of minimal residual disease (MRD) in CLL predicts for improved outcome. Approaches to CLL MRD detection vary in sensitivity and cost. Historically, CD5/CD19 co-expression with demonstration of clonality has been the principal method of MRD evaluation in CLL. However this approach is limited by the identification of normal hemopoietic cells and the inability to demonstrate light chain restriction with very low B cell numbers. More recently, an international standardised approach (ISA) to the assessment of MRD in CLL that utilises 10 monoclonal antibodies in a 4 tube test system and permits a sensitivity of 0.01% (Rawstron et al, Leukemia 2007) was published. Here we compare the ISA to a 10 colour flow cytometric assay incorporating all the mononclonal antibodies utilised in the ISA in a single tube.

Method:

Peripheral blood (n=21) and bone marrow (n=7) was collected from patients at various time points post treatment for CLL (including bone marrow from 2 patients day 30 post-allogeneic stem cell transplant). Immunophenotyping was performed using a Gallios flow cytometer (Beckman Coulter). Monoclonal antibodies were used either according to the ISA (antibody/fluorochrome combination as per Rawstron et al) or in a single tube incorporating all of the following monoclonal antibodies: CD3 ECD, CD5 PercP5.5, CD19 eFluor, CD20 PE CY7, CD81 FITC, CD22 PE, CD43 APC CY7, CD79b APC, CD38 A700, CD45 CO. A whole blood or bone marrow lyse method was used and analysis was performed according to the published ISA methodology or for the single tube using templates based on the ISA approach. Results from the two methods were compared.

Results:

Levels of residual disease in the 28 samples analysed varied from <0.01 to 22%. Sixteen samples showed residual disease of <1%. Analysing all samples showed an excellent correlation between the two methods slope = 0.989, intercept =0.1 and R(2)=0.992. There was also excellent correlation for disease levels below 1.0%, (median 0.03 range <0.01–0.63%, n=16) slope = 1.15, intercept =0.007 and R(2)=0.994. Bland Altman analysis showed a mean of 0.008 +/− 0.058 (2SD) for values below 1%.

Conclusion:

The single tube ten colour flow cytometric assay for detection of MRD in CLL gives equivalent results to the ISA. There is a potential for improved sensitivity resulting from increased event detection since there is no need to divide the sample into multiple tubes, particularly post-treatment when cell numbers are frequently limiting. The single tube assay is also simple, rapid and cost effective.

Disclosures:

No relevant conflicts of interest to declare.
Targeting AKT Signaling in Pediatric Acute Lymphoblastic Leukemia with Sulforaphane
Authors: Suppipat Koramit , Zhu Evelyn , Lacorazza Daniel
Published: October 2019

Abstract 1521

Refractory and relapse acute lymphoblastic leukemia (ALL) is one of the leading causes of cancer-related death in children. Novel agents are still in need as a frontline therapy in high risk patients and salvage agent in relapse patients. Leukemic cells acquire a survival advantage over normal blood cells by losing control of the cell cycle and activating pro-survival signals. There are emerging evidences showing that the PI3K/AKT/mTOR pathway is frequently activated in both T cell acute lymphoblastic leukemia (T-ALL) and precursor B cell acute lymphoblastic leukemia (pre-B ALL) (approximately 30% of ALL patients). Thus, drugs able to inhibit AKT signaling are potential candidates for ALL treatment. Sulforaphane (SF) is a dietary compound found in cruciferous vegetables with well-known chemopreventive and chemotherapeutic function in solid tumors by inducing apoptosis and inhibiting survival and cell division. However, the anti-leukemic capacity of SF in hematologic malignancies has not been extensively investigated. In this study, we found increased cleavage of PARP, caspase-3, caspase-8 and caspase-9 by immunoblots in pre-B and T-ALL cell lines treated with SF (5–10 μM) for 24 –48 hours. In addition, we detected increased expression of p21 and cyclin B that correlated with a G2/M block and DNA damage observed in SF treated cells by analysis of DNA content and H2AXγ expression. Noteworthy, SF-treated pre-B and T-ALL cell lines exhibit lower levels of total and phosphorylated AKT in addition to decreased levels of total and phosphorylated mTOR, which was independent of p53 and PTEN activities. Lower survival signals upon SF treatment correlated with a dose- and time-dependent cytotoxicity in pre-B ALL cell lines (Nalm-6, REH and RS-4) and T-ALL cell lines (Jurkat, RPMI, DND-41 and KOPTK1) compared to EBV-transformed lymphoblasts used as a control. Interestingly, SF showed a synergistic effect with vincristine in pre-B and T-ALL cell lines, suggesting a potential use of SF in adjunctive therapy. Aiming to support future clinical trials, we confirmed that SF is effective in lymphoblasts from pediatric patients suffering with ALL, both pre-B and T, in a time- and dose-dependent manner (95% confidence interval IC50: pre-B ALL = 9.9 – 12.13 μM, T-ALL = 8.3 –11.00 μM). To investigate whether SF is effective in vivo in a xenograft mouse model, we injected nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with Nalm-6 cells transduced with GFP-FFluc for bioluminescent imaging detection. SF treatment (2 mg i.p. daily) significantly reduced tumor burden compared to the control group (injected with vehicle). Collectively, our data show that SF has anti-leukemic properties in ALL, both in primary samples from patients and cell lines, by inducing cell death selectively in leukemic cells likely by inhibiting AKT-dependent survival signals.

Disclosures:

No relevant conflicts of interest to declare.
Thymic Dendritc Cells Generated Ex Vivo Induce Donor Regulatory T Cell Differentiation and Hasten Immune Reconstitution After Hematopoietic Stem Cell Transplantation
Authors: Dallas Mari Hashitate , Langfitt Deanna , Busby Kenneth
Published: October 2019

Abstract 1905

Dendritic cells (DCs) are the first committed cells to engraft in the thymus after hematopoietic stem cell transplantation (HSCT). The critical role of thymic DCs in ensuring efficient tolerance and selection has been well demonstrated, however its role in facilitating donor engraftment has not been reported. Here we show DCs accelerates thymic reconstitution by inducing regulatory T cells (Tregs) differentiation and enhancing T cell recovery after HSCT. Lethally irradiated CD45.2 C57BL/6 control group received 103 CD45.1 lin−sca-1+c-kit+ (LSK) hematopoietic stem cell progenitors while the DCs group received 103 CD45.1 LSK cells along with 103 CD45.2 GFP+ DCs. DCs were generated ex vivo using bone marrow from CD45.2 GFP+ CD57BL/6 mice and cultured for 7 days with GMCSF. At 4 and 7 days after HSCT, the thymus of DC group contained 1.8 and 4.2- fold higher number of thymocytes (p<0.05) and a 3.2 and 7.4-fold, respectively, higher number of donor derived thymoctyes compared to the control group (p<0.05). Moreover, thymuses of the DCs group had GFP+ CD11c+ cells present in the medulla and 5.6-fold increase in the number of donor derived FoxP3+ Tregs compared to control confirmed by immunohistochemistry (IHC). Furthermore, thymic recovery scored by a pathologist blinded to the groups found significant increase in lymphoid regeneration (H&E) and higher number of CD3+ lymphoid aggregates (IHC) in the DC group compared to control group that had severe, diffuse lymphoid depletion. Lastly, at 2 and 4 weeks after HSCT, peripheral blood of DCs group contained 2.6 and 4.8-fold, respectively, higher numbers of CD3+ cells derived from donor LSK cells compared to the control group (p<0.05). Here, we demonstrate that donor DCs efficiently migrate and home to the thymic medulla and hasten thymic recovery as demonstrated by the higher number of total thymoctyes. Furthermore, DCs facilitate thymic engraftment as shown by increase number of donor derived FoxP3+ Tregs and thymocytes. Lastly, recipients of DCs have earlier generation of de-novo donor derived CD3+ T cells in the peripheral blood. By using the GFP+ cells along with donor LSK cells, we were able to confirm that the facilitation of early thymic recovery was due to the increased engraftment of the donor cells rather than autologous recovery of the host. In conclusion, this study demonstrates that DCs committed prior to thymic entry maintains the ability to home to the medullary region and facilitate thymic recovery by enhancing Tregs differentiation. Thus, ex vivo generation of donor DCs to augment a HSC graft may

Disclosures:

No relevant conflicts of interest to declare.
Cost of Autologous and Allogeneic Stem Cell Transplantations for Hematological Disease: A Dutch Multicenter Daily Practice Study
Authors: Blommestein Hedwig M , Verelst Silvia GR , Cornelissen Jan J , Huijgens Peter C , Blijlevens Nicole MA , Groot Carin A Uyl-de
Published: October 2019

Abstract 2079

Background:

Peripheral blood stem cell transplantations (PBSCT) are very expensive life-saving medical procedures carried out in patients with hematological disease. The current tariffs are expected to be too low due to developments in the treatment protocols. A revision of the tariffs is urgently necessary. We calculated the daily practice cost of PBSCT for treating hematological diseases in Dutch practice, to provide a proper basis for revising hospital budgets.

Patients and methods:

From three Dutch university hospitals, we randomly selected 191 who underwent an autologous (auto) or allogeneic (allo) PBSCT in 2008 or 2009. The alloPBSCT were subcategorized into sibling, matched unrelated donor (MUD) and unrelated cord blood (UCB). We obtained data from hospital registrations to study all treatment related activities. Unit prices were based both on real costs and tariffs (base year 2010). Thereafter, the average costs per patient per PBSCT and per period were calculated. The total cost included the selection and harvesting, transplantation and 1-year follow-up.

Results:

The average cost per patient of autoPBSCT were € 45,670. The cost of sibling alloPBSCT were € 101,919. The average cost of transplantations from an unrelated donor was much higher: € 171,478 for MUD and € 254,689 for UCB alloPBSCT. Hospital days, laboratory procedures and donor search were the largest cost components and mainly responsible for differences between the four types of PBSCT. None of the patient characteristics were correlated with average cost. The costs calculated in this study are above current reimbursement. The difference is significant (p=0.043) and depending on the type of PBSCT, the shortfall varied between 2% and 100%.

Conclusion:

Average cost of AutoSCT and alloSCT lay above current reimbursement levels. Appropriate financing is necessary to guarantee the continuation of PBSCT in Dutch patients according to current indications. The costs calculated in this study provide reliable input for economic evaluations and cost-effectiveness studies.

Disclosures:

No relevant conflicts of interest to declare.
Interaction of Multimerin 1 with Collagens: Role in Platelet Adhesion
Authors: Tasneem Subia , Pawlikowska Monika , Bihan Dominique , Farndale Richard W. , Hayward Catherine P. M.
Published: October 2019

Abstract 2207

Multimerin 1 (MMRN1) is a large, homopolymeric adhesive protein stored in platelets and endothelium that binds to activated platelets, endothelial cells and the extracellular matrix after agonist stimulation. MMRN1 supports platelet adhesion by von Willebrand factor (VWF) dependent and independent mechanisms, and also increases platelet adhesion to Horm collagen. Mice deficient in Mmrn1/Snca (α-synuclein) showed defective platelet adhesion to collagen in vitro and in vivo which was corrected by MMRN1. The ability of MMRN1 to support platelet adhesion in vivo and enhance platelet adhesion to collagen ex vivo, led us to explore the molecular basis of MMRN1 interactions with collagen. Solid phase binding assays were used to test MMRN1 binding to human fibril forming collagen (types I, II, III and V) and to types IV and VI collagen. Binding assays were also used to map the MMRN1 binding sites on collagen using collagen peptide toolkits III and II (which has significant similarity to type I collagen). Static adhesion assays were used to test selected collagen peptides for platelet adhesion. Platelet adhesion assays at high shear rates 1500s−1, were used to test the adhesion of washed platelets from normal and VWF-deficient subjects to type I collagen pre-treated or in fluid phase with: BSA (negative control), MMRN1, VWF or their combination. Human collagens types I, II, III and VI (p-values <0.001) but not types IV or V (p-values = 0.84 and 0.09, respectively) supported MMRN1 binding. Peptide toolkits binding studies indicated that MMRN1 bound to a single site on collagen III (peptide III-38) and to two sites on collagen II, with peptide II-9 showing much stronger binding than peptide II-44. Like the VWF binding peptide III-23 (which did not overlap the MMRN1 binding site), peptide III-38 supported platelet adhesion in combination with GFOGER, the peptide with high affinity for platelet α2β1. The possibility that MMRN1 binds to collagen at sites distinct from VWF was supported by the observations that pre-treatment of collagen I matrices, with the combination of MMRN1 and VWF, increased platelet adhesion more than MMRN1 or VWF alone (p-values< 0.001). Moreover, adhesion deficit of VWF-deficient platelets on collagen type I matrix pre-treated with a combination of MMRN1 and VWF was corrected by adding fluid phase VWF but not MMRN1 (p-values < 0.0001). Taken together, our data indicates that MMRN1 binds to different forms of human collagen that support platelet adhesion. As MMRN1 binds to sites on collagen distinct from VWF or integrin α2β1, it may be important for maximizing platelet adhesion at sites of vascular injury.

Disclosures:

No relevant conflicts of interest to declare.
Biochip Array Analysis of Various Mediators of Inflammation in Disseminated Intravascular Coagulation
Authors: Rahman Saud , Hoppensteadt Debra , Cunanan Josephine , Davis Rachael , Sadeghi Nasir , Kaul Inder , Fareed Jawed
Published: October 2019

Abstract 2302

Disseminated intravascular coagulation is a polypathologic syndrome which involves blood, endothelial and target organ dysfunction resulting in the generation of various mediators of vascular dysfunction, hemostatic aberrations and hemodynamic disturbances. In addition, various disorders of target organs such as kidney, liver, heart and brain are observed. Uncontrolled protease generation results in the formation of various mediators of inflammation such as neuron specific enolase (NSE), neutrophil gelatinase associated lipocalin (NGal) and soluble tumor necrosis factor receptor 1 (TNF R1). Endothelial damage results in the generation of thrombomodulin (TM) and endogenous coagulation/fibrinolysis results in D- Dimer formation. In order to study the circulating levels of these mediators, a Biochip array method (Randox, Oceanside, CA) was utilized with samples obtained from clinically diagnosed DIC (n=100) and normal individuals (n=10). The results are summarized in the following table. Circulating levels of these mediators were markedly increased in DIC patients in comparison to normals. The most striking increase was noted in NGal (4–6 fold) and TNF R1 (10 fold). Other mediators were also increased in the DIC group, however the data was broadly scattered. These results indicate that beside the aberration in the hemostatic system NGal and TNF R1 along with other inflammatory mediators may play a major role in the pathophysiology of DIC.MarkerNormal ControlsDIC BaselineCRP (ug/ml)3.47 + 0.913.11 + 0.2*NSE (ng/ml)6.11 + 8.610.78 + 1.6*NGAL(ng/ml)306.45 + 25.51376.56 + 48.5*TNFR1 (ng/ml)0.35 + 0.013.89 + 0.3*DD (ng/ml)232.8 + 74.41318.94 + 80.5*TM (ng/ml)3.4 + 0.33.16 + 0.2

P=<0.05

Disclosures:

No relevant conflicts of interest to declare.
Direct and Continuous Inhibition of ADAM17 Using a Novel Selective Inhibitor Restores Functional Platelet Yield From Human Pluripotent Stem Cells
Authors: Hirata Shinji , Jono-Ohnishi Ryoko , Nishimura Satoshi , Takayama Naoya , Nakamura Sou , Nakauchi Hiromitsu , Murata Takahiko , Eto Koji
Published: October 2019

Abstract 2323

Platelet transfusion is therapeutically important for patients with thrombocytopenia and/or bleeding disorders. Problems associated with a lack of donors and unknown infections in the blood have not been fully resolved, however. In that context, human induced pluripotent stem cells (hiPSCs) are a potentially abundant source of infection-free platelets. The pluripotent state of hiPSCs and their differentiation depend upon appropriate culture conditions defined in part by oxygen and temperature. We therefore initially examined whether temperatures at or below 24°C, which are required for preservation of platelet concentrates ex vivo, allow hiPSC differentiation to generate platelets. We found that only at 37°C were platelets generated. But at 37°C in vitro, platelets are subject to degradation exemplified by the shedding of GPIbα, a receptor for von Willebrand factor (vWF), which is caused by a disintegrin and metalloprotease (ADAM) 17. We therefore developed KP-457, a novel ADAM17 inhibitor that has a reverse hydroxamic acid structure and has been found safe in rats and dogs. Although inhibition of p38 MAP kinase, putatively upstream of ADAM17, reportedly inhibits GPIbα shedding in stored human platelets, even at 37°C, administration of the p38 inhibitor SB203580 induces cytotoxicity during differentiation, leading to a loss of platelet yield from hiPSCs. By contrast, KP-457 significantly protected GPIbα expression in platelets from hiPSCs and in aged human platelets in culture at 37°C. Moreover, iPSC-derived platelets generated in the presence of KP-457 displayed improved hemostatic function when studied using an imaging system that enables characterization of single-platelet kinetics during thrombus formation after laser-induced injury in vivo. We propose this new drug could markedly improve the maintenance of functional platelets generated in culture, particularly those derived from hiPSCs.

Disclosures:

No relevant conflicts of interest to declare.
Use of Genomics for Transfusion Therapy Management in Patients with Sickle Cell Disease
Authors: Westhoff Connie M , Chou Stella T , Smith-Whitley Kim , Friedman David
Published: October 2019

Abstract 2324

A genomic approach to blood group typing is now possible and high-throughput automated platforms have been developed to test for numerous blood group antigens in a single assay. These methods are reproducible and highly correlated with RBC serologic phenotype. We routinely perform a complete RBC phenotype for clinically significant minor red cell antigens on pre-transfusion samples from patients with sickle cell disease, and we antigen match patients for C, E, and K for transfusion.

In this study we compared the historic serologic typing with that predicted from DNA testing for clinically significant antigens in 114 samples from chronically transfused patients with SCD to determine concordance and to evaluate the clinical utility of genotyping for the management of transfusion therapy.

Serologic typing was performed by standard methods with licensed commercial reagents. DNA was isolated from WBCs, and minor antigen genotyping was performed with HEA (human erythrocyte antigen) BeadChip (BioArray, Inc). RH genotyping was by a combination of methods including PCR-RFLP, AS-PCR, exon-specific amplification and sequencing, and, for some, Rh-cDNA amplification and sequencing.

Comparison of serologic typing with DNA-based testing for thirteen blood group antigens, CcEe, Fya/b, K, Jka/b, MN and Ss, in 114 samples found 8 discrepancies in 1,482 antigens analyzed, for 99.5 % concordance. Discrepancies were in several systems (C, Fy, Ss, and M), and at least one has been confirmed to be a serologic recording error. All are under investigation.

DNA-based testing for RH found 54 of 114 patients inherited variant RHD alleles; many also had conventional RHD in trans. Sixteen patients had made anti-D, despite typing as D+. Ten of 35 patients (∼30%) whose RBCs typed as C+ had a hybrid allele encoding variant C antigen. Five had made anti-C, which prompted us to change our protocol so patients with variant C by DNA testing are transfused on a C- protocol. DNA testing found a large amount of diversity in ce-alleles in this population. Seventy-two of 114 patients carried at least one of nine different variant ce-alleles. Ten patients had made anti-e, despite typing as e+, and were homozygous for variant ce-alleles. In total, 49/114 patients with SCD were homozygous for variant RH alleles and were not truly Rh matched for D, C and e antigens by serology.

Similar to the way in which HLA typing by DNA has revolutionized bone marrow transplantation by providing a superior alternative to serological testing, we find that minor blood group antigen typing by DNA improves efficiency, reduces cost, and expands antigen matching, especially in the Rh system. Continuing studies are needed to identify more precisely which variant alleles are associated with clinically significant antibody production to improve antigen matching for patients with sickle cell disease.

Disclosures:

No relevant conflicts of interest to declare.
ADAMTS13 Helps Distinguish Thrombotic Thrombocytopenic Purpura From Other Microangiopathies and Guide Necessity of Plasma Exchange Therapy
Authors: Shah Neil , Matevosyan Karen , Burner James , Sarode Ravindra
Published: October 2019

Abstract 2328

Introduction:

Congenital thrombotic thrombocytopenic purpura (TTP) is caused by genetic mutations in ADAMTS13, a metalloproteinase that cleaves ultra-large VWF multimers to generate normal sized multimers present in circulation. However, acquired deficiency of ADAMTS13 due to auto-antibodies is not universally accepted as diagnostic of idiopathic TTP. Distinguishing idiopathic TTP from other thrombotic microangiopatheis (TMA) is important in guiding treatment as plasma exchange (PE) is only beneficial in TTP and select cases of other microangiopathies such as atypical HUS. Since January 2006 we routinely obtain ADAMTS13 activity performed by FRET assay from The Blood Center of Wisconsin in all patients with TMA. We report the diagnostic utility of ADAMTS13 to distinguish TTP from other forms of TMAs.

Methods:

A retrospective analysis was performed on consecutive patients with TMA who had ADAMTS13 assayed from January 2006 to October 2010. Demographics, presenting clinical and laboratory features are given in the Table. Responses to therapeutic plasma exchange (TPE), diagnoses at discharge, and other underlying clinical conditions were also recorded. Relevant statistical analysis was performed using unpaired t-test to compare means and Fisher's exact method for contingency tables.

Results:

We divided our cases based on severe ADAMTS13 deficiency (<10%) as TTP and non-severe deficiency (>20%) as TMA. TMA causes included quinine induced HUS (1), gemcitabine related HUS (1), malignant hypertension/pre-eclampsia/HEELP (3), sepsis (2), MCTD (5), malignancy (3), ITP (1), HIV/opportunistic infection (5), drugs (1), and multiple (5).

Conclusions:

In our experience severe ADAMTS13 deficiency appears to distinguish TTP from TMA. TMA had better mortality despite either not initiating or discontinuing PE based on ADAMTS13 levels. However, if we had continued PE in TMA then these patients would have been considered as NON-ADAMTS13 deficient TTP who responded well to PE.

Disclosures:

No relevant conflicts of interest to declare.
NAC Improves the Differentiation of IPS Cells Into Hematopoietic Progenitors
Authors: Berniakovich Ina , Laricchia-Robbio Leopoldo , Izpisua Juan Carlos
Published: October 2019

Abstract 2349

In vitro differentiation of human induced pluripotent stem (iPS) cells is a new way to obtain donor material for blood transplantation. However, this is a long process in the course of which cells are exposed to alien environmental factors, capable to change cellular properties and decrease cellular viability. Indeed, cells in vitro are exposed to mechanical stress, artificial growth surface, unnatural gas composition etc. This fact could partially explain why cells obtained during directed iPS cells differentiation have different qualities in comparison with the analogous population from in vivo.

It is supposed that oxidative stress is the major underlying mechanism of negative influence of various in vitro environmental factors on cells. N-acetylcysteine (NAC) is a powerful antioxidant. Due to free radical scavenging ability, the principal function of NAC is rendered to the inhibition of cellular damage and cell death in response to reactive oxygen species. Cytoprotective function of NAC is well demonstrated both in vitro and in vivo.

We have established a protocol to obtain hematopoietic stem cell-like cells from human iPS cells with a pick of their production at three weeks of differentiation. We analyzed how intracellular accumulation of reactive oxygen species and NO, as well as cell viability, apoptosis, stress resistance, mitochondrial membrane potential were changed during this process by comparing status of cultures at 1 and at 3 weeks of differentiation. We found that during differentiation cells progressively accumulated intracellular reactive oxygen species and increased production of NO. The level of apoptosis in culture was significantly higher at 3 weeks of differentiation than at 1 week. Cell viability, on the contrary, decreased from 1 week till 3 weeks of differentiation. Stress resistance quantified through the amount of cells resistant to the H2O2 treatment was also decreased in 3 weeks old cultures. We also demonstrated that during in vitro culture the mitochondrial membrane potential of the cells under basal conditions and upon stimulation with carbonyl cyanide m-chlorophenylhydrazone was decreased at 3 weeks of differentiation in comparison with that at 1 week. All these phenomena were reversed by NAC supplementation. Remarkably, NAC administration also improved the hematopoietic differentiation of human iPS cells in terms of production of CD34, CD45, CD43 positive cells, that showed normal functionality in colony forming unit assay. CD34+ cells obtained from NAC treated cultures also increased their migration towards SDF1, therefore showing an increased ability of our CD34+ cells to home into bone marrow.

Our results suggest that supplementation with NAC is beneficial for the improvement of hematopoietic differentiation of human iPS cells.

Disclosures:

No relevant conflicts of interest to declare.
Interferon-Gamma Impairs the Self-Renewal of Hematopoietic Stem Cells
Authors: de Bruin Alexander M. , Hooibrink Berend , Nolte Martijn A.
Published: October 2019

Abstract 2351

Regulation of hematopoiesis during stress situations, such as bacterial or viral infections, is crucial for the maintenance of sufficient numbers of cells in the blood. It has become clear that activated immune cells provide such feedback signals to the bone marrow. An important mediator in this respect is the pro-inflammatory cytokine Interferon-gamma (IFNγ), which is produced in the bone marrow by activated T cells during the course of an infection. As such, we have previously shown that T cell-derived IFNγ can directly influence the output of myeloid and erythroid cells.

To address whether IFNγ can also influence the function of hematopoietic stem cells (HSCs), we cultured highly purified HSCs from murine bone marrow with or without IFNγ and found that IFNγ strongly reduced the absolute number of HSCs in these cultures, both phenotypically and functionally. We confirmed that the functional impact of IFNγ was due to a direct effect on HSCs and not mediated by more differentiated progenitors. In addition, IFNγ does not directly influence the quiescent state of purified HSC, nor their cell cycle entry. By labeling HSCs with CFSE, we found that IFNγ reduces HSC expansion in vitro by decreasing their proliferative capacity, but not their ability to differentiate. To investigate the impact of IFNγ on HSCs in vivo, we infected WT and IFNγ−/− mice with lymphocytic choriomeningitis virus (LCMV) and found that IFNγ severely impaired HSC recovery upon infection. Finally, to exclude indirect effects of IFNγ on other cell types we generated chimeric mice with bone marrow from both WT and IFNγR−/− mice. Infection of these mixed-chimeric mice with LCMV resulted in decreased recovery of WT HSCs, but not of IFNγR−/− HSCs in the same mouse, which formally demonstrates that IFNγ directly impairs the proliferation of HSCs in vivo.

Based on these experiments we conclude that IFNγ reduces HSC self renewal both in vitro and in vivo. Importantly, we thereby challenge the current concept in literature that IFNγ would induce the proliferation of HSCs (Baldridge et al, Nature 2010). Our findings thus provide challenging new insight regarding the impact of immune activation on hematopoiesis and will contribute significantly to the scientific discussion concerning this process. Moreover, our data also provide an explanation for the occurrence of anemia and bone marrow failure in several human diseases in which IFNγ is chronically produced.

Disclosures:

No relevant conflicts of interest to declare.
Activation of a Mir-181-Targeting HOXA-PBX3 Homeobox Gene Signature Is Associated with Adverse Prognosis of Cytogenetically Abnormal Acute Myeloid Leukemia
Authors: Li Zejuan , Huang Hao , Li Yuanyuan , Jiang Xi , Chen Ping , Arnovitz Stephen , Radmacher Michael D. , Maharry Kati , Elkahloun Abdel , Yang Xinan , He Chunjiang , Price Colles O. , Dohner Konstanze , Zhang Zhiyu , Shen Chen , Neilly Mary Beth , Lussier Yves A , Zhang Yanming , Larson Richard A. , Le Beau Michelle M. , Caligiuri Michael A , Bullinger Lars , Valk Peter , Delwel Ruud , Lowenberg Bob , Liu Paul P. , Marcucci Guido , Bloomfield Clara D. , Rowley Janet D. , Chen Jianjun
Published: October 2019

Abstract 236

Altered expression of microRNAs (miRNAs, a class of small regulatory RNAs) is associated with various types of cancers, including acute myeloid leukemia (AML). We showed previously that increased expression of miR-181a with or without miR-181b was associated with a favorable prognosis for patients with cytogenetically normal AML (CN-AML). However, the prognostic value of miR-181 expression in cytogenetically abnormal AML (CA-AML) remains elusive, even though CA-AML represents the majority of human AML. To investigate the association of expression signatures of miR-181 family members and of their potential target genes with outcome in patients with primary CA-AML, we employed two independent sets of 86 CA-AML patients to investigate the association of expression signatures of miR-181 family members with outcome. We also used four independent sets of 454 CA-AML patients to identify and validate a prognostic signature of miR-181 targets. In addition, we investigated the biological functions of miR-181a/b and target(s) in leukemia cell lines and in a leukemia mouse model. As with CN-AML, we found that both miR-181a and miR-181b expression signatures are significantly (P<.05) associated with favorable overall survival (OS) of patients, but only the miR-181b signature is an independent predictor in multivariable model tests. An overexpressing gene signature of potential targets of miR-181a/b, HOXA7, HOXA9, HOXA11, and PBX 3, derived from a meta-analysis of three independent sets of 183 patients, was an independent predictor of adverse OS, which was confirmed in a validation set of 271 patients. Ectopic expression of miR-181b significantly (P<.05) promoted apoptosis and decreased viability of MONOMAC-6/t(9;11), THP-1/t(9;11), and KOCL-48/t(4;11), and delayed leukemogenesis in our mouse model; such effects could be reversed by forced expression of PBX3. Our data suggest that the silencing of miR-181b and thereby the activation of the four homeobox genes likely contributes to the poor prognosis of adverse CA-AML patients. Thus, restoring expression of miR-181b and/or targeting the HOXA/PBX3 pathways in poor prognosis AML patients may provide new strategies to improve outcome substantially.

Disclosures:

No relevant conflicts of interest to declare.
Cooperating Oncogenes and Their Targets in LMO2-Induced T-Cell Leukemia
Authors: Smith Stephen B. , Tripathi Rati , Cleveland Susan , Mathias Elizabeth , Yi Andrew , Mullighan Charles G , Downing James R. , Copeland Neal G. , Jenkins Nancy A , Dave Utpal P.
Published: October 2019

Abstract 2458

LIM domain Only 2 (LMO2) is an important driver in acute T-cell lymphoblastic leukemia. Enforced expression of LMO2 in mouse models induces T-ALL with long latency suggesting that it requires cooperating oncogenic mutations for leukemia to fully manifest. To identify cooperating oncogenes, we analyzed spontaneous T-ALLs induced by retroviral insertional mutation, and T-ALLs that developed in B6.CD2-Lmo2 transgenic mice. First, we noted that the CD2-Lmo2 transgenic T-ALLs exclusively upregulated the class II basic helix-loop-helix transcription factor, Lyl1, whereas the retrovirally-induced T-ALLs showed upregulation of Tal1. We attributed this difference to the stage at which the Lmo2 activating mutations occurred. Nevertheless, we found that LYL1 protein upregulation is functionally important, as it is part of a macromolecular complex containing LMO2 that binds tandem E boxes as described for TAL1. LMO2 and LYL1 are known to be co-expressed in human T-ALL, but we found that they are part of a larger gene expression signature comprised of NMYC, HHEX, MEF2C, and BCL2 genes. The latter genes are remarkable because they are oncogenic when overexpressed; and, Mef2c and Nmyc are concordant insertions in independent tumors driven by Lmo2 or Hhex in retroviral insertional mutagenesis models. In fact, insertion site analysis suggests that Lmo2 and Hhex may actually operate in the same pathway and that Hhex may be a target of Lmo2. We tested this genetic result by chromatin immunoprecipitation and reporter assays and discovered that Lmo2 and its binding partners, Ldb1 and Lyl1, occupy an enhancer in the first intron of Hhex in T-ALL cells. Furthermore, LMO2 or LYL1 knockdown downregulated expression of HHEX in T-ALL. HHEX activation has significant functional consequences in T-ALL. We found that its upregulation is associated with T-ALL treatment resistance, clinical latency, and differential activation of Notch1 targets. These data offer important insight into the pathogenesis of Lmo2-induced T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
Bioimaging of Vascular Microenvironment Reveals Presence of Fli1a, Not VEGFR2, in Acute Lymphoblastic Leukemia Model in Syngeneic Zebrafish
Authors: Revskoy Sergei , Mizgirev Igor , Corey Seth J.
Published: October 2019

Abstract 2551

Despite great advances in the curability of children with acute lymphoblastic leukemia (ALL), outcomes for pediatric and adult patients with relapsed ALL remain poor. This highlights the need for new approaches to ALL treatment and novel tools for screening of potential therapeutics. Dysregulated angiogenesis has been implicated both in the pathogenesis of leukemia and development of chemoresistance. However, the complexity of the bone marrow microenvironment and the precise contributions by the various components cannot be easily dissected in cell lines, tissue blocks, or mouse models. For instance, leukemia-associated angiogenesis was mainly characterized by immunostaining of bone marrow biopsies. High resolution in vivo bioimaging of fluorescence-tagged tumors and their microenvironment has recently become feasible due to establishment of transplantable tumor models in clonal syngeneic zebrafish. We have applied this model to dissection of cellular mechanisms of angiogenesis during stages of progression of T-ALL, This novel animal model enables us to 1) track leukemic cell proliferation and dissemination in vivo in different temperio-spatial and vascular contexts, and 2) follow up on cellular angiogenic events in response to leukemia progression including those occurring in response to chemotherapy for leukemia. In zebrafish, angiogenesis is similar to that of mammals and has been well characterized by using VEGFR2 and Fli1a transgenic reporter systems. In embryos, the fli1a:EGFP expression pattern mirrors that of VEGFR2 fluorescence in vascular endothelial cells. However, later in the development, in larvae and adult fish, the pattern of Fli1a or VEGFR2 expression diverges. This divergence further extends to angiogenesis in areas adjacent to T-ALL with more prominent development of Fli1a vasculature. Leukemic patches are characterized by a microenvironment where Fli1a is predominant and VEGFR2 is absent. We are now using cyclophosphamide treatment of leukemic fish to dissect the role of microenvironment and whether angiogenic factors are modified. These data yield new insights into molecular mechanisms of leukemogenesis in conjunction with angiogenesis. Furthermore, our findings would have predicted the lack of efficacy of VEGFR inhibitors in leukemia therapy. Our model offers an advantage for cost-efficient in vivo large scale screening system for antiangiogenic drugs for acute leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Unresponsiveness to CHOP Is Associated with Activation of the p38 MAPK Pathway in Patients with DLBCL
Authors: Vega Mario I , Aviles-Salas Alejandro , Chalapud J Ramon , Huerta-Yepez Sara , Hernandez-Pando Rogelio , Martinez-Paniagua Melisa , Martinez-Miguel Bernardo , Hernandez-Atenogenes Miriam , Vega Gabriel G , Chen Haiming , Berenson James R. , Mayani Hector , Martinez-Maza Otoniel , Bonavida Benjamin
Published: October 2019

Abstract 2647

We have reported in B-NHL cell lines that the p38 MAPK was constitutively activated and was involved in the regulation of tumor cell resistance to cytotoxic drugs. Further, inhibition of this pathway reversed drug resistance. Based in these findings we hypothesized that the activation of the p38 MAPK pathway in patients with B-NHL may be associated with unresponsiveness to cytotoxic drug therapy. This study was designed to test this hypothesis. Eighty patients with Diffused Large B Cell Lymphoma (DLBCL) were used for analysis. Freshly derived tumor tissues from these patients were obtained from biopsies prior to any treatment. Tissue microarrays were prepared and examined by immunohistochemistry for the expression of both p38 MAPK and phosphorylated p38 MAPK (active). The antibodies were tested for specificity. The frequency of stained cells <30% was considered negative and the frequency ≥30% was considered positive. The frequency of stained cells was recorded only for the tumor cells in the arrays and the patterns of expressions were compared to adjacent normal tissues. Among the 80 patients who were treated with CHOP, and were (57) responders (R) and (23) non-responders (NR) to therapy. The data collected were analyzed and correlated with both the response to CHOP therapy and to event-free survival (EFS). The findings revealed that 66/80 (82%) tumor tissues expressed p38 MAPK and only 24/80 (30%) expressed phosphorylated p38 MAPK. The expression and activation of p38 MAPK correlated with high score performance status (1≥, p= 0.01). There was a significant correlation between the expression and the activity of p38 MAPK and failure to respond to CHOP therapy (p=0.02 and 0.002, respectively). Also, the failure to respond to CHOP therapy correlated with the nuclear expression of phospho-p38 MAPK (p < 0.05). Analysis of different clinical pathological parameters did not show a significant correlation with the expression of p38 MAPK. Further analysis demonstrated that high phospho p38 MAPK in the tumor significantly correlated with poor EFS (p=0.005). These findings established for the first time that a subset of patients with DLBCL and whose tumors expressed high and active p38 MAPK responded poorly to CHOP and had poor EFS. We propose that the activation of p38 MAPK in DLBCL patients at diagnosis may require a different treatment strategy than CHOP. In addition, inhibitors of the activation of p38 MAPK in DLBCL are potential for therapeutic intervention. The findings also suggested that activation of p38 MAPK is a potential prognostic biomarker.

Disclosures:

No relevant conflicts of interest to declare.