bluuebunny/crossref_metadata_2025 huggingface dataset:
Murine Bone Marrow-Derived Megakaryocytes Are Capable of Antigen Cross-Presentation on Major Histocompatibility Class (MHC) I Molecules
Authors: Zufferey Anne , Speck Edwin R. , Guo Li , Aslam Rukhsana , Kapur Rick , Semple John W.
Published: October 2019

Major Histocompatibility complex (MHC) class I is a complex composed of two proteins, the MHC-encoded alpha heavy chain and the beta2-microglobulin light chain. In the endoplasmic reticulum, it receives an antigen peptide of usually eight to eleven residues in its groove and is exported to the plasma membrane. In classical MHC class I presentation, endogenous antigens are presented to CD8+ T cells. On the other hand, cross-presentation consists in the presentation of exogenously-derived proteins in the context of MHC class I molecules to CD8+ T lymphocytes, inducing a cytotoxic response against the antigen presenting cell (APC). Platelets have been previously shown to be able to cross-present immunogenic ovalbumin (OVA) peptides, as well as bacterial peptides and to trigger CD8+ T lymphocyte activation. It is thought that the antigen cross presenting function of platelets is transmitted from megakaryocytes (MK) during thrombopoiesis, but whether megakaryocytes can cross-present is still unknown. To address this question, we developed an in vitro endocytosis model of murine bone marrow-derived megakaryocytes. C57BL/6 mouse megakaryocytes are enriched by gradient sedimentation and pulsed with fluorescent ovalbumin (OVA) for various times. In addition, we used DQ-OVA, a BODIPY FL-conjugated OVA, which becomes fluorescent upon proteolysis. Cells were then analyzed by flow cytometry or confocal microscopy to assess the OVA megakaryocyte internalization, its proteolytic degradation and its loading onto MHC class I molecules. We can show the endocytosis of exogenous OVA by megakaryocytes, in a pathway involving lysosomes. The endocytosed OVA were subsequently proteolytically cleaved into the immunogenic OVA peptide SIINFEKL and the peptide is inserted into the antigen binding groove of MHC class I molecules. The MHC class I-OVA peptide complexes are stored in the MK alpha-granules and can be translocated to the plasma membrane. Thus, we have shown that MKs can effectively process and present exogenous antigens on MHC class I molecules. This opens a new avenue toward a functional characterization of this pathway and its modulation, leading to new therapeutic approaches in autoimmune diseases targeting megakaryocytes, such as immune thrombocytopenia (ITP).

Disclosures

No relevant conflicts of interest to declare.
Acute Efficacy of a Novel Anti-Tissue Factor Pathway Inhibitor Antibody BAY 1093884 in Hemophilia A Mouse Severe Tail Bleeding
Authors: Xu Yifan , Koellenberger Maria , Laux Volker , Kauser Katalin , Sim Derek
Published: October 2019

Tissue factor pathway inhibitor (TFPI) is the major inhibitor of the tissue factor initiated extrinsic coagulation pathway in blood and is intact in patients with hemophilia. The inhibition of TFPI may restore hemostasis in patients with hemophilia. BAY 1093884 is a fully human monoclonal antibody against TFPI developed as a bypass agent for hemophilia patients with or without inhibitors. It restores thrombin burst for stable clot formation in hemophilic conditions in vitro. The goal of these studies was to determine the in vivo acute efficacy of BAY 1093884 in the hemophilia A (HemA) mouse.

In the first study, the acute efficacy of BAY 1093884 (3−100 mg/kg) was demonstrated and compared with full-length recombinant factor VIII (rFVIII; 10−100 IU/kg) by a HemA mouse tail clip model, in which blood loss from a severed tail tip was measured over 45 minutes after injury (n=12−27 mice/group). Naive C57/BL6 and HemA mice were used as positive and negative controls, respectively. Whereas isotype control antibody−treated HemA mice had median blood loss of 870 μL, increasing doses of BAY 1093884 to 50 and 100 mg/kg significantly reduced blood loss to a median of 55 and 5 μL. The dose required to reduce blood loss by 50% was 18 mg/kg, approximately equivalent to the efficacy of 20 IU/kg rFVIII.

In a second study, we characterized the combined action of BAY 1093884 and activated recombinant factor VII (rFVIIa; n=10−25 mice/group). Low doses of BAY 1093884 (2.5 mg/kg) and rFVIIa (0.5 and 1.0 mg/kg) with minimal efficacies were tested. Untreated HemA mice had median blood loss of 860 μL. As stand-alone treatments, 2.5 mg/kg BAY 1093884, 0.5 mg/kg rFVIIa, and 1 mg/kg rFVIIa provided minimal blood loss protection, with bleeding volume reduced to 675, 830, and 770 μL, respectively. In comparison, the combination of 2.5 mg/kg BAY 1093884 with 0.5 mg/kg rFVIIa or 1.0 mg/kg rFVIIa reduced median blood loss to 215 and 35 μL, respectively. These results showed a combination effect of BAY 1093884 and rFVIIa in this severe acute efficacy model.

These studies demonstrate that BAY 1093884 could potently reduce acute blood loss in HemA mice and may offer a new treatment option for hemophilia patients.

Disclosures

Xu: Bayer HealthCare LLC: Employment. Koellenberger:Bayer Pharma AG: Employment. Laux:Bayer Pharma AG: Employment. Kauser:Bayer HealthCare LLC: Employment. Sim:Bayer HealthCare LLC: Employment.
A Method for Deriving Pharmacokinetic Constants for Factor VIII and IX from Limited Blood Sampling
Authors: Escobar Miguel Antonio , Bond Daniel Preston , Cantini Madeline , Cannon Krishna
Published: October 2019

Introduction

The treatment of hemophilia A and B is based on the replacement of the deficient factor dosed by weight. However, dosing for hemophilia treatment has been arrived at by empiric assessment, essentially "trial and error" based on the pharmacokinetics (PK) of the factors and the characteristics of the replacement product. In the last decade clinical pharmacokinetics has gained popularity in hemophilia so that factor dosage can be adjusted according to the requirement of the individual patient. Factor-specific population pharmacokinetic models have been developed for individualized treatment of patients with hemophilia A and B. The math modeling technique presented in this project relies on minimal blood sampling to derive the constants necessary to predict the peak and trough levels within the commonly excepted error bounds.

Methods

Data that included FVIII, FIX levels and weight were obtained retrospectively from severe hemophilia A and B adult patients and approved by the ethics committee of the University of Texas Health Science Center. A single compartment decay model equation was used in both a custom iOS application and an Excel spreadsheet to calculate the decay constant between any 2 points on a decay curve. Using this local constant as the half-life in the standard decay equation allowed the calculation of the peak at time = 0. This peak combined with dosing information and the subject's weight allowed the calculation of the Recovery. These 2 constants in the equation: Level=(dose*Recovery/weight)*0.5^(time/half-life) allowed the calculation of the level at any point on the curve. Using this method on several example datasets showed that the model is reasonably able to predict peak and trough levels for both factor VIII and IX. Because of the time dependent nature of the local decay constant, better results are obtained using data points after the first half of the expected half-life. This method can also be used to predict steady state levels, peaks and troughs for any prophylaxis schedule.

Conclusion

Appropriate dosing of factor VIII or IX is at best an approximate calculation. The method described in this publication generates a math model that is generally as accurate as a multiple sample PK study with far fewer blood samples taken. Clinical applications of this model can be utilized to predict factor levels after a single infusion of factor VIII/IX in adults that are treated on-demand or prophylaxis. It can also be utilized in individuals that are infusing extended half-life products or in the surgical setting.

Disclosures

Escobar: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bond:Pfizer: Consultancy, Research Funding. Cantini:Pfizer: Research Funding. Cannon:Pfizer: Research Funding.
Mechanisms of Immunosuppression By FVIII-Specific TCR Engineered Human Regulatory T Cells
Authors: Kim Yong Chan , Zhang Ai-Hong , Yoon Jeong Heon , Scott David William
Published: October 2019

Expanded antigen-specific engineered regulatory T cells (Tregs) have been proposed for potential clinical application for the treatment of undesirable immune responses, such as inhibitor responses in hemophilia A patients and autoimmune diseases. By providing an antigen-specific T-cell receptor (TCR) to polyclonal natural Tregs, we suggested that antigen-specific engineered Tregs would migrate specifically to particular target tissues and induce antigen-specific immune tolerance in the local milieu. Previously, we developed FVIII C2-specific Tregs using a long-term stabilization protocol in vitro and demonstrated that these stabilized engineered Tregs successfully modulated FVIII-specific T-cell- and B-cell immune responses. Herein, we examined the mechanism of suppression by antigen-specific engineered Tregs compared to polyclonal normal natural Tregs.

Initially, we tested whether these FVIII-specific engineered Tregs were able to suppress neighboring activated T-cell effectors locally. We found that FVIII C2-specific Tregs strongly suppressed myelin basic protein (MBP)-specific T effectors by presentation of both specific antigens in same APC population. However, we also observed that C2-specific Tregs could suppress MBP-specific T effectors presented on different APCs. These results imply contactless suppressive function of C2-specific engineered Tregs. Using a modified trans-well suppression assay, in which physical distance and clear separation between Tregs and a set of T effectors was created, we found that C2-specific activated Tregs showed significant contactless suppression only when T effectors were also present. In addition, and confirming previous studies with polyclonal Tregs, suppression by FVIII-specific engineered Tregs could be overcome by increasing the dose of IL-2 in co-culture media. This suggests that Tregs act, in part, by usurping IL-2 needed by T effectors to proliferate. Surprisingly, neutralization of CTLA-4 did not interfere with FVIII C2-specific suppression of engineered Tregs in contrast to the reversal seen with anti-CD3e-driven non-specific immunosuppression.

Our data strongly suggest that suppressive function of FVIII-specific engineered Tregs is not restricted to cell-to-cell contact. Rather cross-talk of engineered Tregs and T effectors potentially generate a contactless suppressive mechanism to suppress other FVIII-specific multiple effector cells in the local milieu for effective immune tolerance. Understanding the mechanism of contactless suppression mechanism should provide critical clues to develop more effective engineered Tregs as a therapeutic tool in hemophilia A.

(Supported by NIH grants HL061883 and HL126727)

Disclosures

Kim: Henry Jackson Foundation: Other: patent filed. Zhang:Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.
Signs of Hypofibrinolysis in Patients with Unprovoked Idiopathic Venous Thromboembolism
Authors: Bassora Fernanda Dutra Santiago , Orsi Fernanda Andrade , Alves Graziela Silveira Araujo , Montalvão Silmara Aparecida De Lima , Zapponi Kiara , Romano Anna Virginia Calazans , Colella Marina Pereira , de Paula Erich Vinicius , Annichino-Bizzacchi Joyce Maria
Published: October 2019

Background: The causes for venous thromboembolism (VTE) remain undetermined in at least 30% of patients with unprovoked VTE. Hypofibrinolysis may be associated to VTE, however the occurrence of hypofibrinolysis in patients with unprovoked, idiopathic, VTE is not well stablished.

Aims: To evaluate whether hypofibrinolysis would be associated with unprovoked idiopathic VTE.

Methods: Patients with a history of unprovoked VTE without acquired or inherited thrombophilia were included. Global tests of fibrinolysis, such as euglobolin lysis time (ELT) and lysis area on fibrin plate (LAFP), and specific tests of fibrinolysis, such as plasma activity plasminogen, a-2 antiplasmin (a2AP), plasminogen activator

inhibitor-1 (PAI-1), and thrombin activatable fibrinolysis inhibitor (TAFI), were performed in patients and healthy controls. We also analyzed the plasma activity of factor (F) XIII.

Results: Thirty-one patients and fifty healthy controls were included. ELT results were higher in patients than in controls (median= 295 and IQ= 205-355 minutes vs. median=250 and IQ= 167-295 minutes, respectively, p = 0.006) and LAFP values were lower in patients compared to controls (median=81 and IQ= 56-110 vs. median= 95 and IQ 72-132 respectively, p = 0.0014), suggesting that they were experiencing a hypofibrinolytic state. Plasma activity of plasminogen (median= 131 and IQ= 119-141 vs. median=120 and IQ=111-137, respectively, p = 0.045) and FXIII (median= 103 and IQ 89-127 vs. median= 96 and IQ=80-105, respectively, p = 0.050), were higher in patients than in controls, whereas plasma activity of α-2AP was lower in patients (median= 124 and IQ 114-128 vs. median= 127 and IQ=121-133, p≤0.001). Interestingly, patient's median TAFI activity was lower than in controls (median=16 and IQ 13-18μg/mL vs. median= 19 and IQ= 17-21g/mL, p≤0.001). However, PAI-1 activity did not differ between groups.

Conclusions: Hypofibrinolysis may occur in patients with unprovoked idiopathic VTE and may be detected by global tests of fibrinolysis. It is possible that hypofibrinolysis contribute to the pathogenesis of thrombosis in these selected cases.

Disclosures

No relevant conflicts of interest to declare.
Kras Is Critical for B Cell Lymphopoiesis
Authors: Zheng Yongwei , Chen Yuhong , You Xiaona , Yu Mei , Fu Guoping , Su Xinlin , Zhu Wen , Wu Zhihong , Zhang Jing , Wen Renren , Wang Demin
Published: October 2019

Small GTPases of the Ras subfamily regulate multiple signaling pathways and control numerous biological functions. Although the three major Ras members, Kras, Hras and Nras, are highly homologous, individual Ras gene can have distinct biological functions. Embryonic lethality of Kras-deficient mice precludes study of the biological role of Kras. Here, we generated and examined mice with hematopoietic- and B cell-specific deletion of Kras. In VavCreKrasfl/fl mice with hematopoietic deletion of Kras, the populations of bone marrow (BM) pre-, immature and mature B cells were reduced. The population of peripheral follicular (FO), marginal zone (MZ) and B1 mature B cells were also reduced in VavCreKrasfl/fl relative to control mice. In addition, BM chimeric mice with B cell-specific deficiency of Kras generated by transplantation of T- and B-null Rag1-deficient mice with a mixture of BM cells from VavCreKrasfl/fl mice and B cell-deficient μMT mice displayed a marked reduction of pre-, immature and mature B cells in the BM and mature B cells in the spleen. Thus, Kras deficiency intrinsically impairs early B cell development and late B cell maturation. Further, the effect of Kras deficiency on B-cell proliferation and survival was examined. The 3H-thymidine incorporation rate of Kras-deficient, relative to control, mature B cells in response to anti-IgM or anti-IgM plus IL-4 was markedly reduced. In addition, Kras-deficient B cells displayed a marked decrease of cell cycle entry and increase of cell apoptosis upon anti-IgM or anti-IgM plus IL-4 stimulation. Thus, Kras deficiency impairs BCR-induced B cell proliferation and survival. Lastly, the role of Kras in BCR signaling was studied. The level of total BCR-activated Ras (Ras-GTP) was largely reduced in Kras-deficient B cells. BCR-induced Ca2+ flux was comparable between Kras-deficient and control B cells. BCR-induced phosphorylation of Akt and IkBa was normal in mutant relative to control B cells. However, BCR-induced activation of ERK1/2 but not JNK or p38 was impaired in Kras-deficient relative to control B cells. Consistently, BCR-induced activation of Raf-1 and MEK1/2, the upstream activators of ERK1/2, was markedly reduced in mutant B cells. In addition, pre-BCR-induced ERK1/2 activation was impaired in Kras-deficient pre-B cells. Pre-BCR-induced activation of AP-1, the transcription factors downstream of ERK1/2, was decreased in mutant pre-B cells. Thus, Kras plays an important role in BCR- and pre-BCR-mediated activation of the ERK pathway in B cells. Taken together, these findings demonstrate that Kras is the Ras family member that critically regulates early B cell development and late B cell maturation through controlling the Raf-1/MEK/ERK pathway.

Disclosures

No relevant conflicts of interest to declare.
The NFkB System Regulates Flt3-Mediated Hematopoiesis
Authors: Liu Yi , Hoffmann Alexander
Published: October 2019

FMS-like tyrosine kinase 3 (Flt3) plays pivotal roles in the survival, proliferation and differentiation of hematopoietic stem/progenitor cells (HSPCs). However, how proliferation and differentiation via Flt3 is regulated is poorly understood. In this study, our bioinformatic meta-analysis of existing cancer microarray datasets in Oncomine correlated reduced nfkb1 expression with acute myeloid leukemia (AML). The nfkb1 gene products p105 and p50 are not transcriptional activators but regulators of NFκB activators. Examining bone marrow HSPCs, we found that nfkb1-/- mice produce much higher (~5-8X) total cell yields compared to wild type (WT) when cultured with Flt3 Ligand (Flt3L) ex vivo. Cell cycle analysis and apoptosis assay revealed that nfkb1-/- cells have profoundly increased proliferation and enhanced survival compared to WT cells. Nfkb1-/- cells show skewed differentiation towards plasmacytoid DCs and delayed differentiation of conventional DCs. To examine the molecular mechanism, we investigated how the NFκB system is assembled as HSPCs, which have little NFκB, progress through hematopoiesis to become differentiated immune cells with robust NFκB systems. In nfkb1-/- cells, the distribution of NFκB signaling complexes was altered and resulted in overall elevated RelA expression. Reducing RelA expression in compound rela+/- nfkb1-/- HSPCs partially rescued the myelopoiesis phenotype, suggesting that hyper NFκB activity is the cause. Further in vivo studies are underway to further test NFκB's role in a Flt3L injection model and a Flt3-ITD-mediated mouse leukemia model. In conclusion, our work reveals that precisely timed control of NFκB system assembly is a hallmark of hematopoiesis and that its misregulation perturbs normal Flt3-mediated hematopoiesis and can be a cause of myeloid cancer.

Disclosures

No relevant conflicts of interest to declare.
Characterizing the Function of an RNA Binding Protein, IGF2BP3, in Hematopoiesis
Authors: Tran Tiffany M , Palanichamy Jayanth Kumar , Howard Jonathan , Contreras Jorge R. , Fernando Thilini , Basso Giuseppe , Pigazzi Martina , Sanford Jeremy R , Rao Dinesh S.
Published: October 2019

Post-transcriptional control of gene expression plays important roles in defining normal and pathological cellular phenotypes. Amongst mechanisms of post-transcriptional regulation, RNA binding proteins (RBPs) have recently been shown to play important roles. However, in vivo roles for RBPs are not well understood. Here, we identified the RBP IGF2BP3 to be specifically overexpressed in MLL-rearranged B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and a risk of high relapse. IGF2BP3 was required for the survival of B-ALL cell lines, and knockdown led to decreased proliferation and increase apoptosis. In addition, enforced expression of IGF2BP3 in murine bone marrow transplant assays caused a proliferation of hematopoietic stem and progenitor cells and a skewing of hematopoietic development to the B-cell/myeloid lineage. Using cross-link immunoprecipitation and high-throughput sequencing, we uncovered the transcriptome regulated by IGF2BP3; including novel direct targets, MYC and CDK6. These were regulated following experimental alteration of IGF2BP3 expression in vivo, and are regulated via elements within their 3'untranslated regions. Hence, IGF2BP3 mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism operant in MLL-rearranged B-ALL, highlighting IGF2BP3 and its cognate RNA binding partners as potential therapeutic targets in this disease.

Disclosures

No relevant conflicts of interest to declare.
IGH-FR1 and Leader Analysis By Next Generation Sequencing for the Detection of Clonality and Somatic Hypermutation in Lymphoid Malignancies
Authors: Piccaluga Pier Paolo , Gazzola Anna , Huang Ying , Panganiban Jeff , Hutt Kasey , Klass Michael
Published: October 2019

The standard method to analyze clonality and somatic hypermutation of immunoglobulin genes in B-cell non Hodgkin lymphomas is a multiplex PCR followed by capillary electrophoresis and/or Sanger sequencing. Recently, Next Generation Sequencing (NGS) of immune receptor genes was demonstrated to be highly effective, with increased sensitivity for detecting sequences of interest. Based on that, it was proposed for the diagnosis and monitoring of lymphoid neoplasms. A phase 3 diagnostic accuracy study was designed to compare the value of the first commercially available kit for NGS of IGH/FR1 with the gold standard analysis. One hundred twenty samples were evaluated for IGH rearrangements with both traditional capillary electrophoresis (IdentiClone™ IGH Gene Clonality Assay by Invivoscribe Technologies) and NGS (LymphoTrackTM IGH Assay - MiSeq and LymphoTrack® IGH Somatic Hypermutation Assay - MiSeq by Invivoscribe Technologies on an Illumina MiSeq).

The NGS clonality assay compared to conventional analysis had an overall diagnostic accuracy of 96% (63/66 cases), sensitivity (ST), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 95%, 100%, 100%, and 75%, respectively.

Regarding mutational analysis, conventional detection and NGS gave comparable results. Notably, NGS was capable of analyzing all cases, including those failed by Sanger sequencing. In discrepant cases NGS results were confirmed by a different set of primers that provided coverage of the IGH Leader sequence.

In conclusion NGS was effective for IGH analysis in lymphoid disease (even on FFPE samples). In order to apply them for routine diagnostics, NGS based approaches should be evaluated prospectively and cost-effectiveness assessments should be performed.

Disclosures

Huang: Invivoscribe: Employment. Panganiban:Invivoscribe: Employment. Hutt:Invivoscribe: Employment. Klass:Invivoscribe: Employment.
Preclinical Development of Bispecific Chimeric Antigen Receptor Targeting Both CD19 and CD22
Authors: Qin Haiying , Haso Waleed , Nguyen Sang Minh , Fry Terry J
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Despite advances in upfront therapy, relapsed and refractory ALL remains a primary cause of cancer-related mortality in children. Immunotherapy using genetically engineered T cells expressing chimeric antigen receptors (CARs) targeting CD19 has demonstrated dramatic clinical results in children and young adults with pre-B cell ALL with a 70-90% complete remission rate in multiple clinical trials. However, not all patients respond and at least 10% of patients will relapse due to loss of the targeted CD19 epitope. CD22 is another pan-B cell surface antigen that is broadly expressed on ALL blasts and has been targeted successfully using immunoconjugates. We have recently developed a highly active CD22 CAR currently being tested in a clinical trial. Conceptually, dual targeting both CD19 and CD22 on Pre-B ALL with a single bispecific CAR may result in enhanced avidity and increased T cell activation which may result in additive or synergistic responses compared to targeting a single antigen. In addition, since there are heterogeneous expression levels of CD19 and CD22 on leukemic blasts, dual targeting may also provide a more broadly active therapy. Finally, simultaneously targeting of both CD19 and CD22 on the surface of pre-B cell ALL may reduce the likelihood of antigen escape due to downregulation or deletion. We describe the preclinical development of two novel CD19 and CD22 bispecific CARs assembled with CD19 and CD22 single chain fragment variable regions (scFv) binding domains on a single construct. T cells transduced the CD19/CD22 bispecific CARs have comparable CAR surface expression when compared to T cells expressing either the CD19 or CD22 CAR. T cells expressing the bispecific CAR are active in vitro against ALL cell lines expressing both CD19 and CD22 as measured by interferon gamma production and chromium release. In vitro activity is maintained against K562 cells expressing either CD19 or CD22 alone. Interestingly, the order and the linker between the CD19 and CD22 scFvs in the CAR construct has a major impact on the efficacy of the dual CAR with a membrane-distal CD22 binding scFv demonstrating improved efficacy. Finally, in xenograft models both bi-specific CARs efficiently eradicate ALL cell lines and patient derived xenografts. In vivo treatment of a Crisper-knockdown-CD19-negative leukemia line and a CD19-negative patient-derived xenograft is ongoing. In summary, CD19/CD22 bispecific CARs demonstrate robust pre-clinical activity against pre-B cell ALL and provides a strategy to improve the clinical efficacy of CAR therapy.

Disclosures

No relevant conflicts of interest to declare.
Novel Bromodomain Inhibitors Suppress Proliferation of Multiple Myeloma Cells
Authors: Ashihara Eishi , Oki Ryoko , Imayoshi Natsuki , Yoshioka Makoto , Strovel Jeffrey , Honjo Ayako , Sakai Yumi , Takada Tetsuya , Chauhan Jay , Raje Mithun , Fletcher Steven , Takata Kazuyuki
Published: October 2019

New molecular targeting agents against multiple myeloma (MM) have been developed and the prognosis of patients with MM has been improved. However, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. Bromodomain (BRD4) inhibitors such as JQ-1 and I-BET151 have been shown as candidates for a molecular targeting agent against MM. In this study, we searched for chemical compounds inhibiting BRD4 binding ability and we investigated their effects on MM cell proliferation. We identified novel compounds CA1 and CA2 as a BRD4 inhibitor using an alpha screening assay. The IC50 values of the binding ability of recombinant human BRD4-BD1-BD2 of CA1 and CA2 are 0.44 and 1.1 mM, respectively (Figure 1). We next investigated the effects of CA1 and CA2 on MM cell lines (MM.1s, AMO-1, NCIH929, and OPM-2 cells). Analyses with MTT assay showed that these compounds inhibited the proliferation of MM cells in a dose-dependent manner. c-myc and cyclinD1 mRNA transcripts were decreased by the treatment of CA1 and CA2, while, p21 mRNA transcripts were increased (Figure 2). In flow cytometric analysis, cell cycle arrest occurred in a G1 phase and apoptosis was induced. Western blotting analysis showed the decrease of c-myc protein levels as well as the cleavage of caspase-3. In conclusion, novel BRD4 inhibitors CA1 and CA2 inhibited binding of BRD4 to its ligands, resulting in the suppression of MM cell proliferation and the induction of apoptosis via the caspase activation.

Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures

Yoshioka: ConverGene LLC: Employment. Strovel:ConverGene LLC: Employment.
Survival Outcomes in Blastic Plasmacytoid Dendritic Cell Neoplasm (2001-2012)
Authors: Mosalpuria Kailash , Bhatt Vijaya R. , Nawal Lata
Published: October 2019

Background: BPDCN is a rare subtype of aggressive acute leukemia that usually has an indolent clinical presentation such as isolated solitary or multiple cutaneous lesions. Despite initial clinical responses to systemic chemotherapy, BPDCN follows an aggressive course with a reported median OS of approximately 8-14 months based on single-center studies. We aimed to determine the OS of BPDCN and its predictors utilizing a US population-based database.

Methods: Using International Classification of Disease for Oncology, 3rd edition (ICD-O-3) code 9727/3, SEER 18 database was analyzed to identify adult patients with BPDCN diagnosed from 2001-2012. We divided the entire cohort into 3 groups based on time periods of 4 years each (2001-2004, 2005-2008, 2008-2012) to represent early (E), middle (M) and late (L) cohorts. Age, sex, race, marital status, radiation use, and OS were compared across the three time periods. Multivariate analyses of OS were performed using Cox regression to adjust for significant covariates. A p-value of <0.05 was considered statistically significant.

Results: Of 417 patients, the majority were males (67%), patients aged ≤ 60 years (75%) and whites (82%). A higher proportion of patients were diagnosed in the early time period (E=43% vs. M=29% vs. L = 29%). About a sixth of the cohort (17%) received radiation therapy. Median OS was not reached for the cohort. Five-year OS was higher for patients ≤ 60 years, as compared to patients >60 years (70% vs. 50%, p<0.01). On a multivariate analysis, age >60 years (hazard ratio, HR 2.05; 95% confidence interval, CI 1.28-3.27, p <0.008) and African American ethnicity (HR 1.79; 95% CI 1.10-2.89, p=0.018) were associated with a higher hazard for death whereas OS did not differ based on sex, marital status, time period, or the use of radiation therapy. Of the 106 patients >60 years, the majority were males (68%), whites (93%), married (74%), and did not receive radiation (92%). On a multivariate analysis after adjusting for age, sex, race, time of diagnosis, and radiation therapy, being single (marital status) was associated with a worse OS (HR2.24; 95% CI 1.05-4.77, p=0.034) among patients >60 years.

Conclusions: To our knowledge, this is the largest and most recent population-based study in BPDCN. Compared to prior single-center studies, our results demonstrate much better OS. Older age and African American ethnicity are determined to be negative prognostic factors for OS. Single older patients are at even a higher risk of mortality. The study findings may help in patient counseling and informed decision-making. Racial disparities in OS require further investigation.

Disclosures

No relevant conflicts of interest to declare.
Low Density Granulocytes in Patients after Allogeneic Hematopoietic Stem Cells Transplantation (allo-HSCT): A Distinct Class of Neutrophils in Systemic Alloimmunity
Authors: Mikhaltsova Ekaterina , Savchenko Valeri G. , Kuzmina Larisa A. , Drokov Mikhail , Vasilyeva Vera , Koroleva Olga , Dubnyak Daria , Parovichnikova Elena N.
Published: October 2019

Introduction

It's generally considered that all alloimmune process such as acute graft-versus host disease (aGVHD) after allo-HSCT are mostly controlled by lymphocytes. The role of neutrophils in systemic alloimmunity after allo-HSCT is still illusive. In 1987 a distinct subset of proinflammatory, low-density granulocytes (LDGs) isolated from the peripheral blood mononuclear cell fractions of patients with system lupus erythematosus has been described. There is no LDG's in healthy donors. While the origin and role of LDGs still needs to be fully characterized, we try to describe this population in patients with hematological malignancies after allo-HSCT

Patients and methods.

Peripheral blood samples were collected in EDTA-tubes before allo-HSCT, on day +30,+60,+90 after allo-HSCT and at day of aGVHD from 47 patients with hematological malignancies (AML=22, ALL n=17, LPD=3, MDS =2; CML=2; 17 with active disease, 30 - in CR) after allo-HSCT (from matched unrelated donor n=34, from matched related donor n=13; MAC = 13, RIC=34). Isolation of mononuclear cells from human peripheral blood was made by standard protocol using Lympholyte®-M Cell Separation Media (Cedarlane Labs). The anti-CD66b-PE (Biolegend, USA) antibodies and FSC/SSC were used to determine LDGs cells as FSChigh \SSChigh \CD66b+. 100000 of cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA).

Results. Results of blood evaluation of 47 patients with hematological malignancies, whose blood was examined after allo-HSCT presented in table 1.

Conclusion Despite the fact that we don't get significant differences. LDG's detection in allo-HSCT patients need further investigation.

Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD Table 1. Incidence of LDG after allo-HSCT in patients with and without aGVHD

Disclosures

No relevant conflicts of interest to declare.
Thrombophilic Factors in Retinal Vessel Thrombosis: A Single Center Experience
Authors: Maxey Nicole , Mohammadi Zeinab , Dominguez-Perez Pamela , Cortes Erasto , Antony Nishaal , Landeros Mark M , Arango Carmen , Campos-Gines Angel , Vazquez Carlos W , Herrada Juan
Published: October 2019

Background: Retinal Vessel Thrombosis (RVT) is an ophthalmologic emergency that can result in permanent vision loss. The retinal vessels are considered a rare site for thrombosis, obligating physicians to evaluate the underlying cause. Classic risk factors include hypertension, cardiovascular disease, diabetes, and glaucoma. In addition, there is a variable degree of association with inherited and acquired thrombophilia. Our aim is to describe our experience with eleven patients (pts) evaluated for inherited and acquired thrombophilia who presented with RVT.

Methods: Retrospective analysis.

Results: We reviewed the medical records of 11 consecutive pts seen in our institution between 2001 and 2015 who were referred with the diagnosis of RVT. Eight out of eleven were females and three were males; median age was 47 years, with an age range from 30 to 78. Clinical presentation was transient visual loss in the affected eye in 10 pts and persistent conjunctivitis in 1 pt. Laboratory evaluation of thrombophilia in blood samples showed the following: 1 pt had both factor V Leiden mutation (heterozygous) along with prothrombin-20210A mutation (heterozygous); 3 pts had MTHFR gene mutations (two were heterozygous and one was homozygous); 1 pt had MTHFR gene mutation and positive lupus anticoagulant; 1 pt had antiprothrombin antibodies and homocysteinemia; 1 pt had factor V Leiden (heterozygous); 1 pt had factor V Leiden (heterozygous) and elevated factor VIII; 1 pt had protein S deficiency and elevated factor VIII; 1 pt had anticardiolipin antibodies; and lastly 1 pt had no laboratory abnormalities found.

Conclusion: In our limited pt sample, we have found that retinal vessel thrombosis can be associated with inherited and acquired thrombophilia. The clinical implication of these findings remains incompletely understood and merits further research.

Disclosures

No relevant conflicts of interest to declare.
Developing a Model of Human Pluripotent to Hematopoietic Stem Cell Development in Mistrg Mice
Authors: Astle John , Xiang Yangfei , Rongvaux Anthony , Weibel Carla , Elizabeth Henchey , Halene Stephanie , Park In-Hyun , Flavell Richard
Published: October 2019

De novo generation of HSCs has been described as a "holy grail" of stem cell biology, however the factors required for converting human pluripotent stem cells (PSCs) to true hematopoietic stem cells (HSCs) capable of robust long-term engraftment have yet to be fully characterized. Two groups have shown that injection of PSCs into immunodeficient mice leads to teratomas containing niches producing hematopoietic progenitors capable of long-term engraftment. Once these hematopoietic progenitors and their microenvironments are better characterized, this system could be used as a model to help direct in vitro differentiation of PSCs to HSCs. Toward this end, we have injected human PSCs into immunodeficient mice expressing human rather than mouse M-CSF, IL-3, GM-CSF, and thrombopoietin, as well as both human and mouse versions of the "don't eat me signal" Sirpa (collectively termed MISTRG mice). These cytokines are known to support different aspects of hematopoiesis, and thrombopoietin in particular has been shown to support HSC maintenance, suggesting these mice may provide a better environment for human PSC-derived HSCs than the more traditional mice used for human HSC engraftment. The majority of teratomas developed so far in MISTRG contain human hematopoietic cells, and the CD34+ population isolated from over half of the teratomas contained hematopoietic colony forming cells by colony forming assay. These findings further corroborate this approach as a viable method for studying human PSC to HSC differentiation.

Disclosures

No relevant conflicts of interest to declare.
Decreased Tim-3 Expression Level of Peripheral Blood Natural Killer Cells in Severe Aplastic Anemia
Authors: Fu Rong , Yuan Xin , Liu Chunyan , Liu Hui , Wang Yihao , Chen Tong , Li Lijuan , Song Jia , Wang Guojin , Shao Zonghong
Published: October 2019

Severe aplastic anemia (SAA) is a life threatening disease characterized by severe pancytopenia and bone marrow failure. T-cell immunoglobulin- and mucin domain-containing (Tim)-3 has been initially used to identify dysfunctional T cells, but recent studies have demonstrated that Tim-3 is widely detected on nature killer cells (NK cell) and may serves as a marker for activation and maturation of NK cells. In our study, Tim-3, expressed on peripheral blood of NK cells in SAA patients, was quantitatively analyzed by Flow cytometry before and after immunosuppressive therapy (IST). Results showed that the expression of Tim-3 in patients before IST [(61.11±15.46)%] was significantly lower than that in patients after IST[(74.30±12.63)%, P<0.05] and normal controls[(70.39±12.73)%, p<0.05]. However, no difference was observed between patients before and after IST. Therefore, we concluded that low expression of Tim-3 in NK cells may play a crucial role in early stage of SAA.

Disclosures

No relevant conflicts of interest to declare.
Very Severe Aplastic Anemia Due to Doxycycline Therapy
Authors: Singh Prabhsimranjot , Sinha Ankur , Kamath Anisha , Malhotra Sonali , Chandra Abhinav B
Published: October 2019

Very severe aplastic anemia due to doxycycline therapy

A 42 y/o male presented to the emergency department with 3 weeks history of fatigue and epistaxis. On initial blood work he was found to have pancytopenia with hemoglobin of 4.7g/dl, platelet count of 4,000 and absolute neutrophil count of 160. He has no significant family history. His past medical history was only significant for a skin rash, which was biopsied 3 months ago and reported to be follicular dermatitis. The only home medication he has taking was doxycycline 100mg twice a day for the past few weeks before the current hospital admission. He was transfused with packed red blood cells and platelets to maintain hemodynamic stability. His bone marrow biopsy was reported to be hypocellular (<5% cellularity) with normal morphological features (see figure 1). Paroxysmal nocturnal hemoglobinuria was ruled out by flow cytometry. Workup including HIV, EBV, Hepatitis B and C and parvovirus serology was reported to be negative. His history of long-term Doxycycline use as well as no family history of any similar disorder, it is plausible that doxycycline could have caused his aplastic anemia. In congruence with the above mentioned findings, he is diagnosed with very severe aplastic anemia and is planned to be started on immunosuppressive therapy with equine anti-thymocyte globulin and cyclosporine along with consultation for allogeneic transplant.

Aplastic anemia is a remarkably rare but serious adverse effect of drug therapy like antimicrobials. We report this as the first case, to the best of our knowledge based on the literature review, of possible doxycycline induced severe aplastic anemia. Our patient had history of skin rash for which he was taking doxycycline for few weeks before he presented with pancytopenia. Acquired aplastic anemia is a rare hematological disorder presenting with pancytopenia and a predominantly empty marrow. It is a fatal disease and irrespective of the etiology, without treatment, patients usually succumb due to infection and bleeding. Cases have been reported with tetracycline as a causative agent of aplastic anemia. We hypothesize doxycycline to be the possible etiology of severe aplastic anemia in this patient. Practitioners need to be aware of this rare but fatal complication of this widely used antibiotic.

Figure 1. Hypo-cellular Bone Marrow Figure 1. Hypo-cellular Bone Marrow

Disclosures

No relevant conflicts of interest to declare.
De Novo Nethyltransferases, DNMT3a and DNMT3b Are Underexpressed in Multiple Myeloma
Authors: Latalova Pavla , Minarik Jiri , Smesny Trtkova Katerina
Published: October 2019

Background and aims: Presently, there is growing evidence that along with the important role of genetic abnormalities, epigenetic aberrations are relevant factors in multiple myeloma (MM). As was recently found, genome-wide analysis of DNA methylation reveals epigenetic alterations in plasma cells from patients with MM and individuals with monoclonal gammopathy of undetermined significance (MGUS). MGUS is characterized by predominant hypomethylation. Transformation into MM is accompanied by progressive hypermethylation with maximum methylation seen in relapsed disease. DNA methyltransferases (DNMTs) catalyze DNA methylation through transfer of methyl group to cytosine of the CpG dinucleotides, resulting in 5-methylcytostine. DNMT1 maintains patterns of methylated cytosine residues in human genome. DNMT3A and DNMT3B are de novo DNA methyltransferases, whose role is to maintain new methylation pattern that forms due to formation of the cancer.

Methods: 30 bone-marrow aspirates from individuals with MGUS or MM patients before the treatment initiation were used. The cDNA was synthesized using 100 ng of total RNA in a 20 µl reaction volume (Roche, Diagnostics, Basel, Switzerland). Quantification of DNMT1, DNMT3a and DNMT3b levels by TaqMan® probes (Life Technologies, Grand Island, NY) with Xceed qPCR Master Mix (IAB, BioTech-Europe, Czech Republic) was performed. For normalization, the GAPDH was used.

Results: Although MM is characterized by widespread alterations in DNA methylation, we observed that DNMT3a and DNMT3b de novo methyltransferases were underexpressed in both, MGUS individuals and MM patients when compared to DNMT1 expression level (Figure 1). The transcribed genes have increased levels of 5-hydroxymethylcytosine, then the DNMTs activities might compensate for active hydroxymethylation - demethylation.

Conclusions: Our results confirm that the expression of de novo DNA methyltransferases is deregulated in MM cell lines. The presented analysis is first of its kind that was performed on human myeloma cell lines, especially with the focus on the residual expression of Dnmt3a.

With support of the grant NT14393.

Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients. Figure 1. Quantitative RT-PCR for DNMT1, DNMT3a and DNMT3b in MGUS individuals and MM patients.

Disclosures

No relevant conflicts of interest to declare.
Oroxylin a Heightens Tumor Necrosis Factor a-Induced Differentiation Effects on Acute Myeloid Leukemia Cells Via Modulation on AKT and NF-Kb Pathways Involving RXRa
Authors: Xu Jingyan , Ouyang Jian , Hui Hui
Published: October 2019

Tumor necrosis factor alpha (TNFα) is a complicated cytokine which could induce differentiation of acute myeloid leukemia (AML) cells. We have found that Oroxylin A, a natural compound isolated from Scutellariae radix, markedly enhanced TNFα-induced NBT reduction and CD11b/CD14 expression of AML cells. Besides, Giemsa staining also displayed that the combination group induced U937-MDR cells to differentiate into monocyte-like cells yet NB4 and HL-60-resistant cells to granulocytic-like cells. Further study showed that TNFα induced PI3K subunit p85α binding to N-terminal truncated nuclear receptor RXRα (tRXRα) proteins and activating AKT. On the contrary, these could be inhibited by Oroxylin A through inhibiting interaction between tRXRα and p85α in NB4 and HL-60-resistant cells. Moreover, Oroxylin A inhibited the activation of NF-κB signaling and the DNA binding activity by TNFα proved by EMSA in these two AML cells. All these suggested that Oroxylin A was able to inhibit NF-κB signaling and RXRα-dependent AKT signaling, the negative effects of TNFα for AML therapy, suggesting that combination of Oroxylin A and TNFα might be a novel candidate for differentiation therapy for AML cells and required further investigation.

Disclosures

No relevant conflicts of interest to declare.
Low Dose Steroids Can Alleviate Blinatumomab-Associated Toxicities without Negatively Impacting Treatment Efficacy
Authors: Mori Shahram , Nelson Rebecca Jean , Patel Rushang D. , Ahmed Wesam B.
Published: October 2019

Relapsed/refractory precursor B cell acute lymphoblastic leukemia (B cell ALL) carries a poor prognosis in adults and remains a challenging therapeutic area. Allogeneic stem cell transplantation is the only potentially therapeutic strategy for this group of patients, but its success depends on the disease status at the time of transplant. Blinatumomab is a recently approved bi-specific CD19-directed CD3 T-cell engager (BiTE) antibody in B-cell ALL patients who have failed induction chemotherapy. Its administration however is commonly complicated by grade III-IV toxicities including high-grade fevers and Cytokine Release Syndrome (CRS) defined as elevations in ferritin levels, pro-inflammatory cytokines such as IL-6, fever, chills and vascular leak. These complications can result in in premature treatment interruptions, failures as well as treatment-related mortality. At this time, it is unknown whether the concomitant use of steroids with Blinatumomab can alleviate these symptoms without negatively impacting the efficacy of treatment.

Here we describe two patients who were treated with standard dosing of Blinatumomab starting at 9 mcg/day on days 1-7 and 28 mcg/day on days 8-28. Both patients developed grade III-IV febrile episodes within the first 24 hours of treatment resulting in initial treatment interruption. These febrile episodes were associated with elevations in ferritin level. After a brief supportive measure, Blinatumomab was restarted but this time concurrently with methyl-prednisolone 5-10mg BID for days 2-5. Neither patient required any further treatment interruption or developed CRS as measured by normal IL-6 levels. In both cases a complete remission was achieved and they were successfully bridged to allogeneic stem cell transplantation.

In conclusion, in relapsed and refractory B-cell ALL, Blinatumomab-associated grade III/IV toxicities may be successfully addressed with low dose steroids without inhibiting anti-leukemic effect of BiTE antibody.

Disclosures

No relevant conflicts of interest to declare.