bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Inherited Bleeding Disorders in Women with Pregnancy Loss
Authors: Krause Manuela , Pillitteri Daniele , Pilgrimm Ann-Kathrin , Scholz Thomas , Schwerdtfeger Rainer , Kirchmaier Carl
Published: October 2019

Abstract 4657

Introduction:

Pregnancy is a hypercoagulable state, and thromboembolism is the leading cause of antepartum and postpartum maternal mortality. Women with thrombophilic mutations (factor V leiden, prothrombin, and MTHFR) and inherited bleeding disorders, such as deficiency of factor XIII and fibrinogen, have been shown to be at increased risk of pregnancy loss. However, the risk of miscarriage in women with other inherited bleeding disorders has been discussed controversially. Due to the lack of data, it cannot be determined if the risk of miscarriage is increased in women with von Willebrand disease (vWD).

The aim of our study was to clarify the association between inherited bleeding disorders and pregnancy loss.

Patients and Methods:

Subjects

Concerning this investigation we included 91 female patients with two [n=46] or more [n=45] miscarriages occurring prior to 28 weeks of gestation and/or stillbirth without apparent reason. The median age of the examined group at the time of first fetal loss was 29 years, ranging from 17 to 41 years.

Methods

At first we compiled a detailed clinical history of bleedings of all patients. Subsequently, we performed various tests to gather information regarding coagulation abnormalities and thrombophilic defects. Therefore a molecular and functional assessment of the following data was performed:

Coagulation factors, vWF:Ag, vWF:RCo, phospholipid antibodies, hyperhomocysteinaemia (HHCY), protein S (PS), protein C (PC), antithrombin (AT) and FV-Leiden mutation (G1691A), FII mutation (G20210A) and MTHFR C677T.

Results:

In our investigated population consisting of 91 women we registered 299 pregnancies of which 240 resulted in fetal loss, 232 prior to week 28 of pregnancy and 8 stillbirths.

Seven out of 91 patients (8%) were carriers of inherited coagulation disorders; vWD: n=2 (2%), FVII deficiency: n=3 (3%), thrombocytopathy: n=2 (2%). In our study collective there was no increased rate of patients with vWD. None of the patients showed a FXIII- or fibrinogen deficiency. However, 17 patients (19%) have a bleeding diathesis.

In 55 patients (60%) we could detect the following thrombophilic defects:

FV-Leiden (G1691A): n=10, MTHFR C677T: n=42, PS: n=1, PC: n=1, APS: n=1.

Conclusion:

The incidence of vWD patients in our miscarriage collective is the same as the overall incidence of vWD patients in the general population. Therefore vWD is not associated with an increased risk of fetal loss.

Disclosures:

No relevant conflicts of interest to declare.
Outcome of Splenectomy On Adult Idiopathic Thrombocytopenic Purpura (ITP)
Authors: Dutta Tarun K. , Vy Vishnu , Basu Debdatta
Published: October 2019

Abstract 4690

Aim:

To study outcome of splenectomy on adult ITP patients (both in prospective and retrospective groups)

Patient and Methods:

Two groups of ITP patients were studied. They were as follows:

(i) Retrospective group – Twenty two patients who underwent splenectomy during last twenty one years

(ii) Prospective group – All patients admitted during last three years with a minimum follow-up of six months (29 patients)

Result:

Retrospective group: Twenty two patients had undergone splenectomy during last twenty one years. Median time from diagnosis to splenectomy was 32 months. Median platelet count was 28,000/μl with some form of drug treatment (before undergoing splenectomy). Median post-splenectomy follow-up was 54 months (maximum 252 months). Eleven (50%) patients had either primary failure or relapse following splenectomy during subsequent years. The cumulative break-up figure was as follows:

Primary failure – 3 (13.6%)

At 24 months – 7 (31.8%)

At 5 years – 9 (40.9%)

At 10 years – 11(50%)

Prospective group: Out of 29 patients of ITP enrolled according to inclusion criteria of minimum six months’ follow-up, 20 had complete remission with steroids; nine had primary failure. Out of 20 patients with complete remission, two had relapse within six months of remission. Out of total 11 patients with failure to response to steroids (nine primary failure and two secondary failure), six patients agreed to undergo splenectomy. Three patients had complete remission till last follow-up (maximum follow-up was for 23 months). Two had primary failure following splenectomy and one had relapse. Five patients who did not agree for splenectomy are in complete or partial remission with azathioprine.

Conclusion:

At the end of two years in retrospective group, almost one-third of 22 patients who underwent splenectomy had either primary failure or relapse. Similarly in prospective group, though small in number, at the end of 23 months itself, three of six patients who underwent splenectomy had either no response or had relapsed. Thus this study questions the utility of splenectomy as second-line treatment after steroid failure. Rather, patient may be given a choice to opt for other form of drug treatment after steroid failure and advised splenectomy as and when unavoidable. Nevertheless, any patient opting for splenectomy should be cautioned that there is a fair chance of relapse later in life. Furthermore, it appears incidence of relapse following splenectomy is higher in Indian patients as compared to that in the Western patients.

Disclosures:

No relevant conflicts of interest to declare.
Rapid Anuric Acute Kidney Injury After the Administration of IV Anti-D in a Teenager
Authors: Haggett L. Kristy , Ayyanar Kanyalakshmi , Shoemaker Larry
Published: October 2019

Abstract 4691

Introduction:

A 16 year old male developed dark brown urine and prolonged anuric renal failure requiring hemodialysis after receiving IV anti-D. He was diagnosed with idiopathic thrombocytopenia purpura (ITP) after presenting with diffuse petechiae and a platelet count of 7000/μl, about a week after onset of viral prodrome that had resolved 2 days before. The white cell count, hemoglobin, PT, PTT, INR, and direct Coomb’s screen (IgG and complement) values were normal/negative at initial presentation, as were plasma chemistries, including LDH and uric acid levels. Except for small ketones and small heme reactions, the initial urinalysis was also unremarkable. Following IV anti-D, the patient developed intravascular coagulation concurrent with hemolysis and renal failure.

Method:

In this case report we describe a teenager who presented with ITP and rapidly developed severe anuric acute kidney injury (AKI) associated with mild intravascular hemolysis and coagulopathy. We review previous reports of IV Anti-D associated AKI, in order to identify common risk factors that may be used as a pre-treatment screen.

Result:

Anuric renal failure following IV anti-D has previously been reported and is a profoundly rare complication associated with a high risk of death. There is no reliable screening test, as this case illustrates. The clinical and laboratory course in this teenager, as well as from other reported cases, lead us to speculate that while hemolysis following IV-anti D infusion is necessary for the development of acute renal failure, it is insufficient unless there is a concurrent or antecedent intravascular coagulopathy. We suggest that the D-dimers assay should be formally evaluated as a screen prior to IV anti-D therapy to identify those individuals at risk for this life-threatening adverse reaction.

Conclusion:

IV anti-D is a good option for the treatment of ITP but the risks should be seriously considered prior to administration. A preliminary workup should be initiated prior to therapy. Other forms of treatment may be necessary in patients with evidence of renal insufficiency, hemolysis, recent EBV infection, known positive C-antigen or evidence of coagulopathy. We are planning a prospective study to evaluate the usefulness of obtaining a D-dimer in ITP patients prior to treatment. This will elicit information that is currently unavailable regarding the frequency of positive D-Dimers in patients with ITP prior to treatment.

Disclosures:

No relevant conflicts of interest to declare.
IFN-γ Level Could Be an Important Indicator to Determine Optimal Timing for Treatment or Prevention of GVHD in NSG Xenogeneic Model
Authors: Lee Young-Ho , Jang Yun Kyung , Kim Miyeon , Choi Soo Jin , Jang Seongsoo
Published: October 2019

Abstract 4704

Background:

We established xenograft model for graft-versus-host disease (GVHD) using human peripheral blood mononuclear cells (PBMC) in NSG mice. In addition, we also investigated the optimal timing for treatment or prevention of GVHD by observing the changes of inflammatory cytokines in xeno GVHD-induced mice.

Materials and Methods:

We infused various numbers of hPBMCs intravenously into irradiated NOD.scid gamma (NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJ, NSG) mice and assessed the optimal dose of hPBMCs for GVHD model by measuring GVHD clinical score and histopathologic findings as well as inflammatory cytokines.

Results:

We observed the compatible findings with GVHD by clinical score as well as immunohistochemical analysis which showed abundant invasion of lymphocytes in the lung, liver, kidney, and small intestines in xeno GVHD-induced mice. On administration of 1 × 106 and 2.5 × 106 hPBMCs, all NSG mice died on 41.6±6.8 days and 24.0±10.4 days, respectively. In contrast, 5 out of 6 NSG mice transplanted with 0.5×106 of hPBMCs survived until 89.6±23.2 days. Additionally, the changes of inflammatory cytokines were analyzed in NSG mice using 1×106 of hPBMC. The concentrations of IFN-γ were markedly increased from day 18 to day 36, however, TNF-α, and IL-2 were rarely detected during 36 days. CD8+ T cells were detected 2–3% in only 1 out of 3 mice and CD 56 was not appeared at all.

Conclusion:

These results demonstrated that 2.5×106 of hPBMC was optimal for acute GVHD and 0.5×106 of hPBMCs for chronic GVHD model in NSG mice. In addition, the changes of IFN-γ levels would provide an important evidence for the optimal timing for treatment or prevention of GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Patients with Monosomal Karyotype in Acute Myeloid Leukemia Is Prognostically Worse Than with Complex Karyotype
Authors: Xiaoli Liu , Na Xu , Qingfeng DU , Dan Xu , Fanyi Meng , Lulu Xu , Jinfang Zhang , Bintao Huang , Xujing Luo
Published: October 2019

Abstract 4735

Purpose: Monosomal karyotype (MK) refers to the presence of two or more distinct autosomal monosomies or a single monosomy associated with a structural abnormality. To analyze the prognosis of cytogenetic components of a complex karyotype or Monosomal Karyotype in acute myeloid leukemia (AML) except acute promyelocytic leukemia(APL). Patients and Methods:Cytogenetics and overall survival (OS), Disease free survival(DFS) were analyzed in 551 AML patients age 14 to 60 years in our center.Results: There ware 235 patiets with cytogenetic abnormalities, 25 cases with inv(16)(p13.1q22) or t(16;16)(p13.1;q22),and 63 cases with t(8;21); 31 cases (13.2%)met the criteria for MK and 39 cases (16.6%) had a complex karyotype without monosomies. OS was significantly inferior in patients with MK compared with those with a complex karyotype without monosomies (P<0.01;HR 1.85,95% confidence interval(95%CI),0.95-2.81). There was no difference between MK cases with complex karyotype cases in DFS (P>0.05□GHR 3.42,95% confidence interval(95%CI),2.96-6.70). There was significant difference in regardless of whether OS or DFS between MK+ patients with MK− patients (P<0.01). Conclusion: MK was one of independent risk factor in AML patients.

Disclosures:

No relevant conflicts of interest to declare.
Utility of Blood Cultures in Febrile Patients in a BCSH Level 4 Centre
Authors: Lee Sophie , Kandiah Kesavan , Holyhead Kate , Aung Sandar , Smith Neil , Nikolousis Emmanouil , Paneesha Shankaranarayana
Published: October 2019

Abstract 4779

Objective:

To describe the epidemiology of bacterial organisms in a level 4 haematology centre and to investigate the prevalence of positive blood cultures at various time points in a febrile patient

Methodology:

A retrospective review was performed on 138 haematology patients who had blood cultures taken on ward 19 for febrile episodes at Heartlands Hospital, a BCSH Level 4 centre over a 6 month period from 1/5/2009 until 30/11/2009.

Results:

There were 704 blood cultures performed on 138 patients for 246 ‘febrile’ episodes. There were 59 positive blood cultures.

Patients with AML and DLBCL are most likely to require blood cultures as a result of febrile neutropenia.

Coagulase Negative Staphylococcus is the most common organism isolated from blood cultures in neutropenic patients during ‘febrile’ episodes.

There was only one pathogen cultured that was resistant to the first line antibiotic (Pipercillin/Tazobactam) that was used at this Trust.

The incidence of positive blood cultures at onset of fever is 15% is comparable to that seen in other institutions of similar patient populations. This decreased to 1.5 % at 24 hours, 1.7% at 48 hours and 6.3% at 72 hours.

Conclusion:

Based on the results above, it appears reasonable in stable patients to reduce the number of blood cultures done to 72 hours after the initial culture.

At the time of this review, the antibiotic policy in place at HOE NHS trust is appropriate for the pathogens that normally affect this patient population.

There should be regular audits to observe the epidemiology of microbials that affect haematology patients in this region and also to monitor their antimicrobial resistance to ensure appropriate first line antibiotics are in use.

Disclosures:

No relevant conflicts of interest to declare.
Application of Mass Cytometry to Measure Proliferation During Normal and Malignant Hematopoietic Differentiation
Authors: Behbehani Gregory K. , Bendall Sean C , Clutter Matthew , Medeiros Bruno C. , Fantl Wendy J. , Nolan Garry P.
Published: October 2019

Abstract 4782

Mass cytometry is a recently introduced proteomic technology that permits simultaneous measurements currently of as many as 40 parameters per single cell, including surface markers and intracellular signaling molecules and has recently provided a system-wide view of immune/hematopoietic cell differentiation within human bone marrow (Bendall et al, Science 2011). Given the unprecedented level of detail at the single cell level this approach provides, this technology is ideal for delineating the molecular and cellular basis of tumor heterogeneity. As an extension from our recent mass cytometry studies, we have now developed a method to perform cell cycle analysis by utilizing uridine incorporation, simultaneously with determinations of other cell cycle phase markers such as cyclins A, B1, and D as well as phosphorylation of retinoblastoma protein, histone H3, plus markers of apoptosis and other critical markers that demarcate malignant dysregulation. This allows for cell cycle state characterization that can be employed in both cell lines and primary cells growing in vitro or in vivo. We have combined this new method with a hierarchical clustering algorithm (SPADE) to profile the cell cycle state distribution across the continuum of hematopoietic differentiation in normal bone marrow. We observe the expected quiescence of terminally differentiated cells, hematopoietic progenitor and stem cell populations, and note that the majority of cell proliferation is confined to immunophenotypically immature cell populations of the myeloid, erythroid and monocytic linages. We have begun a broad comparison of these “normal” proliferation states to those observed across punctuated states of malignant progression that vary in “differentiation” of malignant cells in primary human sample in patients with acute myeloid leukemia revealing. The distribution of proliferation across these subpopulations generally parallels that of normal samples with more immunophenotypically “differentiated” AML cell populations having lower proliferative rates, however, not all AML patient samples follow this trend.

Disclosures:

Fantl: Nodality, Inc.: Equity Ownership.
Expression of Kindlins in Acute Myeloid Leukemia
Authors: Mi Yingchang , Wu Wenbin , Zhang Qing , Li Yan , Li Xiaoyan , Tian Zheng , Wang Min
Published: October 2019

Abstract 4910

The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. They comprise of three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, they share considerable sequence and structural similarities. A few of study revealed that Kindlin-2 influences solid tumor cell invasion and resistance. With regard to AML, the influence of Kindlins is still unknown. To evaluate the clinical significance of Kindlin-2 in acute myeloid leukemia (AML), we investigated the expression of Kindlin-2, kindling-3 in AML cells.

1. Materials and methods

K562, KG-1a, HL60, U937, Jurkat cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified atmosphere of 5% CO2. Bone marrow (BM) samples were obtained from 88 patients with de novo AML from Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Samples of 9 normal donors and ITP were used as the control group.

Bone marrow mononuclear cells (BMMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. Expressions of Kindlin-2, Kindlin-3 were detected by RQ-PCR. The following primers for real-time PCR were used: (a) Kindlin-2 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (b) Kindlin-2 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (c) Kindlin-3 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (d) Kindlin-3 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (e) GAPDH sense primer, 5'-GAAGGTGAAGGTCGGAGTC-3'; (f) GAPDH antisense primer, 5'-GAAGATGGTGATGGGATTTC-3'. Analysis was performed using ABI 7500 Sequence Detection software (Applied Biosystems). The expression of Kindlin-2 and Kindlin-3 were showed as RQ value calculated through ΔΔCt method [ΔΔCt = (CtKindlin □ CtGAPDH)sample □ (CtKindlin □ CtGAPDH)calibrator]. The ΔCt (CtKindlin □ CtGAPDH) of K562 was defined as calibrator, and the RQ of calibrator was 1.000. Relationships between Kindlin-2, Kindlin-3 and the patients' clinical data were analyzed.

2. Results

Expression of Kindlins in newly diagnosis AML

The level of Kindlin-2 in AML (0.163±1.665) was significantly lower than that in non-AML (1.683±1.395) controls (p=0.010). No significant difference was found between the AML and controls in levels of Kindlin-3 (p=0.216). Out of the 79 patients who accepted treatment, 61 patients achieved complete remission (CR) and 18 patients were NR. Patients with higher expression of Kindlin-2 had a higher CR rate (86.8% vs 68.3%) (p=0.050). Expression of kindling 3 was unrelated to CR rate. Both of kindling-2 and kindling-3 increased after CR. This finding implicates Kindlin-2 as a potential prognostic factor of AML.

Disclosures:

No relevant conflicts of interest to declare.
Hyper Immunoglobulin E Syndrome (HIES): Report of a New Case Treated with Omalizumab and Dexametasone
Authors: Cortázar-Benítez Luis Fernando , Rojas Sotelo R.M. , Vargas-Viveros Pablo , Hurtado-Monroy Rafael , Romo Aguirre Catalina , Valenzuela Tamariz Jorge , Zárate Alejandra , Masse Sergio
Published: October 2019

Abstract 4934

The classical triad of eczema, recurrent skin and lung infections and high serum IgE, other connective tissue, cardiac and brain abnormalities are the accepted diagnostic criteria for HIES. The treatment of HIES is not established due to the scarce number of published cases, however is limited to skin local measures and supportive care. Herein we inform a severe case with response to Omalizumab, a monoclonal anti IgE antibody in combination with Dexametasone.

A 36 year old male with family history of asthma and atopic dermatitis (AD). Since child, the patient had allergies and AD. At 8 years he presented asthma and recurrent upper airway infections at 2 to 3 times per year and 7 dental pieces removal. In the last 3 years he noted important increase of AD with very intense and disabling pruritus without response to treatment and progression to generalized erithrodermia, nodes in legs and arms, and skin trasudate, axilar and inguinal lymph nodes (6×4 cm). Laboratory positive test were eosinophils 6%, serum IgE 29,280 IU/mL. Skin biopsy showed psoriasis-like dermatitis, micro abscesses and dermatopathic lymphadenopathy. We started treatment with trimetoprim/Sulfamethoxazole 80/400 mg twice a day, oral Dexametasone 20 mg weekly and Omalizumab 200 mg subcutaneously every two weeks. After 15 days of treatment there was a rapid improvement of skin derangement and IgE levels dropped to 2000 IU/mL after 16 weeks of treatment. Patient continues on treatment with no side effects and pruritus disappears.

Omalizumab is a monoclonal antibody against IgE recently introduced to the treatment of HIES in a few patients reported since 2008 with excellent response, along with intensive care of skin lesions, prompt antibiotic and antimycotic treatment for infections are the mainstay of HIES management.

Disclosures:

No relevant conflicts of interest to declare.
Generation of Melanoma Antigen Specific T Cells From Human Ips Cells Using a Feeder Free System.
Authors: Vizcardo Raul , Masuda Kyoko , Yamada Daisuke , Ikawa Tomokatsu , Shimizu Kanako , Fujii Shin-ichiro , Koseki Haruhiko , Kawamoto Hiroshi
Published: October 2019

Abstract 2292

iPS cells can be induced from various types of somatic cells by reprogramming them using Yamanaka factors (Oct4, Sox2, Klf4, and Myc). They are reported to be very similar to ES cells in many respects, such as gene expression pattern and multipotency. They are expected to be used as a cell source for production of various types of cells to be used in regeneration medicine. In this study, we planned to use iPS cells as a progenitor source for immune cell therapy. For this purpose, iPS cells derived from a lymphocyte that shows a certain antigen-specificity are preferable, because antigen specificity is inherited to iPS cells made from the lymphocyte. For example, if iPS cell are produced from cytotoxic T cells specific to a tumor antigen, T cells generated from these T-iPS can be used in cell therapy for patients bearing cancer.

We have succeeded in establishing iPS cells from human mature T cells (T-iPS cells), namely from whole CD3+ cells or CD4−CD8+ cells of cord blood as well of adult peripheral blood. These T-iPS cells were confirmed to bear productively rearranged TCRβ chain gene. These T-iPS cells were differentiated to CD4+CD8+ double positive T cells and eventually into CD8+ single positive T cells expressing αβTCR in an in vitro co-culture system using OP9-DL1 stromal cells. Such T cell induction occurred more efficiently from T-iPS cells than from human ES cells or from iPS cells derived from other cell types. Sequence analysis of their TCRβ chain suggests that these T cells preserved their original antigen specificity. To confirm that T-iPS cells can generate antigen specific T cells we established T-iPS cells from mature cytotoxic T cells specific to MART-1 (Melanoma antigen recognized by T cells 1) epitope. These MART-1 T-iPS cells were differentiated in vitro until CD4+CD8+ double positive T cells. By stimulation with anti-CD3 antibody, DP cells generated in vitro from MART1-T-iPS cells turned into a large number of CD8+ T cells specific to MART-1, which produced a substantial amount of IFNg upon TCR stimulation. The present study thus provides a novel method for cloning and expanding CD8+ T cells specific to a given antigen, which can be potentially applied for cell therapy against cancer.

Disclosures:

No relevant conflicts of interest to declare.
Hematopoietic Lineage Selective Roles for Trib Pseudokinases Post Bone Marrow Transplantation.
Authors: Verma Rakesh , Dev Arvind , Wojchowski Don M.
Published: October 2019

Abstract 2298

Trib's 1–3 are a homologous set of pseudokinases that (through their molecular actions as adaptors for E3 ubiquitin ligases, Akt and/or Mek/Erk) can exert diverse tissue and cellular effects. Upon ectopic expression, Trib2 promotes myeloid leukemia, and is elevated in its expression in melanoma. Trib3 can modulate insulin action, adipogenesis, and cardiomyopathy. Trib1 is implicated in inflammatory cytokine responses, and megakaryocytic leukemia. This illustrates the range of Trib functions, and also points to important regulatory roles for Tribs during hematopoiesis. To date the majority of Trib action studies have applied gain-of-function approaches. To gain new insight into hematopoietic roles of Trib2 and Trib3, we have generated novel knockout mouse models, and have employed a bone marrow transplant approach to assess possible effects of Trib2 or Trib3- deficiency on blood cell development. This approach also brings to bear the frequent requirement of physiological stress for the engagement of Trib pseudokinases. We assessed blood lineage reconstitution via hematological and flow cytometric analyses in irradiated (2 × 450 RAD) and bone marrow transplanted congenic recipient C57BL/B6.Ptprca, Ly-5.1 mice (5 × 105 Trib2-KO, Trib3-KO or wild-type donor cells). In all experiments, full representation of donor derived Ly5.2 hematopoietic cells was confirmed in blood, bone marrow and spleen. Trib2-KO and Trib3-KO transplanted mice each exhibited skewed hematopoietic reconstitution profiles (as compared directly to wild-type BM transplanted controls). Trib2-KO transplanted mice exhibited persistently elevated lymphoid and WBC levels at week 6, 10 and 36 post BMT, and peripheral blood CD19+/B220+/Ly5.2+ B-cells also were elevated. In contrast, Trib3-KO transplanted mice exhibited sustained increases in reticulocyte production, together with heightened splenic erythropoiesis. Within a bone marrow transplantation context, findings reveal novel lineage- specific roles for Trib2 during lymphopoiesis, and for Trib3 during stress erythropoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Activation of Notch Signaling Mediates Ex Vivo Expansion of Multilineage, Engrafting Murine Hematopoietic Progenitors From Embryonic Sources.
Authors: Hadland Brandon K , Varnum-Finney Barbara , Bernstein Irwin D.
Published: October 2019

Abstract 2321

An important goal in the application of pluripotent stem cells (PSC) for therapeutic purposes is the derivation of hematopoietic stem and progenitor cells (HSPC) capable of efficient engraftment in vivo. Fundamental to achieving this goal is improved understanding of key signal pathways required to establish, maintain and expand HSPCs from embryonic sources. Ex vivo activation of Notch signaling in mouse bone marrow and human cord blood-derived HSC can facilitate expansion of rapidly engrafting multilineage progenitors, which has recently been translated for therapeutic purposes. In contrast, similar expansion of engrafting progenitors has not been successful from PSC. This prompted us to evaluate whether embryonic-derived HSPC have capacity to respond to ligand-induced Notch signaling ex vivo, and whether Notch activation could promote expansion of engrafting progenitors from these embryonic sources. We have examined the effects of ex vivo activation of Notch receptors by immobilized, exogenous Notch ligands on highly enriched populations of embryonic HSC and HSC precursors (pre-HSC) at various developmental stages. We find that activation of Notch by the ligand Delta1 within HSC/pre-HSC isolated from embryonic aorta-gonad-mesonephros (AGM) promotes expansion of progenitors with erythromyeloid colony forming potential and T/B-lymphoid potential in vitro, with concurrent expression of surface phenotypes resembling fetal liver-stage HSC. Furthermore, Notch activation in embryonic HSPC also mediates expansion of progenitors with rapidly engrafting myeloid and lymphoid capacity in irradiated mouse models. Our results demonstrate that embryonic stage HSPC have capacity to expand in response to Notch activation, and thus further studies comparing AGM- and PSC-derived hematopoietic precursors are needed to elucidate differences that may account for failure to expand HSPC from PSC.

Disclosures:

Bernstein: Seattle Genetics, Inc.: Consultancy.
Incorporation of a Histone Demethylase, JARID1B, in a Novel ETS-E Box DNA-Binding Complex in Murine Erythroid Progenitors.
Authors: Cai Ying , Nagarajan Lalitha , Curtis David J. , Brandt Stephen J.
Published: October 2019

Abstract 2335

The TAL1/SCL gene, originally discovered from its involvement by a recurrent chromosomal translocation in T-cell acute lymphoblastic leukemia, is important for hematopoietic stem cell and progenitor function and is essential for hematopoietic and vascular development. A member of the basic helix-loop-helix family of transcription factors, TAL1 binds a DNA sequence motif, CANNTG, termed the E box. We and others recently identified a novel TAL1-containing DNA-binding complex that includes an ETS protein, ELF2 (also known as NERF) in erythroid cells, and recognizes a bipartite sequence element containing an adjacent ETS binding site and E box. Our work showed this complex also contains proteins common to other TAL1 DNA-binding complexes described, including a LIM domain protein, LMO2 in erythroid cells, the LIM domain binding protein Ldb1, and putative single-stranded DNA-binding proteins SSBP2 and SSBP3. As both ELF2 and histone demethylase JARID1A, and later the related JARID1B, were identified using the same methodology (yeast two-hybrid analysis) to interact with LMO2, and multiple peptides derived from Jarid1b were identified by mass spectrometry analysis of highly purified Tal1-containing complexes from murine erythroleukemia (MEL) cells, we investigated whether JARID1B was present in the TAL1- and ELF2-containing complex. First, co-immunoprecipitation analysis identified Jarid1b in Tal1- or Elf2-containing immune precipitates and Tal1 in Jarid1b-containing immune precipitates from MEL cell extracts. Further, chromatin immunoprecipitation (ChIP) and re-ChIP analysis showed that Elf2 and Jarid1b co-occupied a region in the fourth intron of the Ssbp3 gene in MEL cells, previously demonstrated to be a physiologic target of the Tal1/Elf2 complex. Finally, knockdown of Elf2 and Jarid1b in MEL cells produced the same phenotype, decreased cellular proliferation. These results suggest a role for the JARID type histone demethylase JARID1B, and by inference histone demethylation, in the function of the ETS-E box DNA-binding complex and in proliferation of late-stage murine erythroid progenitors.

Disclosures:

No relevant conflicts of interest to declare.
Secreted Mediators of Self-Renewal of Hematopoietic Stem Cells Identified Using Bio-Informatic Analysis of Co-Cultures of HSC and Stromal Cells.
Authors: Vilne Baiba , Istvanffy Rouzanna , Eckl Christina , Bock Franziska , da Costa Olivia Prazeres , Stümpflen Volker , Peschel Christian , Mewes Hans-Werner , Oostendorp Robert A.J.
Published: October 2019

Abstract 2353

Hematopoiesis is maintained throughout life by the constant production of mature blood cells from hematopoietic stem cells (HSC). One mechanism by which the number of HSC is maintained is self-renewal, a cell division in which at least one of the daughter cells is a cell with the same functional potential as the mother cell. The mechanisms of this process are largely unknown. We have described cell lines that maintain self-renewal in culture. To study possible mechanisms and mediators involved in self-renewal, we performed co-cultures of HSC model cells: Lineage-negative Sca-1+ c-Kit+ (LSK) cells and HSC maintaining UG26–1B6 stromal cells. Microarray analyses were performed on cells prior to co-culture and cells sorted from the cultures. STEM clustering analysis of the data revealed that most changes in gene expression were due to early cell activation. Functional enrichment analysis revealed dynamic changes in focal adhesion and mTOR signaling, as well as changes in epigenetic regulators, such as HDAC in stromal cells. In LSK cells, genes whose products are involved in inflammation, Oxygen homeostasis and metabolism were differentially expressed after the co-culture. In addition, genes involved in the regulaton of H3K27 methylation were also affected. Interestingly, connective tissue growth factor (CTGF), which is involved in TGF-b, BMP and Wnt signaling, was upregulated in both stromal and LSK cells in the first day of co-culture. To study a possible extrinsic role of CTGF as a stromal mediator, we co-cultured siCTGF knockdown stromal cells with wild-type LSK cells. Since self-renewal requires cell division, we focused on cell cycle regulation of LSK cells. We found that knockdown of CTGF in stromal cells downregulates CTGF in LSK cells. In addition, knockdown of stromal CTGF downregulated Ccnd1, Cdk2, Cdkn1a (p21), Ep300 and Fos. On the other hand, decreased CTGF in stromal cells upregulates Cdkn1b (p27) and phosphorylation of Smad2/3. These results show that stromal CTGF regulates the cell cycle of LSK cells. On a functional level, we found that decreased stromal CTGF results in an increased production of MPP and myeloid colony-forming cells in 1-week co-cultures. We will present data showing whether and how a decrease in CTGF in stromal cells affects the maintenance of transplantable HSC. In summary, our current results indicate that reduced expression of CTGF in stromal cells regulates mediators of cell cycle and Smad2/3-mediated signaling in LSK cells, resulting in an increased production of myeloid progenitors.

Disclosures:

No relevant conflicts of interest to declare.
Maintenance Therapies for Multiple Myeloma (MM): A Network Meta-Analysis
Authors: Mahony Helen , Kumar Ambuj , Mhaskar Rahul , Miladinovic Branko , Wheatley Keith , Djulbegovic Benjamin
Published: October 2019

Abstract 236

Background:

There is little consensus on which maintenance therapy clinicians should choose for their patients. Since 1999, the three novel agents of bortezomib, lenalidomide, and thalidomide have been approved for use among patients with MM. These agents have been increasingly used as maintenance therapy. To date, only two randomized controlled trials of maintenance therapy have examined the efficacy of these novel agents head-to-head. Here, we conduct a network meta-analysis of bortezomib, lenalidomide, and thalidomide to determine which of these novel agents could potentially increase overall survival (OS) and progression-free survival (PFS).

Methods:

A comprehensive literature search of MEDLINE (PubMed), the Cochrane Central Register of Controlled Trials (CENTRAL), and meetings abstracts from American Society of Hematology, American Society of Clinical Oncology, European Society for Medical Oncology and European Hematology Association was undertaken to identify all phase III randomized controlled trials (RCTs) of maintenance therapy published until July 2012. We applied the Bayesian mixed treatment comparison (MTC) method under the random-effects model. The indirect comparisons were constructed from trials that have one treatment in common. For each included RCT, we calculated the hazard ratio (HR) and its corresponding standard error and used this to calculate the indirect estimates of HR and corresponding credible intervals (CrI). We also ranked the treatments according to the probability of best treatment and calculated the surface underneath the cumulative ranking curve (SUCRA). All analyses were conducted in WinBUGS 1.4.3 and Stata 11.2.

Results:

The network, number of trials for each comparison, and number of patients enrolled is shown in Figure 1. The network for OS was based on 12 RCTs enrolling 5542 patients and the network for PFS was constructed from 13 RCTs and 5784 patients. The MTC networks were consistent for both OS and PFS. For both OS and PFS, two comparisons were produced (Figure 2). For OS, the analysis showed that none of the treatments were superior. For PFS, lenalidomide was superior to thalidomide (HR = 0.58, 95% CrI [0.37, 0.94]). The estimates of SUCRA and rank probabilities (Figure 3) suggested that for OS bortezomib was best followed by lenalidomide and thalidomide. For PFS, lenalidomide was best followed by bortezomib and thalidomide.

Conclusion:

Using the MTC method, we found no evidence that any of the novel agents are superior to one another in terms of OS. Lenalidomide was the only novel agent which was superior to another active therapy (thalidomide). While these results provide preliminary evidence to which novel agent may be more beneficial as maintenance therapy, definitive conclusions cannot be reached until large, well designed RCTs evaluating these therapies head-to-head are conducted.

Disclosures:

No relevant conflicts of interest to declare.
Novel Bone Marrow Failure Syndrome Due to a Deletion of the EVI1/Mecom Gene.
Authors: van der Veken Lars T. , Bierings Marc , Maiburg Merel , Groenendaal Floris , Bloem Andries C , Knoers Nine V. , Buijs Arjan
Published: October 2019

Abstract 2368

We report on a patient born with severe congenital abnormalities including macrocephaly, brain MRI abnormalities, bilateral clubfeet, and bone marrow failure with pancytopenia presenting with none but T-lymphocytes in the peripheral blood.

A 824 kb to 868 kb heterozygous deletion on chromosome 3q26.2 was identified by array-CGH on peripheral blood using an Agilent 180k oligo array (Amadid 023363). The deletion truncated the 3' part of EVI1/MECOM and extended into microRNA 551b (MIR551B). The 3' MECOM deletion was confirmed by FISH using a MECOM specific probe on uncultured peripheral blood cells in 10 investigated metaphases and 200 interphase nuclei. To assess whether the deletion was constitutional, FISH analysis was performed on buccal swaps and cultured skin fibroblasts. Loss of one MECOM signal was observed in 200 interphases, and 10 metaphases of cells derived from buccal mucosa and skin fibroblasts, respectively, demonstrating the constitutional nature of the 3' MECOM deletion. Karyotyping and FISH analysis of both parents did not reveal a structural or numerical abnormality of 3q26.2 MECOM, indicating the deletion to be de novo.

EVI1/MECOM is a transcriptional regulator essential for maintaining embryonic and adult hematopoietic stem cells by directly regulating transcription of GATA2. In mouse models, homozygous disruption of MECOM results in embryonic lethality, with hypocellular bone marrow, reduced body size, small or absent limb buds, abnormal development of the nervous system and heart and massive hemorrhaging. Furthermore, MECOM heterozygosity leads to a marked impairment of the self-renewal capacity of hematopoietic stem cells.

In our case the heterozygous deletion results in 3' terminal truncated MECOM lacking the C-terminal acidic amino acid cluster domain (AD) encoding sequences. The AD of MECOM is important for activation of GATA2 transcription in vitro. This supports the notion that haploinsufficiency of MECOM causes bone marrow failure due to loss of critical MECOM transcriptional AD encoding sequences in this case, supposedly by deregulating GATA2 mediated control of hematopoietic stem cell homeostasis.

We will discuss our data in relation to one seemingly similar case deposited recently in the DECIPHER database. In conclusion, we report on a novel 3q26 EVI1/MECOM deletion syndrome with multiple severe congenital abnormalities and bone marrow failure due to EVI1/MECOM haploinsufficiency.

Disclosures:

No relevant conflicts of interest to declare.
The Under-Treatment of Elderly Medicare Diffuse Large B-Cell Lymphoma Patients
Authors: Satram-Hoang Sacha , Skettino Sandra , Hoang Khang , Guduru Sridhar R , Gunuganti Ashok , Reyes Carolina
Published: October 2019

Abstract 237

Introduction:

Prior real-world data show that a large proportion of the elderly population with diffuse large b-cell lymphoma (DLBCL) are not receiving the recommended therapy for their disease. Over half of the patients diagnosed with DLBCL are over the age of 60 and clinical trials show that patients over the age of 60 respond well to current guideline-based therapy. We set out to examine the patient characteristics associated with receiving recommended therapy and the survival outcomes in an older population in routine clinical practice.

Methods:

We conducted a retrospective cohort analysis of 9866 DLBCL patients in the linked Surveillance, Epidemiology, and End Results (SEER)-Medicare database. Patients were diagnosed between January 1, 2000 to December 31, 2007, were >66 years, and continuously enrolled in Medicare Part A and B with no HMO coverage in the year prior to diagnosis. Chi-square test for categorical variables and ANOVA or t-test for continuous variables were used to assess differences in patient and disease characteristics by treatment status. Logistic regression modeling assessed the patient characteristics predictive of not receiving treatment. Kaplan-Meier curves and Cox proportional hazards regression assessed survival by treatment type. Date of last follow-up was December 31, 2009.

Results:

There were 7832 (80%) patients who received treatment and 2034 (20%) who did not receive treatment during the study time period. Untreated patients were older at diagnosis with mean age of 81 compared to 76 years in treated patients (p<.0001). As age increased, the treatment rate significantly decreased especially among patients >80 years (Table 1). Untreated patients were also more likely non-white (15% vs. 12%; p<.01), diagnosed with stage IV disease (33% vs. 29%; p<.0001), had higher comorbidity burden, and lived in areas of lower socioeconomic status than treated patients (p<.0001). In the adjusted logistic regression model, increasing age, non-white race, higher comorbidity score, and lower income were predictive of not receiving treatment. The multivariate survival analysis, adjusting for age, sex, race, stage, histology, comorbidity, income, and year of diagnosis, demonstrated a 3-fold higher risk of death in the untreated patients (HR=2.98; 95%CI=2.81–3.16) compared to treated patients.

Conclusions:

This real-world analysis of elderly DLBCL patients confirmed that 20% of them are not receiving treatment, and the rate of under-treatment is even more pronounced among those >80 years. As a consequence, untreated patients had a 3-fold higher risk of death compared to those who received treatment.

Disclosures:

Satram-Hoang: Genentech, Inc.: Consultancy. Skettino:Genentech, Inc.: Employment, Roche Stock Other. Reyes:Genentech, Inc.: Employment, Roche Stock Other.
Aid-Initiated DNA Lesions Are Differentially Processed in Distinct B Cell Differentiation Stages in a Locus-Dependent Manner.
Authors: Chen Zhangguo , Viboolsittiseri Sawanee , Eder Maxwell , Yuan Shunzong , Wang Jing H.
Published: October 2019

Abstract 2376

Activation induced deaminase (AID) initiates U:G mismatches that are subsequently converted into point mutations or DNA double-stranded breaks. AID-mediated DNA alterations in switch (S) regions at the Igh locus frequently occur in both antigen-stimulated germinal center (GC) B cells and cytokine-activated B cells. To investigate whether AID-initiated U:G lesions are differentially processed in a differentiation stage-specific manner at non-Ig loci to maintain genome stability, we established a knock-in model by inserting an Sg2b region into the first intron of proto-oncogene c-myc. We found that the inserted Sg2b region mutated at an extremely low level and did not enhance genomic instability of c-myc locus in antigen-stimulated GC B cells. In contrast, the inserted Sg2b region mutated more frequently and increased c-myc locus abnormalities in cytokine-activated B cells. Furthermore, uracil glycosylase deficiency led to increased mutation frequency at the c-myc locus. These results reveal that AID-initiated lesions are differentially processed via error-free or error-prone repair in a differentiation stage-specific and locus-dependent manner. Our data might provide mechanistic explanation for differential frequency of AID-mediated genetic alterations in distinct subtypes of common B cell lymphomas.

Disclosures:

No relevant conflicts of interest to declare.
Serum 2-Hydroxyglutarate Levels Predict Isocitrate Dehydrogenase Mutations and Clinical Outcome in Acute Myeloid Leukemia.
Authors: DiNardo Courtney D. , Levine Ross L. , Propert Kathleen J , Loren Alison W. , Paietta Elisabeth , Sun Zhouxin , Straley Kimberly , Yen Katharine , Patel Jay P. , Agresta Samuel , Abdel-Wahab Omar , Perl Alexander E , Fernandez Hugo F , Margolis David , Tallman Martin S. , Luger Selina M. , Carroll Martin
Published: October 2019

Abstract 2481

Purpose:

Cancer-associated IDH mutations produce the metabolite 2-hydroxyglutarate (2HG), but the clinical utility of serum 2HG measurements has not been previously established. We studied whether 2HG measurements in AML patients correlate with the presence of IDH mutations and whether diagnostic or remission 2HG measurements predict survival.

Patients and Methods:

Serum samples from 223 previously untreated adults (≤ 60 years of age) with de novo AML from the Eastern Cooperative Oncology Group E1900 clinical trial (62 IDH mutated, 161 IDH wild-type) were analyzed for 2HG concentration by reverse-phase liquid chromatography coupled to mass spectrometry (GC-MS).

Results:

Pretreatment 2HG levels ranged from 10 to 30000 ng/ml and were significantly elevated in IDH-mutant samples (median 3004.1 ng/ml), as compared to the wild-type cohort (median 61.2 ng/ml) (p < 0.0005). 2HG levels did not differ among the specific IDH1 or IDH2 allelic variants. In ROC analysis, a discriminatory level of 700 ng/ml segregated patients with and without IDH mutations with 86.9% sensitivity and 90.7% specificity. On repeat mutational analysis of 13 IDH wild-type samples with 2HG levels >700 ng/ml, IDH mutations were identified in nine samples, most often at low allele burden. IDH mutant patients with 2HG levels ≤ 200 ng/ml at complete remission experienced improved overall survival compared to those with higher 2HG levels (HR 3.5, p = 0.02) (Figure 1).

Conclusion:

We establish a firm association between IDH mutations and elevated serum 2HG concentration in AML. These data confirm that peripheral blood measurement of an oncometabolite provides useful diagnostic and prognostic information for cancer therapy, and furthermore can inform patient selection of IDH mutant targeted therapies.

Disclosures:

Levine: Agios Pharmaceuticals: Research Funding. Straley:Agios Pharmaceuticals: Employment. Yen:Agios Pharmaceuticals: Employment. Agresta:Agios Pharmaceuticals: Employment. Carroll:Agios Pharmaceuticals: Research Funding.