bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Elements Classified Hematological Malignancies from Healthy Population
Authors: Hagiwara Shotaro , Suzuki Yoshinari , Nakayama Akihiro , Matsunaga Akihiro , Iida Yutaka , Shimura Mari
Published: October 2019

Introduction

It is a little known about how multiple elements interacts and functions in cells, although a specific element had already reported to take a role, enzymatic, transcriptional activities, etc. Although, several metals such as cadmium, nickel, lead, and chrome were known as carcinogen, it is also not known about relations of multiple elements in disease mechanisms. To investigate the impact of multiple elements on the features of hematological malignancies, we analyzed the multiple elements in the serum of patients with hematological malignancies and healthy persons.

Methods

We obtained 47 of peripheral blood samples from 40 patients with hematological malignancies, and 102 of healthy persons: 50 persons who visited to medical check-up and 52 volunteers.

Hematological malignancies were 16 acute myeloid leukemia, 5 acute lymphoblastic leukemia, 9 non-Hodgkin's lymphoma, 9 Multiple myeloma, and 1 Chronic myeloid leukemia.

Samples were centrifuged, and 1.0-1.5ml of plasma was separated. We measured 23 elements were measured using inductively coupled plasma mass spectrometry.

To assess the difference of the element patterns between hematological malignancies and healthy persons, multivariate discrimination analysis was performed. Overall survival of hematological malignancies was assessed by Log-rank test.

Multivariate analysis with the elements highly discriminated hematological malignancies from healthy persons' samples. Elements data increased the accuracy to discriminate hematological malignancies from healthy persons close to 100 % in addition to clinical data such as complete blood count, albumin, total bilirubin, blood urine nitrogen, creatinine. To our knowledge, this is the first report on the successful classifying disease from healthy persons by measurements of multiple elements with more than 90% accuracy using peripheral blood serum (Table 1). Five year overall survival of patients with hematological malignancy was likely dependent on this discriminant score. Data suggested the potential interaction between multiple elements and hematological malignancies, and the possibility future applications on diagnosis and prognosis.

Table 1. Accuracy of cross-validated classification patients with hematological malignancies and healthy persons by discriminant analysis Accuracy Clinical data Multi-elements Multi-elements + Clinical data % 93.7 91.3 98.9

Disclosures

No relevant conflicts of interest to declare.
Neutropenic enterocolitis in adults with hematological malignancy afte intensive cheMotherapy
Authors: Yaping Xie , Huang Xilian , Shi Pengfei , Chen Kuang , Gao Daquan , Qian Shenxian
Published: October 2019

Background. Neutropenic enterocolitis (NE) is a life-threatening complication occurring after intensive chemotherapy; however, only a few data are available for adults with hematological malignancy in China. The aim of this retrospective study was to elucidate the incidence, risk factors, and outcome of NE in adults with hematological malignancy after intensive chemotherapy. Methods. The medical records of 226 adult patients who were diagnosed with hematological malignancy and neutropenic enterocolitis after intensive chemotherapy from 2004 to 2013 were reviewed. Diagnosis of NE was established in case of neutropenic fever, abdominal pain or diarrhea, and bowel wall thickening >4mm on abdominal sonography or CT. Results. Amongst 1804 patients with hematological malignancy treated at the department of hematology, Hangzhou first people's hospital from 2004 to 2013, NE was diagnosed in 226 patients (12.5%), amedian +14(10–19) days after chemotherapy, with a death rate of 11.9%. Bloodstream infections were present in 23 patients, with gram-negative bacteria in 17 patients. Previous therapy with cytarabine and an intestinal wall thickness of ≥10 mm in the ultrasonographic examination affected survival, were associated with greater mortality. In multivariate analysis, the clinical findings of 4 or more symptoms was associated with higher risk of death. Conclusions. NE is a rare but severe complication in patients undergoing intensive chemotherapy. Use of cytarabine, an intestinal wall thickness of ≥10 mm and the clinical findings of 4 or more symptoms were associated with a higher death rate.

Disclosures

No relevant conflicts of interest to declare.
Hematopoietic Stem Cell Transplantation in the Elderly: Nutritional and Geriatric Assessment
Authors: Pereira Andrea Z , Piovacari Silvia MF , Tanaka Marcia , Barrere Ana Paula N , Lucio Fabiana , Silva Juliana Bernardo , Goncalves Polianna M , Koch Ludmila O , Nicastro Mariana , Hamerschlak Nelson
Published: October 2019

Introduction: Hematopoietic stem cell transplantation (HSCT) may improve outcomes of patients with hematologic malignancies not curable with conventional therapies. Being in some diseases the only curative option. HSCT in elderly patients with good performance status and no comorbidities could, in fact, not only survive the transplant with reasonable risk, but also benefit in the same measure as younger patients.

Objectives: To study and correlate nutrition and geriatric assessment in elderly patients undergoing HSCT.

Methods: A retrospective study of 17 elderly patients (>60 years) undergoing HSCT May 2012 to January 2014 in the Hematology-Oncology and Bone Marrow Transplantation Center at Albert Einstein Hospital in São Paulo, Brazil. All patients were evaluated approximately one month prior to HSCT. In the geriatric assessment were done hand-grip strength(HGS), questions about mobility and functional limitation. In the nutrition, we studied the Body Mass Index(BMI) (kg/m2), and serum levels of vitamin D, zinc and albumin.

Results: 17 elderly patients were observed in this study, mean age was 65,5±3,8 years, BMI was 28±6,0 kg/m2, HGS was 28±8,5 kg; serum levels of albumin 3,2±0,5g/dl(normal:3,5-5,0); serum level of vitamin D 23,4±14 ng/ml (normal >20); serum levels of zinc 65,5±18 mg/dl (normal: 66-132,5 mg/dl). We found the negative correlation between BMI and HGS (rp=0,42). There were a significant and positive association between serum levels of zinc and albumin, and HGS and grades of mobility questions(p<0,05). The serum levels of vitamin D weren`t significantly associated with geriatric factors.

Conclusion: Our study showed that the obese patients with more risks of complications in HSCT had more functional limitation. Besides low levels of zinc and albumin were associated with worst results in the geriatric assessment. In the elderly the immobility and weakness can increased the complications after HSCT. The geriatric and nutrition assessment are important to improve HSCT results.

Disclosures

No relevant conflicts of interest to declare.
RUNX1 and GATA2 Regulate the Expression of the SET Oncogene in Acute Myeloid Leukemia
Authors: Pippa Raffaella , Dominguez Ana , Marcotegui Nerea , Malumbres Raquel , Guruceaga Elizabeth , Odero Maria D.
Published: October 2019

INTRODUCTION. The protein SET (I2PP2A), a potent protein phosphatase 2A (PP2A) inhibitor, has been implicated in many cell processes such as DNA replication, chromatin remodeling and gene transcription, differentiation, migration, and cell-cycle regulation. In fact, SET has been described as an oncogene that regulates important signaling pathways. Our group reported that PP2A inhibition is a common event in AML, and that SET is overexpressed in 28% of acute myeloid leukemia (AML) cases, where it is associated with short overall survival. Moreover, the anti-leukemic effects of the FTY720 and OP449 PP2A-activating drugs in AML cells depend on interaction/sequestration of SET. However, despite the importance of SET overexpression and its prognostic impact in both hematological and solid tumors, there are few data about the mechanisms involved in its regulation.

AIM. To characterize the functional promoter region of the SET gene, and to identify transcription factors (TFs) involved in its regulation.

RESULTS. Luciferase reporter assays with five truncatedconstructs allowed us to determine a 163bp-region as the minimal promoter region of SET that contains consensus sites for several TFs. Chromatin immunoprecipitation (ChIP) assays confirmed the binding of RUNX1, GATA2, MYC, and SP1. RUNX1 and GATA2 are two essential TFs in hematopoiesis, and localized on the SET promoter when the acetylation state of both histone H3 and H4 and the tri-methylation on H3K4 is high, confirming that they both could act as positive regulators of SET transcription. In silico analysis in large series of adult patient samples with de novo AML recently published by The Cancer Genome Atlas Research Network showed a significant positive correlation between SET and RUNX1 and GATA2 at mRNA level. Furthermore, knockdown of RUNX1 and/or GATA2 triggered SET downregulation, whereas only a simultaneous overexpression of these two TFs caused a significant up-regulation of SET. Interestingly, RUNX1 interacts with GATA2 in both HL-60 and HEL cell lines. Moreover, we found that SP1 is also part of this transcription complex. Altogether, these results show that RUNX1 and GATA2, together with SP1, regulate the transcription of the SET gene.

CONCLUSIONS We have defined the minimal promoter region of the SET gene, and have demonstrated that RUNX1 and GATA2 regulate its expression in AML. Moreover, our functional studies demonstrate that RUNX1 and GATA2 form a complex with SP1 that activates the transcription of SET in AML cells. This study opens new directions to further understand the mechanisms of SET overexpressing leukemias.

Disclosures

No relevant conflicts of interest to declare.
Dual Inhibition of Bcr-Abl and Hsp90 By C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells
Authors: Wu Lixian , Yu Jing , Liu Yang , Liguang Lou , Wu Yong , Chen Yuanzhong , Xu Jianhua
Published: October 2019

Purpose: Although such tyrosine kinase inhibitors (TKIs) as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of CML patients, TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition.

Experimental Design: Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation and immunoblotting were applied to examine Hsp90 function; Colony-forming unit (CFU), long-term culture-initiating cells (LTC-ICs), and flow cytometry were used to test CML progenitor and stem cells.

Results: Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, inhibited the growth of both imatinib-sensitive and resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34+CD38+ and CD34+CD38- cells in vitro. Moreover, C086 could decreased the number of CD45+, CD45+CD34+CD38+ and CD45+CD34+CD38- cells in CML NOD-SCID mice.

Conclusions: Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl-induced leukemia resistant to TKIs.

Disclosures

No relevant conflicts of interest to declare.
The New Targeted Nanoparticles: Tf-Peg-PLL-PLGA Combined with Daunorubicin Overcome Hypoxia Induced Drug Resistance of K562 Cells
Authors: Liu Ping , Guo Liting , Wang Fei , Xia Guohua , Wang Yonglu , Li Xueming , Song Huihui , Chen Wei , Yin Haixiang , Chen Baoan
Published: October 2019

Drug resistance is a major obstacle in cancer chemotherapy, especially in the treatment of leukemia. Hypoxia is an inseparable component of the solid tumor microenvironment as well as the bone marrow (BM) microenvironment. In this study, we investigated the effect of the new nanoparticles polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid (PLGA) (PEG-PLL-PLGA) modified by transferrin (Tf) combined with daunorubicin (DNR) on K562 cells under hypoxia. We found the IC50 of DNR and DNR-PEG-PLL-PLGA in K562 cells increased under hypoxia, while the IC50 of DNR-Tf-PEG-PLL-PLGA in K562 cells decreased under hypoxia according to the results of MTT assay. The apoptosis of cells and the intracellular concentration of DNR were detected by flow cytometry (FCM). From the datas, we found that the new targeted nanoparticles can overcome hypoxia induced drug resistance of K562 cells. By targeting the transferrin receptor (TfR) on the surface of K562 cells, the new targeted drug led to an increase in intracellular accumulation of DNR. Transcription and Protein of P-glycoprotein (P-gp) and TfR increased in K562 cells under hypoxia, which probably had a relationship with the decreased degradation of the Hypoxia-Inducible Factor-1α (HIF-1α). We further detected the levels of HIF-1α, Bcl-2, Bax and Caspase-3 of K562 cells in transcription and protein under hypoxia regulated by DNR, DNR-PEG-PLL-PLGA and DNR-Tf-PEG-PLL-PLGA, respectively. DNR-Tf-PEG-PLL-PLGA had the best effect when compared with other groups. Furthermore, intravenous injection of DNR-Tf-PEG-PLL-PLGA expressed the best effect of suppressing proliferation of K562 cells injected to nude mice among the three drugs. Therefore, the new drug DNR-Tf-PEG-PLL-PLGA may be a potential cancer therapy for human leukemia.

Disclosures

No relevant conflicts of interest to declare.
Platelet Expression of a Genetically Encoded Calcium Indicator Reveals Platelet Activation Gradients in Real Time during Thrombus Formation in Vivo
Authors: Stalker Timothy J. , Wu Jie , Brass Lawrence F.
Published: October 2019

Introduction: Recent studies have demonstrated that platelet activation during hemostatic thrombus formation in vivo is heterogeneous in time and space. We have previously shown that in response to a penetrating injury in the microcirculation platelet accumulation and activation are driven by gradients of soluble agonists emanating from the site of injury. These gradients result in a characteristic thrombus architecture in which a core of fully activated platelets that have released their alpha granules is overlaid by a shell of less activated platelets that frequently embolize. While dependent on ADP/P2Y12 signaling, the extent and dynamics of platelet activation in the shell region remain poorly understood. Here, we used intravital imaging of platelet cytosolic calcium concentration to examine platelet activation in real time following vascular injury in vivo.

Methods: We generated transgenic mice expressing a genetically encoded calcium indicator (GCaMP3) specifically in megakaryocytes and platelets by crossing PF4-Cre mice with Ai38 mice carrying the GCaMP3 transgene. GCaMP3 fluorescence was visualized in platelets following laser-induced injury in mouse cremaster arterioles using spinning disk confocal intravital microscopy.

Results: Rapid and dynamic changes in GCaMP3 fluorescence were observed during platelet accumulation following vascular injury. Platelets immediately adjacent to the site of injury exhibited a rapid and sustained increase in cytosolic calcium. Peak cytosolic calcium levels in the core region occurred within the first minute post-injury, prior to peak P-selectin expression. Platelets in the shell region were characterized by dynamic changes in cytosolic calcium at the level of individual platelets, with the appearance of transient “waves” of calcium signaling propagating among groups of platelets. At the population level, cytosolic calcium concentration was substantially lower in the shell region as compared to the core.

Conclusions: Expression of a genetically encoded calcium indicator in platelets allows for the visualization of platelet activation events in real time in vivo. Platelets within the core region of a thrombus exhibit a rapid and sustained increase in cytosolic calcium concentration, indicating robust activation. In contrast, platelets in the shell region exhibit transient increases in cytosolic calcium indicating weak activation, consistent with their increased likelihood of embolization. The examination of platelet calcium signaling in vivo provides a sensitive readout of platelet activation that will shed new light on mechanisms regulating hemostasis and thrombosis, and may be useful in assessing the impact of anti-thrombotic agents on platelet function. Research support was provided by the American Heart Association and National Heart, Lung and Blood Institute.

Disclosures

Stalker: Medicines Company: Research Funding. Brass:Medicines Company: Research Funding.
Genome and Epigenome Editing: A Revolution in Science and Medicine
Authors: Joung J. Keith
Published: October 2019

Targeted genome and epigenome editing technologies have recently emerged as important tools for biomedical research and as potential reagents for therapies of gene-based diseases. This talk will focus on our recent work on both improving and defining the genome wide specificities of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases, a robust platform for introducing targeted genome sequence alterations. The talk will also briefly describe the creation and validation of new technologies for modifying specific epigenomic marks on histones and DNA that can be used to induce targeted alterations in endogenous human gene expression. Taken together, these methodologies provide transformative tools for understanding human biology and offer promising pathways forward for developing new classes of therapeutics based on targeted alterations of gene sequence and/or gene expression.

Disclosures

Joung: Editas Medicine: Consultancy, Equity Ownership, Other, Patents & Royalties; Transposagen Biopharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Horizon Discovery: Consultancy, Membership on an entity's Board of Directors or advisory committees.
Dual Face of Immune Microenvironment in Myeloma
Authors: Dhodapkar Madhav V.
Published: October 2019

An important property of human multiple myeloma (MM) is the growth of tumor cells and their precursors in the bone marrow microenvironment. This specialized microenvironment includes a distinct component of immune cells that includes cells of innate as well as adaptive immune system. Growing body of evidence points to a central role for cross-talk between this immune microenvironment and tumor cells in regulating the growth of tumor cells in myeloma. These interactions play a dual role-with some interactions promoting tumor growth while others suppress growth of tumor cells. Cross talk between T and B cells has been implicated in normal B/plasma cell differentiation and may also play a role in pathogenesis of myeloma, explaining increased risk of plasma cell malignancies in certain settings such as Gaucher disease. The bone marrow of MM patients is often infiltrated with innate cells such as dendritic cells (DCs) and cross-talk between myeloid/plasmacytoid DCs or macrophages may promote growth and survival of MM cells. This cross-talk may also have fundamental implications for inducing genomic instability in tumor cells, such as via the induction of cytidine deaminases in tumor cells. Genomic instability is an early feature of human MM and many of the mutations found in MM tumor cells are also present in the precursor states. The immune system has the capacity to recognize these precursor lesions and even in the setting of clinical MM, the tumor bed contains T cells that can be recruited to kill tumor cells. Specific targets of tumor immunity may differ between preclinical and clinical malignancy. Immunity to certain stem cell-associated antigens are in particular associated with reduced risk of progression to clinical MM. With advanced disease, tumor infiltrating T cells encounter several immune inhibitory checkpoints which leads to suppression of function of tumor-infiltrating T cells. In addition to T cells, cells of the innate immune system such as natural killer and natural killer T cells may also be attractive targets of protective tumor immunity in MM. Immunologic approaches may also be combined with other strategies such as other immunomodulatory drugs (IMiDs) already in clinical use in MM. Recent advances in mouse modeling provide new opportunities to better understand how to harness the immune system against human MM. Such approaches have the advantage of the potential for durable control of tumor cells via immunologic memory. Such approaches are now entering the clinic as attractive targets to restore anti-tumor immunity, both in the context of therapy or prevention of MM.

Disclosures

Dhodapkar: Celgene: Research Funding. Off Label Use: checkpoint blockade drugs, imids.
A Humanized Anti FcγRIIa Scfv Inhibits Platelet Activation, Aggregation and Thrombus Formation in Heparin-Induced Thrombocytopenia (HIT)
Authors: Perdomo Jose , New Jaa Yien , Ahmadi Zohra , Jiang Xing-Mai , Chong Beng H
Published: October 2019

Introduction. Heparin is widely used as an anticoagulant to prevent thrombosis and to treat venous thromboembolism and myocardial infarction. A complication of heparin use is the development of heparin-induced thrombocytopenia (HIT), which is a limb- and life-threatening disorder due to associated thrombotic events. HIT arises through the formation of immune complexes between heparin, platelet factor 4 and HIT autoantibodies. These immune complexes engage with FcγRIIa receptors on platelets, leading to platelet activation and aggregation and subsequent initiation of the coagulation pathway. Current HIT treatment consists of cessation of heparin administration and substitution with parenteral anticoagulants such as argatroban and danaparoid. While these anticoagulants are generally beneficial in reducing thrombocytopenia, they are only partially effective since the risk of thrombosis continues due to the underlying FcγRIIa-mediated platelet activation. Thus, alternative anticoagulants do not reduce morbidity and mortality rates, highlighting the need for more effective HIT interventions.

Methods. IV.3 is a monoclonal antibody that recognizes and blocks the FcγRIIa receptor and is used in assays to confirm the presence of HIT antibodies. We derived the VH and VL sequences of IV.3 and constructed a single-chain variable fragment (scFv) antibody in the form of VH-linker-VL. Using a complementarity determining region grafting and point mutation approach the scFv was humanized with the aim of reducing potential immunogenicity for future clinical applications. The molecule was expressed in E. coli and purified by FPLC. We reconstituted the HIT condition in a micro-fluidics device on a Vena8 Fluoro+ biochip coated with vWf using whole blood flowing at 20 dyne/cm2 at 37oC. Whole blood was stained with DiOC6 and the formation of platelet aggregates was monitored by fluorescence microscopy. Video images were acquired at 1 frame every 2 sec for 460 sec.

Results. The purified scFv interacts with FcγRIIa on platelets. Platelet aggregation and serotonin release assays show that the scFv effectively prevents aggregation and activation induced by HIT immune complexes. We demonstrate that in the HIT condition reconstituted in a micro-fluidics system the scFv precludes thrombus deposition in a dose-dependent manner as determined by thrombus coverage area and mean thrombus diameter (Figure 1).

Conclusions. These data provide evidence that a humanized scFv binds and neutralizes FcγRIIa on platelets. This interaction prevents HIT immune complex-induced platelet aggregation and activation in vitro and stops thrombus deposition ex vivo. This molecule, therefore, inhibits a critical initiating event in HIT and may serve as a potential treatment for this condition.

Disclosures

No relevant conflicts of interest to declare.
Structural Basis for PECAM-1 Homophilic Binding
Authors: Paddock Cathy , Zhou Dongwen , Lertkiatmongkol Panida , Newman Peter J. , Zhu Jieqing
Published: October 2019

PECAM-1 is a 130 kDa member of the immunoglobulin gene (Ig) superfamily that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1-mediated homophilic interactions, known to be mediated by its two amino-terminal Ig homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to sense flow, regulate leukocyte transendothelial migration, maintain vascular integrity, and transmit survival signals into the cell. Mutagenesis studies have implicated amino acids on either side of the face of IgD1 in homophilic binding, however the spatial orientation of this domain, the role that IgD2 plays in positioning IgD1 to participate in PECAM-1 homophilic interactions, and the contribution of its sialylated glycans to homophilic PECAM-1/PECAM-1 interactions remain to be critically explored. Complicating things further, PECAM-1 forms dimers and oligomers within the plane of the plasma membrane. The structural features of PECAM-1 responsible for maintaining the PECAM-1/PECAM-1 homophilic contacts necessary for carrying out these activities, however, have heretofore remained unresolved. Given the importance of PECAM-1-mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientation of the PECAM-1-PECAM-1 homophilic binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic binding domain. The structure of IgD1-D2 was determined by multi-crystal anomalous diffraction and refined at 2.8 Å resolution. Interestingly, PECAM-1 crystallized as a strand-swapped dimer, a feature that has been seen in the crystal structures of many other members of the Ig superfamily, including cadherins, NCAM2, CTLA-4, and CD47. The homophilic interface between adjacent IgD1 domains was large, with a total buried interface of 1608 Å2. In addition to the large IgD1/IgD1 interface, interdomain contacts were found between IgD1/IgD2, and IgD2/IgD2 that likely reinforce the PECAM-1/PECAM-1 homophilic interactions that form as a consequence of the large number of PECAM-1 molecules that become concentrated at endothelial cell-cell borders as a result of diffusion trapping. Taken together, these results provide atomic-level detail to the unique architecture of the interface that forms the most abundant component of the endothelial cell intercellular junction.

Disclosures

No relevant conflicts of interest to declare.
Thrombocytopenia in Hospitalized Non-ICU Patients
Authors: Fountain Eric , Arepally Gowthami M
Published: October 2019

Thrombocytopenia is common in patients admitted to the intensive care unit, with a reported prevalence of 8-68% and incidence of 13-44% in published series. Those who develop thrombocytopenia in an ICU have a statistically increased risk of ICU mortality, duration of mechanical ventilation, and platelet, RBC, and FFP transfusions. Few studies have examined the occurrence and risk factors for thrombocytopenia in non-ICU hospitalized patients. The only systematic investigation of thrombocytopenia in the non-ICU hospitalized medical population dates to 1989, predating modern diagnostic assays for a variety of infectious and drug-induced causes of thrombocytopenia. We, therefore, undertook this study to characterize the incidence and causes of thrombocytopenia in the general medical, non-ICU patient population at a tertiary care hospital. For this study, we performed a single-institutional retrospective analysis of patients admitted to a general medical ward at a tertiary care medical center (Duke University Hospital). Inclusion criteria included all adult patients (>18 years) admitted from the emergency department to the general medicine floors during the calendar year defined as 01/2014-01/2015. Exclusion criteria included pre-existing thrombocytopenia, ICU admission, or patients undergoing chemotherapy. Primary endpoints included patients with incident thrombocytopenia, defined as a platelet count on admission greater than 150 x 10^9/L with subsequent platelet counts decreasing to < 150 x 10^9/L. Patients meeting these criteria were evaluated for admission diagnosis, pertinent past medical history, cause of thrombocytopenia (infection, splenic sequestration, drugs, surgery, liver disease), cost of admission, and prognosis. Preliminary evaluation of patients admitted to the general medical service in 2014 reveals ~711 patients who meet inclusion criteria. Of patients developing absolute thrombocytopenia, 56% experienced a platelet count fall of greater than 30%. Those with incident thrombocytopenia greater than 30% had a statistically significant increase in mortality (n=36/399) compared to those with milder thrombocytopenia (n=13/312), (9.0% versus 4.2%, p<0.05), and had an increased length of stay (12.6 days versus 6.8 days, p<0.01). In patients who developed absolute thrombocytopenia, review of diagnosis codes associated with thrombocytopenia reveal a predominance of infectious etiologies (43%; sepsis, severe sepsis, septic shock, bacteremia, urinary tract infection/pyelonephritis, pneumonia, Clostridium difficile colitis). These results are similar to prior published data which suggests infection as a leading cause of incident thrombocytopenia in non-ICU hospitalized patients. Additional studies are underway to delineate non-infectious causes of thrombocytopenia.

Disclosures

No relevant conflicts of interest to declare.
Deciphering Molecular Control of VEGFR2 Regulation in Hematopoietic Progenitors: GATA1-Mediated Repression of VEGFR2 Promotes Optimum Erythropoiesis
Authors: Ganguly Avishek , Aljitawi Omar S. , Paul Soumen
Published: October 2019

VEGFR2 (also known as Flk1) is expressed in hemetopoietic precursors and is essential for both hematopoietic and vascular development. Interestingly, development of differentiated hematopoietic cell from hematopoietic stem cells (HSCs) is associated with VEGFR2 repression, whereas VEGFR2 expression is maintained throughout endothelial development. This differential regulation of VEGFR2 has been implicated as a key step to successfully branch out hematopoietic vs. endothelial development. However, molecular mechanisms that regulate transcriptionally active vs. repressive Vegfr2 chromatin domains in hematopoietic stem/progenitor cells (HSPCs) vs. differentiated hematopoietic cells are incompletely understood. Here, we report that transcription factor GATA1, a master-regulator of erythroid differentiation, is essential to repress VEGFR2 expression in erythroid progenitors. Genetic complementation analysis demonstrated that VEGFR2 expression in maintained in GATA1-null erythroid progenitors and rescue of GATA1-function induces VEGFR2 repression. Mechanistic studies in primary hematopoietic progenitors from mouse fetal liver and differentiating mouse embryonic stem cells (ESCs) identified a repressor element at the (-)88 kb region of the Vegfr2 locus from which GATA1 represses Vegfr2 transcription in erythroid progenitors. Furthermore, CRISPR/Cas9-mediated deletion of the Vegfr2(-)88 kb region results in reduced erythroid differentiation during fetal liver hematopoiesis. These results indicate that GATA1-mediated repression of VEGFR2 could be a determinant of optimum erythropoiesis.

Disclosures

No relevant conflicts of interest to declare.
Gadd45a Loss Enhances MLL Leukemogenesis By Promoting Beta-Catenin Mitochondrial Translocation and Inhibiting Apoptosis
Authors: Voli Florida , Yi Hangyu , Martinez Brendon , D'Andrea Richard J , Wang Jenny Yingzi
Published: October 2019

Gadd45a is a mediator of growth arrest and its expression is suppressed in primary human MLL-rearranged AML (predominantly MLL-AF9) and AML1-ETO AML with increased FLT3 expression (Valk et al., N Engl J Med 2004; Perugini et al., Leukemia 2009). While Gadd45a is frequently silenced in several human cancers, including leukemia, little is know about how Gadd45a loss contributes to leukemogenesis. Our studies have shown that Gadd45a loss promotes MLL-AF9 mediated development in AML in mice and protects leukemic cells from apoptosis via novel β-catenin/mitochondria pathway.

To explore the functional role of Gadd45a loss in MLL-rearranged AML, we retrovirally introduced MLL-AF9 into c-Kithigh progenitors isolated from Gadd45a knockout mouse bone marrow cells. Our results showed that Gadd45a knockout markedly reduced the colony forming capacity of pre-leukemic cells and significantly shortened the latency for MLL leukemia development in mice (p = 0.037, N = 6 mice). As Gadd45a could induce cell death through the mitochondria pathway (Tong et al., Mol Cell Biol 2005), we next examined the expression of proteins involved in the mitochondrial apoptosis in Gadd45a knockout pre-leukemic cells. Our western blot analysis showed that Gadd45a knockout dramatically decreased expression of pro-death proteins Txnip, Bim and Bax but interestingly, significantly increased active β-catenin expression in mitochondria. Given that Gadd45a has been reported to regulate β-catenin distribution (Ji et al., Oncogene 2007), we postulate that Gadd45a loss facilitates the translocation of β-catenin into mitochondria. We have previously demonstrated that β-catenin is highly expressed in MLL-AF9 transformed pre-leukemic cells and is lower in Hoxa9/Meis1 transformed pre-leukemic cells, while increased β-catenin expression is correlated with a poor survival rate in mice. Here, we retrovirally introduced a constitutively active form of β-catenin into Hoxa9/Meis1 pre-leukemic cells. As expected, activation of β-catenin reduced endogenous expression of Txnip and Bim in mitochondria, exhibiting an expression pattern similar to that regulated by Gadd45a knockout. Consistently, both Gadd45a knockout and β-catenin activation not only reduced the basal cellular reactive oxygen species (ROS) levels but also inhibited Antimycin A (an ROS generator)-induced mitochondrial ROS production.

Taken together, our study discovers a previously unrecognized Gadd45a loss/β-catenin/mitochondria pathway that prevents mitochondrial ROS accumulation and protects leukemic cells from cell death in MLL leukemogenesis.

Disclosures

No relevant conflicts of interest to declare.
CD8 Tregs Promote Gvhd Prevention and Restore Impaired GVL Effect Mediated By CD4 Tregs in Mice
Authors: Heinrichs Jessica Lauren , Nguyen Hung , Bastian David , Wu Yongxia , Daenthanasanmak Anusara , Yu Xue-Zhong
Published: October 2019

Adoptive regulatory T-cell (Treg) therapy has enhanced the outcome of patients suffering from graft-versus-host (GVH) disease following allogeneic hematopoietic stem cell transplantation (allo-HCT); however, fear of broad immune suppression and subsequent dampening of the beneficial graft-versus-leukemic (GVL) responses remains a challenge. In order to subvert broad immune suppression, we generated alloantigen-specific induced Tregs (iTregs) from resting CD4 or CD8 T cells and tested the ability of iTregs to suppress GVH and maintain GVL responses. We utilized a clinically relevant murine model of haploidentical-HCT with the addition of host-original leukemia cell line to evaluate the effects of CD4 and CD8 iTregs in GVH and GVL responses. While alloantigen-specific CD4 iTregs were effective in preventing GVHD (Fig. 1 A and C), they completely abrogated the GVL effect against aggressive leukemia resulting in 100% tumor mortality (Fig. 1 B and D). Mechanistically, these CD4 iTregs were found to potently suppress the expansion of effector T cells (Teffs) and their ability to secrete IFNγ and granzyme B in the recipient spleen and liver, which may contribute to the impaired GVL activity. Using similar approach, we generated alloantigen-specific CD8 iTregs and found they express higher levels of granzyme B and CTLA-4 compared to nTreg and CD4 iTregs. In vivo studies showed these CD8 iTregs moderately attenuated GVHD (Fig. 1 A and C)while completely sparing the GVL effect (Fig. 1 B and D). We thus further reasoned that the combination of CD4 and CD8 iTregs could achieve the optimal goal of allo-HCT: GVHD suppression with GVL preservation. Indeed, the combination therapy potently suppressed GVHD resulting in increased survival and decreased pathological injury to target organs than either CD4 or CD8 iTreg singular therapy (Fig. 1 A and C). More importantly, the combination therapy maintained potent GVL responses reflected by significantly decreased tumor mortality and load (Fig. 1 B and D).Mechanistically, we observed addition of CD8 iTregs maintained the suppression of Teff expansion but restored the ability of Teffs in producing inflammatory cytokines (e.g. IFNγ and TFNα) and cytolytic effector molecules (e.g. granzyme B and TRAIL). To our knowledge the current findings are the first to support the use of combinational iTreg therapy to achieve optimal suppression of GVHD while maintaining GVL responses.

This work was supported by NIH grants: R01 CA118116 and R01 CA169116

Disclosures

No relevant conflicts of interest to declare.
Lower Use of DLI Post Relapse of Malignancy May Result in Shorter Post-Relapse Survival (PRS) in Patients Undergoing T Cell Replete Haploidentical Donor Transplantation (HIDT) Versus Matched Related (MRD) or Matched Unrelated Donor (MUD) Hematopoietic Cell Transplantation (HCT)
Authors: Solh Melhem , Zhang Xu , Brown Stacey , Morris Lawrence E. , Holland H. Kent , Solomon Scott R. , Bashey Asad
Published: October 2019

Relapse of hematologic malignancies remains a major cause of treatment failure and mortality among patients receiving allogeneic hematopoietic cell transplantation (HCT). Patients relapsing after a matched related or unrelated donor transplantation may benefit from donor lymphocyte infusion (DLI) as part of their post relapse therapy. DLI post-relapse has less frequently been utilized among recipients of HIDT presumably due to the potential for severe GVHD if high T-cell doss are administered without immunosuppression. However, reluctance to administer DLI may limit the possibility of remission following disease relapse in HIDT. We compared the frequency of use of DLI and survival outcomes for patients relapsing after HIDT using post-transplant cyclophosphamide to those relapsing after MRD or MUD HCT at our institution.

195 consecutive HCT recipients with relapse of hematologic malignancy occurring after HIDT (N=35), MUD (N=74) and MRD (N=81) between 1998 and 2014 were included in this analysis. The median age was 50 years (19-77 years) and median time to relapse was 159 days (25-2465 days) post HCT. Patients receiving HIDT had similar median time to relapse (5.1 vs 5.2 vs 5.2 months, p=0.638), Dana-Farber/CIBMTR disease risk index (DRI), comorbidity index (CMI) scores, longer times to neutrophil engraftment (17 vs 14 vs 14 days, p<0.001), and platelet engraftment (28 vs 19 vs 18 days, p<0.001) when compared to MUD and MRD recipients respectively. Post-relapse survival at 1 year was worse among HIDT recipients when compared to MRD (9% versus 41%, p=0.008) and MUD (9% vs 35%, p=0.025). DLI was used in 3 (8%) relapsed HIDT patients, 48 MRD patients (59%) and 28 (38%) MUD recipients. Among patients relapsing post MRD and MUD HCT, use of DLI was associated with a better post relapse survival (p=0.04). In a multivariate analysis, donor type (HIDT), time to relapse (<3 months versus > 3months post-transplant) and comorbidity index (CMI score >= 3) were all predictive of worse post relapse survival.

This analysis shows that relapse post HIDT carries a worse prognosis than relapse post MRD and MUD. Efforts such as post-transplant maintenance and haploidentical DLI infusions should be investigated further in the HIDT setting.

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

Disclosures

No relevant conflicts of interest to declare.
NON-Cryopreserved Hematopoietic STEM CELL Transplantation in Multiple Myeloma. a Single Center Experience in Oran (ALGERIA)
Authors: Bekadja Amine MA , Talhi Souad ST , Ouldjeriouat Hafida OH , Soufi Osmani OS , Brahimi Mohamed BM , Bouhass Rachid Amar RB , Yafour Nabil , Arabi Abdessamed AA
Published: October 2019

Introduction:

For younger patients under 65 years of age, induction followed by high-dose chemotherapy with autologous stem cell transplantation (ASCT) is the standard treatment in multiple myeloma (MM). There is limited experience with non-cryopreserved autologous hematopoietic stem cell transplantation. We evaluated the efficacy and safety of non-cryopreserved storage of ASCT in patients undergoing ASCT for MM.

Patients and methods:

Autologous stem cell was mobilized using G-CSF alone (10 µg/kg/day for 5 days). Leukapheresis to harvest stem cells were performed on day -2 and -1. The grafts were kept in a conventional blood bank refrigerator at +4°C until reinfusion on day 0. The conditioning regimen consisted of melphalan 200 mg/m2 in all patients.

Results:

From May 2009 to December 2013, 134 patients with MM were treated in our center in Oran. The median age at ASCT was 55 years (range; 27-67). There were 80 males and 54 females. The median harvested CD34+ cell count was 3,5x106/kg (range; 1, 22 to 13, 24). All patients had engraftment on the median of day 10 (range; 7 to 17) and platelet transfusion independence on the median of day 13 (range; 9 to 24). There was no graft failure. Mucositis grade 3/4 was seen in 68% patients. Transplant related mortality at 100 days was 2.9%. The overall response to transplant was 92%.

In the 130 evaluable patients, the median post-transplant overall survival had not been reached. The estimated overall survival at 75 months was 63% with 95% confidence interval and the median post-transplant disease free Survival was 35 months (0.05%). 93 (72%) patients are alive and 75 (81%) without disease activity after a median follow-up of 35 months (range; 3 to 75).

Discussion:

We conclude that high dose chemotherapy and autologous transplant with non cryopreserved ASCT is a simple, effective and safe method for MM with equivalent results, and that cryopreservation is not necessary in the treatment of MM under our work conditions in developing countries

Disclosures

No relevant conflicts of interest to declare.
Grouping of Genetic Risk By the European Leukemia Net Classification Predicts Overall Survival and Relapse Incidence in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation
Authors: Hemmati Philipp G. , Terwey Theis , Na Il-Kang , Jehn Christian , Vuong Lam G. , le Coutre Philipp , Dörken Bernd , Arnold Renate
Published: October 2019

Introduction: In acute myeloid leukemia (AML) genetic risk factors are among the strongest predictors for overall outcome. Recently, the European Leukemia Net (ELN) proposed a revised classification based on the presence or absence of specific cytogenetic and/or molecular aberrations. Here, we evaluated the prognostic significance of this system in patients with AML undergoing allogeneic stem cell transplantation (alloSCT).

Patients and Methods: A total of 363 patients transplanted at our center between 2004 and 2014 was retrospectively evaluated. According to the ELN classification genetic risk was favorable (N=51), intermediate-1 (N=120), intermediate-2 (N=98), or adverse (N=94). Remission status at the time of alloSCT was first complete remission (CR1) (N=204), CR>1 (N=61), or refractory (N=98). In 107 patients standard myeloablative conditioning (MAC) was used, whereas reduced intensity conditioning (RIC) was applied in 256 patients. Grafts were from either related (N=103) or unrelated (matched: N=191, mismatched: N=69) donors. The median age was 52 (range: 18-75) years.

Results: For the surviving patients the median follow-up was 30 (range: 3-129) months. Whereas in the subgroup of patients aged ≥60 years (N=98) no significant differences in disease-free survival (DFS) or cumulative incidence of relapse (CI-R) between the 4 ELN subgroups were found, the ELN classification was highly predictive for in the subgroups aged <60 years. Patients with an adverse risk karyotype had significantly lower DFS as compared to patients with a favorable or an intermediate risk profile, i.e. 30% versus 56% for favorable, 52% for intermediate-1, or 59% for intermediate-2 (p=0.0064). Correspondingly, the CI-R was highest in patients with an adverse risk profile, i.e. 53% at 5 years. In turn, patients with favorable, intermediate-1, or intermediate-2 risk disease had a CI-R of 15%, 40%, or 21% (p<0.001). In the intermediate-1 subgroup, adverse outcome, i.e. relapse, was predominant in the group of FLT3-ITD positive patients, whereas the CI-R in patients lacking a FLT3-ITD was similar to what was observed in the intermediate-2 subgroup.

Conclusions: Taken together, our data suggest that the ELN classification of genetic risk is suitable for predicting relapse and overall survival of patients with AML aged <60 years undergoing alloSCT. The adverse outcome of patients in the intermediate-2 subgroup may be related to presence of a FLT3-ITD. Therefore, further efforts are needed to improve the clinical results in this particular group.

Disclosures

No relevant conflicts of interest to declare.
Disparities in Receipt of Radiotherapy and Survival By Age, Sex and Ethnicity Among Patients with Stage I Follicular Lymphoma
Authors: Shah Binay K. , Bista Amir , Sharma Sandhya
Published: October 2019

Background: Radiotherapy (RT) is a first line treatment option for stage I follicular lymphoma. In this population-based study, we evaluated disparities in receipt of radiotherapy and survival among patients with stage I follicular lymphoma.

Methods: Adult patients (age ≥18 years) with stage I follicular lymphoma, as the first primary cancer, diagnosed between 1992 and 2007 were identified using Surveillance, Epidemiology and End Results (SEER) 18 database. Study population was divided into various groups based on age (<60 years and ≥60 years), sex, ethnicity (Caucasians, African Americans and Others) and marital status (married, single and separated/divorced/widowed). Binomial logistic regression was used to analyze factors associated with receipt of RT. SEER*Stat was used to compute 5 year RS for various groups and compared using Z score. Cox proportional hazard model was used to compare factors associated with 5 year RS.

Results: Of the total 7328 patients (median age: 63 years), 2675 (36.5%) received RT. African Americans were less likely to receive RT compared to Caucasians (adjusted odds ratio [OR] of 0.602;P <0.001). Older patients (≥60yrs) were less likely to receive RT compared to younger patients (adjusted OR of 0.636; P<0.001). Single and S/D/W were less likely to receive RT compared to married (adjusted OR of 0.835 and 0.865 with P of 0.029 and 0.029 respectively). Recent years of diagnosis was independent predictor of omission of RT (adjusted OR for one year increase is 0.975, P <0.001). 5 years RS was significantly better in patients receiving RT (97%+0.7% vs. 88.4%+0.7%, P <0.0002). In multivariate analysis, male sex, age <60years , Caucasian race and married marital status were found to be independent predictor of better RS among patients receiving RT (P <0.000001).

Conclusion: Among patients with stage I follicular lymphoma, receipt of RT is associated with superior survival. Patients belonging to AA and Others race, old age group, single and S/D/W as marital status and recent years of diagnosis were independent predictors for omission of RT. 5 year RS among the patients receiving RT was significantly better for male, younger age, Caucasians and married patients.

Disclosures

No relevant conflicts of interest to declare.