bluuebunny/crossref_metadata_2025 huggingface dataset:
Using High-Throughput Sequencing of Red Blood Cells and Platelets to Identify Micrornas Associated with Hematopoiesis.
Authors: Doss Jennifer , Jima Dereje , Voora Deepak , Dave Sandeep , Chi Jen-Tsan Ashley
Published: October 2019

Abstract 2326

Human mature red blood cells (RBC) and platelets are both terminally differentiated cells lacking nuclei. However, these two cell types do possess a diverse and abundant set of microRNAs (miRNAs), a set of small, non-coding RNAs acting as post-transcriptional regulators. To identify novel microRNAs associated with differentiation of RBCs and platelets from common progenitors, we performed high-throughput sequencing of these differentiated cells types. In particular, these accessible cells may prove valuable to identify disease biomarkers.

We identified an unbiased set of both known and novel microRNAs by preparing small RNA libraries for application to the Illumina GAII high-throughput sequencing platform. We used a modified version of the probabilistic modeling algorithm, miRDeep (Friedländer 2008), to identify many novel and known microRNAs. Genomic loci that overlapped with miRNAs described in miRBase were identified as known miRNAs. The remaining genomic loci were identified as encoding candidate novel miRNAs. In RBCs we identified 253 predicted miRNA precursor loci, with 226 miRNA precursor loci annotated in miRBase (known miRNAs), whereas the remaining 27 precursor loci were identified as novel miRNAs. In platelets we identified 566 predicted miRNA precursor loci, with 488 known miRNAs and 78 novel miRNAs. Other small RNAs that did not pass miRDeep criteria were also analyzed. One of the most abundant RNA sequences in the RBC sample consisted of a distinct fragment of Ro-associated Y4 RNA (hY4). Y RNAs have been shown to be involved in chromosomal DNA replication, and Y1 and Y4 have been shown to be present in mature erythrocytes (O'Brien 1990). These distinct non-coding RNAs may possess a unique role in erythroid cell expansion.

In addition, we assessed dynamic changes in the expression level of several selected microRNAs during human erythropoiesis. We are currently investigating relevant targets and regulatory functions of these microRNAs during erythropoiesis and platelet development. This global analysis will enhance our understanding of events dictating red cell and platelet maintenance and development.

Disclosures:

No relevant conflicts of interest to declare.
Long Noncoding RNAs in Erythropoiesis and Megakaryopoiesis.
Authors: Paralkar Vikram R , Mishra Tejaswini , Luan Jing , Yao Yu , Konuthula Neeraja , Bodine David M. , Hardison Ross C , Weiss Mitchell J.
Published: October 2019

Abstract 2331

Lnc (long noncoding) RNAs are RNA transcripts greater than 200nt that regulate gene expression independent of protein coding potential. It is estimated that thousands of lncRNAs play vital roles in diverse cellular processes and are involved in numerous diseases, including cancer. We hypothesize that multiple lncRNAs regulate erythrocyte and megakaryocyte formation by modulating gene expression. To identify lncRNAs in erythro-megakaryopoiesis, we purified two biological replicates each of murine Ter119+ erythroblasts, CD41+ megakaryocytes and bipotential megakaryocyte-erythroid progenitors (MEPs) [Lin− Kit+, Sca1−, CD16/32−, CD34−]. We performed strand-specific, paired-end, 200nt-read-length deep sequencing (RNA-Seq) to a depth of ∼200 million reads per sample using the Illumina GAII platform. We used the Tophat and Cufflinks suite of bioinformatic tools to assemble and compare de-novo transcriptomes from these three cell types, producing a high-confidence set of 69,488 transcripts. We confirmed that the RNA-seq assemblies accurately reflect gene expression predicted from prior studies. For example, Ter119+ cells were highly enriched for key erythroid transcripts encoding globins, heme synthetic enzymes and specialized membrane proteins. Megakaryocytes expressed high levels of gene encoding lineage-specific integrins and platelet markers. MEPs expressed numerous progenitor genes including Gata2, Kit and Myc. Thus, the RNA-seq data are of high-quality and sufficient complexity to accurately represent erythroid, megakaryocytic and MEP transcriptomes. We used a series of Unix-based bioinformatic filtering tools to identify lncRNAs that are expressed in these transcriptomes. We identified 605 “stringent” lncRNAs, and 813 “potential noncoding” transcripts. 47% of the lncRNAs are novel unannotated transcripts, validating the use of de-novo RNA-Seq in unique cell populations for lncRNA discovery. Among the 605 “stringent” lncRNAs, 103 are erythroid-restricted, 133 are meg-restricted and 280 are MEP-restricted, consistent with reports that lncRNAs exhibit exquisitely cell-type specific expression. Current efforts are aimed at generating a more comprehensive map of lncRNA expression at specific stages of erythroid and megakaryocyte/platelet development, and performing high throughput functional screens to analyze currently identified lncRNAs. Our studies are beginning to define new layers of gene regulation in normal erythro-megakaryopoiesis and are relevant to the pathophysiology of related disorders including various anemias, myeloproliferative and myelodysplastic syndromes and leukemias.

Disclosures:

No relevant conflicts of interest to declare.
A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.
Authors: Senyuk Vitalyi , Zhang Yunyuan , Liu Yang , Ming Ming , Chen Jianjun , Rowley Janet D. , Nucifora Giuseppina , Qian Zhijian
Published: October 2019

Abstract 2332

MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis.

EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9.

It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells.

Disclosures:

No relevant conflicts of interest to declare.
Functional Influence of CXCL12 Regulating miRNAs in Mesenchymal Stem Cells (MSCs).
Authors: Arabanian Laleh S. , Fierro Fernando , Poitz David M. , Strasser Ruth H. , Bornhaeuser Martin , Ehninger Gerhard , Stölzel Friedrich , Illmer Thomas
Published: October 2019

Abstract 2350

CXCL12 is a chemokine known to be critical for the regulation of the interaction between hematopoietic stem cells (HSCs) and their niche in the bone marrow, e.g. mesenchymal stem cells (MSCs). MicroRNAs (miRNAs) are post-transcriptional regulators recently shown to mediate a variety of cellular processes in the bone marrow niche. However, identification of specific miRNAs and their regulatory role in the crosstalk between HSCs and MSCs are still poorly understood.

From a library of 470 miRNAs, 26 miRNAs were shown to downregulate the levels of CXCL12 in the supernatant of the human MSC line SCP-1. Eight of them (miR-23, 130b, 135, 200b, 200c, 216, 222, 602) were chosen for further investigation according to their significant interaction with the 3'UTR of CXCL12 as determined by luciferase assay. Among them, miR-23a,130 and 222 were expressed in 46 human primary MSCs, whereas the other miRs show negligible expression in resting MSCs.

However, we observed, that MSCs that underwent adipogenic and osteogenic differentiation showed strongly decreased CXCL12 protein values early (day 5) and at later stages (day 14). The later drop in CXCL12 expression was clearly associated with an increased expression of miR-23a and miR-200.

We furthermore tested a subset of stimuli (proinflammatory cytokines, cytotoxic drugs, chemokines) for their ability to modulate the described miRNAs. Amongst them, exclusively the application of transforming growth factor ß1 (TGF-ß1), resulted in the induction of miR-23a and at the same time reduction of CXCL12. The effect was counteracted by transfection of anti-miR-23 molecules.

Taken together, we have shown for the first time that CXCL12-targeting miRNAs (in particular miR23a) have a significant potential to regulate the properties of the stem cell niche. Moreover, miR-23 is implicated in the signalling pathway of TGF-ß1 in human MSCs.

Disclosures:

No relevant conflicts of interest to declare.
Immunosuppressive Therapy with Antithymocyte Globulin and Cyclosporine for Fulminant Aplastic Anemia.
Authors: Yagasaki Hiroshi , Watanabe Ken-ichiro , Kudo Kazuko , Tsuchida Masahiro , Shichino Hiroyuki , Morimoto Akira , Kobayashi Ryoji , Yabe Hiromasa , Kikuchi Akira , Ohga Shouichi , Ito Etsuro , Ohara Akira , Nakahata Tatsutoshi , Kojima Seiji
Published: October 2019

Abstract 2364

Background:

Survival after immunosuppressive therapy (IST) for aplastic anemia (AA) depends on disease severity, which is classified according to polymorphic neutrophil (PMN) count as follows: nonsevere AA (nSAA; PMN > 0.5 × 109/L); severe AA (SAA; PMN 0.2 – 0.5 × 109/L); and very severe AA (vSAA; PMN < 0.2 × 109/L). Although patients with fulminant AA, defined as PMN = 0 for > 2 weeks, generally have poor outcome, no data are available for a large cohort. Therefore, we evaluated the outcome of children with fulminant AA who were enrolled in an AA-97 study and received ATG, cyclosporine, and granulocyte colony-stimulating factor (G-CSF).

Patients and Methods:

A total of 288 children newly diagnosed with AA (median age, 8 years) were enrolled into an AA-97 study between 1997 and 2006 and treated with a combination of horse antithymocyte globulin (Lymphoglobulin®) and cyclosporine. For patients with PMN < 0.2 × 109/L, G-CSF was added. Patients were classified into 3 groups according to disease severity as follows: fulminant (PMN 0, n = 35); vSAA (PMN 0 – 0.2 × 109/L, n = 129); and SAA (PMN 0.2 – 0.5 × 109/L, n = 124). Of the 35 patients with fulminant AA, 8 had a history of acute hepatitis at the time of diagnosis. One case was considered to be drug-induced AA. The remaining 26 patients had unknown etiology. We evaluated the response rate (RR) at 6 months, 5-year overall survival (OS), and 5-year failure free survival (FFS). Treatment failure was defined as death due to any cause, a requirement for a secondary therapy, relapse, clonal evolution to myelodysplastic syndrome/acute myelocytic leukemia, and paroxysmal nocturnal hemoglobinuria.

Results:

The children with fulminant AA showed a significantly lower RR than those with vSAA and SAA (40.0%, 62.8%, and 64.5%, respectively; p =.023). Of the 20 non-responders in the fulminant AA group, 11 were rescued by alternative donor stem cell transplantation, and 5 achieved a late response after 6 months. As a result, no significant difference was noted in 5-year OS and 5-year FFS among the children with fulminant AA, vSAA, and SAA (88.5%/48.2%, 95.8%/65.1%, and 96.8%/68.1%, respectively).

Conclusion:

In consideration of favorable OS, the choice of IST is indicated in children with fluminant AA who lack an HLA-matched family donor.

Disclosures:

No relevant conflicts of interest to declare.
Disseminated Mycobacterium Avium Infection Accelerates Hematopoietic Stem Cell Exhaustion,
Authors: King Katherine Y , Zheng Yayun , Goodell Margaret A.
Published: October 2019

Abstract 2365

Background:

Anemia, leucopenia, or pancytopenia is a common presenting sign in a number of chronic infectious diseases such as HIV and atypical mycobacterial infection. In order to understand the pathophysiology of pancytopenia as a complication of chronic infection, we have investigated the responses of hematopoietic stem cells (HSCs) to mycobacterial infection. We previously found that HSCs proliferate in the setting of a Mycobacterium avium infection. We hypothesized that sustained infection impairs self-renewal in HSCs and leads to premature exhaustion of the stem cell compartment.

Method:

Testing HSC self-renewal is nontrivial and requires serial bone marrow transplantation to measure HSC totipotency over time. Three successive rounds of bone marrow transplantation were conducted using HSCs from mice with M. avium infection.

Results:

HSCs from infected mice were significantly less capable of sustaining trilineage peripheral blood production after serial transplantation compared to control HSCs. These findings demonstrate that self-renewal of HSCs is compromised during chronic infection. It follows that the total number of HSCs should decline with time during sustained infection. To test this, serial monthly infection with M. avium was conducted over an 8-month period. Repeatedly infected animals showed a gradual decrease in the number of committed lymphoid and myeloid progenitors and a significant depletion of HSCs in the bone marrow by 6 months following initial infection. Transcriptional profiling of HSCs from M. avium-infected animals by RNAseq revealed previously uncharacterized potential regulators of HSC self-renewal.

Conclusion:

Our results demonstrate that HSC self-renewal is compromised during chronic infection, leading to exhaustion of the stem cell compartment. These findings elucidate both the basis for and possible therapeutic approaches towards hematologic complications in patients with atypical mycobacterial infection, tuberculosis, HIV, and other chronic inflammatory conditions.

Disclosures:

No relevant conflicts of interest to declare.
ATM and 53BP1 Function in Nonoverlapping Pathways to Suppress T Lineage Lymphomagenesis.
Authors: Rybanska Ivana , Chou Jeremy , Prakash Mansi , Huso David , Franco Sonia
Published: October 2019

Abstract 2374

Background:

In response to DNA double-strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that promotes their detection, signaling and repair. Among DDR factors, the Ataxia-Telangiectasia Mutated (ATM) kinase and the 53BP1 adaptor localize to chromatin surrounding DSBs, where 53BP1 is thought to transduce a subset of ATM-dependent reactions. In addition, 53BP1 may have ATM-independent functions via its interactions with the MRE11/RAD50/NBS1 (MRN) complex and others to protect DNA ends from degradation and to promote synapsis of distant DSBs. Both ATM and 53BP1 are tumor suppressors in humans and mice, but whether the combined mutation may aggravate the DSB repair defect and/or accelerate oncogenesis in vivo is currently unknown.

Objectives:

1) To generate a compound mouse model deficient for both ATM and 53BP1 and investigate their genetic interaction during growth and development; 2) To determine whether 53BP1 plays ATM-independent functions in DSB repair and cell cycle checkpoint activation in primary thymocytes; 3) To determine the effect of 53BP1 deficiency on the penetrance and latency of T cell lymphomas in ATM−/− mice.

Design/Methods:

We have generated 53BP1/ATM double knock out (DKO) mice and assessed the kinetics of DSB signaling and repair in primary thymocytes and mature B cells. We have also followed cohorts of DKO and control mice for tumor development and evaluated mechanisms of tumorigenesis using a combination of molecular, biochemical and cytogenetic approaches.

Results:

When compared to ATM−/− mice, DKO mice showed no differences in organismal growth and development, or in lymphoid differentiation. However, DKO mice developed T cell lymphomas with higher penetrance and at younger age than ATM−/− mice, and their survival was markedly reduced (p=0.0001; log-rank test). Like ATM−/− lymphomas, DKO lymphomas contained clonal translocations with breakpoints at the TCRα/δ locus and amplification of upstream DNA sequences, indicating that 53BP1 deficiency markedly accelerates the acquisition of oncogenic translocations in an ATM−/− background. In primary cells, loss of 53BP1 further increases the frequency of ionizing irradiation (IR)-induced chromosomal aberrations in ATM−/− cells. We will present a model for how 53BP1 actions at DSBs suppress genomic instability in ATM-deficient thymocytes, including its effects on checkpoint activation and DSB repair.

Conclusion:

Our studies in a mouse model of T cell lymphomagenesis suggest that mutations in 53BP1 and ATM may be nonmutually exclusive in human lymphomas. The combined mutation may define a subgroup of patients with more aggressive hematological malignancies, and may have implications for their response to therapy with DNA damaging agents.

Disclosures:

No relevant conflicts of interest to declare.
PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells.
Authors: Bera Rabindranath , Liang Der-Cherng , Chiu Ming-Chun , Huang Ying-Jung , Liang Sung-Tzu , Shih Lee-Yung
Published: October 2019

Abstract 2393

Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells.

Disclosures:

No relevant conflicts of interest to declare.
Interaction of c-Myb with p300 Is Required for the Induction of Acute Myeloid Leukemia by Human AML Oncogenes, and Represents a Potential Therapeutic Target.
Authors: Pattabiraman Diwakar R , Shakhbazov Konstantin , Barbier Valerie , McGirr Crystal , Krishnan Keerthana , Mukhopadhyay Pamela , Hawthorne Paula , Trezise Ann , Grimmond Sean , Papathanasiou Peter , Perkins Andrew C. , Levesque Jean-Pierre , Alexander Warren S. , Winkler Ingrid G , Gonda Thomas J
Published: October 2019

Abstract 2402

The MYB oncogene is widely expressed in acute leukaemias and is important for the continued proliferation of leukemia cells, raising the possibility that MYB may be a therapeutic target. However realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal haemopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for Myb's intrinsic transforming activity (1). Here we use haemopoietic cells from the Booreana mouse strain, which carries a mutation in Myb that prevents interaction with CBP/p300 (see Fig. 1 and ref. 2), to examine the requirement for this interaction in myeloid transformation and leukaemogenesis. Using this strain and a strain (plt6) carrying a “complementary” mutation in p300 (3), we show that the Myb-p300 interaction is essential for in vitro transformation by the myeloid leukaemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type (WT) mice, Booreana cells fail to induce leukaemia upon transplantation into irradiated recipients following transduction with an AML1-ETO9a retrovirus (Fig. 2).

These data, as well as reinforcing the notion that MYB is an essential cofactor for myeloid leukaemogenesis, highlight disruption of the Myb-p300 interaction as a potential therapeutic strategy for AML. Furthermore, our results suggest that such a strategy would have a useable therapeutic index since Booreana mice, unlike Myb null mice, are viable. We have started to explore the use of small peptides to provide proof-of-principle for this approach.

We have also begun to explore the molecular basis of the requirement for MYB, and the Myb-p300 interaction, in AML using gene expression profiling. This has highlighted several Myb target genes – identified by our previous ChIP-Seq analysis (4) - that have been previously implicated in myeloid leukaemogenesis, as being differentially expressed between WT and Booreana cells transduced with AML1-ETO9a. One striking example is the apparent Myb-p300 dependent activation of Ets2 and Gfi1, accompanied by repression of Gata2 and Gfi1b; this pattern has previously been reported in MLL-AF9 and MOZ-TIF-induced murine AML (5).

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Elimination of Mixed-Lineage Leukemia Leukemic Stem Cells by a Potent Small Molecule PRC2 Inhibitor.
Authors: Wang Jenny Yingzi , Sprod Owen , Haber Michelle , Norris Murray D
Published: October 2019

Abstract 2456

Leukemia carrying a chromosomal translocation involving the mixed-lineage leukemia (MLL) gene is particular resistant to current therapy, and is associated with frequent relapse. Current evidence points towards self-renewing leukemic stem cells (LSC) as the driver of relapse. The eradication of LSC is thought to be critical for successful anti-leukemia therapy. Recent studies suggest that the multi-protein complex polycomb repressive complex 2 (PRC2) is required for MLL-rearranged AML. PRC2 contains the core proteins EZH2, EED and SUZ12, which have intrinsic histone methyltransferase activity specific to trimethylation of histone H3 on lysine 27. It is involved in the epigenetic silencing of tumor suppressor genes (e.g., p16 and p19). In this study we determined the effect of a PRC2-associated inhibitor (hereafter named PAI) on the PRC2 components in LSC isolated from bone marrow cells of mice with established leukemia induced by the MLL-AF9 oncogene. Treatment with PAI (0.1 to 0.5 μM) for 24 hours in culture media depleted protein levels of EZH2 and EED in LSC. Following treatment, quantitative real-time PCR and immunoblotting analysis demonstrated increased mRNA and protein levels of p16 and p19, as well as inhibition of cell cycle progression. PAI treatment also induced a 10-fold increase of apoptosis in LSC. As a consequence, colony formation assays showed that LSC treated for 24 hours with 0.1 μM PAI completely lost their ability to form compact colonies in methylcellulose. We are currently assessing the effects of PAI in in vivo models of MLL-rearranged leukemia where preliminary data indicate that we are able to achieve pharmacologic inhibition of the PRC2 activity. Collectively, these findings indicate that PAI depletes the levels of PRC2 components, and eliminates LSC, thus representing a potentially promising novel treatment for AML.

Disclosures:

No relevant conflicts of interest to declare.
CD34 Expression On the Blasts of Bone Marrow Is a Novel Predictor of Poor Prognosis Independent of FlT3-ITD in Acute Myeloid Leukemia with NPM1-Mutation.
Authors: Zhu Hong-Hu , Liu Yan-Rong , Jiang Hao , Lu Jin , Qin Yazhen , Jiang Qian , Huang Xiaojun
Published: October 2019

Abstract 2491

Background:

Most studies have demonstrated NPM1-mutated acute myeloid leukemia(NPM1m+AML) has a favorable prognosis, especially in the absence of concomitant FLT3-ITD. However, recent studies showed the prognostic heterogeneity of NPM1m+AML. CD34-expression was demonstrated as a poor prognosis factor of adult patients with AML in most previous reports. The strike immunophenotype of NPM1m+AML is regarded as lacking expression of CD34, which accounts for about 75% patients. Whether CD34-expression on the blasts has independent prognostic value in NPM1-m+AML has yet to be clarified. We therefore systematically evaluated the prognostic relevance of CD34 status in the context of NPM1mutation status and clinical features in 71 patients treated at our institution.

Methods:

Seventy-one NPM1m+AML patients were retrospectively analyzed in our center. Immunophenotype of AML were detected by 4 color flow cytometry. NPM1 mutation and FLT3-mutation were detected by PCR.

Results:

The percentage of CD34-expressed blasts of bone marrow ranged from 0.01% to 93.2%. Patients with CD34 expression>7%(according to ROC analysis) had a lower complete remission(CR) rate after 1 course induction, disease-free survival(DFS) and overall survival(OS) compared those with CD34-expression¡Ü7%(CR, 46.4% vs. 81.4%, p=0.0038; 3 years DFS, 26.2% vs. 68.5%, p=0.001; 3 years OS, 24.4% vs.69.1%, p<0.0001). Multivariate analysis revealed CD34-expression as an independent prognostic factor independent of FlT3-ITD for relapse, DFS and OS. We established a new prognostic model based on CD34 and FLT3 status at diagnosis, which could allow dissection of NPM1m+AML into prognostically different three subgroups: CD34>7% and FLT3-ITD, CD34>7%or FLT3-ITD, CD34¡Ü7% and FLT3-WT(3 years DFS of 0 vs. 58.3% vs. 77%, p=0.002; OS of 0 vs. 59.1% vs. 79.9%, p<0.0001 ) .

Conclusions:

We demonstrated for the first time, that CD34-expression on the blasts is a novel poor predictor independent of FlT3-ITD in patients with NPM1-mutation, and established a new prognostic model based on CD34 and FLT3 status at diagnosis, which could allow immediate clinical utility for prognostic stratification and risk-adapted therapeutic choices.

Disclosures:

No relevant conflicts of interest to declare.
A Phase 2 Trial of HQK-1001 (sodium 2,2-dimethylbutyrate), an Oral Fetal Globin Gene Inducer, in HbE-Beta Thalassemia in Thailand
Authors: Fucharoen Suthat , Chaneiam Nattawara , Patthamalai Beer Poramin , Perrine Susan P
Published: October 2019

Abstract 251

Beta thalassemia intermedia (BTI) syndromes are characterized by globin chain imbalance, ineffective erythropoiesis, hemolytic anemia, and have no therapeutic approved for the underlying pathology. Higher fetal globin (HbF) expression can compensate for beta globin deficiency, reduce globin chain imbalance, and ameliorate phenotype within the same genotypes. HQK-1001 (HemaQuest Pharmaceuticals, San Diego, CA) is an orally bioavailable, non-cytotoxic, short-chain fatty acid derivative which induces fetal globin expression in multiple experimental models. In thalassemic erythroid progenitors in vitro, HQK-1001 prolongs STAT-5 phosphorylation and increases expression of the pro-survival protein Bcl-xL. In a Phase I/II dose-escalation trial in BTI, HQK-1001 administered at 10, 20, 30, and 40 mg/kg/day for 8 weeks was well–tolerated, demonstrated a t½ of 9–11 hours, and treatment resulted in an increase in HbF and total hemoglobin (Hgb), with best results observed at 20 mg/kg.

This open-label Phase 2 trial evaluated HQK-1001 administered orally at 20 mg/kg once daily for 26 weeks in adult patients with HbE-β0 thalassemia (NCT01609595). Ten subjects were enrolled, ages 20–36 years, including 7/10 female and 5/10 splenectomized subjects. The beta globin molecular mutations of the subjects, in addition to Codon 26 (G-A), included Codon 41/42 (-TTCT), Codon 17 (A-T), Codon 110 (T-C), and IVS I-I (G-T). At the time of this analysis, 9 of 10 subjects have completed at least 20 weeks of dosing. HbF has increased in all subjects above baseline levels by a mean of 10% (range 4.3% to 20.9%) and total HbF has increased by a mean of 1.07 g/dL, (range 0.52–2.38 g/dL, with positive changes in total HbF observed in 9/10 subjects. Total Hgb has increased by >0.5 g/dL above baseline (mean of 1.27 g/dL, range 0.7 to 2.2 g/dL) in 3 subjects after 3 to 5 months of dosing. Treatment was well-tolerated. There have been no severe drug-related adverse events, no serious adverse events, and no clinically relevant laboratory abnormalities. One subject developed transient proteinuria and edema without changes in renal function 3 weeks following an episode of pharyngitis. This ongoing trial demonstrates consistent induction of fetal globin expression by HQK-1001 at a well-tolerated dose level in subjects with HbE-beta thalassemia.

Disclosures:

Fucharoen: HemaQuest Pharmaceuticals: Research Funding. Perrine:HemaQuest Pharmaceuticals: Equity Ownership, Patents & Royalties.
Clinical Implications of Reticulin Fibrosis of Bone Marrow in De Novo Acute Myeloid Leukemia.
Authors: Tang Tzung-Chih , Chang Hung , Sun Chien-Feng , Shih Lee-Yung , Dunn Po , Wang Po-Nan , Wu Jin-Hou , Kuo Ming-Chung , Lin Tung-Liang , Hung Yu-Shih , Kao Hsiao-Wen
Published: October 2019

Abstract 2585

Background:

Microenvironment of bone marrow (BM) plays an important role to support proliferation, renewal and differentiation of hematopoietic stem cells. Whether the stroma of BM affects leukemic cells with the same manner, or impacts on the prognosis in leukemia patients, has not been fully investigated. Previous studies have described that increased reticulin content in the BM is associated with poor outcome in patients with acute lymphoblastic leukemia, chronic myeloid leukemia and primary myelofibrosis, but there is no cohort study to determine the clinical correlation between degree of reticulin fibrosis of BM and acute myeloid leukemia (AML). To investigate prognostic impact of reticulin fibrosis on de novo AML, 881 patients diagnosed between Jun 1999 to Dec 2011 in Chang Gung Memorial Hospital and treated with anthracycline-containing induction chemotherapy were retrospectively reviewed.

Patients and methods:

According to the grading of reticulin content in the bone marrow, we categorized the 881 patients into four groups: A. BM easily aspirated without biopsy, n = 698; B. Reticulin grade 0, n = 99; C. Reticulin grade 1–2, n = 51; D. Reticulin grade 3–4, n = 33. The induction failure (IF) rate after treatment with induction chemotherapy, the recovery duration of absolute neutrophil count (ANC) greater than 0.5 × 109/L in patients who achieved the first complete remission, the overall survival (OS) and relapse-free survival (RFS) in four groups were analyzed. Based on the cytogenetic or molecular features, 648 of the patients were stratified into unfavorable, intermediate and favorable risk groups, and the clinical significance of reticulin fibrosis of BM were also examined for various risk groups.

Results:

Of the 881 patients, the patients in group D had a statistically higher IF rate (P = 0.0108) and longer ANC recovery duration (P = 0.0008). But the OS and RFS between four groups were not significantly different (P = 0.5146 and 0.3853, respectively). After risk stratified by cytogenetic and molecular analysis, increased reticulin content of BM (group C or D) had an adverse impact on OS in the intermediate and favorable risk groups (P = 0.006 and 0.0215, respectively).

Conclusion:

Reticulin content of BM influences the IF rate and myeloid recovery for the patients of de novo AML, and affects OS in patients with intermediate or favorable risk factors.

Disclosures:

No relevant conflicts of interest to declare.
Increased Risk of Central Venous Catheter Associated Thrombosis in Acute Promyelocytic Leukemia.
Authors: Grisariu Sigal , Spectre Galia , Kalish Yossef , Gatt Moshe E.
Published: October 2019

Abstract 2588

Background:

Patients with acute leukemia and in particular, patients with acute promyelocytic leukemia (APL), are considered to be at high risk for the development of venous thromboembolism. The presence of central venous catheters (CVC) increases the risk of thrombotic complications; however, previous reports have varied as to the frequency of this complication. Current guidelines do not recommend the routine use of prophylactic anticoagulation for patients with CVC.

Design and methods:

We have suspected an increased incidence of CVC thrombosis in patients with APL and therefore conducted a retrospective survey of 473 patients who were treated for acute leukemia at our institution.

Results:

The overall rate of symptomatic CVC thrombosis was 6.8%. We found a significantly (p< 0.001) increased rate of symptomatic CVC thrombosis (32%) in patients with APL as compared with patients with acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) (6.4% and 4% respectively) as shown in Figure 1. CVC thrombosis was most prevalent following induction treatment, during the period of recovery of blood counts, and was associated with significantly higher platelet counts in patients with APL compared to other AML patients. We therefore relate this higher thrombosis rate to the disease pro-coagulants, the treatment with all-trans-retinoic-acid and the high platelet counts at the time of induction recovery.

Subsequently, following this observation, we treated 13 newly diagnosed patients with APL with low molecular weight heparin prophylaxis during the induction recovery phase of blood counts. None of these patients had a thrombotic event, nor a bleeding complication.

Conclusions:

Although our treatment group was small, we suggest considering prophylaxis for CVC thrombosis during platelet recovery after induction treatment in patients with APL.

Disclosures:

No relevant conflicts of interest to declare.
Absolute Monocyte Count At Diagnosis Is a Poor Prognostic Factor and Impacts Survival in Diffuse Large B-Cell Lymphoma: A Collaborative Multi-Center Study Involving 1026 Patients.
Authors: Tadmor Tamar , Alessia Bari , Sacchi Stefano , Marcheselli Luigi , Liardo Eliana Valentina , Avivi Irit , Benyamini Noam , Attias Dina , Pozzi Samantha , Federico Massimo , Polliack Aaron
Published: October 2019

Abstract 2651

In non-Hodgkin lymphoma (NHL), individual characteristics of the tumor cells alone are not the only parameters which determine prognosis and overall survival. Non-malignant immune cells and the tumor microenvironment also play an important contributory role in determining the eventual clinical outcome. There is an increasingly growing interest in the role of monocytes and their precursors in the pathogenesis of lymphoproliferative disorders. Recently, elevated peripheral blood monocyte counts have been shown to be an independent prognostic parameter associated with poor prognosis and decreased overall survival in patients with both non- Hodgkin lymphoma and Hodgkin. The main aim of this collaborative multicenter study was to re-evaluate and attempt to verify the prognostic significance of monocytosis (> 800 cells/mm3) at diagnosis, in a large cohort of 1026 newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) followed at different medical centers both in Israel and Italy. Additional aims of this study were to define possible correlations with a number of other well recognized prognostic factors in DLBCL.

We reviewed clinical and laboratory data of consecutive untreated patients with DLBCL, followed and eventually treated in different centers in Haifa, Israel and in Italy between 1993–2010. Median age at diagnosis was 59 years (range 18–95 yrs) and the 5-years OS after a median follow up of 51 months (range 0.2–180 months) was 69% for the entire cohort. Monocyte counts were available for 852 patients. Those with peripheral blood monocyte count (PBMC) < 800 mm3 had a 5 years OS of 71%, compared to 59% for those with PBMC > 800 mm3 (p=0.0002). In univariate analysis age, IPI score and monocyte counts were associated with a worse OS. In multivariate analysis, monocytosis retained a negative prognostic value even when adjusted for IPI (HR 1.53, CI95% 1.16–2.02, p=0.003).

Of the 852 patients, 496 (58%) were treated with chemotherapy alone, while 356 patients (42%) received chemo-immunotherapy with similar regimens including rituximab. The addition of rituximab to the combination chemotherapy did not abrogate the negative adverse effect of monocytosis (p=0.0015) observed in patients receiving chemotherapy alone.

This large validation study confirms earlier very recent results and shows that the monocyte level (monocytosis), a simple prognostic parameter, can easily be used routinely to evaluate newly diagnosed patients with DLBCL and identify those with a higher risk of poor survival. The addition of monocytosis as an additional parameter to the basic IPI, may possibly generate a more comprehensive score incorporating both patient, tumor and immune parameters.

Disclosures:

No relevant conflicts of interest to declare.
Constant Activation of the RAF-MEK-ERK Pathway As a Diagnostic and Therapeutic Target in Hairy Cell Leukemia.
Authors: Tiacci Enrico , Schiavoni Gianluca , Martelli Maria Paola , Boveri Emanuela , Pacini Roberta , Tabarrini Alessia , Zibellini Silvia , Santi Alessia , Pettirossi Valentina , Fortini Elisabetta , Ascani Stefano , Arcaini Luca , Inghirami Giorgio , Paulli Marco , Falini Brunangelo
Published: October 2019

Abstract 2657

The BRAF-V600E mutation defines genetically hairy cell leukemia (HCL) among B-cell leukemias and lymhphomas. In solid tumors, BRAF-V600E is known to aberrantly activate the oncogenic MEK-ERK pathway, and targeted BRAF and/or MEK inhibitors have shown remarkable efficacy in clinical trials of melanoma patients. However, the MEK-ERK pathway status in HCL has not been thoroughly investigated so far. Therefore, as a read-out of MEK-ERK pathway activation, we assessed phospho-ERK expression in 37 HCL patients using immunohistochemistry on routine biopsies and/or Western blotting on purified leukemic cells. Beside confirming the constant presence of BRAF-V600E in all patients, we documented ubiquitous phospho-ERK expression in HCL. Conversely, all 44 HCL-like cases in parallel studied (40 splenic marginal zone lymphoma, 2 HCL-variant and 2 splenic lymphoma/leukemia unclassifiable) were devoid of BRAF-V600E and none expressed phospho-ERK. Lack of phospho-ERK expression was also documented in two exceptionally rare cases of non-HCL CD5-negative B-cell lymphoproliferative disorders not otherwise specifiable that were previously described to harbour the BRAF-V600E mutation on allele-specific PCR (Arcaini et al, Blood 2012;119:188–191), pointing to the presence of this mutation in only a small part of the leukemic clone in these cases. Our findings support the use of phospho-ERK immunohistochemistry in the differential diagnosis between HCL and HCL-like neoplasms and establish the MEK-ERK pathway as a rational therapeutic target in HCL.

Disclosures:

Tiacci: Not applicable: Dr. Tiacci filed a patent for the clinical use of BRAF mutations as biomarkers of HCL. Other. Inghirami:OncoEthix SA: Research Funding. Falini:Not applicable: Dr. Falini filed a patent for the clinical use of BRAF mutations as biomarkers of HCL. Other.
Ivig, but Not a Monoclonal Anti-RBC Antibody, Requires the Presence of Gr-1+ Cells to Ameliorate Immune Thrombocytopenia in a Murine Model
Authors: Crow Andrew R. , Lazarus Alan H
Published: October 2019

Abstract 268

Although there are many theories as to the mechanism of action of IVIg in the treatment of autoimmune disease, the exact pathway by which IVIg functions remains unclear. Many cell populations have been implicated in the IVIg pathway, including dendritic cells, which are considered to be one of the central initiators of IVIg effects, and macrophages, which are involved in platelet destruction. In addition, there is evidence from several groups that additional intermediary cell types may be involved. IVIg administration can induce a suppressive effect on peripheral blood neutrophil counts in ITP patients. In fact, alloimmunized thrombocytopenic patients, who display low neutrophil counts, do not respond to IVIg therapy.

Here, we questioned whether Gr-1+cells (consisting primarily of neutrophils) are a critical cell type required for IVIg function, in a murine model of ITP. Another IVIg product which has ameliorative effects similar to IVIg but appears to function via a different mechanism is anti-D. We have previously shown that IVIg and a monoclonal antibody with “anti-D like” activity, TER-119, can successfully ameliorate thrombocytopenia in a murine model of ITP. In human patients as well as murine models of ITP, these 2 therapeutics appear to function via different mechanisms. Some work has shown that IVIg and anti-D work by the same, or overlapping mechanism, while other work shows a notable difference in that IVIg can cause neutropenia under conditions where it works to ameliorate autoimmune inflammation.

Mice pretreated with 50 mg IVIg (∼2g/kg), or 50 ug TER-119 thirty min prior to administration of anti-platelet antibody MWReg30, show protection from thrombocytopenia compared with untreated mice. To assess the potential role for Gr-1+ cells in IVIg vs TER-119 mediated amelioration of murine ITP, we used RB6-8C5, a well described rat antibody for Gr-1+ cell depletion. Mice were injected with RB6-8C5 or control rat IgG 24 hr prior to thrombocytopenia induction. Mice pretreated with RB6-8C5 failed to respond to IVIg therapy compared with control mice. In contrast, Gr-1+ cell depletion had no effect on the ability of TER-119 to ameliorate the thrombocytopenia. This suggests that Gr-1+ cells likely play an essential role in IVIg function. In contrast, TER-119, does not depend on the presence of Gr-1+cells, suggesting that the mechanisms of action for IVIg and RBC specific antibodies are different for this requirement.

In line with these observations, it has been observed that IVIg can modulate neutrophil activity, suggesting that in the murine ITP model, IVIg may function through a neutrophil dependent pathway. Experiments using more specific granulocyte antibodies will help ascertain whether neutrophils or some other Gr-1+ cell population is involved in IVIg function.

Disclosures:

No relevant conflicts of interest to declare.
Mutational Status of Splenic Marginal Zone Lymphoma Revealed by Whole Exome Sequencing.
Authors: Martinez Nerea , Varela Ignacio , Vaque Jose P. , Derdak Sophia , Beltran Sergi , Mollejo Manuela , Sanchez-Beato Margarita , Cereceda Laura , Bayes Monica , Gut Marta , Oscier David , Rodriguez-Peralto Jose L. , Sole Francesc , Fraga Maximo , Garcia Juan F. , Sebastian Elena , Gut Ivo G. , Piris Miguel A.
Published: October 2019

Abstract 2698

Background:

Splenic marginal zone lymphoma (SMZL) is a small B cell neoplasm whose molecular pathogenesis is still unknown. It has a relatively indolent course, but a fraction of the cases may show an aggressive behavior. The lack of comprehensive molecular analysis for SMZL precludes the development of targeted therapy. Here we studied the mutational status of 6 SMZL samples using Whole Exome Next Generation Sequencing.

Methods:

Genomic DNA was extracted from splenic tumor or peripheral blood samples and oral mucosa as the corresponding non-tumor control. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). The variant calling was performed using an in house written software calling potential mutations showing a minimum independent multi-aligner evidence.

Results:

We performed paired-end-76pb whole exome sequencing on 6 SMZL samples and the corresponding normal counterpart. Three of the samples corresponded to CD19 isolated cells from peripheral blood, while other three corresponded to spleen freshly frozen tissue. The mean coverage obtained was 104.07 (82.46–119.59) with a mean of 91.41% (90.41–93.73) of bases with at least 15× coverage. After filtering, 237 substitutions and 21 indels where obtained. No recurrent variation was found. Six of the variations found here were already described in other malignancies. Variations were classified into silent (75), missense (147), nonsense (8), and essential splice (5), according to their potential functional effect, and into tolerated (54) and deleterious (76) according to the “variant effect predictor” tool of Ensembl Genome Browser.

Whole exome sequencing permitted us to identify variations in several genes of TLR/NFkB pathway (Myd88, Peli3), BCR (Myd88, Arid3A) or signal transduction (ARHGAP32), essential pathways for B-cell differentiation. These variations and other involving selected genes, such as the Bcl6 repressor BCOR, were validated by capillary sequencing.

These results were confirmed and expanded in a second series of 10 new cases by exome sequencing.

Conclusions:

SMZL samples contain somatic mutation involving genes regulating BCR signaling, TLR/NFKB pathways and chromatin remodeling.

Disclosures:

No relevant conflicts of interest to declare.