bluuebunny/crossref_metadata_2025_split huggingface dataset:
Osteoclasts From Human Embryonic Stem Cell-Derived Hematopoietic Progenitors Support Development of An in Vitro Niche Microenvironment.
Authors: Islam Mohammad Saiful , Allred Jeremy R , Hill Katherine L , Hexum Melinda K , Kaufman Dan S. 
Published: October 2019

Abstract
Abstract 2517
Poster Board II-494
Osteoclasts are bone resorbing cells located in the bone marrow that play a key role in hematopoiesis and formation of the hematopoietic niche. Previous studies demonstrate osteoclasts arise from the monocyte-macrophage lineage of cells within the bone marrow microenvironment. Osteoclast progenitor cells typically express the monocyte marker CD14, though relatively little is known about the developmental history of these osteoclastogeneic cells in humans. Osteoclast progenitors have been previously isolated from peripheral blood and bone marrow. Previous studies by our group and others demonstrate human embryonic stem cells (hESCs) can differentiate into CD34+CD45+ cells that can be supported to differentiate into many types of hematopoietic cells, though development of osteoclasts have not been previously demonstrated. To better evaluate osteoclastogenesis, hESCs were allowed to differentiate on M210 stromal cells for 19–21 days then sorted for CD34+CD45+ cells. These cells were placed into a secondary co-culture with M210 stromal cells using osteoclast differentiation factors macrophage colony-stimulating factor (hM-CSF) and receptor activator of nuclear factor–kB ligand (hRANKL) for 14–21 days. During this time, mononuclear hematopoietic cells expanded on top of the stromal cells, followed by appearance of multinucleated cells that became adherent and embedded within the stromal cell layer. These adherent multi-nucleated cells were TRAP-positive, consistent with osteoclasts. Q-RT-PCR analysis demonstrated expression of osteoclast genes NFATC1, TRAP, CATHEPSIN K AND MMP-9. Culturing these putative osteoclasts on dentin discs demonstrated dentin resorption and pit formation, consistent with osteoclasts cultured from peripheral blood. No pit formation observed when the same experiments were done without hRANKL and hMCSF. Most remarkable was the extensive proliferation of CD45+CD33+ myeloid cells around the periphery of the adherent, multinucleated osteoclasts in culture, resembling a multi-cellular hematopoietic niche environment. Further studies are underway to better evaluate the interaction of these osteoclast cells with other hESC-derived hematopoietic cells. Together, these studies demonstrate development of functional osteoclasts directly from hESC-derived CD34+CD45+ hematopoietic progenitor population. We also show development of a novel in vitro HSC niche microenvironment utilizing hESC-derived osteoclasts. This system has the potential to now incorporate other hESC-derived cell populations as a model to better define cellular and extracellular mechanisms that mediate human hematopoietic development.

Disclosures:
No relevant conflicts of interest to declare.
Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.
Authors: Katsumoto Takuo , Kitabayashi Issay 
Published: October 2019

Abstract
Abstract 2525
Poster Board II-502
MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells.

Disclosures:
No relevant conflicts of interest to declare.
FGFR1 Is Required for and Elucidates Multiple Mechanisms of HSC Mobilization.
Authors: Ross Jason T. 
Published: October 2019

Abstract
Abstract 2532
Poster Board II-509
Though fibroblast growth factor receptor 1 (FGFR1) is known to be expressed in hematopoietic stem cells (HSCs), and FGF ligands can support their in vitro culture, the function of FGFR1 in HSCs in vivo has not been elucidated. We examined mouse models in which Fgfr1 is conditionally deleted in adult hematopoietic tissues. While they exhibit normal hematopoiesis, FGFR1-null HSCs fail to mobilize to peripheral tissues in response to 5-fluorouracil (5FU) and AMD3100; however, they mobilize at levels similar to control animals when treated with granulocyte-colony stimulating factor (G-CSF). Further investigation revealed that FGFR1-null HSCs cannot migrate in response to the CXCR4 ligand SDF-1 in vitro, and after 5FU treatment they display diminished expression of CXCR4 in bone marrow at time points prior to mobilization. Additionally, HSCs lacking FGFR1 cannot be expanded in culture, indicating they bear proliferation defects. Together, these data support a novel role for FGFR1 in mobilization of HSCs and indicate that activation of FGF signaling may distinguish stress-responsive HSC mobilization from more physiologic mechanisms triggered by cytokines such as G-CSF.

Disclosures:
No relevant conflicts of interest to declare.
CCR2 Mediates Hematopoietic Stem and Progenitor Cell Trafficking to Inflammation Sites to Enhance Organ Repair.
Authors: Si Yue , Charo Israel , Tsou Chia-Lin 
Published: October 2019

Abstract
Abstract 2547
Poster Board II-524
Hematopoietic stem cells (HSCs) are bone-marrow derived, self-renewing pluripotent cells that give rise to terminally differentiated circulating blood cells. Recent work has implicated HSCs in the repair of parenchymal tissue in the setting of inflammation. In response to the antagonist of the chemokine receptor CXCR4, HSCs and their progenitors migrate from bone marrow to the blood. However, little is known about the signals that mediate their trafficking from the blood into peripheral tissues. Recently, we showed that mice genetically deficient in chemokine receptor CCR2 (CCR2–/– mice) have a marked decrease in the number of circulating “inflammatory” (7/4+, Ly6c+) monocytes, but no decrease in myeloid progenitor cells in the bone marrow (Tsou et al, J Clin Invest, 2007, 902). These data indicated that although CCR2 is not necessary for HSCs to differentiate into mature monocytes, it does play a role in monocyte egress from bone marrow to blood. In the current study, we extend this work and investigate the expression of CCR2 on hematopoietic stem cells and progenitor cells (HSPCs), and tested the hypothesis that CCR2 is critical for the recruitment of circulating HSPCs to sites of inflammation. Here we report that the chemokine receptor CCR2 is expressed on subsets of primitive hematopoietic stem cells as well as some early myeloid progenitors but not on the progenitors dedicated to megakaryocyte and erythroid differentiation. CCR2 mediates the chemotaxis of c-Kit+Lin- bone marrow derived cells to MCP-1 (CCL2) and MCP-3 (CCL7), which are known CCR2 ligands. In unchallenged mice, CCR2 appears to play a minimal role in HSCs trafficking. However, following instillation of thioglycollate to cause aseptic inflammation HSPCs were actively recruited to the peritoneum, as demonstrated by both FACS and functional colony formation assays. That these cells were true HSCs was demonstrated by their engraftment into the bone marrow of irradiated recipients. In contrast, mice genetically deficient in CCR2 (CCR2−/−) were profoundly impaired in the recruitment of HSCs to the inflamed peritoneum. Furthermore, in human disease models of acetaminophen induced liver damages, hematopoietic stem and progenitors were increased dramatically at site of injury in WT mice but not in CCR2−/− mice. CCR2 recruited HSPC play a tissue protective role by increase the numbers of M2 macrophages in injured liver. Taken together, these findings document a novel role for CCR2 in the specific homing of primitive hematopoietic stem and progenitors to sites of inflammation and injury, and suggest a new role for chemokines in increasing the numbers of differentiated leukocytes at sites of inflammation. Recruited HSPCs hasten the resolution of the inflammatory response, and promote repair of injured tissue, at least partially due to locally increase the number of tissue reparative macrophages.

Disclosures:
No relevant conflicts of interest to declare.
Pathway Based Evaluation of Cytarabine Pharmacogenetics in Children with Acute Myeloid Leukemia.
Authors: Myers Kasiani C. , Gerbing Robert B. , Alonzo Todd A. , Phillips Christine L , Gamis Alan S. , Radloff Gretchen A. , Perentesis John , Davies Stella M. 
Published: October 2019

Abstract
Abstract 2610
Poster Board II-586

Cytarabine is a key chemotherapeutic agent in the treatment of acute myeloid leukemia (AML). We have taken a pathway based candidate gene approach to select variants likely to be critical to cytarabine metabolism. Single non-synonymous nucleotide polymorphisms and polymorphisms located in promoter regions of genes involved in cytarabine metabolism, with known frequencies > 5% in the Caucasian population were identified. We genotyped 479 children treated for de novo AML on the Children's Cancer Group (CCG) 2941 and 2961 protocols for these polymorphisms. All patients received intensively timed induction therapy with IDA-DCTER (idarubicin, dexamethasone, Ara-C (800 mg/m2 total dose), thioguanine, etoposide and daunomycin) given on days 0 to 3 followed by DCTER given on days 10 to 13. On recovery of ANC and platelet counts, patients were randomized to consolidation therapy with Regimen A: a further course of IDA-DCTER/DCTER or Regimen B: IDA-FLAG (idarubicin, fludarabine, Ara-C (7590 mg/m2 total dose Ara-C) and G-CSF). Patients with a matched family donor received intensification with allogeneic transplant; all others received a single course of high dose Ara-C (24,000 mg/m2 total dose) and L-asparaginase. There were no significant associations between genotypes and clinical characteristics of leukemia, although polymorphism frequencies did differ significantly by race. Significant associations with outcome of therapy in a multivariate analysis adjusted for race are listed below:
The nucleoside transporter gene hENT promoter polymorphism rs747199 influenced 5 year overall survival (OS) from study entry (46 ± 6% in G/G cases versus 58 ± 9% in other patients (p=0.039). Similarly, 5 year OS from end of two courses was improved in children with a C/C genotype at rs693955 (OS 66 ± 7% vs. 54 ± 11%, p= 0.058). No significant differences were found in disease free survival, relapse rate (RR), or treatment related mortality for these variants. Nucleoside transporter polymorphisms may play a critical role in modulating cytarabine transport and influence the outcome of therapy for AML in children.

Disclosures:
No relevant conflicts of interest to declare.
Target Epitopes of Human T Lymphotropic Virus 1 Recognized by Class I MHC-Restricted Cytotoxic T Lymphocytes in HAM/TSP Patients and Infected Patients with Autoimmune Disorders.
Authors: Kozako Tomohiro , Akimoto Masaki , White Yohann , Toji Shingo , Matsushita Kakushi , Kubota Ryuji , Izumo Shuji , Suzuki Shinsuke , Uozumi Kimiharu , Shimeno Hiroshi , Soeda Shinji , Arima Naomichi 
Published: October 2019

Abstract
Abstract 2660
Poster Board II-636

Human T-cell leukemia virus-1 (HTLV-1) causes adult T cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) after long-term infection. A variety of collagen diseases such as Sjögren's syndrome and systemic lupus erythematosus has been reported in HTLV-1-infected individuals, although the precise relationship between these disorders and HTLV-1 infection remains unknown. We have previously reported the decreased frequency and function of HTLV-1 Tax-specific cytotoxic T lymphocytes (CTLs) in ATL patients due to insufficient cytolytic effector molecules compared with asymptomatic carriers (ACs) (ASH annual meeting 2007). However, there is no report on the repertoire of HTLV-1-specific CTLs in HAM/TSP patients or carriers with collagen diseases (CCs), both of which are characterized by abnormal immune states. In order to characterize HTLV-1-specific CTLs in ACs, HAM/TSP patients and CCs, we examined the frequency and diversity of HTLV-1-specific CTLs using 16 distinct HTLV-1 Tax and Env HLA-A*0201 and HLA-A*2402 tetramers. There was no statistically significant difference in the proportion of patients having HTLV-1 Tax specific CTLs among ACs, HAM/TSP and CC patients, but a greater proportion of patients with HAM/TSP or collagen disease had HTLV-1 Env-specific CTLs compared with ACs (Env175-183, Env239-246, Env442-450 for HLA-A*0201 and Env11-19, Env21-29, Env153-161 for HLA-A*2402) (Table 1). Within CD8+ lymphocyte subsets, the percentage of cells binding either Tax11-19/HLA-A*0201 or Tax 301-309/HLA-A*2402 tetramer (Figure 1), was significantly higher for HAM/TSP patients compared to ACs, being lowest among CCs. Additionally, the number of epitope repertoires found on HTLV-1 Env-specific CD8+ cells in ACs was considerably lower than for HAM/TSP patients and CCs (ACs, 1/6; HAM/TSP, 4/6; CCs, 6/6). This study demonstrates the relatively greater importance of CTLs recognizing HTLV-1 envelope tetramers compared to HTLV-1 Tax tetramers, thereby suggesting that the diversity, frequency and repertoire of HTLV-1-specific CTL clones, especially Env-CTLs, may be related to the hyperimmune response in HAM/TSP and carriers with collagen diseases.

Disclosures:
No relevant conflicts of interest to declare.
Interim Report of Phase II Study of Bortezomib Plus CHOP Every 2 Weeks in Patients with Disseminated Stage Diffuse Large B-Cell Lymphoma.
Authors: Kim Jeong Eun , Lee Dae Ho , Lee Soon Il , Lee Jae Hoon , Kim Won Seog , Yoon Bora , Suh Cheolwon 
Published: October 2019

Abstract
Abstract 2688
Poster Board II-664
Rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) and dose-dense CHOP therapy has improved the treatment results of diffuse Large B-cell lymphoma (DLBCL) patients. Nevertheless, a substantial number of patients progress or relapse. Bortezomib is a proteasome inhibitor that is widely used in myeloma treatment and was recently approved for use in mantle cell lymphoma treatment. Its antitumor activity in DLBCL patients was reported in several studies, both as a single agent and in combination with chemotherapy. In this study, we tried to add bortezomib to dose-dense CHOP every 2 weeks to evaluate the complete response (CR) rate and safety of the treatment. Patients with previously untreated disseminated stage DLBCL and age 70 years or less were eligible. All patients received CHOP (Cyclophosphamide 750 mg/m2 IV, doxorubicin 50mg/m2 IV, vincristine 1.4mg/m2 IV on day 1, and prednisolone 100mg PO on days 1 to 5) and granulocyte colony-stimulating factor at a dose of 5μg/kg from days 4 to 13 every 2 weeks. Bortezomib, as recommended by the previous phase I trial, was administered at a dose of 1.6mg/m2 on days 1 and 4 of each cycle. Thirty-five patients were enrolled from March, 2007 to March 2009. Total 188 cycles of treatment were done. Twenty-six patients finished planned 6 cycles of treatment, while 9 patients could not continue all planned treatment due to treatment related toxicities and one patient experienced disease progression after 5 cycles of treatment. Twenty-four patients had a CR (92.4%), one patient had a partial response (3.8%), and one patient had a progressive disease (3.8%). Grade 3 hematologic toxicity episodes occurred including 20 anemia (10.7%), 4 neutropenia (2.1%), and 6 thrombocytopenia (3.2%). Grade 4 hematologic toxicity episodes comprised 2 anemia (1.6%), 11 neutropenia (5.9%), and 6 thrombocytopenia (3.2%). One patient died of severe infection with neutropenia. Among 35 patients, grade 3 non-hematologic toxicity occurred with fatigue (11.4%), nausea (2.9%), vomiting (5.7%), diarrhea (8.6%), abdominal pain (8.6%), and sensory neuropathy (20.0%). One patient experienced grade 4 constipation. This interim analysis shows that bortezomib plus dose-dense CHOP every two weeks showed promising activity in disseminated DLBCL patients as the first-line treatment with acceptable toxicity. Further accrual will be continued till the planned patient enrollment goal for phase II results.

Disclosures:
No relevant conflicts of interest to declare.
Long-Term Outcomes of Patients with Newly Diagnosed NK/T-Cell Lymphoma Treated by EPOCH Regimen.
Authors: Huang Huiqiang , Lin Zexiao , Lin Xubing , Cai Qingqing , Xia Zhongjun , Jiang Wenqi 
Published: October 2019

Abstract
Abstract 2689
Poster Board II-665

Introduction:
Extranodal NK/T-cell lymphoma (ENKL) is a distinct clinicopathological entity of non-Hodgkin's lymphoma, which is characterized by a highly aggressive clinical process and poor prognosis. There are no standard treatment strategies available now. The present study aimed to evaluate the treatment outcome and toxicity of EPOCH regimen for ENKL.

Methods:
From June 2003 to June 2008, 34 patients including 30 nasal ENKL (88.2%) and 4 extranasal ENKL (11.8%) received doxorubicin, vincristine, etoposide over 96 hours infusion with bolus cyclophosphamide and oral predinisone (EPOCH) combination as first-line treatment. By the Ann Arbor lymphoma Staging Classification, 29 patients (85.3%) had stage ±E-IIE disease and 5 patients (14.7%) had stage IIIE-‡WE disease. Median cycles of EPOCH regimen administered were 2.5 (range from 1 to 6 cycles). Additional involved field radiation therapy (IFRT) was administered to patients with localized regional disease after chemotherapy.

Results:
Among 34 patients, 33 were eligible for objective response evaluation. The response rate (RR) for the whole group was 60.6% (20/33) with complete remission (CR) rate of 45.5% (15/33). The RR of patients with nasal ENKL was 66.7% (20/30), the CR rate was 50% (15/30). Of the 4 patients with extranasal ENKL, only one achieved stable disease (SD), the other three had progressive disease (PD) during chemotherapy. After a median follow-up of 22 (2–68) months, the estimated overall 3-year overall survival rate (OS) was 52.2%. For patients with nasal ENKL, the estimated median survival time was not reached, the 3-year OS was 59.4%. For patients with extranasal ENKL, the estimated median survival time was only 7 months. For stage ±E-IIE patients with nasal ENKL who received initial EPOCH chemotherapy followed IFRT, the CR rate was 75.0%, and the 3-year survival rate was 75.0%. 102 cycles of chemotherapy were administered to 34 patients. Major adverse effect was myelosuppression. The incidence of grade III-‡W neutropenia was 30.9% and 60 cycles needed G-CSF support. Other toxicities were mild, no treatment-related mortality occurred.

Conclusion:
EPOCH regiment was effective on ENKL with acceptable toxlicity. EPOCH followed by IFRT yielded promising outcome for patients with localized nasal ENKL. However, patients with extranasal ENKL responded poorly, and more efficacious treatment strategies are urgently needed.

Disclosures:
No relevant conflicts of interest to declare.
Alterations of DCC Gene in B-Cell Malignancies.
Authors: Nagoshi Hisao , Taki Tomohiko , Nishida Kazuhiro , Goto Minako , Kobayashi Satoru , Tsutsumi Yasuhiko , Matsumoto Yosuke , Kuroda Junya , Horiike Shigeo , Otsuki Takemi , Taniwaki Masafumi 
Published: October 2019

Abstract
Abstract 4428
Many genetic alterations have been detected in various tumors to date, and these are concerned with oncogenesis closely. Oligonucleotide array analysis has contributed to detect genomic gains and losses implicated in the development of human malignant tumors, identifying either oncogenes or tumor suppressor genes. In our recent data, oligonucleotide array analysis showed recurrent genomic copy number variations at 18q21-22 in malignant lymphoma and multiple myeloma cell lines. There are a lot of genes related to oncogenesis at 18q21-22, such as SMAD4, DCC, MBD2, TCF4, TXNL1, WDR7, FECH, NARS, MALT1, and BCL2. We studied expression of these genes by reverse-transcriptase polymerase chain reaction (RT-PCR). Although most genes examined expressed in almost all cell lines examined, DCC (deleted in colorectal cancer) could be detected in a small number of malignant lymphoma cell lines.
DCC, which encodes a receptor for netrin-1, would control apoptosis and be concerned in oncogenesis. Loss of one DCC allele or decreased DCC expression occur in many solid tumors, suggesting that DCC inactivation constitutes a critical event in the development of these tumors. Similarly, the inactivation of DCC can have a role in the development of hematologic malignancies, including leukemia and malignant lymphoma. We analyzed DCC expression in 15 multiple myeloma and 9 malignant lymphoma cell lines, by RT-PCR using various primer pairs. However, no products could be detected using any primer pairs containing DCC exon 1 primer in three multiple myeloma cell lines, although other primer pairs could amplify products, suggesting the DCC transcripts lacking exon 1 in these cell lines. This result made us predict the abnormal form of DCC. Therefore, we tried to clone the DCC transcripts fused to unknown sequences instead of exon 1, using bubble PCR method for cDNA in these three cell lines. Some products were obtained and sequenceanalysis revealed that the bubble PCR products contained exon 2 of DCC fused to unknown sequences. BLAST search revealed that these unknown sequences were part of intron 1 of DCC, resulting in the lack of start codon in exon 1. Putative abnormal DCC protein lacks a part of N-terminus, suggesting that structural abnormalities of DCC play some roles in oncogenesis of hematologic malignancies. Although the previous studies described the abnormalities of DCC derived from allelic loss or decreased expression, structural abnormalities of DCC we found in this study might be another critical mechanism for DCC abnormalities even if the normal expression could be detected by RT-PCR which could amplify only limited portion of the gene.

Disclosures:
No relevant conflicts of interest to declare.