bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cross-Talk Between CLL Cells and Endothelial Cells in the Bone Marrow Microenvironment: Role of Signal Transducer and Activator of Transcription (Stat)-3.
Authors: Badoux Xavier C. , Ramos Carlos Bueso , Harris David , Li Ping , Liu Zhiming , Burger Jan , O'Brien Susan , Ferrajoli Alessandra , Keating Michael , Estrov Zeev 
Published: October 2019

Abstract
Abstract 1242
Poster Board I-264
We have recently found that Stat3, constitutively phosphorylated on serine 727 residues, activates genes that are known to be activated by tyrosine phospho-Stat3 in CLL cells (Hazan-Halevi et al. Blood (ASH Annual Meeting Abstracts), Nov 2008; 112: 1058). Because Stat3 modulates VEGF expression in other cellular systems, we asked whether VEGF is a Stat3 target gene in CLL. Using chromatin immunoprecipitation (ChIP) we found that anti-Stat3 antibodies immunoprecipitated DNA of VEGF as well as of Stat3-regulated genes (such as Stat3 and p21). To further delineate this finding, we infected CLL cells with Stat3-shRNA and determined changes in RNA levels by qRT-PCR. In four out of four CLL samples, Stat3-shRNA induced a significant reduction in the RNA levels of VEGF, confirming that serine phosphorylated STAT3 upregulates VEGF gene transcription in CLL. Furthermore, anti-VEGF immunohistochemical staining detected high levels of VEGF in CLL bone marrow cells, and enzyme-linked immunosorbent assay (ELISA) detected significantly higher VEGF levels in the serum of CLL patients (N = 47) compared to serum of normal individuals (N = 14). Serum VEGF levels correlated with the patients' absolute lymphocyte count. Because VEGF is known to induce vasculogenesis, we studied bone marrow biopsies of 10 patients with CLL and demonstrated increased microvascular density in all specimens. We noted also attachment of CLL cells to endothelial and vascular endothelial cells in all studied marrow sections. To clarify the interaction between CLL and endothelial cells we used human umbilical vein endothelial cells (HUVEC) and studied their effect on CLL cell apoptosis by measuring Annexin V using flow cytometry. In co-culture studies, direct contact with HUVEC protected CLL cells from spontaneous apoptosis. This effect was abrogated by insertion of micropore filters between HUVEC and CLL cells, suggesting that the protective mechanisms required direct interaction between CLL and endothelial cells. Similarly, anti-VEGF receptor (VEGFR) antibodies partially reversed the protective effect of HUVECs on CLL cells, consistent with an antiapoptotic effect dependent on VEGF-VEGFR interaction. Taken together, our data suggest that constitutively activated Stat3 mediates the interaction between CLL cells and the bone marrow microenvironment via VEGF, thereby providing a survival advantage to the neoplastic cells.

Disclosures
No relevant conflicts of interest to declare.
The Sal-Like 1 (SALL1) Protein is Highly Expressed in Primary Bone Marrow Cells Derived From Patients with Acute Myeloid Leukemia.
Authors: Konig Heiko , Kiefer Susan , Robbins Lynn , Rauchman Michael , Salman Huda S. 
Published: October 2019

Abstract
Abstract 1285
Poster Board I-307

Background
SALL1 is a zinc finger transcription factor that maps to chromosome 16q12.1 in humans and chromosome 8D in mice. It is a transcription factor required for kidney development, and is mutated in patients with Townes-Brocks syndrome (TBS). We have created a mouse model of TBS that expresses a truncated SALL1 protein and observed that these mice displayed a syndrome consistent with myelodysplastic syndrome (MDS) that accelerates to acute myeloid leukemia (AML). The purpose of this study was to investigate whether SALL1 is expressed in AML patients.

Materials and methods
50 bone marrow (BM) samples obtained from AML patients and 10 BM samples obtained from healthy donors were studied. BM samples were processed immediately upon receipt. Immunohistochemistry was performed on paraffin tissue sections of human BM using an anti-SALL1 antibody. RNA was extracted using the RNeasy Mini Kit (Qiagen). Quantitative RT-PCR was performed using the SYBR Green PCR Master Mix and SALL1 specific primers. SALL1 mRNA expression was normalized to RPL19, a ribosomal housekeeping gene. Western blotting was performed to confirm the presence of SALL1 at the protein level. Two AML cell lines (Kasumi-1 and -3) were also screened for SALL1 expression.

Results
44/50 human BM AML samples displayed positive nuclear staining for SALL1. Samples taken from healthy controls showed no evidence of staining. 9/ 9 human BM AML samples showed increased SALL1 mRNA levels as compared to healthy controls with upregulation of SALL1 mRNA levels ranging from 3-fold to 1630-fold. High interpatient variability was observed. On average, SALL1 expression of AML patients was 225.1-fold greater than that of healthy controls (n=9, p=0.004). Western blotting confirmed upregulated SALL1 protein expression in AML BM with no expression seen in healthy controls. No SALL1 mRNA was detected in Kasumi-1 and -3 AML cell lines.

i.) The majority of patient samples examined in this study demonstrated positive staining for SALL1. ii.) Consistent with its role as a transcription factor SALL1 histochemical staining was nuclear. iii.) SALL1 is highly expressed in human AML BM on the mRNA as well as on the protein level. iv.) No SALL1 mRNA was detected in AML cell lines.

Conclusion
Our data in mice and humans link the overexpression of SALL1 to AML. The absence of SALL1 in cell lines and its presence in the BM may indicate that SALL1 is confined to a relatively primitive cell. It is to be speculated that SALL1 has functional significance for the molecular pathogenesis of AML. Further studies to elaborate on its molecular functions and interactions are therefore clearly warranted and being conducted.

Disclosures
No relevant conflicts of interest to declare.
Genome-Wide Identification of Human Micrornas Located in Leukemia-Associated Genomic Alterations.
Authors: Starczynowski Daniel T. , Morin Ryan , McPherson Andrew , Hirst Martin , Marra Marco A. , Lam Wan L , Karsan Aly 
Published: October 2019

Abstract
Abstract 1287
Poster Board I-309
Cytogenetic alterations, such as amplifications, deletions, or translocations, contribute to myeloid malignancies. MicroRNAs (miRNAs) have emerged as critical regulators of hematopoietic processes and their aberrant expression has been associated with various leukemias. Genomic regions containing sequence alterations and fragile sites in human and mouse cancers are enriched with miRNA genes, however the potentially relevant miRNAs within these regions of genomic instability have not been evaluated on a global basis. Here we investigated miRNAs relevant to acute myeloid leukemia (AML) by: 1) mapping miRNAs within leukemia-associated genomic alterations in models of human AML by high-resolution genome arrays, and 2) evaluated absolute expression of these miRNAs by deep small RNA sequencing. We determined ∼75% (542/706) of miRNAs mapped to leukemia-associated copy-number alterations (CNA) in the cell lines, however, only 20% (99/542) of these miRNAs are expressed at levels above background. Small RNA sequencing allowed us to also identify 28 putative novel miRNAs, 18 of which map to leukemia-associated CNA in the cell lines. Our detailed genomic and small RNA analysis analysis of human leukemic cell lines has identified a subset of leukemia-associated miRNAs warranting further validation.

Disclosures
No relevant conflicts of interest to declare.
Hemophilia Activities List Questionnaire Detects Functional Limitations in Young Adults with Hemophilia A Prior to Physical Examination.
Authors: Brewer Jamie R , Harris Sandra , Zakarija Anaadriana 
Published: October 2019

Abstract
Abstract 1300
Poster Board I-322

Background
Measurement of joint range of motion (ROM) identifies hemophilia patients with arthropathy, but may not reflect functional limitations. Methods to detect early functional decline have not been well-studied, but may be important in improving the care of patients with hemophilia.

Methods
Functional status was determined by Hemophilia Activities List (HAL), a self-assessment questionnaire. Normalized scores were calculated & included activities of upper extremities (UE), basic activities of lower extremities (LE), complex activities of LE, leisure activities & summary score. Scores range from 0-100, with 0 representing poor functional status & 100 the best functional status. Joint range of motion (ROM) was measured by goniometry by a single investigator. Overall joint index is calculated as previously described. (Soucie et al, Blood 2004; 103: 2467) Percent of overall joint ROM limitation is calculated as (patient joint index score/normal joint index score) X 100. T-test was performed to assess statistical significance.

Results
45 adults with Hemophilia A were studied, 33 severe and 12 mild. Mean age was 41, range 21-65. ROM limitations & functional status differed based on disease severity and age. ROM limitations increased and functional status decreased with age among patients with severe hemophilia A (Table), but there was no change in these parameters with age in persons with mild hemophilia (data not shown). Patients with severe hemophilia had decreased functional score in all areas, with lowest score in complex LE activities. The ability to perform basic & complex tasks of LE is decreased in severe hemophilia patients in the 3rd-4th decade when compared to a cohort with mild hemophilia (p<0.001), even though there is no significant decline in LE joint ROM limitations (20% vs 15%, p=0.21).

Conclusions
Self-reported functional limitations are common in severe hemophilia. Functional decline in tasks of the LE begins as early as the third decade of life, & occurs before significant limitations in joint ROM are detected. Therefore a self-reported questionnaire can be useful in detection of early disability in adults with hemophilia. Future studies should focus on early detection and treatment interventions to prevent further functional decline.

Disclosures
No relevant conflicts of interest to declare.
Low Prevalence of Asymptomatic Neutropenia in Thailand.
Authors: Lawasut Panisinee , Nakorn Thanyaphong Na , Rojnuckarin Ponlapat , Intragumtornchai Tanin 
Published: October 2019

Abstract
Abstract 1411
Poster Board I-434

Background:
The prevalence and etiology of asymptomatic neutropenia in Asians are still unknown. Therefore, we conducted the epidemiologic study to survey the frequency and related factors of neutropenia in general Thai population.

Method:
Blood samples from healthy Thai citizens resided in Bangkok and 4 provinces from different areas were collected from October 2008-May 2009. Absolute neutrophil count (ANC) < 1.50 ×109/l was used as the cut-off point for neutropenia.[1]

Results:
Six thousand nine hundred and seventy one samples were analyzed. There were 2,422 male and 4,549 female. The median age was 49 years (range 15-93 years). The subjects from Bangkok (n=4,297) are younger and are more commonly male compared to those from non-urban areas (n=2,674) (p<0.01). The overall mean white blood cell count (WBC) and ANC were 7.57±4.07 ×109/l (mean ± 2SD) and 4.18 ±3.05 ×109/l (mean ± 2SD), respectively. The prevalence of neutropenic cases were 0.43% (30 cases, 95%CI 0.41-0.44). The mean percentage of the circulating neutrophil count was 54.58±17.25% (mean ± 2SD). It was notable that 1.87% of the participants had the circulating WBC ≥ 12.6 ×109/l. There was no influence of age, sex or survey sites on the prevalence of neutropenia. The neutropenia subjects defined as ANC lower than 2SD of the studied populations (1.13 ×109/l) were only 8 cases (0.11%). The prevalence of cases, which ANC < 1.0 and 0.5 ×109/l, were 0.03% and 0% respectively.

Conclusion:
The frequency of asymptomatic neutropenia in Thai was 0.43% defined by ANC < 1.50 ×109/l. Compared with the previous largest population report[1], the prevalence were lower than that of American white (0.79%, 95%CI 0.57-1.01), African American (4.47%95%CI 3.92-5.02) and similar to Mexican American (0.38%, 95%CI 0.24-0.52). The underlying cause of these racial discrepancies remains to be explored.
[1] Hsieh MM, Everhart JE, Byrd-Holt DD, Tisdale JF, Rodgers GP. Prevalence of neutropenia in the U.S. population: age, sex, smoking status, and ethnic differences. Ann Intern Med. 2007 Apr 3;146(7):486-92.

Disclosures:
No relevant conflicts of interest to declare.
Prevalence and Risk Factors of Monoclonal Proteinemia in Thai Population.
Authors: Watanaboonyongcharoen Phandee , Nakorn Thanyaphong Na , Rojnuckarin Ponlapat , Lawasut Panisinee , Intragumtornchai Tanin 
Published: October 2019

Abstract
Abstract 1416
Poster Board I-439

Background:
Monoclonal gammopathy of undetermined significance (MGUS) is associated with progression to multiple myeloma and related hematologic malignancies at the rate of 1% per year in western population. Reliable information on prevalence, risk factors and natural history of MGUS in general population are necessary for designing an early detection strategy for myeloma in Thailand.

Patients and methods:
The study was performed in subjects, 50 years of age or older, in Bangkok, one nearby province and 2 remote provinces of Thailand. The demographic data and suspected risk factor history were collected by questionnaires. Complete blood count as well as blood chemistry were done to exclude underlying hematologic and/or systemic conditions. Serum monoclonal proteins were detected using high-resolution gel electrophoresis.

Results:
Serum samples were obtained from 3,261 participants. There were 1,105 males (33.9%) and 2,156 females (66.1%). The median age was 57. Abnormal protein electrophoresis findings were detected in 76 samples (prevalence 2.3%, 95% confidence interval [CI] 1.8% - 2.8%) showing small M-spikes at gamma-globulin region in 50 (1.5%) or at beta-globulin region in 25 (0.8%) or hypogammaglobulinemia in 1 case (0.03%). The prevalence of MGUS in subjects less than 60, 60-69 and 70 yrs or more was 2.0% (41/1975), 2.6% (22/851) and 3.0% (13/435), respectively. Using multivariate analysis, presence of MGUS was strongly associated with history of drug abuse (odd ratio 4.63, 95%CI 1.14-22.08) and current residences outside Bangkok (odd ratio 2.30, 95%CI 1.18-4.79). Radiation and chemical exposure, hair and nail-coloring products and pesticides were not statistically significant risk factors in our population.

Conclusions:
The overall prevalence of MGUS in Thai population was 2.3%, lower than those of western countries but comparable to what reported from Japan and Taiwan.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition of AKT Signaling Potently Inhibits Growth of MPL-Mutant Cells in the Myeloproliferative Neoplasms.
Authors: Huang Zan , Koppikar Priya , Mercher Thomas , Levine Ross L. , Crispino John 
Published: October 2019

Abstract
Abstract 1896
Poster Board I-919
Somatic mutations in JAK2 and MPL are associated with BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Mutations in these genes have also been identified in rare cases of acute megakaryoblastic leukemia (AMKL). To investigate the role of activated JAK2 and MPL signaling in aberrant megakaryopoiesis associated with MPN and AMKL, we overexpressed JAK2 V617F or MPL W515L in primary murine hematopoietic progenitor cells. We found that expression of mutant MPL, but not JAK2, led to a dramatic increase in the size and number of megakaryocyte colonies. Overexpression of constitutively activated alleles of STAT3 or STAT5 did not, however, enhance megakaryocyte colony formation. Moreover, knock-down of STAT3 or STAT5 failed to inhibit the growth of JAK2 V617F or MPL W515L transduced clones of the G1ME megakaryocytic cell line. Together, these observations argue against the simple model of JAK2 and MPL mutants conferring cytokine independent growth and excessive proliferation solely by inducing STAT signaling. Since TPO is known to activate additional signaling pathways, including MAPK/ERK and PI3K/AKT, we hypothesized that alterations in pathways other than JAK/STAT contribute to MPNs. To determine which of these signaling pathways are involved, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, dramatically suppressed growth of these cells by inducing G1 arrest and apoptosis. Of note, the inhibitory effect of these two small molecules was more pronounced than that caused by JAK2 inhibitor II. Inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC all failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Since triciribine showed the strongest activity against the mutant cell lines, we next assayed its activity on a mouse model of MPL-induced AMKL, on primary murine cells expressing MPL W515L and on human MPN specimens. Twice daily injection with 0.1mg/kg of triciribine significantly delayed AMKL development in mice transplanted with 6133/MPL mutant cells. Triciribine also repressed CFU-Mk formation from MPL W515L transduced mouse bone marrow as well as BFU-E formation from human PV patient samples. These findings demonstrate that AKT is a potential target for therapy of human myeloproliferative neoplasms.

Disclosures:
Levine: Novartis: Research Funding; TargeGen: Consultancy.
Expression of the B-Cell Stimulatory Factors BAFF and APRIL in Gastric MALT Lymphomas.
Authors: Sivanathan Visvakanth , Wündisch Thomas , Kim Theo Daniel , Neubauer Andreas , Steif Johannes N. , Huynh Minh 
Published: October 2019

Abstract
Abstract 1938
Poster Board I-961

Background:
Early gastric MALT (mucosa associated lymphoid tissue) lymphoma is still dependent on antigen presentation as indicated by the remission after Helicobacter pylori (H.p.) eradication. Autocrine and paracrine secretion of B-cell stimulatory factors by inflammatory bystander cells may sustain lymphoma growth and survival. The TNF (tumor necrosis factor) familiy members BAFF (B cell- activating factor of the TNF family) and APRIL (A Proliferation Inducing-ligand) have previously been shown to be expressed in B-cell lymphomas.

Method:
We assessed the expression levels of BAFF and APRIL, as well as their receptors BAFF-R (BAFF receptor) and TACI (Transmembrane Activator and CAML Interactor) in 24 patients with early gastric MALT lymphomas by real-time quantitative PCR of snap-frozen biopsy samples. Distant biopsies with gastric MALT, but without evidence for lymphoma, from the same patients were used for intra-individual comparison. A second independent cohort of 28 patients with H.p.-associated chronic gastritis was used for inter-individual comparison. In selected cases immunohistological staining was performed on paraffin-embedded samples.

Results:
BAFF was significantly up-regulated in lymphoma tissue when compared with chronic gastritis in the inter-individual group (chronic gastritis 0,03 [standard deviation (SD)=0,21] < lymphoma tissue 0,07 [SD=0,16], Mann Whitney test (MWT) p=0,03). APRIL (chronic gastritis 24,01 [SD=19,94]) > lymphoma tissue 10,41 [SD= 6,93], unpaired t-test p=0,007) as well as TACI (chronic gastritis 16,82 [SD=6060,83] > lymphoma tissue 5,34 [SD=8,47], MWT p=0,01) were down-regulated in lymphoma tissue in comparison to the chronic gastritis tissue. No differences could be shown for the same genes in the intra-individual comparison group. No differences were found for BAFF-R between all groups. Immunohistological studies (n=5) confirmed a strong expression of BAFF and APRIL in all MALT lymphoma cases.

Conclusions:
The higher expression of BAFF in both, lymphoma and non-neoplastic gastritis, in gastric MALT lymphoma as compared to lymphoid tissue of chronic gastritis cases suggests an involvement of BAFF in the development of gastric MALT lymphoma. In addition, the lower expression of TACI as a negative regulator of B-cell development may further support the importance of B-cell stimulatory factors and their receptors. However, the absence of significant differences within a given patient with gastric MALT lymphoma indicates the necessity of additional events for full transformation.

Disclosures:
No relevant conflicts of interest to declare.
A Novel GATA1 Mutation (Ter414Arg) in a Family with the Rare X-Linked Blood Group Lu(a-b-) Phenotype.
Authors: Singleton Belinda K , Roxby David , Stirling John , Spring Frances A , Wilson Carolyn , Poole Joyce , Anstee David J 
Published: October 2019

Abstract
Abstract 1979
Poster Board I-1001
The X-borne transcription factor GATA-1 is essential for erythroid and megakaryocyte development. In 1986, Norman et al. (Vox Sang 51:49) described a large family in which the rare Lu(a-b-) blood group phenotype is inherited as the result of an X-borne gene. Serologic and flow cytometric analyses confirmed the suppression of Lutheran blood group antigens on the red blood cells from the original male propositus. Analysis of DNA from the propositus revealed a mutation (1240T>C) in the termination codon of GATA1 converting TGA to a codon for arginine (CGA). The mutation predicts a translated GATA-1 protein containing an additional 41 amino acids at the carboxy terminus. DNA from an unaffected sister of the propositus had a wild-type GATA1 sequence. The GATA1 mutation was not present in 78 random blood donors as determined by restriction fragment length polymorphism (RFLP) analysis using BspHI. The propositus, who is now 64, has an Hb of 122 g/l, a low platelet count (86 × 109/l) with occasional macrothrombocytes (diameter 4-5 um) and a history of bruising easily. Several mutations in the amino terminal zinc finger domain of GATA-1 have been linked with thrombocytopenia and thalassemia or dsyerythropoietic anemia and the presence of macrothrombocytes. Our data provide evidence that mutations outside the zinc finger domain can affect GATA-1 functions in erythroid and megakaryocyte differentiation. In this case, the predicted extended carboxy terminus may interfere with GATA-1 interactions involving other DNA-binding proteins.

Disclosures:
No relevant conflicts of interest to declare.