bluuebunny/crossref_metadata_2025 huggingface dataset:
Population Pharmacokinetics of Enoxaparin in Children at Risk for Symptomatic Thromboembolism.
Authors: Trame Mirjam Nadine , Mitchell Lesley , Male Christoph , Barrett Jeffrey S. , Hempel Georg , Nowak-Gottl Ulrike
Published: October 2019

Abstract 1071

Poster Board I-93

Introduction:

Enoxaparin, a low-molecular-weight-heparin (LMWH), is used off-label in children to prevent symptomatic thromboembolism when acute anticoagulation or secondary prevention is required due to venous thrombosis or stroke. This investigation was conducted because of concerns of altered pharmacokinetics and a lack of safety and efficacy data when used in children.

Patients and Methods:

Data of 126 children and adolescents with a median age of 5.9 years receiving enoxaparin either as a once or twice daily dosing regimen were analyzed. Children < 12 months of age received a starting dose of 1.5 mg/kg followed by a maintenance dose of 1.3 mg/kg. Children > 12 months of age were started on 1 mg/kg followed by a maintenance dose of 1 mg/kg. Blood samples were drawn after patients reached steady-state on their maintenance dose at baseline prior to the next dose, and at 2, 4, 8 and 12 hours after administration. The median enoxaparin concentration in our population resulted in a median anti-factor Xa activity of 0.4 U/ml (range 0 – 1 U/ml anti-factor Xa). By means of population pharmacokinetics using nonlinear mixed-effects modelling (NONMEM) plasma concentration-time data were analyzed. Several covariates such as age, body weight and body surface area were tested on their effects on the pharmacokinetic parameters.

Results:

Using a two-compartment model the enoxaparin kinetics were described sufficiently. By using body weight and age as covariates for clearance (CL) and central volume of distribution (V1) the best results were obtained. The final population estimates of enoxaparin resulted to be: CL 7.11 ml h-1 kg-1 ± 14.3%, V1 7.31 ml kg-1 ± 33.5%, intercompartimental clearance (Q) 194 ml h-1 ± 24.7%, peripheral volume of distribution (V2) 45.1 l ± 52.5% and absorption rate (ka) 0.0799 h-1 ± 21.7% (estimates ± standard errors). Interindividual variability (IIV) was found to be 75.6% for CL and 78.4% for Q, respectively. Figure 1 shows the predicted activity time-course versus the measured activities for a representative patient. The model is capable of describing all aging and dosing groups of our childhood population (neonates, infants to adolescents).

Conclusion:

The high IIV in CL and Q in our population underlines the need for monitoring the activity and individualizing the dose. Further population pharmacokinetic/-dynamic investigations should be conducted to predict target enoxaparin levels or other new antithrombotic drugs for more safety and efficacy during antithrombotic therapy when used in children.

Disclosures:

Off Label Use: Enoxaparin (LMWH) is used off-label in children to prevent symptomatic thromboembolism..
The Use of Angiotensin Converting Enzyme Inhibitors and Angiotensin Receptor Blockers and the Risk of Developing Venous Thromboembolism in Patients with Atherosclerotic Disease.
Authors: Chae Young Kwang , Khemasuwan Danai , Chebrolu Lakshmi , Gupta Shikha , Neagu Stefan , Carpio Alejandra , Kim Jongoh , Friedman Alan L.
Published: October 2019

Abstract 1078

Poster Board I-100

Objectives:

Arterial and venous thrombosis may share common pathophysiology involving the activation of platelets and inflammatory mediators. A growing body of evidence suggests prothrombotic effect of renin angiotensin system (RAS) including vascular inflammation and platelet activation. We hypothesized that the use of angiotensin converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs), therefore, plays a role in protecting against venous thromboembolism (VTE) in patients with history of atherosclerosis. Whether ACEIs and ARBs actually prevents VTE has never been studied in a clinical setting.

Methods:

We conducted a retrospective study, reviewing 596 consecutive patients admitted to Albert Einstein Medical Center (AEMC), Philadelphia with a diagnosis of either myocardial infarction or ischemic stroke during September 2007 to January 2009. Patients were followed up to maximum of 30 months. Patients who had been treated with anticoagulation therapy before or after the first visit at AEMC were excluded. The occurrence of VTE during the follow up period, risk factors for VTE on admission, and use of ACEIs and ARBs during the follow up period were recorded.

Results:

The mean age of the entire study population was 68.1 years. 52.0% of the patients were female and 76.5% were African American. The overall incidence of VTE was 13.4% (n=80); and 68.8% (n= 410) were on RAS inhibitors [either ACEIs only (n=348, 58.4%) or ARBs only (n=89, 14.9%) or both (n=27, 4.5%)]. Among patients on RAS inhibitors, 11.0 % (45/410) developed a VTE, compared with 18.8% (35/186) in the nonuser group [OR (Odd ratio), 0.53; 95% CI (confidence interval), 0.33 – 0.86; P=0.01]. Even after controlling for factors related to VTE (smoking, history of cancer, and immobilization, hormone use) and diabetes, the use of RAS inhibitors was still associated with lower risk of developing VTE [OR, 0.55; 95% CI, 0.34 – 0.90; P=0.02]. Although statitistically not significant due to small sample size, the OR of VTE for ACEI only users were 0.66 [CI, 0.37-1.16, p=0.15], whereas the OR for ARB only users was 0.75 [CI, 0.30-1.85, p=0.53]. Interestingly, Among patients using both ACEI and ARB, no one developed VTE (0/27), compared with 10.9% (38/348) in ACEI only users and 7.9% (7/89) in ARB only users.

Conclusions:

The use of RAS inhibitors appears to be associated with a reduction in the risk of VTE. This possible antithrombotic effect of antagonizing RAS warrants further prospective clinical investigation.

Disclosures:

No relevant conflicts of interest to declare.
Three Novel Alternative Splicing Mutations in BCR-ABL1 detected in CML Patients with Resistance to Kinase Inhibitors.
Authors: Ma Wanlong , Kantarjian Hagop , Cortes Jorge , Zhang XI , Wang Xiuqiang , Zhang Zhong , Yeh Chen-Hsiung , Sferruzza Anthony , O'Brien Susan , Albitar Maher
Published: October 2019

Abstract 1107

Poster Board I-129

The BCR-ABL1 tyrosine kinase is the target for kinase inhibitors in patients with chronic myeloid leukemia (CML). Multiple types of mutations in the BCR-ABL1 kinase domain have been reported. We previously reported a common alternatively spliced BCR-ABL mRNA with a 35-nucleotide insertion (35INS) between ABL1 kinase domain exons 8 and 9 that is expressed at various levels in CML patients with resistance to kinase inhibitors. Here we report 3 novel alternative splicing mutants expressed as the dominant transcripts in patient with CML and resistance to kinase inhibitors. We initially screened for ABL1 kinase domain mutations in approximately 200 patients with resistance to more than one of the three kinase inhibitors (imatinib, nilotinib, or dasatinib). Screening was conducted by first amplifying the fusion BCR-ABL1 transcript, to ensure that normal ABL1 was not amplified, then sequencing the ABL1 kinase domain coding region. We discovered 3 not previously described splice mutants, present in one patient each. All 3 showed >90% mutant transcript. The first resulted from the insertion of 79 nucleotides into the ABL1 exon 8-9 junction. The inserted sequence in fact contained a sequence from regions of intron 8, located 120 bp apart: the 35-nucleotide sequence previously described, and an additional 44-nucleotide segment downstream from 35INS. The combined 79-nucleotide insertion splice mutant showed the same protein change as 35INS (p C475YfsX11). The second splice mutation comprised an 84-nucleotide sequence from intron 7 inserted into the ABL1 exon7-8 junction, also causing a frameshift and protein truncation (p A424EfsX18). The third splice derived from a 231–nucleotide sequence from intron 4 retained in the ABL1 exon 4-5 junction. This insertion added 40 intron-encoded amino acids after K274, leading to a frameshift and early termination of BCR-ABL1 in the p-loop (p E275LfsX41). Exposure to kinase inhibitors may lead to the expression of alternatively spliced mRNA that is significantly elongated but causes a truncated fusion BCR-ABL1 that confers resistance to kinase inhibitors. These findings, when combined with the data on 35INS, support the concept that loss of the C-terminus of BCR-ABL1 is associated with significant resistance to kinase inhibitors; this mechanism appears to be a major source of resistance to kinase inhibitors.

Disclosures

No relevant conflicts of interest to declare.
Old Age Affects Survival but Not Response in Philadelphia Positive (Ph+) Chronic Myeloid Leukemia (CML) Patients Treated with Imatinib (IM): A Study of the GIMEMA CML WORKING PARTY.
Authors: Gugliotta Gabriele , Castagnetti Fausto , Palandri Francesca , Breccia Massimo , Amabile Marilina , Soverini Simona , Testoni Nicoletta , Marzocchi Giulia , Baldazzi Carmen , Annunziata Mario , Usala Emilio , Sica Simona , Tiribelli Mario , Bocchia Monica , Gozzini Antonella , Russo Sabina , Palmieri Fausto , Meneghini Vittorio , D'Emilio Anna , Alimena Giuliana , Martinelli Giovanni , Pane Fabrizio , Saglio Giuseppe , Baccarani Michele , Rosti Gianantonio
Published: October 2019

Abstract 1118

Poster Board I-140

Background

The median age of an unselected population of Ph+ CML patients is close to 60 years. In the prognostic classifications (Sokal, Blood 1984; Hasford, JNCI 1998) that were elaborated before the introduction of IM, age was a significant and important prognostic factor. The most recent IM studies have not clarified the prognostic importance of age and IM therapy is still denied to several elderly patients.

Aim

to asses the relationship between age (less and more than 65 years) and outcome, in CML patients treated front-line in early chronic phase (ECP).

Methods

We analyzed the data of 559 previously untreated ECP patients who were assigned to receive IM 400 mg daily (76%) or 800 mg daily (24%) in three controlled, prospective studies of GIMEMA (Clin Trials Gov. NCT00514488 and NCT00510926; and an observational study of IM 400 mg). The median follow-up is currently 42 (extremes 1 – 64) months. There were 115 patients more than 65 years old (median age 71 years), while 444 (79%) were less than 65 years (median age 46 years). The proportion of patients who were treated with IM 800 mg daily was the same in both age groups.

Results

The cumulative complete cytogenetic and major molecular response rates were identical in the two age groups (88% vs 88% and 82% vs 83%, respectively). However, overall survival (86% vs 93%, p = 0.01), failure-free survival (72% vs 81%, p=0.03) and particularly event-free survival (calculated based on the intention-to-treat principle, where events were any failure [according to the European LeukemiaNet criteria – Baccarani, Blood 2006] and treatment discontinuation for any cause) (60% vs 71%, p=0.006) were significantly inferior in the older age group. All these difference were mainly due to comorbidities leading to more deaths in CP (table).

Conclusions/Methods

These data show that response to IM was not affected by old age. Survival curves were affected because of age-related complications and comorbidities. Age should never be a contraindication to IM treatment. Acknowledgments: European LeukemiaNet, COFIN, University of Bologna and BolognAIL.

Disclosures

Saglio: Novartis: Honoraria. Baccarani:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau.
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 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 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 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 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 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 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 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 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 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.
Erythrocyte Dematin Regulates Glucose Transport Via Akt Phosphorylation and 14-3-3zeta Association.
Authors: Mohseni Morvarid , Khan Anwar , Chishti Athar H.
Published: October 2019

Abstract 1990

Poster Board I-1012

Erythrocyte dematin is a widely expressed actin-binding and bundling protein, and functions as a suppressor of RhoA signaling in fibroblasts (Mohseni and Chishti, Molecular Cell Biology 28: 4712-4718, 2008). Dematin is a substrate of multiple protein kinases, and its actin bundling activity is regulated by cAMP dependent protein kinase. Recently, we identified a novel interaction between dematin and glucose transporter-1 (GLUT1) that is critically important for erythrocyte shape and membrane mechanical properties (Khan et al., Journal of Biological Chemistry 283:14600-14609, 2008). Since homologues of dematin and GLUT1 exist in many non-erythroid cells, we proposed that a conserved mechanism might couple related sugar transporters, such as the insulin-responsive glucose transporter-4 (GLUT4), to the actin cytoskeleton via dematin. Immunocytochemistry established the presence of dematin in 3T3-L1 adipocytes, and a small pool of dematin and GLUT4-containing vesicles co-localized in 3T3-L1 cells under both basal and insulin-stimulated conditions. Plasma membrane sheet assays indicate that upon insulin stimulation, dematin translocates to the plasma membrane along with GLUT4, resulting in partial co-localization at the plasma membrane. Furthermore, dematin RNAi treated 3T3-L1 cells show reduced GLUT4 protein expression, suggesting that dematin may regulate a sub-population of GLUT4 via the lysosomal degradation pathway in adipocytes. Importantly, glucose transport was reduced by ∼28% in 3T3-L1 adipocytes depleted of dematin, and by ∼15% in the dematin headpiece knockout (HPKO) mouse primary adipocytes. Since a significant amount of dematin did not co-localize with GLUT4 in the cytosol and plasma membrane, biochemical interaction between dematin and GLUT4 could not be verified using immunoprecipitation and transfection assays. Although dematin does not bind directly to GLUT4 under these conditions, a possibility existed that this interaction may be transient and mediated through an adaptor protein. Interestingly, dematin contains seven 14-3-3 binding sites, and 14-3-3 adaptor has been shown to be functionally involved in GLUT4 trafficking. We demonstrate that phosphorylated dematin binds to 14-3-3 in 3T3-L1 adipocytes under both basal and insulin stimulated conditions. Mutagenesis studies identify serine-85 on dematin as the primary phospho-binding site for 14-3-3zeta. Furthermore, using pharmacological inhibitors, Akt is identified as the likely protein kinase that phosphorylates dematin to mediate the biochemical interactions between dematin and 14-3-3zeta. Together, our results identify erythrocyte dematin as a potential regulator of glucose transporter trafficking and degradation pathways in adipocytes with functional implications for glucose homeostasis, diabetes, and obesity.

Disclosures:

No relevant conflicts of interest to declare.
Deficiency of MnSOD in Hematopoietic Stem Cells Causes a Sideroblastic Anemia-Like Phenotype.
Authors: Case Adam John , Wagner Brett A. , Buettner Garry R. , Domann Frederick E.
Published: October 2019

Abstract 1991

Poster Board I-1013

Introduction:

Sideroblastic anemia is caused by an inappropriate incorporation of iron into hemoglobin, and as such a phenotype demonstrating ringed sideroblasts, improper iron deposition, and malignancy is observed. The only known hereditary cause of this disease is a mutation in delta-aminolevulinate synthase 2 (ALAS2), which is a mitochondrially located enzyme that catalyzes the first step in heme synthesis, while the causes of the acquired or idiopathic forms remain unknown. With the knowledge that the free radical superoxide (O2•-) is able to displace iron from heme-containing proteins, it is hypothesized that the loss of the mitochondrial anti-oxidant enzyme manganese superoxide dismutase (MnSOD), encoded by the SOD2 gene, may cause disruption of proper heme synthesis and as such may contribute to a sideroblastic anemia-like phenotype. Methods: Due to the early lethality of the constitutive MnSOD knock-out mouse, a floxed (flanked by loxP) MnSOD mouse was created. Homozygous hematopoietic stem cell specific SOD2 knock-outs were generated by breeding the floxed MnSOD mice with mice expressing Cre-recombinase under the control of the hematopoietic stem cell specific promoter Vav. Results: Mice harboring the hematopoietic knock-out of MnSOD are born viable, and are able to live to maturity. Complete blood count (CBC) analysis of knock-out mice demonstrated a decrease in red blood cells (RBC), thymic white blood cells, and splenic white blood cells. The knock-outs additionally showed a decrease in hemoglobin to 5 g/dL compared to 11 g/dL in controls, a reduction in hematocrit to 32% versus 43.5% in controls, and a reticulocyte count of 31.6% compared to only 7.9% in controls. CBC analysis also revealed many RBC morphology flags including hypochromasia, ansiocytosis, and hematocrit variance. These findings were visualized in blood smears (see below). Finally, electron paramagnetic resonance spectrometry of plasma from the knock-out mice showed a ∼2-fold decrease in transferrin levels compared to controls. Conclusions: The loss of MnSOD in the hematopoietic stem cell population of a mouse creates a viable organism, but with an apparent sideroblastic anemia phenotype. Additionally, the white blood cells within these organisms also may be affected due to the decrease in total cellularity, and thus could contribute to immunodeficiency and/or malignancy later in the animal's life. Overall, this phenotype is very comparable to those observed in human patients, and as such MnSOD may play an undiscovered role in acquired or idiopathic sideroblastic anemia. Future work will focus on further characterization of the animals, examination of human blood samples for MnSOD mutations, and the use of SOD mimetics to potentially reverse the observed phenotype. This work was supported by NIH grants CA73612 and CA115438

Disclosures:

No relevant conflicts of interest to declare.
Pediatric-Based Therapy for Young Adults with Newly Diagnosed Lymphoblastic Leukemia.
Authors: Rytting Michael , Thomas Deborah A. , Franklin Anna R , Jabbour Elias , Wierda William , Faderl Stefan , Wells Robert , Kadia Tapan , Ravandi Farhad , Ferrajoli Alessandra , Cortes Jorge , O'Brien Susan , Kantarjian Hagop
Published: October 2019

Abstract 2037

Poster Board II-14

Adolescents 14 to 21 years of age with de novo acute lymphoblastic leukemia (ALL) have improved outcomes if treated on pediatric chemotherapy regimens as opposed to adult regimens. Young adults with ALL may also benefit from chemotherapy modeled after pediatric regimens, though toxicities may be limiting. We report on 48 young adult patients between the ages of 12 to 40 years with de novo Philadelphia chromosome negative ALL treated with the augmented Berlin-Frankfurt-Munster (BFM) chemotherapy regimen. All patients have completed at least the initial 28 days of therapy (induction) consisting of high dose prednisone, pegylated asparaginase (PEG-asp), vincristine, daunorubicin and intrathecal treatments. The median age of the group was 20 yrs (14-36); 40 patients had pre-B ALL and 8 T-ALL, No infectious deaths were observed during induction. 45 (95%) patients achieved a remission by 29 days and 2 achieved remission after a 2 week extension of induction. One patient was refractory to therapy. 39 (81%) patients achieved a morphological marrow remission (<5% blasts) by day 15 of treatment (rapid early responders). Minimal residual disease (MRD) assessed by flow cytometry was negative at the end of induction for 30 (62%), positive in 10 (21%), suspicious in 6 (12%) and not available for 2 patients. In the 41 patients who completed 12 weeks of therapy, MRD was negative in 35 (85%) and positive in 6 (15%). 7 (15%) patients have relapsed or have refractory disease; 1 patient died in CR. Admission for fever of unknown origin with neutropenia occurred in 10 (21%) patients, an additional 10 (21%) patients had documented infections. Grade III-IV hepatic toxicity has been prominent: 22 (45%) increased transaminase, 14 (29%) hyperbilirubinemia. 9 (19%) patients had allergic reactions to PEG-asp, and 10 (21%) had thrombotic events. The majority of grade III-IV adverse events have been reversible. The median complete remission duration is 57 weeks (range 5-143). The overall survival at two years is 82%. In summary, this pediatric-based regimen for young adults with ALL effectively induces remission, both by morphology and by flow cytometry. Toxicity has been significant, but mostly transient and tolerable. Longer follow-up is needed to determine the long-term efficacy of this regimen in young adults with ALL.

Disclosures:

Rytting: Enzon: Speakers Bureau. Jabbour:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.
Factor Xa Dimerization and Prothrombinase Complex Formation Are Competitive Process On a Membrane Surface.
Authors: Chattopadhyay Rima , Koklic Tilen , Lentz Barry
Published: October 2019

Abstract 2123

Poster Board II-100

Exposure of phosphatidylserine (PS) molecules on activated platelet membranes is a crucial event in blood coagulation. Binding of PS to a specific site on factors Xa and on Va promotes prothrombinase assembly and enhances proteolytic activity of factor Xa. PS binding produces significant structural changes in both proteins. Recent studies demonstrate formation of inactive Xa dimer by both soluble, short-chain PS and PS-containing membranes at high Ca+2 concentration (Chattopadhyay et al.,2009, Biophys. J, 974). Association of factor Xa with factor Va in the presence of soluble PS relieves this inactivation. From kinetic measurements, we found that apparent Kd's for the Xa-Va interaction in the presence of 50 mM PS/PC (25/75) membranes increased in with increasing factor Xa concentrations, suggesting that factor Va must compete with factor Xa dimer in order to form the Xa-Va complex on the membrane surface. The fluorescence anisotropy of active site labeled factor Xa, FEGR (Fluorescein-GLU-GLY-ARG-chloromethylketone)-Xa, is lowered in the presence of PS membrane on which it forms dimer (Koklic et al., October 2009, Biophys. J., V-97, Issue 8) We report now that addition of factor Va to FEGR-Xa/membrane samples produced a saturating increase in anisotropy, clearly indicating that factor Va competed with factor Xa dimer formation. This experiment was repeated for several Ca2+ concentrations and yielded increasing apparent Kd's for Xa-Va interaction with increasing Ca2+concentration. FEGR-Xa fluorescence anisotropy as well as DEGR-Xa (factor Xa labeled at the active site with Dansyl-GLU-GLY-ARG-chloromethylketone) fluorescence intensity at varying membrane and factor Va concentrations for both 23 nm and 120 nm vesicles were analyzed globally to obtain surface Kd's of dimerization and factor Va-factor Xa interaction. This showed that membrane discreteness (slow distribution of factor Xa between discrete membrane vesicles) had almost no effect on the competition between factor Xa dimer formation and Xa-Va complex formation. We conclude that prothrombinase complex formation can overcome PS-induced factor Xa inhibition through factor Xa dimer formation at Ca2+ concentrations just above plasma concentration. In this way, one can imagine that factor Va is required to overcome factor Xa dimerization and amplify thrombin production in the early platelet plug.

Supported by USPHS grant HL072827 to BRL.

Disclosures:

No relevant conflicts of interest to declare.
Epitope Mapping of a Monoclonal Antibody against VWF Which Diminishes the Proteolytic Cleavage of VWF by ADAMTS13 Under Flow.
Authors: Zhang Jingyu , Ma Zhenni , Dong Ningzheng , Su Jian , Wang Anyou , Ruan Changgeng
Published: October 2019

Abstract 2133

Poster Board II-110

Introduction: In our former study, we have found that SZ-34, a monoclonal antibody to Von Willebrand factor (VWF), can inhibit the proteolysis of VWF by ADAMTS13 under shear stress. But the precise epitope of this antibody (SZ-34) on VWF is not clear for it is generated by immunizing mouse with native full-length VWF purified from pooled human normal plasmas. Thus, the objective of this study is to map the epitope of SZ-34 and to explore the effect of VWF structrue on the proteolytic activity by ADAMTS13. Materials and Methods: Firstly we constructed and expressed a series of recombinant proteins of different domains or polypeptide fragments of human VWF in prokaryotic cell expression system, including A1A2A3, D′D3, A1, A2, A3, A1A2, A2A3 and five sub-fragments of A2 domain. Then native VWF and these recombinant proteins or polypeptide fragments were subjected to polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blots with SZ-34. Results: Different recombinant proteins of VWF were successfully expressed and purified. Results of Western blot showed that SZ-34 could bind specifically some recombinant proteins, such as full-length VWF, A1A2A3, A2 and GST-D1459D1596 in which the last was a fusion protein of a sub-fragment of A2 domain with GST. But SZ-34 couldn't bind to others, including A1, A3, D′D3, GST-D1459E1554, GST-E1554D1596, GST-D1596R1668 (VWF73) and GST- E1554R1668. In addition, the reacting activity of SZ-34 with native VWF was significantly stronger than with unfolded VWF, such as heat-treated or 1.5M guanidine hydrochloride-treated VWF. Conclusions: The epitope of SZ-34 is located within N-terminal region fore-VWF73 inside VWF-A2 domain. Besides, SZ-34 maybe is a conformation-specific monoclonal antibody. Combining with our former findings that SZ-34 inhibits the proteolytic cleavage of VWF by ADAMTS13, we can conclude that N-terminal region fore-VWF73 inside VWF-A2 domain also regulates the proteolytic activity of VWF by ADAMTS13, although VWF73 is considered as the minimal substrate for ADAMTS13.

Disclosures:

No relevant conflicts of interest to declare.