bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of the V617F Mutation of JAK2 Using a Novel Fully Automated SNP Super-Rapid Detector.
Authors: Tanaka Ruriko , Kuroda Junya , Ashihara Eishi , Ishikawa Takayuki , Taki Tomohiko , Hirai Mitsuharu , Majima Satoshi , Taniwaki Masafumi , Maekawa Taira , Kimura Shinya
Published: October 2019

The JAK2 V617F substitution mutation (JAK2V617F) is one of the genetic hallmarks of chronic myeloproliferative diseases (CMPDs), such as polycythemia vera (PV), essential thrombocythemia (ET), or chronic idiopathic myelofibrosis (CIMF). Accurate and rapid detection of this mutation is essential for diagnosing and treating CMPDs today. We have developed a novel, rapid, sensitive and fully-automated single nucleotide polymorphism (SNP) detection system, termed ARKRAY SNP Detection System (ASDS), and used it to detect JAK2V617F in patients with CMPDs. With ASDS, diagnosis requires only 100ml of whole blood (or DNA), and the system automatically performs DNA extraction and PCR. The detection of both wild type and mutant jak2 alleles from PCR amplicons was measured by the increase in fluorescence produced by the dissociation of a JAK2V617F-specific guanine-quenching probe, and was completed within 75 minutes. In dilution assays of HEL cells (a JAK2V617F-positive leukemia cell line) using MYL cells (a chronic myelogenous leukemia (CML)-derived cell line with wild type JAK2), the system reliably quantified the mutation in a cell population containing as few as 1.0% mutant cells (Figure). We tested 44 samples from CMPDs patients using ASDS and direct sequencing (DS) (13 PV, 23 ET, 3 CIMF, 1 chronic myelomonocytic leukemia (CMMoL), 1 chronic neutrophilic leukemia (CNL), 3 unclassifiable CMPD (uCMPD)), which included samples from 3 post-allogenic bone marrow transplantation (BMT) patients with CIMF or uCMPD. Using ASDS, we detected JAK2V617F in 12/13 PV, 13/23 ET, 0/1 CIMF without BMT, 1/1 CMMoL, 0/1 CNL and 1/3 uCMPD. Overall, these results were comparable to previous results using relatively sensitive detection strategies, such as allele-specific PCR (AS-PCR). One of the 3 post-BMT CIMF samples was positive for JAK2V617F, which indicated that there were residual disease clones after BMT. ASDS detected JAK2V617F in one PV and eight ET patients, while DS failed to detect the mutation in these same samples, which clearly indicated that ASDS has a higher sensitivity than DS. JAK2V617F was absent in all samples from secondary erythrocytemia and healthy volunteers. Collectively, these results demonstrate that ASDS is a powerful and convenient tool for detecting JAK2V617F. With its associated high sensitivity, convenience and rapidity, this system will enable “Point-of-care” testing in clinical laboratories and “Patient-oriented” therapy for CMPDs. ASDS could also be applied to the detection of other point mutations relevant to cancer treatment, such as mutations in the BCR-ABL kinase domain that are associated with CML.

Figure Figure
Serum Free Light Chain Assessment in 311 Patients with Gammopathy.
Authors: Trigo Fernanda , Guimaraes Cristina , Bodas Abilia , Teixeira-Pinto Armando , Guimaraes Jose E.
Published: October 2019

Serum free light chain (FLC) levels are a useful multiple myeloma (MM) marker and a indicator of tumour burden both for diagnosis and follow up purposes. A total of 311 patient samples were assayed in our laboratory for FLC and the kappa/lambda (κ/λ) chain ratio was calculated and compared with the classical methods for characterization of gammopathy (immunofixation, IMF, immunoglobulin levels and total light chain levels and respective ratio). Ig (A, G, M) and total κ and λ chain levels were assayed by nephlometry (Dade-Behring BNII). Immunofixation was performed in a Hydrasys (Sebia) setting. FLC assay was done using Binding Site reagents (Dade-Behring). Statistical analysis was performed by SPSS® for Windows v. 15. Concordance between IMF results and free κ/λ chain ratio was calculated. Sensitivity and specificity of the free κ/λ chain ratio in the identification of positive and negative IMF were also determined. Reference intervals used for free κ/λ and total κ/λ chain ratios were [0.26; 1.65] and [1.35; 2.65], respectively. Out of 311 patients with gammopathy studied, 235 had absence of monoclonality as defined by the immunoelectrophoretic profile. Inclusively, only 51% of the 53 patients with suspected MM and 66% of the 41 patients with a diagnosis of MGUS were IMF positive. Sensitivity and specificity of total κ/λ chain ratio for identification of positive or negative IMF were respectively 70% and 91% with a global concordance of 86%. In 215 (70%) patients, IMF and free κ/λ chain ratio were in agreement. However, 74 (32%) of IMF negative patients had abnormal free κ/λ chain ratio: 18% had a final diagnosis of chronic renal failure, 13% of CLL or NHL, 9% of MGUS, 7% of MM and 3% of amyloidosis; the remainder 50% were diagnosed as having a disease other than lymphoplasmacytic disorder. These results stress the value of free light chain determination in the diagnosis and follow up of gammopathies and its usefulness as a marker for multiple myeloma and associated monoclonal gammopathies.
The Role of AKT and ERK in Regulating D-Cyclin Translation Inhibition and G1 Arrest in Multiple Myeloma Cells Treated with mTOR Inhibitors: Rationale for Combination Thereapy.
Authors: Frost Patrick J. , Shi YiJiang , Bardalaban Carolyne , Hoang Bao , Lichtenstein Alan
Published: October 2019

In a previous study, we showed that heightened AKT activity sensitized multiple myeloma (MM) cells to the in vivo anti-tumor effects of CCI-779. To test the mechanism of AKT’s regulatory role, we studied isogenic U266 MM cell lines transfected with an activated AKT allele or empty vector. The AKT-transfected cells were markedly more sensitive to cytostasis induced in vitro by rapamycin or in vivo by CCI-779. In contrast, cells with quiescent AKT were completely resistant. The ability of rapamycin and CCI-779 to inhibit D-cyclin expression was also significantly greater in AKT-transfected MM cells and this was, in part, due to a greater ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. As ERK/p38 activity can facilitate IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of rapamycin sensitivity. AKT-transfected cells demonstrated significantly decreased ERK and p38 activity, suggesting their involvement. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells while a p38 inhibitor had no effect. Furthermore, the combination of rapamycin and the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. These data support a scenario where ERK facilitates D-cyclin IRES function and heightened AKT activity down regulates this ERK-dependent phenomenon. Thus ERK and AKT activity are potential predictors of responsiveness to mTOR inhibitors.
The Cyclin Interactor p26INCA1 Regulates the Hematopoietic Stem Cell Pool Via CDK Inhibition.
Authors: Baeumer Nicole , Diederichs Sven , Koschmieder Steffen , Skryabin Boris V. , Zhang Feng , Stehling Martin , Koehler Gabriele , Berdel Wolfgang E. , Serve Hubert , Mueller-Tidow Carsten
Published: October 2019

Cell cycle progression is driven by the kinase activity of cyclin/CDK complexes. Dysregulation of the cell cycle leads to altered cell growth and contributes to tumorigenesis. Recently, we identified p26INCA1 as novel interaction partner of Cyclin A1/CDK2. Here, we characterize the phenotype of Inca1-null mice to uncover the cellular and molecular function of Inca1. Inca1-knockout mice were viable and fertile. FACS analyses revealed that aging mutant animals harbored an increased hematopoietic stem cell (HSC) pool. Bone marrow cells of young mice exhibited enhanced clonogenic replating efficiency in colony formation assays as compared to wildtype mice. Weekly administration of the myeloablative agent 5-fluorouracil (5-FU) led to a significantly shorter life span of Inca1−/ − mice compared to wildtype littermates. The increased 5-FU toxicity might thus be related to a higher number of cycling HSC in Inca1−/ − bone marrow. Analysis of the impact of Inca1 on cell cycle regulation demonstrated that the fraction of Inca1−/ − embryonic fibroblasts (MEFs) in S phase was significantly increased. Ectopic INCA1 expression reduced proliferation and colony formation of proliferating cells such as primary bone marrow cells, HeLa, HuTu80 and 32D cell lines. Serum starvation rapidly induced and mitogenic signals inhibited Inca1 expression providing a further link to cell cycle regulation. To identify the molecular mechanism of cell cycle regulation by Inca1, we investigated the influence of Inca1 on the direct inhibition of CDK2. In spleen lysates from Inca1-deficient mice, cellular CDK2 kinase activity towards Histone H1 was significantly induced compared to lysates of wildtype littermates. In in vitro kinase assays, recombinant INCA1 strongly inhibited CDK2 activity. In addition, we hypothesized that other cyclin kinase inhibitors (CKI) could partially compensate in vivo for the loss of Inca1 function. p21cip1/waf1 mRNA and protein expression were induced in Inca1−/ − MEFs compared to wildtype cells hinting at a partial compensation of the loss of Inca1 by induction of p21. Loss of Inca1 combined with p21 knockdown synergistically increased S-phase. These results indicate that Inca1 could be functionally related to p21 and that the rather mild phenotype observed in Inca1−/ − mice and the modest differences in Cdk activity observed in cell lysates lacking Inca1 could be due to compensatory induction of the CKI p21. In summary, loss of Inca1 increased cell proliferation, replating efficiency, S-phase progression, and Cdk2 activity whereas gain of Inca1 suppressed these cell functions. Inca1 expression was induced during cell cycle arrest. We conclude that Inca1 could be a novel cell cycle suppressor regulating the quiescence of HSCs through the inhibition of Cdk2.
Manipulation of Oxygen Tension and Medium Composition To Enhance CD34+ Cell Numbers and Megakaryocytic Potential of Cord Blood Progenitors.
Authors: Kidd Shannon M. , Brouard Nathalie , Cao Kevin , Robinson Simon N. , Thomas Michael , Nguyen Tuongvan , Qiao Wei , Thall Peter , Zweidler-McKay Patrick , Shpall Elizabeth J. , Simmons Paul J.
Published: October 2019

A major disadvantage of cord blood (CB) hematopoietic stem cell transplant is delayed engraftment. Cell dose has been identified as the key variable limiting neutrophil and platelet reconstitution. This has lead to the development of culture systems promoting the ex-vivo expansion of CB hematopoietic stem and progenitor cells (HSC/HPC). Typically such systems involve suspension cultures with cytokine supplementation, where the selected media supplies differing essential nutrients. Physiologically, HSCs are regulated by their interactions with the osteoblastic stem cell niche, a region characterized by low oxygen tension. We therefore investigated the effects of oxygen tension as well as differing medium composition on the ex-vivo expansion of CB HSC/HPC.

METHODS: Replilcate cultures of fresh CB CD34+ (3,000 cells/mL) cells were established at 5% (hypoxia) or 20% O2 (normoxia) in αMEM medium supplemented with 20% fetal bovine serum (FBS) or in CellGro serum-free medium. 100ng/mL stem cell factor (SCF), Fms-like tyrosine kinase 3 ligand (Flt3-L), thrombopoietin (Tpo) and granulocyte- colony stimulating factor (G-CSF) were added to each medium. The cellular output was evaluated after 7 and 14 days by counting total nucleated cells (TNC) and flow cytometric analysis measuring HSC/HPC (CD34), myeloid (CD11b), and megakaryocytic (CD41) cell progeny.

RESULTS: Cultures established under hypoxic conditions demonstrated a consistent increase in TNC (range 1.15 to 2.27-fold; N=8, p= 0.02) compared to those grown in normoxia. In addition, six of eight CB CD34+ samples showed equivalent or greater TNC production in CellGro versus αMEM/FBS. In accord with this, cultures initiated in CellGro at 5% O2 demonstrated a nearly 2-fold higher incidence (10.1% vs. 5.5%) and content (2.5 ± 0.5 x104vs. 1.3 + 0.3 x104, p=0.001) of CD34+ cells at day 7 than in αMEM/FBS. However the most striking difference between the two culture media was their capacity to support megakaryocyte differentiation in 5% O2. At day 14, a mean of 4.3% of cells cultured in CellGro expressed CD41 corresponding to a mean of 1.8± 0.3 x105 CD41+ cells/culture compared to only 0.12 ± 0.08 x105 CD41 cells (0.03% of cells) in αMEM/FBS cultures (p =0.0001). This reveals a 15-fold difference in megakaryocytic cell production. These data demonstrate that significant increases in TNC, CD34+ and CD41+ fractions can be gained by modifying oxygen tension and medium composition for ex-vivo expansion of CB progenitors. The increase in megakaryocytic cells may be of particular importance in ameliorating bleeding complications and the need for extensive platelet transfusions as a consequence of thrombocytopenia following CB transplant.

Figure Figure
Detection of the mRNA Transcription Level of Several Genes of the HIF and NO Metabolic Pathways in PBMCs of Sickle Cell Disease Patients Using Quantitative RT-PCR Assay.
Authors: Ammosova Tatyana , Xu Min , Nekhai Sergei , Kato Gregory J. , Gladwin Mark T. , Castro Oswaldo L. , Gordeuk Victor R.
Published: October 2019

Background. Pulmonary hypertension is a common complication of sickle cell disease but the pathophysiology is not completely understood.

Methods. Using quantitative RT-PCR we studied mRNA from PBMCs of seven patients with sickle cell disease having pulmonary hypertension, five patients without pulmonary hypertension and 10 controls with neither sickle cell disease nor pulmonary hypertension. We investigated transcripts of several HIF regulated genes including endothelin-1 (ET1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS), several genes of the NO metabolic pathway including cGMP-specific phosphodiesterase 5A (PDE5), soluble guanylate cyclase large subunit (sGC), and arginase 2 (Arg2), and a gene implicated in primary pulmonary hypertension, bone morphogenetic protein receptor type II (BMPR2). β-actin and diacylglycerol kinase alpha (DGKA) were used as housekeeping gene controls.

Results. HIF-regulated genes: Levels of ET1 transcripts were higher in patients with sickle cell disease and pulmonary hypertension than the other two groups while VEGF transcripts were higher in both sickle cell disease groups than controls and levels of iNOS transcripts were low and did not differ among the groups. Upregulation of ET1 is associated with the development of hypoxic pulmonary hypertension in both experimental animals and humans. NO pathway genes: PDE5, sGC, Arg2 transcripts were higher in patients with sickle cell disease and PH than the other two groups. Upregulation of PDE5 and Arg2 might be expected to contribute to reduced NO signaling. Primary pulmonary hypertension gene: BMPR2 transcripts were higher in patients with sickle cell disease and PH than the other two groups.

Discussion. These results are consistent with the possibility that complex molecular pathways, including dysregulation of HIF and NO pathways, may contribute to the development of pulmonary hypertension in sickle cell disease.
No Correlation between Thromboxane A2 Synthesis and Platelet Activation in Aspirin Treated Patients Undergoing Coronary Artery Bypass Graft Surgery.
Authors: Favreau Frederic , Jayle Christophe , Bosc Laure , Christiaens Luc , Macchi Laurent
Published: October 2019

Introduction: Aspirin usage before coronary artery bypass graft (CABG) surgery is associated with a lower risk of postoperative in-hospital mortality and appears to be safe (1). The aim of this study is to evaluate the biological effect of aspirin in these conditions. We evaluated thromboxane A2 synthesis, indirect markers of platelet activation levels and inflammatory process in aspirin treated patients undergoing CABG surgery.

Methods: Twenty aspirin treated (160 mg/day) patients were enrolled prospectively. We evaluated the association between aspirin usage for more than 1 month and urine 11-dehydro thromboxane B2 (11DHTB2) level, as well as soluble P-Selectin (sPsel) and soluble CD40 ligand (sCD40L) serum levels during the 7 days following surgery. Considering the variation of platelet count following CABG surgery and the known relation between sCD40L level and platelet mass (2), we reported sPsel and sCD40L serum levels to the platelet count. Blood and urine samples were collected at H0 (before surgery) and at H36, 72, 168 post surgery. Inflammatory process was also investigated by C-reactive protein and fibrinogen level determinations.

Results: Main data are expressed in table 1. 11DHTB2 (metabolite of thromboxane A2) increased at H36 following surgery and decreased progressively until day 7 without reaching the preoperative level. 11DHTB2 didn’t correlate with the inflammatory process (C-reactive protein and fibrinogen levels). Only concomitant increase of leukocytes count and 11DHTB2 level was observed. In the other hand, at H36 post surgery, sPsel and sCD40L levels slightly increased. Soluble P-selectin level progressively decreased until H168 while sCD40L level remain stable up to H168. At least, no correlation was found between sPsel, sCD40L and 11DHTB2.

Conclusion: These data indicated that, under aspirin treatment, a slight and transitional platelet activation occurred after CABG surgery. According to the lake of correlation between sPsel, sCD40L levels and 11DHTB2 level, we hypothesized that 11DHTB2 is produced by an another source (i.e leukocytes) which occurred independently to platelet activation.

Biological data. H0 (n = 20) H36 (n = 20) H72 (n = 20) H168 (n = 20) All data are expressed as mean ± 1SD. 11DHTB2: 11-dehydro thromboxane B2; sCD40L: soluble CD40 ligand. Platelet count (109/L) 247 ± 46 176 ± 49 175 ± 59 319 ± 89 Leukocytes count (109/L) 8.1 ± 3.1 12.5 ± 2.9 9.8 ± 4.1 9.7 ± 3.8 C-reactive protein (mg/L) 11.5 ± 20.4 113.1 ± 80 207 ± 94 87 ± 93.3 Fibrinogen (g/L) 4.2 ± 1.2 4.4 ± 1.4 7.8 ± 1.6 8.5 ± 1.9 11DHTB2 (ng/mmol creatinin) 24.5 ± 21.1 104.8 ± 36.8 42.5 ± 31.7 33.9 ± 42.7 sCD40L (ng/108 platelets) 2.3 ± 1 3.3 ± 1.1 3.5 ± 1 3.3 ± 0.9 sP-selectin (ng/108 platelets) 55 ± 34 99 ± 64 81 ± 48 59 ± 27
Complete Remission Status at Autologous Transplantation Is an Important Prognostic Factor in Multiple Myeloma Patients Received Upfront Single Autologous Transplantation.
Authors: Kim Jin Seok , Cheong June-Won , Min Yoo-Hong , Suh Cheolwon , Kim Hawk , Jo Deog Yeon , Ryoo Hun Mo , Kim Kihyun , Yoon Sung Soo , Lee Jae Hoon ,   the Korean Multiple Myeloma Working Party (KMMWP)
Published: October 2019

Upfront high dose myeloablative chemotherapy followed by single ASCT is a standard therapy for the newly diagnosed MM patients under 65 years. Disease status after the induction chemotherapy is variable, so we evaluated the prognostic effect of the disease status at ASCT especially in the initial chemo-sensitive MM patients. We retrospectively analyzed the initial chemo-sensitive MM patients (≥ PR) enrolled from KMMWP web based registration (www.myeloma.or.kr). Between Nov 1996 and Jan 2007, 197 MM patients (median age 53) were treated with induction chemotherapy followed by single ASCT at 8 institutions in Korea. All the patients received PBSCs support after conditioning with melphalan alone. Some patients with no detectable M-protein without testing immunofixation or positive immunofixation result were classified as CR. Median follow-up time was 21.2 months (0.4–96.0) from the day of ASCT. CR and PR at ASCT were achieved in 63 (32%) and 134 (68%), respectively. The initial DS stage was not correlated with disease status at ASCT, but initial low stage according to the ISS & SWOG staging system were correlated with CR status at ASCT (P=0.017, P=0.044, respectively). Overall survival from the day of ASCT (OS) was significantly correlated with CR status at ASCT comparing with PR status at ASCT (P=0.0015). OS according to the status at ASCT among the patients who received VAD induction chemotherapy (N=161) showed same results (P=0.002). CR status after ASCT also predict OS (P=0.0135), but the MM patients with continued CR after ASCT showed significantly higher OS after ASCT compared with induced CR patients after ASCT who had PR status before ASCT (P=0.0178). Multivariate analysis indicated that status at ASCT (CR versus PR) was a significant prognostic factor for prediction of OS after ASCT (P=0.012 by Cox proportional hazard analysis, OR=2.83, 95% CI, 1.25–6.37). We concluded that MM patients with CR at ASCT had better OS comparing with PR at ASCT. And continued CR status after ASCT may be a very important prognostic factor. So we should try more intensive induction chemotherapy regimens including new anti-myeloma agents to achieve maximum response status just before ASCT.

Figure. (A) OS according to status at ASCT, (B) OS according to status after ASCT. (C) OS according to status after ASCT only in the MM patients who achieved CR after ASCT (N = 147). Figure. (A) OS according to status at ASCT, (B) OS according to status after ASCT. (C) OS according to status after ASCT only in the MM patients who achieved CR after ASCT (N = 147).
Bortezomib Resistant Multiple Myeloma Cells Are Tumor Initiating Cells That Have a Stem Cell-Like Genetic Signature.
Authors: Park Juwon , Lee Chansu , Oh Jeong-In , Kim Byung-Su , Kwon Jung Hye , Kim Byoung Kook , Ahn Kwang-Sung , Yoon Sung-Soo
Published: October 2019

Abstract 2805

Poster Board II-781

Bortezomib, a proteasome inhibitor, effectively suppresses survival and growth of multiple myeloma (MM) cells. It can inhibit activation of nuclear factor-kB (NF-kB) and has been used in the treatment of MM. However, recent studies reported that patients with relapsed or refractory MM acquired resistance against bortezomib during disease progression. Therefore, we need to elucidate the molecular mechanism by which resistance emerges and this is crucial for improving treatment outcome by potentiating therapeutic efficacy of bortezomib in MM. Here, we established bortezomib resistant MM cells (U266/vel R) using soft-agar assay and identified the major molecular events that are related with acquired resistance against bortezomib. MTT assay showed that U266/Vel R cells have a growth advantage over bortezomib and the endogenous levels of pERK and pSTAT-3, -5 in U266/vel R were higher than those in parental cells. The expression of a set of genes associated with cancer stem cells was up-regulated in U266/vel R. FACS analysis showed that side population was gradually increased when U266 cells were serially exposed to bortezomib. In addition, increasing number of CD138 negative U266 cells were found in U266/ vel R cell population. It suggested that surviving cells in the presence of bortezomib could be MM cancer stem cells. Our data strongly suggest that biologic characteristics of bortezomib resistant MM cells are mediated by these potential cancer stem cells.

Disclosures:

No relevant conflicts of interest to declare.
Bortezomib Reverts Stroma-Mediated Resistant to APO2L/TRAIL in Multiple Myeloma.
Authors: Parquet Nancy , Mead Mark , Anasetti Claudio , Dalton William S. , Perez Lia
Published: October 2019

Abstract 2806

Poster Board II-782

Hematopoietic stroma provides a sanctuary for multiple myeloma (MM) cells and promotes resistance to immune control mediated by APO2L/TRAIL in part by increasing synthesis of the anti-apoptotic protein c-FLIP (J Immunol, 180: 3; 2008). Bortezomib, a reversible 26S proteosome inhibitor, sensitizes cancer cells to APO2L/TRAIL induced apoptosis in a variety of malignancies including MM. In addition, it has been well established that bortezomib interferes with the protective interaction between cancer cells and the bone marrow stroma. In this study we tested whether bortezomib can reverse the APO2L/TRAIL environmental mediated-immune resistant (EM-IR). Using MM cell lines (RPMI 8226 and U266) or CD138+ positive selected cells from MM patient's bone marrow, we found that exposure to HS5 stroma cells by direct adhesion or in a transwell system induced resistance to bortezomib (10nM for 24 hours) and that pretreatment with bortezomib (10nM for 20 hours) effectively overcomes APO2L/TRAIL resistance (10ng/mL for 4 additional hours). Conditioned medium made from a 14-day culture of MM patient's bone marrow stroma induced resistance to bortezomib and pretreatment with bortezomib sensitized cells to APO2L/TRAIL induced cell death as shown with HS5 cells. Bortezomib showed not to be cytotoxic to HS5 stroma cells and only MCP-2-3 and IL-10 levels were altered in the stroma-MM milieu within cytokines measured by ELISA array. IL-6, a cytokine shown to induce APO2L/TRAIL mediated resistance, remained unchanged with bortezomib treatment. We found that bortezomib increased expression of TRAIL receptors (DR5, DCR1 and DCR2), but such expression did not predict for sensitivity to apoptosis as DR4 demonstrated to be the receptor responsible for activation of APO2L/TRAIL. Soluble factor(s) released by HS5 stroma increased expression of c-FLIP and induced STAT-3 and ERK phosphorylation in myeloma cells. However, only c-FLIP protein expression was effectively reduced by bortezomib. c-FLIP quantitative-PCR found that HS5 increased transcription only after 20 hours bortezomib treatment and significantly reduces soluble factor(s) induced c-FLIP transcription. In a transwell assay, HS5 stroma cells induced NF-κB activation and the addition of bortezomib diminished its activation at 20 hours. These findings provide the rationale to combine bortezomib and APO2L/TRAIL to disrupt the influence of the stroma microenvironment on myeloma cells.

Disclosures:

Off Label Use: sirolimus for graft-versus-host disease.
Involvement of Ets Factor Spi-B, E Protein E2-2, and Id2 in Waldenström's Macroglobulinemia.
Authors: Zhou Yangsheng , Liu Xia , Xu Lian , Hunter Zachary , Sun Jenny , Yang Guang , Ciccarelli Bryan , Patterson Christopher J , Garrett-Sinha Lee A. , Treon Steven
Published: October 2019

Abstract 2946

Poster Board II-922

Waldenström's macroglobulinemia (WM) is an incurable B cell disorder with a lymphoplasmacytic infiltrate in the bone marrow (BM) and IgM monoclonal gammopathy. WM tumor cells show variable differentiation, ranging from mature B-cells to plasma cells, which likely results from failure to fully undergo differentiation. In this study, we analyzed the expression of several genes involved in B cell differentiation by real time RT-PCR, such as Ets factors, the basic helix-loop-helix (bHLH) E proteins, as well as the inhibitors of DNA binding (Id) proteins which antagonize E protein activity. Comparison of BM CD19+ B cells obtained from 13 WM patients with 6 age-matched healthy donors showed that expression of the Ets factor Spi-B was increased four-fold, while Id2 was decreased three-fold. However, transcript levels of E proteins were similar between the two groups. Transduction of Spi-B in BCWM.1 WM cells resulted in two-fold higher levels of Id2 and five-fold lower levels of E2-2 compared with control. Id2 transduced BCWM.1 cells expressed two-fold lower levels of E2-2 and Spi-B. Taken together, these results implicate that increased expression of Spi-B alone cannot suppress Id2 transcription in the absence of E2-2 activity. Interestingly, overexpressing Spi-B while concomitantly knocking down Id2 increased the expression of the XBP-1 splicing isoform 2.5-fold without changing levels of Blimp-1 and IRF4. Moreover, inhibition of Spi-B expression by RNA interference or forced expression of Id2 in transduced BCWM.1 cells induced a significant decrease of anti-apoptotic Bcl-2. Importantly, we also showed that Spi-B co-immunoprecipated with Blimp-1 in nuclear extracts. Collectively, these data suggest that the regulatory network of the Spi-B, E2-2, and Id2 plays an essential role in B cell differentiation as well as the pathogenesis of WM, and suggests that Spi-B overexpression may block WM cell differentiation by sequestration of Blimp-1 while promoting tumor cell survival though up-regulation of Bcl-2.

Disclosures:

No relevant conflicts of interest to declare.
High Soluble P-Selectin and Low Platelet Count as Thrombosis Risk Markers in Glioma Patients.
Authors: Vormittag Rainer , Marosi Christine , Ay Cihan , Simanek Ralph , Schwarzinger Ilse , Preusser Matthias , Elandt Katarzyna , Zielinski Christoph C , Pabinger Ingrid
Published: October 2019

Abstract 2985

Poster Board II-961

Background

Glioma patients are at high risk for venous thromboembolism (VTE). However, predictive laboratory parameters have not been identified. High platelet count (PLC) and increased soluble P-selectin (sP-selectin) have been reported as risk factors in cancer patients, so we investigated sP-selectin and PLC as risk markers in glioma patients.

Methods

The Cancer and Thrombosis Study (CATS) is a prospective observational study, whose endpoint is the occurrence of objectively confirmed VTE. sP-selectin was measured in the third week after neurosurgical intervention using a human sP-selectin Immunoassay (R&D Systems®, Minneapolis, USA). Multivariable Cox regression analysis was applied to calculate hazard ratios (HR) for VTE, including PLC, sP-selectin, age, sex and type of surgery.

Results

140 patients with newly diagnosed high grade glioma were analysed (52 women; median age 54.5 years [interquartile range (IQR): 42.8-5.1]) during a median observation time of 309 (range: 3-1664) days. Twenty patients developed VTE (6 women, 14 men), of which 2 events were fatal pulmonary embolisms. The cumulative probability of VTE was 10% at six and 15% at twelve months. sP-selectin levels (ng/mL) were higher in patients with VTE compared to those without (median=51.8, IQR: 36.9–66.0 versus median=38.8, IQR: 30.7–52.1, p=0.011). Interestingly, PLC (G/l) was significantly lower in patients with (median=214, IQR: 166-248) than in those without VTE (median=255, IQR: 200-327; p=0.011). In multivariable regression analysis high sP-selectin (75th percentile: 55.1ng/mL) and low PLC (25th percentile: 198G/L) were significant risk markers of VTE (HR=3.4, 95% CI 1.3-9.0, and HR=3.3, 95% CI 1.2-8.8, respectively).

Conclusion

Our study revealed two strong predictive markers for VTE in glioma patients. Elevated sP-selectin is associated with a three-fold increased risk of thrombosis. In contrast to patients with other solid tumours, in glioma patients low PLC is associated with increased thrombosis risk.

Disclosures:

Pabinger: Amgen Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees.
Tissue Type Plasminogen Activator Regulates Myeloid-Cell Dependent Neoangiogenesis During Tissue Regeneration.
Authors: Hattori Koichi , Ohki Makiko , Ohki Yuichi , Tashiro Yoshihiko , Nakauchi Hiromitsu , Heissig Beate
Published: October 2019

Abstract 3052

Poster Board II-1028

The fibrinolytic system comprises an inactive proenzyme, plasminogen (Plg), which is converted by activators such as tPA to the active enzyme, plasmin. Treatment with tissue type plasminogen activator (tPA) can dissolve blood clots and can ameliorate the clinical outcome in ischemic diseases (e.g. such as pulmonary embolism, myocardial infarction and stroke). Better survival in tPA-treated vs. placebo patients have been attributed to its effect on thrombus lysis. But the underlying mechanism how tPA improves ischemic tissue regeneration is not well understood. Bone marrow (BM)-derived hematopoietic cells have been shown to promote neoangiogenesis during tissue regeneration or cancer growth. Here we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin mobilizes CD45+/CD11b+ pro-angiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signalling. To study the role of tPA for cell-driven tissue regeneration, we choose a model of hindlimb ischemia. tPA improves cell incorporation of CD11b+ cells into ischemic tissues, and increases expression of neoangiogenesis-related genes including VEGF-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b+ cells and VEGFR-1+ cells, but not the same number of carrier-mobilized CD11b+ cells or CD11b− cells, accelerates neovascularization and ischemic tissue regeneration, showing that tPA administration had qualitatively changed CD11b+ cells and made them more angiogenic. Inhibition of VEGF-signalling suppresses tPA-induced neovascularization in a model of hindlimb ischemia. Thus, tPA mobilizes CD11b+ cells from the BM, increases systemic and local (cellular) VEGF-A, which can promote angiogenesis locally during ischemic recovery. We provide clinically relevant evidence that administration of the single agent tPA promotes ischemic tissue regeneration by recruiting pro-angiogenic CD11b+ myeloid cells, which qualitatively are more angiogenic then their PBS stimulated counterparts, and which incorporate with high efficiency into peripheral ischemic tissues. These data introduce a new paradigm in cell biology whereby fibrinolytic enzymes mediate systemic and localized effects on hematopoietic cells thereby modulating their tissue integration potential via integrin modulation and cytokine release and thus these data establish a novel role for tPA in the growing field of regenerative medicine.

Disclosures

No relevant conflicts of interest to declare.
A Cohort Study of Warfarin Administered to Achieve An INR of 2.0 to 3.0 for the Prevention of Recurrent Venous Thrombosis in Patients with An Antiphospholipid Antibody.
Authors: Song Madeline , Kovacs Michael J. , Crowther Mark A.
Published: October 2019

Abstract 3123

Poster Board III-60

Introduction

Despite recent evidence, controversy remains regarding the optimal method of recurrent thrombosis prevention in patients with antiphospholipid antibodies (APLA) and prior thrombosis. Specifically, although two randomized trials have supported the efficacy of “usual intensity warfarin” (administered to a target INR of 2.0 to 3.0) there remains a dearth of prospective data examining this treatment.

The aim of our study was to confirm that patients with prior thrombosis and an APLA had a low risk of recurrent thrombosis while being treated with warfarin administered to achieve an INR of 2.0 to 3.0.

Methods

We examined the annual rate of recurrent thrombosis in patients with prior thrombosis and APLA who had all been treated with OAT (oral anticoagulant therapy) to achieve a target INR of 2.0 to 3.0.

Eligible patients had to have had at least one prior objectively confirmed arterial or venous thrombosis and at least one positive standardized test for an APLA. Patients were selected from the Victoria Hospital, and follow up appointments were conducted through the hospital's thrombosis clinic. Consenting patients underwent a baseline clinical examination with the intent to be followed every six months for evidence of recurrent thrombosis, adverse consequences of anticoagulation therapy, or until death.

Results

Eighty three patients (mean age 50.5 years; 50 females) were enrolled; one patient was lost to follow up and did not contribute data to the analyses. Median follow up was 12 months (Range 5-18 months). No patients experienced a recurrent thrombotic event. 10 patients (12.2%) had a minor bleeding event (5 had significant ecchymosis, two had rectal bleeding, two had vaginal bleeding, and one had hematuria). No major bleeding events occurred. One patient had a presumed transient ischemic attack (TIA). One patient (1.2%) died during follow up due to multi-system organ failure in the context of a failing hepatic allograft.

Conclusion

Patients with previous thrombosis and an APLA have a low risk of recurrent thrombosis while having a target INR of 2.0 to 3.0. “Usual intensity warfarin” therapy appears to be an adequate method of thromboprophylaxis in these patients.

Disclosures

Crowther: BI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Leo Pharma: Consultancy, Honoraria, Research Funding; Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Artisan Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees.
Results of the EmbraceAC Trial: A Head-to-Head Comparison of Warfarin with Tecarfarin, A New Anticoagulant.
Authors: Garcia David , Milner Peter G , Canafax Daniel M , Ellis David J
Published: October 2019

Abstract 3135

Poster Board III-72

EmbraceAC was conducted to evaluate whether tecarfarin (T) is superior to optimized warfarin (W) as measured by interpolated time in therapeutic range (TTR). T is a novel vitamin K antagonist free of CYP-based metabolism.

EmbraceAC was a 6-9 month randomized, double-blind, multicenter study of optimized W vs. T using a dose control center (DCC) and prospective genotyping in oral anticoagulation patients. Data from weekly INR monitoring was analyzed on an ITT basis. A total of 612 patients from 47 sites (94.3% warfarin experienced; 74.5% treated for AF, 15.7% for DVT/PE, 14.2% for cardiomyopathy/MI, and 15.5% for heart valve) were randomized (305 to W, 307 to T). Mean interpolated TTR was 73.2% for W and 74.0% for T (p, 0.51). Pre-specified subgroup analyses of warfarin experienced patients, wild-type CYP2C9 patients, and on-treatment analysis all showed statistically significant superiority of T over W. For the 31% of patients taking drugs that inhibit CYP2C9, the observed TTR for W decreased, whereas the TTR for T remained steady. There was no significant difference in the composite of clinical outcomes (death, stroke, MI, DVT, PE, or major hemorrhage): 6 for W and 8 for T (p, 0.41). Tolerability was good with both treatments and there were no differences in overall AE rate. TTR for W was exceptionally high, perhaps as a result of frequent monitoring, availability of accurate and timely genotype information, close surveillance by the DCC and early dose adjustment for potential interacting drugs. Statistical superiority of T was not established in the primary end point. As hypothesized for a drug designed to avoid CYP metabolism, T showed a significant advantage over W for patients exposed to CYP2C9 inhibitors.

DG - Consultant to Bristol Meyers Squibb, CSL Behring, ARYx Therapeutics; PM, DC, DE - ARYx Therapeutics employees.

Disclosures

Garcia: Bristol Meyers Squibb: Consultancy; CSL Behring: Consultancy; ARYx Therapeutics: Consultancy. Milner:ARYx Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Canafax:ARYx Therapeutics: Employment, Equity Ownership. Ellis:ARYx Therapeutics: Employment, Equity Ownership.
Use of Rituximab in Patients with Congenital Bleeding Disorders and High Titre Inhibitors.
Authors: Curtin Julie , Teo Juliana , Shoemark Robyn , Tatjana Kilo , Harris Catherine , Webster Boyd , Lammi Ahti
Published: October 2019

Abstract 3501

Poster Board III-438

The development of high titre inhibitors remains a serious complication of treatment in patients with congenital bleeding disorders. We describe a single centre experience of the use of Rituximab (monoclonal anti-CD20 antibody) in 10 patients with congenital bleeding disorders and high titre inhibitors. Nine of the patients have severe haemophilia A (Factor VIII <1%) and 1 patient has type 3 von Willebrand disease (VWD). Six patients had received previous immune tolerance therapy with various regimens prior to receiving Rituximab. Four patients with risk factors predictive of poor response to tolerisation received Rituximab as part of their initial therapy. Three of these four patients received additional immunosuppression (including intravenous immunoglobulin (IVIG) & prednisone (n=2), and IVIG, prednisone and vincristine (n=1)). Patients received cycles of weekly doses of 375 mg/m2 over 2 – 4 weeks. Four patients responded after one cycle of Rituximab therapy. The remaining 6 patients received repeated cycles of Rituximab with the frequency of cycles being between three and 12 months. These patients have to date received between 2 and 13 cycles of Rituximab over a period of up to 6 years. All patients have responded with a fall in their inhibitor titre and improvement in factor recovery. Five patients had a complete response so that they can be managed with second daily factor prophylaxis. One of these patients (the type 3 VWD patient) had a relapse 12 months after his initial response to Rituximab. He has responded to a second course of 4 doses of Rituximab. Three of the remaining five patients remain on immune tolerance induction (ITI) between 8 months and 6 years after starting Rituximab therapy. All of these patients have improved factor VIII recovery with marked reduction in bleeding frequency. Of the other two patients, 1 patient moved interstate and was no longer treated with Rituximab and the other had ITI stopped after 15 months because of poor compliance with daily factor therapy. At this time his inhibitor was undetectable and he had improved factor recovery and survival. The Rituximab was well tolerated in all patients. Four patients developed hypogammaglobulinaemia, 1 patient developed transient neutropenia, three patients had port infections, though the frequency of infection was no greater than before Rituximab therapy. This single centre of experience shows that repeated doses of Rituximab are well tolerated and has a role in the tolerisation of patients with poor response to more traditional methods of ITI.

Disclosures:

Off Label Use: Rituximab is a monoclonal antibody against CD20 that is being used for its immunomodulatory properties.
Improvement In Venous Thromboembolism (VTE) Prophylaxis In Hospitalized Cancer Patients with a Continuing Medical Educational Program In a University Hospital In South America
Authors: Conte Guillermo , Figueroa Gaston , Aranda Sandra , Araos Daniel , Gonzalez Nestor , Torres Carlos , Picarte Javier
Published: October 2019

Abstract 2565

Introduction:

Cancer patients have a 4 to 6-fold higher risk of suffering Venous Thromboembolism (VTE), the second cause of death in hospitalized cancer patients. These patients are considered as high risk for VTE by the ACCP guidelines, so the pharmacological prophylaxis is indicated if there is no contraindication. A study carried out at our hospital between 2003 and 2004 showed a low use of thromboprophylaxis in this group of patients. The implementation of diverse strategies may improve this prophylaxis. A continuing medical education program was introduced to medical interns and internal medicine residents between 2005 and 2009 with this purpose. The objective of this study was to evaluate changes in the use of thromboprophylaxis in cancer patients hospitalized between 2008 and 2009.

Methods:

A six-month seminar program was implemented for medical interns and internal medicine residents enhanced by daily visits to the patient rooms by the medical staff who supervised the correct use of thromboprophylaxis.

The results were based on a retrospective analysis of clinical data of medical and surgical patients diagnosed with cancer hospitalized at the University of Chile Clinical Hospital in the 2003–2004 period (Group A) and in the period 2008–2009 (Group B) (Table 1).

Exclusion Criteria at Admission: VTE diagnosis, use of anticoagulants, thromboprophylaxis contraindication and age <18 years.

Conclusion:

The risk for VTE is very high in hospitalized cancer patients and is associated with an increased risk of recurrent thrombosis. Several international medical organizations (NCCN, ASCO, ESMO, ASH) call for improving thromboprophylaxis in this high-risk population.

The introduction of an active medical educational program addressed to medical interns and internal medicine residents resulted in a significant increase in the proportion of hospitalized cancer patients who received appropriate prophylaxis in accordance with the ACCP guidelines from 65.8% at the start of the study to 86.4% (p< 0.001) after 24 months.

Disclosures:

No relevant conflicts of interest to declare.
ATG Use Is Associated with Frequent Occurrence of Positive Blood Cultures in Patients in the Early Phase After Hematopoietic Stem Cell Transplantation
Authors: Seweryn Marek , Jarosz Urszula , Krawczyk-Kulis Malgorzata , Markiewicz Miroslaw , Helbig Grzegorz , Bober Grazyna , Kyrcz-Krzemien Slawomira
Published: October 2019

Abstract 4564

Background:

Infectious complications remain an important cause of morbidity and mortality in the early phase after hematopoietic stem cell transplantation (HSCT).

Aim:

The aim of this study was to assess the frequency of positive blood cultures and its potential correlation with different studied parameters in large patient population studied in the first 30 days after HSCT.

Material and methods:

431 patients at median age of 47 years (range 18–85) transplanted between 2009–2011 for hematological and non-hematological malignancies were included in our analysis. There were 242 males and 189 females.

Results:

The indications for autologous and allogeneic HSCT were following: AML – 105 (24%), NHL – 86 (20%), MM – 75 (17,5%), HL – 48 (11%), ALL – 40 (9%), MDS – 17 (4%), AA – 15 (3,5%), CML – 12 (2,8%), PNH – 11 (2,6%), connective tissue diseases – 5 (1,2%), CLL – 3 (0,7%) and other – 14 (3,2%). The following transplant procedures were performed: ABCT – 213 (49%), ABMT – 3 (0,7%), alloBCT – 56 (13%), alloBMT – 21 (5%), URDBCT – 87 (20%), URDBMT – 51 (12%). Pre-transplant ATG and anti-CD52 antibody were used in 142 (33%) and 5 (1.2%) patients, respectively. Amongst 431 transplanted patients, 495 blood cultures were collected; range 0–8 (median 1). Eighty seven blood samples were positive (17,6%). The following pathogens were detected: gram-positive bacteria in 48% (n=42), gram-negative bacteria in 38% (n=33), fungi in 1% (n=1) and both G(+) and G(−) bacteria in 13%(n=11). The gram-positive bacteria included: Staphylococcus epidermidis: 21 (50%), Micrococcus spp: 4 (9%), Enterococcus faecium: 3 (7%), Enterococcus faecalis: 3 (7%), Streptococcus haemolyticus: 3 (7%). The following gram-negative bacteria were found: Enterobacter cloacae: 10 (30%), Escherichia coli: 7 (21%), Pseudomonas aeruginosa: 5 (15%), Klebsiella pneumonia: 5 (15%). Candida albicans was detected only in one case. The use of ATG was associated with higher number of total blood draw and positive blood cultures. No significant correlation was found between the specific pathogen and the use of ATG. Male gender was associated with significantly higher number of blood sampling and with tendency to higher number of positive blood cultures. The type of conditioning regimen, the source of stem cell and the donor origin (auto vs sibling vs unrelated) did not influence the number of positive blood culture. There was tendency to higher number of blood intake, but not positive blood culture in patients transplanted in NR if compared to PR or CR.

Conclusions:

Positive blood cultures were positive in about 20% of patients after HSCT. Only pre-transplant ATG use was associated with the higher number of positive blood culture.

Disclosures:

No relevant conflicts of interest to declare.