bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Significance of Circulating Ki-67 Protein and Caspase-3 Activity Levels in Patients with Acute Myeloid Leukemia.
Authors: Bruey Jean-Marie , Kantarjian Hagop M. , Estrov Zeev , Ma Wanlong , Yeh Chen-Hsiung , Sferruzza Anthony , Estey Elihu , Albitar Maher
Published: October 2019

Abstract 3104

Poster Board III-41

Ki-67 and caspase-3 are widely accepted as proliferation and apoptosis markers in human tumors. We recently reported that Ki-67 can be detected as a circulating protein (cKi-67) in the plasma of patients with acute lymphoblastic leukemia (ALL), and that higher levels in plasma correlated with more aggressive disease. Here we investigated the levels of cKI-67 in patients with acute myeloid leukemia (AML). Since both apoptosis and proliferation are interconnected in defining the biology of a cancer, we also evaluated the level of apoptosis in AML as determined by caspase-3 activity in plasma. The study included a group of 106 newly diagnosed AML patients and 98 normal control subjects. We used the Meso Scale Discovery system for determining the levels of cKi-67. Apoptosis was determined by measuring caspase-3 activity (DEVD) in the plasma using a standard enzymatic fluorogenic assay. Median (range) levels of cKi-67 were significantly higher in patients with AML than in control subjects (1301 [0-6789] ng/mL vs 339 [35.76-2830.65] ng/mL; P<0.0001), as were caspase-3 activity levels (11.9 [5.8-218.2] pmol/min vs 8.27 [4.54-34.30] pmol/min; P<0.0001). Neither cKi-67 level nor caspase-3 activity correlated with white cell count, hemoglobin, platelet count, lactate dehydrogenase, blast count, age, cytogenetic grouping, or performance status. However, high levels (above the upper quartile of 2100 ng/mL) of cKi-67 were significantly associated with longer survival among patients >70 years of age (n=84; P=0.02) and those with unfavorable cytogenetics (n=38; P=0.006). On the other hand, high levels of caspase-3 activity (above upper quartile of 14 pmol/min) were associated with shorter survival (P=0.02) irrespective of age or cytogenetic grouping. In conclusion, proliferation and apoptosis as determined by plasma cKi-67 level and caspase-3 activity are unusual in AML. In that, higher proliferation and lower apoptosis are associated with better survival, most likely reflecting that cells progressing through the cell cycle may respond better to chemotherapy. These measurements should be considered in new therapeutic approaches that target cell cycle.

Disclosures

No relevant conflicts of interest to declare.
Hypoxia Suppresses Arsenic Trioxide-Induced Apoptosis in Human Acute Myeloid Leukemia Cells.
Authors: Yuk Yen Ju , Seo Young Jin , Kang Hyoung Jin , Jang Mi Kyoung , Lee Ji Won , Kim Hye Ry , Han Eun Jong , Shin Hee Young , Ahn Hyo Seop
Published: October 2019

Abstract 3109

Poster Board III-46

Acute myeloid leukemia (AML) is a class of prevalent hematopoietic malignancies, which often remains incurable because of the development of drug resistance. Leukemic cells originated from hypoxic condition in bone marrow that gives a benefit to leukemic cells by protecting them from anti-cancer drugs through physical barrier and by induction of some survival signal mediators. As the development of drug resistance is a key element in the failure of chemotherapy for leukemia, it has been studied about many factors make leukemic cells resist to chemotherapeutic drugs. However, effect of hypoxia on drug induced apoptosis is still poorly understood. Here, we investigated whether hypoxia affects the resistance to drugs in leukemic cells and whether hypoxia has some role in drug-induced apoptosis. To demonstrate whether hypoxia influences the apoptosis of human leukemic cells induced by chemotherapeutic drugs, the human AML cell lines (U937, HL-60, CCRF-CEM and K562) were treated with arsenic trioxide (ATO), actinomycin-D, 17-AAG, valproic acid and cytrabine under hypoxic condition and hypoxiamimetic agent cobalt chloride (CoCl2). Cellular proliferation was evaluated by methyl thiazolyl tetrazolium (MTT) assay. Subsequently, FACS analysis and western blot were performed to investigate the apoptosis related proteins. Among AML cell lines, HL-60 cells became resistant to apoptosis induced by chemotherapeutic drug, especially ATO, under the hypoxic condition. It was demonstrated through FACS analysis that level of Annexin-V staining in hypoxia (13.58%) was lower than in normoxia (29.07%). Also, among many apoptosis related molecules, activation of HIF-1 alpha under hypoxic condition was associated with the cell survival (HSP70) and apoptosis (BAX). Expression of HSP70 was increased and the level of BAX was dramatically down-regulated by HSP70 in ATO treated HL-60 cells. These data show that the hypoxia increases resistance to ATO induced apoptosis and the effect was mediated by HSP70/BAX dependent pathway. Collectively, this results provided several lines of direct evidence for the role of hypoxia on apoptosis of AML cells, in which HSP 70 and BAX elicit a role as an effector downstream to HIF-1alpha. These discoveries would shed new insights for understanding underlying mechanisms of hypoxia and designing new therapeutic strategy for AML.

Disclosures

No relevant conflicts of interest to declare.
The Potential Role of Galectin-1 in Hypoxia Induced AML Cell Differentiation through C/EBPαa Transcription Regulation.
Authors: Zhao Meng , Zhao Xu-Yun , Liao Shi-Hua , Zhao Ke-Wen , Wang Li-Shun , Chen Guo-Qiang
Published: October 2019

Abstract 3120

Poster Board III-57

Recently we reported that cobalt chloride-simulated hypoxia and mild hypoxia could induce the differentiation of human acute myeloid leukemic (AML) cells probably through hypoxia inducible factor-1 alpha (HIF-1αa) independent of its transcription activity. In this study, we used differential gel electrophoresis to compare the HIF-1αa regulated proteins between conditional induction and absence of HIF-1αa protein in U937T cells. Among all the up-regulated proteins indentified, the mRNA and protein level of galectin-1 was most remarkably changed. Knockdown of galectin-1 partially blocked the differentiation induced by HIF-1αa overexpression in U937T cells as assessed by morphological criteria and differentiation associated-antigens. Notably, reduced expression of C/EBPαa, a critical transcription factor for granulocyte differentiation, could inhibit the up-regulation of galectin-1 induced by HIF-1αa, and inducible expression of C/EBPαa in U937T cells also increased galectin-1 expression. Furthermore, by using the luciferase assay and chromatin immunoprecipitation we localized the binding site of C/EBPαa in the promoter of galectin-1. Finally the role of galectin-1 during chloride-simulated hypoxia and mild hypoxia was addressed by decreasing the expression of galectin-1 with small interfering RNA. Taken together, these results conclude that galectin-1 is required for hypoxia triggered C/EBPαa dependent AML cell differentiation.

Disclosures

No relevant conflicts of interest to declare.
Bleeding Complications of Fondaparinux Versus Nadroparine in Knee Replacement Surgery Patients.
Authors: Dijkstra Margriet , Gerritsma Carina LE , van Raay Jos JAM , van den Akker Scheek Inge , van Hulst Marinus
Published: October 2019

Abstract 3127

Poster Board III-64

An increase in bleeding complications was observed after the introduction of fondaparinux for prevention of deep-venous thrombosis (DVT) in knee-replacement surgery. 18 weeks after introduction fondaparinux was switched to nadroparine due to safety concerns.

Objectives

To investigate if fondaparinux (2.5mg once-daily starting 6 hours post-surgery) is associated with a higher rate of major bleeding compared to nadroparine (2,850ie once-daily starting 6 hours post-surgery) given for DVT prophylaxis after total knee-replacement in the Martini Hospital (Groningen, the Netherlands). Also, the rate of hematomas without clinical consequences was evaluated.

Methods

An observational study of all patients treated with fondaparinux after total knee replacement in 2008 and comparison with an equal number of patients treated with nadroparine after the switch. 181 Total knee replacements were included; 90 treated with fondaparinux and 91 treated with nadroparine. Major bleeding was defined as: allogenic blood transfusion during DVT prophylaxis; > 2g/dl hemoglobin drop; bleeding or hematoma requiring prolonged hospital stay, re-intervention or extra outpatient clinic visits. Hematomas not fulfilling the definition of major bleeding were regarded as hematomas without clinical consequences.

Results

Both groups were equal in gender, age and BMI. There was no significant difference in major bleeding (fondaparinux x vs nadroparine y, p=z). Also, no significant difference was found between both groups in: allogenic blood transfusion during DVT prophylaxis; number of patients with a > 2g/dl hemoglobin drop; rate of bleeding or hematoma requiring prolonged hospital stay, re-intervention or extra outpatient clinic visits. There was a significant difference in hematomas without clinical consequences (fondaparinux n=20 vs. nadroparine n=3, p=< 1*10-6 ).

Conclusions

We found no difference in major bleeding complications between fondaparinux and nadroparine. Significant more hematomas without clinical consequences were found for fondaparinux.

Disclosures

No relevant conflicts of interest to declare.
Enoxaparin Produces Sustained Antithrombotic Effects in Comparison to Fondaparinux and Rivaroxaban: Clinical Implications.
Authors: Jeske Walter , Masse Nicholas , Emanuele Joseph , Zhu He , McGeehan Elizabeth , Fareed Jawed , Bakhos Mamdouh , Walenga Jeanine M.
Published: October 2019

Abstract 3128

Poster Board III-65

Enoxaparin (Lovenox®), fondaparinux (Arixtra®) and rivaroxaban (Xarelto®) have been shown to be effective in mediating antithrombotic effects in post-surgical indications. Because of marked differences in the pharmacokinetic and pharmacodynamic behavior of these drugs we hypothesized that after the last dosage, the duration of the residual antithrombotic activity of the various drugs also differs. In order to compare the duration of effect of these three drugs, a rabbit stasis-thrombosis model (RSTM) utilizing FEIBA as the thrombogenic challenge and a rabbit ear bleeding model (REBM) were employed. Individual groups of rabbits (n=5) were treated with doses to mimic prophylactic, therapeutic and supratherapeutic (5x therapeutic) drug levels. Enoxaparin and fondaparinux were administered subcutaneously and rivaroxaban was administered by oral gavage. Blood samples were collected at baseline and at 16-18 hours post-administration of the last dose on day 1 or after 4 days of repeated dosing in the RSTM or at 3 hours post-administration in the REBM. At the 16-18 hour time point, circulating anti-Xa levels were not observed in any of the treatment groups. Despite this, enoxaparin treated animals exhibited a strong antithrombotic response following administration of the therapeutic dose (clot score = 1.3 ± 0.6 vs. 2.7 ± 0.6 for saline controls). Dosing once daily for four days did not enhance the antithrombotic activity of enoxaparin. In the plasmatic thrombin generation assays, a reduction in thrombin generation was noted in samples from enoxaparin-treated animals. For enoxaparin, no increase in bleeding was observed at prophylactic or therapeutic doses when compared to saline. An increase in bleeding (∼5 fold vs. saline) was observed at 3 and 6 hours post-administration of the ‘overdose’. A dose-dependent increase in anti-Xa activity was observed in blood samples collected 3 hours post-administration. For fondaparinux, no increase in bleeding was observed with any of the doses tested. There was a moderate increase in anti-Xa activity in the samples from rabbits treated with the ‘overdose’ of fondaparinux. This level was comparable to that observed with the therapeutic dose of enoxaparin. These studies suggest that enoxaparin produces a sustained antithrombotic effect in contrast to rivaroxaban and fondaparinux. These observations underscore that residual/sustained antithrombotic effects of enoxaparin may be partly responsible for the prolonged antithrombotic actions associated with the clinical use of enoxaparin.

Disclosures

No relevant conflicts of interest to declare.
Age of Blood as a Limitation for Transfusion: Potential Impact On Blood Inventory and Availability.
Authors: Fontaine Magali J. , Chung Yenho T. , Erhun Feryal , Goodnough Lawrence
Published: October 2019

Abstract 3142

Poster Board III-79

Background

Evolving concerns about storage lesions for red blood cells (RBC) and their effect on patient outcomes have led to ongoing trials evaluating the benefits of transfusing fresher blood (less than 14 days) to acutely ill patients. If improved outcomes with fresh blood are clearly demonstrated, current inventory management strategies may have to be revisited.

Methods

We evaluated several RBC maximum shelf lives (MSLs) and their impact on RBC availability. First, we determined the average age of the RBC units in our inventory based on the ABO/Rh blood type by analyzing the data set of 18,987 RBCs transfused from April 08, 2008 to April 09, 2009. Secondly, we determined the feasibility of issuing RBC of designated age to patients using the same data set.

Results

We found that 80% of the RBC inventory was delivered before 14 days of age (DoA), and that 42% versus 65% was issued before 14 DoA versus 21 DoA, respectively. RBC units spent on average 8.6 days on the shelf with a mean age at delivery of 10.2 days and a mean age at issue for transfusion of 18.8 days which varied depending on the ABO blood type as described in Table 1.

We then defined four scenarios where RBCs have different MSL: Scenario #1, #2, and #3 used a MSL of 7, 14, and 21 days, respectively. Scenario #4 used a combination of different MSLs depending on the category of patients, as follows: 7 days for 12% of patients (i.e., neonates), 14 days for 15% of patients (i.e., adult cardiovascular surgical patients), and 42 days for the 73% remaining patients receiving RBCs for transfusion. The simulations were performed using the original 18,987 data sets and results are summarized in Table 2 showing a significant decrease in the availability of RBC units by 51%, 20%, 10% and 0% for Scenario #1, #2, #3, and #4, respectively. Furthermore, the outdate rate would increase to 3.2%, 2.2%,1.0%, and 4.4% for the respective scenario.

Conclusion

Changing the maximum shelf life for red blood cell units will require novel approaches to RBC inventory management in order to meet hospital demands without risking a major shortage and putting patients at risk.

Disclosures

No relevant conflicts of interest to declare.
Covalent Antithrombin-Heparin Complex Catalysis of Activated Protein C Inhibition by Protein C Inhibitor.
Authors: Van Walderveen Maria Christina , Berry Leslie R. , Chan Anthony
Published: October 2019

Abstract 3167

Poster Board III-107

Introduction

Thrombin (IIa), when bound to thrombomodulin (TM), readily converts protein C (PC) into activated PC (APC). APC functions as an anticoagulant by inactivating activated factors V (FVa) and VIII (FVIIIa), and indirectly reduces IIa generation. Once formed, APC activity is controlled through inhibition by PC inhibitor (PCI), a reaction that is catalyzed by heparin (H). Chan et al. developed a covalent antithrombin-heparin (ATH) complex with increased anticoagulant activity compared to H. The current investigation looked at the role and mechanism by which ATH affects the inhibition of APC by PCI.

Methods

Discontinous second order rate constant inhibition assays of APC+PCI, in the presence of ATH or unfractionated H (UFH), were performed. These experiments were repeated in the presence of low molecular weight H (LMWH) or ATH (LMWATH), or a high molecular weight ATH (HMWATH). The affinity of ATH or UFH for APC or PCI was assessed using native electrophoresis and APC or PCI immobilized onto agarose beads.

Results

Second order rate experiments revealed that, at peak values, ATH (k2=2.0 × 107 ± 1.2 × 106) was a significantly slower catalyst of APC inhibition by PCI than UFH (k2= 3.0 × 107 ± 2.0 × 106; p=0.005). The peak reaction rate value for UFH occurred at a higher catalyst concentration (300nM) compared to ATH (60nM). LMWH was a poor catalyst of APC inhibition by PCI (k2= 4.2 × 105 ± 8.2 × 104). However, both LMWATH and HMWATH had high catalytic function (k2= 4.1 × 107 ± 2.1 × 106 and k2= 2.7 × 107 ± 4.8 × 105, respectively). Although UFH and ATH were able to bind to either APC or PCI at a pH of 7.3, increasing the pH to 8.8 eliminated all binding affinity, except that of ATH for APC.

Conclusion

UFH functions mechanistically through a template-mediated effect by bridging APC with PCI. The catalytic function of UFH is dependent upon H chain length, as LMWH was ineffective at enhancing APC inhibition by PCI. On the other hand, ATH may act mainly through conformational APC activation. There was no H chain length dependence for ATH, as LMWATH was able to significantly catalyze PCI inhibition of APC. We speculate that ATH's AT moiety may assist interaction with APC but prevents the H component of ATH from bridging the enzyme with the inhibitor. Compared to UFH, ATH is a more potent inhibitor of IIa, but is less effective at inhibiting APC and thereby decreasing IIa generation. These results add to the growing evidence that ATH is a superior anticoagulant to UFH.

Disclosures

No relevant conflicts of interest to declare.
Stat5 Is Essential for BCR-ABL-Transformed Chronic Myeloid Leukemia (CML) Associated with Increased CCN3 Gene Expression.
Authors: Zhu Bing-Mei , Wickre Mark , Na Risu , Klover Peter , Martin Cyril , Kimura Akiko , Hennighausen Lothar
Published: October 2019

Abstract 3271

Poster Board III-1

Introduction:

Signal transducers and activators of transcription5 (Stat5) proteins are involved in many cellular processes through mediated cytokine, hormone, and growth factor signaling. But its role in disease pathogenesis has not been fully elucidated. Recently, activation of BCR-ABL has been reported to regulate a novel gene, CCN3 in cell lines and primary cells derived from chronic myeloid leukemia (CML) patients. To investigate the function of Stat5 in CML initiation and maintenance and determine the downstream target genes on Jak-Stat5 pathway, we developed a BCR-ABL - induced CML - like disease model by using retro-viral infection in Cre-mediated Stat5 knockout transgenic mice and analyzed the progress of CML. We also used Stat5 knockout (Stat5 KO) mice to perform gene profiling. Results: Our study showed that loss of Stat5 resulted in increased survival rate and remission of CML. Microarray analysis showed that CCN3 expression was down-regulated in KL cells derived from Stat5 KO mice. BCR-ABL-activated Stat5 increased expression level of CCN3 in CML cells. We further determined that Stat5 binds to CCN3 promoter region in IL-3 stimulated 32D cells and BCR-ABL-induced CML cells. Conclusions: Our study suggested that Stat5 is essential for BCR-ABL transformed CML and that CCN3 is involved in normal hematopoiesis and CML development. Further study will be necessary to uncover the function of CCN3 and more targets of Stat5 pathway in CML development and discover the therapeutic significance.

Disclosures:

No relevant conflicts of interest to declare.
T/NK Lymphocytosis in CML Ph+ Patients During Dasatinib Therapy.
Authors: Rossi Antonella Russo , Ricco Alessandra , Carluccio Paola , Delia Mario , Leo Manuela , Casieri Paola , Stagno Fabio , Minervini Angela , Liso Vincenzo , Specchia Giorgina
Published: October 2019

Abstract 3279

Poster Board III-1

Background.

Some authors reported that Natural Killer (NK) cells from CML patients are defective in NK cell activity and NK cell number decrease as the disease progresses to the advanced phase and probably the abnormal BCR/ABL gene causes abnormal NK cell differentiation.TK inhibitors reduce BCR/ABL transcription and could restore NK cell numbers and/or function.Moreover Dasatinib,by the blockade of SRC Kinases,could affect the development of NK cells as well as T-lymphocytes.Our aim was to verify the impact of Dasatinib treatment on T CD8+ and NK cells modulation.

Methods.

We evaluated 24 patients with CML resistant/intollerant to Imatinib and treated with Dasatinib at a starting dose of 70 mg/BID or 100 mg/QD.Blood count were monitored; lymphocytosis has been definited by an increased number of peripheral blood lymphocyte counts ≥ 3.0×10(e)9/L and by the predominance of LGLs in peripheral blood smear. Immunophenotyping was done with flow-cytometry using antibodies against the following antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD16, and CD56.

Results.

With a median of 19 mo. of Dasatinib therapy (range 3–43), 15/24 cases (62.5%) developed peripheral blood lymphocytosis. Median onset of lymphocytosis was 3 months after the initiation of Dasatinib therapy (range 1–12) and duration was 14 months (range 6–40).Lymphocytosis was CD3+/CD8+/Cytotoxic T Cell in 9 patients (60%) and CD3-/CD16+/CD56+/NK Cell in 6 patients (40%).In all 15 patients no symptoms or signs suggestive of LGL leukemia or viral infections were documented.There was no significant difference in terms of the frequency of severe adverse events, including pleural effusion between patients with and without lymphocytosis. 11(73%) of the 15 patients who developed lymphocytosis achieved MMolR and 4/11 presented Bcr/Abl mutation at the time of imatinib treatment (F317L, E255K, F359V, E255K),whereas only 3(33%) of the 9 patients without lymphocytosis achieved MMolR and 1/3 presented Bcr/Abl mutation (F359V). Moreover molecular response was earlier in the group of patients with lymphocytosis (8vs12 mo.).

Conclusions.

The development of lymphocytosis in our patients seems to be associated to an improved response to dasatinib in terms of molecular response and time to response. The assessment of higher frequency lymphocytosis requires further analysis; a larger patients'cohort should be needed to explore the biological role of lymphocytosis and the impact on the long term outcome in patients treated with dasatinib.

Disclosures:

No relevant conflicts of interest to declare.
Role of Gene Mutations in Adult Acute Myeloid Leukemia Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Chou Sheng-Chieh , Tang Jih-Luh , Lin Liang-In , Hou Hsin-An , Chen Chien-Yuan , Hsu Szu-Chun , Ko Bo-Sheng , Huang Shang-Yi , Chou Wen-Chien , Yao Ming , Tsay Woei , Chen Yao-Chang , Tien Hwei-Fang
Published: October 2019

Abstract 3373

Poster Board III-261

Purpose

Several gene mutations had been found to have clinical implications in patients with acute myeloid leukemia (AML), especially in those with normal karyotype. However, the role of such gene mutations for AML patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) was unclear and inconclusive. We retrospectively evaluated the prognostic impact of 8 gene mutations in adult AML patients undergoing allo-HSCT.

Materials & Methods

From 1995 to 2007, a total of 463 consecutive adult patients with de novo non-M3 AML had comprehensive gene mutation analyses at the National Taiwan University Hospital. Three hundred and twenty five patients who received conventional induction chemotherapy were enrolled in this study. Those who received only low dose chemotherapy or palliative treatment were excluded. The genetic alterations analyzed included NPM1, FLT3/ITD, FLT3/TKD, CEBPA, AML1/RUNX1, RAS, MLL/PTD, and WT1. The clinical implication of these genetic alterations in the patients receiving allo-HSCT was analyzed, and the result was compared with that in patients without allo-HSCT.

Results

The clinical characteristics in the patients receiving allo-HSCT (n=100) and those without (n=225) were similar with the exception of age, being younger in the former group (35.4 years vs. 49.5 years p<0.001). In univariate analysis, older age (Age > 45 years), higher initial WBC count (WBC>50K/μL), elevated LDH level, unfavorable karyotype, FLT3/ITD, mutations of AML1/RUNX1 were significantly associated with poorer overall survival (OS) in patients not receiving allo-HSCT; While NPM1mut/FLT3ITDneg and CEBPA mutations served as significantly good prognostic indicators. In multivariate analysis, age, WBC count, karyotype, FLT3/ITD, AML1/RUNX1, CEBPA and NPM1mut/FLT3ITDneg remained to be independent prognostic factors in non-allo-HSCT patients. However, in patients receiving allo-HSCT, only unfavorable karyotype and disease status (refractory or remission) at the time of transplantation were associated with poorer OS both in univariate and multivariate analyses. The similar prognostic impact of FLT3/ITD, CEBPA, AML1/RUNX1 and NPM1 on OS was not seen in patients receiving allo-HSCT. Furthermore, in contrast to its poor prognostic impact in non-allo-HSCT patients, mutation of AML1/RUNX1 was a significant good prognostic factor for relapse free survival (p=0.046), although not for OS, in allo-HSCT group.

Conclusion

FLT3/ITD, mutations of AML1/RUNX1, CEBPA and NPM1 have great prognostic implication for OS in AML patients not receiving allo-HSCT. However, their impact on OS is ameliorated in patients receiving allo-HSCT. The results need to be confirmed by further studies on more patients.

Disclosures:

No relevant conflicts of interest to declare.
High CD34 Yield Not Impaired by Melphalan 10mg/m2 Test-Dosing for Pharmacogenomics in M-VTD-PACE as Induction Therapy in Total Therapy 4 (TT4) for Low-Risk and Total Therapy 5 (TT5) for High-Risk Newly Diagnosed Multiple Myeloma (MM).
Authors: van Rhee Frits , Alsayed Yazan , Cottler-Fox Michele H. , Haessler Jeff , Waheed Sarah , Nair Bijay , Crowley John , Anaissie Elias , Barlogie Bart
Published: October 2019

Abstract 3411

Poster Board III-299

Introduction:

Based on profound outcome differences in Total Therapy 3 (TT3) among patients with gene expression profiling (GEP)-defined low- and high-risk groups, TT4 for low-risk MM randomizes patients between standard and light version of TT3, whereas TT5 for high-risk MM applies dose-dense treatment sequences. Both trials employ M-VTD-PACE combination therapy for induction followed by HPC collection with the objective of collecting a minimum of 20 × 106 CD34 cells per kg body weight. The M-VTD-PACE regimen includes M (melphalan, 10mg/m2) as a test-dose after a preceding bortezomib test-dose (1.0mg/m2), each of which is followed 48hr later by GEP studies to examine prognostically relevant and drug-unique GEP alterations. There was obvious concern that the M test-dose might interfere with HPC collection. Here, we examined the CD34 yield and the days required in the first 84 patients in comparison to predecessor TT3 protocols.

Methods:

Box-plots were used to display the total CD34 collections and the CD34 collection days. Comparisons were made using the Wilcoxon Rank Sum Test.

Results:

As is evident from the appended Figure, the addition of M did not adversely affect CD34 total yield or the kinetics of procurement. Results will be presented on the engraftment kinetics and early outcome results for this first personalized medicine trial in MM.

Conclusion:

We were able to introduce M pharmaco-genomic investigations in this first-in-kind risk-directed front-line protocol for MM without compromising HPC mobilization.

Disclosures:

van Rhee: Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Bryostatin Enhances the Cytotoxic Effects of Anti-CD22 Immunotoxins in CLL by Two Distinct Mechanisms: Implications for a Sequential Therapy.
Authors: Ringshausen Ingo , Decker Thomas , Biberacher Viola , Meyer zum Buschenfelde Christian , Oelsner Madlen , Wagner Michaela , Bogner Christian , Peschel Christian
Published: October 2019

Abstract 3429

Poster Board III-317

Recently, treatment of CLL has been improved by the addition of Rituximab, a monoclonal antibody directed against CD20. Although Rituximab displays modest activity when used as monotherapy, the combination of Rituximab and chemotherapy has demonstrated impressive efficacy in CLL. However, even though remissions can be achieved in the majority of patients, CLL still remains an incurable disease for most patients. Therefore, alternative treatment options are needed. BL22 is a recombinant protein composed of the variable region of a monoclonal antibody, which binds to CD22 on the surface of normal and malignant B-cells and of PE38, a truncated Pseudomonas exotoxin. Previously, we demonstrated that BL22 induced cell death in CLL, involving the intrinsic apoptotic pathway. However, apoptosis induction correlates with expression of CD22 on the surface of CLL cells and was only moderate in CD22low expressing cells. The aim of this study was to increase BL22 cytotoxicity by modulating the surface expression of CD22 on CLL cells. Bryostatin is a macrocyclic lactone which structurally mimics the PKC-activating second messenger diacylglycerol. This PKC modulator has demonstrated anti-leukemic effects in CLL by itself in vivo. Here we show that Bryostatin up-regulates the expression of CD22 on CLL cells in a PKC dependent pathway, thereby increasing the cytotoxic effects of BL22. However, PKC-mediated up-regulation of Mcl-1 attenuated the anti-leukemic effects of BL22. We unravel that CD22 and Mcl-1 up-regulation by Bryostatin occurs in a dose dependent manner. Importantly, higher doses of Bryostatin prevented the undesirable up-regulation of Mcl-1 by inhibition of PKC in spite of CD22 up-regulation. Therefore, combined treatment of BL22 and Bryostatin seems to be a promising approach to enhance the cytotoxic effects of the immunotoxin by modulating PKC activity and CD22 up-regulation. In addition, our data provide evidence for a sequential therapy with both drugs, since CLL cells can be primed with Bryostatin. After a single dose of Bryostatin, PKC-modulation lasts for several days, allowing to administer BL22 at later time points, thereby decreasing the risk of potential drug interactions. Finally we demonstrate that stromal cell mediated drug resistance in CLL can be overcome by Bryostatin/ BL22 therapies. Conclusively, Bryostatin enhances the anti-leukemic effects of BL22 in CLL. Clinical trails are needed to prove if such an approach has clinical impact on the treatment and prognosis of B-CLL.

Disclosures

No relevant conflicts of interest to declare.
Enhanced In Vivo activation of Adoptively Transferred Genetically Targeted T Cells Following Cyclophosphamide Chemotherapy: Initial Results From a Phase I Clinical Trial Treating CLL Patients with Autologous CD19-Targeted T Cells.
Authors: Brentjens Renier J. , Hollyman Daniel , Park Jae , Santos Elmer , Yeh Raymond , Stefanski Jolanta , Taylor Clare , Weiss Mark A. , Filippa Daniel , Riviere Isabelle , Sadelain Michel W
Published: October 2019

Abstract 3436

Poster Board III-324

Patient T cells may be genetically modified to express chimeric antigen receptors (CARs) targeted to antigens expressed on tumor cells. We have initiated a clinical trial treating chemotherapy-refractory chronic lymphocytic leukemia (CLL) patients with autologous T cells modified to express the 19-28z CAR targeted to the CD19 antigen expressed on most B cell malignancies. In the first cohort of this trial, patients were infused with the lowest planned dose of modified T cells alone. All patients treated in this cohort experienced low-grade fevers following modified T cell infusion, and 2 of 3 treated patients exhibited subjective and laboratory evidence of transient reductions in tumor burden. The first patient treated on the second cohort of this study received prior cyclophophamide chemotherapy followed by the same dose of modified T cells administered to the first cohort of patients. This patient experienced persistent fevers, dyspnea, hypotension, renal failure, and died 44 hours following modified T cell infusion, likely secondary to sepsis. Modified T cells were not detectable in the peripheral blood of treated patients at 1 hour following completion of T cell infusion. However, post mortem analyses revealed a rapid infiltration of targeted T cells into anatomical sites of tumor involvement. Serum levels of the inflammatory cytokines IL-5, IL-8, and GM-CSF, but not TNFα, markedly and rapidly increased following infusion of genetically targeted T cells in this patient, mirroring the in vitro cytokine secretion profile of this patient's T cells, and consistent with marked in vivo activation of the modified T cells. Similar cytokine signatures were not found in patients from the first cohort. Significantly, serum cytokine analyses from the second cohort patient revealed a marked increase in the pro-proliferative cytokines IL-2, IL-7, IL-12, and IL-15 following cyclophosphamide therapy, in contrast to the baseline levels found in the first cohort. This report demonstrates the high efficiency trafficking of CD19-targeted T cells and in vivo activation of T cells encoding a second generation CD28/zeta chain-based chimeric antigen receptor. Furthermore, these data highlight mechanisms whereby cyclophosphamide may generate an in vivo milieu that enhances the anti-tumor efficacy of autologous tumor targeted T cells.

Disclosures

No relevant conflicts of interest to declare.
Rituximab Combining with Fresh Frozen Plasma for the Treatment of Patients with Refractory Advanced CLL.
Authors: Xu Wei , Li Jianyong , Miao Kourong , Hong Ming , Qiao Chun , Wu Yu-Jie , Fan Lei , Liu Peng
Published: October 2019

Abstract 3450

Poster Board III-338

Chronic lymphocytic leukaemia (CLL) is characterized by a progressive accumulation of monoclonal B lymphocytes in the bone marrow, lymphatic organs and blood. Even with the use of newer therapies, many patients with CLL develop progressive, treatment-resistant disease. Rituximab (RTX), a monoclonal antibody, targets CD20 on B lymphocytes and widely used in indolent B cell neoplasms. The lower density of CD20 on CLL cells may play a role, however, the causes of the lesser susceptibility of CLL to RTX remain poorly understood. Complement deficiencies have been identified in many CLL patients and appear to be more pronounced in patients with more advanced disease. Since complement mediated cell lysis is one of the major mechanisms of RTX action, we hypothesized that the therapeutic effect of RTX in CLL may be enhanced by the provision of complement through concurrent administration of fresh frozen plasma (FFP). Between August 2008 and April 2009, twelve patients with refractory advanced CLL were enrolled in this open clinical trial. All had been previously treated with fludarabine and two also failed treatment with RTX. The median age patients was 57 years (range, 48-82 years). According to the Binet staging system, 2 patients were in Binet B, 10 patients in Binet C. Florescence in situ hybridization (FISH) studies identified del(13q14) in 8 patients, and 4 patients with del(13q14) as the sole abnormality. Trisomy 12 was found in 3 patients, del(11q22.3) in 2 paitents, del(17p13) in 2 patients, del(6q23) in 1 patient, and IgH translocation in 1 patient, respectively. By flow cytometry, CD38 was positive in 9 patients, and ZAP-70 positive in 8 patients. Immunoglobulin heavy chain variable gene (IgVH) mutational status was detected by multiplex PCR, and six patients had unmutated VH. Two units of FFP followed with RTX 375 mg/m2 as a single agent, repeated every 1-2 weeks up to a total of 2-5 cycles. Complete response (CR) was achieved in 4 patients (33.3%), partial remission (PR) in 6 patients (50.0%). A major improvement of clinical signs and symptoms was achieved in all patients. Lymphocyte counts dropped markedly followed by shrinkage of lymph nodes and spleen and improvement of the anaemia and thrombocytopenia. This could be maintained over 9 months (range, 3-11 months) without progression. Toxicity was minimal and the treatment was well tolerated in all cases. It was show that RTX combining with FFP may provide a useful therapeutic option in patients with refractory advanced CLL.

Disclosures

No relevant conflicts of interest to declare.
BTG1, a Gene Frequently Deleted in Pre-B ALL, Controls Glucocorticoid Receptor-Mediated Gene Expression.
Authors: van Leeuwen Frank N. , van Galen Joost Casper , Kuiper Roland P. , van Emst Liesbeth , Levers Marloes R , Tijchon Esther , Scheijen Blanca , Waanders Esmee , van Reijmersdal Simon V , Gillissen Christian , van Kessel Ad Geurts , Hoogerbrugge Peter M.
Published: October 2019

Abstract 3458

Poster Board III-346

Background

By genome wide profiling we have found that about 10 % of pediatric pre-B ALL cases contain a (single copy) deletion of the B cell translocation gene 1 (BTG1) gene. BTG1 belongs to a family of potential tumor suppressor genes, which include BTG2, BTG3, TOB and TOB2. Proteins encoded by members of this gene family have been implicated in the induction of growth arrest or apoptosis in a variety of model systems. Moreover, BTG1 associates with and regulates the activity of the arginine methyl transferase PRMT1, a coactivator of nuclear receptor-mediated transcription. Hence we hypothesized that loss of BTG1 function, for instance due to deletion, may affect glucocorticoid induced therapy responses in ALL.

Results

Using RNA interference, we find that loss of BTG1 expression decreases sensitivity of pre-B ALL cells to the apoptosis-inducing effects of synthetic GCs about 10,000 fold (Figure). This acquired GC resistance is accompanied by a greater than 10 fold reduction in GR protein expression as well as a (near complete) loss of GC-induced gene expression. Conversely, re-expression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression. By chromatin immunoprecipitations using anti PRMT1 antibodies we show that PRMT1 is recruited to the GR gene promoter in a BTG1-dependent manner, consistent with a role for this arginine methyl transferase in the regulation of GR-mediated gene expression.

Conclusions

Together, our results demonstrate the importance of the BTG1/PRMT1 complex in regulating GR-mediated gene expression and reveal how deregulation of the this complex can give rise to GC resistance. Targeting of these coactivators as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC based therapies in ALL as well as other hematological malignancies.

Disclosures

No relevant conflicts of interest to declare.
Comparison of Differential Gene Expression Profiles of Relapsed Patients with Acute Promyelocytic Leukemia Treated with Arsenic Trioxide with the Profile of An Arsenic Trioxide Resistant NB4 Cell Line.
Authors: Chendamarai Ezhilarasi , Shaji Ramachandran , Viswabandya Auro , George Biju , Srivastava Alok , Chandy Mammen , Mathews Vikram
Published: October 2019

Abstract 3460

Poster Board III-348

Arsenic trioxide (ATO) has proven efficacy in the management of patients with acute promyelocytic leukemia (APL). Approximately 20% of patients treated with an ATO based regimen relapse. The long term remission rate with ATO in relapsed patients is significantly inferior to that seen in newly diagnosed cases. The biological mechanisms of resistance to ATO in these patients have not been evaluated. We undertook a gene expression profile study to address the potential mechanisms of resistance in patients with relapsed APL. The study included 8 patients at diagnosis and 8 patients at relapse from whom total RNA was extracted from bone marrow mononuclear cells with > 90% promyelocytes (enriched with lineage depletion cocktail as required). This was then subjected to a one color 44K Agilent cDNA array. Data was normalized, filtered and analyzed using Gene Spring software (Ver.10). Using unpaired t-Test, 1744 genes were identified to be differentially expressed (>2 fold) between samples at diagnosis and at relapse. Out of the 865 up regulated genes, the collagen family genes COL5A1 and COL2A1, haematopoietic lineage specific genes CD34 and TdT, C-Lectin family COLEC1 and TRIM15 were found to be 10 fold higher in relapse. About 880 genes were down regulated in relapse which included ABCA1, LEP, TUSC1 and XIST. We generated an ATO resistant NB4 cell line by chronic and gradual exposure to ATO over a period of 1 year. Gene expression profile of naïve and resistant NB4 cell lines arrayed in replicates were obtained. Comparison of relapsed patients and the ATO resistant NB4 cell line data revealed 51 genes were up regulated and 41 were down regulated commonly and in the same direction between these 2 groups in comparison to newly diagnosed patients and naïve NB4 cell line. By gene ontology (GO) analysis of these 92 common genes using Biointerpreter software the following biological processes were identified, namely; differentiation (47 genes), cell adhesion (14 genes), cell proliferation (17 genes), transcription factors and gene regulation (33 genes), apoptosis (11 genes), ECM remodeling (10 genes) and cell migration (5 genes). Of interest in the up-regulated gene list were anti-apoptotic gene BAG4, cell adhesion genes COL2A1 and JAM3 and differentiation gene COLEC11. The prominent down regulated genes included the ABC transporter family gene ABCA1, TNF receptor super family gene TNFRSF14 and the aldehyde dehydrogenase family gene ALDH8A1. These data provide the first evidence of the possible mechanisms of resistance to ATO in APL but needs further evaluation and validation.

Disclosures

No relevant conflicts of interest to declare.
Patients with Severe Factor XI Deficiency Have a Reduced Incidence of Venous Thromboembolism.
Authors: Salomon Ophira , Zivelin Ariella , Tamarin Ilia , Steinberg David M. , Varon David , Seligsohn Uri
Published: October 2019

Abstract 3491

Poster Board III-428

Patients with severe factor XI deficiency (level ≤15U/dL) were recently found to be protected against ischemic stroke but not against myocardial infarction (Blood 2008, 111:4113). Regarding venous thrombosis, several clinical and experimental observations suggest that factor XI deficiency might confer protection. Thus, only 2 patients with severe factor XI deficiency have been reported with unprovoked venous thromboembolism, and protection against FeCl3 Cinduced thrombosis in the vena cava was observed in factor XI deficient mice (J Thromb Haemost 2006, 4:1982). Other studies established that an increased level of factor XI is associated with an increased risk of venous thromboembolism. In the present study, we investigated whether patients with severe factor XI deficiency are protected against venous thromboembolism. We interviewed 222 unrelated patients with severe FXI deficiency and reviewed their medical charts. In the cohort, there were 126 females and 96 males aged 20-94 years of whom 137 were 50 years or older. The expected incidence rates of venous thromboembolism in the general population for males and females at different age groups were extracted from a population-based study of venous thromboembolism (J Thromb Haemost 2007,5:692). The expected incidence rate of venous thromboembolism in the factor XI deficiency cohort was 4.67 (2.37 in females and 2.30 in males), while the observed incidence rate was 0/222. This difference was statistically significant taking into account age and sex by the Poisson test (p=0.036). The prevalence of the common inherited thrombophilias was examined in 194 of the 222 patients. Seven (3.6%) patients harbored factor V Leiden, and 17 (8.8%) harbored the prothrombin G20210A mutation; the corresponding proportions in the general Israeli population are 3.9% for factor V Leiden, and 5.4% for prothrombin G20210A mutation. Thus, there was no decreased prevalence of the common thrombophilias in the factor XI deficient cohort. Collectively, the data suggest that patients with severe factor XI deficiency are protected against venous thromboembolism.

Disclosures:

No relevant conflicts of interest to declare.
Type of Factor VIII Product as Inhibitor Risk Factor in Patients with Severe Hemophilia A and Null Mutations.
Authors: Mancuso Maria Elisa , Mannucci Pier Mannuccio , Rocino Angiola , Garagiola Isabella , Tagliaferri AnnaRita , Margaglione Maurizio , Santagostino Elena
Published: October 2019

Abstract 3500

Poster Board III-437

Aim

This retrospective study evaluates the interactions of known determinants of the inhibitor risk in severe and moderate hemophiliacs followed-up at 3 Italian Hemophilia Centers.

Methods

clinical data up to inhibitor development or 150 exposure days (EDs) were collected in 393 patients with Factor VIII <5% (FVIII <1%: 331, 84%) and median age of 27 years (interquartile range, IQR:15-36) first treated with plasma-derived (pdFVIII, 70%) or recombinant products (rFVIII, 30%). Exposure to blood components for more than 4 days was an exclusion criterion.

Results

the F8 genotype was known in 334 patients (85%), 57% with null mutations (nonsense mutations, inversions and large deletions). Inhibitors (titer ≥ 0.6 BU/ml on at least two consecutive occasions and FVIII recovery <66% of expected) were detected in 112 patients (28%) after a median of 17 EDs (IQR:10-29), being high responding (HR, peak titer ≥ 5 BU/ml) in 23% and transient in 7%. Table 1 shows the crude and adjusted hazard ratios of potential determinants of inhibitor development in this cohort. Intensive treatment was defined as treatment duration ≥ 3 consecutive days at a dose ≥ 50 IU/kg/day. Sub-analyses were performed in 272 patients who never switched class of product (pdFVIII and rFVIII) and in 314 patients who did not started prophylaxis (≥ 1 regular infusion per week): in both cases the use of rFVIII at first exposure was associated with an increased inhibitor risk (adjusted hazard ratio 5.1, 95%CI 1.5-16.9 and 4.1, 95%CI 1.8-9.4, respectively). Similar results were obtained also excluding 103 patients (26%) who were first exposed to blood components (adjusted hazard ratio 3.5, 95%CI 1.5-7.9). Interestingly, patients with null mutations treated with rFVIII had a higher inhibitor risk than those first treated with pdFVIII (adjusted hazard ratio 3.3, 95%CI 1.4-7.6), while no difference was found in patients with non null mutations treated with rFVIII (hazard ratio 1.4, 95%CI 0.5-3.7). These results were confirmed including only high-responding inhibitors in the analysis.

Conclusions

our results show the influence of the interaction between type of FVIII product and F8 genotype on inhibitor risk, being the use of rFVIII associated with an higher risk in patients with null mutations. In our cohort this association was confirmed also after adjustment for the other known genetic and environmental risk factors of inhibitor development.

Disclosures:

No relevant conflicts of interest to declare.