bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of the Novel Non-Quinone Based HSP-90 Inhibitor PU-H71 in JAK2V617F and MPLW515L-Induced Murine Models of Myeloproliferative Neoplasms.
Authors: Koppikar Priya , Marubayashi Sachie , Taldone Tony , Abdel-Wahab Omar , West Nathan , Gozman Alexander , Ahn James , Rodina Anna , Bhagwat Neha , Hedvat Cyrus V. , Bradner James E. , Chiosis Gabriela , Levine Ross L.
Published: October 2019

Abstract 3898

Poster Board III-834

The discovery of mutations in the JAK-STAT signaling pathway in the majority of patients with myeloproliferative neoplasms (MPN) has led to the development of JAK2 kinase inhibitors for the treatment of these disorders. Although JAK2 inhibitors demonstrate efficacy in preclinical models and in early phase clinical trials, to date, JAK2 inhibitor treatment has not resulted in molecular responses or in improvements in blood counts. We therefore have investigated the effects of additional therapies that might provide benefit to patients with myeloproliferative disorders, including Hsp90 inhibition, which has been shown to abrogate oncogenic signaling pathways in other human malignancies. We analyzed the effect of PU-H71, a novel non-quinone based Hsp-90 inhibitor, in MPN cell lines, primary patient samples, and in animal models. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth in isogenic cell lines expressing JAK2/MPL mutations, JAK2V617F-positive leukemia cell lines, and primary MPN patient samples, which was associated with induction of apoptotic cell death at clinically achievable concentrations. Further, we observed JAK2 degradation in cell lines and primary samples with PU-H71 treatment, and immunoprecipitation experiments documented association of JAK2 with HSP90 and with PU-H71, demonstrating that JAK2 is a client of the HSP90 chaperone complex. PU-H71 potently inhibited downstream signaling pathways, including STAT signaling, MAPK signaling, and AKT signaling in JAK2/MPL positive cell lines and primary samples. Most importantly, in vivo therapy with PU-H71 in mice expressing JAK2V617F or MPLW515L normalized peripheral blood counts, attenuated extramedullary hematopoiesis, and improved survival compared to vehicle treated mice. We observed reduction in total JAK2 expression in target organs from PUH-71 treated mice, and noted in vivo inhibition of signaling pathways in a manner analogous to in vitro studies. Taken together, these data indicate that Hsp90 inhibition, either alone or in combination with JAK2 inhibitors, may prove useful against human MPN.

Disclosures:

Levine: Novartis: Research Funding; TargeGen: Consultancy.
Prognostic Significance of Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia (GCH) and Lymphomas of Germinal Center Origin.
Authors: Paz-Carreira Jose M , Garcia-Rivero Arantxa , Losada Raquel , Mendez Jose C , Albors Manuel , Miras Fatima , Alvarez Augusto , Bal Fernando , Alba Jesus , Deben Guillermo
Published: October 2019

Abstract 3950

Poster Board III-886

INTRODUCTION

Germinal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. GC have been known to be a major source of B-cell lymphomas, including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCBLs. Much less is known about the role of methylation in GCH. We determined the methylation status of 5 tumor-suppressor genes in 50 patients with lymphoma of GC origin and 50 GCH in order to find any differences between the pathological and the physiological state as well as its prognostic significance. MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 30 DLCL, 20 FL and 50 GCH were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14 and MGMT. Methylation status of each gene was correlated with clinicopathological status. Overall survival (OS) rates were calculated by the Kaplan-Meier method and differences were compared with the log-rank test. RESULTS. Median age was 65 in patients with lymphoma and 19 for GCH. Sex distribution was similar in all entities (60% females). Both lymphoma groups were balanced with respect to the presence of B symptoms, bulky disease, bone marrow infiltration, advanced stage and high IPI/FLIPI. DAP-k promoter methylation was present in more patients with lymphoma (89 and 87%) than with BFH (37%) p<0.0001. RaRB was methylated with higher frequency in FL (60%) than in DLCL (23%) and FH (12%) p<0.0001. SHP1 was more frequently methylated in FL (67%) and GCH (58%) than in DLCL (20%) p=0.01. Promoter hypermethylation of SHP1 was significantly associated with longer OS (p=0.021). Methylation of RaRB, p14 and MGMT were associated with shortened OS but the differences were not statistically significant. Those patients with DAPK methylated live longer but not significantly. In multivariate analysis hypermethylation of none of the genes studied remained an independent prognostic factor. CONCLUSIONS. Inactivation of DAP-K, and Rarβ is present in GC lymphomas with significantly higher frequency than in BFH. Thus, it may have pathogenic significance. SHP1 is methylated more frequently if FL and BFH than in DLCL, therefore that gene may be associated with aggressive disease. Methylation of DAP-k, SHP1, Rarβ, p14 and MGMT has no significant impact on overall survival. Markers for aberrant methylation may represent a promising way to monitor the onset and progression of malignancies but more extensive and prospective trials are needed to precisely define its role.

Disclosures:

No relevant conflicts of interest to declare.
ETO2 Controls Hematopoietic Stem Cell Expansion.
Authors: Barakat Stephane , Lambert Julie , Sauvageau Guy , Hoang Trang
Published: October 2019

Abstract 396

Hematopoietic stem cells that provide short term reconstitution (ST-HSCs) as well as hematopoietic progenitors expand from a small population of long term hematopoietic stem cells (LT-HSCs) that are mostly dormant cells. The mechanisms underlying this expansion remain to be clarified. SCL (stem cell leukemia), is a bHLH transcription factor that controls HSC quiescence and long term competence. Using a proteomics approach to identify components of the SCL complex in erythroid cells, we and others recently showed that the ETO2 co-repressor limits the activity of the SCL complex via direct interaction with the E2A transcription factor. ETO2/CBF2T3 is highly homologous to ETO/CBFA2T1 and both are translocation partners for AML1. We took several approaches to identify ETO2 function in HSCs. We initially found by Q-PCR that ETO2 is highly expressed in populations of cells enriched in short-term HSC (CD34+Flt3-Kit+Sca+Lin-) and lympho-myeloid progenitors (CD34+Flt3+Kit+Sca+Lin-) and at lower levels in LT-HSCs (CD34-Kit+Sca+Lin- or CD150+CD48-Kit+Sca+Lin-). Next, the role of ETO2 was studied by overexpression or downregulation combined with transplantation in mice. Ectopic ETO2 expression induces a 100 fold expansion of LT-HSCs in vivo in transplanted mice associated with differentiation blockade in all lineages, suggesting that ETO2 overexpression overcomes the mechanisms that limit HSC expansion in vivo. We are currently testing the role of the NHR1 domain of ETO2 in this expansion. Conversely, shRNAs directed against ETO2 knock down ET02 levels in Kit+Sca+Lin- cells, causing a ten-fold decrease in this population after transplantation, associated with reduced short-term reconstitution in mice. Finally, proliferation assays using Hoechst and CFSE indicate that ETO2 downregulation affects cell division (CFSE) and leads to an accumulation of Kit+Sca+Lin-cells in G0/G1 state (Hoescht). In conclusion, we show that ETO2 is highly expressed in ST-HSCs and lymphoid progenitors, and controls their expansion by regulating cell cycle entry at the G1-S checkpoint. In addition, ETO2 overexpression converts the self-renewal of maintenance into self-renewal of expansion in LT-HSCs.

Disclosures:

No relevant conflicts of interest to declare.
Myeloproliferative Disease or Myeloid Leukemia Caused by Cbl Mutants in a Mouse Transplantation Model.
Authors:   Bülent Sargin on behalf of the Study Alliance Leukemias (SAL), Bandi Srinivasa Rao , Rensinghoff Marion , Grundler Rebekka , Tickenbrock Lara , Köhler Gabriele , Duyster Justus , Berdel Wolfgang E , Serve Hubert , Brandts Christian , Müller-Tidow Carsten
Published: October 2019

Abstract 3973

Poster Board III-909

Purpose

Somatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold for several signaling adaptor molecules. We recently identified the first c-Cbl mutation in human disease in an AML patient, called Cbl-R420Q.

Results

We transduced primary murine bone marrow retrovirally with c-Cbl mutants and transplanted it into lethally irradiated mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. Eleven out of 13 mice and 8 out of 11 mice died in the Cbl-R420Q group and Cbl-70Z group, respectively. Two animals succumbed to a myeloid leukemia, the other mice developed a myeloproliferative disease. The leukemic mice showed a leukocytosis of up to 140.000/μL. They developed a splenomegaly with massive expansion of myeloid cells in liver and spleen. Histology sections of spleen, liver and bone marrow and FACS analyses of spleen, bone marrow and peripheral blood showed extensive infiltration of myeloid cells.

Conclusion

Thus, transplantation of bone marrow cells expressing Cbl mutants leads to a myeloid leukemia or to a myeloproliferative disease with long latency and high penetrance.

Disclosures:

No relevant conflicts of interest to declare.
Promyelocytic Leukemia-Retinoic Acid Receptor α Binds and Prevents Activation of the Fas Receptor and Suppresses Fas-Mediated Apoptosis.
Authors: Tao Rong-Hua , Berkova Zuzana , Wise Jillian , Daniluk Urszula , Samaniego Felipe
Published: October 2019

Abstract 3976

Poster Board III-912

Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas killing observed in some normal and cancer cells, we have screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified a group of peptides derived from promyelocytic leukemia (PML). PML enhances pro-apoptotic signaling, while the promyelocytic leukemia–retinoic acid receptor α (PMLRARα) activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα, which typically operates in a dominant-negative manner, blocks Fas-mediated apoptosis. Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937-PR9 cells and in APL primary cells. The binding of PMLRARα to Fas was mapped to the B-box domain of PMLRARα. Flow cytometry analysis of propidium iodide-stained and Annexin-V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody (CH-11) indicated that the presence of PMLRARα was associated with blocked Fas-mediated apoptosis at early and late stages. The knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Expression of PMLRARα in U937-PR9 cells prevented Fas-mediated cleavage of procaspase-8 and also prevented procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11). Further analysis indicated that PMLRARα bound to FLIPL/S and forms an apoptotic inhibitory complex with Fas, which prevents Fas activation. The data suggest that tissue-specific inhibitors of Fas such as PMLRARα block Fas-mediated apoptosis and thus can contribute to cancer development. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which we have shown to occur by direct regulation. We have identified an attractive potential target to the regulation of apoptosis at the PMLRARα-Fas and PML-Fas interfaces. By neutralizing the effect of death receptor inhibitors such as PMLRARα and other potential inhibitors, we can improve on the success of the many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells.

Disclosures:

No relevant conflicts of interest to declare.
Ineffective Erythropoiesis Caused by Phenylhydrazine Activates the Expression of GDF15 in Maturing Erythroblasts.
Authors: Tanno Toshihiko , Sripichai Orapan , Noh Seung-Jae , Byrnes Colleen , Meier Emily Riehm , Lee Y. Terry , Miller Jeffery L.
Published: October 2019

Abstract 4032

Poster Board III-968

Several types of ineffective erythropoiesis (thalassemia, congenital dyserythropoietic anemia type I, and refractory anemia with ringed sideroblasts) develop anemia and eventually manifest secondary hemochromatosis even in the absence of transfusion therapy. The highly elevated levels of GDF15 in those patients may contribute to iron loading due to suppression of hepcidin. However, the underlying mechanism for high-level expression of GDF15 in association with ineffective erythropoiesis remains vague. Phenylhydrazine (PHZ), which causes hemoglobin denaturation and alpha-globin precipitation, was utilized in CD34+ cultures as an ex vivo model of ineffective erythropoiesis. All experiments were performed in triplicate with cells from three separate human donors. Initial studies were performed to determine the effects of phenylhydrazine upon reactive oxygen species (ROS) production and apoptosis in the erythroblasts. In dosed titrations (0-10 uM), PHZ was added to the culture medium before and after erythroblast hemoglobin accumulation on culture days 3-6 and 11-14, respectively. PHZ addition prior to the accumulation of hemoglobin had little effect on ROS production. In contrast, PHZ added to hemoglobinized erythroblasts resulted in significant and dosed increases in ROS production. In addition, apoptosis cascades that are downstream of ROS production were studied including activation of caspase-3, EGR-1 transcription, p53 stabilization, and phosphatidylserine on the cell surface (detected by Annexin V). There was a dose-dependent increase in caspase-3, p53 stabilization, and Annexin V positive cells that mirrored the ROS production after PHZ treatment only among the more mature erythroblasts. However, the PHZ addition did not cause a significant increase in the nuclear localization of EGR-1. Since GDF15 is a known target of p53, PHZ effects upon GDF15 expression were also measured. Production of GDF15 was not observed on culture days 3-6 in the absence or presence of PHZ. In contrast, dosed increases in GDF15 expression were detected among the hemoglobinized erythroblasts (culture days 11-14) in response to phenylhydrazine (PHZ = 0 uM, GDF15 = 16 ± 3 pg/1×104cells; PHZ = 1 uM, GDF15 = 23 ± 5 pg/1×104cells; PHZ = 5 uM, GDF15 = 48 ± 9 pg/1×104cells [p<0.01]; PHZ = 10 uM, GDF15 = 75 ± 22 pg/1×104cells [p<0.01]). Of note, GDF15 expression was not detected after enucleation in peripheral blood erythrocytes exposed to PHZ despite increased ROS. According to these data, it is proposed that ineffective erythropoiesis may be caused by increased ROS production during the later stages of erythroblast maturation that activates caspase-3, apoptosis and p53-mediated expression of GDF15.

Disclosures:

No relevant conflicts of interest to declare.
Dynamic Changes in Membrane Protein Expression During Murine and Human Erythropoiesis: Resolving the Distinct Stages in Terminal Erythroid Differentiation.
Authors: An Xiuli , Liu Jing , Chen Ke , Heck Sussane , Chasis Joel Anne , Mohandas Narla
Published: October 2019

Abstract 4039

Poster Board III-975

Erythropoiesis is the process by which nucleated erythroid progenitors proliferate and differentiate to generate, every second, millions of non-nucleated red cells with their unique discoid shape and membrane material properties. In the present study, we examined the time-course of appearance of individual membrane proteins during erythropoiesis to enhance our understanding of the evolution of the unique features of the red cell membrane. We found that all the major transmembrane and all the skeletal proteins in mature red blood cells, except actin, accrued progressively during terminal differentiation of murine erythroid progenitors. In marked contrast, during the same time course, accumulation of various adhesion molecules decreased. In particular, the adhesion molecule, CD44 exhibited a progressive and a dramatic 30-fold decrease from proerythroblast to reticulocyte; this enabled us to devise a new strategy for distinguishing unambiguously between erythroblasts at successive developmental stages during murine erythopoiesis. By contrast, CD71, which is routinely being used as a surface marker for this purpose, proved markedly less effective than CD44 in identifying erythroblasts at successive developmental stages. Indeed, we found that CD71 expression decreased only fourfold during terminal erythroid differentiation, and not in the progressive manner observed for CD44. A similar pattern of membrane protein expression was noted during terminal differentiation of human erythroid progenitors. These findings provide new insights into the genesis of red cell membrane function during erythroblast differentiation, and also offer a means of defining stage-specific defects in erythroid maturation in inherited and acquired red cell disorders and in bone marrow failure syndromes.

Disclosures:

No relevant conflicts of interest to declare.
A Comparison of Serum Hepcidin and GDF15 Expression in Cases of Iron Depletion Due to Blood Loss.
Authors: Tanno Toshihiko , Rabel Antoinett , Lee Y. Terry , Leitman Susan , Miller Jeffery L.
Published: October 2019

Abstract 4043

Poster Board III-978

Iron depletion affects billions worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. Hepcidin is a negative regulator of iron absorption in the body. Low serum hepcidin levels provide a physiological response to iron demand in patients with iron deficiency. Based on a recent discovery of elevated growth differentiation factor 15 (GDF15) as a pathological suppressor of hepcidin in cases of thalassemia (Tanno et al. 2007), it was hypothesized that GDF15 may be elevated under physiological circumstances such as iron deficiency, but this area of research remains controversial. Here GDF15 and hepcidin levels were measured in the peripheral blood of subjects with iron-depleted erythropoiesis, and monitored in patients receiving iron replacement therapy. Initially, clinical studies were performed to measure GDF15 in the serum of iron-depleted blood donors (n = 10) and healthy volunteers (n = 10). Iron depletion was defined for this study as serum ferritin level (<10 ng/ml) and transferrin saturation (<15%). When compared to the control group, hemoglobin and RBC counts were also significantly reduced (p< 0.01) among the iron-depleted group. Serum hepcidin levels were also significantly reduced in the iron-depleted group (<5 ng/ml) compared to healthy volunteers (46 ± 41 ng/ml, p < 0.01). However, the GDF15 levels were not elevated in the iron-depleted group. Instead, iron-depletion was associated with a slight decrease in serum GDF15 (304 ± 90 pg/ml) compared to that in the control group (386 ± 104 pg/ml, p = 0.084). In three additional patients with iron depletion due to menstrual blood loss, serum GDF15 and hepcidin levels were monitored during oral iron replacement. In association with normalization of the ferritin and transferrin saturation, hepcidin levels increased in each patient, but GDF15 levels remained stable (202 ± 15 pg/ml versus 215 ± 23 pg/ml). In summary, iron depletion due to blood loss was not associated with increased GDF15, and there was no obvious correlation between GDF15 and hepcidin expression at the time of depletion or during iron replacement therapy. The data support the hypothesis that GDF15 elevation contributes to the pathological suppression of hepcidin and iron overload in cases of ineffective erythropoiesis rather than the physiological suppression of hepcidin in cases of iron depletion.

Disclosures:

No relevant conflicts of interest to declare.
Role of HDAC9 in γ-Globin Gene Regulation.
Authors: Muralidhar Shalini A , Pace Betty
Published: October 2019

Abstract 4074

Poster Board III-1009

Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies will likely involve chromatin modification in the presence of histone deacetylase (HDAC)/protein complexes to promote γ-globin gene activation. The role of various HDACs in globin transcription is not very well understood therefore, the objective of our study was to identify HDACs involved in γ-gene regulation. Screening studies were performed in K562 erythroleukemia cells to determine transcription levels for HDAC genes in the absence or presence of HbF induction. Treatment with butyrate (2mM), trichostatin A (0.5μM) and the non-HDAC inhibitor control hemin (50μM) significantly reduced mRNA levels of HDAC9 and its splice variant HDRP (histone deacetylase related protein) lending indirect evidence for their involvement in drug-mediated γ-globin regulation. Subsequent studies were performed to delineate whether HDAC9 can directly modulate γ-globin gene transcription since a role for HDAC9 in hematopoiesis has been previously demonstrated. Furthermore, consensus binding sites for GATA-1 are present in the HDAC9 gene proximal promoter. Initially, we performed siRNA knockdown using Oligofectamine (Invitrogen) in K562 cells and measured γ-globin levels by real time quantitative PCR analysis. Treatment with siHDAC9 (Dharmacon) produced dose-dependent γ-globin gene silencing over an 80-320nM range; control siRNA molecules had no effect. When HDAC9 was over-expressed in K562 cells using pTarT-HDAC9 at 10-50μg concentrations, a dose dependent 2.5-fold increase in γ-globin mRNA (p<0.05) was produced. These data support a positive regulatory role for HDAC9 in γ-gene regulation. To confirm the physiological relevance of HDAC9, similar studies were performed in human primary erythroid progenitors using a two-phase liquid culture system. The 320nM siHDAC9 concentration produced 48% and 60% decrease in γ-globin mRNA at day 11 (early progenitors) and day 28 (late progenitors) respectively. Enforced HDAC9 expression increased γ-globin by 2.5-fold (p<0.05) at both days. ELISA was performed to quantify HbF protein and cytospin preps were made to visualized hemoglobin by fluorescent staining with anti-γ-FITC antibody. HDAC9 enforced expression for 72 hrs produced a 7-fold increase in HbF and γ-FITC positive cells increased >50%. Collectively these data support a positive role for HDAC9 in γ-globin regulation. Chromatin immunoprecipitation assays will be completed to elucidate the contribution of HDAC9 in maintaining an active chromatin domain in the γ-globin promoter. We will also define interactions of GATA-1 in the HDAC9 gene to coordinate expression during erythroid maturation.

Disclosures:

No relevant conflicts of interest to declare.
Imatinib Mesylate and Hyper-CVAD (IM-HCVAD) for Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL).
Authors: Arellano Martha , Muringampurath-John Disni , Shenoy Pareen J. , Winton Elliott F. , Bryan Beverly , Langston Amelia , Sinha Rajni , McLemore Morgan L. , Heffner Leonard , Khoury H. Jean
Published: October 2019

Abstract 4103

Introduction

The addition of IM to hyper-CVAD led to higher rates and longer duration of complete remissions in Ph+ ALL. The purpose of this study is to report single institution experience with IM-HCVAD followed by maintenance IM or allogeneic transplantation in newly diagnosed adult Ph+ ALL treated between 10/02 and 08/08.

Methods

IRB-approved retrospective analysis of the institutional hematological database.

Results

Thirty-three patients, median age 51 (22-72), with 8 (24%) older than 60, presented with a median WBC of 24,000/mm3 (range, 2-200) and LDH of 2.6 ULN (1.3-11). Four (12%) had extra-medullary/CSF leukemia. Patients received a median of 7 (range, 1-8) cycles of IM-HCVAD followed by maintenance IM +/- POMP as tolerated (Blood 2004;103:4396-4407). After 2 cycles, 1 patient had primary refractory disease and expired, and 32 (97%) achieved complete hematological and cytogenetic remission. Twenty-four of 32 (75%) subsequently achieved complete molecular remission (CMR). Among those who achieved CMR, 6/24 relapsed with preceding loss of CMR in 4. Four of 8 who did not achieve CMR relapsed. One patient in CR was lost to follow-up after induction. Thirteen (39%) were allografted in CR1 using TBI-based myeloablative (9) or non-myeloablative (4) conditioning and 18 received maintenance IM-based therapy. With a median follow-up of 18.3 months (range, 4.4-76), 10 patients (32%) relapsed (1 post-transplant and 9 during IM maintenance) and received salvage therapy leading to CR2 in 4, of which 1 was successfully allografted, and 3 remain in CR2 with a follow-up of 20, 36, and 60 months. Eleven patients died (6 from GVHD/infection, and 5 from relapsed refractory leukemia). For the 13 allograft recipients in CR1 disease-free (DFS) and overall survival (OS) were 15, and 18 months; and 15 months and 20 months for the 18 IM maintenance patients, respectively (Figure). WBC > 30,000/mm3 and residual disease detected by flow cytometry after 2 cycles were associated with decreased OS (p < 0.03). A trend for lower survival was noted among African American patients (p= 0.07).

Conclusion

The addition of IM to hyper-CVAD is associated with high rates of CR (97%) in newly diagnosed Ph+ ALL. In this relatively small cohort with a short follow-up, peripheral blood PCR did not predict outcomes. Ph+ALL patients are candidates for novel maintenance regimens.

Disclosures:

Off Label Use: Imatinib combined with the hyper-CVAD chemotherapy. Khoury:Novartis Oncology: Honoraria.
Inhibition of Platelet Function by Aspirin in Insulin Dependent Diabetics without Vascular Disease.
Authors: Schwartz Kenneth A. , Schwartz Dianne E , Myneni Anjana , Nelson Peggy , Gossain Ved
Published: October 2019

Abstract 4172

Diseases like diabetes that are associated with increased platelet production are reported to have decrease aspirin (ASA) induced platelet inhibition. ASA inhibition of platelet function is mediated via acetylation of platelet cylooxygenase, which is completed approximately 30 minutes after ingestion. Thereafter production of new non-acetylated platelets increases the proportion of normal platelets gradually reversing ASA platelet inhibition. We tested the hypothesis that insulin dependent diabetics (type 1) without clinical evidence of occlusive vascular disease have less aspirin platelet inhibition than normal controls. The amount of ASA (325mg) platelet inhibition was measured using the slope (S) of platelet prostaglandin agonist (PPA) stimulated light aggregation curve that decreases after ASA. Aggregation was measured at 3 time points: before ASA ingestion, time 0 (T0); 2 hours post ASA (T2); and 24 hours after ASA (T24). Percent recovery from aspirin inhibition was calculated using the formula (T24–T2)/ (T0-T2) x100. Eight adult insulin dependent diabetic patients without clinical evidence of vascular disease and who were not taking aspirin were compared with 11 nondiabetic controls. 24 hours after aspirin ingestion diabetic patients recovered 10% of their maximal ASA platelet inhibition compared to 13% in controls (p=0.69). We conclude that recovery of platelet function from ASA in insulin dependent diabetics without clinical vascular disease occurs at the same rate as normal controls, suggesting that the previously reported decrease in platelet inhibition in diabetic patients may be related to increased platelet production associated with clinically evident occlusive vascular disease.

Disclosures:

No relevant conflicts of interest to declare.
Biological Activity of Low-Molecular-Weight Heparin Increases Over 9 Days in Patients Treated for Acute Venous Thromboembolism.
Authors: Harenberg Job , Bauer Kai , Abletshauser Claudia
Published: October 2019

Abstract 4176

Subcutaneous (s.c.) body-weight adjusted as well as fixed dose low-molecular-weight heparin (LMWH) for treatment of acute venous thromboembolism (VTE) has been proven to be at least as effective and safe as intravenous unfractionated heparin (UFH). We hypothesized that the anticoagulant effects of LMWH may accumulate during a 9 days fixed dose therapy in patients with acute VTE.

Ten patients received 8,000 IU LMWH certoparin bid s.c. for 9±1 days after having given written informed consent. The local ethics committee accepted the study protocol. Serial blood and urine were collected at days 2 and 9 and daily before and after the morning administration of the anticoagulant. The pharmacodynamic parameters were analysed on the anti-factor Xa S2222 method (aXa), heptest, thrombin generation inhibition assay (TGIA), and tissue factor pathway inhibitor activity (TFPI). The area under the activity time curves (AUC) of the parameters was compared at days 2 and 9.

LMWH reached steady state levels of the S2222 and heptest assay within 24 hrs. aXa, heptest, TGIA and TFPI were 22%, 38%, 13% and 22% higher at day 9 compared to day 2. The elimination half-lives of aXa and heptest and the aXa excreted into the urine did not differ between days 2 and 9, respectively. The AUC of the aXa did not correlate with the body weight of the patients.

Fixed dose, body weight-independent subcutaneous LMWH accumulated to some extend after 9 days of treatment in patients with acute VTE. However, the results of the clinical trials with the LMWH certoparin did not show more bleeding complications compared to UFH. Therapy with LMWH for more than 10 days may require dose reduction.

Disclosures:

Harenberg: Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Honoraria; Roche Diagnostics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bayer Health Care: Consultancy, Honoraria. Abletshauser:Novartis: Employment.
Survey On Directed Family Cord Blood Banking for Transplantation Among the National Cord Blood Bank Network in Italy.
Authors: Saracco Paola , Lombardini Letizia , Screnci Maria , Salvaneschi Laura , Tamburini Anna , Saccardi Riccardo , Pupella Simonetta , Pagliarino Mauro , Bargamaschi Paola , Urbani Serena , Fazio Luigina , Bosi Alberto , Grazzini Giuliano , Costa Alessandro Nanni
Published: October 2019

Abstract 4195

Directed family cord blood (DCB) storage provides hemopoietic stem cell source for transplantation (HSCT) for families with an existing or a potentially future recipient with HSCT curable disease (D). The National CB Banks Network in Italy (ITCBN) has a leading role in providing public DCB service for high-risk families, in compliance to GITMO directives for eligibility criteria (HSCT curable D: malignant MD, non MD, inherited ID). To provide best cost-effective practices recommendations it is important to report on DCB procedures and HSCT rate (HSCT-R) among public Banks. By 12.12.2008 almost 1800 DCB units were stored in 18 Italian Banks and 104 (9%) issued for HSCT. The present survey aims at summarizing the over 15 yrs DCB experience among 5 ITCBN Banks active since 1997 (range 1990-1997), and including 670 DCB units.

Results

Preliminary analysis reports a 94% overall compliance to eligibility criteria directives, and overall HSCT- R for an alive sibling of 12% (63/522); the 63 HSCT were 97% matched, for curing ID in 84% and with 72 % overall survival outcome. Different policies among Banks were compared (Bank vs others: 1) eligibility criteria distribution : Bank PV06 DCB for MD <50% (42% vs 55-67%), Banks Rm04,R04 significant DCB for non sibling recipient (parent with MD) (17-24% vs 0.5%-2%), Bank T02 with significant (53/261) DCB for future sibling with ID (20% vs 2-5%); 2) Timing of HLA typing: Banks T02,FI03,PV06 with DCB following prenatal HLA (20-27% vs 0%), Banks FI03,Rm04,PV06 with CB HLA typed at birth (61-73% vs 23-25%). Significant HSCT-R (37/177=21 %) was reported by PV06 (HLA selection based storage). Lowest HSCT-R by Rm04,R04 (3/101=3%-5/98=5%) (parental mismatch). At T02 when excluding DCB for future sibling (low birth rate), and autologous (11 ID) HSCT-R increased from 6% (15/261) to 9% (15/170)

Conclusion.

Advice for public long-term DCB storage should depend on HLA compatibility, potential recipient, disease progression, and likelihood/ timing of using DCB; Italian CB Banks are actively cohoperating to find best public banking practices to ensure a CDB storage system that is ethical, cost effective and responsive to patient needs.

Disclosures:

No relevant conflicts of interest to declare.
Measurement of Thrombin-Alpha-2-Macroglobulin Complexes Generated in Plasma.
Authors: Vetr Helga , Geiter Sabine , Graf Markus , Knapp Silvia , Binder Bernd R
Published: October 2019

Abstract 4205

The aim of this study is to evaluate a new assay (TECHNOZYM®TAM Activity) for measurement of thrombin-alpha2-macroglobulin (TAM) complexes. Furthermore, we asked the question whether the acute phase nature of a2Macroglobulin would be reflected in increased TAM levels in plasma of sepsis patients.

Thrombin generation was measured using TECHNOTHROMBIN®TGA which does not use any inhibitors of clot formation. Thus, clots formed during the reaction need to be removed either by centrifugation or filtration prior to sample transfer to the TAM assay which is a combined immuno-activity assay specifically detecting a2M bound thrombin.

Pre-existing TAM levels in various haemostatic samples did not differ significantly from those in normals, with one exception. Surprisingly, Factor X deficient samples expressed about 3 fold higher levels of TAM than normals. Approximately 4% of total thrombin generated was found in TAM complexes (TAM/AUC ratio 3,6 % ± 1, TAM concentration 73nM ±14). No significant difference in TAM values could be found for the different methods of clot removal (n=8; p>0,3). As expected, thrombin generation in normal as well as in factor deficient samples lead to approximately 10-fold increase of TAM. Only in FX-deficient and Coumadin samples no increase in TAM was observed. This is consistent with the small amount of total thrombin generated in these samples. TAM levels in plasma of sepsis patients were significantly higher (19.2nM ± 1.7) than in normals (9.7nM ±0.5; n=40, p<0,0001). Levels of TGA triggered TAM correlate with total amount of thrombin generated and TAM measurement in TGA samples is not necessary for routine TGA measurements. Furthermore, TAM levels are increased in the plasma of sepsis patients and do not correlate with acute phase markers in these patients. Thus TAM might be suitable as a new marker to differentiate a (sub)group of sepsis patients.

Disclosures:

Vetr: Technoclone GmbH: Employment. Geiter:Technoclone GmbH: Employment. Graf:Technoclone GmbH: Employment.
Cross-Reactivity of Rabbit Anti-Bovine Thrombin IgGs with Human α-Thrombin and a Recombinant Version of Human Thrombin (Recothrom™).
Authors: Zhu He , Hoppensteadt Debra , Wahi Rakesh , Paterson Craig , Fareed Jawed
Published: October 2019

Abstract 4211

It has been reported that patients exposed to topical bovine thrombin preparations may develop antibodies to bovine thrombin, factor V or various other proteins. In some cases these antibodies can cross-react with the corresponding human coagulation proteins. The present study was undertaken to determine if the antibodies induced by bovine thrombin antigens in rabbits could also cross-react with human a-thrombin and RecothromTM. Bovine crude thrombin and its purified versions, thrombin 4A and 4B, were administered to individual groups of rabbits on 0, 21, 42, 91, 123 and 151 days. Blood was drawn from each rabbit on days 30, 50, 105, 137 and 165, respectively. The antiserum from each rabbit and the pooled antisera from individual groups were purified to obtain the IgGs. Utilizing western blotting method, the possible cross-reactivity of each IgG with human thrombin and RecothromTM was explored using serial diluted human α-thrombin (20μg, 10μg, and 5μg) and RecothromTM (10U, 5U, and 2.5U). No cross-reactivity with either human α-thrombin or RecothromTM was observed with both anti-bovine crude thrombin IgGs and thrombin 4B IgGs collected on day 30 and day 165. However, anti-bovine thrombin 4A IgGs showed cross-reactivity with both human α-thrombin and RecothromTM. Cross-reactivity of anti-bovine thrombin 4A IgGs with human α-thrombin and RecothromTM was augmented with time. The minimum concentration of 4A IgG required to exhibit cross-reactivity with human α-thrombin and RecothromTM varied considerably among individual rabbits. These results demonstrate that rabbit anti-bovine thrombin 4A IgG could cross-react with both human a-thrombin and RecothromTM in a time/concentration-dependent manner.

Disclosures:

Paterson: King Pharmaceuticals: Employment.
Fludarabine Plus Cyclosporine for Refractory/Relapse Aplastic Anemia.
Authors: He Guangsheng , Wang Xiuli , Depei Wu , Sun Aining , Jin Zhengming , Qiu Huiying , Miao Miao
Published: October 2019

Abstract 4221

Objective

To evaluated the efficiency of fludarabine plus cyclosporine for refractory/ relapse aplastic anemia (AA).

Methods

We treated 7 refractory/ relapse aplastic anemia (AA) patients by fludarabine plus cyclosporine (fludarabine 25 mg/m2, day 1-5; cyclosporine 3mg/kg/d) from November 2003 to April 2008. The concentration of cyclosporine was maintained between 200-300ng/ml. The AA patients who was refractory to immunosuppressive therapy was defined as did not get transfusion dependence 6 months after treatment with ATG (anti-thymocyte globulin, ATG) and cyclosporine, who become transfusion dependent again after transfusion dependence were relapse.

In the patient group, 5 were males and 2 was female, with ages ranging from 12 to 45 years (median=22.5 years). 3 patients previously were treated by ATG plus cyclosporine, 3 were only received cyclosporine, and one was treated by Allo-HSCT (Table 1).

Results

The reponse rate was 57.1%(4/7), all were partial remission with count of neutrophil and levels of hemoglobin achieved normal levels while the number of platelet was lower than 100°Á109/L. No serious infection was found.

Conclusion

Fludarabine plus cyclosporine as a new immunosuppressive therapy for refractory/ relapse aplastic anemia patients is an effective regimen, it was worth to explore in clinical research.

Disclosures:

No relevant conflicts of interest to declare.
Apheresis of Hematopoietic Progenitor Cell (HPC) From Allogeneic Donors ≥60 Years of Age.
Authors: Janssen William E. , Rahn Darlene , Field Teresa , Kharfan-Dabaja Mohamed A , Ayala Ernesto , Ochoa Jose L , Hackett Martha , Anasetti Claudio , Fernandez Hugo F.
Published: October 2019

Abstract 4226

There has been reluctance to collect apheresis HPC grafts from donors of age 60 and beyond. This is predicated on concern over relative frailty, poor venous access, reduced potential for HPC mobilization, and reduced potential for stable engraftment of HPC transplant. We have expanded our transplant eligibility into patients beyond the age of 60, with the concomitant acceptance of matched sibling donors of similar age. We have reviewed 96 consecutive sibling donors, 19 of whom were of age ≥60 years at the time of collection. All donors were collected using Gambro Spectra apheresis instruments, and the volume of blood pheresed was gauged to target a CD34 dose of 5-10 million per Kg of recipient weight. We have found the following:

Age < 60 (n=77) Age ≥ 60 (n=19) p Blood volumes (Mean±Std) 4.9±3.3 6.1±3.1 .85 CD34/RecipKg E6 (Mean±Std) 8.1±2.8 5.8±1.7 .02 CD34/Blood vol E7. (Mean±Std) 18.0±11.7 10.9±7.8 .06 < 5E6 CD34/Kg Collected (n) 5 3 .21 > 1 collection (n) 12 7 .04 Catheter required (n) 23 6 .54 Grade 3+ pheresis complications 2 3 .47 ANC500 Median days (Min-Max) 16 (10-28) 16 (13-75) .14 Plt20K Median days (Min-Max) 16 (8-77) 16 (13-89) .30

These results suggest that donors of age ≥60 years may be successfully collected, and that the resultant grafts can be expected to produce successful transplants. There is not a higher toxicity rate or need for catheter insertion associated with the collections in the older age group. There is a clear trend, however, to reduced mobilization of CD34+ cells, as reflected in the need for more collections, and fewer CD34+ cells per blood volume leukopheresed. In spite of this, sufficient cells to produce functional grafts were collected from all donors, although a limited number of donors in both age categories failed to collect a full 5e+6 CD34/Kg. We conclude that in the context of an aging demographic, allograft donors of age ≥60 may be successfully employed for HPC collection. Further, we propose that the application of plerixafor in ≥60 year old donors should be investigated in the context of a growing need to collect allografts from donors in this age group.

Disclosures:

Off Label Use: sirolimus for graft-versus-host disease.
Characterization of the Putative Stem Cells From Umbilical Cord Blood.
Authors: Gaikwad Amos S. , Cubbage Michael , Goltsova Tatiana , Threeton Christopher , Ty Maria , Hollinger Alvin , Perlaky Laszlo , Hurwitz Mary
Published: October 2019

Abstract 4231

Cord blood (CB) is a rich source of hematopoietic stem cells (HSC) with long-term repopulating activity necessary for allogeneic stem cell transplantation. CD34+ stem cells are considered sufficient for transplantation, however recent progress in stem cell biology indicates that cells with other surface markers such as CD133 or cells expressing high aldehyde dehydrogenase activity with low side scatter (ALDHhigh/SSClow) or a rare side population (SP) of cells that exclude the Hoechst 33342 vital dye via multi drug transporters have been shown to possess stem cell properties. We characterized CD34+, CD133+, ALDH+ and SP in mononuclear cells (MNC) isolated from human CB. While the SP cell population is rare and detectable in few CB-MNC examined, we found abundant CD34+ and CD133+ cells (1.0+/-0.5 and 0.8+/-0.4 per 100 CD45+ MNC cells, respectively) following the ISHAGE protocol. A distinct ALDH+ cell population (median of 0.26%; range of 0.1 to 0.5%) was also present in all of the CB-MNC analyzed. Over 90% of the ALDH+ cells were also CD34+ and CD133+. The ability of CB-MNC to form colonies in methocult semi-solid media supplemented with cytokines yielded myeloid, lymphoid and erythroid colonies. The clonogenic potential of CB-MNC ranged from 16-48%. We are assessing the colony forming ability of purified stem cell fractions using flow cytometry. The clonogenic efficiency of these individual putative stem cells will be discussed.

Disclosures:

No relevant conflicts of interest to declare.
Chronic Myeloid Leukemia and BCR/ABL Signal Pathway Are Not Associated with AKT1 Pleckstrin Homology Domain(E17K) Mutations.
Authors: He Yanli , Zheng Jine , Hu Yanjie , Xiao Hong , Li Huiyu , Liu Jun , Li Xiaoqing , Du Wen , Chen Xiangjun , Hu Yu , Huang Shiang
Published: October 2019

Abstract 4262

AKT1 E17K mutation was recently found in several human cancers. PI3K/AKT is one of major downstream signal pathways of BCR-ABL oncogene and essential for CML leukemogenesis and IM resistance. More recently, it has been shown that increased PI3K/AKT activity in CML contributes to the development of drug resistance of malignant cells and combination of PI3K/AKT pathway inhibitor and rapamycin can efficiently kill imatinib-resistant BCR-ABL-expressing cells, highlighting the potential of PI3K/AKT as the new targets for CML therapy. Considering the central role of AKT in CML, we performed a screening study to investigate the possible role of AKT1 E17K mutation in CML in the Chinese population. To study the frequency of AKT1 E17K mutation in CML patients in Chinese population as well as its possible relation to IM sensitivity, we recruited 86 patients with CML revealed the presence of Philadelphia chromosome at diagnosis. At molecular level, B2a2, B3a2, B2a3 and B3a3 were identified by RT-PCR in 41,43,1 and 1 cases, respectively. Molecular response to IM was evaluated by real-time PCR at 6 months and 12 months after IM administration. The CCR, MMR and CMR were achieved in 74, 39 and 27 cases, respectively. No AKT1 E17K mutation was found in all patients. We also studied the AKT1 E17K genotype in 7 CML patients at blast stage. The additional genetic abnormalities were summarized. For these patients, CD34+ leukemic cells were purified by FACS from PBMNCs before DNA extraction. Again, we failed to detect any AKT1 E17K mutant in this group of patients.

Conclusion

No mutation was found in all patients with different phases, genotypes and IM responses. The results indicated that the acquisition of AKT1 E17K mutation should not be the major event in IM resistance or CML blasts.

Disclosures:

No relevant conflicts of interest to declare.
Comparison of Response Over 6 Years Between Patients with Chronic Myeloid Leukemia Who Received Imatinib as Initial Therapy and Those Who Received Imatinib as Secondary Therapy.
Authors: Jiang Hao , Huang Xiaojun , Chen Shanshan , Jiang Bin , Jiang Qian , Qin Yazhen , Lai Yueyun
Published: October 2019

Abstract 4281

Objective

To compare responses in a single center over 6 years between patients with Philadelphia-positive chronic myeloid leukemia (CML) who received imatinib as initial therapy and those who received imatinib as secondary therapy.

Patients and Methods

We reviewed 171 cases of patients with Philadelphia chromosome (Ph)-positive CML who received imatinib for 72 months: 73 patients in an initial therapy group (time from diagnosis of CML less than 6 months) and 98 patients in a secondary therapy group (time from diagnosis of CML greater than 6 months).

Results

Cumulative rates of complete cytogenetic response (CCyR) at 6, 12, and 36 months after imatinib treatment in the initial therapy group and secondary therapy group were 75%, 89% and 96%, and 48%, 77% and 84%, respectively (P=0.0002). The median time to CCyR in initial and secondary therapy groups was 6 months (95%CI, 3.3–8.3) and 9 months (95%CI, 6.4–11.6), respectively (P=0.0002). Cumulative rates of major molecular response (MMoR) at 9, 12 and 18 months after imatinib treatment in initial therapy and secondary therapy groups were 26%, 42% and 49%, and 16%, 25% and 36%, respectively (P=0.048). The median time to MMoR in initial and secondary therapy groups was 19 months (95%CI, 11.7–26.3) and 36 months (95%CI, 19.7–52.4), respectively (P=0.048). Estimated rates of freedom from progression to accelerated phase (AP) and blastic phase (BP) in initial and secondary therapy groups were 99% and 89%, respectively (P=0.08). Estimated 6-year progression-free survival (PFS) rates in cases with CCyR and without CCyR were 93% and 62%, respectively (P=0.0012). Estimated 6-year event-free survival (EFS) rates in initial and secondary therapy groups were 96% and 68%, respectively (P=0.04). The event was defined as progression to accelerated AP and BP, loss of CCyR or CHR.

Conclusion

The time to CCyR and MMoR in the initial therapy group was significantly shorter than that in the secondary therapy group. There was no difference in PFS rates between the two groups in the 72-month long-term follow-up. The EFS rates indicated that patients receiving imatinib as initial therapy achieved more stable remissions than those receiving it as secondary therapy.

Disclosures:

No relevant conflicts of interest to declare.