bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of HIV Resistant and Functional Macrophages From Hematopoietic Stem Cell Derived Induced Pluripotent Stem Cells
Authors: Kambal Amal , Mitchell Gaela , Cary Whitney , Gruenloh William , Jung Yunjoon , Nacey Catherine , Kalomoiris Stefanos , Lindsey Matt , Bauer Gerhard , Nolta Jan A. , Anderson Joseph S.
Published: October 2019

Abstract 562

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV infected patients. In this study, we have utilized human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were de-differentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 shRNA and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs towards the hematopoietic lineage, a robust quantity (>35%) of colony forming CD133+ HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC derived macrophages exhibited strong protection (>3 logs) from HIV-1 infection. Here we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.

Disclosures:

No relevant conflicts of interest to declare.
The Activated K-RAS Protein Accelerates Human Derived-MLL/AF4 Induced Leukemo-Lymphomogenicity In Transgenic Mice Model
Authors: Tamai Hayato , Miyake Koichi , Takatori Miyuki , Miyake Noriko , Yamaguchi Hiroki , Dan Kazuo , Shimada Takashi , Inokuchi Koiti
Published: October 2019

Abstract 61

Acute lymphoblastic leukemia (ALL) with MLL/AF4 fusion is a common infant leukemia associated with a poor prognosis. Research into the molecular mechanisms underlying ALL with MLL/AF4 has been hampered by the absence of a good animal model: there is a large discrepancy between the time to disease onset in infants and the long latency seen in previously established murine models (human derived-AF4 knock-in and inverter model). We speculated that the absence of associated K-RAS mutations could explain the long latency seen in murine models of ALL with MLL/AF4. To establish a model combining MLL/AF4 fusion and activated K-RAS, we first established MLL/AF4+ transgenic (Tg) mice through overexpression of human MLL/AF4 fusion protein. These MLL/AF4+ Tg mice exhibited splenomegaly and lung tumors at 8–12 months of age (median, 12 months), and histopathological analysis showed disruption of the spleen structure and infiltration of the liver, lung and spleen by lymphoma cells consisted of pro B-cells (CD45R/B220+CD43+). Western blot analysis showed expression of both MLL/AF4 and HoxA9 was upregulated in all of the tissues tested. Infiltration of tumor cells into the peripheral blood was observed after a long latency period in MLL/AF4+ Tg mice (median, 14 months), at which time abnormal lymphocytes accounted for 7.2±1.2% (n=5) of white blood cells. FACS analysis showed these abnormal lymphocytes to be CD45R/B220+CD43+CD19+ cells, which is the same phenotype as the lymphoma cells. To develop a more aggressive oncogenic model mouse, we generated MLL/AF4+K-RAS mutation+ Tg mice by crossing MLL/AF4+ Tg mice and K-RAS mutation (G12D)+ Tg mice. The resultant mice developed lymphoma significantly earlier (median, 6 months) than MLL/AF4+ Tg mice (median LFS: 5.5 months vs. 12 months, P=0.001). Infiltration of tumor cells into the peripheral blood was observed after a short latency period in MLL/AF4+K-RAS mutation+ Tg mice (median age, 6 months), at which time leukemia cells accounted for 3.2±0.9% of WBCs, suggesting the collaborative acceleration of leukemo-lymphomogenicity through overexpression of MLL/AF4 and mutant K-RAS. We anticipate that the MLL/AF4+K-RAS mutation+ Tg mouse model will be useful for studying the molecular mechanisms of MLL/AF4 leukemogenesis and for developing new therapies against MLL-related malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Tyrosine 201 Is Critical for Transformation Mediated by JAK2V617F
Authors: Yan Dongqing , Mohi M. Golam
Published: October 2019

Abstract 626

The JAK2V617F mutation is associated with most cases of Ph-negative myeloproliferative neoplasms (MPNs). In normal hematopoietic cells, JAK2 becomes activated upon cytokine stimulation. The JAK2V617F protein is a constitutively activated tyrosine kinase that leads to activation of several intracellular signaling pathways and transformation of hematopoietic cells to growth factor-independence. Previous studies have shown several tyrosine residues within JAK2 are phosphorylated upon growth factor stimulation. However, the role of these tyrosine residues in signaling and transformation mediated by JAK2V617F remains unclear. Tyrosine 201 lies within a YXX(L/V) motif, which is a potential binding site for Src homology 2 (SH2) domain containing protein(s). Here, we sought to determine the role of tyrosine 201 in JAK2V617F-induced hematopoietic transformation by introducing a tyrosine-to-phenylalanine point mutation (Y201F) at this site. We observed that Y201F mutation significantly inhibited the cytokine-independent cell growth and induced apoptosis in Ba/F3-EpoR cells expressing JAK2V617F. The Y201F mutation also resulted in significant inhibition of JAK2V617F-mediated transformation of hematopoietic cell lines and bone marrow progenitors in vitro. Biochemical analyzes revealed that Y201F mutation almost completely inhibited the phosphorylation/activation of JAK2V617F. We found that Shp2 binds to the Y201 site of JAK2 and mediates the interaction between JAK2V617F and EpoR. In addition, we observed constitutive phosphorylation of Erk1/2 and p70S6 kinase mediated by JAK2V617F was inhibited by the Y201F mutation. Treatment of Ba/F3-EpoR-JAK2V617F cells with JAK inhibitor I or mTOR inhibitor rapamycin inhibited the phosphorylation of p70S6 kinase. Moreover, inhibition of p70S6 kinase by rapamycin treatment (0.5-1 nM) significantly impaired the proliferation and clonogenic growth of Ba/F3-EpoR-JAK2V617F cells. Together, these results suggest that p70S6 kinase is a downstream target of JAK2V617F, and mTOR inhibitor could be useful in treating JAK2V617F-evoked MPN. Finally, using a bone marrow transduction/transplantation approach, we found that Y201 plays an important role in efficient induction of MPN mediated by JAK2V617F.

Disclosures:

No relevant conflicts of interest to declare.
Autologous (auto) Versus Allogeneic (allo) Hematopoietic Cell Transplantation (HCT) for T-NHL: A CIBMTR Analysis
Authors: Smith Sonali , Burns Linda J , van Besien Koen , LeRademacher Jennifer , He Wensheng , Laport Ginna G. , Montoto Silvia , Maloney David G , Hari Parameswaran
Published: October 2019

Abstract 689

T-cell lymphomas (T-NHL) are aggressive, heterogeneous malignancies, marked by chemoresistance with fewer than one-third of patients achieving disease control with non-transplant therapies. There are no large comparative studies of autoHCT and alloHCT in T-NHL. We analyzed post-transplant outcomes of 241 recipients (≤ 60 yrs) of autoHCT (n=115) and alloHCT (n=126, 76 matched siblings) for T-NHL between 1996–2006 reported to the CIBMTR. Median age was 43 yrs for autoHCT and 38 yrs for alloHCT. In the alloHCT cohort: 59% received myeloablative conditioning, 60% had matched sibling donors. Histology was heterogeneous (Table 1).

Median time to transplant after diagnosis was similar in both groups (10m vs. 11m). AutoHCT, compared to alloHCT, was used more in ALCL (53% vs. 40%, p=0.04) and in those with less advanced and more sensitive disease: first complete remission (CR1) (35% vs. 14%, p=0.001), chemosensitive disease (86% vs. 60%, p<0.0001), and ≤ 2 lines prior therapy (65% vs. 44%, p<0.001). Median follow-up was 71m for autoHCT and 49m for alloHCT. Treatment related mortality (TRM) at 100 days in the autoHCT and alloHCT cohorts was 2% and 17%, respectively. For those beyond CR1, overall survival (OS) at 1 yr was 62% vs. 52% and at 3-yrs 53% vs. 41% for the autoHCT and alloHCT cohorts, respectively (Table 2). Relapsing T-NHL was the cause of death in 73% of autoHCT and 44% of alloHCT pts.

In multivariate analysis, alloHCT and more pretransplant chemotherapy regimens were associated with a higher risk of TRM. Relapse risk was higher with chemotherapy resistant disease and those not in CR. PFS and OS were similar after autoHCT or alloHCT with a greater number of prior chemotherapy regimens and chemotherapy resistance impacting survival outcomes adversely. Among 101 peripheral T cell lymphoma (PTCL) pts, the risk of relapse was significantly lower with alloHCT (RR 0.5, 95% CI 0.26–0.97) but countered by increased TRM for alloHCT (RR 3.03, 95% CI 1.02 –8.96). PFS and OS after autoHCT or alloHCT for PTCL were similar and adversely affected by pretransplant chemotherapy resistance. Among alloHCT recipients, there were no differences in outcomes based on donor source, conditioning regimen type or intensity.

In summary, pts undergoing autoHCT for T cell NHL appear to be selected for less advanced disease and greater chemosensitivity, making direct outcome comparisons with alloHCT difficult. AlloHCT is an effective strategy for high risk patients, albeit with significant TRM. Higher numbers of chemotherapy regimens prior to transplant adversely impacted both TRM and survival, suggesting that HCT should be considered earlier in the disease course.

Disclosures:

No relevant conflicts of interest to declare.
Trends In Mortality Rates and Age of Death In Sickle Cell Disease (SCD): 1979–2005
Authors: Lanzkron Sophie , Haywood Carlton
Published: October 2019

Abstract 736

While improvements in care have resulted in significant decreases in mortality for children with SCD, it is unclear if similar decreases in mortality rates have occurred in adults with the disease. The purpose of this research is to describe mortality rates and trends in age of death for those with SCD over the last 3 decades. We used the National Center for Health Statistics Multiple Cause of Death (MCOD) files to examine age at death and calculate mortality rates from 1979–2005. ICD9 and ICD10 codes for SCD were used as appropriate to identify sickle cell related deaths. Mortality rates were calculated as deaths per 100,000 African American population. The number of African Americans each year was determined using available census data. Trends in mortality rates were examined using negative binomial regression and age of death was examined using t-tests and linear regression. After excluding certificates with codes for sickle trait and those with multiple sickle codes we identified 16,654 sickle-related deaths. The age range was 0 to 107 years. Mean age of death was significantly different for men (33.4, 95% CI [33.0, 33.7]) and women (36.9, 95% CI [36.5, 37.4]). SCD was the most common listed underlying cause of death (COD) at 62.8%. Infection was the second most common COD (5.9%). Controlling for sex and the presence of infection as COD, the mean age of death increased significantly by 0.08 years (p<0.001) each year over the time period studied, with men on average dying 4.3 years earlier than women (p<0.001). The mean age of death in 2005 was 43 yrs for women and 37 yrs for men. Those with COD of pulmonary hypertension, stroke and renal disease had a significantly older age of death than those without those diagnoses, while having infection as the underlying COD was associated with a younger age at death. The overall mortality rate increased by 0.7% (p<0.001) each year over the time period studied. Mortality rates for adults and children over time are shown in Figure 1. The adult (>19 yrs) mortality rate increased by 1% (p<0.001) each year over the time period studied. The pediatric mortality rate decreased by 3% (p<0.001) each year over the time period studied. When controlling for the pediatric mortality rate the adult mortality rate increased by 1.6% (p<0.001) each year. This data confirms prior studies showing a significant decrease in mortality for children with sickle cell disease over the last 30 yrs. The mortality rate for the adult population appears to be steadily increasing over the same time period. It seems unlikely that this is due merely to an influx of younger patients surviving to adulthood. Further investigation as to the cause of the increasing mortality rate in adults is needed.

Disclosures:

No relevant conflicts of interest to declare.
Statin Treatment Inhibits FLT3 Signaling by Preventing Glycosylation of the Mature Receptor and Leads to Cell Death In Mutant FLT3 Expressing Cells
Authors: Williams Allen , Nguyen Bao , Li Li , Brown Patrick , Levis Mark J. , Small Donald
Published: October 2019

Abstract 779

FLT3 is a tyrosine kinase receptor expressed on the surface of hematopoietic stem cells/progenitors and contributes to leukemogenesis as evidenced by its frequent mutation in acute myeloid leukemia (AML). FLT3 normally undergoes N-linked glycosylation to produce an immature 130 Kd protein in the endoplasmic reticulum followed by further processing in the Golgi to form the final 160 Kd mature glycoprotein that translocates to the surface. At the surface, FLT3 binds to FLT3 ligand (FL) resulting in phosphorylation and activation of multiple signaling cascades. FLT3 activating mutations primarily occur as internal tandem duplications (ITD) within the juxtamembrane domain or as point mutations in the kinase domain and have become the target of numerous small molecule tyrosine kinase inhibitors (TKI), several of which have entered clinical trials for AML. However, clinical trials to date have shown limited success due to a variety of reasons including the emergence of resistance mediated by alternative pathway activation, the acquisition of point mutations, expression of increased FLT3 ligand levels and poor inhibition of FLT3 kinase activity for a number of reasons. Statins are a well-defined class of drugs that have been safely used for treatment of hypercholesterolemia. Here, we show for the first time that statins can inhibit constitutive activation of both kinase domain and ITD mutants. Western blotting demonstrates that fluvastatin inhibits FLT3 phosphorylation by preventing receptor glycosylation, initially leading to loss of the mature receptor followed by instability of the receptor and subsequent degradation of FLT3 protein and ultimately, cell death. MTT analysis indicates that BaF3 cells transfected with FLT3/ITD are killed in a dose-dependent fashion by fluvastatin with an IC50 of <1 uM, a level which has been achieved in clinical trials. Annexin V binding confirms that BaF3/ITD cells undergo apoptosis when treated with Fluvastatin. Fluvastatin treatment causes a reduction in surface FLT3 expression as measured by flow cytometry that parallels induction of apoptosis. This finding is consistent with results obtained by immunofluoresence microsopy showing increased intracellular retention of FLT3/ITD. Additionally, fluvastatin eliminates the 2–3 fold shift in the dose-response to TKI seen when FLT3/ITD expressing cells are stimulated by FL. Also, a number of mutations that confer high level resistance to TKI are overcome by fluvastatin. These results demonstrate that statins, a class of drugs already clinically approved, might be useful in the management of AML cases in which FLT3 is over expressed, mutated or resistant to other clinically available inhibitors, either alone or more likely in combination with FLT3 TKI.

Disclosures:

Levis: Ambit Biosciences, Inc: Consultancy.
Statin Treatment Reduces the Incidence of Recurrent Pulmonary Embolism: a Population Based Case-Control Study
Authors: Biere-Rafi Sara , Gerdes Victor , Hutten Barbara , Squizzato Alessandro , Ageno Walter , Souverein Patrick , de Boer Anthonius , Buller Harry Roger , Kamphuisen Pieter
Published: October 2019

Abstract 808

Background:

Recurrence of pulmonary embolism (PE) has a high incidence of 5–10% and is associated with increased mortality. Long-term anticoagulant treatment is however associated with an increased bleeding risk. It is therefore essential to investigate other treatment strategies to reduce the long-term recurrence of PE. Statins seem to reduce a first episode of thrombosis, but whether they also have an effect on recurrent pulmonary embolism (PE) is unknown.

Methods:

A case-control study was conducted using data from the PHARMO Record Linkage System, a Dutch population-based registry of pharmacy records linked with hospital discharge records. Cases were patients hospitalized with a primary diagnosis of PE between 1998 and 2006. Four controls without a history of PE were matched to each case for age, gender and geographic region.

Results:

The study population consisted of 4.495 PE cases and 16.802 controls. The median age of the study population was 60 years (range 18–96) and 57% was female. Overall, 9% of both cases and controls used statins (odds ratio (OR) 0.95; 95% confidence interval (CI) 0.84–1.07); only rosuvastatin use reduced PE (OR 0.47; 95% CI 0.24–0.92). During a median follow-up period of 4.1 years (range 2.2–6.6), 396 (11%) patients had a recurrent PE. Statin treatment strongly reduced the incidence of recurrent PE (HR 0.57; 95% CI 0.42–0.79), which persisted after adjustment for vitamin K antagonists, duration of statin use, type of VTE or cardiovascular history.

Conclusions:

Statin treatment seems to be highly effective in the reduction of recurrent pulmonary embolism. Considering the low rate of side effects, statins may be an interesting option for long-term secondary prevention in patients with PE.

Disclosures:

No relevant conflicts of interest to declare.
CXCR4-SDF-1 Signaling Mediated up-Regulation of Survivin Expression In Mesenchymal Progenitor Cells Is Critical for Their Proliferation and Maintenance of Osteoblastic Niche
Authors: Singh Pratibha , Speth Jennifer , Hu Peirong , Pelus Louis M.
Published: October 2019

Abstract 941

Hematopoietic stem cells reside in osteoblastic and vascular niches within the bone marrow. The osteoblastic niche is composed of mesenchymal stem cell derived progenitor cells (MPC) and osteoblasts and are the main sources of the CXC chemokine CXCL12/SDF-1 in the bone marrow microenvironment. Several published studies suggest that the interaction between CXCR4 expressed on hematopoietic stem cells with SDF-1 produced in the bone marrow microenvironment is important for their retention in the bone-marrow. However, the role of SDF-CXCR4 signaling in formation and maintenance of osteoblastic niches in the bone marrow is not known. In this study, we examined the role of CXCR4 signaling in MPC proliferation and differentiation and its effects on hematopoietic stem cell (HSC) function. Flow cytometry analysis demonstrated that CXCR4 is expressed on the phenotypically defined MPC. Deletion of CXCR4 in tamoxifen cre inducible CXCR4flox-flox mice (verified by PCR and flow cytometry; 90% gene deletion and surface CXCR4 expression) results in significantly decreased numbers of Lin- CD45- CD31- Sca-1+ ALCAM- MPC (39±4.2%) and Lin- CD45- CD31- Sca-1-CD51+ osteoblasts (25±2.6%) in bone marrow 15 days after tamoxifen treatment. SDF-1 induced proliferation of CXCR4 deficient MPC was decreased by 4-fold compared to control, measured by the colony forming unit-fibroblast (CFU-F) assay. To determine, whether CXCR4 deficiency in bone marrow stromal cells affects SDF-1 induced HSC proliferation, we cultured FACS sorted wild-type SLAM SKL (103 cells) on CXCR4 deficient stroma for 5 days and total SLAM SKL cell numbers were counted by flow-cytometey analysis. CXCR4 deficient stroma failed to support optimal HSC proliferation and 48±5.2% less SLAM KSL cells was observed on CXCR4 deficient stroma compared to wild-type stroma. To investigate the mechanisms through which CXCR4-SDF-1 signaling regulates MPC proliferation, we evaluated the effect of SDF-1 treatment on expression of the anti-apoptotic and cell-cycle regulator protein, Survivin, in MPC. Multivariate intracellular flow cytometry demonstrated that Survivin expression increased by 23±4.2% in wild-type MPC after SDF-1 treatment (50ng/ml), however no significant increased was demonstrated in CXCR4 deficient MPC cells. CFU-F formation was reduced by 2.5 fold when the Survivin gene was conditionally deleted in MPC. Moreover, fewer SLAM SKL cells were detected on Survivin deficient stroma compared to wild-type stroma after SDF-1 treatment for 5 days. In conclusion, our data suggest that CXCR4-SDF-1 signaling mediated Survivin expression in MPC is important for their proliferation and maintenance of the bone-marrow hematopoietic niche.

Disclosures:

No relevant conflicts of interest to declare.
A Unique New Humanized Mouse Model for Multiple Myeloma (MM): Opportunities for Studying MM In Its Natural Environment and for Preclinical Testing
Authors: Groen Richard W , Hofhuis Frans M , Aalders Linda , van Kessel Berris , de Weers Michel , Mutis Tuna , Lokhorst Henk , Martens Anton C
Published: October 2019

Abstract 981

Here we report the development of a unique mouse model to study the pathobiology of multiple myeloma (MM) by implementing a technology for creating a natural human bone environment in the immune deficient RAG2γ-/-c-/- mouse. To this end we combined a procedure to culture-expand human BM-derived mesenchymal stromal cells (MSC) that were seeded on biphasic calcium phosphate (BCP) particles and subsequently implanted subcutaneously in RAG2γ-/-c-/- mice. Within 6 weeks this leads to the formation of so-called ossicles that contain substantial amounts of human bone, while the remaining open spaces are filled with mouse hematopoietic cells and blood vessels, creating an environment that strongly resembles the human bone marrow. A striking finding was that this humanized environment in the mouse acts as a 3-D natural “niche” for patient MM (pMM) cells. Intrascaffold injection of pMM cells resulted in engraftment and outgrowth of tumor cells in close contact with the human bone layer in the ossicles. In addition, intracardial injection, revealed that these primary tumor cells were also capable of homing to the implanted artificial BM-niches, while no tumor cells were detected in the mouse BM. The outgrowth of pMM in this model is accompanied by an increase in osteoclast number on the bone surface, indicating the presence of bone resorption, one of the most important clinical sequelae of MM. Interestingly, by gene-marking pMM cells with luciferase and using bioluminescent imaging, we were able to follow myeloma outgrowth in time, and visualize the effect of treatment. Hence, this novel humanized mouse model provides the first opportunity to investigate patient-derived MM plasma cells in their natural environment, which may lead to better insights in the pathogenesis of this disease. Furthermore, it could serve as a model for preclinical testing of new therapeutic approaches for the treatment of MM patients.

Disclosures:

de Weers: Genmab bv: Employment.
MicroRNA Regulation of Immune Cell Development and Function
Authors: Rao Dinesh S.
Published: October 2019

Abstract SCI-31

The NF-kappa B pathway is a central mediator of inflammation and plays an important role in myeloid cell development. During activation of macrophages by LPS, three microRNAs (miRNAs) were initially identified as being upregulated in an NF-kappa B dependent manner. Subsequent efforts to characterize two of these miRNAs, namely miR-155 and miR-146a, have revealed important roles not only in inflammation but also in hematopoietic development. Curiously, these two miRNAs seem to play opposing roles in promoting immune cell and progenitor proliferation, and the phenotypes induced in gain and loss-of-function contexts will be discussed in detail. Specifically, miR-155 overexpression and miR-146a deficiency lead to myeloid proliferations with pathologic consequences. These differential roles are largely explained by the targets of these microRNAs, which include signal transduction regulators: SHIP1 and SOCS1 in the case of miR-155, and TRAF6 and IRAK1 in the case of miR-146a. The roles of these miRNAs as effector and feedback regulator of the NF-kappa B pathway, respectively, will be discussed in the context of myeloid development. More broadly, these studies, as well as studies of miRNAs in B-cell development, have revealed that understanding miRNA roles in these processes will illuminate new aspects of biology and pathology. For example, the regulation of hematopoietic development by these miRNAs has revealed important interconnections between pathways that previously may have been considered disparate. Moreover, these studies have begun to underscore the importance of identifying specific targets of a miRNA in a given physiologic or pathologic context.

Disclosures:

No relevant conflicts of interest to declare.
Pathways Regulating Blood Cell Production
Authors: Hilton Douglas J.
Published: October 2019

Abstract SCI-41

Our current research takes genetic, genomic and bioinformatic approaches to identify gene networks important in regulating haemopoiesis; the production of nine lineages of blood cells from multipotential, self-renewing stem cells. In this presentation I will discuss our unpublished experiments aimed at elucidating pathways that regulate megakaryocyte-lineage commitment.

Disclosures:

Hilton: CSL: Research Funding; Murigen: Equity Ownership.
CEBPA Single Nucleotide Polymorphism (SNP) rs34529039 As An Independent Adverse Prognostic Factor in Acute Myeloid Leukemia Treated with Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Aljurf Mahmoud , Abalkhail Hala , Owaidah Tarek , Almhareb Fahed , Chaudhri Naeem A. , Walter Claudia Ulrike , Toulimat Mohammed , Al Zahrani Hazzaa , Mohamed Said , Rasheed Walid , Alsharif Fahad , Albitar Maher
Published: October 2019

Abstract 1465

The gene for the transcription factor CCAAT/enhancer binding protein α (CEBPA) is important for the differentiation of granulocytes and has been reported to be mutated in patients with acute myeloid leukemia (AML). Double mutation of CEBPA has been shown to be associated with good outcome in the cytogenetic intermediate-risk subgroup. Multiple single nucleotide polymorphisms (SNPs) have been reported in this gene in addition to a 6-nucleotide insertion. Most of the studies of prognostic relevance of CEBPA have been reported in patients treated with standard chemotherapy focusing on the mutations in the CEBPA gene. The prognostic significance of CEBPA is not fully studied in AML patients treated with intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (HSCT) in their first complete remission (CR1). We studied the prognostic value of the CEBPA mutations and the SNPs in patients with AML treated with HSCT in CR1.

Methods:

Samples from 69 patients with AML were analyzed for CEBPA mutations by direct sequencing of the entire coding sequence of the gene. All patients were diagnosed with AML, treated with intensive chemotherapy, then received HSCT in CR1. The group included intermediate (N=42) and adverse (N=27) cytogenetic risk groups.

Results:

Double CEBPA mutations were detected in 7 patients (10%). However, silent G to T (690g>t) transition in rs34529039 SNP (silent T230) was detected in 15 patients (22%). The same genotype was detected in 17% of normal control individuals. The prevalence of rs34529039 T genotype in our patient population was similar to that detected in the United State population (Quest Diagnostics data: 17% in normal controls and 28% in AML patients). Outcome in patients with double mutations did not differ significantly in event free survival (EFS) or overall survival (OS) in this group of patients, although the number of patients with double mutation is too small. In contrast, the T rs34529039 genotype was significantly associated with shorter EFS (P=0.03) and time-to-relapse (P=0.006). The same trend is seen for OS, although the P-value is not significant due to very few events. When we excluded patients with adverse cytogenetic abnormalities, similar results were obtained and EFS was significantly poor in patients with T phenotype (P=0.04 for EFS).

Conclusion:

The data suggests that HSCT after intensive chemotherapy in CR1 may neutralize the prognostic value of CEBPA double mutations. However, the rs34529039 is a very important independent adverse prognostic factor in patients with AML and should be used in stratifying patients.

Disclosures:

No relevant conflicts of interest to declare.
Early Clearance of Peripheral Blood Blasts but Not White Blood Cells Is a Powerful Prognostic marker for complete Response and Overall Survival in Patients with Acute Myeloid Leukemia (AML) receiving Induction Chemotherapy
Authors: Quintas-Cardama Alfonso , Kantarjian Hagop , Garcia-Manero Guillermo , Brandt Mark , Pierce Sherry , Borthakur Gautam , Cortes Jorge E.
Published: October 2019

Abstract 1553

Background:

Most pts with AML will achieve complete remission (CR) following therapy with cytarabine and an anthracycline. Early clearance of blasts in peripheral blood (PB) has been shown to be an important prognostic factor in acute lymphoblastic leukemia. We explored the dynamics of PB blasts in pts with AML undergoing induction chemotherapy with AI (ara-C 1.5g/m2x3d and idarubicin 12mg/m2x3d) at our institution and their impact in long-term outcomes.

Patients & Methods:

We reviewed the dynamics of PB blasts (i.e. PB blasts=0%) or WBC (i.e. WBC≤0.1×109/dL) clearance in pts with AML receiving AI (n=486). Patients with acute promyelocytic leukemia were excluded. Pt characteristics were as follows: median age 55 yrs (range, 19–73), pts <60 yrs 44 (30%), diploid cytogenetics (n=54, 36%), poor cytogenetics (n=57, 38%), median WBC 4.9 (range, 0.3–97.4), platelets 39 (range, 2–581), hemoglobin 8.4g/dL (range, 3.3–13.2), PB blasts (15% (range, 1–96), BM blasts 45% (1–96).

Results:

The overall response rate (ORR=CR+CRp) was 65% (58%+7%). Forty-four (30%) pts were resistant to induction therapy. To evaluate the dynamics of PB WBC and PB blast clearance, we divided all pts in 5 groups depending on the day they cleared their WBC or blasts from PB: group 1 (0–1 days), 2 (2–3 days), 3 (4–5 days), 4 (6–8 days), and 5 (beyond day 8). A total of 21, 34, 25, 4, and 3 pts were included in groups 1 through 5. No differences in CR rates were observed across all 5 groups according to the time of clearance of PB WBC (p=0.653). Likewise, similar median overall survival (OS) rates were observed in all 5 groups regardless of the timing of PB WBC clearance (0.2). However, the timing of PB blast clearance was associated with a distinct probability of achieving CR, being 73%, 74%, 65%, 24%, AND 60% for groups 1 through 5 (p=0.003). This translated into distinctly different median OS for the 5 groups (p=0.03) (Figure 1A). When groups 1, 2, and 3 were merged and compared with the merge of groups 4 and 5, the ORR for the resulting two new groups was 71% and 32% (p<0.001) and the corresponding median OS was 40 weeks and 75 weeks (p=0.038) (Figure 1B), suggesting that early PB clearance predicts long-term outcomes.

Conclusion:

We have shown in a large cohort of uniformly treated pts with AML that early clearance of PB blasts (but not WBC) is an important risk factor for achievement of CR and for OS. Clearance of PB blasts should be considered in prognostication schemas for pts with AML.

Disclosures:

No relevant conflicts of interest to declare.
Selective Small Molecule Inhibition of P110α and δ Isoforms of PI3 Kinase Is Cytotoxic to Human AML Progenitors
Authors: Xing Yan , Gerhard Brigitte , Hogge Donna E.
Published: October 2019

Abstract 1556

P110α, β, γ and δ isoforms are catalytic domains of phosphoinosityl-3-kinase (PI-3K) which is frequently constitutively active in blast cells from acute myeloid leukemia (AML) patients. RNA and protein from all 4 isoforms were ubiquitously expressed in 32 AML samples which also showed expression of p-Akt Ser473, indicating PI-3K activation. Treatment of AML patient blast cells (n=45) with the p110α selective inhibitor PI3-Kinase alpha inhibitor 2 and p110δ selective inhibitor PCN5603 (Piramed Pharma,UK) caused dose dependent kill of AML colony forming cells (CFC) and inhibition of p-Akt Ser473 expression. In contrast, the P110β and p110γ selective inhibitors TGX-221 and AS-604850 showed little AML CFC kill. AML samples were more sensitive to PI3-Kinase alpha inhibitor 2 and PCN5603 killing than normal bone marrow or normal peripheral blood (Median IC50=1.8μM for 45 AML-CFC and 4.3μM for 8 normal CFC for PI3-Kinase alpha inhibitor 2. Median IC50=1.9μM for 45 AML-CFC and 6.2μM for 10 normal CFC for PCN5603). There was no significant difference between the IC50 and IC90 of p110α and δ inhibitors against AML CFC from samples with or without the FLT3 ITD. There was a weak correlation between the IC50 of the PI3-Kinase alpha inhibitor 2 or the IC50 of PCN5603 and the ratio of pAkt/total Akt (R2 = 0.23 and 0.24, respectively, p<0.05). RNAi inhibition of p110α and p110δ decreased the corresponding isoform's RNA and protein expression and caused AML CFC kill and inhibition of p-Akt Ser473 expression. There was a strong correlation between the percent AML CFC kill and RNA knockdown (R2 = 0.67) and between inhibition of p-Akt and p110 protein expression (R2 = 0.90 for α and 0.82 for δ). Thus, p110α and δ inhibition may achieve selective targeting of malignant rather than normal CFCs. Furthermore, treatment of AML cells with PCN5603, decreased survival of AML long-term culture- initiating cells (AML LTC-IC). In contrast, less toxicity toward normal bone marrow LTC-IC was observed (IC50=0.08μM, ≤2μM and 1.33μM for 3 AML samples and IC50=3.09μM, 2.63μM, >10μM and >10μM for 4 normal bone marrows). Selective inhibition of p110α and δ isforms of PI-3K kills AML progenitors including candidate leukemic stem cells (AML LTC-IC) with relative sparing of analogous normal cells suggesting that these molecules could be therapeutic targets in AML.

Disclosures:

No relevant conflicts of interest to declare.
Retherapy with Bendamustine-Containing Regimens in Patients with Relapsed/Refractory CLL and Indolent Lymphomas Achieves High Response Rates
Authors: Weide Rudolf , Feiten Stefan , Friesenhahn Vera , Heymanns Jochen , Kleboth Kristina , Thomalla Jörg , van Roye Christoph , Köppler Hubert
Published: October 2019

Abstract 1615

Purpose: Bendamustine-containing regimens have shown high response rates and long lasting remissions in relapsed/refractory indolent B-cell-malignancies. Here we have evaluated the efficacy of Bendamustine-containing regimens as retherapy in this patient population.

Patients and methods:

Patients with CLL or indolent B-cell-malignancies (NHL) who previously had been treated with Bendamustine and were retreated with either Bendamustine (90mg/m2, day 1+2, q day 29 (1–6 cycles)) (B) or Bendamustine (90mg/m2, day 1+2) + Mitoxantrone (6–10mg/m2, day 1), q day 29 (1–4 cycles) (BM) or Bendamustine (90mg/m2, day 1+2) + Rituximab (375mg/m2, day 1), q day 29 (1–6 cycles) (BR) or Bendamustine (90mg/m2, day 1+2) + Mitoxantrone (6mg/m2 day 1) + Rituximab (375mg/m2, day 8,15,22,29) (BM was repeated on day 36 × 1–3 every 4 weeks) (BMR) between 2000–2010 were analyzed retrospectively. Data were collected from patient files into a database and analyzed statistically using SPSS.

Results:

88 patients (57 CLL, 31 NHL) received a Bendamustine-retherapy-regimen. The median age at the first Bendamustine retherapy was 72 years (50–88). The mean number of therapies per patient was 6.0 in CLL and 7.0 in NHL; the mean number of Bendamustine therapies was 3.0 in CLL and 2.6 in NHL. Bendamustine-containing retherapy consisted of B in 4%, BM in 12%, BR in 29% and BMR in 55%. The overall response rate of Bendamustine retherapy was 79% (9% CR, 70% PR). ORR according to regimen was as follows: B: 57% (14% CR, 43% PR), BM: 70% (4% CR, 66% PR), BR: 55% (10% CR, 45% PR), BMR: 84% (7% CR, 78% PR). Main toxicity was a reversible grade 3 or 4 hematotoxicity in 35% of retherapies! No therapy associated death was observed. Overall survival since first diagnosis was 134 months in CLL (23–187) and 131 months in NHL (20–339). Overall survival since start of Bendamustine-containing retherapy was 33 months in CLL (2–129+) and 23 months in NHL (2–62+).

Conclusion:

Bendamustine-containing retherapy-regimens achieve high response rates in patients with relapsed/refractory CLL and NHL. Hematotoxicity is relevant but reversible.

Disclosures:

No relevant conflicts of interest to declare.
Loss of Stress Sensor GADD45a Accelerates BCR-ABL-Driven Leukemogenesis
Authors: Mukherjee Kaushiki , Sha Xiaojin , Bhatia Ravi , Hoffman Barbara , Liebermann Dan
Published: October 2019

Abstract 1668

The bcr-abl fusion oncogene causes chronic myelogenous leukemia (CML) in human. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, is upregulated during myeloid lineage terminal differentiation. It is involved in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or gadd45a null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR-ABL fusion oncoprotein. It was observed that loss of gadd45a accelerates BCR-ABL driven CML resulting in the development of a more aggressive AML like disease. BCR-ABL transformed GADD45A deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulation of p30C/EBPα and MCL-1 expression. Since Gadd45a functions as a tumor suppressor in murine BCR-Abl driven leukemia, the status of Gadd45a mRNA expression levels was also investigated in human CML samples by utilizing real time PCR analysis. It was found that Gadd45a transcript levels were significantly upregulated in chronic phase CML samples. However in accelerated and blast phase samples, expression was significantly downregulated relative to normal controls. Thus, Gadd45a expression was observed to be altered in human CML samples correlating with disease progression. These results provide novel evidence that gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis. These data also provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to a more aggressive leukemic phenotype.

Disclosures:

No relevant conflicts of interest to declare.
A Cytotoxic Human Monoclonal Antibody Recognizing Cell Surface WT1 Peptide/HLA-A2 Complexes
Authors: Scheinberg David A. , Dao Tao , Yan Su , Zhou Liang , Korontsvit Tatyana , Zakhaleva Victoriya , Curcio Michael , Liu Cheng
Published: October 2019

Abstract 1677

The Wilms' tumor oncogene protein (WT1) is an attractive target for immunotherapy for a wide range of leukemias and cancers. WT1 is an intracellular transcription factor that is widely expressed in acute and chronic leukemias, but not appreciably in normal adult tissues. WT1 has been implicated in oncogenesis and appears to be expressed in the CML stem cell. Thus far, the intracellular location of WT1 has limited its use as a cancer target to T cell-based vaccine therapies. Several such vaccines directed to WT1 peptides are in clinical trials worldwide. However, such approaches is likely to be weaker and slower acting than typical antibody therapies that act directly to kill cells, especially for a leukemia that is not in remission. The WT1 peptide (RMFPNAPYL) is presented by HLA-A0201 and induces cytotoxic CD8 T cells capable of killing WT1+ leukemia cells. Therefore, we hypothesized that a monoclonal antibody (mAb) specific for WT1 peptide/HLA-A2 complexes on the cell surface (a “TCR-like antibody”) could be an effective direct therapeutic agent alone, or armed with potent anti-cancer agents. In addition, such an antibody would provide a potential tool for identifying appropriate patients for clinical trials of both WT1 vaccine and WT1 mAb therapies, or as a prognostic tool. Using phage-display technology, we selected a number of human scFv phage mAbs specific for WT1 peptide RMFPNAPYL, when bound in the context of HLA-A0201 molecules. Bivalent, full-length human IgG1 mAbs were next constructed and tested for biochemical and immunological functions. The scFv phage mAbs bound specifically to T2 cells (TAP deficient) pulsed with WT1 peptide RMFPNAPYL, but not other HLA-A0201-binding peptides, demonstrating specific recognition of the WT1peptide/HLA-A0201 complex. Flow cytometry and radio-immunoassay was used to quantify sites, determine avidity, and show specificity. The IgG1 mAb showed high binding affinity (Kd= 0.2 nM) for the WT1 peptide/A0201 complex on live cells. The IgG1 mAb also recognized leukemia and tumor cell lines (n= 8 of 12) that expressed WT1, in an HLA-A0201, WT1 peptide-restricted manner, but not WT1-negative or HLA-A02-negative cells (n=13). The mAb was capable of killing WT1 peptide-pulsed T2 cells, as well as un-pulsed WT1+, A0201 + cancer cell lines, demonstrating presentation of the epitope in sufficient numbers for therapeutic attack. Complement mediated killing of cells was not seen. Alanine scanning of the epitope pointed to peptide position #1 as critical. In conclusion, cytotoxic human monoclonal antibodies targeting WT1 peptide/HLA complexes represent a novel therapeutic approach to target leukemias and solid tumors that over-express the intracellular WT1 oncoprotein.

Disclosures:

Yan: eureka therapeutics: Employment. Zhou:eureka therapeutics: Employment. Liu:eureka therapeutics: Employment.
Protein Disulfide Isomerase Plays An Important Role in Beta2 Integrin-Mediated Neutrophil Recruitment During Vascular Inflammation
Authors: Hahm Eunsil , Huh Sungjin , Rogelj Snezna , Cho Jaehyung
Published: October 2019

Abstract 17

Cell surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, plays an important role in regulating integrin-mediated cellular functions. Although surface PDI has been reported to regulate L-selectin shedding on neutrophils, its role in neutrophil recruitment during vascular inflammation has not been explored. In this paper, we present novel findings on how neutrophil PDI regulates neutrophil recruitment into the site of vascular inflammation. Using real-time microscopy with cell-impermeable inhibitors for PDI (bacitracin or function-blocking antibodies), we demonstrated that inhibition of PDI reduces adhesion of human blood neutrophils to TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under venous shear. There was no additive effect on neutrophil adhesion in a combination of blocking anti-PDI and anti-beta2 antibodies, suggesting that surface PDI regulates beta2 integrin-mediated neutrophil adhesion to TNF-alpha-activated HUVECs under shear. Further, bacitracin and blocking anti-PDI antibodies diminished alphaMbeta2 integrin-mediated neutrophil adhesion to intercellular adhesion molecule-1 (ICAM-1)-coated surfaces under static conditions. When PDI gene is knocked down in neutrophil-like HL60 cells by shRNA, alphaMbeta2 activation and cell adhesion to TNF-alpha-activated HUVECs under shear were significantly reduced. High resolution confocal microscopy and immunoprecipitation assay revealed that surface PDI interacts with alphaMbeta2 integrin and that such interaction was enhanced by neutrophil activation and inhibited by bacitracin, a thiol isomerase inhibitor. Exogenous PDI equivalently bound to CHO cells stably expressing alphaMbeta2 or alphaLbeta2 when those cells were treated with Mn2+, whereas PDI binding was significantly reduced when the active site CGHC residues on PDI were mutated. Using multi-channel fluorescence intravital microscopy, we demonstrated that inhibition of PDI by infusion of PDI inhibitors impairs stable adhesion of neutrophils to the TNF-alpha-inflamed cremaster muscle venule wall in living mice. These findings suggest the potentially fundamental but poorly understood role for surface PDI in the regulation of beta2 integrin function in neutrophils. Therefore, our results provide the first evidence that inhibition of PDI may be a therapeutic target for the neutrophil-mediated vascular inflammation.

Disclosures:

No relevant conflicts of interest to declare.
Immunoglobulin Free Light Chain Levels Predict Survival in Primary Myelofibrosis and De Novo Myelodysplastic Syndromes
Authors: Pardanani Animesh , Lasho Terra L , Finke Christy , Rajkumar S. Vincent , Singh Preet Paul , Ketterling Rhett P , Hanson Curtis A. , Katzmann Jerry A , Tefferi Ayalew
Published: October 2019

Abstract 1756

Background:

We hypothesized that surrogate markers of host immune response to clonal myeloproliferation predict survival in myeloid malignancies. Because of immediate applicability to current practice, we chose plasma immunoglobulin free light chain (FLC) concentration (B-cell activation marker) as the biomarker of interest.

Patients and Methods:

Two independent cohorts of patients with primary myelofibrosis (PMF) or myelodysplastic syndromes (MDS) were studied. Both groups were fully annotated for karyotype. Kappa (κ) and lambda (λ) FLC were measured by a quantitative nephelometric assay. Patients with monoclonal FLC were excluded.

Results:

Above upper normal-limit values for κ or λ FLC was documented in 33% of 240 study patients with PMF and 46% of 74 patients with MDS. Multivariable analysis revealed significant associations between increased FLC and elevated creatinine, as well as advanced age in PMF (p=0.0004) and hemoglobin <10 g/dL in MDS (p=0.005). Increased FLC predicted shortened survival in both PMF and MDS, independent of age, creatinine, and other conventional risk factors. Receiver-operating characteristic analysis-based cutoff levels for κ plus λ total FLC delineated risk groups with highly significant differences in overall survival (Figure); International Prognostic Scoring System-adjusted HR (95% CI) in PMF was 1.9 (1.3-2.7) and in MDS 6.3 (2.7-16.6). No correlations were seen with leukemia-free survival, karyotype or JAK2, MPL or IDH mutations.

Conclusions:

Elevated plasma FLC concentration predicts inferior survival in both PMF and MDS. Its lack of correlation with leukemia-free survival and tumor-specific genetic markers suggests a primarily host-driven biological phenomenon that might be applicable to other malignant and non-malignant conditions.

Disclosures:

No relevant conflicts of interest to declare.