bluuebunny/crossref_metadata_2025 huggingface dataset:
Dysregulated Choline Metabolism in T-Cell Lymphoma and Its Relationship to Oncogenesis
Authors: Xiong Jie , Wang Li , Zhao Weili
Published: October 2019

T-cell lymphoma (TCL) is the malignant proliferation of CD3+ lymphocytes and represents the main type of non-Hodgkin's lymphoma. TCL patients are generally resistant to conventional chemotherapy and have poor clinical outcome. Therefore, biomarker commonly expressed and closely related to tumor progression needs to be further investigated in TCL, helping to develop targeted therapeutic approaches and to eventually improve prognosis of the patients.

Growing evidences show that metabolic enzymes regulate key oncogenic signaling pathways and play an essential role on tumor progression. Here serum metabolomic analysis was performed in 45 patients with T-cell lymphoma (TCL) and 50 healthy volunteers, and the results showed that dysregulation of choline metabolism occurred in TCL. (Figure 1A-B) Further verified by real-time PCR, choline kinase-¦Á (CHKA), the main regulatory enzyme of choline metabolism, was overexpressed in patients with TCL, but without significant difference in the TCL subtypes. (Figure 1C-D)

In T-lymphoma cells, pharmacological and molecular silencing of Chok¦Á significantly decreased Ras-GTP activity, AKT and ERK phosphorylation, MYC oncoprotein expression (Figure 2A-C), leading to induction of tumor cell apoptosis and necropotosis (Figure 2D-H). Clinically, gene expression profile analyzed in tissue samples of TCL patients showed that altered genes were enriched in PI3K and MAPK pathways according to CHKA expression of the TCL cases (Figure 2I).

In a T-lymphoma xenograft murine model, Chok¦Á inhibitor CK37 remarkably retarded tumor growth (Figure 3A), and SUV intensity of the CK37-treated tumors was significantly decreased on the figure of 18F-FDG small-animal PET/CT (Figure 3B). As in vitro study, comparing with the untreated group, p-AKT, p-ERK, MYC expression, and activity of Ras-GTP were significantly decreased, while in situ cell apoptosis/necropotosis increased in the tumor of the CK37 group. (Figure 3C-F)

To our knowledge, our report is the first time to analyze serum metabolomic profile in TCL patients, and provides direct evidence of aberrant choline metabolism in this disease. Importantly, dysregulation of choline metabolism was present in all histological types of TCL studied, consistent with acute myeloid leukemia that metabolomic status is independent on morphological classification or genetic feature. Therefore, manifested with a heterogeneous group of disease, TCL may share the similar profile of disturbed choline metabolism and this metabolic pathway can be exploited as a source of biomarkers and a potential therapeutic target in TCL.

Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3.

Disclosures

No relevant conflicts of interest to declare.
Increasing TCR Zeta Expression and Maintaining the Clonality of T Cells from AML Patients after IL-2, IL-7 and IL-12 Induction
Authors: Shi Li , Chen Shaohua , Yang Lijian , Lu Yuhong , Luo Gengxin , Zhu Kanger , Li Yangqiu
Published: October 2019

T-cell immunodeficiency is a common feature in patients with leukemia, lower activation of T cells was one of reasons. The TCR zeta chain has emerged as a key subunit of the T-cell antigen receptor, which plays a central role in the signal-transducing events leading to T cell activation. The proliferation and activation of T cells may be inducted by T cell related cytokines. In this study, we explored the change of TCR zeta gene expression and the clonality of T cells after induction with different immune cytokines, including IL-2, IL-7 or IL-12. CD3+ T cells sorted from peripheral blood of 4 cases with AML were induced with different immune cytokines, including IL-2, IL-7, IL-12, anti-CD3 and anti-CD28 antibodies in vitro. The expression levels of TCR zeta gene and related genes in T cells before and after induction were then analyzed by fluorescence quantitative RT-PCR. The distribution and clonality of TCR Vβ subfamily T cells were analyzed by RT-PCR and Genescan techniques. Increasing expression levels of TCR zeta gene and zap-70 (TCR zeta chain associated-protein) gene in CD3+T cells from AML patients were found after induction with single stimulating factor or the combination with different cytokines, while the expression of FcεRIγ (TCR ζ gene complementary factor) was down-regulated. We further compared the T cell clonality in CD3+T cells from AML patients after cytokine induction, eight to 22 TCR V β subfamilies could be detected in T cells from AML cases, most of them displayed polyclonal expansion. The number of expressed TCR Vβ subfamilies was increased without the change of clonality in T cells induced by CD3+CD28+IL7. In conclusion, TCR zeta gene and its related genes could be upregulated through induction with different cytokine combination such as IL-2, IL-7 and IL-12, therefore to improve the T cell activation in patients with AML. And the main effect of cytokines might to maintain the T cell clonality and nonspecific amplification of T cell clones. Further investigation can be designed to amplify the specific anti-AML TCR Vβ clones using AML associated antigens and such cytokine combination.

Disclosures

Shi: National Natural Science Foundation of China (no. 81100353, 81270604), the Fundamental Research Funds for the Central Universities (No. 21611447, 21612116), And Medical Science Foundation of Guangdong Province(A2011325). : Research Funding.
Treatment Reality of Patients with Immune Thrombocytopenia (ITP) in Routine Care
Authors: Weide Rudolf , Feiten Stefan , Friesenhahn Vera , Heymanns Jochen , Kleboth Kristina , Thomalla Jörg , van Roye Christoph , Köppler Hubert
Published: October 2019

Introduction: New treatment options like thrombopoietin receptor agonists (TRAs) and rituximab have been introduced into the clinic which have found their way into national and international treatment guidelines. The aim of this study was to answer the following questions concerning diagnosis and treatment of patients with ITP in routine care: How are patients diagnosed and treated?Which sequences of therapy are applied?How many patients die due to bleeding complications?

Methods: All patients with ITP diagnosed between 06/1995-06/2014 in a community-based oncology group practice in Germany were analyzed retrospectively.

Results: 402 patients with a median age of 55 (7-90) were evaluated. 57% were female and 43% male. 357 (89%) were classified as primary ITP and 311 (77%) as having chronic ITP. In 234 patients (58%) a bone marrow biopsy was part of the diagnostic work up. Only 191 patients (48%) needed therapy. First line therapy (n=191) were steroids in 81%, intravenous immunoglobulins (ivIgG) in 12% and ivIgG plus steroids in 6%. Second line therapy (n=102) were ivIgG in 49%, steroids in 23%, ivIgG plus steroids in 17%, other immunosuppressive agents in 11% and splenectomy in 5%. Third line therapy (n=63) was splenectomy in 22%, other immunosuppressive agents in 27%, steroids in 19%, ivIgG in 16%, 11% combination therapy, rituximab in 10% and TRAs in 5%. Fourth line therapy (n=38) consisted of steroids in 26%, splenectomy in 26%, immunosuppressive agents in 34%, ivIgG in 13%, rituximab in 11% and TRAs in 3%. Patients received a median of 2 lines (1-10) of therapy. Treatment modalities most frequently used were steroids in 93%, immunoglobulins in 56%, splenectomy in 21% and other immunosuppressive agents in 21% of patients. Rituximab and TRAs were used in 10% and 5% only. 75% of patients received a durable remission (complete or partial) after their last therapy. 10% showed no response, in 15% remission couldn't be evaluated due to external treatments. 146 patients (76%) are off treatment. 1 patient (0.2%) died due to bleeding complications.

Conclusions: Bone marrow biopsy is used as a diagnostic procedure in 58% of patients. Treatment modalities most frequently used are steroids, immunoglobulins, splenectomy and other immunosuppressive agents. Rituximab and TRAs are used infrequently. A high percentage of ITP-patients achieve durable remissions and ITP-related mortality is low.

Disclosures

No relevant conflicts of interest to declare.
Targeted DNA Sequencing for Hemophilia A, B, and von Willebrand Disease: Analytical Precision in Clinical Laboratories and Improved Disease Characterization
Authors: Severino Patricia , Oliveira Liliane Santana , Torres Natalia , Guerra Joao Carlos , Hamerschlak Nelson , Rezende Suely M
Published: October 2019

Hemophilia A, B, and von Willebrand disease correspond to more than 90% of all inherited bleeding disorders associated with coagulation factor deficiencies. Symptoms between these deficiencies may vary greatly and yet are often phenotypically similar. Bleeding episodes can range from mild to severe, at times with life threatening hemorrhages. Currently, biochemical assays are performed to assess the function of each coagulation factor, but diagnosis remains cumbersome and prone to multiple sources of variability between laboratories. Genetic evaluation allows for the examination of multiple coagulation factor genes simultaneously and may quickly identify possible causes to the disease. Additionally, genetic testing should be more reproducible and readily comparable between clinical laboratories. In this work we evaluate the potential use of targeted sequencing of three coagulation factors genes – F8, F9 and VWF – for the concurrent diagnosis and characterization of hemophilia A, B, and von Willebrand disease samples. For targeted DNA sequencing we selected specific DNA probes using genomic coordinates spanning the complete intronic and exonic regions of the three genes, as well as flanking gene sequences. Eleven hemophilia A samples and four hemophilia B samples, clinically characterized and submitted to Sanger sequencing for F8 and F9 genes coding regions, respectively, were included in this study. Our results indicate that even though DNA quality may be ideal for traditional DNA sequencing, enrichment techniques require more intact fragments, as reflected by variations in sequencing coverage between samples: quadruplicate results per sample showed 100X coverage varying from 80% of sequenced regions to less then 20%. Point substitutions found in F9 genes by Sanger sequencing were confirmed by targeted sequencing, but results for F8 gene were less satisfactory, in agreement with probe design limitations at this point. Of interest for hemophilia A patients, four samples possessed, in addition to the alterations in F8, point mutations in VWF. Probe design and sequencing parameters did not allow for the identification of F8 intron 1 and intron 22 inversions, frequent alterations in hemophilia A, but optimization procedures are currently underway. We conclude that targeted sequencing approach may be a viable and more complete solution for the diagnosis and management of hemophilia A, B and von Willebrand disease.

Disclosures

No relevant conflicts of interest to declare.
The PUP-GCP Clinical Trial: A Low Inhibitor Rate in Previously Untreated Patients with Severe Hemophilia a Treated with Octanate
Authors: Knaub Sigurd , Klukowska Anna , Komrska Vladimir , Laguna Pawel , Vladimir Vdovin , Jansen Martina
Published: October 2019

Background :

Octanate is a highly purified, double virus inactivated, human plasma-derived factor VIII (FVIII) concentrate with all coagulation FVIII bound to its natural stabilizer VWF in a VWF:RCo/FVIII:C ratio of approximately 0.4. A prospective clinical trial has been initiated in 2000 in order to assess the immunogenicity, efficacy and tolerability of Octanate in previously untreated patients (PUPs).

Materials and Methods:

Patients with severe hemophilia A without previous exposure to FVIII or FVIII-containing products were enrolled. Inhibitor screening, using the modified Bethesda method, was performed prior to treatment, starting every 3-4 EDs (ED 1-20), and afterwards every 10 EDs (ED 21-100), but at least every three months. Efficacy and tolerability were assessed by a 4-point verbal rating scale.

Results:

The study is clinically completed, after fifty-one subjects with severe haemophilia A, at screening 0,01-5,61 years old (median 0,65 years; median 0,99 years at treatment start) have been enrolled. Three of them developed clinically relevant high titer inhibitors over the course of the study. Another two displayed transient inhibitors that disappeared spontaneously without changing the dose or dosing interval. All inhibitors developed under on-demand treatment and before ED 50. For 4 inhibitor patients an intron 22 inversion, for 1 inhibitor patient a large deletion of exons 7 – 12 was found.

From 51 subjects, 45 exceeded 50 EDs. Octanate was well-tolerated and the adverse event profile was consistent with the population studied. The haemostatic efficacy in prophylaxis and treatment of bleeding episodes was generally rated as “excellent” and no complication was reported for any surgical treatment.

Conclusion:

Despite frequent inhibitor testing and predominant on-demand treatment, the data indicate a low overall inhibitor rate for Octanate in patients who exceeded 50 EDs (5/45) of which only 3 (6.7%) were clinically relevant.

Disclosures

Knaub: Octapharma AG: Employment. Klukowska:Octapharma AG: Investigator Other. Komrska:Octapharma AG: Investigator Other. Laguna:Octapharma AG: Investigator Other. Vladimir:Octapharma AG: Investigator Other. Jansen:Octapharma AG: Employment.
Revealing the Immune Population Hierarchy Using Novel Interactive Visualization Tools, a Comparison of Methods: Sunburst, viSNE, and Spade on Mass Cytometry Data
Authors: Chen Tiffany J , Kotecha Nikesh
Published: October 2019

The development of new technologies for high-parameter data has resulted in a critical bottleneck: identification of immune subsets is restricted to expert-based analysis, focusing on post-acquisition characterization of cell populations. Identification of cell subsets in flow cytometry has primarily focused on manual analysis, despite the fact that computational tools have proven useful for high-parameter and cross-sample comparisons. Sharing well-annotated data improves transparency and facilitates vital reproduction of results by external groups. Adoption of these new tools for immune subset discovery requires thorough collaborative investigation and validation of identified cell populations. To this end, in this study we compare the ease of discovery of immune subsets by comparing analysis through the use of three visualization tools: the sunburst hierarchy, the SPADE tree, and dimensionality reduction using viSNE. The sunburst hierarchy is a visual and interactive representation of traditional manual gating, whereas the SPADE tree is a semi-automated clustering and visualization tool for identification of cell subsets. viSNE allows interaction with high parameter data in the context of two-dimensional space where gating can be accomplished. In this study, we demonstrate the ability to automatically elucidate many immune subsets using Cytobank via an iterative analytic approach, combining computational tools (viSNE and SPADE) to recapitulate manually derived cell subsets.

Disclosures

Chen: Cytobank, Inc: Employment, Equity Ownership. Kotecha:Cytobank, Inc: Employment, Equity Ownership.
Histone Demethylase LSD1-Mediated Repression of GATA-2 Is Critical for Erythroid Differentiation
Authors: Guo Yidi , Li Yue , Huang Tianrui , Liang Dongxue , Li Zhe , Hu Xin
Published: October 2019

GATA-2 transcription factor is predominantly expressed in hematopoietic stem and progenitor cells (HS/PCs), and counteracts erythroid specific transcription factor GATA-1 to modulate proliferation and differentiation of hematopoietic cells. During hematopoietic cell differentiation, GATA-2 exhibits dynamic expression patterns, regulated by collaborations between GATA-1 and epigenetic regulators. Here, we show that histone specific demethylase1 (LSD1) regulates the expression of GATA-2 gene during erythroid differentiation. Knockdown of LSD1 leads to increased GATA-2 expression and inhibits induced MEL and embryonic stem cell differentiation. Furthermore, we demonstrate that LSD1 binds at the 1S promoter of GATA-2 gene locus, and suppresses GATA-2 expression through histone demethylation. Thus, our data reveals that LSD1 mediates erythroid cell differentiation by epigenetic modification of GATA-2 gene locus.

Disclosures

No relevant conflicts of interest to declare.
Hemogenic Endothelium-like Changes Were Derived to an Adult Human Dermal Fibroblast By Electrical Stimulation
Authors: Matsuo Takuji , Shirasaki Ryosuke , Yoko Oka , Yamamoto Tadashi , Ooi Jun , Matsumoto Kennsuke , Shirafuji Naoki
Published: October 2019

Background and Aims: We previously reported that when an adult human dermal fibroblast (HDF) was cultured with interleukin (IL)-1-beta (b) and erythropoietin (EPO), hematopoiesis-related molecules expressed. And, lymphatic duct-neogenesis genes also expressed, and vascular endothelial growth factor (VEGF)-A, and -C were produced. When anti-human VEGF-C antibody (Ab) was added to the cultures, hematopoiesis-related genes expressed; however, morphologic changes were not observed (54th ASH, 18th EHA). Reported findings on nuclear transfer reveal that electrical stimulation induces nuclear fusion and changes cell-fate. We observed the effect of electrical stimulation to IL-1-b-stimulated HDF.

Materials and Methods: HDF was cultured with IL-1-b, EPO, VEGF-A, and anti-human VEGF-C Ab for 14 days. Then, cells were suspended in electrical stimulation-buffer (0.25 M d-sorbitol, 0.1 mM Ca-acetate, 0.5 mM Mg-acetate, 1 mg/mL fatty-acid-free BSA, 0.5 mM HEPES), and incubated on ice for 10 minutes. Cells were stimulated at 110V 20mA (0.2 cm electrode, gap), and were further cultured with k/o DMEM containing 20% KSR and SCF, IL-6, FL, IGF-2, and VEGF-A. Morphological changes and expressions of vascular endothelial cell-related genes, and hematopoiesis-related ones were observed.

Results: When HDF was stimulated electrically, a few cells showed vascular endothelial cell-morphology after two days. When cells were further cultured in a hematopoietic stem cell-culturing condition, a part of endothelial cell-morphology changed to that of non-adherent hematopoietic cells or blastic colonies, in which increased expression-levels of SCL, CD41, GATA-2, CD34, and CD45 were accepted with significant statistical difference.

Discussion: Recent reports reveal that a kind of vascular endothelial cells, called hemogenic endothelium, can convert to a hematopoietic cell, and works an important role in hematopoietic stem cell-generation. We hypothesized that when cells expressed a kind of specific transcripts, some stimulation triggerred to convert morphologic changes and cell-fate as are observed in nuclear fusion. And, our observations indicated that when IL-1-b-stimulated HDF, expressing hematopoiesis-related transcripts, were stimulated electrically, cellular morphology changed. Currently, we are precisely analyzing electrically stimulated HDF’s biological characteristics.

Disclosures

No relevant conflicts of interest to declare.
Hypomethylation and Overexpression of CD70 in CD4+ T Cells of the Patients with Immuno-Related Pancytopenia
Authors: Shao Zonghong , Ren Yue , Fu Rong
Published: October 2019

Objective To study the expression of CD 70 and the methylation level of CD70 promoter in immuno-related pancytopenia (IRP) patients, and explore the role of CD70 in the pathogenesis of IRP.

Methods Thirty-five IRP patients and fifteen healthy donors were enrolled in this study. Peripheral blood mononuclear cells were isolated from venous blood by density gradient centrifugation and CD4+ T cells were isolated by immunomagnetic beads.The mRNA level and the percentage of CD70 of CD4+ T cells were measured by real-time quantitative polymerase chain reaction (RT-PCR) and flow cytometry respectively. Methylation-Specific PCR (MSP) was performed to determine the methylation level of CD70 promoter.

Results The percentage of CD70 expression on CD4+ T cells of untreated IRP patients[(7.46±1.51)%] was significantly higher than that of recovered IRP patients [(5.95±1.34) %] and normal controls [(1.83±0.6)%] and the result of recovered IRP patients was significantly higher than that of normal controls (P<0.05).The relative expressions of CD70 mRNA in CD4+ T cells were [2.314(0.200~6.084)]A[1.021 (0.135~3.434)]A [0.353 (0.008~2.258)] in three groups respectively. The differences between untreated IRP patients,recovered IRP patients and normal controls were significant (P<0.05). The CD70 promoter methylation level in CD4+ T cells of all IRP patients was significantly lower than that of normal controls (p<0.05). The expression of CD70 positively correlated to the ratio of CD5+ B cells (r=0.533, p<0.001).

Conclusions The overexpression of CD70 may lead to immunologic disarrangement and patients and it may play important role in the pathogenesis of IRP.

Disclosures

No relevant conflicts of interest to declare.
Chromatin State Shaping the Gene Expression Profiling in the Inducible Differentiation of Promyelocytic Leukemia Cells
Authors: Song Chunhua , Chung Lisa , Pan Xiaokang , Zhanjun Li , Ding Yali , Sachdev Mansi , Muthusami Sunil , Gowda Chandrika S. , Ge Zheng , Steffens Sadie , Tan Bi-Hua , Kendziorski Christina , Newton Michael , Dovat Sinisa
Published: October 2019

Background Epigenetic changes in DNA and chromatin are regarded as emerging major players for hematopoietic stem cells development and lineage differentiation. Epigenetic deregulation of gene expression leads to leukemia and reversibility of epigenetic modifications makes DNA and chromatin changes attractive targets for therapeutic intervention. The promyelocytic leukemia HL-60 cell can differentiate into microphage, granulocyte and monocyte with stimulation of phorbol myristic acid (PMA), dimethylsulfoxide (DMSO), and Vitamin D3, respectively, by affecting the gene expression on cell cycle and cell differentiation. However, how epigenetic regulation on gene expression in the inducible differentiation is still undetermined.

Methods and Results We did the microarray analysis to identify the genome-wide gene expression profile of HL-60 cells in various time points(0h, 6h, 1d,2d,4d and 6d) under the treatment of PMA, DMSO and D3 and found that around 3000 genes are significantly altered commonly in the cells upon the 3 treatments. The percentage of down-regulated genes in the all commonly altered genes is significantly higher than that of up-regulated genes, and the significantly altered genes showed significantly physical clustering on chromosome loci, indicating the epigenetic regulation involved in the regulation of the gene expression. We did observed the expression changes of epigenetic enzymes in the process and further did the ChIP-on-ChIP analysis by using the customer array tiling the selected changed genes and 5 genomic loci hybridizing with ChIP’d DNA of histone markers (H3K4me3, H3K9me3 and H3K27me3). We found that the obvious decrease of H3K4me3 binding and increase of H3K9me3 and H3K27me3 are observed in the promoter of the commonly down-regulated genes. These data indicated histone H3K4, H3K9 and H3K27 marker play important roles in shaping the chromatin state and regulating gene expression.

Conclusion We identified the signature genes controlling the inducible differentiation of promyelocytic leukemiacells and found the epigenetic mechanism regulating the gene expression in the process.

Disclosures

No relevant conflicts of interest to declare.
Leukemia Cell Differentiation upon Targeted Therapy Revealed By a Systems Biology Approach
Authors: Li Yulin , Seita Jun , Felsher Dean W , Dill David
Published: October 2019

Accurately determining the differentiation status of hematopoietic cells is crucial for understanding tumorigenesis and developing novel therapies for human leukemia. However, most current methods are subject to various biases. In this paper, we have developed a novel systems biology method, called the Lineage Maturation Index, to define the changes in differentiation state of hematopoietic cells based on comprehensive gene expression profiles. The changes in gene expression profiles of a specific hematopoietic lineage are shown asthe “lineage vector” from a point representing the gene expression of a hematopoietic stem cell (HSC) to another point representing a mature cell. By projecting the gene expression profile of a specific hematopoietic cell type onto the “lineage vector”, we can compute the LMI and define the relative differentiation state of this cell type. A shift in LMI from HSCs towards mature cell types indicates differentiation. We have confirmed the LMI method can track the differentiation and transdifferentiation of normal hematopoietic cells. Applying this method, we have shown that differentiation is a major consequence of various targeted therapies against leukemia irrespective of the driving oncogenes. Furthermore, we have used the method to screen a drug response library and discovered several drugs currently in clinical use for other purposes have potent differentiation activities in leukemia cells. Therefore, the LMI method is a robust computational approach to characterize differentiation of the hematopoietic system and discover novel differentiation therapy for leukemia.

Disclosures

No relevant conflicts of interest to declare.
Novel FLT3 Mutation Shows Dominant Negative Effects on the Wild-Type FLT3 Receptor
Authors: Bauer Julia , Sandhöfer Nadine , Hiddemann Wolfgang , Spiekermann Karsten
Published: October 2019

Background

The FMS-like tyrosine kinase-3 (FLT3) gene encodes for a receptor tyrosine kinase playing an important role in hematopoiesis. In acute leukemias it is one of the most frequently mutated genes. In this study we functionally characterized a novel frameshift deletion mutation of FLT3 found in a relapsed patient with acute myeloid leukemia (AML). The frameshift leads to a premature stop codon resulting in a truncated form of the receptor lacking most of the intracellular domains.

Material and Methods

FLT3 cDNA was expressed in the IL-3 dependent pro-B cell line Ba/F3 via a retroviral expression vector. The transduced cell lines were sorted by fluorescence-activated cell sorting (FACS). Stable expression of the receptor was confirmed on mRNA level by polymerase chain reaction (PCR) and on protein level by western blotting and cell surface expression of the receptor by flow cytometry. Cell proliferation assays were performed in presence or absence of IL-3 or FLT3-ligand. By western blotting receptor activation and its downstream signaling were analyzed. Ligand-binding of the receptor was shown via biotinylated and fluorescent FLT3-ligand-receptor-complexes by flow cytometry.

Results and Discussion

Stable expression of FLT3 Q569Vstop does not enable Ba/F3 cells to grow IL-3-independent. FLT3-ligand can still be bound by the mutated receptor but is not able to stimulate receptors´ signaling and growth of the cells. Furthermore coexpression of wild-type (WT) and mutant FLT3 receptor also abolishes the ability to stimulate the WT receptor with its ligand. This is confirmed by analyzing downstream signaling in the cells as MAPK is less phosphorylated in the FLT3-WT/Q569Vstop coexpressing cells than in FLT3-WT expressing cells alone.

Conclusion

Most of the FLT3 mutations are activating mutations leading to a constitutive activation of the receptor and ligand-independent growth. In our study we characterized a novel FLT3 mutation found in an AML patient which has not been described before. The resulting truncated receptor is still integrated in the plasma membrane and binds its ligand but its ability to be fully activated is completely lost. Furthermore coexpressed with a FLT3-WT receptor, it even prevents stimulation and activation of the WT receptor, thus acting in a dominant negative manner. How the truncated form of the receptor contributes to progression of acute leukemia is of great interest and will be further investigated.

Disclosures

No relevant conflicts of interest to declare.
Decreased SATB1 Expression in ALT and Promoted Invasion of Jurkat Cell in Vitro
Authors: Xu Lihua , Tan Huo , Yang Shaojiang
Published: October 2019

BACKGROUND: Special AT-rich sequence-binding protein-1 (SATB1) is critical for genome organizer that reprograms chromatin organization and transcription profiles, and associated with tumor growth and metastasis in several cancer types cancer. However, their expression patterns and function values for adult T-cell leukemia (ALT) are still largely unknown.

METHODS: Expression of SATB1 in normal and ALT peripheral blood leucocytes (n=50 for normal, n=30 for ALT) were evaluated by RT-qPCR in mRNA level. Expression of protein SATB1 in leukemia cell lines were evaluated by Weston Blot.SATB1-shRNA was constructed in psuper-retro-puro retrovirus vector. Jurkat-SATB1-sh and control stable cell lines were established after virus infection and puromycin selection. Cellular proliferation and invasion of Jurkat-ctr and SATB1 knockdown Jurkat cells were evaluated by CCK-8 kit and transwell matrigel invasion assay, respectively.

RESULTS: SATB1 mRNA expression was decreased in ALT (p<0.001) compared with normal peripheral blood leucocytes cells. SATB1 protein expression in Jurkat cell was higher than other leukemia cell lines. SATB1-shRNA was constructed successfully. Weston Blot show Jurkat-SATB1-sh and control stable cell lines were established successfully. Knockdown of SATB1 expression significantly enhanced invasion of Jurkat cell in vitro but no effect in proliferation

CONCLUSIONS: Decreased expression of SATB1 in ALT was associated with cell invasion.

Disclosures

No relevant conflicts of interest to declare.
Multidrug Resistance Gene (MDR1) Polymorphism and Response to Nilotinib in Egyptian Patients with Chronic Myeloid Leukemia (CML)
Authors: Mattar Mervat , Ellithy Hend , Elnahas Yasser , Azzazi Mohamed osman
Published: October 2019

Introduction: Inappropriate expression of the multidrug resistance (MDR1) gene I in chronic phase chronic myeloid leukemia cases (CML-CP) encodes P glycoprotein (Pgp)that may cause resistance to second generation tyrosine kinase inhibitors (TKIs)

Patients and methods: Thirty-one upfront CML-CP patients, planned to receive nilotinib, were included. Detection of MDR1 gene polymorphism C3435T, using PCR Restriction Fragment Length Polymorphisms (PCR-RFLP) was done initially for every patient. We prospectively followed up the patients between February 2012 and February 2014 with PCR for BCR-ABL1 transcripts every 3 months. The molecular response to nilotinib, according to the level of BCR-ABL1 by PCR, was compared to the different MDR1 3435 genotypes.

Results: The majority of the patients carried the MDR1 3435CC genotype of Molecular response was optimal in 56%, 60% and 80% of the patients at month 3, 6 and 12 respectively.There was no statistically significant difference between MDR- C3435T genotypes and the molecular response to treatment with nilotinib.

Disclosures

No relevant conflicts of interest to declare.
Synergistic Effect of Quinacrine with Chemotherapeutics or TRAIL in Hematopoietic Malignant Cells
Authors: Wang Wenge , Lulla Amriti R. , Hernandez-Borrero Liz J. , Dicker David T. , Teye Emmanuel K. , Talekar Mala K. , Dolloff Nathan G. , Pu Jeffrey J. , El-Deiry Wafik S.
Published: October 2019

Quinacrine is a bioactive acridine derivative which has been used for treatment of malaria, giardiasis, systemic lupus erythematosus, and rheumatoid arthritis. In searching for p53 pathway activating agents for cancer therapy, we found that quinacrine stabilizes p53 and induces p53-dependent and p53-independent tumor cell death. Quinacrine also induces expression of TRAIL Death Receptor 5 (DR5) and reduces expression of anti-apoptotic Mcl-1 in tumor cells. These activities predict synergies with TRAIL (tumor necrosis factor-related apoptosis inducing ligand) and chemotherapeutic agents in inducing extrinsic and intrinsic pathway mediated apoptosis. In addition, quinacrine suppresses NFkB activity in tumor cells. Clinical trials have been ongoing for treatment of solid tumors including colon cancer, renal cancer, prostate cancer, and non-small cell lung cancer with quinacrine in combination with chemotherapy or tyrosine-kinase inhibitors, however, the therapeutic potential of quinacrine in blood cancer cells has not been established. We tested quinacrine on hematopoietic malignant cells, which included cell lines of myeloid leukemia, lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and multiple myeloma. We found that quinacrine induces massive cell death in the cell lines tested, at concentrations from as less as 1 microM to 5 microM, 2-10 times lower than required to induce solid tumor cell death. Quinacrine synergizes with TRAIL in inducing cell death of TRAIL-sensitive cells and reverses resistance in TRAIL-resistant cells. Quinacrine also synergizes with chemotherapeutic agents, such as antimetabolites, alkylating agents, and tyrosine kinase inhibitors, in inducing apoptosis of hematopoietic cancer cell lines. Our work supports translational efforts to advance the use of quinacrine from bench to clinic and provides rationale for combination chemotherapeutic regimes for treatment of hematopoietic malignancies.

Disclosures

No relevant conflicts of interest to declare.
Influence of Adriamycin and Wogonin on the Expression of Mir-155 in Burrkit Lymphoma in Vitro
Authors: Wu Xue , Wang Fei , Xia Guohua , Chen Yanping , Chen Baoan
Published: October 2019

Objective:

In this study, we investigated the influence of adriamycin and wogonin to the expression of miR-155 in Burrkit lymphoma in vitro. We focused on exploring the efficiency and pathway of wogonin to induce the apoptosis of lymphoma cells through the transcription and translation level of miR-155 compared with ADM and seeking for the evidence of the synergistic effect between wogonin and ADM.

Methods:

(1) Use drugs to interfere the growth of the Raji cells (Adriamycin (ADM, chemical name:doxorubicin), wogonin, ADM+wogonin), observe the cells through the microscope.

(2) The apoptosis of Raji cells was tested by FCM.

(3) The transcription of miRNA-155 of Raji cells was tested by real-time PCR.

(4) The translation of miRNA-155 of Raji cells was tested by Western blot.

Results:

(5) After being disposed for 24 hours,the experimental groups developed to apoptosis.The statistic significance existed between groups of experiment and control and groups of each experiment.The effect of inducing apoptosis from strong to weak was group of ADM+wogonin,ADM and wogonin.

(6) After being disposed for 24 hours,the transcription level of miRNA-155 of the experimental groups was lower than the control group.Compared with ADM,the transcription level of miRNA-155 of wogonin group seemed to be stronger,while the combination of the two drugs was the weakest.

(7) After being disposed for 24 hours,the translation level of p85α of the experimental groups was lower than the control group. Compared with ADM, the translation level of p85α of wogonin seemed to be stronger, while the combination of the two drugs was the weakest.

Conclusion:

The Chinese materia medica preparation wogonin could induce the apoptosis of lymphoma cells and enhance the effect of ADM through PI3K/AKT pathway.

Disclosures

No relevant conflicts of interest to declare.
Febrile Neutropenia in Children with Acute Lymphoblastic Leukemia Treated with BFM Protocols
Authors: Ozdemir Gul Nihal , Tuysuz Gulen , Apak Hilmi , Ozkan Alp , Yildiz Inci , Celkan Tiraje
Published: October 2019

Aim: Overall survival from leukemia is less in low and middle-income countries than in high-income countries. The aim of this study is to review febrile neutropenia (FN) episodes in children with acute lymphoblastic leukemia (ALL) treated with BFM (Berlin-Franfurt-Münich) protocols at a single center in Turkey.

Methods: Retrospective data were extracted from medical records of children (1-18 years old) with newly diagnosed and relapsed ALL and who were treated between 1995-2010 at Istanbul University, Cerrahpasa Medical Faculty, Pediatric Hematology Oncology Dept.

Results: Two hundred -forty-five FN episodes were detected in 96 evaluable children (mean age: 5.9±3.7 years; M/F: 50/46). According to BFM risk groups; 32 were SRG (standard risk group), 56 were MRG (medium risk group) and 8 were HRG (high risk group) patients. Fifteen patients had relapsed on follow-up and 2 of them had a second relapse. The mean number of FN episodes was 2.5 (±1.58; 0-8) per newly diagnosed patient and 3.6 (±1.88; 1-8) per relapsed patient. Febrile neutropenia episodes were more frequent at high risk patients compared to standart risk patients (p≤0.05). Patients diagnosed before year 2000 had less FN episodes compared to patients diagnosed after 2000 (p≤0.05). Fifty-seven of the FN episodes were fever of unknown origin, 16% were clinically documented infection and 27% were microbiologically documented infection. Of the microbiologically documented infections, 44% of them were caused by gram positive bacteria and 43% were by gram negative bacteria. Eight of 96 patients developed invasive fungal infection during initial treatment and 4 out of 15 patients who relapsed. Of 9 patients who expired on follow-up, the cause of death was fungal infections in 4 and FN in 1.

Conclusion: The high rate of infectious deaths in leukemia compared to that reported in high-income countries, suggests that improvements in infection care and prevention are necessary to improve survival in patients with leukemia. We showed that over the years FN episodes increased at our center in contrast to better quality of patient care. This increase in infections may be atrributed to more intensive treatment regimens. We also showed that high risk and relapsed patients who are treated more aggresively have increased infection rate. The most common cause of death was fungal infections.

Disclosures

No relevant conflicts of interest to declare.
A Dual Bcl-2/xL Inhibitor Induces Tumor Cell Apoptosis in a Hematopoietic Xenograft Model
Authors: Adam Ammar , Byth Kate , Secrist Paul , Schuller Alwin , Reimer Corinne , Lawson Deborah , MacIntyre Terry , Powell Francoise
Published: October 2019

Over-expression of the antiapoptotic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional standard of care therapies. Because Bcl-2 family members play key, but partially overlapping, roles as regulators of apoptosis, simultaneous inhibition of both Bcl-2 and Bcl-xL could serve as a promising anticancer strategy. Clinical validation of this concept has been demonstrated with multiple agents targeting different Bcl2 family members. For example, recent Phase I/II trials of the dual Bcl-2 / Bcl-xL inhibitor Navitoclax demonstrated a 20% response rate in relapsed, refractory CLL patients. However, in this study dosing was often limited by chronic thrombocytopenia, a well established side effect of inhibiting Bcl-xL.

Here we disclose for the first time a novel and potent BH3 mimetic (Bcl2-32), with nanomolar binding affinity for Bcl-2 and Bcl-xL, (Ki = 3.3 and 8.5nM respectively). In contrast to Navitoclax which requires daily oral administration for activity, Bcl2-32 has shown promising efficacy on an intermittent dosing schedule that allows full platelet recovery between doses. In this poster, we describe the in vitro and in vivo activity profile of Bcl2-32 in a variety of cell types that highlights its potential as a dual inhibitor of Bcl2 and BclXL. In addition to exhibiting potent single agent anti-cancer activity in a sensitive Acute Lymphocytic Leukemia (RS4; 11) model with (TGI >100%, P < 0.001), Bcl2-32 also potentiates the effectiveness of standard chemotherapeutic agents in less sensitive DLBCL xenograft models. Taken together, these data suggest that Bcl2-32 represents an exciting development opportunity as single agent or in combination in a broad range of hematopoietic malignancies.

Disclosures

Adam: Astrazenenca: Employment. Secrist:AstraZeneca: Employment.
EVI1 Expression Predicts Poor Survival in Acute Myeloid Leukemia Patients Undergoing Allogeneic Hematopoietic Cell Transplantation with Myeloblastic Conditioning
Authors: He Xuefeng , Chen Suning , Depei Wu , Sun Aining
Published: October 2019

The ecotropic viral integreation site 1 (EVI1 or MECOM) gene mapping to chromosome 3q26 exerts significant oncogene biological effects of anti-apoptosis, proliferation and differentiation on hematopoietic cells. It is considered as a poor prognostic factor in acute myeloid leukemia (AML). EVI1 positive AML accounts for about 10% of de novo AML pts. The remission rate and long time survive are poor. Whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) could salvage such kind of malignancy is still unknown. We conducted a survey of 161 AML pts (median age, 35 years[y]; range 8-60y) after first complete remission undergoing myeloblastic allo-HSCT at the first affiliated hospital of Soochow University. Source of hematopoietic stem cells came from match or partial matched related, unrelated or umbilical cord blood (UCB) donors (Table 1). Pretreated specimens were obtained and real-time polymerase chain reaction was performed to detect expression of EVI1-1d (one major splice of EVI1) and common EVI1 (cEVI) (normalized to PBGD gene). Standard plasmids of EVI1 and PBGD were prepared as calibrators. 13 nonmalignant single lineage cytopenia pts were tested as baseline. Quantities of EVI1-1d and cEVI ranges from negative to 154.2%PBGD and negative to 1214.3%PBGD in each group. Above five and ten folds to baseline in EVI1-1d and cEVI respectively were considered as positive. Accordingly 27 pts were EVI1 positive while 134 pts were negative (16.8% vs 83.2% separately). Unfavorable cytogenetic abnormalities showed a high proportion in EVI1 pos (11/27) than neg group (12/134). 5 and 40 mutations were detected in each group. Three cytogenetic abnormality concerning 11p15 pts expressed high level of EVI1.

Positive expression of EVI1 indicated significant shorter progress-free survival and overall survival (P= 0.0352 and 0.0436 respectively) comparing with negative group (Figure 1).

Table 1. Sources of donors Matched (n) Partial mathed (n) siblings 82 5 Parents or sons/daughters / 30 Unrelated PBSC 26 10 Unrelated UCB 3 5

Figure 1 Figure 1.

Figure 2 Figure 2.

Progress-free Survival (upper) and Overall Survival (below) in myeloblastic allo-HSCT pts according to EVI1 expression

In conclusion, high level of evi1 expression predicts worse prognosis even after myeloblastic allo-HSCT. New treatment strategies post allo-HSCT are needed to improve long time survival in such kinds of pts.

Disclosures

No relevant conflicts of interest to declare.
Identification of Leukemic Stem Cells in Acute Myeloid Leukemia Patients
Authors: Elmasry Manal M W , Elhaddad Alaa
Published: October 2019

Background: Acute myeloblastic leukemia (AML) can be viewed as newly formed, abnormal hemopoeitic tissue initiated by few leukemic stem cells (LSCs). Recognizing the LSC and identifying their behavior, plays a pivotal role in the approach of a targeted therapy.Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R promotes mononuclear phagocytes survival, proliferation and differentiation.

Aim of the work: Defining the self-renewing [Thy1-, CD34+, CD38-] LICs in AML cases before and after induction chemotherapy as a predictor for relapse and to determine how CSF1R (Fms) and CD34 markers affect the growth and survival of human leukemic cells in the CD38- Thy1- population.

Patients and methods: This study was carried out on 30 samples from the peripheral blood of adult patients with de-novo acute myeloid leukemia. The majority of the patients were monocytic AML

Samples were sorted into four populations (Fms+CD34-, Fms+CD34+, Fms-CD34+ and Fms-CD34-) according to the surface markers of the cells. Cells were cultured on mouse stromal cells transfected with a plasmid containing human CSF-1. Samples were cultured using Iscove's modified Dulbecc's medium (IMDM).The cultures were assessed for survival of leukemic cells in days.

Results: The mean survival in days of the cells was 13.9 before chemotherapy and 14.1 after chemotherapy. The difference in growth was insignificant (p>0.05). The Fms-CD34+ population in all but two samples tested had the longest survival time in culture.

Conclusion: Our results suggest that leukemic stem cells may survive chemotherapy mainly due to their quiescence.

Human CSF-1 was shown to increase the number of leukemic cells in co-culture with mouse stroma after 5 weeks. A novel leukemic stem cell (Fms-CD34+) has been identified and is the cell responsible for the growth and maintenance of the leukemic bulk.

Disclosures

No relevant conflicts of interest to declare.
Higher Than Reported Response Rates to Decitabine Observed in Indian AML Patients - Is There a Genomic Basis
Authors: Damodar Sharat , Nataraj KS , Niveditha SR , Purnaiya Rajeev , Kumar Madan , Nagaraj Ashwin , Ram Prahlad
Published: October 2019

Between May 2012 and May 2014, 15 elderly patients (median 64yrs, range 54-80yrs) who presented with acute myeloid leukemia (AML) with comorbidities preventing them from receiving standard induction, were treated with decitabine at a dose of 20 mg/m2 for 5 days at monthly intervals. All patients received a minimum of 3 cycles of decitabine with a range of 3-9 cycles. Bone marrow examination was conducted at the end of 3 cycles to assess response. As opposed to the 40% CR rate reported by Ritchie et al (2013) in an elderly western population, we observed a response rate of 60% in our cohort. 9 out of 15 patients achieved remission, with 7 of the 9 patients regaining normal blood counts. The median clinical follow-up was 375 days with a range of 74-800 days. All 9 patients who responded positively to decitabine are alive and well.

This 50% higher response rate to decitabine in Indian patients led us to examine the genetic basis for this observed difference. We conducted an observational prospective study of 14 Indian AML patients either at diagnosis or at relapse. Whole genome expression data from bone marrow aspirate samples was analyzed using PeCaSo™ (APT Life Sciences, Bangalore, India). PeCaSo is a genetic interaction network analysis platform that provides a ranked list of chemotherapeutic agents, as well as other approved pharmaceutical agents which can potentially be used as adjuvant therapy. For the 14 patients enrolled in the study, we looked at their response to decitabine as predicted by PeCaSo (see figure). As a comparison, PeCaSo was also used to retrospectively analyze whole genome expression data of Caucasian AML patients (n=16) available at The Cancer Genome Atlas (TCGA).

Figure 1 Figure 1.

PeCaSo’s genomic network analysis indicates a higher response rate to decitabine for the Indian AML cohort when compared with a Caucasian cohort, strongly suggesting a potential genetic basis for the clinically observed difference.

This study provides supportive evidence for considering decitabine for induction therapy of AML in Indian patients. Our study’s genomic approach confirms that the heterogeneity and genetic basis of cancer has to be factored in prescribing personalized protocols for the treatment of cancer.

Disclosures

Damodar: APT Life Sciences, Bangalore, India: Research Funding. KS:APT Life Sciences, Bangalore, India: Research Funding. Purnaiya:APT Life Sciences, Bangalore, India: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kumar:APT Life Sciences, Bangalore, India: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Nagaraj:APT Life Sciences, Bangalore, India: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ram:APT Life Sciences, Bangalore, India: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.