bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss Of p53 Promotes Transformation Of Oncogenic Nras-Induced Chronic Myelomonocytic Leukemia To An Acute Phase
Authors: Zhang Jingfang , Liu Yangang , Kong Guangyao , Chang Yuan-I , Ranheim Erik A. , Wang Jinyong , Du Juan , Damnernsawad Alisa , Zhang Jing
Published: October 2019

Chronic myelomonocytic leukemia (CMML) primarily occurs in the elderly with the median age ranging from 65 to 75 years. As defined by WHO, CMML is characterized by persistent monocytosis in peripheral blood, hepatosplenomegaly, and the absence of BCR-ABL fusion gene. CMML is a devastating cancer for multiple reasons, one of which is that approximately 20% of CMML cases evolve into acute myeloid leukemia (AML) soon after their first diagnosis. However, little is known about the cellular and molecular mechanisms underlying this malignant transformation.

Recently, our lab developed a CMML mouse model induced by oncogenic NrasG12D/+ expressed from its endogenous locus. Above 90% of recipient mice with NrasG12D/+ bone marrow cells developed CMML-like phenotypes with a median survival of ∼56 weeks. Interestingly, none of these mice spontaneously transform to AML. To identify the pathogenetic origins underlying CMML transformation to AML, we further deleted p53 expression in NrasG12D/+ bone marrow cells using p53fl/fl allele and Mx1-Cre because deletion of p53 is a common genetic event observed in oncogenic Ras-driven cancers. We found that ERK1/2 is significantly hyperactivated in NrasG12D/+; p53-/- hematopoietic stem/progenitor cells (enriched for myeloid progenitors) in the absence of cytokines or in the presence of low concentration of GM-CSF. Concomitantly, the mutant myeloid progenitors show significantly increased self-renewal in a serial replating assay in vitro.

We transplanted NrasG12D/+, p53-/-, or NrasG12D/+; p53-/- bone marrow cells into lethally irradiated mice. Unlike recipients with p53-/- cells that died of a T-cell disease with 100% penetrance and a median survival of 24 weeks, ∼70% of recipients with NrasG12D/+; p53-/- cells died of AML or acute myeloid sarcoma with a median survival of 16 weeks. These malignant myeloid diseases are transplantable in secondary recipients. Interestingly, only mutant hematopoietic stem cells (HSCs) could initiate and maintain leukemia phenotypes in the NrasG12D/+ induced CMML model, whereas both NrasG12D/+; p53-/- HSCs and myeloid progenitors could initiate AML or acute myeloid sarcoma.

Our results indicate that deletion of p53 cooperates with NrasG12D/+ mutation to transform CMML into an acute phase. This malignant transformation is initiated by mutant myeloid progenitors, which show increased self-renewal and potentially serve as leukemia initiating cells.

Disclosures:

No relevant conflicts of interest to declare.
Increased Expression Of Fgf2 Induces Cytokine Hypersensitivity In Leukemia Stem Cells With Translocations Involving The MLL Gene
Authors: Shah Chirag , Wang Hao , Bei Ling , Eklund Elizabeth
Published: October 2019

Leukemias with chromosomal translocation or partial tandem duplications involving the MLL (mixed lineage leukemia) gene have exceptionally poor prognosis (referred to as 11q23-leukemias). At the molecular level, 11q23-leukemias are characterized by increased expression of a set of homeodomain transcription factors in CD34+ hematopoietic stem cells (HSC) and differentiating CD34- myeloid progenitor cells. This transcription factor set includes HoxB3, B4, A7-11 and Meis1. Although many elegant studies have identified the precise molecular mechanisms by which MLL-fusion proteins increase HOX gene transcription, less is known about the downstream effects of increased Hox expression and potential implications for leukemogenesis. In prior studies, we determined that the gene encoding fibroblast growth factor 2 (Fgf2 or basic Fgf) is a HoxA10 target gene, and production of Fgf2 is increased in HoxA10 overexpressing primary bone marrow cells. In the current studies, we determine that FGF2 is also activated by HoxA9; identifying the only common target gene for HoxA9 and HoxA10 where both proteins activate transcription. We also find Mll-Ell (an MLL-fusion protein) increases FGF2 transcription and Fgf2 expression in a HoxA9 and HoxA10 dependent manner. Importantly, autocrine production of Fgf2 by Mll-Ell expressing HSC and myeloid progenitor cells increases cytokine-induced proliferation and enhances cell survival. In additional studies, we determine that these effects of Fgf2 are mediated by phospho inositol 3 kinase (PI3K) dependent stabilization of beta catenin. We find that autocrine production of Fgf2 results in increased expression of key beta catenin target genes in Mll-Ell expressing cells, including c-myc, survivin and cyclinD1. Beta catenin target genes also include the genes encoding CDX4 and HOXA10. Additionally, Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates the CDX4 gene. Therefore, increased Fgf2 also establishes a positive feedback loop that enhances transcriptional activation of HOXA9 and HOXA10 by MLL-fusion proteins. 11q23-AML is generally refractory, but the molecular mechanisms for this are unknown. Our studies suggest that increased Fgf2 expression in 11q23-AML contributes to cytokine hypersensitivity and sustained survival of leukemia stem cells. Targeting the effects of Fgf2 might increase chemotherapy sensitivity in this form of leukemia. Small molecule inhibitors of Fgf-receptors are available, but have not yet been tested in this form of AML. We suggest that this would be a rational therapeutic approach to this disease.

Disclosures:

No relevant conflicts of interest to declare.
Pharmacological Validation Of Potentiating Targets From SAHA RNA-Interference Modifier Screens In Acute Myeloid Leukemia
Authors: Bogenberger James M , Rudd James , Chow Donald , Kassner Michelle , Fauble Veena , Mesa Ruben A. , Yin Hongwei , Greene Casey , Tibes Raoul
Published: October 2019

The lysine deacetylase inhibitor suberoylanilide acid (SAHA) exhibits promising but limited activity in the treatment of acute myeloid leukemia (AML). To identify potential targets for rational combination therapies that increase the efficacy of SAHA in AML, we performed a functional RNA-interference (RNAi) drug modifier screen to identify genes that, when inhibited, potentiate or antagonize the in vitro anti-leukemic activity of SAHA. A total of 901 kinase, phosphatase and closely associated signaling genes were silenced in TF-1, HEL and THP-1 cells, with four different siRNA sequences per gene, both alone and in combination with SAHA treatment. Screen hit lists for each cell line were over-laid on an integrated functional relationship network. A community detection algorithm was then applied to this sub-network and siRNA sensitive modules were identified. Each module represents a highly connected set of genes in the integrated network. To identify pathways represented by each module, enrichment was evaluated using the National Cancer Institute (NCI) Protein Interaction Database (PID) pathways. Both cell line-specific and universal sensitizing targets, grouped into a small number of pathways, emerged from these screens. Seven pharmacologic inhibitors interfering with these pathways have been assessed in a panel of four or five AML cell lines, including SET-2, TF-1, HEL, THP-1, and OCI-AML3. Of the seven tested inhibitors targeting SAHA sensitizing networks, one combination exhibiting universal SAHA sensitization in all AML cell lines tested was further assessed in drug dose response assays using ex vivo cultures of bone marrow mononuclear cell populations derived from patients with various myeloid malignancies. The combination demonstrated “one-sided” SAHA EC50 fold-enhancement, as well as strong synergy by “two-sided” CalcuSyn combination index determination in all ex vivo specimens examined to date (N=6), including polycythemia vera (PV), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndrome, MDS transformed to AML, and de novo AML samples. Thus, this first reported large-scale SAHA RNAi modifier screen in AML has identified a specific pathway/target whose inhibition broadly synergizes with HDAC inhibition in myeloid malignancies. A novel compound putatively targeting this respective pathway/target is currently in early clinical development and has shown limited single-agent activity. With the full dataset presented, we will propose a novel rational combination to enhance the activity of HDAC inhibition in AML and other myeloid malignancies that could be translated into design of a clinical trial.

Disclosures:

No relevant conflicts of interest to declare.
Genomic Signature Predicts Resistance To Busulfan In AML Cell Lines
Authors: Patel Pritesh R. , Mahmud Dolores , Zhang Wei , Garcia Joe GN , Rondelli Damiano
Published: October 2019

The alkylating agent Busulfan (Bu) is commonly used in the pre-transplant conditioning regimen for patients with acute myeloid leukemia (AML). Relapse after transplant is in part due to resistance to Bu. Our study was aimed at identifying a genomic signature that could predict resistance to Bu in AML cells.

In order to obtain a list of candidate genes which may correlate with increasing resistance to Bu we performed a linear regression analysis controlled for cancer type on NCI60 Stanford cDNA data and GI50 values. This produced a list of 111 genes correlating with increasing Busulfan GI50 (p<0.01, FDR<50%). The ability of these 111 genes to predict GI50 was subsequently tested based upon their expression in a second independent gene expression platform (Affymetrix U133 plus 2.0). This analysis yielded 6 genes (KCNH2, CD74, CD53, HCLS1, ERC2 and HLA-DQB2) predicting higher GI50 values (p<0.05). We then tested the ability of these genes to predict Bu GI50 in a panel of 6 AML cell lines (K562, HL60, HL60-MX1, KG1, THP1 and NB4). To obtain GI50 values on AML cell lines we treated the cells with doses of Bu ranging from 0-200mcg/ml. At 24 hours, cells were washed and resuspended in fresh medium. Proliferation of cells was measured by standard 3H-thymidine uptake assay at 48 hours. Sigmoidal dose response curves and GI50 values were then calculated. GI50 values varied from 13.9 - 70.4 micromoles/ml. RNA quantitation for the 6 identified genes was performed using the Quantigene assay (Panomics, Santa Clara, CA). Expression was normalized to control gene levels (RPLP0). Two genes (ERC2 and HLA-DQB2) showed low or undetectable expression and were excluded from further analysis. Logistic regression analysis showed that a combined overexpression of any 2 of the remaining 4 genes was significantly associated with increasing resistance to Bu GI50 in AML cells (p=0.006). Of the 6 cell lines tested, the 2 most resistant (THP1 and K562) both displayed this pattern of gene overexpression.

To our knowledge, this is the first attempt to obtain a list of genomic biomarkers to predict Bu resistance in AML using a genome wide approach. This data provides a rationale for validation of these 4 genes in a clinical dataset with the potential future application of predicting relapse after Bu based high dose therapy.

Disclosures:

No relevant conflicts of interest to declare.
Expression Of Tissue Transglutaminase Is Associated With Glucocorticoid Resistance In T-Cell Acute Lymphoblastic Leukemia
Authors: Jung Hyun Joo , Lee Seong Wook , Park Jun Eun
Published: October 2019

Glucocorticoids are core drugs in the treatment of T-cell acute lymphoblastic leukemias (T-ALL) because they induce apoptosis and cell-cycle arrest in leukemia lymphoblasts. The treatment response to glucocorticoid is the most important predictive factor for treatment outcome of T-ALL. Development of secondary glucocorticoid resistance is lethal to the patients at relapse. To improve survival for glucocorticoid-resistant T-ALL patients, it is necessary to develop new therapeutic strategies to overcome glucocorticoid resistance. In the present study, we used biochemical and molecular methodologies to demonstrate that tissue transglutaminase (TG2) confers glucocorticoid resistance in T-ALL. TG2 is a calcium-dependent protein crosslinking enzyme reported to be overexpressed in various metastatic or chemotherapy-resistant cancer cells including lymphoma cells. We found that T-ALL cells expressed elevated levels of TG2. We generated a model of glucocorticoid-adapted subclones of T-ALL cell lines that were extremely less sensitive to glucocorticoid than the parent cells. Glucocorticoid-adapted subclones not only showed the change of ABC transporters activity but also increased TG2 expression and activity than the parent cells in T-ALL. Inhibition of TG2 suppressed glucocorticoid resistance and improved the cytotoxicity of glucocorticoid in glucocorticoid-adapted subclones of T-ALL. Our study is the first to show the expression of TG2 and the contribution of TG2 to glucocorticoid resistance in T-ALL. The modulation of TG2 expression could be a new therapeutic target to overcome glucocorticoid resistance in T-ALL. Furthermore, change of TG2 expression could serve as markers of secondary glucocorticoid resistance in T-ALL.

Disclosures:

No relevant conflicts of interest to declare.
Loss Of IKZF1 Function Mediates Resistance Towards Glucocorticoid-Induced Apoptosis
Authors: Havinga Jorn , Yuniati Laurensia , Demkes Marc , Schenau Dorette van Ingen , Kuiper Roland P. , Hoogerbrugge Peter M. , van Leeuwen Frank N. , Scheijen Blanca
Published: October 2019

Background

Glucocorticoids (GCs) such as prednisolone and dexamethasone are critical components of multi-agent chemotherapy regimens used in the treatment of acute lymphoblastic leukemia (ALL). Children with ALL are stratified into risk groups based on diagnostic features (i.e. age and cytogenetics) and therapy response. It has been established that the initial response to prednisolone is a major prognostic factor. Moreover, at relapse, de novo or acquired resistance to GCs is common and represents an important determinant in treatment failure. Recent studies performed by us and others have identified IKZF1 gene deletions and mutations as an independent prognostic factor that predicts prognosis and treatment outcome of children with B cell precursor ALL (BCP-ALL). These monoallelic IKZF1 gene deletions either affect the whole gene or may result in expression of dominant-negative IKZF1 isoforms due to intragenic deletions. However, it has not been established whether loss of IKZF1 function directly impacts the response to glucocorticoids.

Results

We examined whether haplodeficiency for Ikzf1 gene expression in mouse lymphocytes affects glucocorticoid-induced apoptosis. Splenocytes from Ikzf1+/- knockout mice were activated with lipopolysaccharide (LPS) and treated with increasing concentrations of either prednisolone or dexamethasone for 48 hours. B-lymphocytes haplodeficient for IKZF1 showed a significantly enhanced survival after treatment with GCs compared to wild type cells, as measured in an MTS assay and by AnnexinV staining. In case of prednisolone, the inhibitory concentration (IC50) was about ∼200-fold higher in the Ikzf1+/- splenocytes as compared to the wild-type cells. Gene expression analysis revealed that Ikzf1+/- splenocytes displayed lower overall expression levels as well as diminished transcriptional activation of several glucocorticoid receptor (GR)-induced target genes (i.e. Sgk1, Irs2, Zfp36L2). Furthermore, in luciferase reporter assays we established that IKZF1 overexpression enhances GR-mediated transcriptional activation in response to prednisolone. Finally, lentivirus-mediated IKZF1-shRNA expression in Nalm6 cell line, which reduces endogenous IKZF1 protein levels to around 50%, inhibits prednisolone and dexamethasone-induced apoptosis, demonstrating that also in human leukemia cells reduced IKZF1 expression levels protect against GC-induced cell death.

In conclusion, our data provide evidence that loss of IKZF1 function mediates resistance to glucocorticoid-induced apoptosis, which may contribute to the poor outcome of IKZF1-deleted BCP-ALL.

Disclosures:

No relevant conflicts of interest to declare.
Targeting CD96 For Antibody Based Elimination Of Leukemic Stem Cells In AML: A New Strategy In Stem Cell Transplantation
Authors: Staudinger Matthias , Kellner Christian , Peipp Matthias , Schub Natalie , Humpe Andreas , Gramatzki Martin
Published: October 2019

Although the mortality of autologous stem cell transplantation in contrast to allogeneic is low, in AML patients the lack of immune surveillance as well as contamination of the transplant with residual leukemic stem cells (LSC) limits its use. Therefore, elimination of LSC by targeted therapy may represent a promising therapeutic approach. Recently, CD96 was identified as marker antigen on AML-LSC (Hosen et al., PNAS 104: 11008, 2007). Here, by addressing CD96 with magnetic cell sorting (MACS) or using antibody dependent cellular cytotoxicity (ADCC), new strategies for engineering autologous stem cell grafts or for in vivo targeting of residual AML stem cells are presented.

To evaluate the efficacy of depletion of LSC by MACS technology, grafts containing hematopoietic stem cells were spiked with CD96 positive AML cells. Using biotinylated CD96 antibody TH111 raised in our laboratory in combination with anti-biotin-micro beads (Miltenyi Biotech, Bergisch Gladbach, Germany) up to a 1000-fold depletion of targeted cells was achieved. The viability, cell count and the potential of hematopoietic progenitor cells (HPC) to proliferate and differentiate were not affected by this procedure as documented by flow cytometry and colony forming assays.

As residual LSC residing within the patient may also account for AML relapse after high-dose chemotherapy and subsequent SCT, eradication of AML stem cells in vivo is desirable. To target CD96+ AML-LSC by ADCC, chimeric antibodies containing wild type or affinity maturated variable regions in combination with an optimized human IgG1Fc were generated by recombinant DNA technologies. Both recombinant antibodies were expressed in Hek 293 cells enriched to homogeneity by affinity chromatography and analyzed for their functional properties. As shown by flow cytometry, the antigen binding affinity of the maturated antibody was enhanced (EC50 0.6 μg/ml vs. 2 μg/ml). Moreover, as analyzed in standard ADCC assays, NK cell mediated lytic properties against CD96-positive target cells were elevated (maximum lysis: 52%) using the affinity maturated chimeric CD96 antibody (EC50: 0.02 μg/ml vs. 0.15 μg/ml).

Thus, this CD96 purging strategy avoids unwanted transplantation of AML-LSC and may help to revitalize autologous stem cell transplantation in this indication. Although, specific side effects by CD96 application will have to be considered, this may allow for an additional therapeutic avenue to eliminate in vivo residual AML-LSC in autologous as well as in allogeneic situations.

Disclosures:

No relevant conflicts of interest to declare.
The Distinct Stem Cell Population Derived From Chorionic Plate Of Human Placenta Which Has Differentiation Potential Into Erythroid Lineage Cells
Authors: Park Yong , Kim Ji Hye , Jung Ji Hye , Kang Ga-won , Kim Dae Sik , Kim Seok Jin , Kim Byung Soo
Published: October 2019

Background/Aims

Placenta is a specialized organ which has a property of fetal and adult tissues simultaneously. Mesenchymal stem cell and hematopoietic stem cells have been founded in the parenchyma of placenta tissue contributed by fetal side. Recently, it was reported that placenta is a major hematopoietic organ contributing to both generation and expansion of multipotent hematopoietic stem and progenitor cells. We hypothesized that hematopoietic stem cells in placenta might be originated from another placental stem cells which has been so-called “placenta mesenchymal stem cells”. To prove this hypothesis, this study is designed to induce in vitro differentiation of placental stem cells into the hematopoietic stem cells without genetic transduction.

Methods

Placental stem cells was obtained from surgically isolated placental chorionic plates from healthy women who had undergone abortion at 6-8 weeks of gestation. To prevent the contamination of hematopoietic stem cells, CD34-CD44+ cells were purified by flowcytometric isolation. After mesenchymal differentiation potential was identified in these cells by induction of adipogenic and osteogenic differentiation, they were cultured in vitro with serum-free media including cytokine cocktail which was known to induce differentiation of embryonic stem cells into hematopoietic lineage (erythropoietin, Flt-3/Flk-2 ligand, G-CSF, GM-CSF, IL-6/7/15, stem cell factor, and thrombopoietin). In vitro differentiation was performed in conventional cell culture dish (group 1) and Ultra-low attachment dish (group2) for 3 weeks, respectively (Figure 1). Expression of hematopoietic stem and progenitor cell markers were analyzed at every 1 week.

Results

In group 1 (conventional cell culture dish), no hematopoietic differentiation was identified regardless of the concentrations of cytokine cocktail. In group 2 (Ultra-low attachment dish), expression of pan-hematopoietic markers, CD34 and CD45 was also not observed. However, after 2 weeks of differentiation induction, red cells aggregates and expression of erythroid-lineage markers (CD235a and CD38) were detected in cells cultured using high concentration cytokines (Media 3 in Figure 1). The proportion of cells expressing erythroid-lineage markers became increased during in vitro differentiation (Figure 2).

Conclusion

Although this study failed to show the differentiation of placenta-derived stem cells into hematopoietic stem cells, the results demonstrated that placenta-derived stem cells could be differentiated into the erythroid lineage under certain condition (i.e. liquid culture + high concentration cytokines). Therefore, placenta-derived stem cells which have been so-called “placenta mesenchymal stem cells” is considered to be a distinct stem cell population including property of erythroid progenitor cells.

Disclosures:

No relevant conflicts of interest to declare.
The Hematopoietic Stem Cells Could Be Switched From Leukemic Cell Lines
Authors: Hu Jianda , Yang Ting , Zhu Liangfang , Luo Xiaofeng , Yuan Xiaohong , Chen Zhizhe
Published: October 2019

Background

The hematopoietic potential from induced pluripotent stem cells (iPS) has been explored for some years. However, the direct reprogramming from the blood cells to hematopoietic progenitors bypass the pluripotency is even more attractive for the clinical practice.

Methods

The lentiviral vector encoding human reprogramming factors (Oct-4, Sox2, c-Myc, Klf4FOSCK) were transfected along with packaging plasmid pCMV-dR8.91 and envelope plasmid pCMV-VSV-G in 293T package cells to produce lentiviral particles. The construction was then confirmed by RT-PCR and restriction enzyme digestion anaysis, and the lentivirus titer was determined using GFP/DAPI based on cell count by Image J software. Primary mouse embryo fibroblast (PMEF) feeder cells were isolated and prepared from CF-1 inbred mouse strain to help maintain pluripotency and to provide a cellular matrix for stem cells growing. Lastly, the lentivirus carrying OSCK factors was used totransduce HL-60 cells in three consecutive rounds of spin-infection on the prepared feeder layers with an interval of 12 hours, while the lentivirus carrying GFP served as a negative control. The cell clusters were picked based on morphology and alkaline phosphatase(AP) staining. The stem cell properties were tested by the expression of cell surface antigens using Flow cytometry. and the mRNA expression of OCT4, SOX2, C-MYC,KLF4,etc. by QT-PCR.

Results

The lentiviral particles were successful packaged and used to infect HL-60 cells. We occasionally observed some Human embryonic stem cells (hES) cell-like colonies in between the cells around 14 days after infection. These cell colonies also showed similarity to hES cells in feeder dependency. Detection of cell surface CD34 by FCM showed that HL-60 cells were switched to be a hematopoietic fate, expression of CD34 from 1.77% to 98.42%. Simultaneously, expressions of the myeloid antigen CD13, CD117 decreased. The gene expressions of OCT4, SOX2 indicated that the exogenous gene were down-regulation or silence while the endogenous gene were up-regulation. However, the cell colonies can survive only for a short time which might due to the absence of the survival factors they require or the first hematopoietic microenvironment, like the yolk sac.

Conclusion

After being reprogrammed, the HL-60 cell derived colonies showed the similarity to hES cells in morphology, feeder dependency and the expression of stem cell antigens. However, they may need an appropriate microenvironment at different time points to be programmed into primitive hematopoietic stem cells.

Disclosures:

No relevant conflicts of interest to declare.
Value Of Magnetic Resonance Imaging In The Study and Follow-Up Of Paroxysmal Nocturnal Hemoglobinuria
Authors: Villegas Ana , Gonzalez Ataulfo , Matute Fatima , Nieto Jorge Martinez , de la Fuente Felix
Published: October 2019

Introduction

Renal damage is relatively frequent in Paroxysmal Nocturnal Hemoglobinuria (PNH). Iron accumulation from hemolysis in the renal tubules has been found to be one of the triggering factors. Hemosiderin accumulates in the epithelial cells of the proximal renal tubules localized in the renal cortex, and they can be measured (and monitoring) using magnetic resonance imaging (MRI).

Methods

We studied 7 PNH patients with MR using T2-weighted gradient echo and multiecho sequences. We quantified renal, hepatic and myocardial iron with T2* relaxometry model. Four of these patients were studied at diagnosis and after 1 year of treatment with eculizumab. In 2 of them the MRI was done after 2 and 3 years respectively of treatment with eculizumab. One patient was studied after 8 months on eculizumab, and MRI was repeated at 12 and 24 months. Liver and myocardial iron deposition was quantified in all cases.

Results

At diagnosis the median hemoglobin of the 7 patients was 8.9 g/dl (8.2-12-4), the PNH median PNH clone size was 77% (60-90) and the median LDH level was 2340 U/L (1100-3600). Three patients presented an important accumulation of iron in the renal cortex, with much lower signal intensity than the medulla in the T2-weighted MR images. One patient with mild hemolysis (Hb 12.4 g/dl, 20% reticulocytes and 1,100 LDH) did not present iron accumulation in the renal cortex, and neither did the 2 patients studied after 2 a 3 years of eculizumab treatment. All the patients improved, presenting median of Hb of 11,2 g/dl ( 9,5-12,9) and of LDH of 520 U/L ( 430- 721). The patient studied after 8 months showed an increase of iron in the renal cortex that persisted, despite the improvement with the treatment with eculizumab, with increase of the Hb and decrease of the LDH levels; this patients had a C hepatitis and positivity on the hemochromatosis genes C282Y/H63D, with important iron hepatic overload (11-17 mgFe/g). In the rest of the patients the hepatic iron was normal, with values from 0.5 to 2 mgFe/g, except in one case where the levels were higher (10 mgFe/g) due to previous transfusions used to treat an aplastic anemia pre-PNH. Myocardial iron was normal in the 7 patients, with values ranging from 33 to 60 ms in T2. Only one of the patients presented several episodes of acute renal insufficiency, related with hemolytic crisis, with residual proteinuria, that disappeared when treated with eculizumab.

Conclusions

A diffuse signal loss in the renal cortex without liver and spleen alteration is indicative of intravascular hemolysis, suggesting PNH. Serial MRI studies of iron overload in the renal cortex can be used for the diagnosis, and for monitoring the effects of treatment.

Disclosures:

No relevant conflicts of interest to declare.
Comparison Of Flow Cytometric and Clinical Findings In Patients With Paroxysmal Nocturnal Hemoglobinuria
Authors: Demirer Taner , Baslar Suphi , Uysal Vasif akin , Beksac Meral , Ozen Mehmet , Konuk Nahide , Ilhan Osman , Arslan Onder , Ozcan Muhit , Topcuoglu Pervin , Gurman Gunhan
Published: October 2019

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare disorder of blood cells associated with mutations of X-linked gene called phosphatidylinositol glycan class A, and presenting as hemoglobinuria, signs of cytopenias (fatigue, easy bleeding) or thrombosis.

In this study, 39 patients with PNH were retrospectively evaluated with regard to disease findings, laboratory investigations, complications and relationship between those and clone size. Median follow-up time was 26 months. Patients were divided into two groups. There were 24 classical PNH and 15 Aplastic Anemia (AA) or Myelodisplastic Syndrome (MDS) associated PNH patients. Evident signs of hemolysis at the time of diagnosis and thrombosis were seen in classical PNH patients. Sign of hemolysis developed in two AA patients. Hemoglobinuria, hemolysis test (increased reticulocyte and lactate dehydrogenase) were determined to be correlated with clone size. There was association between clone size and thrombosis. No patients with clone size smaller than 50% developed thrombosis and all patients with thrombosis were in classical PNH subtype which has greater clone size. PNH is a rare disease; therefore the effect of small number of patients on the statistical parameters must be taken into consideration. Over the entire course of follow-up time 8 patients died, 6 due to complications of allogeneic stem cell transplantation and 2 due to conditioning regimen before transplantation. Numbers of deaths are equal in each subtype.

In summary, in this study there was a linear correlation between hemoglobinuria and LDH levels with clone size, which was statistically significant (p=0,031 and p=0,001 respectively). Other clinical signs did not correlate with clone size. Thrombotic complications were seen only in classical type PNH patients. No patients with clone size smaller than 50% developed thrombosis, MPV levels were significantly higher than others (p=0,04). There was also statistically significant correlation between reticulocyte and LDH levels with thrombotic events (p=0,009 and 0,003 respectively). In addition, after allogeneic stem cell transplantation 9 patients were evaluated for PNH clone and in 7 the clone was disappeared.

Disclosures:

No relevant conflicts of interest to declare.
Memory B Cells Display Changes In The Expression Pattern Of Genes Related To Cell Survival In Elderly Individuals
Authors: Baez Alicia , Alvarez-Laderas Isabel , Piruat Jose Ignacio , Caballero-Velazquez Teresa , Millan-Ucles Africa , Barbado María Victoria , Medrano Mayte , Garcia-Guerrero Estefania , Sanchez-Abarca Luis Ignacio , Perez-Simón Jose Antonio , Espigado Ildefonso
Published: October 2019

Introduction

Memory B cells (MBCs) have a very long half-life, remaining viable in a quiescent state for years. However, elderly individuals have less amount of MBCs and they produce lower amounts of antibodies. Thus, in aged individuals immunization become less efficient in terms of quantity and quality. Furthermore, several hematologic malignancies involving B-cell lineage, such as non-Hodgkin lymphomas, chronic lymphocytic leukemia or multiple myeloma, are increasingly common in aging population. With this background, we analyzed the gene expression patterns of naïve B cells (NBCs) versus MBCs in both younger and older subjects, in order to identify genes related to longevity that could be altered in the elderly population and related to an increased risk of developing certain lymphoid malignancies.

Methods

NBCs and MBCs were obtained by immunomagnetic separation from buffy coats of 10 healthy donors: 5 young (20-25 years) and 5 elderly (65-70 years). By microarray techniques we analyzed the expression of 45000 genes in all samples. Unsupervised hierarchical clusters of gene expression data were performed using the average linkage and the Euclidean distance. To identify differentially expressed genes between experimental groups we applied non-parametric Mann-Whitney test. The differences in expression with a p value < 0.05 were considered significant. All analyses were performed using the Multi-experiment Viewer 4.7.1 software.

Results

Firstly, we confirmed that the expression pattern of NBCs versus MBCs is significanly different in 3548 genes in young and 2145 genes in elderly individuals. Next, in order to evaluate the effect of age in NBCs and MBCs, we compared the gene expression pattern of young versus aged subjects in both cell populations. Interestingly, we did not find significant differences in the naïve population between young and aged individuals, whereas we found 1593 genes differentially expressed in young versus elderly subjects within MBCs; therefore, age affects the gene expression pattern of MBCs but not NBCs. Furthermore, we identified 467 genes which displayed a higher or lower expression in MBCs from aged as compared to younger individuals and NBCs, most of them involved in proliferation and cell cycle, apoptosis, cell survival and hematological diseases (Table 1).

Conclusions

Gene expression profiles of NBCs and MBCs are different. While there are no significant differences in NBCs of young versus elderly individuals, we detected significant differences in expression patterns of MBCs between both age groups, which indicates that MBCs could be more susceptible to age related alterations.

Disclosures:

No relevant conflicts of interest to declare.
JAK2V617F Mutation In Hematologic Malignancies In Childhood and Myeloproliferative Disorders In Adulthood
Authors: Yigin Akin , Antmen Bulent Ali , Guvenmez Hatice Korkmaz , Yilmaz Mustafa , Sasmaz Ilgen , Kilinc Yurdanur
Published: October 2019

Introduction

Mutations in JAK2 have been implicated in polycythemia vera (PV), essential thrombocythemia (ES), primary myelofibrosis (PMF) as wll as other myeloproliferative disorders.

Aim

In this study we aimed to investigate the frequency of JAK2V617F mutation on 216 patients with hematologic malignancies in childhood and 176 patients with myeloproliferative disorders in adulthood.

Materials and method

Patient group consist of 164 ALL and 52 AML patients in childhood and 79 PV, 51 ES, 22 chronic myeloid leukemia patients (CML) and 24 PMF patients in adulthood. These patients followed by Cukurova University, Departments of Pediatric and Adult Hematology, are included in this study. Blood samples were collected in these patients group and DNA was isolated using high pure template preparation kit (Roche) and stored -80oC. Gene mutations were studied using TMB (TıbMolBiol) LightMix Kit JAK2V617F genomic and analyzed by Light Cycler 2.0 Roche Diagnostic, GmBh, Germany in both groups.

Findings

JAK2V617F mutation was found 1 of 164 ALL patients (0,6%), 0 of 52 AML patients (0%) in childhood. Nevertheless, JAK2V617F mutation was also found 71 of 79 PV patients (89,8%), 22 of 51 ET patients (43,1%), 1 of 22 CML patients (4,5%) and 15 of 24 PMF patients (62,5%) in adulthood.

Result

As a result we found high frequency of JAK2V617F mutation in PV patients than the other myeloproliferative disorders. JAK2V617F mutation was significantly high in myeloproliferative disorders in adulthood comparing with childhood acute leukemias (p<0,01). Also, this mutation was significantly high in PV patients comparing with the other myeloproliferative disorders (P<0,05). We found statistically significance between genotype and allels distribution of JAK2V617F mutation (p<0.001) and determined that T-allels may be risk of disease in terms of allel distrubition (p<0.001). Thsi study is one of the widest series to investigation of JAK2V617F mutation frequency especially in childhood hematologic malignancies and we showed that this mutation is almost all 0% in this age group. On the other hand, we showed that JAK2V617F mutation with using realtime PCR test is very useful test for diagnosing PV and ET desase.

Disclosures:

No relevant conflicts of interest to declare.
Role Of Stromal Cell-Mediated Notch Signaling In Hematological Malignancies
Authors: Bassi Giulio , Kamga Paul Takam , Kamdje Armel Nwabo , Stradoni Roberta , Malpeli Giorgio , Amati Eliana , Nichele Ilaria , Carusone Roberta , Zanoncello Jasmina , Pizzolo Giovanni , Krampera Mauro
Published: October 2019

Stromal cells are essential components of the bone marrow (BM) microenvironment regulating and supporting the survival of different tumors, including B-cell acute and chronic lymphocytic leukemia (B-ALL and CLL), and acute myeloid leukemia (AML). In this study, we investigated the role of Notch signalling in human BM-mesenchymal stromal cell (hBM-MSC)-promoted ALL, CLL and AML survival and chemoresistance. The block of Notch signalling through γ-secretase inhibitor (GSI) XII reverted the protective effect mediated by co-culture with BM-MSC. The treatment with combinations of anti-Notch neutralizing Abs resulted in the decrease of B-ALL cell survival, either cultured alone or cocultured in presence of BM-MSC from normal donors and B-ALL patients. The inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the GSI XII. The same Notch receptors are involved in CLL survival except for Notch-1 that, in CLL, mediates a synergistic effect with other Notch receptors in inducing the anti-apoptotic phenotype. Some preliminary data showed that Notch system is involved in survival and chemoresistance of acute myeloid leukemia blasts. Overall, our ﬁndings show that stromal cell-mediated Notch signaling has a role in promoting ALL, CLL and AML survival and resistance to chemotherapy. Therefore, the target of Notch pathway activation may represent a useful strategy to overcome drug resistance and improve the efficacy of conventional treatments.

Disclosures:

No relevant conflicts of interest to declare.
Outcome Of Patients With Acute Myeloid Leukemia/High Risk Myelodysplastic Syndrome Based On Bone Mineral Density: Data From a Single center 2000-2012
Authors: Anand Ankit , Al-sharif Hussam , Abdelfatah Mohamed , Haller Nairmeen , Al-Ameri Ali
Published: October 2019

Background

Acute myeloid leukemia results from clonal proliferation of myeloid precursors, resulting in accumulation of leukemic blasts in the bone marrow. The effect of leukemic blasts in the bone have not been well studied. It is hypothesized that leukemic blasts may affect the osteoblast and osteoclast balance.

Aim

The aim of this study is to describe the effect of osteoporosis/osteopenia on the survival of patients with AML/ high risk MDS

Methods

A retrospective study of all AML and high-risk MDS patients treated at Akron General Medical Center, Ohio during 2001-2012. Following IRB approval, patients' charts were reviewed for information on demographics, cytogenetics, and treatment. Patients were classified into low risk, intermediate risk and high risk MDS based on WHO criteria. Charts were reviewed for a diagnosis of osteoporosis/osteopenia. Overall survival rates were determined by Kaplan-Meier survival analysis. Patients were classified into osteopenia/osteoporosis based on bone density scans. An additional three patients were identified with osteopenia on the basis of lumbar X rays and one patient was diagnosed with osteoporosis on the basis of typical compression fracture for osteoporosis.

Results

During the study period, 187 patients were identified with AML/high risk MDS. A total of 31 patients were found to have osteopenia/osteoporosis based on radiological findings. Eight patients had osteoporosis and 24 patients had osteopenia. A survival analysis was performed on the study population based on bone mineral density. Group one (osteoporosis) included eight patients; group two (osteopenia) included 23 patients and group three (no radiological evidence of osteoporosis/osteopenia) included 156 patients. Results are depicted in the survival curve below.

Conclusions

Bone mineral density remains an unidentified factor in patient's survival, suffering from AML/ High risk MDS. It appears that patients with normal bone density may live longer than patients with osteopenia/osteoporosis, but this is not confirmed here statistically. Future studies with a larger patient sample may be helpful in further exploring this aspect and the role of bisphosphonates in patients with AML/high risk MDS.

Disclosures:

No relevant conflicts of interest to declare.
Creation Of Patient Derived AML Xenografts Displaying Distinct Phenotypes and Geneotypes
Authors: Li Henry Qixiang , Liu Jingping , An Xiaoyu , Wang Na , Huang Liang , Wu Ran , Cai Jie , Wery Jean-Pierre
Published: October 2019

We have recently successfully engrafted leukemic cells from bone marrow of several AML patients into immunocompromised NOD/SCID mice. One of them, AM7577, was reported in the last year ASH1. This model displayed typical aggressive AML disease of M5 subtype, which starts at bone marrow and gradually expand to peripheral (spleen, lymphonode and peripheral blood…). AM7577 also harbors interesting genotypes of mutations for IDH2-R140Q, FLT3-ITD, DNMT3A R882H and NPM1.

Our second AML PDX model, AM8096, was established similarly using bone marrow from a recurrent patient with AML-M2 disease. AML8096 displayed similarly aggressive disease and the same 100% mortality, including typical symptoms (BW loss, hunched, inactivity, labored breathing, ruffled coat and eventual mortality) and with abundant leukemic cells in bone, etc. The leukemic cells can serially be passed in mice with 100% take-rate and cause consistent disease (even with < 1e4 cells). This also creates a renewable and potentially unlimited source of leukemia cells. The leukemic cells in mice are identical to those of the original patient leukemic cells (CD45+, CD33+, CD13+, CD123+, and CD19-. However, some aspects of disease presentations are vastly different from that of AM7577. First, the peripheral symptom is significant less characterized by lower leukemic counts in peripheral blood and only slightly enlarged spleen and smaller lymphonode. Second, the leukemic cell morphology is also rather different with AM8096 demonstrating less differentiated phenotype. Third, the genotype, in contrast to AM7577, is wild-types for all the above oncogenes. While we have not identified the likely leukemogenesis driver gene for this model, we are currently performing RNAseq of AM8096, along with AM7577, in order to explore the underlying molecular mechanisms that drive both diseases. In addition, we are also investigating the drug response to standard of care (SOC), as compared to those AM7577.

In summary, the two AML models could serve as useful experimental models to investigate the diverse leukemogenesis, including molecular mechanisms by genetic profiling. Ultimately, they can be used to help to identify new treatment strategies for AML.

1. Liu, e.a. A unique leukemia mouse model established from AML patient with IDH2 R140Q and FLT3-ITD mutations among other common AML mutations. ASH-2012 Annual Meeting (2012).

Disclosures:

Li: Crown Bioscience: Employment. Liu:Crown Bioscience: Employment. An:Crown Bioscience: Employment. Wu:Crown Bioscience: Employment. Cai:Crown Bioscience: Employment. Wery:Crown Bioscience: Employment.
Identification and Characterization Of a Potent FLT3 Inhibitor
Authors: Chen Yun , Guo Yao , Ho Wanting , Joe Zhizhuang
Published: October 2019

Acute myeloid leukemia (AML) is a malignant myeloid disorder for which there is no effective treatment. Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in AML patients. This makes FLT3 an attractive therapeutic target. Currently, several potent FLT3 inhibitors have been developed. However, their clinical efficacy is limited largely due to their poor effectiveness toward the FLT3-D835 mutants which are often present in AML or acquired after treatment of FLT3-ITD-positive AML with tyrosine kinase inhibitors. Needless to say, more potent FLT3 inhibitors targeting both FLT-ITD and FLT3-D835 mutants are needed. In addition, combinations of tyrosine kinase inhibitors with drugs targeting other signaling pathways represent a new trend in anti-cancer drug development.

To establish an effective kinase assay for FLT3 inhibitor screening, we generated a protein substrate designated GST-FLT3S which was expressed in E. coli cells as a glutathione S-transferase fusion protein. The protein substrate together with recombinant proteins containing the catalytic domain of wild type and mutant forms of FLT3 expressed in baculovirus was used in biochemical screening of inhibitors. Several potent inhibitors were obtained. Importantly, one of the inhibitors with an oxindole core structure inhibited FLT3 and D835 FLT3 mutants equally well with nanomolar IC50 values. We further analyzed the potency of the inhibitor by performing cell-based assays. The cells used included FLT3-ITD-positive cell line MV-4-11 and an EPO-dependent erythroleukemia cell line transformed by retrovirus mediated expressions of FLT3-ITD and FLT3-D835 mutants. At nanomolar concentrations, the inhibitor blocked growth factor signaling and effectively caused apoptosis and cell cycle arrest. It showed significant advantage over the current available FLT3 inhibitor, sorafenib.

Loss-of-function mutations of tumor suppressor p53 are common in solid tumors but relatively rare in AML although its expression is often suppressed. This makes p53 a potential target for anti-AML drug development. We employed MDM2 inhibitor nutlin-3 which blocks the degradation of p53. Importantly, at sub-nanomolar concentrations, FLT3 inhibitors and nutlin-3 synergistically inhibited growth of cells containing FLT3-ITD or FLT3-D835 mutants.

Altogether, we developed an effective substrate for screening of FLT3 inhibitors and identified one compound with high potency toward both FLT3-ITD and FLT3-D835. We further demonstrated that targeting FLT3 and p53 simultaneously greatly increases drug potency.

Disclosures:

No relevant conflicts of interest to declare.
The Survival Of The Elderly Acute Myeloid Leukemia Patients Do Not Depend On The Intensity Of Induction Chemotherapy. Retrospective Study Results
Authors: Grosicki Sebastian , Bodzenta Ewa , Kriegler Marek , Szypula Ilona , Fejklowicz Marcin , Twardosz Maja , Kurzawa Teresa , Barchnicka Agnieszka , Melisz Aleksandra
Published: October 2019

The aims of this study were comparison of overall survival (OS) and complete remission rate (CR) in whole group of acute myeloid leukemia (AML) >60 patients, and in younger older (61 – 70) AML patients in dependency of qualification to risk group according to PALG stratification and a schedule of treatment.

Patients with AML >60 after diagnosis were qualified into the risk groups according to PALG stratification for AML >60. The patients from group 1 patients were receiving intensive induction chemotherapy DAC (daunorubicin, cytarabine, cladribine) or DA (daunorubicin, cytarabine), those from group 2 were receiving cytarabine+thioguanine induction chemotherapy, and those from group 3 chemotherapy with low doses of cytarabine. Patients from first group, who reached CR after induction were receiving consolidation with mitoxantrone + standard dose of cytarabine, and next they were qualified to HSCT or maintenance phase. Patients from group 2 and 3 independently of results of induction were receiving successive cycles of chemotherapy like in first course every 4 weeks to 2 years with very intensive supportive care.

One hundred sixteen patients with untreated AML treated In period 02.2009-12.2012, age 73 (61-89), men 50% were included to this study. Thirty three patients were qualified to group 1, 50 to second and 33 to third. After preliminary analysis, because of similar early and late outcomes of the therapy in group 2 and 3, those groups of patients were joint for next analysis.

Disclosures:

No relevant conflicts of interest to declare.