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TEL-ABL fusion genes using a multi-fusion gene QRT-PCR screening method in acute lymphoblastic leukemia
S88
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
treated the AML1/ETO positive cell line Kasumi-1 with 5-aza-2 deoxycytidine (DAC) and detected the expression level and the promoter activity of CST7. We found that both the CST7 mRNA level and the CST7 promoter activity significantly increased in DAC-treated Kasumi-1 cells. The data suggested that AML1/ETO might repress the gene transcription by changing the DNA methylation status of its target genes. Collectively, our study provides evidence that AML1/ETO suppresses CST7 promoter activity not only through directly binding to the promoter regions, but also depended on the DNA methylation status affected by AML1/ETO.
3098 - DETECTION OF TEL-AML1/TEL-ABL FUSION GENES USING A MULTI-FUSION GENE QRT-PCR SCREENING METHOD IN ACUTE LYMPHOBLASTIC LEUKEMIA Yufeng Liu1, Jingyu Zhang2, Shuting Mao2, Shufang Su2, and Linlin Wei2 1 The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; 2 the First Affiliated Hospital of Zhengzhou University, Henan, China As an important hematopoietic regulatory component, TEL rearrangement is observed in a variety of hematological malignancies including myelodysplastic syndromes, acute leukemias, and myeloproliferative disorders. TEL-ABL fusion is a rare gene rearrangement event, and cases where TEL-AML1 and TEL-ABL fusions occur concurrently have not been reported in the literature. Here, we describe a case of a 3-year-old girl with acute lymphoblastic leukemia (ALL) with concurrent TEL-AML1 and TEL-ABL rearrangements. The girl was admitted to the hospital with a week-long episode of intermittent abdominal pain. Physical examination revealed skin bleeding, lymphadenectasis and hepatomegaly. Bone marrow aspiration showed diffuse lymphoblast infiltration. Immunophenotyping indicated that bone marrow cells are predominately hemocytoblasts. G-band karyotyping analysis of the patient reveals a normal 46,XX karyotype .Wright-Giemsa staining of bone marrow blood smear indicated a predominance of primative and immature lymphoid cell populations. The patient was admitted B-ALL. Chromosomal gene fusion and consequent TEL-ABL transcript levels were quantified by multi-fusion gene qRT-PCR screening. Through the use of this method, we successfully detected the presence of TEL-ABL and TEL-AML1 fusion transcripts. Treatment with VDLP induction chemotherapy was administered, TEL-AML1 transcript levels were found to be reduced and TEL-ABL transcripts were undetectable, accompanied by hematological remission. This study demonstrates the efficacy of multi-fusion gene qRT-PCR screening methods to accurately detect TEL-related gene fusion, and documents the rare occurrence of two TEL fusion events in ALL. Due to early detection and prescribed induction chemotherapy treatment, the patient achieved hematological remission with reduction of both TEL-AML1 and TEL-ABL transcripts. Importantly, TEL-ABL levels were reduced to undetectable levels following treatment. As t(9;12) chromosomal rearrangements may not be detected using cytogenetic methods, this case underscores the efficacy of qRT-PCR detection methods to accurately identify gene fusions such as the TEL-AML1/TEL-ABL double fusion described above. Future identification of more concurrent double gene fusion events may give indication to the prognostic value of multiple fusions in ALL.
3099 - EARLY B CELL DIFFERENTIATION FROM FEEDER-FREE HES COLONIES Guojie Li and Sandeep Dave Duke University, Durham, USA The pluripotent stem cells are a powerful tool to study the molecular mechanisms of development and malignancy of hematological system. We adopted a method to generate CD34+CD38-CD90+ hematopoietic stem/progenitor cells (HSPCs) from feeder free hES cells colonies. These cells can form erythroid and myeloid lineage cells in the colony forming cell (CFC) assays. They also express HSPCs transcription factors such as RUNX1, PU.1. We found that when the colonies begin to produce HSPCs, if the cytokine cocktail in the media was changed to favor lymphoid conditions for 7-10 days, we observed CD45+CD19+ cells in the population. Further analysis demonstrated that they expressed EBF1, PAX5, IL7Ra and VPREB, which support they are at the pro-/pre- B cell stage. This method provides a quicker and alterative way to differentiate early B cells from pluripotent stem cells.
3100 - ANALYSIS OF HEMATOPOIETIC CELLS GENERATED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS DIFFERENTIATING IN TERATOMAS Margarita MacAldaz1,2,3, Paul Miller2, Melanie Kardel4, Connie Eaves2, Annelise Bennaceur-Griscelli5, and Ali Turhan5 1 Terry Fox Laboratory, Vancouver, Canada; 2BC Cancer Agency, Vancouver, Canada; 3University of British Columbia, Vancouver, Canada; 4 STEMCELL Technologies Inc, Vancouver, Canada; 5Institut National U935 de la Sante et de la Recherche Medicale, Hopitaux Universitaires Paris Sud Bicetre, Universite Paris-Sud 11, Paris, France Much progress has been made in characterizing human hematopoietic cells that can sustain the long-term output of mature blood cells in vivo. However, a method for their expansion ex vivo has remained elusive. One approach is to derive these cells from induced pluripotent stem cells (iPSCs), although a reproducible in vitro protocol for this has also not yet been achieved. Recently, however, some success has been reported in teratomas produced from human iPSCs transplanted into immunodeficient mice. We are now exploring the possibility that this latter approach may be improved using immunodeficient NOD-Rag1-null- IL2Rgc-null mice with a c-kit (W41/W41) deficiency (NRG-W41 mice) with or without a transgenic source of human IL3, GMCSF and SCF (NRG-W41-3GS mice). Initial experiments showed nonirradiated NRG-W41 mice support higher levels of multi-lineage human hematopoietic cell reconstitution from transplants of normal cord blood or bone marrow CD34+ cells compared to NRG controls. Thus, in our first teratoma experiments, we compared the output of human hematopoietic (CD45+) cells in NRG-W41 and NRG-W413GS mice injected subcutaneously with 103-106 human iPSCs 6 mouse fibroblasts engineered to produce human FLT3-L, SCF, IL3 and IL6 in Matrigel. Teratomas were consistently produced from 105 human iPSCs. Following their enzymatic dissociation into single viable cell suspensions, and FACS analysis of the cells with various human-specific antibodies, we found CD34+ cells were detected in all teratomas with O0.1% CD45+ human cells. Initial experiments demonstrated that teratomas produced w40-fold more CD34+CD45+ cells when generated in NRG-W41-3GS versus NRG-W41 with yields of up to 7x106 CD45+ cells from a teratoma containing 4x108 cells. The co-injection of human growth factor-producing mouse fibroblasts also increased the overall production of human CD34+and CD34+CD45+ cells by w6fold even in the NRG-W41-3GS mice. These teratoma derived cells also produced granulopoietic (GM), erythroid (E) and mixed (GEM) colonies in standard growth factor-supplemented methylcellulose cultures demonstrating the generation of functionally competent human hematopoietic progenitors in iPSC derived teratomas. These experiments lay the foundation for more detailed investigation of the conditions that will promote the generation of hematopoietic stem cells and their properties.
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
treated the AML1/ETO positive cell line Kasumi-1 with 5-aza-2 deoxycytidine (DAC) and detected the expression level and the promoter activity of CST7. We found that both the CST7 mRNA level and the CST7 promoter activity significantly increased in DAC-treated Kasumi-1 cells. The data suggested that AML1/ETO might repress the gene transcription by changing the DNA methylation status of its target genes. Collectively, our study provides evidence that AML1/ETO suppresses CST7 promoter activity not only through directly binding to the promoter regions, but also depended on the DNA methylation status affected by AML1/ETO.
3098 - DETECTION OF TEL-AML1/TEL-ABL FUSION GENES USING A MULTI-FUSION GENE QRT-PCR SCREENING METHOD IN ACUTE LYMPHOBLASTIC LEUKEMIA Yufeng Liu1, Jingyu Zhang2, Shuting Mao2, Shufang Su2, and Linlin Wei2 1 The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; 2 the First Affiliated Hospital of Zhengzhou University, Henan, China As an important hematopoietic regulatory component, TEL rearrangement is observed in a variety of hematological malignancies including myelodysplastic syndromes, acute leukemias, and myeloproliferative disorders. TEL-ABL fusion is a rare gene rearrangement event, and cases where TEL-AML1 and TEL-ABL fusions occur concurrently have not been reported in the literature. Here, we describe a case of a 3-year-old girl with acute lymphoblastic leukemia (ALL) with concurrent TEL-AML1 and TEL-ABL rearrangements. The girl was admitted to the hospital with a week-long episode of intermittent abdominal pain. Physical examination revealed skin bleeding, lymphadenectasis and hepatomegaly. Bone marrow aspiration showed diffuse lymphoblast infiltration. Immunophenotyping indicated that bone marrow cells are predominately hemocytoblasts. G-band karyotyping analysis of the patient reveals a normal 46,XX karyotype .Wright-Giemsa staining of bone marrow blood smear indicated a predominance of primative and immature lymphoid cell populations. The patient was admitted B-ALL. Chromosomal gene fusion and consequent TEL-ABL transcript levels were quantified by multi-fusion gene qRT-PCR screening. Through the use of this method, we successfully detected the presence of TEL-ABL and TEL-AML1 fusion transcripts. Treatment with VDLP induction chemotherapy was administered, TEL-AML1 transcript levels were found to be reduced and TEL-ABL transcripts were undetectable, accompanied by hematological remission. This study demonstrates the efficacy of multi-fusion gene qRT-PCR screening methods to accurately detect TEL-related gene fusion, and documents the rare occurrence of two TEL fusion events in ALL. Due to early detection and prescribed induction chemotherapy treatment, the patient achieved hematological remission with reduction of both TEL-AML1 and TEL-ABL transcripts. Importantly, TEL-ABL levels were reduced to undetectable levels following treatment. As t(9;12) chromosomal rearrangements may not be detected using cytogenetic methods, this case underscores the efficacy of qRT-PCR detection methods to accurately identify gene fusions such as the TEL-AML1/TEL-ABL double fusion described above. Future identification of more concurrent double gene fusion events may give indication to the prognostic value of multiple fusions in ALL.
3099 - EARLY B CELL DIFFERENTIATION FROM FEEDER-FREE HES COLONIES Guojie Li and Sandeep Dave Duke University, Durham, USA The pluripotent stem cells are a powerful tool to study the molecular mechanisms of development and malignancy of hematological system. We adopted a method to generate CD34+CD38-CD90+ hematopoietic stem/progenitor cells (HSPCs) from feeder free hES cells colonies. These cells can form erythroid and myeloid lineage cells in the colony forming cell (CFC) assays. They also express HSPCs transcription factors such as RUNX1, PU.1. We found that when the colonies begin to produce HSPCs, if the cytokine cocktail in the media was changed to favor lymphoid conditions for 7-10 days, we observed CD45+CD19+ cells in the population. Further analysis demonstrated that they expressed EBF1, PAX5, IL7Ra and VPREB, which support they are at the pro-/pre- B cell stage. This method provides a quicker and alterative way to differentiate early B cells from pluripotent stem cells.
3100 - ANALYSIS OF HEMATOPOIETIC CELLS GENERATED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS DIFFERENTIATING IN TERATOMAS Margarita MacAldaz1,2,3, Paul Miller2, Melanie Kardel4, Connie Eaves2, Annelise Bennaceur-Griscelli5, and Ali Turhan5 1 Terry Fox Laboratory, Vancouver, Canada; 2BC Cancer Agency, Vancouver, Canada; 3University of British Columbia, Vancouver, Canada; 4 STEMCELL Technologies Inc, Vancouver, Canada; 5Institut National U935 de la Sante et de la Recherche Medicale, Hopitaux Universitaires Paris Sud Bicetre, Universite Paris-Sud 11, Paris, France Much progress has been made in characterizing human hematopoietic cells that can sustain the long-term output of mature blood cells in vivo. However, a method for their expansion ex vivo has remained elusive. One approach is to derive these cells from induced pluripotent stem cells (iPSCs), although a reproducible in vitro protocol for this has also not yet been achieved. Recently, however, some success has been reported in teratomas produced from human iPSCs transplanted into immunodeficient mice. We are now exploring the possibility that this latter approach may be improved using immunodeficient NOD-Rag1-null- IL2Rgc-null mice with a c-kit (W41/W41) deficiency (NRG-W41 mice) with or without a transgenic source of human IL3, GMCSF and SCF (NRG-W41-3GS mice). Initial experiments showed nonirradiated NRG-W41 mice support higher levels of multi-lineage human hematopoietic cell reconstitution from transplants of normal cord blood or bone marrow CD34+ cells compared to NRG controls. Thus, in our first teratoma experiments, we compared the output of human hematopoietic (CD45+) cells in NRG-W41 and NRG-W413GS mice injected subcutaneously with 103-106 human iPSCs 6 mouse fibroblasts engineered to produce human FLT3-L, SCF, IL3 and IL6 in Matrigel. Teratomas were consistently produced from 105 human iPSCs. Following their enzymatic dissociation into single viable cell suspensions, and FACS analysis of the cells with various human-specific antibodies, we found CD34+ cells were detected in all teratomas with O0.1% CD45+ human cells. Initial experiments demonstrated that teratomas produced w40-fold more CD34+CD45+ cells when generated in NRG-W41-3GS versus NRG-W41 with yields of up to 7x106 CD45+ cells from a teratoma containing 4x108 cells. The co-injection of human growth factor-producing mouse fibroblasts also increased the overall production of human CD34+and CD34+CD45+ cells by w6fold even in the NRG-W41-3GS mice. These teratoma derived cells also produced granulopoietic (GM), erythroid (E) and mixed (GEM) colonies in standard growth factor-supplemented methylcellulose cultures demonstrating the generation of functionally competent human hematopoietic progenitors in iPSC derived teratomas. These experiments lay the foundation for more detailed investigation of the conditions that will promote the generation of hematopoietic stem cells and their properties.