HSC GENETIC HETEROGENEITY DETERMINES CLONAL DYNAMICS IN PMF
(Abstract release date: 05/19/16)
EHA Library. Triviai I. 06/11/16; 135211; S455
Dr. Ioanna Triviai
Contributions
Contributions
Abstract
Abstract: S455
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall A3
Background
Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm of stem cell origin, characterized by sequential distinctive waves of aberrant myeloid differentiation. Atypical erythroid-megakaryocytic progenitors expand at the initial stages of PMF and perish in the course of PMF development and/or the expansion of pre-leukemic clones. The continuum of clonal dynamics dominating the chronic phase disease, as exhibited in the variability of divergent myeloid progeny in PMF progression, reflects the molecular heterogeneity of malignant HSCs that sustain fluctuations in clone propagation.
Aims
In our previous work, we described a CD133+ stem cell population circulating in PMF patient peripheral blood, which exhibits multi-lineage differentiation capacity in vivo and in vitro and induces the PMF phenotype in the first xenotransplantation model of PMF. Single cell analysis of CD133+ patient-derived HSCs revealed multi-clonal lineage restricted differentiation potential of the stem cell pool in PMF. The correlation of the molecular burden of individual stem cells with their differentiation potential sheds light on the order of genetic lesions orchestrating the sequence of impaired stem cell function.
Methods
CD133+ HSCs from 100 PMF patients were molecularly characterized by whole exon sequencing. Sorted HSC cells were functionally analyzed at a single cell level for variable myeloid colony formation. 2230 colonies were phenotypically characterized and isolated. Analysis of the PMF HSC clonogenic potential indicates that the presence of mutations in the epigenetic regulator EZH2 correlates with granulo/monocytic differentiation but limited erythroid colony formation potential (0-0,05%), as determined in three different patient samples (2 JAK2-V617F+, 1 CALR-fs*+). Transplantation of these patient samples gave the highest engraftment in our mouse model and in one case, EZH2muJAK2wt leukemic transformation.
Results
CD133+ HSC-derived single colony analysis indicated 8 different genotypic clones of HSC, which exhibit variable granulo/monocytic differentiation capacity in vitro. From a total of 569 formed colonies, 538 were CFU-GM,-G,-M and 31 BFU-E. PCR analysis of colonies for JAK2-V617F and Sanger sequencing for EZH2-D265H and EZH2-Y733C mutations indicates that the presence of JAK2-V617F in hetero- or homozygosity can occur in the EZH2-D265H background without influencing the granulo/monocytic commitment of these mutated HSCs. Interestingly, the limited BFU-Es that arose contained only single JAK2-V617F mutations in the same patient. Moreover, the presence of single EZH2-D265H heterozygous clones, single JAK2-V617F hetero- or homozygous clones, as well as double mutated clones indicate two independent mutational events affecting the same locus and nucleotide have occurred in this patient. In view of the overall high frequency of JAK2-V617F mutations, we predict that the EZH2 mutation was the first mutation in double mutant clones in this patient. The presence of homozygous EZH2-Y733C mutations in other patients was detected irrespectively of JAK2-V617F mutation and is correlated with granulocytic-monocytic committed progenitors.
Conclusion
Our results indicate that mutations in epigenetic regulators precede the expanding mutations of the chronic phase PMF (JAK2, CALR). Thus, they confer the genomic instability connected with subsequent emergence of following mutations and they shape the genomic landscape supporting the expansion of pre-leukemic clones.
Session topic: Myeloproliferative neoplasms - Clinical 2
Keyword(s): AML, Bone Marrow Fibrosis, Epigenetic, Hematopoietic stem cell
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall A3
Background
Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm of stem cell origin, characterized by sequential distinctive waves of aberrant myeloid differentiation. Atypical erythroid-megakaryocytic progenitors expand at the initial stages of PMF and perish in the course of PMF development and/or the expansion of pre-leukemic clones. The continuum of clonal dynamics dominating the chronic phase disease, as exhibited in the variability of divergent myeloid progeny in PMF progression, reflects the molecular heterogeneity of malignant HSCs that sustain fluctuations in clone propagation.
Aims
In our previous work, we described a CD133+ stem cell population circulating in PMF patient peripheral blood, which exhibits multi-lineage differentiation capacity in vivo and in vitro and induces the PMF phenotype in the first xenotransplantation model of PMF. Single cell analysis of CD133+ patient-derived HSCs revealed multi-clonal lineage restricted differentiation potential of the stem cell pool in PMF. The correlation of the molecular burden of individual stem cells with their differentiation potential sheds light on the order of genetic lesions orchestrating the sequence of impaired stem cell function.
Methods
CD133+ HSCs from 100 PMF patients were molecularly characterized by whole exon sequencing. Sorted HSC cells were functionally analyzed at a single cell level for variable myeloid colony formation. 2230 colonies were phenotypically characterized and isolated. Analysis of the PMF HSC clonogenic potential indicates that the presence of mutations in the epigenetic regulator EZH2 correlates with granulo/monocytic differentiation but limited erythroid colony formation potential (0-0,05%), as determined in three different patient samples (2 JAK2-V617F+, 1 CALR-fs*+). Transplantation of these patient samples gave the highest engraftment in our mouse model and in one case, EZH2muJAK2wt leukemic transformation.
Results
CD133+ HSC-derived single colony analysis indicated 8 different genotypic clones of HSC, which exhibit variable granulo/monocytic differentiation capacity in vitro. From a total of 569 formed colonies, 538 were CFU-GM,-G,-M and 31 BFU-E. PCR analysis of colonies for JAK2-V617F and Sanger sequencing for EZH2-D265H and EZH2-Y733C mutations indicates that the presence of JAK2-V617F in hetero- or homozygosity can occur in the EZH2-D265H background without influencing the granulo/monocytic commitment of these mutated HSCs. Interestingly, the limited BFU-Es that arose contained only single JAK2-V617F mutations in the same patient. Moreover, the presence of single EZH2-D265H heterozygous clones, single JAK2-V617F hetero- or homozygous clones, as well as double mutated clones indicate two independent mutational events affecting the same locus and nucleotide have occurred in this patient. In view of the overall high frequency of JAK2-V617F mutations, we predict that the EZH2 mutation was the first mutation in double mutant clones in this patient. The presence of homozygous EZH2-Y733C mutations in other patients was detected irrespectively of JAK2-V617F mutation and is correlated with granulocytic-monocytic committed progenitors.
Conclusion
Our results indicate that mutations in epigenetic regulators precede the expanding mutations of the chronic phase PMF (JAK2, CALR). Thus, they confer the genomic instability connected with subsequent emergence of following mutations and they shape the genomic landscape supporting the expansion of pre-leukemic clones.
Session topic: Myeloproliferative neoplasms - Clinical 2
Keyword(s): AML, Bone Marrow Fibrosis, Epigenetic, Hematopoietic stem cell
Abstract: S455
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall A3
Background
Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm of stem cell origin, characterized by sequential distinctive waves of aberrant myeloid differentiation. Atypical erythroid-megakaryocytic progenitors expand at the initial stages of PMF and perish in the course of PMF development and/or the expansion of pre-leukemic clones. The continuum of clonal dynamics dominating the chronic phase disease, as exhibited in the variability of divergent myeloid progeny in PMF progression, reflects the molecular heterogeneity of malignant HSCs that sustain fluctuations in clone propagation.
Aims
In our previous work, we described a CD133+ stem cell population circulating in PMF patient peripheral blood, which exhibits multi-lineage differentiation capacity in vivo and in vitro and induces the PMF phenotype in the first xenotransplantation model of PMF. Single cell analysis of CD133+ patient-derived HSCs revealed multi-clonal lineage restricted differentiation potential of the stem cell pool in PMF. The correlation of the molecular burden of individual stem cells with their differentiation potential sheds light on the order of genetic lesions orchestrating the sequence of impaired stem cell function.
Methods
CD133+ HSCs from 100 PMF patients were molecularly characterized by whole exon sequencing. Sorted HSC cells were functionally analyzed at a single cell level for variable myeloid colony formation. 2230 colonies were phenotypically characterized and isolated. Analysis of the PMF HSC clonogenic potential indicates that the presence of mutations in the epigenetic regulator EZH2 correlates with granulo/monocytic differentiation but limited erythroid colony formation potential (0-0,05%), as determined in three different patient samples (2 JAK2-V617F+, 1 CALR-fs*+). Transplantation of these patient samples gave the highest engraftment in our mouse model and in one case, EZH2muJAK2wt leukemic transformation.
Results
CD133+ HSC-derived single colony analysis indicated 8 different genotypic clones of HSC, which exhibit variable granulo/monocytic differentiation capacity in vitro. From a total of 569 formed colonies, 538 were CFU-GM,-G,-M and 31 BFU-E. PCR analysis of colonies for JAK2-V617F and Sanger sequencing for EZH2-D265H and EZH2-Y733C mutations indicates that the presence of JAK2-V617F in hetero- or homozygosity can occur in the EZH2-D265H background without influencing the granulo/monocytic commitment of these mutated HSCs. Interestingly, the limited BFU-Es that arose contained only single JAK2-V617F mutations in the same patient. Moreover, the presence of single EZH2-D265H heterozygous clones, single JAK2-V617F hetero- or homozygous clones, as well as double mutated clones indicate two independent mutational events affecting the same locus and nucleotide have occurred in this patient. In view of the overall high frequency of JAK2-V617F mutations, we predict that the EZH2 mutation was the first mutation in double mutant clones in this patient. The presence of homozygous EZH2-Y733C mutations in other patients was detected irrespectively of JAK2-V617F mutation and is correlated with granulocytic-monocytic committed progenitors.
Conclusion
Our results indicate that mutations in epigenetic regulators precede the expanding mutations of the chronic phase PMF (JAK2, CALR). Thus, they confer the genomic instability connected with subsequent emergence of following mutations and they shape the genomic landscape supporting the expansion of pre-leukemic clones.
Session topic: Myeloproliferative neoplasms - Clinical 2
Keyword(s): AML, Bone Marrow Fibrosis, Epigenetic, Hematopoietic stem cell
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 12:15 - 12:30
Location: Hall A3
Background
Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm of stem cell origin, characterized by sequential distinctive waves of aberrant myeloid differentiation. Atypical erythroid-megakaryocytic progenitors expand at the initial stages of PMF and perish in the course of PMF development and/or the expansion of pre-leukemic clones. The continuum of clonal dynamics dominating the chronic phase disease, as exhibited in the variability of divergent myeloid progeny in PMF progression, reflects the molecular heterogeneity of malignant HSCs that sustain fluctuations in clone propagation.
Aims
In our previous work, we described a CD133+ stem cell population circulating in PMF patient peripheral blood, which exhibits multi-lineage differentiation capacity in vivo and in vitro and induces the PMF phenotype in the first xenotransplantation model of PMF. Single cell analysis of CD133+ patient-derived HSCs revealed multi-clonal lineage restricted differentiation potential of the stem cell pool in PMF. The correlation of the molecular burden of individual stem cells with their differentiation potential sheds light on the order of genetic lesions orchestrating the sequence of impaired stem cell function.
Methods
CD133+ HSCs from 100 PMF patients were molecularly characterized by whole exon sequencing. Sorted HSC cells were functionally analyzed at a single cell level for variable myeloid colony formation. 2230 colonies were phenotypically characterized and isolated. Analysis of the PMF HSC clonogenic potential indicates that the presence of mutations in the epigenetic regulator EZH2 correlates with granulo/monocytic differentiation but limited erythroid colony formation potential (0-0,05%), as determined in three different patient samples (2 JAK2-V617F+, 1 CALR-fs*+). Transplantation of these patient samples gave the highest engraftment in our mouse model and in one case, EZH2muJAK2wt leukemic transformation.
Results
CD133+ HSC-derived single colony analysis indicated 8 different genotypic clones of HSC, which exhibit variable granulo/monocytic differentiation capacity in vitro. From a total of 569 formed colonies, 538 were CFU-GM,-G,-M and 31 BFU-E. PCR analysis of colonies for JAK2-V617F and Sanger sequencing for EZH2-D265H and EZH2-Y733C mutations indicates that the presence of JAK2-V617F in hetero- or homozygosity can occur in the EZH2-D265H background without influencing the granulo/monocytic commitment of these mutated HSCs. Interestingly, the limited BFU-Es that arose contained only single JAK2-V617F mutations in the same patient. Moreover, the presence of single EZH2-D265H heterozygous clones, single JAK2-V617F hetero- or homozygous clones, as well as double mutated clones indicate two independent mutational events affecting the same locus and nucleotide have occurred in this patient. In view of the overall high frequency of JAK2-V617F mutations, we predict that the EZH2 mutation was the first mutation in double mutant clones in this patient. The presence of homozygous EZH2-Y733C mutations in other patients was detected irrespectively of JAK2-V617F mutation and is correlated with granulocytic-monocytic committed progenitors.
Conclusion
Our results indicate that mutations in epigenetic regulators precede the expanding mutations of the chronic phase PMF (JAK2, CALR). Thus, they confer the genomic instability connected with subsequent emergence of following mutations and they shape the genomic landscape supporting the expansion of pre-leukemic clones.
Session topic: Myeloproliferative neoplasms - Clinical 2
Keyword(s): AML, Bone Marrow Fibrosis, Epigenetic, Hematopoietic stem cell
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