FACS SORTED PERIPHERAL BLOOD CELLS IDENTIFY CALR MUTATIONS IN B-AND T CELLS
(Abstract release date: 05/19/16)
EHA Library. Kjær L. 06/09/16; 132882; E1333
Mr. Lasse Kjær
Contributions
Contributions
Abstract
Abstract: E1333
Type: Eposter Presentation
Background
Somatic mutations in exon 9 of the calreticulin gene (CALR) are found in the majority of the JAK2V617F negative Philadelphia-negative myeloproliferative neoplasms (MPNs) essential thrombocythemia (ET) and primary myelofibrosis (PMF). A hallmark of the mutated CALR protein is its preferential expression in megakaryocytes, and so far, mutations in CALR have only been found in cells of myeloid lineage. However, mutations in the CALR gene are considered an early event and thus expected to be present in cells of the lymphoid compartment as well. The mutant allele burden has been suggested to have an impact on the disease phenotype, but patients with high mutant allele burden appear to be rare and it is unknown if homozygous sub-clones is common in patients.
Aims
Here we wish to determine if the CALR mutations are present in B- and T- lymphocytes and investigate if homozygous clones are present in the patients.
Methods
In this study, we separated the myeloid and lymphoid cell fractions from 11 CALR mutated patients (4 ET, 1 pre-fibrotic primary myelofibrosis (pre-PMF), 5 PMF, and 1 post-ET MF) using fluorescent activated cell sorting (FACS). The mutations included the type 1 and type 2 subtypes, and two different 34 base-pair deletions. The allele burden was quantified for type 1 and 2 mutations using allele specific qPCR and the 34 base-pair deletions quantified by fragment analysis. Granulocytic and erythroid cell colonies from 15 patients (4 ET, 3 pre-PMF, 7 PMF, and 1 post-ET MF) were cultured in the presence of erythropoietin and after expansion individual colonies was isolated and analyzed for the CALR mutant allele burden.
Results
The mutant allele burden was determined in whole-blood (median: 41%, range: 3-47%), granulocytes (CD16+ median: 51%, range: 4.8-63%), and monocytes (CD14+; median: 47%, range: 5.6-61%). In 3 of 11 patients (27%), the type 1 mutation was found in T-lymphocytes (CD3+; median: 6.4%, range: 5-7.8%), and in 2 of 11 patients (11%) in B-lymphocytes (CD19+; median: 25%, range: 18-31%). One patient had the type 1 mutation in both cell types. The type 2 mutation was found in T-lymphocytes of a single patient (16%). Isolated granulocytes and erythroid cells from cultured cell colonies revealed the CALR positive clones from all patients to be heterozygous for the mutations.
Conclusion
The presence of the CALR mutations in both myeloid and lymphoid lineages indicates the CALR mutation to be an early event in a multipotent hematopoietic stem cell expanding as a heterozygous mutated clone of the myeloid lineage to become dominant in the majority of CALR mutated MPN patients.
Session topic: E-poster
Keyword(s): Flow cytometry, Mutation analysis, Myeloproliferative disorder, Quantitative RT-PCR
Type: Eposter Presentation
Background
Somatic mutations in exon 9 of the calreticulin gene (CALR) are found in the majority of the JAK2V617F negative Philadelphia-negative myeloproliferative neoplasms (MPNs) essential thrombocythemia (ET) and primary myelofibrosis (PMF). A hallmark of the mutated CALR protein is its preferential expression in megakaryocytes, and so far, mutations in CALR have only been found in cells of myeloid lineage. However, mutations in the CALR gene are considered an early event and thus expected to be present in cells of the lymphoid compartment as well. The mutant allele burden has been suggested to have an impact on the disease phenotype, but patients with high mutant allele burden appear to be rare and it is unknown if homozygous sub-clones is common in patients.
Aims
Here we wish to determine if the CALR mutations are present in B- and T- lymphocytes and investigate if homozygous clones are present in the patients.
Methods
In this study, we separated the myeloid and lymphoid cell fractions from 11 CALR mutated patients (4 ET, 1 pre-fibrotic primary myelofibrosis (pre-PMF), 5 PMF, and 1 post-ET MF) using fluorescent activated cell sorting (FACS). The mutations included the type 1 and type 2 subtypes, and two different 34 base-pair deletions. The allele burden was quantified for type 1 and 2 mutations using allele specific qPCR and the 34 base-pair deletions quantified by fragment analysis. Granulocytic and erythroid cell colonies from 15 patients (4 ET, 3 pre-PMF, 7 PMF, and 1 post-ET MF) were cultured in the presence of erythropoietin and after expansion individual colonies was isolated and analyzed for the CALR mutant allele burden.
Results
The mutant allele burden was determined in whole-blood (median: 41%, range: 3-47%), granulocytes (CD16+ median: 51%, range: 4.8-63%), and monocytes (CD14+; median: 47%, range: 5.6-61%). In 3 of 11 patients (27%), the type 1 mutation was found in T-lymphocytes (CD3+; median: 6.4%, range: 5-7.8%), and in 2 of 11 patients (11%) in B-lymphocytes (CD19+; median: 25%, range: 18-31%). One patient had the type 1 mutation in both cell types. The type 2 mutation was found in T-lymphocytes of a single patient (16%). Isolated granulocytes and erythroid cells from cultured cell colonies revealed the CALR positive clones from all patients to be heterozygous for the mutations.
Conclusion
The presence of the CALR mutations in both myeloid and lymphoid lineages indicates the CALR mutation to be an early event in a multipotent hematopoietic stem cell expanding as a heterozygous mutated clone of the myeloid lineage to become dominant in the majority of CALR mutated MPN patients.
Session topic: E-poster
Keyword(s): Flow cytometry, Mutation analysis, Myeloproliferative disorder, Quantitative RT-PCR
Abstract: E1333
Type: Eposter Presentation
Background
Somatic mutations in exon 9 of the calreticulin gene (CALR) are found in the majority of the JAK2V617F negative Philadelphia-negative myeloproliferative neoplasms (MPNs) essential thrombocythemia (ET) and primary myelofibrosis (PMF). A hallmark of the mutated CALR protein is its preferential expression in megakaryocytes, and so far, mutations in CALR have only been found in cells of myeloid lineage. However, mutations in the CALR gene are considered an early event and thus expected to be present in cells of the lymphoid compartment as well. The mutant allele burden has been suggested to have an impact on the disease phenotype, but patients with high mutant allele burden appear to be rare and it is unknown if homozygous sub-clones is common in patients.
Aims
Here we wish to determine if the CALR mutations are present in B- and T- lymphocytes and investigate if homozygous clones are present in the patients.
Methods
In this study, we separated the myeloid and lymphoid cell fractions from 11 CALR mutated patients (4 ET, 1 pre-fibrotic primary myelofibrosis (pre-PMF), 5 PMF, and 1 post-ET MF) using fluorescent activated cell sorting (FACS). The mutations included the type 1 and type 2 subtypes, and two different 34 base-pair deletions. The allele burden was quantified for type 1 and 2 mutations using allele specific qPCR and the 34 base-pair deletions quantified by fragment analysis. Granulocytic and erythroid cell colonies from 15 patients (4 ET, 3 pre-PMF, 7 PMF, and 1 post-ET MF) were cultured in the presence of erythropoietin and after expansion individual colonies was isolated and analyzed for the CALR mutant allele burden.
Results
The mutant allele burden was determined in whole-blood (median: 41%, range: 3-47%), granulocytes (CD16+ median: 51%, range: 4.8-63%), and monocytes (CD14+; median: 47%, range: 5.6-61%). In 3 of 11 patients (27%), the type 1 mutation was found in T-lymphocytes (CD3+; median: 6.4%, range: 5-7.8%), and in 2 of 11 patients (11%) in B-lymphocytes (CD19+; median: 25%, range: 18-31%). One patient had the type 1 mutation in both cell types. The type 2 mutation was found in T-lymphocytes of a single patient (16%). Isolated granulocytes and erythroid cells from cultured cell colonies revealed the CALR positive clones from all patients to be heterozygous for the mutations.
Conclusion
The presence of the CALR mutations in both myeloid and lymphoid lineages indicates the CALR mutation to be an early event in a multipotent hematopoietic stem cell expanding as a heterozygous mutated clone of the myeloid lineage to become dominant in the majority of CALR mutated MPN patients.
Session topic: E-poster
Keyword(s): Flow cytometry, Mutation analysis, Myeloproliferative disorder, Quantitative RT-PCR
Type: Eposter Presentation
Background
Somatic mutations in exon 9 of the calreticulin gene (CALR) are found in the majority of the JAK2V617F negative Philadelphia-negative myeloproliferative neoplasms (MPNs) essential thrombocythemia (ET) and primary myelofibrosis (PMF). A hallmark of the mutated CALR protein is its preferential expression in megakaryocytes, and so far, mutations in CALR have only been found in cells of myeloid lineage. However, mutations in the CALR gene are considered an early event and thus expected to be present in cells of the lymphoid compartment as well. The mutant allele burden has been suggested to have an impact on the disease phenotype, but patients with high mutant allele burden appear to be rare and it is unknown if homozygous sub-clones is common in patients.
Aims
Here we wish to determine if the CALR mutations are present in B- and T- lymphocytes and investigate if homozygous clones are present in the patients.
Methods
In this study, we separated the myeloid and lymphoid cell fractions from 11 CALR mutated patients (4 ET, 1 pre-fibrotic primary myelofibrosis (pre-PMF), 5 PMF, and 1 post-ET MF) using fluorescent activated cell sorting (FACS). The mutations included the type 1 and type 2 subtypes, and two different 34 base-pair deletions. The allele burden was quantified for type 1 and 2 mutations using allele specific qPCR and the 34 base-pair deletions quantified by fragment analysis. Granulocytic and erythroid cell colonies from 15 patients (4 ET, 3 pre-PMF, 7 PMF, and 1 post-ET MF) were cultured in the presence of erythropoietin and after expansion individual colonies was isolated and analyzed for the CALR mutant allele burden.
Results
The mutant allele burden was determined in whole-blood (median: 41%, range: 3-47%), granulocytes (CD16+ median: 51%, range: 4.8-63%), and monocytes (CD14+; median: 47%, range: 5.6-61%). In 3 of 11 patients (27%), the type 1 mutation was found in T-lymphocytes (CD3+; median: 6.4%, range: 5-7.8%), and in 2 of 11 patients (11%) in B-lymphocytes (CD19+; median: 25%, range: 18-31%). One patient had the type 1 mutation in both cell types. The type 2 mutation was found in T-lymphocytes of a single patient (16%). Isolated granulocytes and erythroid cells from cultured cell colonies revealed the CALR positive clones from all patients to be heterozygous for the mutations.
Conclusion
The presence of the CALR mutations in both myeloid and lymphoid lineages indicates the CALR mutation to be an early event in a multipotent hematopoietic stem cell expanding as a heterozygous mutated clone of the myeloid lineage to become dominant in the majority of CALR mutated MPN patients.
Session topic: E-poster
Keyword(s): Flow cytometry, Mutation analysis, Myeloproliferative disorder, Quantitative RT-PCR
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