MUTATIONAL PROFILE BY NEXT-GENERATION SEQUENCING IN ACUTE MYELOID LEUKEMIA (AML): A SINGLE CENTER EXPERIENCE
(Abstract release date: 05/19/16)
EHA Library. Monne M. 06/09/16; 134537; PB1637
Maria Monne
Contributions
Contributions
Abstract
Abstract: PB1637
Type: Publication Only
Background
AML is a clonal disorder of hematopoietic stem and progenitor cells caused by acquired and occasionally inherited genetic alterations. Cytogenetic aberrations together with gene mutations are powerful prognostic markers and the mutational profile can be incorporated in the decision making processes to treatment.
Aims
A real-life cohort of AML patients from Sardinia (Italy) was analyzed to evaluate the usefulness of a next-generation sequencing (NGS) gene panel for the detection of mutations associated with AML in a context of routine diagnostic molecular workup.
Methods
Thirty AML patients, 15 males and 15 females with median age of 60 years (range 18-88 years) were enrolled for the study. DNAs were screened with IonAmpliseq AML panel (ThermoFisher) on IonPGM platform. The panel includes hotspots mutations of the genes: ASXL1 (exon 12), BRAF (V600E),CBL(exons 8-9), FLT3(codons 676,830-850), IDH1(exon 4), IDH2(exon 4), JAK2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons3-8), WT1 (exons 7,9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53 genes.The panel design does not include the FLT3-ITD gene region. Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) chemistry and library clonal amplification was performed by emulsion PCR using Ion PGM™Template HiQ OT2 Reagents. Combination of 5 libraries on an Ion 318™Chip resulted in >2500X average coverage depth with >97% of the target bases covered at 500X. Genomic variants were filtered and analysed through AmpliseqAML single 5.0 workflow.
Results
The mean count of sequencing reads obtained per sample was 0.73 million and the mean sequencing depth was over 2500X with 91% uniformity. Seventy-five mutations in 19 genes were detected in 31 samples with an average of 2.3 mutations per sample. TP53 mutations were found in 4 patients of whom one with unfavorable cytogenetics.The AML panel detected 15 mutations (20%) in NPM1, 6 (8%) TET2, 10 (13%) DNMT3A, 6 (8%) CEBPA, 5 (7%)TP53, 3 (4%) RUNX1,4 (5%) FLT3, 4 (5%) IDH2, 2 (3%) ASXL1, 5 (7%) GATA2, 6 (8%) PTPN11 and 3 (4%) KIT genes. Only one patient remained wild-type for these genes. Based on mechanism of action, genes involved in signal transduction and DNA methylation were the most frequently mutated, accounting for the 39 % and 32% of detected mutations, respectively. The AML gene panel identified 100% of previously identified mutations by conventional molecular biology techniques in FLT3(TKD), NPM1, IDH1-2 and CEBPA genes. Mutational profiles obtained by NGS allowed to refine cytogenetic classification in the normal karyotype subgroup shifting a large proportion of patients (24 out of 30) from the intermediate to poor risk category.In one patient we were able to assess clonal dominance over the course of the disease treatment. A female 50 y.o. diagnosed with AML-M2 presented at diagnosis IDH2 p.R172K mutation, DNMT3A p.F640fs*11 and CEBPA p.P196_P197insHP at frequencies of 42%, 49% and 20%, respectively. Monitoring of mutation levels in two consecutive bone marrow samples during treatment according to AML1310 protocol, demonstrated treatment associated clonal responses. IDH2 mutation had a frequency of 7.3% after 4 weeks of therapy and resulted absent after 8 weeks (not detected over 6500 reads), while DNMT3A and CEBPA mutations were present at the levels of 30% and 47%, respectively. Longitudinal monitoring of three mutations indicated the presence of independent clones behaving discordantly during therapy.
Conclusion
A targeted multi gene panel analysis can provide in a short turnaround time a pretreatment mutational profile as well as the MRD status helping decision of intensity and type of induction therapy and modulation of post remission strategies.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, MRD, Mutation analysis
Type: Publication Only
Background
AML is a clonal disorder of hematopoietic stem and progenitor cells caused by acquired and occasionally inherited genetic alterations. Cytogenetic aberrations together with gene mutations are powerful prognostic markers and the mutational profile can be incorporated in the decision making processes to treatment.
Aims
A real-life cohort of AML patients from Sardinia (Italy) was analyzed to evaluate the usefulness of a next-generation sequencing (NGS) gene panel for the detection of mutations associated with AML in a context of routine diagnostic molecular workup.
Methods
Thirty AML patients, 15 males and 15 females with median age of 60 years (range 18-88 years) were enrolled for the study. DNAs were screened with IonAmpliseq AML panel (ThermoFisher) on IonPGM platform. The panel includes hotspots mutations of the genes: ASXL1 (exon 12), BRAF (V600E),CBL(exons 8-9), FLT3(codons 676,830-850), IDH1(exon 4), IDH2(exon 4), JAK2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons3-8), WT1 (exons 7,9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53 genes.The panel design does not include the FLT3-ITD gene region. Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) chemistry and library clonal amplification was performed by emulsion PCR using Ion PGM™Template HiQ OT2 Reagents. Combination of 5 libraries on an Ion 318™Chip resulted in >2500X average coverage depth with >97% of the target bases covered at 500X. Genomic variants were filtered and analysed through AmpliseqAML single 5.0 workflow.
Results
The mean count of sequencing reads obtained per sample was 0.73 million and the mean sequencing depth was over 2500X with 91% uniformity. Seventy-five mutations in 19 genes were detected in 31 samples with an average of 2.3 mutations per sample. TP53 mutations were found in 4 patients of whom one with unfavorable cytogenetics.The AML panel detected 15 mutations (20%) in NPM1, 6 (8%) TET2, 10 (13%) DNMT3A, 6 (8%) CEBPA, 5 (7%)TP53, 3 (4%) RUNX1,4 (5%) FLT3, 4 (5%) IDH2, 2 (3%) ASXL1, 5 (7%) GATA2, 6 (8%) PTPN11 and 3 (4%) KIT genes. Only one patient remained wild-type for these genes. Based on mechanism of action, genes involved in signal transduction and DNA methylation were the most frequently mutated, accounting for the 39 % and 32% of detected mutations, respectively. The AML gene panel identified 100% of previously identified mutations by conventional molecular biology techniques in FLT3(TKD), NPM1, IDH1-2 and CEBPA genes. Mutational profiles obtained by NGS allowed to refine cytogenetic classification in the normal karyotype subgroup shifting a large proportion of patients (24 out of 30) from the intermediate to poor risk category.In one patient we were able to assess clonal dominance over the course of the disease treatment. A female 50 y.o. diagnosed with AML-M2 presented at diagnosis IDH2 p.R172K mutation, DNMT3A p.F640fs*11 and CEBPA p.P196_P197insHP at frequencies of 42%, 49% and 20%, respectively. Monitoring of mutation levels in two consecutive bone marrow samples during treatment according to AML1310 protocol, demonstrated treatment associated clonal responses. IDH2 mutation had a frequency of 7.3% after 4 weeks of therapy and resulted absent after 8 weeks (not detected over 6500 reads), while DNMT3A and CEBPA mutations were present at the levels of 30% and 47%, respectively. Longitudinal monitoring of three mutations indicated the presence of independent clones behaving discordantly during therapy.
Conclusion
A targeted multi gene panel analysis can provide in a short turnaround time a pretreatment mutational profile as well as the MRD status helping decision of intensity and type of induction therapy and modulation of post remission strategies.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, MRD, Mutation analysis
Abstract: PB1637
Type: Publication Only
Background
AML is a clonal disorder of hematopoietic stem and progenitor cells caused by acquired and occasionally inherited genetic alterations. Cytogenetic aberrations together with gene mutations are powerful prognostic markers and the mutational profile can be incorporated in the decision making processes to treatment.
Aims
A real-life cohort of AML patients from Sardinia (Italy) was analyzed to evaluate the usefulness of a next-generation sequencing (NGS) gene panel for the detection of mutations associated with AML in a context of routine diagnostic molecular workup.
Methods
Thirty AML patients, 15 males and 15 females with median age of 60 years (range 18-88 years) were enrolled for the study. DNAs were screened with IonAmpliseq AML panel (ThermoFisher) on IonPGM platform. The panel includes hotspots mutations of the genes: ASXL1 (exon 12), BRAF (V600E),CBL(exons 8-9), FLT3(codons 676,830-850), IDH1(exon 4), IDH2(exon 4), JAK2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons3-8), WT1 (exons 7,9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53 genes.The panel design does not include the FLT3-ITD gene region. Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) chemistry and library clonal amplification was performed by emulsion PCR using Ion PGM™Template HiQ OT2 Reagents. Combination of 5 libraries on an Ion 318™Chip resulted in >2500X average coverage depth with >97% of the target bases covered at 500X. Genomic variants were filtered and analysed through AmpliseqAML single 5.0 workflow.
Results
The mean count of sequencing reads obtained per sample was 0.73 million and the mean sequencing depth was over 2500X with 91% uniformity. Seventy-five mutations in 19 genes were detected in 31 samples with an average of 2.3 mutations per sample. TP53 mutations were found in 4 patients of whom one with unfavorable cytogenetics.The AML panel detected 15 mutations (20%) in NPM1, 6 (8%) TET2, 10 (13%) DNMT3A, 6 (8%) CEBPA, 5 (7%)TP53, 3 (4%) RUNX1,4 (5%) FLT3, 4 (5%) IDH2, 2 (3%) ASXL1, 5 (7%) GATA2, 6 (8%) PTPN11 and 3 (4%) KIT genes. Only one patient remained wild-type for these genes. Based on mechanism of action, genes involved in signal transduction and DNA methylation were the most frequently mutated, accounting for the 39 % and 32% of detected mutations, respectively. The AML gene panel identified 100% of previously identified mutations by conventional molecular biology techniques in FLT3(TKD), NPM1, IDH1-2 and CEBPA genes. Mutational profiles obtained by NGS allowed to refine cytogenetic classification in the normal karyotype subgroup shifting a large proportion of patients (24 out of 30) from the intermediate to poor risk category.In one patient we were able to assess clonal dominance over the course of the disease treatment. A female 50 y.o. diagnosed with AML-M2 presented at diagnosis IDH2 p.R172K mutation, DNMT3A p.F640fs*11 and CEBPA p.P196_P197insHP at frequencies of 42%, 49% and 20%, respectively. Monitoring of mutation levels in two consecutive bone marrow samples during treatment according to AML1310 protocol, demonstrated treatment associated clonal responses. IDH2 mutation had a frequency of 7.3% after 4 weeks of therapy and resulted absent after 8 weeks (not detected over 6500 reads), while DNMT3A and CEBPA mutations were present at the levels of 30% and 47%, respectively. Longitudinal monitoring of three mutations indicated the presence of independent clones behaving discordantly during therapy.
Conclusion
A targeted multi gene panel analysis can provide in a short turnaround time a pretreatment mutational profile as well as the MRD status helping decision of intensity and type of induction therapy and modulation of post remission strategies.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, MRD, Mutation analysis
Type: Publication Only
Background
AML is a clonal disorder of hematopoietic stem and progenitor cells caused by acquired and occasionally inherited genetic alterations. Cytogenetic aberrations together with gene mutations are powerful prognostic markers and the mutational profile can be incorporated in the decision making processes to treatment.
Aims
A real-life cohort of AML patients from Sardinia (Italy) was analyzed to evaluate the usefulness of a next-generation sequencing (NGS) gene panel for the detection of mutations associated with AML in a context of routine diagnostic molecular workup.
Methods
Thirty AML patients, 15 males and 15 females with median age of 60 years (range 18-88 years) were enrolled for the study. DNAs were screened with IonAmpliseq AML panel (ThermoFisher) on IonPGM platform. The panel includes hotspots mutations of the genes: ASXL1 (exon 12), BRAF (V600E),CBL(exons 8-9), FLT3(codons 676,830-850), IDH1(exon 4), IDH2(exon 4), JAK2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons3-8), WT1 (exons 7,9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53 genes.The panel design does not include the FLT3-ITD gene region. Genomic libraries were prepared using Ion AmpliSeq™2.0 (ThermoFisher) chemistry and library clonal amplification was performed by emulsion PCR using Ion PGM™Template HiQ OT2 Reagents. Combination of 5 libraries on an Ion 318™Chip resulted in >2500X average coverage depth with >97% of the target bases covered at 500X. Genomic variants were filtered and analysed through AmpliseqAML single 5.0 workflow.
Results
The mean count of sequencing reads obtained per sample was 0.73 million and the mean sequencing depth was over 2500X with 91% uniformity. Seventy-five mutations in 19 genes were detected in 31 samples with an average of 2.3 mutations per sample. TP53 mutations were found in 4 patients of whom one with unfavorable cytogenetics.The AML panel detected 15 mutations (20%) in NPM1, 6 (8%) TET2, 10 (13%) DNMT3A, 6 (8%) CEBPA, 5 (7%)TP53, 3 (4%) RUNX1,4 (5%) FLT3, 4 (5%) IDH2, 2 (3%) ASXL1, 5 (7%) GATA2, 6 (8%) PTPN11 and 3 (4%) KIT genes. Only one patient remained wild-type for these genes. Based on mechanism of action, genes involved in signal transduction and DNA methylation were the most frequently mutated, accounting for the 39 % and 32% of detected mutations, respectively. The AML gene panel identified 100% of previously identified mutations by conventional molecular biology techniques in FLT3(TKD), NPM1, IDH1-2 and CEBPA genes. Mutational profiles obtained by NGS allowed to refine cytogenetic classification in the normal karyotype subgroup shifting a large proportion of patients (24 out of 30) from the intermediate to poor risk category.In one patient we were able to assess clonal dominance over the course of the disease treatment. A female 50 y.o. diagnosed with AML-M2 presented at diagnosis IDH2 p.R172K mutation, DNMT3A p.F640fs*11 and CEBPA p.P196_P197insHP at frequencies of 42%, 49% and 20%, respectively. Monitoring of mutation levels in two consecutive bone marrow samples during treatment according to AML1310 protocol, demonstrated treatment associated clonal responses. IDH2 mutation had a frequency of 7.3% after 4 weeks of therapy and resulted absent after 8 weeks (not detected over 6500 reads), while DNMT3A and CEBPA mutations were present at the levels of 30% and 47%, respectively. Longitudinal monitoring of three mutations indicated the presence of independent clones behaving discordantly during therapy.
Conclusion
A targeted multi gene panel analysis can provide in a short turnaround time a pretreatment mutational profile as well as the MRD status helping decision of intensity and type of induction therapy and modulation of post remission strategies.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, MRD, Mutation analysis
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