MUTATIONAL ANALYSIS OF ACUTE MYELOID LEUKEMIA WITH A NEXT-GENERATION SEQUENCING PANEL
(Abstract release date: 05/19/16)
EHA Library. Alonso C. 06/09/16; 134525; PB1625
Dr. Carmen Alonso
Contributions
Contributions
Abstract
Abstract: PB1625
Type: Publication Only
Background
In the last years numerous recurrently mutated genes have been described for acute myeloid leukemia (AML). Although only CEBPA, NPM1 and FLT3-ITD have been incorporated to risk stratification algorithms, other genes like RUNX1, ASXL1, DNMT3A or TP53 are currently being evaluated and could be soon incorporated. Furthermore, FLT3, IDH1/2 or KRAS are being tested as potential therapeutic targets. Next generation sequencing (NGS) assays allows a parallel detection of these genes.
Aims
The aim of this study is to characterize the mutational status of a series of “de novo” AML patients using a NGS panel that includes the hotspots of most recurrently mutated genes and other potentially actionable targets.
Methods
We studied 130 AML patients diagnosed from June 1999 to June 2014 in a single centre. Inclusion criteria were age <65 years, diagnosed with non-promyelocytic AML, treated according PETHEMA AML clinical protocols and with available DNA sample at diagnosis. Ion Ampliseq AML community (IAAC) panel was employed for mutation detection. This panel includes hotspots of ASXL1 (exon 12), BRAF (V600E), CBL (exons 8-9), FLT3 (codons 676, 830-850), IDH1 (exon 4), IDH2 (exon 4), JAK 2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons 3-8) and WT1 (exons 7 and 9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53. Libraries were amplified with Ion Torrent Ampliseq 2.0 beta and sequenced in the Ion PGM or Proton platforms. Variant annotation was carried out with the Ion Reporter Software (Life Technologies). Polymorphisms, synonymous, low depth read mutations (<100X) and low read % variants (<5%) were filtered out. Moreover, regions with intrinsic strand bias or low coverage were curated with the Integrative Genomics Viewer (IGV). FLT3-ITD mutations, which are not included in the panel design, were detected by capillary electrophoresis (Thiede 2002 Blood).
Results
Regarding cytogenetics, 9 patients showed favourable risk, 82 with intermediate risk (70 normal karyotype) and 23 unfavourable risk. For the remaining 6 patients cytogenetics could not be assessed.The IAAC panel found 322 variants in 125 patients. In brief, 59 NPM1 mutations, 49 in DNMT3A (30/49 mutations (61,22%) were the R882 mutation), 36 in TET2, 31 in CEBPA (13 patients showed biallelic mutations), 21 in RUNX1, 18 in NRAS, 16 in IDH2, 15 in FLT3, 15 in TP53, 11 in GATA2, 11 in PTPN11, 11 in ASXL1, 8 in WT1, 7 in IDH1, 7 in KRAS, 4 in KIT, 2 in CBL and 1 mutation in BRAF (Figure 1). Moreover, 31 FLT3-ITD mutations were detected by conventional techniques. The average of mutations per patient was 2.71 (range 0-8). Only 5 (3.8%) remained wild type.NPM1, DNMT3A and FLT3 (including FLT3-ITD) mutations were significantly enriched in the intermediate risk group (p<0.001 p=0.002, p=0.004, respectively), while TP53 mutations aggregated in the unfavourable karyotype group (p<0.001). NPM1 mutations significantly concurred with DNMT3A and FLT3 mutations (p<0.0001, p<0.0001, respectively), and these three genes are mutually exclusive with TP53 (p<0.001; p=0.005, p= 0.011) and RUNX1 (p=0.001, p=0.018, p=0.047) mutations. NPM1 and FLT3 were also mutually exclusive with GATA2 (p=0.010 and p=0.016). NPM1 and CEBPA were mutually exclusive (p=0.036), as well as FLT3 with NRAS (p=0.014), and ASXL1 with NRAS (p=0.038). NPM1 and PTPN11 mutations showed concurrence (p=0.012) (Figure 2).
Conclusion
IAAC panel detects mutations with validated prognostic relevance (NPM1 and CEBPA) and other mutations with probable diagnostic or prognostic value and/or potential therapeutic targets are also studied and identified in one assay. The parallel study of numerous mutations allows the identification of recurrence and exclusivity patterns.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, AML, Mutation analysis, Mutation status
Type: Publication Only
Background
In the last years numerous recurrently mutated genes have been described for acute myeloid leukemia (AML). Although only CEBPA, NPM1 and FLT3-ITD have been incorporated to risk stratification algorithms, other genes like RUNX1, ASXL1, DNMT3A or TP53 are currently being evaluated and could be soon incorporated. Furthermore, FLT3, IDH1/2 or KRAS are being tested as potential therapeutic targets. Next generation sequencing (NGS) assays allows a parallel detection of these genes.
Aims
The aim of this study is to characterize the mutational status of a series of “de novo” AML patients using a NGS panel that includes the hotspots of most recurrently mutated genes and other potentially actionable targets.
Methods
We studied 130 AML patients diagnosed from June 1999 to June 2014 in a single centre. Inclusion criteria were age <65 years, diagnosed with non-promyelocytic AML, treated according PETHEMA AML clinical protocols and with available DNA sample at diagnosis. Ion Ampliseq AML community (IAAC) panel was employed for mutation detection. This panel includes hotspots of ASXL1 (exon 12), BRAF (V600E), CBL (exons 8-9), FLT3 (codons 676, 830-850), IDH1 (exon 4), IDH2 (exon 4), JAK 2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons 3-8) and WT1 (exons 7 and 9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53. Libraries were amplified with Ion Torrent Ampliseq 2.0 beta and sequenced in the Ion PGM or Proton platforms. Variant annotation was carried out with the Ion Reporter Software (Life Technologies). Polymorphisms, synonymous, low depth read mutations (<100X) and low read % variants (<5%) were filtered out. Moreover, regions with intrinsic strand bias or low coverage were curated with the Integrative Genomics Viewer (IGV). FLT3-ITD mutations, which are not included in the panel design, were detected by capillary electrophoresis (Thiede 2002 Blood).
Results
Regarding cytogenetics, 9 patients showed favourable risk, 82 with intermediate risk (70 normal karyotype) and 23 unfavourable risk. For the remaining 6 patients cytogenetics could not be assessed.The IAAC panel found 322 variants in 125 patients. In brief, 59 NPM1 mutations, 49 in DNMT3A (30/49 mutations (61,22%) were the R882 mutation), 36 in TET2, 31 in CEBPA (13 patients showed biallelic mutations), 21 in RUNX1, 18 in NRAS, 16 in IDH2, 15 in FLT3, 15 in TP53, 11 in GATA2, 11 in PTPN11, 11 in ASXL1, 8 in WT1, 7 in IDH1, 7 in KRAS, 4 in KIT, 2 in CBL and 1 mutation in BRAF (Figure 1). Moreover, 31 FLT3-ITD mutations were detected by conventional techniques. The average of mutations per patient was 2.71 (range 0-8). Only 5 (3.8%) remained wild type.NPM1, DNMT3A and FLT3 (including FLT3-ITD) mutations were significantly enriched in the intermediate risk group (p<0.001 p=0.002, p=0.004, respectively), while TP53 mutations aggregated in the unfavourable karyotype group (p<0.001). NPM1 mutations significantly concurred with DNMT3A and FLT3 mutations (p<0.0001, p<0.0001, respectively), and these three genes are mutually exclusive with TP53 (p<0.001; p=0.005, p= 0.011) and RUNX1 (p=0.001, p=0.018, p=0.047) mutations. NPM1 and FLT3 were also mutually exclusive with GATA2 (p=0.010 and p=0.016). NPM1 and CEBPA were mutually exclusive (p=0.036), as well as FLT3 with NRAS (p=0.014), and ASXL1 with NRAS (p=0.038). NPM1 and PTPN11 mutations showed concurrence (p=0.012) (Figure 2).
Conclusion
IAAC panel detects mutations with validated prognostic relevance (NPM1 and CEBPA) and other mutations with probable diagnostic or prognostic value and/or potential therapeutic targets are also studied and identified in one assay. The parallel study of numerous mutations allows the identification of recurrence and exclusivity patterns.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, AML, Mutation analysis, Mutation status
Abstract: PB1625
Type: Publication Only
Background
In the last years numerous recurrently mutated genes have been described for acute myeloid leukemia (AML). Although only CEBPA, NPM1 and FLT3-ITD have been incorporated to risk stratification algorithms, other genes like RUNX1, ASXL1, DNMT3A or TP53 are currently being evaluated and could be soon incorporated. Furthermore, FLT3, IDH1/2 or KRAS are being tested as potential therapeutic targets. Next generation sequencing (NGS) assays allows a parallel detection of these genes.
Aims
The aim of this study is to characterize the mutational status of a series of “de novo” AML patients using a NGS panel that includes the hotspots of most recurrently mutated genes and other potentially actionable targets.
Methods
We studied 130 AML patients diagnosed from June 1999 to June 2014 in a single centre. Inclusion criteria were age <65 years, diagnosed with non-promyelocytic AML, treated according PETHEMA AML clinical protocols and with available DNA sample at diagnosis. Ion Ampliseq AML community (IAAC) panel was employed for mutation detection. This panel includes hotspots of ASXL1 (exon 12), BRAF (V600E), CBL (exons 8-9), FLT3 (codons 676, 830-850), IDH1 (exon 4), IDH2 (exon 4), JAK 2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons 3-8) and WT1 (exons 7 and 9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53. Libraries were amplified with Ion Torrent Ampliseq 2.0 beta and sequenced in the Ion PGM or Proton platforms. Variant annotation was carried out with the Ion Reporter Software (Life Technologies). Polymorphisms, synonymous, low depth read mutations (<100X) and low read % variants (<5%) were filtered out. Moreover, regions with intrinsic strand bias or low coverage were curated with the Integrative Genomics Viewer (IGV). FLT3-ITD mutations, which are not included in the panel design, were detected by capillary electrophoresis (Thiede 2002 Blood).
Results
Regarding cytogenetics, 9 patients showed favourable risk, 82 with intermediate risk (70 normal karyotype) and 23 unfavourable risk. For the remaining 6 patients cytogenetics could not be assessed.The IAAC panel found 322 variants in 125 patients. In brief, 59 NPM1 mutations, 49 in DNMT3A (30/49 mutations (61,22%) were the R882 mutation), 36 in TET2, 31 in CEBPA (13 patients showed biallelic mutations), 21 in RUNX1, 18 in NRAS, 16 in IDH2, 15 in FLT3, 15 in TP53, 11 in GATA2, 11 in PTPN11, 11 in ASXL1, 8 in WT1, 7 in IDH1, 7 in KRAS, 4 in KIT, 2 in CBL and 1 mutation in BRAF (Figure 1). Moreover, 31 FLT3-ITD mutations were detected by conventional techniques. The average of mutations per patient was 2.71 (range 0-8). Only 5 (3.8%) remained wild type.NPM1, DNMT3A and FLT3 (including FLT3-ITD) mutations were significantly enriched in the intermediate risk group (p<0.001 p=0.002, p=0.004, respectively), while TP53 mutations aggregated in the unfavourable karyotype group (p<0.001). NPM1 mutations significantly concurred with DNMT3A and FLT3 mutations (p<0.0001, p<0.0001, respectively), and these three genes are mutually exclusive with TP53 (p<0.001; p=0.005, p= 0.011) and RUNX1 (p=0.001, p=0.018, p=0.047) mutations. NPM1 and FLT3 were also mutually exclusive with GATA2 (p=0.010 and p=0.016). NPM1 and CEBPA were mutually exclusive (p=0.036), as well as FLT3 with NRAS (p=0.014), and ASXL1 with NRAS (p=0.038). NPM1 and PTPN11 mutations showed concurrence (p=0.012) (Figure 2).
Conclusion
IAAC panel detects mutations with validated prognostic relevance (NPM1 and CEBPA) and other mutations with probable diagnostic or prognostic value and/or potential therapeutic targets are also studied and identified in one assay. The parallel study of numerous mutations allows the identification of recurrence and exclusivity patterns.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, AML, Mutation analysis, Mutation status
Type: Publication Only
Background
In the last years numerous recurrently mutated genes have been described for acute myeloid leukemia (AML). Although only CEBPA, NPM1 and FLT3-ITD have been incorporated to risk stratification algorithms, other genes like RUNX1, ASXL1, DNMT3A or TP53 are currently being evaluated and could be soon incorporated. Furthermore, FLT3, IDH1/2 or KRAS are being tested as potential therapeutic targets. Next generation sequencing (NGS) assays allows a parallel detection of these genes.
Aims
The aim of this study is to characterize the mutational status of a series of “de novo” AML patients using a NGS panel that includes the hotspots of most recurrently mutated genes and other potentially actionable targets.
Methods
We studied 130 AML patients diagnosed from June 1999 to June 2014 in a single centre. Inclusion criteria were age <65 years, diagnosed with non-promyelocytic AML, treated according PETHEMA AML clinical protocols and with available DNA sample at diagnosis. Ion Ampliseq AML community (IAAC) panel was employed for mutation detection. This panel includes hotspots of ASXL1 (exon 12), BRAF (V600E), CBL (exons 8-9), FLT3 (codons 676, 830-850), IDH1 (exon 4), IDH2 (exon 4), JAK 2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons 3-8) and WT1 (exons 7 and 9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53. Libraries were amplified with Ion Torrent Ampliseq 2.0 beta and sequenced in the Ion PGM or Proton platforms. Variant annotation was carried out with the Ion Reporter Software (Life Technologies). Polymorphisms, synonymous, low depth read mutations (<100X) and low read % variants (<5%) were filtered out. Moreover, regions with intrinsic strand bias or low coverage were curated with the Integrative Genomics Viewer (IGV). FLT3-ITD mutations, which are not included in the panel design, were detected by capillary electrophoresis (Thiede 2002 Blood).
Results
Regarding cytogenetics, 9 patients showed favourable risk, 82 with intermediate risk (70 normal karyotype) and 23 unfavourable risk. For the remaining 6 patients cytogenetics could not be assessed.The IAAC panel found 322 variants in 125 patients. In brief, 59 NPM1 mutations, 49 in DNMT3A (30/49 mutations (61,22%) were the R882 mutation), 36 in TET2, 31 in CEBPA (13 patients showed biallelic mutations), 21 in RUNX1, 18 in NRAS, 16 in IDH2, 15 in FLT3, 15 in TP53, 11 in GATA2, 11 in PTPN11, 11 in ASXL1, 8 in WT1, 7 in IDH1, 7 in KRAS, 4 in KIT, 2 in CBL and 1 mutation in BRAF (Figure 1). Moreover, 31 FLT3-ITD mutations were detected by conventional techniques. The average of mutations per patient was 2.71 (range 0-8). Only 5 (3.8%) remained wild type.NPM1, DNMT3A and FLT3 (including FLT3-ITD) mutations were significantly enriched in the intermediate risk group (p<0.001 p=0.002, p=0.004, respectively), while TP53 mutations aggregated in the unfavourable karyotype group (p<0.001). NPM1 mutations significantly concurred with DNMT3A and FLT3 mutations (p<0.0001, p<0.0001, respectively), and these three genes are mutually exclusive with TP53 (p<0.001; p=0.005, p= 0.011) and RUNX1 (p=0.001, p=0.018, p=0.047) mutations. NPM1 and FLT3 were also mutually exclusive with GATA2 (p=0.010 and p=0.016). NPM1 and CEBPA were mutually exclusive (p=0.036), as well as FLT3 with NRAS (p=0.014), and ASXL1 with NRAS (p=0.038). NPM1 and PTPN11 mutations showed concurrence (p=0.012) (Figure 2).
Conclusion
IAAC panel detects mutations with validated prognostic relevance (NPM1 and CEBPA) and other mutations with probable diagnostic or prognostic value and/or potential therapeutic targets are also studied and identified in one assay. The parallel study of numerous mutations allows the identification of recurrence and exclusivity patterns.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, AML, Mutation analysis, Mutation status
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