AMPLICON BASED PANEL TARGETED RESEQUENCING WITH THE TRUSIGHT MYELOID PANEL IN 100 PEDIATRIC AML PATIENTS
(Abstract release date: 05/19/16)
EHA Library. Walter C. 06/09/16; 134539; PB1639
Ms. Christiane Walter
Contributions
Contributions
Abstract
Abstract: PB1639
Type: Publication Only
Background
Acute myeloid leukemia (AML) is one of the most threatening malignancies in children and adolescents. The accumulation of mutations in leukemia stem cells (LSC) is believed to lead to the development of leukemia. Cyto- and molecular genetics already identified several aberrations which are relevant in leukemogenesis, prognosis and therapy. Nevertheless, the molecular landscape and clonal evolution of AML and its clinical relevance, especially for pediatric patients, is not yet well described. Next Generation Sequencing as an emerging sequencing technology provides the possibility to generate sequence data of high quality and detect genetic aberrations in a minimum of time.
Aims
The aim of this study was to apply amplicon based panel targeted resequencing by using the TruSight Myeloid Panel (Illumina) on a MiSeq Dx System (Illumina) to analyse 100 children with suspicion of AML at the time of initial diagnosis to detect the genetic variants.
Methods
All the patients analysed in this study were screened in order confirm or exclude the diagnosis of AML. With the confirmation of AML, the patients were treated according to the AML-BFM therapy protocols. Next Generation sequencing (NGS) with the TruSight Myeloid panel on a MiseqDX was performed. The sequencing panel is designed to identify somatic mutations associated to myeloid malignancies in 54 genes. Validation was initially performed using data for 10 patients obtained from Sanger-Sequencing from 7 diagnostically relevant mutations and compared to the data obtained from NGS. The genes actually relevant for prognosis and treatment stratification are CEBPA, FLT3, GATA1, KIT, NRAS, NPM1 and WT1.
Results
Every variant detected with Sanger Sequencing could be recovered in the NGS data. In 4 patients we could even detect additional mutations. Variants were detected and analysed using two different analysis software (Variant studio vs. Sophia DDM) and the results were compared. Almost all variants were detectable in both software, although great insertions and deletions are detectable only by Sophia DDM. We could detect CEBPA mutations in 16 patients, FLT3 ITD in 10 patients, FLT3 TKD mutations in 2 patients, GATA1 mutations in 6 patients, KIT mutations in 25 patients, NPM1 mutations in 7 patients, NRAS mutations in 14 patients and WT1 mutations in 24 patients. In 26 patients, we could not detect any mutation in these 7 genes. 23 patients had two different mutations at the same time and even three mutations could be detected in 4 patients. The analysis of variants detected in the 47 other genes covered in the panel is still ongoing.
Conclusion
Amplicon based panel targeted resequencing with the TruSight Myeloid panel provides the possibility to detect mutations in 54 genes associated to myeloid malignancies within 3 days after the sample arrived at our lab. The aim now is to adapt the report of the clinical findings to the detected variants, especially for variants in genes that were not yet diagnostically relevant. The confirmation of pathogenicity of variants in a broad range of genes could promote the possibilities of personalized targeted therapy.
Session topic: E-poster
Keyword(s): AML, Mutation analysis, Pediatric
Type: Publication Only
Background
Acute myeloid leukemia (AML) is one of the most threatening malignancies in children and adolescents. The accumulation of mutations in leukemia stem cells (LSC) is believed to lead to the development of leukemia. Cyto- and molecular genetics already identified several aberrations which are relevant in leukemogenesis, prognosis and therapy. Nevertheless, the molecular landscape and clonal evolution of AML and its clinical relevance, especially for pediatric patients, is not yet well described. Next Generation Sequencing as an emerging sequencing technology provides the possibility to generate sequence data of high quality and detect genetic aberrations in a minimum of time.
Aims
The aim of this study was to apply amplicon based panel targeted resequencing by using the TruSight Myeloid Panel (Illumina) on a MiSeq Dx System (Illumina) to analyse 100 children with suspicion of AML at the time of initial diagnosis to detect the genetic variants.
Methods
All the patients analysed in this study were screened in order confirm or exclude the diagnosis of AML. With the confirmation of AML, the patients were treated according to the AML-BFM therapy protocols. Next Generation sequencing (NGS) with the TruSight Myeloid panel on a MiseqDX was performed. The sequencing panel is designed to identify somatic mutations associated to myeloid malignancies in 54 genes. Validation was initially performed using data for 10 patients obtained from Sanger-Sequencing from 7 diagnostically relevant mutations and compared to the data obtained from NGS. The genes actually relevant for prognosis and treatment stratification are CEBPA, FLT3, GATA1, KIT, NRAS, NPM1 and WT1.
Results
Every variant detected with Sanger Sequencing could be recovered in the NGS data. In 4 patients we could even detect additional mutations. Variants were detected and analysed using two different analysis software (Variant studio vs. Sophia DDM) and the results were compared. Almost all variants were detectable in both software, although great insertions and deletions are detectable only by Sophia DDM. We could detect CEBPA mutations in 16 patients, FLT3 ITD in 10 patients, FLT3 TKD mutations in 2 patients, GATA1 mutations in 6 patients, KIT mutations in 25 patients, NPM1 mutations in 7 patients, NRAS mutations in 14 patients and WT1 mutations in 24 patients. In 26 patients, we could not detect any mutation in these 7 genes. 23 patients had two different mutations at the same time and even three mutations could be detected in 4 patients. The analysis of variants detected in the 47 other genes covered in the panel is still ongoing.
Conclusion
Amplicon based panel targeted resequencing with the TruSight Myeloid panel provides the possibility to detect mutations in 54 genes associated to myeloid malignancies within 3 days after the sample arrived at our lab. The aim now is to adapt the report of the clinical findings to the detected variants, especially for variants in genes that were not yet diagnostically relevant. The confirmation of pathogenicity of variants in a broad range of genes could promote the possibilities of personalized targeted therapy.
Session topic: E-poster
Keyword(s): AML, Mutation analysis, Pediatric
Abstract: PB1639
Type: Publication Only
Background
Acute myeloid leukemia (AML) is one of the most threatening malignancies in children and adolescents. The accumulation of mutations in leukemia stem cells (LSC) is believed to lead to the development of leukemia. Cyto- and molecular genetics already identified several aberrations which are relevant in leukemogenesis, prognosis and therapy. Nevertheless, the molecular landscape and clonal evolution of AML and its clinical relevance, especially for pediatric patients, is not yet well described. Next Generation Sequencing as an emerging sequencing technology provides the possibility to generate sequence data of high quality and detect genetic aberrations in a minimum of time.
Aims
The aim of this study was to apply amplicon based panel targeted resequencing by using the TruSight Myeloid Panel (Illumina) on a MiSeq Dx System (Illumina) to analyse 100 children with suspicion of AML at the time of initial diagnosis to detect the genetic variants.
Methods
All the patients analysed in this study were screened in order confirm or exclude the diagnosis of AML. With the confirmation of AML, the patients were treated according to the AML-BFM therapy protocols. Next Generation sequencing (NGS) with the TruSight Myeloid panel on a MiseqDX was performed. The sequencing panel is designed to identify somatic mutations associated to myeloid malignancies in 54 genes. Validation was initially performed using data for 10 patients obtained from Sanger-Sequencing from 7 diagnostically relevant mutations and compared to the data obtained from NGS. The genes actually relevant for prognosis and treatment stratification are CEBPA, FLT3, GATA1, KIT, NRAS, NPM1 and WT1.
Results
Every variant detected with Sanger Sequencing could be recovered in the NGS data. In 4 patients we could even detect additional mutations. Variants were detected and analysed using two different analysis software (Variant studio vs. Sophia DDM) and the results were compared. Almost all variants were detectable in both software, although great insertions and deletions are detectable only by Sophia DDM. We could detect CEBPA mutations in 16 patients, FLT3 ITD in 10 patients, FLT3 TKD mutations in 2 patients, GATA1 mutations in 6 patients, KIT mutations in 25 patients, NPM1 mutations in 7 patients, NRAS mutations in 14 patients and WT1 mutations in 24 patients. In 26 patients, we could not detect any mutation in these 7 genes. 23 patients had two different mutations at the same time and even three mutations could be detected in 4 patients. The analysis of variants detected in the 47 other genes covered in the panel is still ongoing.
Conclusion
Amplicon based panel targeted resequencing with the TruSight Myeloid panel provides the possibility to detect mutations in 54 genes associated to myeloid malignancies within 3 days after the sample arrived at our lab. The aim now is to adapt the report of the clinical findings to the detected variants, especially for variants in genes that were not yet diagnostically relevant. The confirmation of pathogenicity of variants in a broad range of genes could promote the possibilities of personalized targeted therapy.
Session topic: E-poster
Keyword(s): AML, Mutation analysis, Pediatric
Type: Publication Only
Background
Acute myeloid leukemia (AML) is one of the most threatening malignancies in children and adolescents. The accumulation of mutations in leukemia stem cells (LSC) is believed to lead to the development of leukemia. Cyto- and molecular genetics already identified several aberrations which are relevant in leukemogenesis, prognosis and therapy. Nevertheless, the molecular landscape and clonal evolution of AML and its clinical relevance, especially for pediatric patients, is not yet well described. Next Generation Sequencing as an emerging sequencing technology provides the possibility to generate sequence data of high quality and detect genetic aberrations in a minimum of time.
Aims
The aim of this study was to apply amplicon based panel targeted resequencing by using the TruSight Myeloid Panel (Illumina) on a MiSeq Dx System (Illumina) to analyse 100 children with suspicion of AML at the time of initial diagnosis to detect the genetic variants.
Methods
All the patients analysed in this study were screened in order confirm or exclude the diagnosis of AML. With the confirmation of AML, the patients were treated according to the AML-BFM therapy protocols. Next Generation sequencing (NGS) with the TruSight Myeloid panel on a MiseqDX was performed. The sequencing panel is designed to identify somatic mutations associated to myeloid malignancies in 54 genes. Validation was initially performed using data for 10 patients obtained from Sanger-Sequencing from 7 diagnostically relevant mutations and compared to the data obtained from NGS. The genes actually relevant for prognosis and treatment stratification are CEBPA, FLT3, GATA1, KIT, NRAS, NPM1 and WT1.
Results
Every variant detected with Sanger Sequencing could be recovered in the NGS data. In 4 patients we could even detect additional mutations. Variants were detected and analysed using two different analysis software (Variant studio vs. Sophia DDM) and the results were compared. Almost all variants were detectable in both software, although great insertions and deletions are detectable only by Sophia DDM. We could detect CEBPA mutations in 16 patients, FLT3 ITD in 10 patients, FLT3 TKD mutations in 2 patients, GATA1 mutations in 6 patients, KIT mutations in 25 patients, NPM1 mutations in 7 patients, NRAS mutations in 14 patients and WT1 mutations in 24 patients. In 26 patients, we could not detect any mutation in these 7 genes. 23 patients had two different mutations at the same time and even three mutations could be detected in 4 patients. The analysis of variants detected in the 47 other genes covered in the panel is still ongoing.
Conclusion
Amplicon based panel targeted resequencing with the TruSight Myeloid panel provides the possibility to detect mutations in 54 genes associated to myeloid malignancies within 3 days after the sample arrived at our lab. The aim now is to adapt the report of the clinical findings to the detected variants, especially for variants in genes that were not yet diagnostically relevant. The confirmation of pathogenicity of variants in a broad range of genes could promote the possibilities of personalized targeted therapy.
Session topic: E-poster
Keyword(s): AML, Mutation analysis, Pediatric
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