Contributions
Abstract: PB1700
Type: Publication Only
Background
Internal tandem duplication (ITD) in the FLT3 tyrosine kinase gene is a somatic genetic alteration frequently found in acute myeloid leukemia (AML) and is associated with an aggressive phenotype. The accurate detection of FLT3-ITD together with evaluation of the mutant-to-wild-type allelic ratio are essential due to the strong prognostic and the potential therapeutic relevance of this marker in AML. The gold standard method for the detection of FLT3-ITD consists of PCR and electrophoresis-based product sizing. In the last years, as the number of relevant genetic alterations in myeloid neoplasm is increasing, next generation sequencing (NGS) has started to replace the conventional PCR methods in clinical diagnostic labs. However, NGS methods are usually not very performant for the detection of medium sized or long insertions or deletions.
Aims
The aim of this study was to evaluate the possibility to use NGS for accurate detection of FLT3-ITD in the daily practice.
Methods
We have developed a custom Haloplex NGS panel (Agilent) to sequence 29 genes related to myeloid neoplasm, among which FLT3 exons 14, 15 and 20. We used this panel on a MiSeq (Illumina) sequencing platform on 40 AML samples (23 FLT-ITD negative samples and 17 FLT-ITD positive samples) and compared the performance for FLT3-ITD detection of several analysis tools, both technical (different sequencing cartridges and analytical (Pindel, SOPHiA DDM and SeqNext).
Results
Best results for FLT3-ITD detection were obtained using 500v2 cartridge instead of 300v2. Regarding analysis software Pindel and SOPHiA DDM gave the best results in terms of detection (more than 88%). However, large differences were observed between NGS and fragment analysis regarding the mutant-to-wild-type ratio.
Conclusion
These results demonstrate that FLT3-ITD can be detected reliably with NGS. However, optimization is still needed to accurately determine the mutant-to-wild-type ratio since this ratio is an important aspect of the prognostic value of FLT3-ITD.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Flt3-ITD
Abstract: PB1700
Type: Publication Only
Background
Internal tandem duplication (ITD) in the FLT3 tyrosine kinase gene is a somatic genetic alteration frequently found in acute myeloid leukemia (AML) and is associated with an aggressive phenotype. The accurate detection of FLT3-ITD together with evaluation of the mutant-to-wild-type allelic ratio are essential due to the strong prognostic and the potential therapeutic relevance of this marker in AML. The gold standard method for the detection of FLT3-ITD consists of PCR and electrophoresis-based product sizing. In the last years, as the number of relevant genetic alterations in myeloid neoplasm is increasing, next generation sequencing (NGS) has started to replace the conventional PCR methods in clinical diagnostic labs. However, NGS methods are usually not very performant for the detection of medium sized or long insertions or deletions.
Aims
The aim of this study was to evaluate the possibility to use NGS for accurate detection of FLT3-ITD in the daily practice.
Methods
We have developed a custom Haloplex NGS panel (Agilent) to sequence 29 genes related to myeloid neoplasm, among which FLT3 exons 14, 15 and 20. We used this panel on a MiSeq (Illumina) sequencing platform on 40 AML samples (23 FLT-ITD negative samples and 17 FLT-ITD positive samples) and compared the performance for FLT3-ITD detection of several analysis tools, both technical (different sequencing cartridges and analytical (Pindel, SOPHiA DDM and SeqNext).
Results
Best results for FLT3-ITD detection were obtained using 500v2 cartridge instead of 300v2. Regarding analysis software Pindel and SOPHiA DDM gave the best results in terms of detection (more than 88%). However, large differences were observed between NGS and fragment analysis regarding the mutant-to-wild-type ratio.
Conclusion
These results demonstrate that FLT3-ITD can be detected reliably with NGS. However, optimization is still needed to accurately determine the mutant-to-wild-type ratio since this ratio is an important aspect of the prognostic value of FLT3-ITD.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Flt3-ITD