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Updated: Oct 5, 2026
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in
Elisa Zuffa1, Eugenia Franchini1, Cristina Papayannidis1
1"Seràgnoli" Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.
Abstract:
FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2-2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.
Insights
Ultra-deep sequencing detected small FLT3 internal tandem duplication (ITD)+ clones at diagnosis in Acute Myeloid Leukemia (AML) patients who later developed overt ITD+ disease. This sensitive method aids in understanding mutation timing and monitoring minimal residual disease.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- FLT3 internal tandem duplication (ITD) is a frequent mutation in Acute Myeloid Leukemia (AML).
- FLT3 ITD status can change during disease progression, complicating diagnosis and treatment monitoring.
- Detecting low-abundance mutations is crucial for understanding AML clonal evolution.
Purpose of the Study:
- To develop and apply a highly sensitive ultra-deep sequencing (UDS) method for FLT3 mutational screening.
- To investigate the early occurrence and clonal dynamics of FLT3 ITD in cytogenetically normal (CN-) AML.
- To assess the utility of UDS for minimal residual disease (MRD) detection in AML.
Main Methods:
- Development of an amplicon-based ultra-deep sequencing (UDS) approach for FLT3 mutation screening.
- Retrospective analysis of diagnosis, relapse, and follow-up samples from 5 CN-AML patients initially FLT3 wild-type but ITD+ at relapse.
- Quantification of FLT3 ITD clone abundance from diagnosis to disease progression.
Main Results:
- All analyzed patients harbored a small, undetectable FLT3 ITD+ clone at diagnosis (0.2-2% abundance).
- UDS revealed the clonal evolution of FLT3 ITD+ clones from diagnosis to relapse, influenced by treatment pressure.
- The dynamics of ITD+ clones indicated their persistence and evolution over time.
Conclusions:
- UDS is a valuable tool for sensitive FLT3 mutational screening in AML.
- Early detection of small FLT3 ITD+ clones is possible, impacting prognostic assessment.
- UDS can aid in monitoring minimal residual disease (MRD) and understanding treatment resistance in CN-AML.
