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Multiclonal Profiling of FLT3-ITD in AML Using MinION Sequencing: A Tailored Clustering Approach to Enhance Subclonal
Jordi Martínez-Serra1, Aser Alonso-Carballo1, Ángel Horrillo1
1Hematology Department, Son Espases University Hospital, IdISBa, Palma, Spain.
Background:
Internal tandem duplications (FLT3-ITDs) in the FLT3 gene constitute a key driver mutation in acute myeloid leukemia (AML), strongly associated with poor prognosis and therapeutic resistance. Although general-purpose structural variant callers such as Sniffles have been used to detect FLT3-ITDs, their limitations in resolving clonal diversity and low-frequency variants can lead to underrepresentation of minor clones. These shortcomings highlight the need for a dedicated bioinformatics pipeline.
Materials And Methods:
We developed a custom clustering-based pipeline to overcome the constraints of generic SV callers, leveraging Oxford Nanopore's MinION for sequencing. Our method focuses on FLT3-ITDs by grouping near-identical insertions into biologically meaningful subclones, thereby allowing accurate variant detection of even low-frequency events. The pipeline was benchmarked against capillary electrophoresis (CE) and Sniffles at various thresholds (including 10%, 20%, and 50% allele-frequency cutoffs), with results validated via IGV inspection and cross-mapping.
Results:
The pipeline successfully detected FLT3-ITDs across all tested samples, including low-frequency variants and diverse subclones that Sniffles overlooked. Analyses uncovered complex multiclonal architectures composed of dominant clones (~20-25% of reads) plus multiple minor subclones differing in length, sequence, and breakpoint. Crucially, our approach identified duplications as short as 15 bp-events often dismissed by conventional SV callers. Comparative analyses showed that Sniffles failed to call several biologically validated ITDs detected by our custom pipeline.
Conclusion:
Third-generation sequencing combined with a tailored clustering strategy enhances the detection of FLT3-ITDs and clonal diversity in AML compared to generic variant callers. This method provides critical insights into subclonal populations driving relapse and therapeutic resistance-particularly in relapsed/refractory AML-underscoring the importance of specialized pipelines for precision medicine in leukemia.
Insights
A new bioinformatics pipeline accurately detects FLT3-ITDs, including low-frequency variants and complex subclones in acute myeloid leukemia (AML). This method improves upon generic callers, offering crucial insights for precision medicine in AML treatment.
Area of Science:
- Genomics
- Bioinformatics
- Molecular Biology
Background:
- FLT3-ITDs are key drivers in acute myeloid leukemia (AML), linked to poor prognosis and treatment resistance.
- Generic structural variant callers struggle to detect low-frequency FLT3-ITDs and resolve clonal diversity.
- There is a need for specialized bioinformatics tools to accurately identify FLT3-ITDs in AML.
Purpose of the Study:
- To develop a custom bioinformatics pipeline for sensitive and accurate detection of FLT3-ITDs in AML.
- To improve the characterization of clonal architecture and subclonal diversity in AML patients.
- To overcome the limitations of existing general-purpose structural variant callers for FLT3-ITD detection.
Main Methods:
- Developed a clustering-based bioinformatics pipeline leveraging Oxford Nanopore MinION sequencing.
- Focused on grouping near-identical FLT3-ITD insertions to identify subclones and low-frequency variants.
- Benchmarked the custom pipeline against capillary electrophoresis and the Sniffles caller, validating results with IGV inspection.
Main Results:
- The custom pipeline successfully detected FLT3-ITDs in all samples, including low-frequency variants missed by Sniffles.
- Identified complex multiclonal architectures with dominant and minor subclones exhibiting sequence and breakpoint variations.
- Detected very short duplications (as small as 15 bp) often overlooked by conventional callers.
Conclusions:
- Third-generation sequencing with a tailored clustering strategy enhances FLT3-ITD and clonal diversity detection in AML.
- This specialized approach provides critical insights into subclonal populations driving relapse and therapeutic resistance.
- The findings underscore the importance of dedicated pipelines for precision medicine in leukemia, especially for relapsed/refractory cases.
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