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Published on: August 4, 2016
Fusion Gene Detection and Quantification by Asymmetric Capture Sequencing (aCAP-Seq)
Guillaume Gricourt1, Violaine Tran Quang2, Jean-Michel Cayuela3
1Department of Hematology and Immunology, Henri Mondor University Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Creteil, France.
Abstract:
Several fusion genes such as BCR::ABL1, FIP1L1::PDGFRA, and PML::RARA are now efficiently targeted by specific therapies in patients with leukemia. Although these therapies have significantly improved patient outcomes, leukemia relapse and progression remain clinical concerns. Most myeloid next-generation sequencing (NGS) panels do not detect or quantify these fusions. It therefore remains difficult to decipher the clonal architecture and dynamics of myeloid malignancy patients, although these factors can affect clinical decisions and provide pathophysiologic insights. An asymmetric capture sequencing strategy (aCAP-Seq) and a bioinformatics algorithm (HmnFusion) were developed to detect and quantify MBCR::ABL1, μBCR::ABL1, PML::RARA, and FIP1L1::PDGFRA fusion genes in an NGS panel targeting 41 genes. One-hundred nineteen DNA samples derived from 106 patients were analyzed by conventional methods at diagnosis or on follow-up and were sequenced with this NGS myeloid panel. The specificity and sensitivity of fusion detection by aCAP-Seq were 100% and 98.1%, respectively, with a limit of detection estimated at 0.1%. Fusion quantifications were linear from 0.1% to 50%. Breakpoint locations and sequences identified by NGS were concordant with results obtained by Sanger sequencing. Finally, this new sensitive and cost-efficient NGS method allowed integrated analysis of resistant chronic myeloid leukemia patients and thus will be of interest to elucidate the mutational landscape and clonal architecture of myeloid malignancies driven by these fusion genes at diagnosis, relapse, or progression.
Insights
A new next-generation sequencing (NGS) method accurately detects and quantifies leukemia fusion genes. This advance aids in understanding myeloid malignancy clonal architecture and patient treatment.
Area of Science:
- Hematology
- Molecular Biology
- Genetics
Background:
- Targeted therapies for leukemia fusion genes (e.g., BCR::ABL1) have improved outcomes.
- Leukemia relapse and progression persist as clinical challenges.
- Current myeloid next-generation sequencing (NGS) panels often fail to detect or quantify these critical fusion genes, hindering clonal architecture analysis.
Purpose of the Study:
- To develop and validate a sensitive and cost-efficient NGS method for detecting and quantifying key leukemia fusion genes.
- To enable comprehensive analysis of clonal architecture and dynamics in myeloid malignancies.
Main Methods:
- Development of an asymmetric capture sequencing strategy (aCAP-Seq) coupled with a bioinformatics algorithm (HmnFusion).
- Targeting of 41 genes within an NGS panel to detect BCR::ABL1, PML::RARA, and FIP1L1::PDGFRA fusions.
- Analysis of 119 DNA samples from 106 patients using the developed NGS panel and comparison with conventional methods.
Main Results:
- The aCAP-Seq method demonstrated 100% specificity and 98.1% sensitivity for fusion detection.
- A limit of detection as low as 0.1% was achieved, with linear quantification from 0.1% to 50%.
- NGS-identified breakpoint locations and sequences were highly concordant with Sanger sequencing results.
Conclusions:
- The novel NGS method provides sensitive, specific, and cost-efficient detection and quantification of leukemia fusion genes.
- This approach facilitates integrated analysis of myeloid malignancies, including resistant chronic myeloid leukemia.
- The method is valuable for elucidating the mutational landscape and clonal architecture at various disease stages.
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