Fusion Gene Detection and Quantification by Asymmetric Capture Sequencing (aCAP-Seq)

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.