A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing

Area of Science:

• Oncology
• Molecular Biology
• Genetics

Background:

• Oncogenic gene fusions are critical drivers in numerous cancer types.
• Clinical specimens often have limited quantity and quality, necessitating sensitive detection methods.
• Existing methods for gene fusion detection can be time-consuming or require specific targets.

Purpose of the Study:

• To develop and validate a rapid, sensitive next-generation sequencing (NGS) approach for detecting a broad spectrum of gene fusions in solid tumors.
• To assess the utility of 3'/5' expression ratios of oncogenic kinases as a complementary method for inferring gene fusions.
• To evaluate the concordance of the novel NGS panel with established diagnostic techniques.

Main Methods:

• Development of a multiplex PCR-based amplicon panel targeting 19 driver genes and 94 partners in solid tumors.
• Inclusion of control assays to evaluate 3'/5' expression ratios for 12 oncogenic kinases.
• Validation using 40 known fusion-positive specimens and 59 fusion-negative specimens, with comparison to fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other NGS panels.

Main Results:

• The NGS panel demonstrated good concordance with other methods, correctly identifying all 40 known gene fusion specimens.
• No fusion reads were detected in 59 negative specimens, indicating high specificity.
• 3'/5' expression ratios were informative for ALK, RET, and NTRK1 fusions, but less so for BRAF or ROS1. Four specimens with elevated ratios, negative for known fusions, suggest potential novel fusion events requiring confirmation.

Conclusions:

• The developed multiplex PCR-based NGS panel is a sensitive and specific method for detecting clinically relevant gene fusions in solid tumors.
• The 3'/5' expression ratio assay can provide supplementary information for identifying potential fusion events, particularly for ALK, RET, and NTRK1.
• Gene fusions predicted solely by 3'/5' read ratios require confirmatory testing, highlighting the need for integrated diagnostic approaches.