A Highly Sensitive XNA-Based RT-qPCR Assay for the Identification of ALK, RET, and ROS1 Fusions in Lung Cancer

Area of Science:

• Oncology
• Molecular Biology
• Genetics

Background:

• Lung cancer is frequently driven by oncogenic tyrosine kinases, including ALK, RET, and ROS1 fusions.
• These specific gene fusions are present in a subset of non-small cell lung cancer (NSCLC) patients.
• Detection of these fusions is critical for guiding targeted therapy with tyrosine kinase inhibitors.

Purpose of the Study:

• To develop and validate a multiplexed assay for the quantitative detection of ALK, ROS1, and RET gene fusions.
• To utilize xeno nucleic acid (XNA) molecular clamping technology for enhanced detection accuracy.
• To assess the assay's performance on FFPE samples and its adaptability to degraded RNA.

Main Methods:

• Development of a multiplexed reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay.
• Incorporation of xeno nucleic acid (XNA) molecular clamping for specific fusion detection.
• Validation using synthetic templates, reference FFPE samples, cell lines, and 77 clinical FFPE samples.

Main Results:

• The assay quantitatively detects thirteen ALK, seven ROS1, and seven RET gene fusions.
• Established sensitivity with a limit of detection of 50 copies of synthetic template.
• Successfully identified fusion transcripts in FFPE samples and cell lines, and demonstrated effectiveness in 77 clinical patient samples.

Conclusions:

• The developed XNA-based RT-qPCR assay is effective for detecting multiple lung cancer-associated gene fusions in FFPE samples.
• The assay demonstrates high sensitivity and adaptability to low-input, degraded RNA, crucial for clinical applications.
• Future work will focus on broader clinical sample testing and cell-free RNA analysis.