A highly sensitive XNA-based RT-qPCR assay for the identification of ALK, RET, and ROS1 fusions in lung cancer.

In lung cancer, the progression of tumors is often fueled by genetic alterations leading to the expression of oncogenic tyrosine kinases. Specifically, chimeric tyrosine kinases involving ALK, RET, and ROS1 are observed in approximately 5-7%, 1-2%, and 1-2% of NSCLS patients, respectively. The presence of these ALK, RET, and ROS1 fusion genes determines the response to tyrosine kinase inhibitors. Consequently, accurate detection of these gene fusions is crucial in the realm of precision medicine. To address this need, we have developed a multiplexed RT-qPCR assay based on xenonucleic acids (XNA) molecular clamping technology for detecting lung cancer fusions. This assay is designed to quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. Following this validation, we tested a total of 77 lung cancer patient FFPE samples, demonstrating the effectiveness of our XNA-based fusion gene assay with clinical samples. Notably, this assay is adaptable to highly degraded RNA samples with low input amounts. Our next phase involves expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability.