Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5–7%, 1–2%, and 1–2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can 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 has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability.

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.