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

Abstract

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.

Keywords: ALK; RET; ROS1; RT-qPCR; XNA; fusion; lung cancer.

Figure 1

Enhancing assay specificity and sensitivity with XNA. (a) XNA enhanced specificity by preventing non-specific amplification of abundant wild-type sequences. (b) XNA improved assay sensitivity by inhibiting the wild-type backgrounds.

Figure 2

Evaluating Qfusion^TM assay performance. (a) Each Qfusion^TM assay was tested with the corresponding cell lines: A549, H2228 (EML4-ALK), LC-2/ad (CCDC6-RET), and HCC78 (SLC45A-ROS1). The A549 was a fusion-negative cell line and Cq was not determined (ND). (b,c) FFPE RNA reference standard was diluted with normal FFPE RNA and analyzed with each Qfusion^TM assay. The x-axis represents the logarithm base 10 value of tumor percentage. Cq values were determined for 15% of tumor samples.

Figure 3

Consistency between OptiSeq^TM lung cancer fusion NGS panel and Qfusion^TM ALK, RET, or ROS1 fusion detection assay results. (a) Analysis of twenty FFPE samples using the OptiSeq^TM lung cancer fusion NGS panel revealed the presence of SLC34A2-ROS1 fusion in two patient samples. The Y-axis represents normalized reads, and the error bars indicate the standard deviations of three replicates. (b) The same twenty FFPE samples were subjected to analysis using Qfusion^TM assays. The results confirmed ROS1 fusion positivity in two patients with SLC34A2-ROS1 fusion. Qfusion^TM ALK and RET fusion detection assays showed no ALK and RET fusions. The error bars depict the standard deviations of three replicates.

Figure 4

Assessment of Qfusion^TM assays’ performance utilizing clinical samples. (a) Results from the analysis of 57 FFPE lung cancer patient samples using the Qfusion^TM ALK, RET, or ROS1 fusion detection assay. The data reveals the identification of one patient with ALK and RET fusions and one patient with ROS1 fusion. (b) Validation of distinct fusion events through singleplex RT-qPCR. The identified ALK fusions included EML4-ALK V1/V7, while the RET fusion involved CCDC6-RET. Additionally, a single ROS1 fusion event, CD74-ROS1, was confirmed.