A mass spectrometry assay to simultaneously analyze ROS1 and RET fusion gene expression in non-small-cell lung cancer

Abstract

Introduction: ROS1 and RET gene fusions were recently discovered in non-small-cell lung cancer (NSCLC) as potential therapeutic targets with small-molecule kinase inhibitors. The conventional screening methods of these fusions are time-consuming and require samples of high quality and quantity. Here, we describe a novel and efficient method by coupling the power of multiplexing polymerase chain reaction and the sensitivity of mass spectrometry.

Methods: The multiplex mass spectrometry platform simultaneously tests samples for the expression of nine ROS1 and six RET fusion genes. The assay incorporates detection of wild-type exon junctions immediately upstream and downstream of the fusion junction to exclude false-negative results. To flag false-positives, the system also comprises two independent assays for each fusion gene junction.

Results: The characteristic mass spectrometric peaks of the gene fusions were obtained using engineered plasmid constructs. Specific assays targeting the wild-type gene exon junctions were validated using complimentary DNA from lung tissue of healthy individuals. The system was further validated using complimentary DNA derived from NSCLC cell lines that express endogenous fusion genes. The expressed ROS1-SLC34A2 and CCDC6-RET gene fusions from the NSCLC cell lines HCC78 and LC-2/ad, respectively, were accurately detected by the novel assay. The assay is extremely sensitive, capable of detecting an event in test specimens containing 0.5% positive tumors.

Conclusion: The novel multiplexed assay is robustly capable of detecting 15 different clinically relevant RET and ROS1 fusion variants. The benefits of this detection method include exceptionally low sample input, high cost efficiency, flexibility, and rapid turnover.

Figure 1. Schematic depicting the major steps of the assay and primers designed to detect the fusion junctions and the adjacent wild-type exon boundary junctions

(A) Cartoon generalizing multiplexed primer targets. (B) List of upstream exon junction targeted primers for the 15 fusion genes and their corresponding masses after the primer extension. (C) List of fusion junction targeted extension primers and the respective mass peaks from both forward and reverse directions. (D) List of downstream exon junction targeted primers and the respective mass peaks.

Figure 2. Detection of endogenous expression of the SLC34A2-ROS1 (SL4; R32) fusion gene in cDNA derived from the HCC78 cell line

When the fusion gene is present, the characteristic mass spectrometry peak will appear in the analytical output of the MassARRAY system (blue arrow) along with the location of depleted extension primer (red arrow). If peaks from other reactions not directly related to the reaction lie in the same framed area of the spectrum, they are shown in gray (inactive mode). The results for the four reactions relevant to testing for this fusion gene are shown: (A) peak corresponding to the extension primer reaction targeting the fusion junction in the forward direction; (B) peak corresponding to the extension primer targeting the fusion junction in the reverse direction; (C) peak corresponding to the control extension primer targeting the upstream exon junction; (D) peak at predicted mass corresponding with control extension primer reaction targeting the downstream exon junction.

Figure 3. Detection of CCDC6-RET (C1; R12) fusion gene expression in LC-2/ad cell line cDNA

The characteristic mass spectra are shown for: (A) extension primer reaction targeting the fusion junction in the forward direction; (B) extension primer targeting the fusion junction in the reverse direction; (C) control extension primer targeting the upstream exon junction; (D) control extension primer reaction targeting the downstream exon junction.

Figure 4. The sensitivity of the assay was tested by analyzing a series of dilutions of ROS1-SLC34A2 (SL4; R32) fusion gene positive HCC78 cell line cDNA

HCC78 cell line cDNA was spiked into healthy lung cDNA at the indicated ratios to recapitulate a heterogeneous cell population that is presented in patient specimens. The left arrow indicates the predicted mass corresponding to the unextended primer. The right arrow indicates the peak corresponding to the mass of an extension primer reaction accurately detecting the fusion gene junction.