A Mass Spectrometry Assay to Simultaneously Analyze ROS1 and RET Fusion Gene Expression in Non–Small-Cell Lung Cancer Priyanga Wijesinghe, PhD, Gerold.

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A Mass Spectrometry Assay to Simultaneously Analyze ROS1 and RET Fusion Gene Expression in Non–Small-Cell Lung Cancer Priyanga Wijesinghe, PhD, Gerold Bepler, MD, PhD, Aliccia Bollig-Fischer, PhD Journal of Thoracic Oncology Volume 10, Issue 2, Pages 381-386 (February 2015) DOI: 10.1097/JTO.0000000000000337 Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

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. PCR, polymerase chain reaction. Journal of Thoracic Oncology 2015 10, 381-386DOI: (10.1097/JTO.0000000000000337) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

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 complimentary DNA (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. Journal of Thoracic Oncology 2015 10, 381-386DOI: (10.1097/JTO.0000000000000337) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Detection of endogenous expression of the SLC34A2-ROS1 (SL4; R32) fusion gene in complimentary DNA 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 to control extension primer reaction targeting the downstream exon junction. Journal of Thoracic Oncology 2015 10, 381-386DOI: (10.1097/JTO.0000000000000337) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Detection of CCDC6-RET (C1; R12) fusion gene expression in LC-2/ad cell line complimentary DNA. 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. Journal of Thoracic Oncology 2015 10, 381-386DOI: (10.1097/JTO.0000000000000337) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions