A Single-Tube Multiplexed Assay for Detecting ALK, ROS1, and RET Fusions in Lung Cancer Maruja E. Lira, Yoon-La Choi, Sun Min Lim, Shibing Deng, Donghui.

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A Single-Tube Multiplexed Assay for Detecting ALK, ROS1, and RET Fusions in Lung Cancer Maruja E. Lira, Yoon-La Choi, Sun Min Lim, Shibing Deng, Donghui Huang, Mark Ozeck, Joungho Han, Ji Yun Jeong, Hyo Sup Shim, Byoung Chul Cho, Jhingook Kim, Myung-Ju Ahn, Mao Mao The Journal of Molecular Diagnostics Volume 16, Issue 2, Pages 229-243 (March 2014) DOI: 10.1016/j.jmoldx.2013.11.007 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 ALK, ROS1, and RET fusion transcript assay design. Schematic representation of ALK (A), ROS1 (C), and RET (E) mRNAs and locations of gene expression probe sets relative to known fusion junction sites. Junction sites are depicted as filled flags (commonly occurring) and unfilled flags (less frequent). Representative expression profiles are shown for select patient samples positive for ALK (B), ROS1 (D), and RET (F) fusion transcripts. The Journal of Molecular Diagnostics 2014 16, 229-243DOI: (10.1016/j.jmoldx.2013.11.007) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Detection of fusion transcripts from minimal tumor cell content and RNA input. Reporter counts for cancer cell lines NCIH3122 (ALK positive) (A) and HCC78 (ROS1 positive) (B) RNA serially diluted with increasing amounts of A549 RNA (ALK negative and ROS1 negative). A: Fusion reporter counts as a function of varying RNA percentages of fusion-positive cell lines in a background (bkgrd) of wild-type cell line RNA. B: Geometric means of four 5′ and four 3′ expression probes as a function of varying percentages of fusion-positive lines. C and D: Expression profiles obtained from clinical samples previously confirmed positive for ALK, ROS1, and RET fusions (C) and corresponding reporter counts (D) with decreasing RNA input amounts. The Journal of Molecular Diagnostics 2014 16, 229-243DOI: (10.1016/j.jmoldx.2013.11.007) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Assay performance in clinical specimens. A, C, and E: Geometric means of 3′ and 5′ expression probes for ALK (A), ROS1 (C), and RET (E) transcripts. Background threshold levels are denoted by dashed lines. B, D, and F: Results were obtained from a dual strategy for ALK (B), ROS1 (D), and RET (F) profiling. Dashed lines denote thresholds applied for both fusion and 3′/5′ ratios. Fusion-positive and fusion-negative samples are depicted in red and black, respectively, and are noted by cohorts of origin (sets 1 to 4). SMC152 is ALK IHC 1+ but NanoString and FISH negative. SMC153 has an in-frame CUX1-RET fusion. The Journal of Molecular Diagnostics 2014 16, 229-243DOI: (10.1016/j.jmoldx.2013.11.007) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 CUX1-RET fusion. A: The tumor consists of adenocarcinoma, showing an acinar and papillary pattern. The tumor was positive for TTF-1 (data not shown). B: The break-apart RET FISH shows separate red and green signals in tumor cells. C: RT-PCR confirmation of CUX1-RET fusion. D: Sequence chromatogram of the CUX1-RET fusion junction sequence with protein sequence to demonstrate the in-frame reading frame. E: Schematic representation of the predicted CUX1-RET fusion protein. Original magnification (H&E), ×100 (A). CC, coiled-coil domain; CR, CUT DNA-binding domain; HD, homeobox domain. The Journal of Molecular Diagnostics 2014 16, 229-243DOI: (10.1016/j.jmoldx.2013.11.007) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions