Multiplexed Gene Expression and Fusion Transcript Analysis to Detect ALK Fusions in Lung Cancer Maruja E. Lira, Tae Min Kim, Donghui Huang, Shibing Deng,

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Multiplexed Gene Expression and Fusion Transcript Analysis to Detect ALK Fusions in Lung Cancer Maruja E. Lira, Tae Min Kim, Donghui Huang, Shibing Deng, Youngil Koh, Bogun Jang, Heounjeong Go, Se-Hoon Lee, Doo Hyun Chung, Woo Ho Kim, Eric F.P.M. Schoenmakers, Yoon-La Choi, Keunchil Park, Jin Seok Ahn, Jong-Mu Sun, Myung-Ju Ahn, Dong-Wan Kim, Mao Mao The Journal of Molecular Diagnostics Volume 15, Issue 1, Pages 51-61 (January 2013) DOI: 10.1016/j.jmoldx.2012.08.006 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Detection of ALK fusion transcripts and quantification of ALK 5′ and 3′ expression levels using nCounter assay. A: Schematic representation of ALK fusion probe sets and sequences recognized. B: ALK fusion variants are identified by a common ALK exon 20 reporter probe and frequencies (Freq) in NSCLCs. C: Schematic representation of ALK 5′ and 3′ expression assay and location of probe sets relative to ALK fusion junction. Ex, exon. The Journal of Molecular Diagnostics 2013 15, 51-61DOI: (10.1016/j.jmoldx.2012.08.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Representative expression profiles of selected samples using an ALK fusion transcript assay. A–C: ALK-positive samples, as determined by FISH and IHC. D: Sample negative for ALK rearrangement by FISH but positive for ALK expression by IHC. E–G: Corresponding sequence chromatograms of samples are represented in A, B, and C, showing EML4-ALK V1, V2, and V3a, respectively. H: Sequence chromatogram of sample depicted in D showing an uninterrupted ALK sequence at the canonical junction site. The Journal of Molecular Diagnostics 2013 15, 51-61DOI: (10.1016/j.jmoldx.2012.08.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Performance of ALK fusion transcript assay on experimental set and control cancer cell lines. A: ALK 3′ overexpression, as measured by ALK 3′/5′ score; a background threshold of twofold is denoted by a thick horizontal line. B: ALK exon 20 reporter counts; a background threshold of 60 reporter counts is denoted by a thick horizontal line. C: Concordance with FISH and IHC data, EML4-ALK variant type as determined by RT-PCR/DNA sequencing. Tumor percentage is determined by H&E staining. n/a, not applicable; NC, negative control; nd, not determined; PC, positive control. The Journal of Molecular Diagnostics 2013 15, 51-61DOI: (10.1016/j.jmoldx.2012.08.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Performance of ALK fusion assay on SNUH validation set and control cancer cell lines. A: ALK 3′ overexpression as measured by ALK 3′/5′ score; a background threshold of twofold is denoted by a thick horizontal line. B: ALK exon 20 reporter counts; a background threshold of 60 reporter counts is denoted by a thick horizontal line. C: ALK fusion calls using analysis criteria formulated on experimental set, EML4-ALK variant type as determined by RT-PCR/DNA sequencing. Tumor percentage as determined by H&E staining. Samples SN41 and SN43 are from the same patient but from different FFPE tissue blocks. AM, adrenal metastasis; BM, bone metastasis; BrM, brain metastasis; CL, cell line; C/R, crizotinib-acquired resistance; N, treatment naïve; n/a, not applicable; nd, not determined; P, primary NSCLC; PC, positive control; PE, pleural effusion. The Journal of Molecular Diagnostics 2013 15, 51-61DOI: (10.1016/j.jmoldx.2012.08.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Performance of ALK fusion assay on SMC validation set and control cancer cell lines. A: ALK 3′ overexpression, as measured by ALK 3′/5′ score; a background threshold of twofold is denoted by a thick horizontal line. B: ALK exon 20 reporter counts; a background threshold of 60 reporter counts is denoted by a thick horizontal line. C: Predicted call based on NanoString result and FISH/IHC data; tumor percentage as determined by H&E staining. CR, complete response; N, treatment naïve; n/a, not applicable; NC, negative control; nd, not determined; PC, positive control; PD, progressive disease; PR, partial response. The Journal of Molecular Diagnostics 2013 15, 51-61DOI: (10.1016/j.jmoldx.2012.08.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions