CRISPR/Cas9 Technology–Based Xenograft Tumors as Candidate Reference Materials for Multiple EML4-ALK Rearrangements Testing  Rongxue Peng, Rui Zhang, Guigao Lin, Xin Yang, Ziyang Li, Kuo Zhang, Jiawei Zhang, Jinming Li  The Journal of Molecular Diagnostics  Volume 19, Issue 5, Pages 766-775 (September 2017) DOI: 10.1016/j.jmoldx.2017.06.003 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Generation of EML4-ALK rearrangements by CRISPR/Cas9 system. A: Schematic of the strategy for generating EML4-ALK rearrangements. B–D: Detection of EML4-ALK fusions and fusion transcripts by PCR and RT-PCR on total DNA/RNA extracted from HEK293T cells transfected with corresponding vectors. E: Sequences of the RT-PCR products showing the correct EML4-ALK fusions. F: Western blotting for EML4-ALK protein expression in single clones. H3122 and H2228 were selected as positive controls, whereas HEK293T was selected as a negative control. The sequence of EML4 sgRNA is marked in yellow, and the sequence of ALK sgRNA is marked in green. Chr, chromosome. The Journal of Molecular Diagnostics 2017 19, 766-775DOI: (10.1016/j.jmoldx.2017.06.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Hematoxylin and eosin (H&E)–morphology, ALK-FISH, and ALK-IHC of 1-F8-G9, 9-E11-G5, 14-F9-E8, clinical specimens, and wild-type control. 1-F8-G9, 9-E11-G5, and 14-F9-E8 FFPE samples all display a high amount of tumor cells (A–C), detectable ALK breaks (F–H), and unequivocal ALK protein expression (K–M). The results are concordant with the native ALK-positive NSCLC tumors (D, I, and N). The wild-type control (HEK293T cells) displays negative results (E, J, and O). Red arrows indicate typical tumor cells stained by H&E, representative fluorescence signal of ALK breaks, and positive staining of IHC. Scale bars: 100 μm (A–E); 10 μm (F–J); 50 μm (K–O). Original magnification: ×200 (A–E); ×1000 (F–J); ×400 (K–O). The Journal of Molecular Diagnostics 2017 19, 766-775DOI: (10.1016/j.jmoldx.2017.06.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Sample validation and homogeneity and stability assessment of the novel FFPE reference materials by RT-qPCR. A: Sample validation by RT-qPCR of the novel FFPE reference materials. Data shown in the form of Cq ratio (EML4-ALK Cq/GAPDH Cq). B: Homogeneity and stability assessment of the novel FFPE reference materials showing that no significant trends in the Cq ratio are detectable within the detection limits of the assays. The mean values of all samples are consistent with their individual target values. The Journal of Molecular Diagnostics 2017 19, 766-775DOI: (10.1016/j.jmoldx.2017.06.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Integrative Genomics Viewer (IGV) screenshots of the breakpoints in the EML4 and ALK genes detected by capture-based NGS. A: Fusion of EML4-ALK by capture-based DNA sequencing. The green line signifies the EML4-ALK variant 1, the orange line signifies the EML4-ALK variant 2, and the purple line signifies the EML4-ALK variant 3. B: The IGV screenshot of EML4-ALK rearrangement variant 1. C: The IGV screenshot of EML4-ALK rearrangement variant 2. D: The IGV screenshot of EML4-ALK rearrangement variant 3. Chr, chromosome. The Journal of Molecular Diagnostics 2017 19, 766-775DOI: (10.1016/j.jmoldx.2017.06.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 T7EI assay for each targeted genomic region. T7EI assay for EML4 sgRNA 1 and ALK sgRNA (A), EML4 sgRNA 2 and ALK sgRNA (B), and EML4 sgRNA 3 and ALK sgRNA (C). HEK293T cells were transfected with either the px330 vector expressing Cas9 endonuclease and specific sgRNAs or the px330 vector alone. For each sgRNA target site, the surrounding genomic region was amplified by PCR and subjected to the T7EI assay to evaluate the formation of insertions/deletions. The exons of the EML4 gene are marked in yellow, and exons of the ALK gene are marked in green. The cleavage sites are labeled as scissor symbols. The Journal of Molecular Diagnostics 2017 19, 766-775DOI: (10.1016/j.jmoldx.2017.06.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Construction of s.c. xenograft mouse models. The s.c. xenograft model was prepared by injecting edited cells (1-F8-G9, 9-E11-G5, and 14-F9-E8) into the flanks of nude mice. At 4 weeks after injection, the average volumes of xenografted tumors were approximately 1000 mm3. The Journal of Molecular Diagnostics 2017 19, 766-775DOI: (10.1016/j.jmoldx.2017.06.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions