Rapid Detection of the Epidermal Growth Factor Receptor Mutation in Non-Small-Cell Lung Cancer for Analysis of Acquired Resistance Using Molecular Beacons

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Rapid Detection of the Epidermal Growth Factor Receptor Mutation in Non-Small-Cell Lung Cancer for Analysis of Acquired Resistance Using Molecular Beacons Young-Hee Oh, Youngwook Kim, Young-Pil Kim, Soo-Won Seo, Tetsuya Mitsudomi, Myung-Ju Ahn, Keunchil Park, Hak-Sung Kim The Journal of Molecular Diagnostics Volume 12, Issue 5, Pages 644-652 (September 2010) DOI: 10.2353/jmoldx.2010.090208 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic representation of the detection principle using a molecular beacon. A: Molecular beacon-based real-time PCR. Molecular beacons complementary to a target DNA bind at the annealing temperature, dissociating the stem, and generate a fluorescence signal during real-time PCR. With an increasing number of PCR cycles, fluorescence intensity increases, enabling the detection of the mutation in real-time. B:In situ fluorescence imaging of individual cells carrying a specific mutation. The molecular beacons bind to a target mRNA in the fixed cells, dissociating the stem, and restore a fluorescent signal. The Journal of Molecular Diagnostics 2010 12, 644-652DOI: (10.2353/jmoldx.2010.090208) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: Thermal denaturing profiles of molecular beacons in the presence of mismatched DNA, target DNA, or no target. B: Effect of molecular beacon concentration on the fluorescence signal in MB-based real-time PCR. Concentrations of the MB were 50 nmol/L (diamond), 200 nmol/L (circle), and 500 nmol/L (triangle). C: Effect of amplicon size on the sensitivity of the real-time PCR assay. Genomic DNA was amplified with two different primer sets: (circle) 120-bp amplicon and (diamond) 300-bp amplicon. Closed and open symbols represent genomic DNA from the cell lines H1975 and PC9, respectively. The Journal of Molecular Diagnostics 2010 12, 644-652DOI: (10.2353/jmoldx.2010.090208) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Molecular beacon-based real-time PCR. A: Real-time PCR in the presence of different templates or absence of template as a control. B: Electrophoresis gel image after real-time PCR. Lane 1: PCR with only MB. Lane 2: PCR without template. Lane 3: PCR with genomic DNA from PC9 cells (PC9 gDNA). Lane 4: PCR with H1975 genomic DNA (H1975 gDNA). The Journal of Molecular Diagnostics 2010 12, 644-652DOI: (10.2353/jmoldx.2010.090208) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 A: Detection sensitivity to template concentration. B: standard curve of the real-time PCR assay. Serial diluted H1975 genomic DNA was used as template for the real-time PCR assay; (circle) the PC9 cell line was used as a negative control; (diamond) 1000 ng of H1975 genomic DNA; (square) 100 ng of H1975 genomic DNA; (asterisk) 10 ng of H1975 genomic DNA; (triangle) 1 ng of H1975 genomic DNA; (dashed line) 0.5 ng of H1975 genomic DNA. The Journal of Molecular Diagnostics 2010 12, 644-652DOI: (10.2353/jmoldx.2010.090208) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 A: PCR with a different portion of genomic DNA from H1975 with the secondary T790M mutation. A total of 200 ng of a mixture of genomic DNAs from PC9 and H1975 were used as template. The portion of genomic DNA from H1975 was as follows: 100% (triangle); 50% (asterisk); 10% (circle); 2% (+); PCR without template (diamond); and with PC9 genomic DNA (square) as negative controls. B: Standard sequencing results of PCR products from A. Arrowheads indicate the mutation site. The Journal of Molecular Diagnostics 2010 12, 644-652DOI: (10.2353/jmoldx.2010.090208) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Molecular beacon-based real-time PCR for patient samples with acquired resistance: PC9 genomic DNA (asterisk), H1975 genomic DNA (diamond), sample 1 genomic DNA (square), and sample 2 genomic DNA (triangle) were used as a template. The Journal of Molecular Diagnostics 2010 12, 644-652DOI: (10.2353/jmoldx.2010.090208) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 7 Fluorescence microscope imaging of cells using T790M specific molecular beacons: Target T790M-MB was labeled with FAM fluorophore and control-MB (scrambled molecular beacon) was conjugated with Cy5 fluorophore. Two types of molecular beacons were incubated with H1975 cancer cells that had the secondary mutation in EGFR mRNA (top row) and with the PC9 cancer cells without the secondary mutation (bottom row). FAM: fluorescent images filtered by 550 to 560 nm wavelength band pass (A and D), and Cy 5 fluorescence images were obtained filtered by 650 nm wave length long pass (B and E). DAPI (4’,6-diamidino-2-phenylindole) fluorescent image after DAPI staining; all images were magnified ×10, except Merge ×60, which were magnified ×60 (C and F). The Journal of Molecular Diagnostics 2010 12, 644-652DOI: (10.2353/jmoldx.2010.090208) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions