Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma Cynthia Spittle, M. Renee Ward, Katherine.

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Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma Cynthia Spittle, M. Renee Ward, Katherine L. Nathanson, Phyllis A. Gimotty, Eric Rappaport, Marcia S. Brose, Angelica Medina, Richard Letrero, Meenhard Herlyn, Robin H. Edwards The Journal of Molecular Diagnostics Volume 9, Issue 4, Pages 464-471 (September 2007) DOI: 10.2353/jmoldx.2007.060191 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Detection of V600E in melanoma cell lines. Pyrograms generated for BRAF wild-type (WM3208) (A), heterozygous mutant (WM 902B) (B), and homozygous mutant (WM39) (C) cell lines distinguish T versus A peaks (yellow shading). Nucleotide dispensation order is given below each pyrogram. Numerical position for each nucleotide is indicated at the top. Dispensations of C at positions 1 and 6 are included as controls for misincorporation. Dideoxy cycle sequence tracings for each cell line are shown for comparison. The Journal of Molecular Diagnostics 2007 9, 464-471DOI: (10.2353/jmoldx.2007.060191) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Detection of BRAF variant mutations. Pyrograms for two variant mutations, V600R (B) and V600K (C), show distinct patterns compared with V600E (A). Changes in relative A/G peak heights at pyrogram positions 2, 3, 5, 7, and/or 8 reflect dinucleotide substitutions given at right. The specific variants are confirmed by dideoxy cycle sequencing. The Journal of Molecular Diagnostics 2007 9, 464-471DOI: (10.2353/jmoldx.2007.060191) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Applicability of pyrosequencing assay to FFPE tissue. Pyrograms generated from a V600E mutant tumor (A) and a V600K mutant tumor (B) are identical for paired frozen and FFPE tumors. The Journal of Molecular Diagnostics 2007 9, 464-471DOI: (10.2353/jmoldx.2007.060191) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Cell line DNA mixing study. Theoretical peak heights, calculated from initial percent mutant A peak in undiluted heterozygous V600E mutant tumor, are correlated with actual peak heights generated for each dilution (Pearson's correlation = 0.96), indicating a linear relationship. The Journal of Molecular Diagnostics 2007 9, 464-471DOI: (10.2353/jmoldx.2007.060191) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Analytic sensitivity for V600E detection in a heterozygous tumor. Pyrograms generated from dilution series for two separate experiments (day 1 and day 2) are shown with corresponding dideoxy sequence tracings for day 1 data. The percent tumor corresponding to each data set is indicated at left. The Journal of Molecular Diagnostics 2007 9, 464-471DOI: (10.2353/jmoldx.2007.060191) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Comparison of pyrosequencing data with SNP microarrays. A: SNP XbaI 50K microarray data for chromosome 7 displayed with CNAT 3.0 software. Melanoma cell lines analyzed and location of BRAF gene are indicated. For each cell line, genome smoothed chromosome copy number data are shown at the top and the LOH score is shown at the bottom. Chromosome location in megabases is given at the bottom of the data set for each cell line. Scale for copy number and LOH score indicated at right. B: Corresponding BRAF pyrosequencing data including percent T and A peaks are indicated for each cell line. The Journal of Molecular Diagnostics 2007 9, 464-471DOI: (10.2353/jmoldx.2007.060191) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions