Low Incidence of Minor BRAF V600 Mutation-Positive Subclones in Primary and Metastatic Melanoma Determined by Sensitive and Quantitative Real-Time PCR

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Low Incidence of Minor BRAF V600 Mutation-Positive Subclones in Primary and Metastatic Melanoma Determined by Sensitive and Quantitative Real-Time PCR Thomas Kristensen, Ole Clemmensen, Lise Hoejberg The Journal of Molecular Diagnostics Volume 15, Issue 3, Pages 355-361 (May 2013) DOI: 10.1016/j.jmoldx.2012.12.003 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 qPCR amplification plots. A: Amplification of a fourfold dilution series of BRAF V600E mutation-positive HTB-38 cell line DNA into wild-type DNA by the V600E mutation-specific assay. The undiluted sample contained genomic DNA from approximately 50,000 mutation-positive cells. The calculated number of mutation-positive alleles in each dilution is indicated at the curve. The PCR efficiency was approximately 100%. B: Amplification of a fourfold dilution series of BRAF V600K 50% defined allelic standard into wild-type DNA by the V600K mutation-specific assay. C: Amplification of a fourfold dilution series of wild-type DNA into water by the wild-type–specific assay. D: Amplification of a BRAF V600E and V600K mutation-negative FFPE sample by the wild-type–specific and mutation-specific assays. The mutation-specific assays produced amplification as a result of a weak nonspecific cross-reaction with the wild-type allele, corresponding to ≤0.006% mutation-positive alleles. ΔRn values represent the fluorescence produced by the reporter dye (FAM), corrected for variation in baseline fluorescence and passive reference dye (ROX) fluorescence. ΔRn increases during qPCR because TaqMan probes are hydrolyzed during DNA synthesis. The Journal of Molecular Diagnostics 2013 15, 355-361DOI: (10.1016/j.jmoldx.2012.12.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 qPCR standard curve, with the CT value as a function of the number of BRAF V600E mutation-positive alleles. The standard curve was generated from the dilution series producing the amplification curves shown in Figure 1A. The slope of the standard curve shown is −3.37, and the y-intercept is 42.9. Three standard curve experiments were performed for each of the V600E, V600K, and wild-type–specific assays, and similar results were observed for all experiments, with slopes averaging −3.32 to −3.38 (which corresponds to PCR efficiencies of approximately 100%) and y-intercepts averaging 42. Standard curve experiments using FFPE-derived DNA confirmed PCR efficiencies of approximately 100% also in FFPE samples. The Journal of Molecular Diagnostics 2013 15, 355-361DOI: (10.1016/j.jmoldx.2012.12.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The reproducibility of the BRAF V600E mutation analysis was determined by repeated analysis of four mutation-positive samples in five different qPCR runs. Low coefficients of variation were observed in all four samples, and no pair of results differed by more than 21% in any of the samples. The Journal of Molecular Diagnostics 2013 15, 355-361DOI: (10.1016/j.jmoldx.2012.12.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Percentage BRAF V600E mutation-positive alleles measured by real-time qPCR versus pyrosequencing. A close correlation between the results produced by the two quantitative methods was observed (R2 = 0.96). No significant difference between the results produced by the two methods was detected (P = 0.10, paired t-test). The line representing the equation y = x, which represents a perfect fit between the two methods, is included to show the close association of the experimental data to this line. The Journal of Molecular Diagnostics 2013 15, 355-361DOI: (10.1016/j.jmoldx.2012.12.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Percentage BRAF V600E mutation-positive alleles measured by real-time qPCR in samples of mutation-positive primary melanoma (light gray bars; n = 9) or metastases (striped light gray bars; n = 30). The analytical sensitivity was calculated from the CT value produced by the BRAF wild-type–specific assay in samples of primary melanoma (dark gray bars; n = 11) and metastases (striped dark gray bars; n = 32) that did not fulfill criteria of positivity (as detailed under Results). A dichotomous pattern is observed, with most samples testing either as mutation-positive in a large fraction of alleles or as mutation-negative with a high analytical sensitivity. The Journal of Molecular Diagnostics 2013 15, 355-361DOI: (10.1016/j.jmoldx.2012.12.003) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions