Ashleigh C. McEvoy, Benjamin A. Wood, Nima M. Ardakani, Michelle R

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Presentation transcript:

Droplet Digital PCR for Mutation Detection in Formalin-Fixed, Paraffin-Embedded Melanoma Tissues Ashleigh C. McEvoy, Benjamin A. Wood, Nima M. Ardakani, Michelle R. Pereira, Robert Pearce, Lester Cowell, Cleo Robinson, Fabienne Grieu-Iacopetta, Alexander J. Spicer, Benhur Amanuel, Melanie Ziman, Elin S. Gray The Journal of Molecular Diagnostics Volume 20, Issue 2, Pages 240-252 (March 2018) DOI: 10.1016/j.jmoldx.2017.11.009 Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Two-dimensional Droplet Digital PCR plots of discordant BRAF–mutant tumors. BRAF V600E (A–D) and BRAF V600K (E–G) detection. Blue and orange dots represent the presence of mutant circulating tumor DNA. The Journal of Molecular Diagnostics 2018 20, 240-252DOI: (10.1016/j.jmoldx.2017.11.009) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Representative traces of concordant mutations detected by all platforms tested. Sanger sequencing, pyrosequencing, and Droplet Digital (dd)PCR BRAF results from tumors 33 (BRAF V600E), 25 and 10 (both, BRAF V600K). A, D, and G: Sanger sequencing showing the BRAF mutations, indicated by arrows. B, E, and H: ddPCR result for BRAF mutations. C, F, and I: Pyrosequencing results showing the BRAF mutations as blue shading. The Journal of Molecular Diagnostics 2018 20, 240-252DOI: (10.1016/j.jmoldx.2017.11.009) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Two-dimensional Droplet Digital PCR plots of discordant NRAS Q61K–mutant tumors. Blue and orange dots represent the presence of mutations in circulating tumor DNA. The Journal of Molecular Diagnostics 2018 20, 240-252DOI: (10.1016/j.jmoldx.2017.11.009) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 TERT promoter mutations detected only by Droplet Digital PCR. A–J: TERT C228T (A–H) and TERT C250T (I–J) mutation detection. Blue and orange dots represent the presence of mutations in circulating tumor DNA. The Journal of Molecular Diagnostics 2018 20, 240-252DOI: (10.1016/j.jmoldx.2017.11.009) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Distribution of mutations in BRAF, NRAS, and TERT promoter genes as detected by Droplet Digital PCR. The distribution of mutations in the BRAF, TERT promoter, and NRAS genes in 40 formalin-fixed, paraffin-embedded melanoma tissues are indicated by different colors. Other clinical characteristics are specified, such as the percentage of tumor load, whether the tumor tissue analyzed was derived from a primary or metastatic (Met) lesion, the Breslow thickness, tumor in situ (Tis), as well as the sex and age of the patient at the time of tumor resection. The asterisk indicates detection by pyrosequencing. The Journal of Molecular Diagnostics 2018 20, 240-252DOI: (10.1016/j.jmoldx.2017.11.009) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Comparison of mutant allele frequencies detected by Droplet Digital (dd)PCR and pyrosequencing. Tumors with <50% tumor cellularity showed Spearman r = 0.29, P > 0.05; ≥50% tumor cellularity, r = 0.96, P < 0.0001. The Journal of Molecular Diagnostics 2018 20, 240-252DOI: (10.1016/j.jmoldx.2017.11.009) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions