A Multicenter Blinded Study Evaluating EGFR and KRAS Mutation Testing Methods in the Clinical Non–Small Cell Lung Cancer Setting—IFCT/ERMETIC2 Project.

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A Multicenter Blinded Study Evaluating EGFR and KRAS Mutation Testing Methods in the Clinical Non–Small Cell Lung Cancer Setting—IFCT/ERMETIC2 Project Part 1 Michèle Beau-Faller, Hélène Blons, Caroline Domerg, Dorota Gajda, Nicolas Richard, Fabienne Escande, Jérôme Solassol, Marc G. Denis, Anne Cayre, Isabelle Nanni-Metellus, Sylviane Olschwang, Sarab Lizard, Fabienne Piard, Jean-Luc Pretet, Florence de Fraipont, Ivan Bièche, Patricia de Cremoux, Isabelle Rouquette, Pierre-Paul Bringuier, Jean Mosser, Michèle Legrain, Anne-Claire Voegeli, Patrick Saulnier, Franck Morin, Jean-Pierre Pignon, Gérard Zalcman, Jacques Cadranel The Journal of Molecular Diagnostics Volume 16, Issue 1, Pages 45-55 (January 2014) DOI: 10.1016/j.jmoldx.2013.07.009 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Percentage of centers detecting the mutation in diluted cell line DNA. A: EGFR exon 18, p.G719S (SW48). B: EGFR exon 19, p.E746_750del (H1650). C: EGFR exon 20, p.T790M (H1975). D: EGFR exon 21, p.L858R (H1975). E: KRAS exon 2, p.G12C (H358). MASO, multiplex allele-specific oligonucleotide. The Journal of Molecular Diagnostics 2014 16, 45-55DOI: (10.1016/j.jmoldx.2013.07.009) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Box plot of the percentage of nonamplificable (NA) NSCLC paraffin-embedded samples recovered by alternative techniques of the 74 tested. The bottom and top of the box are the 25th and 75th percentiles (the lower and upper quartiles), respectively, and the horizontal line near the middle of the box is the 50th percentile (the median). The error bars are defined by the lowest datum still within 1.5 interquartile range (IQR) of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile. The number of experiments in which NA recovery rates were computed is indicated in parentheses for each alternative technique. One experiment corresponds to a set of amplicons analyzed for a given exon and a given technique in one center. FA, fragment analysis; HRM, high resolution melting; PYRO, pyrosequencing; TT, targeted techniques. The Journal of Molecular Diagnostics 2014 16, 45-55DOI: (10.1016/j.jmoldx.2013.07.009) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Box plots of kappa values by exon. The number of kappas computed, one for each experiment defined by the combination of exon, technique, and center, is indicated in parentheses for each alternative technique. Kappa SQC, between direct sequencing and alternative molecular techniques (P < 0.001); Kappa SYNTH, between alternative molecular techniques and a reference based on all alternative techniques (P = 0.04). e18, EGFR exon 18; e19, EGFR exon 19; e20, EGFR exon 20; e21, EGFR exon 21. See the legend to Figure 2 for a description of the elements of the box and whisker plot. The Journal of Molecular Diagnostics 2014 16, 45-55DOI: (10.1016/j.jmoldx.2013.07.009) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Box plots of Kappa SYNTH values by groups of alternative techniques. The number of kappas is indicated in parentheses for each alternative technique. Results between alternative techniques were not significantly different (P = 0.3). TT, targeted techniques; FA, fragment analysis; HRM, high resolution melting; PYRO, pyrosequencing. See the legend to Figure 2 for a description of the elements of the box and whisker plot. The Journal of Molecular Diagnostics 2014 16, 45-55DOI: (10.1016/j.jmoldx.2013.07.009) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions