Simultaneous Detection of Clinically Relevant Mutations and Amplifications for Routine Cancer Pathology  Marlous Hoogstraat, John W.J. Hinrichs, Nicolle J.M. Besselink, Joyce H. Radersma-van Loon, Carmen M.A. de Voijs, Ton Peeters, Isaac J. Nijman, Roel A. de Weger, Emile E. Voest, Stefan M. Willems, Edwin Cuppen, Marco J. Koudijs  The Journal of Molecular Diagnostics  Volume 17, Issue 1, Pages 10-18 (January 2015) DOI: 10.1016/j.jmoldx.2014.09.004 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Full coverage of TP53 using the OncoAmp Panel. The bar plot indicates the sequence coverage over TP53, divided into forward reads (red) and reverse reads (blue), with the coverage indicated on the y axis. The TP53 transcript is visualized at the bottom, with black boxes indicating the exons; the white box on the left is the untranslated exon 1 in the 5′ untranslated region of the gene. The vertical red lines in these exons indicate mutations detected in several samples sequenced with the OncoAmp Panel. The blue horizontal lines above the transcript indicate the placement of amplicons. The Journal of Molecular Diagnostics 2015 17, 10-18DOI: (10.1016/j.jmoldx.2014.09.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Gene amplification detection using the OncoAmp Panel and in situ hybridization. A: The z scores of all amplified genes detected in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) samples sequenced with the OncoAmp Panel. B: The z scores per amplicon of EGFR amplification in a brain metastasis of a non–small cell lung carcinoma; the z scores of CDK6 located on the same chromosome have no aberrant copy number. C: The z scores per amplicon of amplified KRAS and FGFR1 in an adenocarcinoma of the stomach; ERBB3 located on the same chromosome as KRAS has no aberrant copy number. D: The z scores per amplicons of amplified ERBB2 in an invasive ductulolobular breast carcinoma; the z scores of TOP2A located nearby have no aberrant copy number. E: Fluorescent in situ hybridization (FISH) (DAPI counterstain) performed on the non–small cell lung carcinoma sample in B, with arrows indicating several of the clusters of EGFR gene copies indicative of gene amplification. F: FISH (DAPI counterstain) performed on the adenocarcinoma of the stomach in C, with arrows indicating clusters of FGFR1 gene copies indicative of gene amplification. G: Chromogenic in situ hybridization performed on the invasive ductulolobular breast carcinoma in D, with arrows indicating several examples of ERBB2 gene clusters. Original magnification: ×100 (E and F); ×40 (G). The Journal of Molecular Diagnostics 2015 17, 10-18DOI: (10.1016/j.jmoldx.2014.09.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Allele frequencies and amplifications in cell line dilution series. A: Observed and expected allele frequencies of 40 variant positions with differential mutation status between the reference sample and the UACC-812 cell line. The symbols represent the data points from various dilution fractions. A line with an intercept of 0 and a slope of 1 indicates the expected trend. B: The z scores of reported amplifications of ERBB2, MDM2, and TOP2A in the different dilutions. The different symbols represent the three genes. The Journal of Molecular Diagnostics 2015 17, 10-18DOI: (10.1016/j.jmoldx.2014.09.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions