Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays

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Presentation transcript:

Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays Sabah Kadri, Bradley C. Long, Ibro Mujacic, Chao J. Zhen, Michelle N. Wurst, Shruti Sharma, Nadia McDonald, Nifang Niu, Sonia Benhamed, Jigyasa H. Tuteja, Tanguy Y. Seiwert, Kevin P. White, Megan E. McNerney, Carrie Fitzpatrick, Y. Lynn Wang, Larissa V. Furtado, Jeremy P. Segal The Journal of Molecular Diagnostics Volume 19, Issue 1, Pages 43-56 (January 2017) DOI: 10.1016/j.jmoldx.2016.07.012 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 University of Chicago Medicine OncoPlus tiered design: A: The capture panel genes are split in two tiers: tier 1 has 316 genes of high clinical significance, 3× more probe than tier 2 (896 genes), and approximately 53% of the coding reads. B: The sorted average depth per position is shown for each tier. The Journal of Molecular Diagnostics 2017 19, 43-56DOI: (10.1016/j.jmoldx.2016.07.012) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: Mutant allele frequency (MAF) concordance: Scatter plot of University of Chicago Medicine (UCM) OncoPlus versus UCM amplicon assay MAFs for all variants across 78 clinical (54 malignant and 24 nonmalignant) samples, showing >10% signal via either assay. The red points highlight two additional variants detected by UCM-OncoPlus not previously detected by OncoHeme. The blue points represent two mutations with higher amplicon MAFs, whereas the green points represent two SRSF2 mutations with higher MAFs in UCM-OncoPlus. B and C: Specificity of UCM-OncoPlus in malignant samples: the average MAFs of all non–single-nucleotide polymorphism (SNP) positions across all solid tumor (B) and malignant peripheral blood or bone marrow (C) samples. The vertical lines show the SDs, whereas the red points show the maximum MAF at each position across all compared samples. D: MAFs of 119 SNP variants in the mixed control sample across 20 sequencing runs. Each MAF is plotted as a red point. The black line represents the average across replicates. E: Average and SDs of MAFs at each non-SNP position in the mixed normal control across replicates. The red points show the maximum signal at each position across all replicates. n = 20 (B and D and E, replicates); n = 34 (C). FFPE, formalin fixed, paraffin embedded. The Journal of Molecular Diagnostics 2017 19, 43-56DOI: (10.1016/j.jmoldx.2016.07.012) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 FLT3 internal tandem duplication (ITD) mutant allele frequency (MAF) comparison: University of Chicago Medicine (UCM) OncoPlus versus OncoHeme FLT3 ITD MAFs for 42 samples (34 malignant and 8 nonmalignant), including 11 positives and 2 low positives (UC25 and UC26). The x axis shows the highest MAF in the UCM-OncoPlus FLT3 ITD module across BWA MEM/Abra, Pindel, and ITD Hunter, whereas the y axis shows the highest OncoHeme MAFs among alignment-based and Amplicon Indel Hunter results for each sample. Dotted lines indicate analytical sensitivity of UCM-OncoPlus. The Journal of Molecular Diagnostics 2017 19, 43-56DOI: (10.1016/j.jmoldx.2016.07.012) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Copy number alterations: A: Log2 average fold change across seven independent nonmalignant blood samples using the baseline historical normal. No false-positive changes were detected as expected. B: Log2 of fold changes in a Foundation Medicine sample (VAL5) using a baseline historical normal. The analysis shows copy number changes in the expected genes, EGFR, MET, RAF1, CCND1, MYC, FGF3, and FGF19, in addition to few other genes on our panel. Dotted lines indicate the fold change cutoffs. The Journal of Molecular Diagnostics 2017 19, 43-56DOI: (10.1016/j.jmoldx.2016.07.012) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Gene fusions: A: Histogram of percentage of fusion read pairs identified via the University of Chicago Medicine fusion algorithm across all read pairs mapped to ALK, RET, and ROS1. There is a clear distinction between the 16 fusion-positive and the 24 nonmalignant clinical samples. The x axis is in log10 scale. B: An example of KIF5B and RET fusion reads shown in Integrated Genomics Browser.29 The highlighted reads are paired-end mates that map across the fusion junction. The Journal of Molecular Diagnostics 2017 19, 43-56DOI: (10.1016/j.jmoldx.2016.07.012) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions