Tumor Mutational Load assessment of FFPE samples using an NGS based assay
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The document describes an Oncomine tumor mutation load assay that assesses tumor mutational load in FFPE samples using a targeted Ion Ampliseq panel covering approximately 1.7 Mb of genomic DNA. The assay is optimized for speed and reproducibility, allowing for detailed reports on mutation counts and signatures that correlate well with whole exome sequencing. This research tool may aid in identifying predictive biomarkers for cancer immunotherapy and improve the understanding of responses to immune checkpoint blockade inhibitors.
Tumor Mutational Load assessment of FFPE samples using an NGS based assay
- 1. Ruchi Chaudhary, Warren Tom, Iris Casuga, Dinesh Cyanam, Vinay Mittal, Nick Khazanov, Paul Williams, Asha Kamat, Dumitru Brinza, Janice Au-Young, Seth Sadis, Fiona C. L. Hyland. Thermo Fisher Scientific, South San Francisco, CA 94088 RESULTS Figure 1. Oncomine Tumor Mutation Load Assay Workflow Tumor Mutational Load assessment of FFPE samples using an NGS based assay Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com ABSTRACT Understanding the molecular determinants of response to immune checkpoint blockade inhibitors is a critical unmet need for translational oncology research. Research tools to characterize the mutational landscape of cancers may potentially help identify predictive biomarkers for immuno-therapy that can be tested in future studies. Herein, we describe a targeted Ion AmpliSeq assay to determine the mutational load and signature of cancer research samples. METHODS A new Ion AmpliSeq targeted panel, derived from the Comprehensive Cancer Panel, was developed that covers approximately 1.7 Mb of genomic DNA and 400 genes. The assay requires up to 20 ng of input DNA and can leverage manual or automated library and templating on the Ion Chef. The informatics workflow utilizes a custom variant calling and germline variant filtering algorithm to accurately estimate somatic variants in cancer research samples. A detailed report is provided that includes the normalized mutation load (mutations/MB), mutation signatures of the somatic variants, and the percentage of mutations consistent with UV damage, tobacco smoke damage, de-amination, and specific substitutions. CONCLUSIONS • The Oncomine Tumor Mutation Load Assay provides an accurate estimate of tumor mutation burden, which is useful for cancer immuno-therapy research and future biomarker development. • Detailed in silico analyses show that the assay correlates well with whole exome sequencing (WES) results. • This assay was highly reproducible in FFPE samples. • Up to 20 ng input DNA and only ~60 minutes hands-on time from DNA to data is required. • The assay workflow was optimized for up to 8 samples to be analyzed simultaneously. • A detailed report provides normalized mutation count per MB as well as mutation signatures. SITC 2017 ABSTRACT - P398 Figure 2. In Silico –Correlation between TML Panel and Exome Figure 3. TML – Correlation between Tumor Only and Tumor/Normal Figure 4. Reproducibility DNA extraction from FFPE AmpliSeq™ Library creation Templating Sequencing Analysis Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Oncomine™ Tumor Mutation Load Assay Ion Chef™ System Ion S5™ System Torrent Suite™ with Ion Reporter™ Two-day workflow * 409 genes (1.7Mb) for broad coverage 2 pool assay (DNA only) – 20 ng DNA in total Configured for manual and automated library preparation Optimized on Ion S5 and Ion 540 chip (8 samples/chip) ~60 Minutes Hands-On Time from DNA to Data, only 2 pipetting steps UI ReportAnalysis Ion Reporter™ Variant Calling Germ-line Filtering Ion Reporter™ QC Run Input BAM from TS Mutation Load per Mb PDF Report, Variant TSV 10ng DNA / pool A somatic variant dataset was derived from COMSIC v80 containing 21,056 exomes derived from 22 major cancer types. The total number of somatic variants observed per sample was highly concordant to the total number of variants intersecting with the genomic regions covered by the Oncomine™ Tumor Mutation Load Assay. For Research Use Only r2 = 0.80 Mutation Count through Exome MutationCountthroughTML Figure 7 . Reports The assay uses proven AmpliSeq technology to generate libraries from FFPE tumor samples that are sequenced on the Ion S5 sequencer. Up to 8 samples are processed simultaneously for sequencing. The workflow includes optimized somatic variant calling and a detailed report. Somatic mutation counts in FFPE samples determined using the TML workflow were highly concordant to somatic mutation counts determined by matched tumor/normal sequencing. Somatic mutation counts estimated with the TML workflow were highly reproducible. Sample types included cell lines, FFPE and FF lung and melanoma samples. Detailed reports provide information with respect to samples, QC and allele ratio distribution. Base substitution classes and mutation signatures are also reported. Figure 6 . Melanoma WES and TML Higher somatic mutation counts were associated with clinical response to immune checkpoint blockade inhibitors in the Rizvi et al, Snyder et al, and Van Allen et al studies. Figures 5 & 6 show an in silico analysis with the variant results reported in the Rizvi and Snyder studies (similar results were seen in the Van Allen study but were not presented due to space limitations). We determined the somatic mutation counts intersecting with the TML panel and plotted those against the clinical response data. The results suggest the TML panel is sufficiently large to predict potential clinical outcome. P-values were determined using the Mann-Whitney Exact test with no assumptions. Rizvi et al. 2016 Science. 348:124-128. Snyder et al. 2014 N Eng J Med. 371:2189-2199. Van Allen et al. 2015. Science 350:207-211. Figure 5 . NSCLC WES and TML Figure 5 & 6. WES p = 0.00084 TML p = 0.0057 WES p = 0.00023 TML p = 0.00035