Development and validation of a scalable next-generation sequencing system for assessing recurrent somatic alterations in solid tumors

Abstract

INTRODUCTION: Treating cancer effectively requires an understanding of the molecular alterations driving each patient's tumor. Prevalent and recurrent cancer-associated somatic alterations have been defined using whole genome, exome, and transcriptome sequencing across thousands of cases. Targeted sequencing efforts that characterize prevalent somatic alterations and require limited sample input may provide an effective diagnostic approach in the future.MATERIALS AND METHODS: Herein, we describe the design and initial characterization of the Oncomine Cancer Research Panel (OCP) that includes recurrent somatic alterations in solid tumors derived from the Oncomine™ cancer database. Using Ion AmpliSeq™, we designed a DNA panel that includes assays for 73 oncogenes with 1, 826 recurrent hotspot mutations, 26 tumor suppressor genes enriched for deleterious mutations, as well as 75 genes subject to recurrent focal copy gain or loss. A complementary RNA panel includes 183 assays for relevant gene fusions involving 22 fusion driver genes. Recommended sample inputs were 20 ng of DNA and 10 ng of RNA. Sequencing libraries were analyzed on an Ion Torrent Personal Genome Machine.RESULTS: Initial testing revealed an average read depth of > 1, 500X with > 95% uniformity and on target frequency. The panel was shown to reliably detect known hotspots, insertions/deletions, gene copy changes, and gene fusions in molecular standards, cell lines and formalin-fixed paraffin embedded clinical specimens. Retrospective analysis of large sample cohorts has been completed and the results of analysis of 100 lung cancer and 100 prostate cancer samples will be summarized. In addition, a cohort of 100 tumor samples from the University of Michigan Molecular Diagnostics laboratory, which were submitted for molecular testing (KRAS, BRAF, EGFR, ALK), were also profiled with OCP. Overall, we achieved >95% sensitivity and specificity for detection of KRAS, BRAF, and EGFR mutations, as well as ALK gene fusions.CONCLUSIONS: We designed OCP to include prevalent and relevant somatic alterations to aid in cancer research exploring whether targeted NGS panels may have utility in future diagnostic applications.