Clinical Validation of KRAS, BRAF, and EGFR Mutation Detection Using Next-Generation Sequencing. Issue 6 (1st June 2014)

• Record Type: Journal Article
• Authors: Lin, Ming-Tseh; Mosier, Stacy L.; Thiess, Michele; Beierl, Katie F.; Debeljak, Marija; Tseng, Li-Hui; Chen, Guoli; Yegnasubramanian, Srinivasan; Ho, Hao; Cope, Leslie; Wheelan, Sarah J.; Gocke, Christopher D.; Eshleman, James R.
• Is Part Of: American journal of clinical pathology. Volume 141:Issue 6(2014)
• Page Start: 856
• Page End: 866
• Publication Date: 2014-06-01
• Subjects: Next-generation sequencing -- Validation -- KRAS -- BRAF -- EGFR -- Read depth -- Deamination; Diagnosis, Laboratory -- Periodicals; Pathology -- Periodicals; 616.07
• DOI: 10.1309/AJCPMWGWGO34EGOD
• Languages: English
• ISSNs: 0002-9173

Abstract

Abstract: Objectives: To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth. Methods: We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA). Results: We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations. Conclusions: Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts.