Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing
Journal
American Journal of Clinical Pathology
Journal Volume
141
Journal Issue
6
Pages
856-866
Date Issued
2014
Author(s)
Lin M.-T.
Mosier S.L.
Thiess M.
Beierl K.F.
Debeljak M.
Chen G.
Yegnasubramanian S.
Ho H.
Cope L.
Wheelan S.J.
Gocke C.D.
Eshleman J.R.
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. ? American Society for Clinical Pathology.
Subjects
BRAF; Deamination; EGFR; KRAS; Next-generation sequencing; Read depth; Validation
Other Subjects
B Raf kinase; epidermal growth factor receptor; formaldehyde; K ras protein; paraffin; article; bottleneck population; gene mutation; gene sequence; genetic procedures; genome; genotype; human; human cell; human tissue; major clinical study; next generation sequencing; point mutation; priority journal; pyrosequencing; single nucleotide polymorphism; BRAF; Deamination; EGFR; KRAS; Next-generation sequencing; Read depth; Validation; Deamination; High-Throughput Nucleotide Sequencing; Limit of Detection; Molecular Diagnostic Techniques; Mutation; Neoplasms; Paraffin Embedding; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; ras Proteins; Receptor, Epidermal Growth Factor; Reproducibility of Results; Sensitivity and Specificity; Tumor Markers, Biological
Publisher
American Society of Clinical Pathologists
Type
journal article