Clinical Validation of KRAS, BRAF, and EGFR Mutation Detection Using Next-Generation Sequencing

Am J Clin Pathol. 2014 Jun;141(6):856-66. doi: 10.1309/AJCPMWGWGO34EGOD.

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.

Keywords: BRAF; Deamination; EGFR; KRAS; Next-generation sequencing; Read depth; Validation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Biomarkers, Tumor / genetics*
  • Deamination
  • ErbB Receptors / genetics*
  • High-Throughput Nucleotide Sequencing / methods*
  • Limit of Detection
  • Molecular Diagnostic Techniques
  • Mutation
  • Neoplasms / diagnosis
  • Neoplasms / genetics*
  • Paraffin Embedding
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins B-raf / genetics*
  • Proto-Oncogene Proteins p21(ras)
  • Reproducibility of Results
  • Sensitivity and Specificity
  • ras Proteins / genetics*

Substances

  • Biomarkers, Tumor
  • KRAS protein, human
  • Proto-Oncogene Proteins
  • EGFR protein, human
  • ErbB Receptors
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins