Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing.
OBJECTIVE
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.
From the Departments of Pathology and.
Ming-Tseh
MT
Lin
From the Departments of Pathology and.
Stacy L
SL
Mosier
From the Departments of Pathology and.
Michele
M
Thiess
From the Departments of Pathology and.
Katie F
KF
Beierl
From the Departments of Pathology and.
Marija
M
Debeljak
Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
Li-Hui
LH
Tseng
From the Departments of Pathology and.
Guoli
G
Chen
Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD; and.
Srinivasan
S
Yegnasubramanian
From the Departments of Pathology and.
Hao
H
Ho
Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD; and.
Leslie
L
Cope
Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD; and.
Sarah J
SJ
Wheelan
From the Departments of Pathology and Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD; and.
Christopher D
CD
Gocke
From the Departments of Pathology and Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD; and jeshlema@jhmi.edu.
James R
JR
Eshleman
D014408:Biomarkers, Tumor / Q000235:genetics*; D003641:Deamination; D066246:ErbB Receptors / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D057230:Limit of Detection; D025202:Molecular Diagnostic Techniques; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics*; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
BRAF; Deamination; EGFR; KRAS; Next-generation sequencing; Read depth; Validation