title: Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers.
abstract: Activating mutations in downstream genes of the epidermal growth factor receptor (EGFR) pathway may cause anti-EGFR resistance in patients with colorectal cancers. We present performance characteristics of a next-generation sequencing assay designed to detect such mutations. In this retrospective quality assessment study, we analyzed mutation detected in the KRAS, NRAS, BRAF, and PIK3CA genes by a clinically validated next-generation sequencing assay in 310 colorectal cancer specimens. Tumor cellularity and mutant allele frequency were analyzed to identify tumor heterogeneity and mutant allele-specific imbalance. Next-generation sequencing showed precise measurement of mutant allele frequencies and detected 23% of mutations with 2-20% mutant allele frequencies. Of the KRAS mutations detected, 17% were outside of codons 12 and 13. Among PIK3CA mutations, 48% were outside of codons 542, 545, and 1047. The percentage of tumors with predicted resistance to anti-EGFR therapy increased from 40% when testing for only mutations in KRAS exon 2 to 47% when testing for KRAS exons 2-4, 48% when testing for KRAS and NRAS exons 2-4, 58% when including BRAF codon 600 mutations, and 59% when adding PIK3CA exon 20 mutations. Right-sided colorectal cancers carried a higher risk of predicted anti-EGFR resistance. A concomitant KRAS mutation was detected in 51% of PIK3CA, 23% of NRAS, and 33% of kinase-impaired BRAF-mutated tumors. Lower than expected mutant allele frequency indicated tumor heterogeneity, while higher than expected mutant allele frequency indicated mutant allele-specific imbalance. Two paired neuroendocrine carcinomas and adjacent adenomas showed identical KRAS mutations, but only PIK3CA mutations in neuroendocrine carcinomas. Next-generation sequencing is a robust tool for mutation detection in clinical laboratories. It demonstrates high analytic sensitivity and broad reportable range, and it provides simultaneous detection of concomitant mutations and a quantitative measurement of mutant allele frequencies to predict tumor heterogeneity and mutant allele-specific imbalance.
authors.affiliation: Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Haley
authors.affiliation: Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.|Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Li-Hui
authors.initials: LH
authors.lastname: Tseng
authors.affiliation: Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Zheng
authors.affiliation: Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.|Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Dudley
authors.affiliation: Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.
authors.forename: Derek A
authors.initials: DA
authors.lastname: Anderson
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.
authors.forename: Nilofer S
authors.initials: NS
authors.lastname: Azad
authors.affiliation: Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.|Department of Oncology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.
authors.forename: Christopher D
authors.identifier: http://orcid.org/0000-0002-9541-2192
authors.initials: CD
authors.lastname: Gocke
authors.affiliation: Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.|Department of Oncology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
authors.affiliation: Departments of Pathology, Johns Hopkins University School of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.
authors.forename: Ming-Tseh
authors.initials: MT
authors.lastname: Lin
mesh_terms: D058534:Class I Phosphatidylinositol 3-Kinases; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D012189:Retrospective Studies
chemical_list: C117307:KRAS protein, human; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)