Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers
Journal
Modern Pathology
Journal Volume
28
Journal Issue
10
Pages
1390-1399
Date Issued
2015
Author(s)
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. ? 2015 USCAP, Inc All rights reserved.
SDGs
Other Subjects
B Raf kinase; epidermal growth factor receptor kinase inhibitor; K ras protein; NRAS protein; phosphatidylinositol 4,5 bisphosphate 3 kinase; phosphatidylinositol 4,5 bisphosphate 3 kinase catalytic subunit alpha; protein; unclassified drug; B Raf kinase; BRAF protein, human; guanosine triphosphatase; KRAS protein, human; membrane protein; NRAS protein, human; phosphatidylinositol 3 kinase; PIK3CA protein, human; protein p21; adenoma; allele; Article; cancer diagnosis; cancer resistance; codon; colorectal cancer; controlled study; exon; gene frequency; gene mutation; human; human tissue; invasive carcinoma; invasive neuroendocrine carcinoma; major clinical study; next generation sequencing; prediction; priority journal; quality control; retrospective study; colorectal tumor; dna mutational analysis; drug resistance; genetics; high throughput sequencing; pathology; procedures; Colorectal Neoplasms; DNA Mutational Analysis; Drug Resistance, Neoplasm; GTP Phosphohydrolases; High-Throughput Nucleotide Sequencing; Humans; Membrane Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Retrospective Studies
Publisher
Nature Publishing Group
Type
journal article