Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers

• Published in: Modern pathology Vol. 28; no. 10; pp. 1390 - 1399
• Main Authors: Haley, Lisa, Tseng, Li-Hui, Zheng, Gang, Dudley, Jonathan, Anderson, Derek A, Azad, Nilofer S, Gocke, Christopher D, Eshleman, James R, Lin, Ming-Tseh
• Format: Journal Article
• Language: English
• Published: United States Elsevier Limited 01.10.2015
• Subjects: Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms - genetics; Colorectal Neoplasms - pathology; DNA Mutational Analysis - methods; Drug Resistance, Neoplasm - genetics; GTP Phosphohydrolases - genetics; High-Throughput Nucleotide Sequencing - methods; Humans; Membrane Proteins - genetics; Phosphatidylinositol 3-Kinases - genetics; Proto-Oncogene Proteins B-raf - genetics; Proto-Oncogene Proteins p21(ras) - genetics; Retrospective Studies
• ISSN: 0893-3952; 1530-0285
• DOI: 10.1038/modpathol.2015.86

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.