Dataset Information

Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing.

ABSTRACT: To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth.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).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.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.

SUBMITTER: Lin MT

PROVIDER: S-EPMC4332779 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing.

Lin Ming-Tseh MT Mosier Stacy L SL Thiess Michele M Beierl Katie F KF Debeljak Marija M Tseng Li-Hui LH Chen Guoli G Yegnasubramanian Srinivasan S Ho Hao H Cope Leslie L Wheelan Sarah J SJ Gocke Christopher D CD Eshleman James R JR

American journal of clinical pathology 20140601 6

<h4>Objectives</h4>To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth.<h4>Methods</h4>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).<h4>Results</h4>We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can res ...[more]

PMID: 24838331

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Project description: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.

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Dataset Information

Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing.

Publications

Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing.

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