title: Routine Clinical Mutation Profiling of Non-Small Cell Lung Cancer Using Next-Generation Sequencing.
abstract: BACKGROUND
  The availability of massive, parallel-sequencing technologies makes possible efficient, simultaneous detection of driver and druggable mutations in cancer.
  
  
  OBJECTIVE
  To develop an amplicon-based, next-generation sequencing, mutation-detection assay for lung cancer using the 454 GS Junior (Roche Applied Science, Indianapolis, Indiana) platform.
  
  
  METHODS
  Fusion primers incorporating target sequence, 454 adaptors, and multiplex identifiers were designed to generate 35 amplicons (median length 246 base pairs) covering 8.9 kilobases of mutational hotspots in AKT1, BRAF, EGFR, ERBB2, HRAS, KRAS, NRAS, PIK3CA, and MAP2K1 genes and all exons of the PTEN gene.
  
  
  RESULTS
  The assay was validated on 23 formalin-fixed, paraffin-embedded lung cancer specimens. A minimum number of reads was consistently achieved with overall median read depth of 529× per amplicon. In total, 25 point mutations and 4 insertions/deletions (indels) with a frequency of 5.5% to 93.1% mutant alleles were detected. All EGFR, ERBB2, KRAS, PIK3CA, KRAS, and PTEN mutations, as detected by next-generation sequencing, were confirmed by pyrosequencing, with the exception of 3 point mutations in a tumor sample with low mutant-allele burden (below the pyrosequencing limit of detection).
  
  
  CONCLUSIONS
  The GS Junior-based, targeted, resequencing assay for a focused set of non-small cell lung cancer driver genes allows for quick and sensitive detection of point mutations and indels for the most relevant therapeutic genes in this type of cancer.
authors.forename: Kristin K
authors.initials: KK
authors.lastname: Deeb
authors.forename: Colleen M
authors.initials: CM
authors.lastname: Hohman
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: Risch
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Metzger
authors.affiliation: From the Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, New York. Dr Deeb is now with the University Hospitals Case Medical Center, Cleveland, Ohio; Mr Risch is now with Life Technologies, Grand Island, New York; and Dr Starostik is now with the Department of Pathology, Immunology and Laboratory Medicine, College of Medicine University of Florida, Gainesville.
authors.forename: Petr
authors.initials: P
authors.lastname: Starostik
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor