title: Assessment of the clinical application of detecting EGFR, KRAS, PIK3CA and BRAF mutations in patients with non-small cell lung cancer using next-generation sequencing.
abstract: BACKGROUND
  Next-generation sequencing (NGS) has been widely applied in clinical research, while its application in routine clinical molecular testing requires careful validation. The aim of our study was to assess the clinical usefulness of the NextDaySeq Lung panel on Ion Torrent™ PGM in mutation detection of actionable genes in lung cancer.
  
  
  METHODS
  The NextDaySeq assay was evaluated by blinded comparisons to Quantitative Real-Time PCR (qPCR) assays with 188 consecutive samples from Chinese patients with non-small cell lung cancer (NSCLC) to detect mutations in EGFR, KRAS, PIK3CA and BRAF. Discordant variants were further validated by Sanger sequencing and independent qPCR and NGS assays.
  
  
  RESULTS
  Our results showed 93.3% concordance of reportable variants mutually covered in both NGS and qPCR assays, with a clinical sensitivity of 89.9%, specificity of 97.5%. Through the comparison, the NGS assays demonstrated its advantages in offering more clinical relevant information, such as detecting non-hotspot mutations and providing mutation allele frequencies (MAF) and accurate mutation sequences. The analytical sensitivity of NGS to detect mutations with low MAF needs further improvement.
  
  
  CONCLUSIONS
  The NextDaySeq Lung panel exhibited good clinical performance, strongly supporting the implementation of the NGS assay in routine clinical use to facilitate therapeutic decision-making for lung cancer patients.
authors.affiliation: a Department of Thoracic Surgery , Shanghai Pulmonary Hospital, Tongji University , Shanghai , P. R. China ;
authors.forename: Xinnan
authors.initials: X
authors.lastname: Xu
authors.affiliation: a Department of Thoracic Surgery , Shanghai Pulmonary Hospital, Tongji University , Shanghai , P. R. China ;
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: b Beijing ACCB Biotech Ltd , Haidian District , Beijing , P. R. China ;
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.affiliation: b Beijing ACCB Biotech Ltd , Haidian District , Beijing , P. R. China ;
authors.forename: Zhao
authors.initials: Z
authors.lastname: Chen
authors.affiliation: a Department of Thoracic Surgery , Shanghai Pulmonary Hospital, Tongji University , Shanghai , P. R. China ;
authors.forename: Gening
authors.initials: G
authors.lastname: Jiang
authors.affiliation: c Lung Cancer Diagnosis & Treatment Center , Shanghai Pulmonary Hospital, Tongji University , Shanghai , P. R. China.
authors.forename: Ke
authors.initials: K
authors.lastname: Fei
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D058534:Class I Phosphatidylinositol 3-Kinases; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Comparison; DNA mutation analysis; next-generation sequencing; non-small cell lung cancer; real-time PCR