title: Validation of targeted next-generation sequencing for RAS mutation detection in FFPE colorectal cancer tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR.
abstract: To validate the targeted next-generation sequencing (NGS) platform-Ion Torrent PGM for KRAS exon 2 and expanded RAS mutations detection in formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) specimens, with comparison of Sanger sequencing and ARMS-Scorpion real-time PCR.
  
  
  
  Beijing, China.
  
  
  
  51 archived FFPE CRC samples (36 men, 15 women) were retrospectively randomly selected and then checked by an experienced pathologist for sequencing based on histological confirmation of CRC and availability of sufficient tissue.
  
  
  
  RAS mutations were detected in the 51 FFPE CRC samples by PGM analysis, Sanger sequencing and the Therascreen KRAS assay, respectively. Agreement among the 3 methods was assessed. Assay sensitivity was further determined by sequencing serially diluted DNA from FFPE cell lines with known mutation statuses.
  
  
  
  13 of 51 (25.5%) cases had a mutation in KRAS exon 2, as determined by PGM analysis. PGM analysis showed 100% (51/51) concordance with Sanger sequencing (κ=1.000, 95% CI 1 to 1) and 98.04% (50/51) agreement with the Therascreen assay (κ=0.947, 95% CI 0.844 to 1) for detecting KRAS exon 2 mutations, respectively. The only discrepant case harboured a KRAS exon 2 mutation (c.37G>T) that was not covered by the Therascreen kit. The dilution series experiment results showed that PGM was able to detect KRAS mutations at a frequency of as low as 1%. Importantly, RAS mutations other than KRAS exon 2 mutations were also detected in 10 samples by PGM. Furthermore, mutations in other CRC-related genes could be simultaneously detected in a single test by PGM.
  
  
  
  The targeted NGS platform is specific and sensitive for KRAS exon 2 mutation detection and is appropriate for use in routine clinical testing. Moreover, it is sample saving and cost-efficient and time-efficient, and has great potential for clinical application to expand testing to include mutations in RAS and other CRC-related genes.
authors.affiliation: Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
authors.forename: Huanwen
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
authors.forename: Huanli
authors.initials: H
authors.lastname: Duan
authors.affiliation: Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
authors.forename: Junliang
authors.initials: J
authors.lastname: Lu
authors.affiliation: Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Liang
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics*; D005091:Exons; D011905:Genes, ras; D006801:Humans; D009154:Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C117307:KRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)