Validation of targeted next-generation sequencing for RAS mutation detection in FFPE XXXX tissues: comparison with Sanger sequencing and ARMS-Scorpion real-time PCR.

@entity1 @entity36006 @entity984 @entity406 @entity14 @entity46789
OBJECTIVE: To validate the targeted next-generation sequencing (NGS) platform-Ion Torrent @entity46789 for @entity984 exon 2 and expanded RAS mutations detection in @entity406 -fixed paraffin-embedded (FFPE) @entity14 ( @entity14 ) specimens, with comparison of Sanger sequencing and ARMS-Scorpion real-time PCR. SETTING: Beijing, China. @entity1 : 51 archived FFPE @entity14 samples (36 @entity1 , 15 @entity1 ) were retrospectively randomly selected and then checked by an experienced pathologist for sequencing based on histological confirmation of @entity14 and availability of sufficient tissue. METHODS: RAS mutations were detected in the 51 FFPE @entity14 samples by @entity46789 analysis, Sanger sequencing and the Therascreen @entity984 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. RESULTS: 13 of 51 (25.5%) cases had a mutation in @entity984 exon 2, as determined by @entity46789 analysis. @entity46789 analysis showed 100% (51/51) concordance with Sanger sequencing (k=1.000, 95% CI 1 to 1) and 98.04% (50/51) agreement with the Therascreen assay (k=0.947, 95% CI 0.844 to 1) for detecting @entity984 exon 2 mutations, respectively. The only discrepant case harboured a @entity984 exon 2 mutation ( @entity36006 ) that was not covered by the Therascreen kit. The dilution series experiment results showed that @entity46789 was able to detect @entity984 mutations at a frequency of as low as 1%. Importantly, RAS mutations other than @entity984 exon 2 mutations were also detected in 10 samples by @entity46789 . Furthermore, mutations in other @entity14 -related genes could be simultaneously detected in a single test by @entity46789 . CONCLUSIONS: The targeted NGS platform is specific and sensitive for @entity984 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 @entity14 -related genes.
