Competitive allele specific TaqMan PCR for KRAS, BRAF and EGFR mutation detection in clinical formalin fixed paraffin embedded samples

BackgroundThe development of targeted therapies has created a need for robust molecular characterization of cancer and it has become a challenge to validate methods to ensure accuracy in tumor mutation testing.MethodsThe current study was designed to evaluate KRAS, BRAF and EGFR genotyping by Competitive Allele Specific hydrolysis probes (TaqMan) PCR technology (CAST), on suboptimal formalin fixed paraffin embedded (FFPE) tumor samples. Assays were calibrated on FFPE samples and a minimal quantification cycle (Cq) cut-off was determined to standardize analyses and avoid over-interpretation of degraded material. Sensibility, specificity and blinded clinical sample screenings (n = 63) were evaluated.ResultsCAST PCR allowed efficient amplification of FFPE samples, probes were highly specific and all assays had a sensibility inferior to 1% except for the EGFR p.T790M assay. 60/63 samples were correctly typed. The three missed mutations were EGFR exon 19 deletions that were not recognized by the DEL19 assays that were used.ConclusionsThis technology is less laborious and prevent crossover of PCR products as compared to multistep methods. TaqMan® Mutation Detection assay is an important technology to consider in the field of mutation detection for KRAS, BRAF and EGFR point mutation screening. Assay calibration on FFPE samples may prevent erroneous interpretations that will ultimately harm clinical oncology practice.

► Accurate tumor mutation testing is a challenge in clinical oncology practice.
► KRAS, BRAF and EGFR were genotyped using CAST qPCR technology on FFPE material.
► A mutation cut-off was determined to avoid over-interpretation of degraded material.
► Mutated alleles were identified up to 1% and FFPE samples were efficiently amplified.
► CAST-PCR is a one step to diagnosis method that prevents crossover of PCR.