Locked Nucleic Acid and TaqMan Probes to Detect KRAS Mutations by Droplet Digital PCR in Colorectal Carcinoma Formalin Fixed Paraffin Embedded Tissue Samples

摘要

BACKGROUND: Metastatic colorectal carcinoma (CRC) tumors with KRAS mutations in exon 2, codons 12 and 13, do not benefit from anti-epidermal growth factor receptor (EGFR) antibody therapy. Current best practice recommends that KRAS mutation analysis be included in patients with CRC as a prerequisite for treatment with cetuximab or panitumumab. We selected 50 CRC formalin fixed paraffin embedded (FFPE) samples to compare a qPCR assay (QIAGEN therascreen? KRAS RGQ PCR Kit) to droplet digital PCR (ddPCR). Wild type and six single nucleotide mutations in KRAS codons 12 and 13 were tested. Methods. DNA was extracted and used in assays employing primers paired with locked nucleic acid (LNA) or TaqMan probes. Droplets were generated and streamed through a droplet reader (BioRad Laboratories, CA). The QuantaSoft software (BioRad Laboratories) was used to analyze the ddPCR data. Results. In limit of detection studies, ddPCR detected 0.002 cpd of known mutant DNA in a background of 1 cpd of wt DNA. Specific KRAS mutations were detected in 16 (of 50) samples using LNA probes. Of the 16 samples tested by LNA probes, 13 were also tested using TaqMan probes, confirming results from the LNA assays. Results from ddPCR assays were compared to results from an outside commercial laboratory. The commercial lab detected and identified mutations in 10 of the 50 FFPE samples tested. ddPCR assays also detected mutations in 10 of these samples. An additional five samples were determined to be positive by commercial qPCR but no specific mutation was reported. ddPCR detected and identified specific mutations in three of these as well as in three samples not identified by commercial qPCR. Conclusions. ddPCR allele specific assays compared well to current commercially available KRAS mutation detection assays. Some cross reactivity was noted in ddPCR assays employing LNA probes but not TaqMan probes. Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities.