Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients

Abstract

BackgroundPatients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations.MethodsWe developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.ResultsOverall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12.ConclusionThe MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

“…In contrast, our multiplex assay simultaneously analyzes 7 mutant primers and probes in the same reaction tube, reducing the amount of clinical material required, which is important in FFPE tissue where the proportion of mutant relative to wild type DNA is likely to be low [ 22 , 31 , 32 ]. Although another multiplex allele specific PCR employing standard end-point PCR and SYBR green I nucleic acid gel stain was recently used to detect KRAS mutant in clinical samples [ 10 ], the latter method requires post-PCR manipulations risking laboratory nucleic acid contamination, since it includes end-point PCR followed by an electrophoretic separation to detect short 64 bp or 68 bp stained products corresponding respectively to the G12S and to the G12V KRAS mutations. Although these small products may be real [ 10 ], in some samples they may correspond to non-specific small molecular weight sub-products of end-point PCR, especially since no specific KRAS hybridization probing of this molecule was provided [ 10 ].…”

“…Although another multiplex allele specific PCR employing standard end-point PCR and SYBR green I nucleic acid gel stain was recently used to detect KRAS mutant in clinical samples [ 10 ], the latter method requires post-PCR manipulations risking laboratory nucleic acid contamination, since it includes end-point PCR followed by an electrophoretic separation to detect short 64 bp or 68 bp stained products corresponding respectively to the G12S and to the G12V KRAS mutations. Although these small products may be real [ 10 ], in some samples they may correspond to non-specific small molecular weight sub-products of end-point PCR, especially since no specific KRAS hybridization probing of this molecule was provided [ 10 ]. Moreover, electrophoretic analysis and Sybr Green I stain used [ 10 ] limits high throughput analysis.…”

“…Although these small products may be real [ 10 ], in some samples they may correspond to non-specific small molecular weight sub-products of end-point PCR, especially since no specific KRAS hybridization probing of this molecule was provided [ 10 ]. Moreover, electrophoretic analysis and Sybr Green I stain used [ 10 ] limits high throughput analysis. In contrast, our multiplex allele-specific Taqman real-time PCR assays not only has the potential to detect low copy numbers of mutated DNA, but the use of fluorescently labeled probes, increase sensitivity, specificity and reproducibility.…”

“…This multiplex allele specific assay potentially allows inexpensive and rapid high throughput screening of the most clinically frequent KRAS mutations in one step, followed a single tube use of seven allele specific primer (i.e, G12S, G12R, G12C, G12D, G12A, G12V and G13D) and common reverse primer and TaqMan probe. In contrast with reports using KRAS plasmid DNA [ 10 , 21 ] to define assay sensitivity, this multiplex allele specific assay was standardized using human reference DNA from clinical samples or from tumor cell lines, permitting multiplex detection of at least 0.15% of mutant DNA (35 pg approximately, equivalent to 5 copies of mutant KRAS DNA) mixed with wild type genomic DNA. Preliminary rapid multiplex mutation detection was followed by precise identification of the specific mutation performed by seven individual allele specific assays.…”

“…The European Medicines Agency (EMA), the National Comprehensive Cancer Network (NCCN), the American Society of Clinical Oncology (ASCO) and the US Food and Drug Administration (FDA) recommend that treatment of cetuximab and panitumumab to target EGFR, requires that CRC patients posses a wild type KRAS. Hence, simple and sensitive screening for KRAS mutations prior to treatment with an anti-EGFR antibody therapy is indispensable [ 2 , 10 ]. Early detection in clinically available tissue is difficult in cases with low abundance mutations relative to wild type DNA.…”