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

Background: Patients 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.

Methods: We 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.

Results: Overall, 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.

Conclusion: The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

Fig 1. Target region of KRAS gene at codons 12 and 13 for amplification and the design of primers.

The positions of primers are illustrated. AS primers share the same forward primer or reverse primer. Solid arrows indicate the forward and reverse primers. The 3′ terminal base of each AS primers was adapted according to its corresponding mutation, and is bold and underlined. Codons and mutated bases are underlined.

Fig 2. MAS-PCR assay of KRAS gene.

Assay distinguished wild-type KRAS gene codons 12 and 13 from different mutants using AS primers. Lane M: Low molecular weight DNA ladder; Lane 1: negative control; Lane 2: wild-type; Lane 3: G12S mutant; Lane 4: G12R mutant; Lane 5: G12C mutant; Lane 6: G12D mutant; Lane 7: G12A mutant; Lane 8: G12V mutant; Lane 9: G13D mutant.

Fig 3. Sensitivity of MAS-PCR assay for identifying KRAS gene mutations.

A representative gel is shown. Dilutions of G12R mutant plasmid and wild-type plasmid DNA (from 100%, 50%, 25%, 10%, 5%, 2%, 1% and 0.1% mutated alleles) Lane M: Low molecular weight DNA Ladder; Lane 1: negative control; Lane 2–9: corresponded to PCR products from 100% to 0.1%.