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

PloS One
Sirirat SeekhuntodNuntaree Chaichanawongsaroj

Abstract

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. 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. 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. The MAS-PCR assay provides a rapid, cost-...Continue Reading

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Citations

Nov 30, 2016·Expert Review of Molecular Diagnostics·Michelangelo FiorentinoRodolfo Montironi
Apr 12, 2017·Sensors·Maria GuarnacciaSebastiano Cavallaro
Mar 25, 2020·Journal of Clinical Laboratory Analysis·Qing-Lin WangKai Li
Apr 18, 2020·Metabolomics : Official Journal of the Metabolomic Society·Dorna VarshaviJeremy R Everett
Mar 18, 2021·Scientific Reports·Ana-Luisa SilvaBarnaby William Balmforth

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Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

SPSS

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