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.
Highlights
Colorectal cancer (CRC) is the most common cancer and the third leading cause of cancer death in the world [1]
Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer
Only the CRC with wild-type KRAS proto-oncogene responds to anti-EGFR antibodies treatment, whereas no therapeutic response occurs in CRC with KRAS mutations [9,10,11]
Summary
Colorectal cancer (CRC) is the most common cancer and the third leading cause of cancer death in the world [1]. One of the major molecular pathways in CRC development is the induction of an activating mutation in the proto-oncogene KRAS (Kirsten rat sarcoma viral oncogene) [4, 5]. Cancer therapies have been developed using monoclonal antibodies, including cetuximab and panitumumab, to target the EGFR [7, 8]. These agents are designed to block ligand-induced EGFR tyrosine kinase activation and, inhibit downstream signaling [9]. 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.
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