Monoclonal anti-epidermal growth factor receptor antibodies bind to the epidermal growth factor receptor and inhibit receptor kinase activity. Clinical trials have indicated that evaluation of the mutational status of KRas and BRaf is necessary to exclude patients who are resistant to the clinical benefit of anti-epidermal growth factor receptor therapy.
To develop a multiplex polymerase chain reaction-based assay for the evaluation of KRas and BRaf mutational status.
A sample-saving and cost-effective, multiplex polymerase chain reaction-based assay to detect somatic mutations in KRAS exon 2 and exon 3 as well as in BRAF exon 15 was developed. The same primer pairs could be successfully used in amplification of a single DNA fragment under the same conditions.
DNA isolated from 20 retrospective formalin-fixed, paraffin-embedded samples of colorectal cancer was screened for mutations using the multiplex polymerase chain reaction assay followed by dideoxy-termination sequencing. Five samples bearing mutations-p.G12D (identified twice), p.G12V, p.G12S, and p.G13D, all encoded in KRAS exon 2-were identified. Three samples were found bearing amino acid substitution p.V600E of BRaf. The detected KRas and BRaf mutations were found to be mutually exclusive.
A multiplex polymerase chain reaction-based amplification followed by dideoxy-termination sequencing may be used advantageously for the evaluation of KRas and BRaf mutational status from formalin-fixed, paraffin-embedded samples.