KRas and BRaf Mutational Status Analysis From Formalin-Fixed, Paraffin-Embedded Tissues Using Multiplex Polymerase Chain Reaction–Based Assay

Abstract

Abstract Context.—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. Objective.—To develop a multiplex polymerase chain reaction–based assay for the evaluation of KRas and BRaf mutational status. Design.—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. Results.—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-termin...