KRas and BRaf mutational status analysis from formalin-fixed, paraffin-embedded tissues using multiplex polymerase chain reaction-based assay.
BACKGROUND
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.


METHODS
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-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.


CONCLUSIONS
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.
Jakubauskas
Arturas
A
Department of Molecular and Regenerative Medicine, Oncology and Transfusion Medicine Center, Vilnius University Hospital, Santariskiu Clinics, Lithuania. arturas.jakubauskas@santa.lt
Griskevicius
Laimonas
L
D019943:Amino Acid Substitution; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000628:therapy; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics*; D005091:Exons; D005557:Formaldehyde; D011905:Genes, ras; D006801:Humans; D020125:Mutation, Missense; D016612:Paraffin Embedding; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA / Q000379:methods; D016707:Tissue Fixation
D017931:DNA Primers; D004273:DNA, Neoplasm; D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf