Abstract

Aims: The c-kit D816V activating mutation is found in >80% of cases of systemic mastocytosis (SM) and represents a potential drug target. Furthermore, because D816V is one of the diagnostic criteria for SM, it is clinically relevant to determine whether the mutation is present. Traditional techniques such as DNA sequencing are often not sensitive enough to detect mutations in low-abundance tumor cells, including SM. Here we describe an allele-specific assay to detect the D816V mutation in DNA from archived formalin-fixed paraffin-embedded tissues.

Methods: A 2-tube PCR format was employed to amplify c-kit exon 17 as a control and an allele-specific reaction to selectively amplify the D816V mutant allele using standard oligos. A D816V-mutant plasmid control was generated by site-directed mutagenesis of wild-type cells. Fourteen cases of SM, 1 D816V-positive seminoma sample, and 35 cases without SM were analyzed using our assay.

Results: Our assay successfully amplified D816V in the mutant plasmid control, 13/14 cases of SM, and confirmed D816V in a seminoma sample. In addition, D816V was not amplified in 35/35 cases without SM. Serial dilution experiments demonstrated sensitivity down to <1%.

Conclusion: We have developed a sensitive, specific, and cost-effective assay to detect the D816V mutation in archived FFPE cases of SM.