Background: The 2447 A>T pathogenic variation at codon 816 of exon 17 (D816V) in the KIT gene, occurring in systemic mastocytosis (SM), leads to constitutive activation of tyrosine kinase activity and confers resistance to the tyrosine kinase inhibitor imatinib mesylate. Thus detection of this variation in SM patients is important for determining treatment strategy, but because the population of malignant cells carrying this variation is often small relative to the normal cell population, standard molecular detection methods can be unsuccessful.
Methods: We developed 2 methods for detection of KIT D816V in SM patients. The first uses enriched sequencing of mutant alleles (ESMA) after BsmAI restriction enzyme digestion, and the second uses an allele-specific competitive blocker PCR (ACB-PCR) assay. We used these methods to assess 26 patients undergoing evaluation for SM, 13 of whom had SM meeting WHO classification criteria (before variation testing), and we compared the results with those obtained by direct sequencing.
Results: The sensitivities of the ESMA and the ACB-PCR assays were 1% and 0.1%, respectively. According to the ACB-PCR assay results, 65% (17/26) of patients were positive for D816V. Of the 17 positive cases, only 23.5% (4/17) were detected by direct sequencing. ESMA detected 2 additional exon 17 pathogenic variations, D816Y and D816N, but detected only 12 (70.5%) of the 17 D816V-positive cases. Overall, 100% (15/15) of the WHO-classified SM cases were codon 816 pathogenic variation positive.
Conclusion: These findings demonstrate that the ACB-PCR assay combined with ESMA is a rapid and highly sensitive approach for detection of KIT D816V in SM patients.