The mutation A2447T (D816V) in the c-kit gene found in systemic mastocytosis (SM) leads to constitutive activation of tyrosine kinase activity and confers resistance to the tyrosine kinase inhibitor imatinib mesylate (IM) at standard doses. Other c-kit point mutations affecting codon 816 have been reported to have a similar dose-response to IM as the D816V mutation. Molecular studies of SM are difficult to perform as the population of malignant cells in the available tissues which carry the mutation is often small relative to the normal cell population. Using methods like direct sequencing is not ideal as the mutant signal is often obscured by the wild-type signal, particularly in heterozygous individuals. More sensitive methods are required when the mutant cells comprise only a small percentage of the total cell population.


To develop rapid and sensitive methods to more accurately determine the c-kit exon 17 mutation status of patients and subsequent eligibility for a Phase II molecularly targeted protocol with IM (INDY study, Novartis Pharmaceuticals, Australia).


Two methods were developed and compared with direct sequencing. The first method utilises enriched sequencing following BsmAI restriction enzyme digestion. The wild-type PCR product is cleaved by BsmAI, but the point mutation A2447T destroys the recognition sequence (GAGAC>GAGTC) and a larger fragment is generated. This fragment (enriched for the mutant sequence) can be excised from the gel and sequenced. The second method is a modification of the allele-specific competitive blocker PCR (ACB-PCR) method in which three primers are combined: (i) a normal primer that targets the normal allele is tagged with a 3′ phosphate to block amplification, (ii) a mutant primer that targets the mutant allele with mismatches to ensure that the normal allele is not amplified and (iii) a common reverse primer.


By analysing DNA extracted from the D816V carrying human leukemic cell line (HMC-1) diluted on a background of normal DNA, enriched sequencing following BsmAI digestion and ACB-PCR were sensitive to the level of 1% and <0.1% respectively. No false positives were observed in 19 different control samples obtained from normal and non-SM haematological malignancy peripheral blood samples.

Skin, spleen or bone marrow samples were obtained from 24 patients; 21 cases fulfilled the WHO criteria for SM and 3 cases were under investigation for SM. All 24 cases were analysed by the ACB-PCR method, enriched sequencing and standard direct sequencing. Using ACB-PCR, 67% (16/24) patients were positive for the D816V mutation. Of the 16 positive cases, direct sequencing detected only 19% (3/16). Enriched sequencing detected 2 additional mutations D816Y and D816N, but only detected (69%) 11/16 of the D816V positive cases. Using both ACB-PCR and enriched sequencing, 86% (18/21 of the WHO classified SM cases) were positive for a point mutation in the c-kit gene, 15 more cases than by direct sequencing alone.


These results indicate that the ACB-PCR method enhances D816V mutation detection in systemic mastocytosis patients compared to both direct sequencing and enriched sequencing. However, as the ACB-PCR is specific only for the D816V mutation, the other mutations D816Y and D816N could only be detected by enriched sequencing. These findings support the need for both methods as a combined approach to SM mutation detection.

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