Sensitive Detection of KIT D816V in Patients with Mastocytosis

Abstract

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

Systemic mastocytosis involving the gastrointestinal tract: clinicopathologic and molecular study of five cases

“…An enrichment procedure is often necessary in systemic mastocytosis because neoplastic mast cells may represent only a minor fraction of the cells present in biopsy samples. Enriched sequencing of mutant alleles, 25 a quick and cost-effective technique is one of several sensitive techniques developed to overcome this limitation. 25,44 In conclusion, this case series and literature review consolidates and expands current knowledge of the clinical, pathologic and molecular spectrum of systemic mastocytosis involving of the gastrointestinal tract.…”

“…Enriched sequencing of mutant alleles, 25 a quick and cost-effective technique is one of several sensitive techniques developed to overcome this limitation. 25,44 In conclusion, this case series and literature review consolidates and expands current knowledge of the clinical, pathologic and molecular spectrum of systemic mastocytosis involving of the gastrointestinal tract. Pathologists should consider this condition when evaluating gastrointestinal biopsies from patients with a constellation of symptoms that include diarrhea, abdominal pain, vomiting and weight loss and when evaluating small bowel biopsies for malabsorption.…”

“…Molecular analysis for the D816V KIT mutation was performed on all cases in which archival material was available (4 of 5 cases). Direct sequencing of exon 17 of the c-kit gene was performed initially and this was followed by enriched sequencing of mutant alleles 25 in cases that were negative.…”

“…The latter was excised from the agarose and purified with QIAquick Gel Extraction Kit (Qiagen, Mississauga, ON, Canada) and subjected to a second round of seminested PCR reaction as previously described. 25 The PCR sequencing conditions were the same as described for the exon 17 pre-sequencing PCR reaction except that the forward primer BSMAI was used. The product, enriched for the mutant allele, was then analyzed by sequencing.…”

“…In two of four patients the D816V KIT mutation was detected by direct sequencing while the remaining two cases were only demonstrated following enriched sequencing of the mutant alleles. 25 …”