One-Step Detection of c-kit Point Mutations Using Peptide Nucleic Acid-Mediated Polymerase Chain Reaction Clamping and Hybridization Probes

Abstract

The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.

Systemic mastocytosis in adults: 2015 update on diagnosis, risk stratification, and management

“…Of note, there is a high correlation between KIT mutation detection and the proportion of lesional cells in the sample, as well as the sensitivity of the screening method employed [65]. Sensitivity of detection may be enhanced by enriching lesional MC by laser capture microdissection, or magnetic bead-or FACSbased cell sorting, respectively, [20,29,66] or through the use of highly sensitive PCR techniques [32]. Outside of a research setting, it is currently not standard practice to screen for KIT mutations other than those involving D816.…”

Chronic mast cell leukemia (MCL) with KIT S476I: a rare entity defined by leukemic expansion of mature mast cells and absence of organ damage

“…Routine examinations were performed at the time of diagnosis and in the follow up, according to generally accepted recommendations and standards [5,9,[16][17][18][19][20][21]. The patient provided written informed consent before physical examination was performed and BM and blood samples were obtained.…”

“…Isolated BM cells were also examined by conventional karyotyping and fluorescence in situ hybridization (FISH). BM studies were performed according to generally accepted standards [15,[18][19][20][21].…”

“…In all 6 patients examined, BM and blood cells were tested for the presence of codon 816 mutations according to published methods [18,22,23]. In the patient with chronic MCL, all coding exons of the KIT gene were analyzed by Sanger sequencing.…”

Aberrant expression of CD30 in neoplastic mast cells in high-grade mastocytosis

“…37,38 The human mast cell leukemia cell line HMC-1.2, which is known to display the KIT mutation D816V, served as a positive control. 21 HMC-1 cells were kindly provided by Dr Butterfield of the Mayo Clinic, Rochester, MN, USA.…”