Improved Detection of the KIT D816V Mutation in Patients with Systemic Mastocytosis Using a Quantitative and Highly Sensitive Real-Time qPCR Assay

Abstract

The vast majority of patients with systemic mastocytosis (SM) carry the somatic D816V mutation in the KIT gene. The KIT D816V mutation is one of the minor criteria for a diagnosis of SM according to the 2008 World Health Organization classification of myeloproliferative neoplasms. In the present study, we present a real-time qPCR assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. A total of 61 samples from 31 cases of SM were included in the study. We detected the mutation in skin or bone marrow in 95% of the cases of SM. We demonstrate the clinical relevance of the assay by identifying as little as 0.03% mutation-positive cells in bone marrow aspirates from SM patients and calculate the analytical sensitivity of negative samples to determine the reliability of the result. We further demonstrate that this method also detects the KIT D816V mutation in peripheral blood in 81% of the mutation-positive cases with SM. The method also allows comparison of mutation-positive and mast cell fractions to determine whether the mutation is present in non-mast cells, a parameter that has recently been reported to be of prognostic importance in patients with indolent SM. Finally, the assay is suitable for use in prospective studies of the KIT D816V allele burden as a treatment endpoint in SM.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications

“…6,17 Based on the results obtained with this method on purified bone marrow cell populations, all patients were further subclassified as having either systemic mastocytosis with KIT D816V mutation restricted to bone marrow mast cells (mast cell-restricted systemic mastocytosis) or systemic mastocytosis with multilineage involvement of bone marrow hematopoiesis by the KIT mutation (multilineage systemic mastocytosis), following the previously defined criteria. 6,10 In a subset of 161 patients, detection of the KIT D816V mutation was also performed in parallel in aliquots of the same samples containing the same amount of gDNA (200 ng per sample for peripheral blood leukocytes, and 50 ng per sample for purified bone marrow neutrophils and/or CD34 + hematopoietic stem and precursor cells) using a previously described allele-specific oligonucleotide-qPCR assay; 13 for this purpose, an incubation at 95°C for the activation of the DNA polymerase (10 min) followed by 45 cycles of PCR amplification (denaturation at 95°C for 10 s followed by annealing at 60°C for 30 s and elongation at 72°C for 1 s) was performed in a LightCycler 2.0 thermocycler using the LightCycler TaqMan Master kit reagents (Roche Diagnostics).…”

“…All hybridization probes required for the peptide nucleic acid-mediated PCR technique, 6 as well as the primers used in both techniques, were obtained from Sigma-Aldrich, whereas the allele-specific oligonucleotide-qPCR TaqMan probe 13 was purchased from Applied Biosystems (Foster City, CA, USA). As negative and positive controls, gDNA from an adult healthy donor and the KIT D816V mutation-positive HMC-1 560+/816+ cell line (proved to have two copies of the KIT gene, including a KIT D816V-mutated allele and a wild-type KIT allele) 23 were systematically studied in parallel to both the patient and control samples.…”

“…6,12 Thereby, the KIT mutation should also be detectable in peripheral blood from a significant percentage of systemic mastocytosis cases. 10 In line with this hypothesis, new highly sensitive methods have been described, 13 which allowed detection of the KIT D816V mutation in peripheral blood leukocytes from most mastocytosis cases, including 66/69 (96%) indolent systemic mastocytosis and 3/4 cutaneous mastocytosis investigated. 9,14 Despite this, the potential relationship between the presence of the KIT D816V mutation in peripheral blood and multilineage involvement of bone marrow hematopoiesis has not been investigated so far.…”

“…13 In addition, we also investigated the presence of multilineage bone marrow involvement by the KIT D816V mutation in highly purified bone marrow CD34 + hematopoietic stem and precursor cells and maturing neutrophils using both methods. Our aim was to determine the precise frequency of peripheral blood involvement by the KIT D816V mutation, its relationship with the degree of involvement of bone marrow hematopoiesis in indolent systemic mastocytosis and the potential diagnostic implications of these findings.…”

Splenomegaly, elevated alkaline phosphatase and mutations in the SRSF2/ASXL1/RUNX1 gene panel are strong adverse prognostic markers in patients with systemic mastocytosis

“…5 Activating mutations in the receptor tyrosine kinase KIT, usually KIT D816V, are pathogenetically relevant somatic point mutations, detected in >80-90% of patients with SM. 6,7 The multi-lineage expansion by KIT D816V and the KIT D816V allele burden (AB) have an important impact on disease phenotype and prognosis. 6,[8][9][10] Furthermore, the presence and number of molecular aberrations, e.g.…”