Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis

Abstract

The recent progress in sensitive KIT D816V mutation analysis suggests that mutation analysis of peripheral blood (PB) represents a promising diagnostic test in mastocytosis. However, there is a need for systematic assessment of the analytical sensitivity and specificity of the approach in order to establish its value in clinical use. We therefore evaluated sensitive KIT D816V mutation analysis of PB as a diagnostic test in an entire case-series of adults with mastocytosis. We demonstrate for the first time that by using a sufficiently sensitive KIT D816V mutation analysis, it is possible to detect the mutation in PB in nearly all adult mastocytosis patients. The mutation was detected in PB in 78 of 83 systemic mastocytosis (94%) and 3 of 4 cutaneous mastocytosis patients (75%). The test was 100% specific as determined by analysis of clinically relevant control patients who all tested negative. Mutation analysis of PB was significantly more sensitive than serum tryptase >20 ng/mL. Of 27 patients with low tryptase, 26 tested mutation positive (96%). The test is furthermore readily available and we consider the results to serve as a foundation of experimental evidence to support the inclusion of the test in diagnostic algorithms and clinical practice in mastocytosis.

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

“…the KIT D816V mutation in both peripheral blood leukocytes and purified bone marrow cell populations, in line with the higher frequencies of KIT mutated cases reported in the literature with the former technique. 9,29 In contrast, both methods showed an overall similar accuracy to predict for mast cell-restricted vs multilineage bone marrow involvement by the KIT mutation based on peripheral blood screening, whenever quantitative (the KIT allele burden) instead of qualitative (negative vs positive peripheral blood for the KIT mutation) allele-specific oligonucleotide-qPCR results were used. Of note, similar results were observed when we restricted the analysis to indolent systemic mastocytosis patients grouped according to the presence vs absence of skin lesions, although both a lower sensitivity and overall accuracy for the detection of the KIT D816V mutation were observed among indolent systemic mastocytosis without skin lesions vs indolent systemic mastocytosis with skin lesions cases for the qualitative allele-specific oligonucleotide-qPCR vs both the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated PCR method.…”

“…However, careful analysis of the literature shows discrepant results regarding both the frequency of systemic mastocytosis cases who are KIT D816V + in peripheral blood and the prognostic impact of the KIT D816V allele burden. Thus, while Kristensen et al 9,29 reported positive rates in peripheral blood samples of indolent systemic mastocytosis cases between 96% and 100%, Erben et al 30 only found 46% of indolent systemic mastocytosis patients to be positive in peripheral blood using a different allele-specific quantitative real-time PCR method. In turn, whereas Erben et al 30 suggested that a greater amount of KIT D816V + cells in peripheral blood is associated with a poorer outcome, the Danish group 15 did not find any correlation between the allele burden for the KIT D816V mutation and the clinical manifestations of indolent systemic mastocytosis.…”

“…As the level of infiltration of bone marrow by tumor mast cells is frequently low 7 (eg o 0.1% of bone marrow-aspirated samples), highly sensitive molecular approaches have long been required for reliable detection of the KIT D816V mutation, even after purification of bone marrow mast cells. 8,9 Despite this, it is now well established that, in at least onethird of indolent systemic mastocytosis and all aggressive systemic mastocytosis patients, bone marrow myeloid and/or lymphoid cells other than mast cells also carry the KIT D816V mutation (multilineage systemic mastocytosis); 6 such patients have a greater risk for disease progression. 10 Among multilineage systemic mastocytosis patients, the presence of the KIT D816V mutation is typically detected in genomic DNA (gDNA) of CD34 + hematopoietic stem and precursor cells, eosinophils, monocytes and maturing neutrophils, and, to a less extent, also in T lymphocytes, in addition to bone marrow mast cells.…”

“…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. In this regard, particularly intriguing is the potential lack of association between the allele burden for the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide polymerase chain reaction (allelespecific oligonucleotide-qPCR), and the clinical and biological features of indolent systemic mastocytosis.…”

“…In this regard, particularly intriguing is the potential lack of association between the allele burden for the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide polymerase chain reaction (allelespecific oligonucleotide-qPCR), and the clinical and biological features of indolent systemic mastocytosis. 9,15 Thereby, the precise biological and clinical significance of the presence of circulating peripheral blood KIT-mutated cells in systemic mastocytosis still remains to be elucidated.…”