KIT mutation analysis in mast cell neoplasms: recommendations of the European Competence Network on Mastocytosis

Abstract

Although acquired mutations in KIT are commonly detected in various categories of mastocytosis, the methodologies applied to detect and quantify the mutant type and -burden in various tissues and cells are poorly defined. We here propose a consensus on methodologies used to detect KIT mutations in patients with mastocytosis at diagnosis and in the follow up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly-sensitive assays, KIT D816V can be detected in peripheral blood leukocytes in most patients with systemic mastocytosis (SM) which is a major step forward in screening and SM detection. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy in these patients. Our recommendations should greatly facilitate diagnostic and follow up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early detection of SM, and help avoid unnecessary referrals.

Clinical presentation and management practice of systemic mastocytosis. A survey on 460 Italian patients

“…The cKITD816V mutation was detected in BM samples of 92.2% of 438 patients, consistent with previous reports [19]. Two D816V-negative patients had different cKIT mutations, D816H and K546K.…”

“…Conversely, the same methodologies were able to detect the mutation in 92% of BM samples, where mutation burden is higher, in line with others' experience. Therefore, as recently suggested, standardized and highly sensitive method of detection need to be devised and implemented to allow the convenient use of peripheral blood samples as a screening tools in patients suspected to have SM [19,27,28].…”

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

“…Moreover, virtually all aggressive systemic mastocytosis cases, as well as around one-fourth of indolent systemic mastocytosis patients, 6 present multilineage involvement of bone marrow cell compartments other than mast cells by the KIT mutation, 11,12,25,26 this being considered as the most powerful prognostic factor for disease progression among adult indolent systemic mastocytosis patients. 7,10,27 Recently, several groups 9,13,14 have confirmed the feasibility of detecting the KIT mutation in peripheral blood leukocytes; consequently, sensitive allele-specific oligonucleotide-qPCR approaches for peripheral blood detection of the KIT D816V mutation 13,28 have been included in recent consensus diagnostic algorithms 18 owing to the less invasive diagnostic approach in peripheral blood vs bone marrow. 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.…”

“…Overall, such apparently discrepant results could be explained by the fact that different approaches were used to assess peripheral blood involvement by the KIT mutation (analysis on gDNA 9,13 vs cDNA 30 ), which therefore might have different sensitivities. Of note, no study has been reported so far in which paired peripheral blood and bone marrow samples had been analyzed in large series of systemic mastocytosis cases by two or more (highly sensitive) PCR methods, 28 to clarify such discrepancies. Furthermore, the precise relationship between the presence of KIT D816V + cells in peripheral blood and multilineage involvement of bone marrow hematopoiesis by the KIT mutation also remains unknown.…”

“…In this study, we compared the performance of the two most widely accepted methods 28 (peptide nucleic acid-mediated PCR-clamping technique 17 vs allele-specific oligonucleotide-qPCR 13 ), which are currently used to detect the KIT D816V mutation in clinical diagnostic laboratories, on the same gDNA samples obtained from paired peripheral blood leukocytes and highly purified bone marrow CD34 + hematopoietic stem and precursor cells and maturing neutrophils, from a large cohort of systemic Figure 2 Frequency and degree of involvement of bone marrow isolated cells from indolent systemic mastocytosis patients grouped according to the presence of mast cell-restricted vs multilineage involvement of hematopoiesis by the KIT D816V + as assessed by the allele-specific oligonucleotide-qPCR method. Frequency and degree of involvement by the KIT D816V mutation of FACS-purified bone marrow CD34 + hematopoietic stem and precursor cells and maturing neutrophils from indolent systemic mastocytosis patients as assessed by the allele-specific oligonucleotide-qPCR technique.…”