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 allele burden in various cells and tissues are poorly defined. We here propose a consensus on methodologies used to detect KIT mutations in patients with mastocytosis at diagnosis and during 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 from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. 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 diagnosis of SM and help to avoid unnecessary referrals.

Figure 1. Structure of normal KIT (KIT Wild-Type)

The KIT gene, located on chromosome 4q12 in humans, contains 21 exons transcribed/translated into a transmembrane receptor tyrosine kinase (RTK) of 145 kDa and 976 amino acids. KIT structure is characterized by 5 Ig-like subunits in the extracellular domain (ECD) which contains a ligand binding site (SCF for KIT) and a dimerization site, and is linked to a cytoplasmic region by a single transmembrane helix. The cytoplasmic region of KIT consists of an autoinhibitory juxta-membrane domain (JMD) and a kinase domain (KD) arranged in a proximal (N−) and a distal (C−) lobe linked by a hinge region. The C-lobe of RTKs type III includes a large Kinase Insert Domain (KID) of ~ 60-100 residues. The red star represents the position 816 where an Asp to Val point mutation (KIT D816V) is found in > 80% of adult systemic mastocytosis patients. The receptor is presented under its monomeric form, whereas a dimer results from stem cell factor (SCF) ligation. ECD: Extracellular domain; JMD: Juxtamembrane domain; KID: Kinase insert domain; PTD: phosphotransférase domain; TK: tyrosine kinase; TMD: transmembrane domain.

Figure 2. Representation of the structure of KIT, illustrating the localization of the more frequently observed mutations in the KIT sequence in pediatric and adult patients with mastocytosis

The receptor is presented under its monomeric form, whereas its wild-type counterpart dimerizes upon ligation with SCF before being activated in normal cells. In children,he KIT D816V PTD mutant (in red) is found in nearly 30% of the patients, whereas the ECD mutants (in blue) are found in nearly 40% of the affected children. In adults, depending of the categoty of mastocytosis, the KIT D81V mutant is found in at least 80% of all patients. The complete list of KIT mutants retrieved in the literature for mastocytosis is depicted here. In children, the structure of KIT is found WT in around 25% of the patients analyzed, whereas in adults, KIT is found WT in less than 20% of all patients analyzed so far. Some of the mutations (in black) are found only in a very few number of patients. Del: deletion; ECD: Extracellular domain; Ins: Insertion; ITD: Internal tandem duplication; JMD: Juxtamembrane domain; KI: Kinase insert; PTD: phosphotransferase domain; TMD: Transmembrane domain. ±: mutation found in less than 1% of the patients; +: mutation found in 1 to 5% of the patients; ++: mutation found in around 30% of pediatric patients and in > 80% of all adult patients.

Figure 3. Proposed algorithm for the use of KIT D816V allele-specific quantitative PCR or RT-PCR (ASO-qPCR) in peripheral blood (PB), together with serum tryptase measurement, for the screening of adult patients with suspected mastocytosis

In case of suspected mastocytosis in an adult patient, the first step is the search for clear signs of mastocytosis in the skin (MIS). If MIS is present, we recommend that a BM examination should be performed directly. If there is no MIS, we recommend to measure serum tryptase level, together with a KIT D816V ASO-qPCR using PB cells. If the serum tryptase level is > 15 ng/mL and/or the KIT D816V ASO-qPCR detects the mutation, a bone marrow examination is recommended, including a KIT D816V ASO-qPCR. If the BM studies are compatible with the diagnosis SM, and the KIT D816V ASO-qPCR is positive, then the final diagnosis is KIT D816V+ SM. If the BM studies are compatible with the diagnosis SM, and the KIT D816V ASO-q(RT)PCR is negative, sequencing of the entire KIT gene should be considered on a BM sample, in order to determine the KIT structure in this D816V-negative patient. In MIS-negative patients who have a serum tryptase level < 15 ng/mL and who are negative for the search of the KIT D816V mutant by ASO-qPCR on PB, we recommend to follow up the patients and to perform a BM investigation only if there is an increase in tryptase or clinical symptoms/signs suggesting the presence of mastocytosis. Otherwise, alternative explanations and differential diagnoses should be considered. ASO-qPCR: allele specific-quantitative PCR on RNA or DNA; BM: bone marrow; CM: cutaneous mastocytosis; MIS: “mastocytosis in the skin”; PB: peripheral blood; SM: systemic mastocytosis; sTryptase: serum total tryptase level.