Comparison of gDNA‐based versus mRNA‐based KIT D816V mutation analysis reveals large differences between blood and bone marrow in systemic mastocytosis

Abstract

Comparison of gDNA-based versus mRNA-based KIT D816V mutation analysis reveals large differences between blood and bone marrow in systemic mastocytosisMost adults with mastocytosis carry the activating somatic KIT D816V mutation. Mutation levels may be very low, thus requiring highly sensitive mutation analysis. KIT D816V mutation analysis may be based on genomic DNA (gDNA) or the expressed messenger RNA (mRNA). However, very limited experimental evidence comparing these two approaches exists, and the optimal technical approach therefore remains unclear.We therefore systematically compared gDNA-based and mRNA-based KIT D816V mutation analysis of peripheral blood (PB) and bone marrow (BM) aspirate from 82 systemic mastocytosis (SM) patients {76 indolent SM [ISM, including 3 BM mastocytosis (BMM) and 4 smouldering SM] and 6 SM with associated clonal haematological nonmast cell lineage disease}. These patients were also included in a previous study (Kristensen et al, 2014), except for one BMM patient where material was unavailable for mRNAbased analysis. In all patients, the first PB sample received for KIT D816V mutation analysis was included [DNA-based PB results previously reported in Kristensen et al (2014)]. BM aspirate was also available from 65 patients and included. The study was performed in accordance with the National Committee on Health Research Ethics and the Helsinki Declaration.A previously described highly sensitive KIT D816V mutation-specific quantitative polymerase chain reaction assay (Kristensen et al, 2011) was used for both gDNA-based and mRNA-based analysis (hereafter gDNA-qKIT and mRNAqKIT, respectively). gDNA-qKIT was performed with gDNA from 50 000 cells per reaction and mRNA-qKIT was performed with cDNA from mRNA from 50 000 cells, thus allowing non-biased comparison of the two approaches. Method details are provided in Data S1.In mRNA-qKIT, PB from 24 of the 82 patients (29%) fulfilled criteria of positivity. In gDNA-qKIT, 77 of the 82 (94%) samples initially tested D816V-positive, thus demonstrating a higher diagnostic yield of gDNA-qKIT in PB samples (v 2 -test: P < 0Á001; Fig 1; Table I). The DNA-based D816V-positive allele burden was significantly higher in PB samples that tested positive in mRNA-qKIT compared to samples that were negative in mRNA-qKIT (median allele burden 2Á8% vs. 0Á051%; Mann-Whitney U-test: P < 0Á001). One of the 5 PB samples that initially tested D816V-negative in gDNA-qKIT produced a borderline result (1 of 3 triplicate reactions producing a Ct-value <44). In mastocytosis patients, a sample producing a borderline result in gDNAqKIT typically produces a positive result when reanalysed using increased amounts of gDNA (Kristensen et al, 2014). The sample producing a borderline result tested positive in reanalysis, thus increasing the diagnostic yield of gDNA-qKIT from 94% to 95%. In addition to the 24 D816V-positive PB samples in mRNA-qKIT, 11 samples produced a borderline result. Due to limited available material, these samples were not reanalysed by mRNA-qKIT. H...

Prospective evaluation of the diagnostic value of sensitive KIT D816V mutation analysis of blood in adults with suspected systemic mastocytosis

“…Of these 14 patients, five were diagnosed with SM in the BM investigation. These five patients all tested D816V-positive in BM aspirate (two only in mRNA-based analysis as previously described 25 ). One of the five patients presented with skin lesions, and the remaining four presented with anaphylaxis (hymenoptera venom-induced in three of four).…”

Adverse Prognostic Impact of the KIT D816V Transcriptional Activity in Advanced Systemic Mastocytosis

“…Although sensitivity and specificity are comparable, only limited data exist upon the comparability between KIT D816V VAF and EAB [10,14,19]. We investigated a large cohort of patients with ISM and AdVSM unveiling an excellent correlation in ISM but not in AdvSM.…”

Clinical significance of droplet digital PCR quantitative monitoring of KIT gene mutation levels in core binding factor leukemia

“…9 The mechanism and detection of KIT gene mutation in patients with CBF-AML has been widely investigated. 10,11 At present, the fusion gene transcription level and RT-qPCR is mainly used to monitor the MRD of CBF-AML-CK patients. 12 In this study, we used ddPCR to absolute quantitatively detect the KIT gene mutation level in 20 CBF-AML-CK patients and discussed the significance of KIT gene mutation level as an MRD index for prognosis.…”