The KIT D816V expressed allele burden for diagnosis and disease monitoring of systemic mastocytosis

Abstract

The activating KIT D816V mutation plays a central role in the pathogenesis, diagnosis, and targeted treatment of systemic mastocytosis (SM). For improved and reliable identification of KIT D816V, we have developed an allele-specific quantitative real-time PCR (RQ-PCR) with an enhanced sensitivity of 0.01-0.1 %, which was superior to denaturing high-performance liquid chromatography (0.5-1 %) or conventional sequencing (10-20 %). Overall, KIT D816 mutations were identified in 146/147 (99 %) of patients (D816V, n = 142; D816H, n = 2; D816Y, n = 2) with SM, including indolent SM (ISM, n = 63, 43 %), smoldering SM (n = 8, 5 %), SM with associated hematological non-mast cell lineage disease (SM-AHNMD, n = 16, 11 %), and aggressive SM/mast cell leukemia ± AHNMD (ASM/MCL, n = 60, 41 %). If positive in BM, the KIT D816V mutation was found in PB of all patients with advanced SM (SM-AHNMD, ASM, and MCL) and in 46 % (23/50) of patients with ISM. There was a strong correlation between the KIT D816V expressed allele burden (KIT D816V EAB) with results obtained from DNA by genomic allele-specific PCR and also with disease activity (e.g., serum tryptase level), disease subtype (e.g., indolent vs. advanced SM) and survival. In terms of monitoring of residual disease, qualitative and quantitative assessment of KIT D816V and KIT D816V EAB was successfully used for sequential analysis after chemotherapy or allogeneic stem cell transplantation. We therefore conclude that RQ-PCR assays for KIT D816V are useful complimentary tools for diagnosis, disease monitoring, and evaluation of prognosis in patients with SM.

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

“…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.…”

“…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. 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.…”

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

“…6 Nextgeneration deep amplicon sequencing by 454 FLX amplicon chemistry (Roche, Penzberg, Germany) was performed to investigate 18 genes recurrently mutated in myeloid neoplasms as previously described. 9 …”

“…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.…”

“…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. in SRSF2, ASXL, or RUNX1 (S/A/R gene panel), have a strong adverse impact on disease phenotype and OS in patients with advSM.. 9,11 One of several WHO criteria to classify SM includes enlargement of visceral organs, e.g.…”