III. Medizinische Klinik, Universitätsmedizin Mannheim, Theodor-Kutzer-Ufer 1-3, 68167, Mannheim, Germany.
Ann Hematol. 2014 Jan;93(1):81-8. doi: 10.1007/s00277-013-1964-1. Epub 2013 Nov 27.
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.
KIT D816V 激活突变在系统性肥大细胞增多症（SM）的发病机制、诊断和靶向治疗中起着核心作用。为了提高和可靠地识别 KIT D816V，我们开发了一种等位基因特异性实时定量 PCR（RQ-PCR），其灵敏度提高到 0.01-0.1%，优于变性高效液相色谱（0.5-1%）或常规测序（10-20%）。总体而言，在 147 例 SM 患者（D816V，n=142；D816H，n=2；D816Y，n=2）中鉴定出 KIT D816 突变，包括惰性 SM（ISM，n=63，43%）、冒烟型 SM（n=8，5%）、伴血液非肥大细胞谱系疾病的 SM（SM-AHNMD，n=16，11%）和侵袭性 SM/肥大细胞白血病±AHNMD（ASM/MCL，n=60，41%）。如果在 BM 中为阳性，KIT D816V 突变存在于所有晚期 SM（SM-AHNMD、ASM 和 MCL）患者的 PB 中，也存在于 46%（23/50）ISM 患者的 PB 中。KIT D816V 表达等位基因负荷（KIT D816V EAB）与通过基因组等位基因特异性 PCR 从 DNA 获得的结果之间存在很强的相关性，也与疾病活性（例如血清胰蛋白酶水平）、疾病亚型（例如惰性与侵袭性 SM）和生存相关。在监测残留疾病方面，KIT D816V 和 KIT D816V EAB 的定性和定量评估成功用于化疗或异基因干细胞移植后的连续分析。因此，我们得出结论，RQ-PCR 检测 KIT D816V 是诊断、疾病监测和评估 SM 患者预后的有用补充工具。
