Department of Anatomical Pathology, Labtests Auckland Limited, New Zealand.
Department of Anatomical Pathology, Labtests Auckland Limited, New Zealand.
Pathology. 2017 Dec;49(7):776-783. doi: 10.1016/j.pathol.2017.08.011.
Melanoma patients with BRAF V600E and V600K mutations show complete or partial response to vemurafenib. Detection assays often scan for the common V600E mutation rather than the rare V600K variant, although this mutation can be found in a high proportion of melanoma patients in the South Pacific. Herein, we describe a BRAF high resolution melting (HRM) assay that can differentiate low level of V600E and V600K mutations using formalin fixed, paraffin embedded (FFPE) reference standards for assay validation. The assay is based on the competitive amplification of differentially melting amplicons (CADMA principle) and has a limit of detection of 0.8% mutant allele for V600K and 1.4% mutant allele for V600E. A differentiation between the two mutations based on the melting profile is possible even at low mutation level. Sixty FFPE specimens were scanned and mutations could be scored correctly as confirmed by castPCR. In summary, the developed HRM assay is suitable for detection of V600K and V600E mutations and proved to be reliable and cost effective in a diagnostic environment.
携带 BRAF V600E 和 V600K 突变的黑色素瘤患者对威罗菲尼有完全或部分反应。检测方法通常会扫描常见的 V600E 突变，而不是罕见的 V600K 变体，尽管这种突变在南太平洋的很大一部分黑色素瘤患者中都能找到。在此，我们描述了一种 BRAF 高分辨率熔解（HRM）检测方法，该方法可以使用福尔马林固定、石蜡包埋（FFPE）参考标准来验证检测，区分低水平的 V600E 和 V600K 突变。该检测方法基于差异化熔解扩增子的竞争扩增（CADMA 原理），对 V600K 的检测限为 0.8%突变等位基因，对 V600E 的检测限为 1.4%突变等位基因。即使在低突变水平下，基于熔解曲线的两种突变之间的区分也是可能的。对 60 个 FFPE 标本进行了扫描，突变可以通过 castPCR 正确评分，证实为阳性。总之，开发的 HRM 检测方法适用于 V600K 和 V600E 突变的检测，并在诊断环境中被证明是可靠且具有成本效益的。
