Detection of BRAF p.V600E Mutations in Melanomas Comparison of Four Methods Argues for Sequential Use of Immunohistochemistry and Pyrosequencing
Detection of BRAF p.V600E Mutations in Melanomas Comparison of Four Methods Argues for Sequential Use of Immunohistochemistry and Pyrosequencing
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DOI：
10.1016/j.jmoldx.2012.09.001
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发表时间：
2013-01-01
影响因子：
4.1
通讯作者：
Emile, Jean-Francois
中科院分区：
文献类型：
--
作者：
Colomba, Emeline;Helias-Rodzewicz, Zofia;Emile, Jean-Francois
BRAF p.V600突变检测最近成为用维罗非尼治疗转移性黑色素瘤患者的必要条件。本研究比较了BRAF突变的不同检测方法。对来自111名患者的黑色素瘤样本进行了BRAF突变分析，其中89人的结果采用以下四种方法获得：桑格测序、实时PCR、免疫组织化学和焦磷酸测序。所有样品含有至少60%的肿瘤细胞。PCR产物的定向桑格测序未能检测到40例p.V600 E突变病例中的3例（7.5%）（灵敏度，92.5%; 95%CI，78.5%至98.0%）。BRAF p.V600E特异性实时PCR鉴定了40例p.V600E突变病例中的39例（97.6%）（灵敏度，97.5%; 95%CI，87.1%至99.6%）和所有39例野生型（WT）病例，并且令人惊讶地，6/6例p.V600K也为阳性（特异性，87.8%; 95%CI，75.8%至94.3%）。然而，未检测到其他突变，p.V600R（n = 1），p.K601E（n = 2）和p.600_601delinsE（n = 1）。VE 1免疫组化（p.V600E特异性）鉴定了所有p.V600E和WT病例（灵敏度，100%; 95% CI，91.2%至100%），但对所有其他BRAF突变均为阴性。焦磷酸测序成功鉴定了所有WT和突变病例。免疫组织化学对p.V600E具有高度特异性，可用作一线方法，如目前用于HER 2扩增检测的方法。焦磷酸测序被证明是检测黑色素瘤中BRAF突变的最有效方法，并且可以在VE 1阴性或无法解释的病例中进行。（J Mol Diagn 2013，15：94-100; http：//dx.doi.org/10.1016/j.jmoldx.2012.09.001）
BRAF p.V600 mutation detection recently became necessary to treat metastatic melanoma patients with vemurafenib. This study compares different methods of detection of BRAF mutations. Melanoma samples from 111 patients were analyzed for BRAF mutations, and for 89 of them, results were obtained with the four following methods: Sanger sequencing, real-time PCR, immunohistochemistry, and pyrosequencing. All samples contained at least 60% of tumor cells. Directional Sanger sequencing of PCR products failed to detect 3 of 40 p.V600E-mutated cases (7.5%) (sensitivity, 92.5%; 95% CI, 78.5% to 98.0%). BRAF p.V600E-specific real-time PCR identified 39 of 40 p.V600E-mutated cases (97.6%) (sensitivity, 97.5%; 95% CI, 87.1% to 99.6%) and all 39 wild-type (WT) cases and surprisingly was also positive for 6/6 p.V600K (specificity, 87.8%; 95% CI, 75.8% to 94.3%). However, other mutations, p.V600R (n = 1), p.K601E (n = 2), and p.600_601delinsE (n = 1), were not detected. Immunohistochemistry with VE1, specific for p.V600E, identified all p.V600E and WT cases (sensitivity, 100%; 95% CI, 91.2% to 100%) but was negative for all other BRAF mutations. Pyrosequencing successfully identified all WT and mutated cases. Immunohistochemistry is highly specific for p.V600E, and could be used as a first-line method, as is currently performed for HER2 amplification detection. Pyrosequencing proved to be the most efficient method to detect BRAF mutations in melanomas and could be performed on VE1-negative or uninterpretable cases. (J Mol Diagn 2013, 15: 94-100; http://dx.doi.org/10.1016/j.jmoldx.2012.09.001)