Dinges Hanns C, Capper David, Ritz Olga, Brüderlein Silke, Marienfeld Ralf, von Deimling Andreas, Möller Peter, Lennerz Jochen K
*Division of Molecular Diagnostics, Institute of Pathology, University Ulm, Ulm †Department of Neuropathology and Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Institute of Pathology, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany.
Appl Immunohistochem Mol Morphol. 2015 May-Jun;23(5):382-8. doi: 10.1097/PAI.0000000000000092.
Detection of BRAF V600E has diagnostic, prognostic, and therapeutic relevance. The recently developed BRAF V600E mutation-specific antibody has evolved into a feasible alternative to DNA analysis. The plethora of immunohistochemical protocols makes implementation tedious and, here we tested a set of manual and automated protocols and compared test performance with sequencing results. For assays, we employed formalin-fixed, in part decalcified, and paraffin-embedded tissue samples. Empiric testing of manual protocols included 10 variables in 17 protocols. Automated immunohistochemical staining and BRAF pyrosequencing served as independent test methods. Test performance measures were compared without considering 1 method as a standard. Four well-fixed samples (2WT/2Mut) were used for testing of all protocols and indicated 2 correctly classifying procedures. Practical performance assessment employed 33 independent tissue samples, composed of 27 leukemias (by pyrosequencing: 8 wild-type; 18 mutated; 1 noninformative) and 6 melanomas (V600E; V600K; wild-type, 2 each). Manual V600E staining was positive in 20 cases (19 of 20 V600E-containing samples plus the 1 sample that was noninformative), whereas all wild-type and V600K cases were immunonegative. Manual or automated staining as well as pyrosequencing would have missed an equal number of V600E-mutated cases and the correlation coefficient for these methods was 0.75 to 0.93 (substantial to almost perfect); the Youden index was 0.95. Detection of V600E-mutated BRAF at the protein level in routine and decalcified tissue samples is possible, and the presented manual protocols should expedite implementation in routine diagnostic practice. Our results indicate that both molecular techniques should be considered complementary.
BRAF V600E的检测具有诊断、预后和治疗意义。最近开发的BRAF V600E突变特异性抗体已发展成为DNA分析的一种可行替代方法。大量的免疫组织化学方案使得实施过程繁琐，在此我们测试了一组手动和自动方案，并将测试性能与测序结果进行了比较。对于检测，我们使用了福尔马林固定、部分脱钙和石蜡包埋的组织样本。手动方案的经验测试包括17种方案中的10个变量。自动免疫组织化学染色和BRAF焦磷酸测序作为独立的测试方法。在不将一种方法作为标准的情况下比较测试性能指标。使用四个固定良好的样本（2个野生型/2个突变型）对所有方案进行测试，结果表明有两种方案分类正确。实际性能评估使用了33个独立的组织样本，包括27例白血病（通过焦磷酸测序：8例野生型；18例突变型；1例无信息）和6例黑色素瘤（V600E；V600K；野生型，各2例）。手动V600E染色在20例中呈阳性（20例含V600E样本中的19例加上1例无信息样本），而所有野生型和V600K病例均为免疫阴性。手动或自动染色以及焦磷酸测序遗漏的V600E突变病例数量相同，这些方法的相关系数为0.75至0.93（从显著到几乎完美）；约登指数为0.95。在常规和脱钙组织样本中检测蛋白质水平的V600E突变BRAF是可行的，所提供的手动方案应能加快在常规诊断实践中的实施。我们的结果表明，这两种分子技术应被视为互补的。
