Pisareva Ekaterina, Gutkina Nadezhda, Kovalenko Sergei, Kuehnapfel Sarah, Hartmann Arndt, Heinzerling Lucie, Schneider-Stock Regine, Lyubchenko Lyudmila, Shamanin Vladimir A
aInstitute of Molecular Biology and Biophysics bBioLink Ltd, Novosibirsk cN.N.Blokhin Russian Cancer Research Center, Moscow, Russia dBioron GmbH, Ludwigshafen eInstitute of Pathology fDepartment of Dermatology, University Hospital Erlangen, Erlangen, Germany.
Melanoma Res. 2014 Aug;24(4):322-31. doi: 10.1097/CMR.0000000000000090.
Mutations at BRAF codon V600 are used as predictive biomarkers for targeted therapy of skin melanoma. Here, a simple sensitive test to detect mutations of BRAF-V600 was developed using real-time PCR with allele-specific primers and TaqMan probes. Two versions of the test using sense and antisense allele-specific primers were designed and evaluated. The test detected 1% mutant allele V600E/K in 10 ng DNA standard made from wild-type human DNA spiked with BRAF-V600E or the V600K plasmid. The test was validated on clinical formalin-fixed paraffin-embedded samples of skin melanoma using pyrosequencing as a reference method. In the clinical samples, we detected the common mutation V600E, as well as the rare mutations V600K, V600E2 (codon GAA), V600E2 K601del, V600D-K601del, and V600R. In comparison with pyrosequencing, both versions of the test had 100% specificity with sensitivities of 97 and 86% for sense and antisense allele-specific primers, respectively. Using the PCR test with sense allele-specific primers, mutations in V600 were found in 33 of 51 Russian patients (64.7%) with cutaneous melanoma. This closed-tube real-time PCR test can be used as a simple and sensitive assay for mutations of BRAF-V600 in cutaneous melanoma.
BRAF基因第V600密码子的突变被用作皮肤黑色素瘤靶向治疗的预测生物标志物。在此，我们开发了一种简单灵敏的检测BRAF-V600突变的方法，该方法使用了带有等位基因特异性引物和TaqMan探针的实时PCR技术。我们设计并评估了使用正义和反义等位基因特异性引物的两种测试版本。该测试在由野生型人类DNA掺入BRAF-V600E或V600K质粒制成的10 ng DNA标准品中检测到了1%的突变等位基因V600E/K。我们使用焦磷酸测序作为参考方法，对皮肤黑色素瘤的临床福尔马林固定石蜡包埋样本进行了验证。在临床样本中，我们检测到了常见的V600E突变，以及罕见的V600K、V600E2（密码子GAA）、V600E2 K601del、V600D-K601del和V600R突变。与焦磷酸测序相比，两种测试版本均具有100%的特异性，正义和反义等位基因特异性引物的灵敏度分别为97%和86%。使用带有正义等位基因特异性引物的PCR测试，在51名俄罗斯皮肤黑色素瘤患者中的33名（64.7%）中发现了V600突变。这种闭管实时PCR测试可作为一种简单灵敏的检测皮肤黑色素瘤中BRAF-V600突变的方法。
