Detection of mutant BRAF alleles in the plasma of patients with metastatic melanoma
Detection of mutant BRAF alleles in the plasma of patients with metastatic melanoma
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DOI：
10.2353/jmoldx.2007.060135
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发表时间：
2007-04-01
影响因子：
4.1
通讯作者：
Polsky, David
中科院分区：
文献类型：
--
作者：
Yancovitz, Molly;Yoon, Joanne;Polsky, David
在40 - 70%的人转移性黑色素瘤组织中报告了BRAF癌基因600位氨基酸的突变，并且已经确定了BRAF在黑色素瘤生物学中的关键作用。对血液隔室进行采样以检测实体瘤的突变状态代表了癌症医学的高度创新性进展，这种方法可能比基于组织的技术更具优势。我们报告的发展，荧光为基础的聚合酶链反应（PCR）检测突变BRAF等位基因，在血浆中。优化突变体特异性PCR测定以特异性扩增突变体BRAF等位基因而不扩增野生型等位基因。进行了将来自BRAF突变黑素瘤细胞系的DNA与野生型人胎盘DNA以不同比例混合的实验，以确定该测定的阈值并将其与常规DNA测序进行比较。然后将该测定法应用于转移性黑素瘤患者的组织和血浆标本。该检测方法检测到0.1 ng突变体DNA混合在100 ng野生型DNA中，灵敏度比DNA测序高500倍。该测定法在26例转移性黑色素瘤患者中的14例（54%）的血浆样本中检测到突变BRAF等位基因。这些数据证明了在转移性黑色素瘤患者中进行BRAF突变血液检测的可行性。
Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles; in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients.