Quantitative analysis of the BRAF V600E mutation in circulating tumor-derived DNA in melanoma patients using competitive allele-specific TaqMan PCR
Quantitative analysis of the BRAF V600E mutation in circulating tumor-derived DNA in melanoma patients using competitive allele-specific TaqMan PCR
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DOI：
10.1007/s10147-016-0976-y
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发表时间：
2016-10-01
影响因子：
3.3
通讯作者：
Okuyama, Ryuhei
中科院分区：
文献类型：
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作者：
Ashida, Atsuko;Sakaizawa, Kaori;Okuyama, Ryuhei
BRAF（V600E）是黑色素瘤中常见的突变，BRAF抑制剂可有效治疗BRAF突变阳性黑色素瘤。携带这种突变的 DNA 从黑色素瘤细胞中释放到循环系统中。因此，外周血中循环的肿瘤衍生 DNA (ctDNA) 代表了一种用于评估癌症患者肿瘤特征的新型生物标志物。然而，ctDNA 在外周血中的含量非常低，这使得检测该 DNA 中的特定突变成为一项挑战。竞争性等位基因特异性 TaqMan PCR (castPCR) 是一种简单的市售检测方法，是一种用于定量少量 DNA 的灵敏技术。通过castPCR 对来自 6 名黑色素瘤患者的 26 个连续血浆样本中的 BRAF (V600E) ctDNA 水平进行定量。castPCR 检测使用 BRAF (V600E) DNA 和 BRAF（野生）DNA 的混合物进行，结果发现能够检测 BRAF (V600E)，在 2 至 10 ng 基因组 DNA 样本中，aeyen0.5 % 的分数丰度。然后从六名携带 BRAF (V600E) 突变的黑色素瘤患者的外周血样本中提取游离 DNA。在三名患者中检测到 BRAF (V600E) ctDNA，其丰度占总 BRAF 无细胞 DNA 的 1.28% 至 58.0%。通过计算机断层扫描成像确定，BRAF (V600E) ctDNA 的丰度与肿瘤负荷相关。在两个病例中，BRAF (V600E) ctDNA 水平先于临床症状恶化而增加。castPCR 检测可以检测和定量含有大量 BRAF（野生）无细胞 DNA 的样品中的少量 BRAF (V600E) ctDNA。因此，我们建议castPCR检测适用于监测黑色素瘤患者血浆中的ctDNA。
BRAF (V600E) is a common mutation in melanoma, and BRAF inhibitors are effective in treating of BRAF mutation-positive melanoma. DNA carrying this mutation is released from melanoma cells into the circulation. As such, circulating tumor-derived DNA (ctDNA) in peripheral blood represents a novel biomarker for evaluating tumor features in cancer patients. However, ctDNA is present in the peripheral blood at very low levels, which makes the detection of specific mutations in this DNA a challenge. Competitive allele-specific TaqMan PCR (castPCR), a straightforward commercially available assay, is a sensitive technique for quantitating a small amount of DNA.The level of BRAF (V600E) ctDNA was quantified by castPCR in 26 consecutive plasma samples from six melanoma patients.The castPCR assay was performed using a mixture of BRAF (V600E) DNA and BRAF (wild) DNA and found to be able to detect BRAF (V600E) at a fractional abundance of aeyen0.5 % in 2- to 10-ng samples of genomic DNA. Cell-free DNA was then extracted from peripheral blood samples collected from six patients with melanoma harboring the BRAF (V600E) mutation. BRAF (V600E) ctDNA was detected in three patients, at a fractional abundance of between 1.28 and 58.0 % of total BRAF cell-free DNA. The abundance of BRAF (V600E) ctDNA correlated with tumor burden, as determined by computed tomography imaging. In two cases, an increase in the level of BRAF (V600E) ctDNA preceded exacerbation of clinical symptoms.The castPCR assay can detect and quantitate small amounts of BRAF (V600E) ctDNA in samples containing large amounts of BRAF (wild) cell-free DNA. Thus, we suggest that the castPCR assay is suitable for monitoring ctDNA in the plasma of melanoma patients.