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首页> 美国卫生研究院文献>American Journal of Translational Research >Droplet digital PCR for detection of BRAF V600E mutation in formalin-fixed paraffin-embedded melanoma tissues: a comparison with Cobas® 4800 Sanger sequencing and allele-specific PCR
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Droplet digital PCR for detection of BRAF V600E mutation in formalin-fixed paraffin-embedded melanoma tissues: a comparison with Cobas® 4800 Sanger sequencing and allele-specific PCR

机译：用于在福尔马林固定石蜡包埋的黑色素瘤组织中检测BRAF V600E突变的液滴数字PCR：与Cobas®4800Sanger测序和等位基因特异性PCR的比较

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Cutaneous melanoma has the worst prognosis of all skin cancers. Although emerging targeted therapies, such as B-Raf kinase inhibitor vemurafenib, improve prognosis they require an accurate and sensitive means of detecting the pathogenic BRAF V600E mutation. We compared the sensitivity of four BRAF V600E detection methods in formalin-fixed, paraffin-embedded melanoma biopsies from 87 consecutive melanoma patients with Breslow stage I-V disease (staging based on the depth of tumor of invasion). The methods assessed were the widely used Cobas® 4800 system based on real-time PCR amplification, Sanger sequencing, allele-specific PCR (AS-PCR), and droplet digital PCR (ddPCR). The BRAF V600E mutation was found in 8 (9.2%), 23 (26.4%), 23 (26.4%) and 31 (35.6%) biopsies, respectively. The limit of detection (LoD) was determined by three different methods: Poisson confidence limits, calibration regression and Tzonev’s method. Pair-wise agreement between the methods was as follows: Cobas vs. Sanger, P = 0.33; Cobas® 4800 vs. AS-PCR, P = 0.33; Cobas® 4800 vs. ddPCR, P = 0.65; Sanger vs. AS-PCR, P = 1; Sanger vs. ddPCR, P = 0.08; AS-PCR vs. ddPCR, P = 0.06. Multinomial logistic regression was used for predictive modeling of the Breslow-Clark score; ddPCR emerged as the best predictor, the other predictors were mitotic activity, type of malignant melanoma and patient’s age. Our results demonstrate that ddPCR is the most sensitive method of detecting the BRAF V600E mutation.
机译：皮肤黑色素瘤在所有皮肤癌中预后最差。尽管新兴的靶向疗法（例如B-Raf激酶抑制剂vemurafenib）改善了预后，但仍需要一种准确而灵敏的手段来检测病原性BRAF V600E突变。我们比较了87例连续Breslow I-V期黑色素瘤患者的福尔马林固定石蜡包埋的黑色素瘤活检中四种BRAF V600E检测方法的敏感性（分期根据浸润深度）。评估的方法是基于实时PCR扩增，Sanger测序，等位基因特异性PCR（AS-PCR）和液滴数字PCR（ddPCR）的广泛使用的Cobas ® 4800系统。分别在8（9.2％），23（26.4％），23（26.4％）和31（35.6％）的活检组织中发现了BRAF V600E突变。检测限（LoD）由三种不同的方法确定：泊松置信限，校准回归和Tzonev方法。方法之间的成对一致性如下：Cobas vs. Sanger，P = 0.33； Cobas ® 4800与AS-PCR相比，P = 0.33； Cobas ® 4800对ddPCR，P = 0.65； Sanger vs.AS-PCR，P = 1； Sanger vs.ddPCR，P = 0.08； AS-PCR与ddPCR相比，P = 0.06。多项逻辑回归用于Breslow-Clark得分的预测模型； ddPCR成为最好的预测因子，其他预测因子是有丝分裂活性，恶性黑色素瘤类型和患者年龄。我们的结果表明，ddPCR是检测BRAF V600E突变的最灵敏方法。

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