How-Kit Alexandre, Lebbé Céleste, Bousard Aurélie, Daunay Antoine, Mazaleyrat Nicolas, Daviaud Christian, Mourah Samia, Tost Jörg
Laboratory for Functional Genomics, Fondation Jean Dausset - CEPH, 75010, Paris, France.
Anal Bioanal Chem. 2014 Sep;406(22):5513-20. doi: 10.1007/s00216-014-7975-5. Epub 2014 Jun 27.
A number of molecular diagnostic methods have been developed for the detection and identification of mutations in tumor samples, which are important for the choice of treatment in the context of personalized medicine. For the treatment of metastatic melanoma, Vemurafenib is recommended for patients with BRAF V600 activating mutations. However, the different assays developed to date for the detection of these mutations lack sensitivity or specificity or do not allow a sequencing-based identification or validation of the mutation. Recently, enhanced improved and complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) has been developed as a sensitive method for the detection and identification of mutations in KRAS codons 12/13. Here, we present the first E-ice-COLD-PCR assay for the detection and identification of BRAF codon 600 mutations, which has a large dynamic range, as 25 pg to 25 ng can be used as DNA input without any reduction in mutation enrichment efficiency, and which can detect down to 0.01 % of mutated alleles in a wild-type background. The assay has been validated on fresh frozen, formalin-fixed paraffin-embedded (FFPE), and plasma samples of melanoma patients and has allowed the detection and identification of BRAF mutations present in samples appearing as wild type using standard pyrosequencing, endpoint genotyping, or Sanger sequencing. Thus, the BRAF V600 E-ice-COLD-PCR assay is currently one of the most powerful molecular diagnostic tools for the ultrasensitive detection and identification of BRAF codon 600 mutations.
已经开发出多种分子诊断方法用于检测和鉴定肿瘤样本中的突变，这对于个性化医疗背景下的治疗选择非常重要。对于转移性黑色素瘤的治疗，推荐将维莫非尼用于具有BRAF V600激活突变的患者。然而，迄今为止开发的用于检测这些突变的不同检测方法缺乏敏感性或特异性，或者不允许基于测序的突变鉴定或验证。最近，已经开发出低温变性温度下增强改进的完全富集共扩增聚合酶链反应（E-ice-COLD-PCR）作为检测和鉴定KRAS密码子12/13突变的灵敏方法。在此，我们展示了首个用于检测和鉴定BRAF密码子600突变的E-ice-COLD-PCR检测方法，该方法具有很大的动态范围，因为25 pg至25 ng的DNA输入量都可使用，且突变富集效率不会降低，并且在野生型背景下能够检测低至0.01%的突变等位基因。该检测方法已在黑色素瘤患者的新鲜冷冻、福尔马林固定石蜡包埋（FFPE）和血浆样本上得到验证，并且能够使用标准焦磷酸测序、终点基因分型或桑格测序检测和鉴定在标准检测中显示为野生型的样本中存在的BRAF突变。因此，BRAF V600 E-ice-COLD-PCR检测方法目前是用于超灵敏检测和鉴定BRAF密码子600突变的最强大的分子诊断工具之一。
