Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department, Institut Curie, PSL Research University, Paris, France.
CNRS UMR144, Institut Curie, PSL Research University, Paris, France.
Clin Chem. 2018 Feb;64(2):317-328. doi: 10.1373/clinchem.2017.272518. Epub 2017 Nov 9.
Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown remains limited.
We established 2 ddPCR assays detecting all genomic alterations within exon 2 and exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan oligoprobes. The assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events.
The and assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different or mutations.
This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.
液体活检领域的进展，结合液滴数字 PCR（ddPCR）的发展，使得能够以高检测准确性对突变进行非侵入性监测。然而，目前的检测方法每次反应只能检测有限数量的突变。ddPCR 是一种公认的检测肿瘤组织中先前特征化的改变的方法，但当其突变未知时，其作为发现工具的用途仍然有限。
我们建立了 2 种 ddPCR 检测方法，使用独特的 TaqMan 寡核苷酸探针，检测结直肠癌和肺癌中具有临床意义的外显子 2 和外显子 19 突变热点中的所有基因组改变。该检测方法可检测密码子 12/13 中最常见的 7 种突变，但也可检测该区域中发现的所有其他突变。该检测方法筛选了外显子 19 所有的框内缺失，这是常见的 EGFR 激活事件。
检测方法高度特异性，在突变等位基因频率<0.1%时均可达到检测极限。我们进一步在多个含有不同或突变的血浆和福尔马林固定石蜡包埋肿瘤样本上验证了它们的性能。
该方法通过单次 ddPCR 反应检测更多突变的优势，同时最大限度地减少患者样本的消耗。这在液体活检方面尤其有用，因为循环肿瘤 DNA 的量通常较低。当肿瘤组织不可用时，该方法可用作发现工具，或在治疗期间监测疾病。
