Université Paris Sorbonne Cité, INSERM UMR-S775, Centre Universitaire des Saints-Pères, Paris, France;
Clin Chem. 2013 Dec;59(12):1722-31. doi: 10.1373/clinchem.2013.206359. Epub 2013 Aug 12.
Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample.
We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR.
Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected.
This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.
多重数字 PCR（dPCR）能够非侵入性且灵敏地检测循环肿瘤 DNA，其性能是当前分子检测方法无法实现的。此外，皮升级数字 PCR 有助于从同一样本中同时筛选多种突变。
我们研究了多重 dPCR 用于从转移性结直肠癌患者的血浆样本中筛选 KRAS（Kirsten 大鼠肉瘤病毒致癌基因同源物）致癌基因 12 和 13 密码子中 7 种最常见突变的效用。对 50 份来自患者的血浆样本进行了检测，这些患者的原发性肿瘤活检组织 DNA 已通过定量 PCR 进行了特征描述。
肿瘤特征表明，19 例患者肿瘤存在 KRAS 突变。对这些样本的血浆 DNA 进行多重 dPCR 分析，鉴定出 14 个与肿瘤中鉴定出的突变相匹配的样本，1 个样本包含不同的 KRAS 突变，4 个样本未检测到突变。在 KRAS 野生型肿瘤样本中，有 2 个 KRAS 突变在相应的血浆样本中被鉴定出来。还使用双 dPCR（即野生型和单突变检测）分析了 KRAS 突变肿瘤患者和预期含有 BRAF（v-raf 鼠肉瘤病毒致癌基因同源物 B）V600E 突变的 5 个血浆样本。双分析结果与 KRAS 突变样本的多重分析结果相匹配，并且由于其更高的灵敏度，能够检测到 2 个具有低水平 KRAS 突变 DNA 的额外样本。所有 5 个带有 BRAF 突变的样本均被检测到。
这项工作证明了多重 dPCR 在临床上可用于同时筛选多种突变，其灵敏度足以检测非侵入性血液采集获得的循环 DNA 中的突变。
