Multiplex Picodroplet Digital PCR to Detect KRAS Mutations in Circulating DNA from the Plasma of Colorectal Cancer Patients
01 Dec 2013-Clinical Chemistry (American Association for Clinical Chemistry)-Vol. 59, Iss: 12, pp 1722-1731
TL;DR: 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 is demonstrated.
Abstract: BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
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Katholieke Universiteit Leuven1, University of Valencia2, Radboud University Nijmegen3, Sheba Medical Center4, University of Turin5, Hospital Clínico San Carlos6, Université Paris-Saclay7, University of Pisa8, Mayo Clinic9, University of São Paulo10, The Chinese University of Hong Kong11, University of Oxford12, Helsinki University Central Hospital13, University of Helsinki14, Institute of Cancer Research15, Bank of Cyprus16, University of Ioannina17, Odense University Hospital18, University of Amsterdam19, Otto-von-Guericke University Magdeburg20, Geneva College21, Medical University of Vienna22, Martin Luther University of Halle-Wittenberg23, Hebron University24, Imperial College Healthcare25
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References
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28 Jul 2005
TL;DR: PfPMP1）与感染红细胞、树突状组胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作�ly.
Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。
18,940 citations
TL;DR: Findings are discussed with a specific focus on the clinical utility of cell-free nucleic acids as blood biomarkers for cancer screening, prognosis and monitoring of the efficacy of anticancer therapies.
Abstract: 1described the presence of cell-free nucleic acid (cfNA) in human blood for the first time. This attracted little attention in the scientific community and it was not until 1994 that the importance of cfNA was recognized as a result of the detection of mutated RAS gene fragments in the blood of cancer patients 2,3 (TIMELINE). In 1996, microsatellite alterations on cell-free DNA (cfDNA) were shown in cancer patients 4
2,427 citations
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Abstract: Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associat...
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