Abstract
Targeted anticancer therapies rely on the identification of patient subgroups most likely to respond to treatment. Predictive biomarkers play a key role in patient selection, while diagnostic and prognostic biomarkers expand our understanding of tumor biology, suggest treatment combinations, and facilitate discovery of novel drug targets. We have developed a high-throughput microfluidics method for mutation detection (MUT-MAP, mutation multi-analyte panel) based on TaqMan or allele-specific PCR (AS-PCR) assays. We analyzed a set of 71 mutations across six genes of therapeutic interest. The six-gene mutation panel was designed to detect the most common mutations in the EGFR, KRAS, PIK3CA, NRAS, BRAF, and AKT1 oncogenes. The DNA was preamplified using custom-designed primer sets before the TaqMan/AS-PCR assays were carried out using the Biomark microfluidics system (Fluidigm; South San Francisco, CA). A cross-reactivity analysis enabled the generation of a robust automated mutation-calling algorithm which was then validated in a series of 51 cell lines and 33 FFPE clinical samples. All detected mutations were confirmed by other means. Sample input titrations confirmed the assay sensitivity with as little as 2 ng gDNA, and demonstrated excellent inter- and intra-chip reproducibility. Parallel analysis of 92 clinical trial samples was carried out using 2–100 ng genomic DNA (gDNA), allowing the simultaneous detection of multiple mutations. DNA prepared from both fresh frozen and formalin-fixed, paraffin-embedded (FFPE) samples were used, and the analysis was routinely completed in 2–3 days: traditional assays require 0.5–1 µg high-quality DNA, and take significantly longer to analyze. This assay can detect a wide range of mutations in therapeutically relevant genes from very small amounts of sample DNA. As such, the mutation assay developed is a valuable tool for high-throughput biomarker discovery and validation in personalized medicine and cancer drug development.
Highlights
Biomarkers have assumed a central role in oncology, enabling the detection, characterization, and targeted treatment of a range of cancer types [1]
We have developed a high-throughput method for mutation detection (MUT-MAP, mutation multi-analyte panel) based on TaqMan and allele-specific PCR (AS-PCR) assays using a microfluidic chip-based technology
The AS-PCR assays incorporate the use of an engineered Thermus species Z05 DNA polymerase (AS1) and, in some cases, covalently modified primers to enhance the specificity of allelespecific quantitative PCR (qPCR) [19,20]
Summary
Biomarkers have assumed a central role in oncology, enabling the detection, characterization, and targeted treatment of a range of cancer types [1]. The detection and validation of tumor biomarkers is critical for the ongoing development of personalized healthcare, both through the support of effective and robust drug trials, and the effective employment of targeted therapies in the clinic [2]. Predictive biomarkers allow the response to a particular line of treatment to be anticipated, based on the known mode of action of the chosen therapy and an understanding of the underlying tumor biology. Prognostic biomarkers enable the prediction of disease progression in the absence of treatment, and have been used to identify signaling pathways that are potential drivers of disease, and putative drug targets [3]
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