title: Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.
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.
authors.affiliation: Oncology Biomarker Development, Genentech Inc., South San Francisco, California, United States of America. rajeshdp@gene.com
authors.forename: Rajesh
authors.initials: R
authors.lastname: Patel
authors.forename: Alison
authors.initials: A
authors.lastname: Tsan
authors.forename: Rachel
authors.initials: R
authors.lastname: Tam
authors.forename: Rupal
authors.initials: R
authors.lastname: Desai
authors.forename: Jill
authors.initials: J
authors.lastname: Spoerke
authors.forename: Nancy
authors.initials: N
authors.lastname: Schoenbrunner
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Myers
authors.forename: Keith
authors.initials: K
authors.lastname: Bauer
authors.forename: Edward
authors.initials: E
authors.lastname: Smith
authors.forename: Rajiv
authors.initials: R
authors.lastname: Raja
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D005557:Formaldehyde / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D016612:Paraffin Embedding; D010957:Plasmids / Q000235:genetics; D057285:Precision Medicine; D011379:Prognosis; D015203:Reproducibility of Results; D016707:Tissue Fixation
chemical_list: D015415:Biomarkers; D005557:Formaldehyde