title: Next generation MUT-MAP, a high-sensitivity high-throughput microfluidics chip-based mutation analysis panel.
abstract: Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multi-analyte panel (MUT-MAP), a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR (AS-PCR) and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development.
authors.affiliation: Oncology Biomarker Development, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Erica B
authors.initials: EB
authors.lastname: Schleifman
authors.affiliation: Oncology Biomarker Development, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Rachel
authors.initials: R
authors.lastname: Tam
authors.affiliation: Oncology Biomarker Development, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Patel
authors.affiliation: Chemistry and Innovation Technology, Pleasanton, California, United States of America.
authors.forename: Alison
authors.initials: A
authors.lastname: Tsan
authors.affiliation: Oncology Biomarker Development, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Teiko
authors.initials: T
authors.lastname: Sumiyoshi
authors.affiliation: Oncology Biomarker Development, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Ling
authors.initials: L
authors.lastname: Fu
authors.affiliation: Oncology Biomarker Development, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Rupal
authors.initials: R
authors.lastname: Desai
authors.affiliation: Chemistry and Innovation Technology, Pleasanton, California, United States of America.
authors.forename: Nancy
authors.initials: N
authors.lastname: Schoenbrunner
authors.affiliation: Program in Core Research, Roche Molecular Systems Inc., Pleasanton, California, United States of America.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Myers
authors.affiliation: Program in Core Research, Roche Molecular Systems Inc., Pleasanton, California, United States of America.
authors.forename: Keith
authors.initials: K
authors.lastname: Bauer
authors.affiliation: Program in Core Research, Roche Molecular Systems Inc., Pleasanton, California, United States of America.
authors.forename: Edward
authors.initials: E
authors.lastname: Smith
authors.affiliation: Oncology Biomarker Development, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Rajiv
authors.initials: R
authors.lastname: Raja
mesh_terms: D058534:Class I Phosphatidylinositol 3-Kinases; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D044085:Microfluidics / Q000379:methods*; D009369:Neoplasms / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics; D015203:Reproducibility of Results; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics; D018631:ras Proteins / Q000235:genetics
chemical_list: D058534:Class I Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D020558:GTP Phosphohydrolases; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D011518:Proto-Oncogene Proteins; D048493:Proto-Oncogene Proteins B-raf; D051057:Proto-Oncogene Proteins c-akt; D019009:Proto-Oncogene Proteins c-kit; D019859:Proto-Oncogene Proteins c-met; D016283:Proto-Oncogene Proteins p21(ras); D051498:Receptor, Fibroblast Growth Factor, Type 3; D051941:fms-Like Tyrosine Kinase 3; D018631:ras Proteins; C117307:KRAS protein, human; C484760:PIK3CA protein, human; C512478:EGFR protein, human; C496365:FGFR3 protein, human; C497970:FLT3 protein, human; C491743:MET protein, human; C494918:AKT1 protein, human; C482119:BRAF protein, human; C579846:NRAS protein, human; C501009:HRAS protein, human