Next generation MUT-MAP, a high-sensitivity high-throughput microfluidics chip-based mutation analysis panel

PLoS One. 2014 Mar 21;9(3):e90761. doi: 10.1371/journal.pone.0090761. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Class I Phosphatidylinositol 3-Kinases
  • DNA Mutational Analysis*
  • ErbB Receptors / genetics
  • GTP Phosphohydrolases / genetics
  • Membrane Proteins / genetics
  • Microfluidics / methods*
  • Neoplasms / genetics
  • Phosphatidylinositol 3-Kinases / genetics
  • Polymerase Chain Reaction
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins B-raf / genetics
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-kit / genetics
  • Proto-Oncogene Proteins c-met / genetics
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Receptor, Fibroblast Growth Factor, Type 3 / genetics
  • Reproducibility of Results
  • fms-Like Tyrosine Kinase 3 / genetics
  • ras Proteins / genetics

Substances

  • KRAS protein, human
  • Membrane Proteins
  • Proto-Oncogene Proteins
  • Class I Phosphatidylinositol 3-Kinases
  • PIK3CA protein, human
  • EGFR protein, human
  • ErbB Receptors
  • FGFR3 protein, human
  • FLT3 protein, human
  • MET protein, human
  • Proto-Oncogene Proteins c-kit
  • Proto-Oncogene Proteins c-met
  • Receptor, Fibroblast Growth Factor, Type 3
  • fms-Like Tyrosine Kinase 3
  • AKT1 protein, human
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins c-akt
  • GTP Phosphohydrolases
  • NRAS protein, human
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins

Grant support

This study was funded by Genentech, Inc. The funders were responsible for the study design, data collection and analysis, decision to publish, and preparation of the manuscript.