Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

PLoS One. 2016 Jul 14;11(7):e0159094. doi: 10.1371/journal.pone.0159094. eCollection 2016.

Abstract

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

MeSH terms

  • DNA Mutational Analysis / methods
  • DNA, Neoplasm / genetics
  • ErbB Receptors / genetics*
  • Humans
  • Lung Neoplasms / genetics
  • Lung Neoplasms / pathology
  • Multiplex Polymerase Chain Reaction / methods*
  • Mutation
  • Proto-Oncogene Proteins p21(ras) / genetics*
  • Sensitivity and Specificity
  • Tumor Suppressor Protein p53 / genetics*

Substances

  • DNA, Neoplasm
  • KRAS protein, human
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • EGFR protein, human
  • ErbB Receptors
  • Proto-Oncogene Proteins p21(ras)

Grants and funding

This work was supported by the Ministère de l’Enseignement Supérieur et de la Recherche, the Université Paris-Descartes, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Institut National du Cancer (INCA, no. 2009-1-RT-03-US-1 and 2009-RT-03-UP5-1), the Agence Nationale de la Recherche (ANR Nanobiotechnologies; no. ANR-10-NANO-0002-09), the SIRIC CARPEM and canceropole funding (no. 2011-1-LABEL-UP5-2). Eleonora Zonta receives salary through the INSERM Physicancer program (no. PC201423). Fanny Garlan thanks the Fondation Servier for a fellowship within the Frontiers in Life Science PhD program (FdV). Karla Perez-Toralla receives salary through the Merieux Institution (Merieux Institute Advanced research Grant to VT/PLP). Ouriel Caen receives salary through the the Association pour la recherche contre le cancer (ARC, no. SL220100601375). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Coren Milbury is employed by RainDance Technologies. RainDance Technologies provided support in the form of salary for author CM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section.