title: Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.
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.
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Zonta
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.
authors.forename: Fanny
authors.initials: F
authors.lastname: Garlan
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.|Medical oncology, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique Hôpitaux de Paris (AP-HP), Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Pécuchet
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.
authors.forename: Karla
authors.initials: K
authors.lastname: Perez-Toralla
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.
authors.forename: Ouriel
authors.initials: O
authors.lastname: Caen
authors.affiliation: RainDance Technologies, Billerica, Massachusetts, United States of America.
authors.forename: Coren
authors.initials: C
authors.lastname: Milbury
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Didelot
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.|Medical oncology, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique Hôpitaux de Paris (AP-HP), Paris, France.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Fabre
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.|Department of Biochemistry, Unit of pharmacogenetic and molecular oncology, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique Hôpitaux de Paris (AP-HP), Paris, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Blons
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.|Department of Biochemistry, Unit of pharmacogenetic and molecular oncology, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique Hôpitaux de Paris (AP-HP), Paris, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Laurent-Puig
authors.affiliation: Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Taly
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C512478:EGFR protein, human; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras)