title: International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.
abstract: This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.
authors.affiliation: Molecular and Cell Biology Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Alexandra S
authors.identifier: 0000-0002-6240-6642
authors.initials: AS
authors.lastname: Whale
authors.affiliation: Molecular and Cell Biology Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Alison S
authors.initials: AS
authors.lastname: Devonshire
authors.affiliation: Digital Biology Center, Bio-Rad Laboratories , 5731 West Las Positas, Pleasanton, California 94588, United States.
authors.forename: George
authors.initials: G
authors.lastname: Karlin-Neumann
authors.affiliation: Digital Biology Center, Bio-Rad Laboratories , 5731 West Las Positas, Pleasanton, California 94588, United States.
authors.forename: Jack
authors.initials: J
authors.lastname: Regan
authors.affiliation: Digital Biology Center, Bio-Rad Laboratories , 5731 West Las Positas, Pleasanton, California 94588, United States.
authors.forename: Leanne
authors.initials: L
authors.lastname: Javier
authors.affiliation: Statistics Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Simon
authors.initials: S
authors.lastname: Cowen
authors.affiliation: Molecular and Cell Biology Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Ana
authors.initials: A
authors.lastname: Fernandez-Gonzalez
authors.affiliation: Molecular and Cell Biology Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Gerwyn M
authors.initials: GM
authors.lastname: Jones
authors.affiliation: Molecular and Cell Biology Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Redshaw
authors.affiliation: Chronix Biomedical , Goetheallee 8, 37073 Goettingen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Beck
authors.affiliation: Department of Internal Medicine I, Ulm University , Albert-Einstein-Allee 23, 89081, Ulm, Germany.
authors.forename: Andreas W
authors.initials: AW
authors.lastname: Berger
authors.affiliation: Laboratoire de Recherche Translationnelle, Centre Léon-Bérard , Lyon, F-69008, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Combaret
authors.affiliation: Sjællands Universitetshospital , Klinisk Biokemisk Afdeling, Molekylærenhed, Sygehusvej 10, 4000 Roskilde, Denmark.
authors.forename: Nina
authors.initials: N
authors.lastname: Dahl Kjersgaard
authors.affiliation: Genoptix Inc. , 1811 Aston Avenue, Carlsbad, California 92008, United States.
authors.forename: Lisa
authors.initials: L
authors.lastname: Davis
authors.affiliation: Service de Transfert d'Oncologie Biologique, Laboratoire de Biologie Médicale, Faculte de médecine Nord , Boulevard Pierre Dramard, Marseille 13916 cedex 20, France.
authors.forename: Frederic
authors.initials: F
authors.lastname: Fina
authors.affiliation: UCL Cancer Institute , Paul O'Gorman Building, 72 Huntley Street, London WC1E 6DD, United Kingdom.
authors.forename: Tim
authors.initials: T
authors.lastname: Forshew
authors.affiliation: Department of Clinical Immunology and Biochemistry, Vejle Hospital , Kabbeltoft 25, 7100 Vejle, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Fredslund Andersen
authors.affiliation: Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute , via Olgettina 60, 20132 Milano, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galbiati
authors.affiliation: Department of Pediatrics and CIMA LAB Diagnostics, Clínica Universidad de Navarra , Avenida Pío XII 36, 31008 Pamplona, Spain.
authors.forename: Álvaro
authors.initials: Á
authors.lastname: González Hernández
authors.affiliation: Michael Smith Laboratories, University of British Columbia , 301 Michael Smith Building, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Haynes
authors.affiliation: The University of Texas MD Anderson Cancer Center , Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), 1515 Holcombe Boulevard 0455, Houston, Texas 77030, United States.
authors.forename: Filip
authors.initials: F
authors.lastname: Janku
authors.affiliation: Solid Tumours Genomics Unit, Tenon Hospital, APHP and Université Pierre et Marie Curie , 4 rue de la Chine, 75970 Paris, France.
authors.forename: Roger
authors.initials: R
authors.lastname: Lacave
authors.affiliation: Johns Hopkins , 1650 Orleans St., Baltimore, Maryland 21287, United States.
authors.forename: Justin
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Cancer Studies, University of Leicester , Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom.
authors.forename: Vilas
authors.initials: V
authors.lastname: Mistry
authors.affiliation: Lung Cancer Group, Division of Molecular Pathology, The Institute of Cancer Research , 123 Old Brompton Road, London SW7 3RP, United Kingdom.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Pender
authors.affiliation: Institut Claudius Regaud - IUCTO , Laboratoire de Biologie Medicale Oncologique, 1 avenue Irène Joliot-Curie, Toulouse 31059 cedex 9, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Pradines
authors.affiliation: Institut Curie, PSL Research University, SiRIC , Laboratory of Circulating Tumor Biomarkers, 26 rue d'Ulm, 75005 Paris, France.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Proudhon
authors.affiliation: Department of Oncology and Pathology, Lund University , Scheelevägen 2, MV 404-B2, SE-22381, Lund, Sweden.
authors.forename: Lao H
authors.initials: LH
authors.lastname: Saal
authors.affiliation: Department of Pediatrics, Benioff Children's Hospital, UCSF Helen Diller Family Comprehensive Cancer Center , 1450 Third Street, San Francisco, California 94158, United States.
authors.forename: Elliot
authors.identifier: 0000-0001-7032-4623
authors.initials: E
authors.lastname: Stieglitz
authors.affiliation: Dana Farber Cancer Institute , Belfer Center for Applied Cancer Science and Department of Medical Oncology, Boston, Massachusetts 02115, United States.
authors.forename: Bryan
authors.initials: B
authors.lastname: Ulrich
authors.affiliation: Molecular and Cell Biology Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Foy
authors.affiliation: Molecular and Cell Biology Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Parkes
authors.affiliation: Digital Biology Center, Bio-Rad Laboratories , 5731 West Las Positas, Pleasanton, California 94588, United States.
authors.forename: Svilen
authors.initials: S
authors.lastname: Tzonev
authors.affiliation: Molecular and Cell Biology Team, LGC , Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.|School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey , Guildford, Surrey GU2 7XH, United Kingdom.
authors.forename: Jim F
authors.identifier: 0000-0002-0945-1911
authors.initials: JF
authors.lastname: Huggett
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: C117307:KRAS protein, human; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras)