International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant

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 thera...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2017-02, Vol.89 (3), p.1724-1733
Main Authors: Whale, Alexandra S, Devonshire, Alison S, Karlin-Neumann, George, Regan, Jack, Javier, Leanne, Cowen, Simon, Fernandez-Gonzalez, Ana, Jones, Gerwyn M, Redshaw, Nicholas, Beck, Julia, Berger, Andreas W, Combaret, Valérie, Dahl Kjersgaard, Nina, Davis, Lisa, Fina, Frederic, Forshew, Tim, Fredslund Andersen, Rikke, Galbiati, Silvia, González Hernández, Álvaro, Haynes, Charles A, Janku, Filip, Lacave, Roger, Lee, Justin, Mistry, Vilas, Pender, Alexandra, Pradines, Anne, Proudhon, Charlotte, Saal, Lao H, Stieglitz, Elliot, Ulrich, Bryan, Foy, Carole A, Parkes, Helen, Tzonev, Svilen, Huggett, Jim F
Format: Article
Language:English
Subjects:
Analytical chemistry
Calibration
Cancer
Digital
DNA - chemistry
DNA - metabolism
Genetics
Humans
Laboratories
Markers
Molecules
Polymerase chain reaction
Polymorphism, Single Nucleotide
Proto-Oncogene Proteins p21(ras) - genetics
Real-Time Polymerase Chain Reaction - methods
Reproducibility
Reproducibility of Results
Sequence Analysis, DNA
Stratification
Therapy
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.6b03980