Alexandra S Whale1, Gerwyn M Jones1, Jernej Pavšič2,3, Tanja Dreo2, Nicholas Redshaw1, Sema Akyürek4, Müslüm Akgöz4, Carla Divieto5, Maria Paola Sassi5, Hua-Jun He6, Kenneth D Cole6, Young-Kyung Bae7, Sang-Ryoul Park7, Liesbet Deprez8, Philippe Corbisier8, Sonia Garrigou9, Valérie Taly9, Raquel Larios10, Simon Cowen11, Denise M O'Sullivan1, Claire A Bushell1, Heidi Goenaga-Infante10, Carole A Foy1, Alison J Woolford1, Helen Parkes1, Jim F Huggett12,13, Alison S Devonshire12. 1. Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK. 2. National Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia. 3. Jožef Stefan International Postgraduate School, Ljubljana, Slovenia. 4. TUBITAK National Metrology Institute (TUBITAK UME), Bioanalysis Laboratory, Gebze, Kocaeli, Turkey. 5. INRIM Istituto Nazionale di Ricerca Metrologica, Turin, Italy. 6. Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD. 7. Center for Bioanalysis, KRISS, Yuseong-gu, Daejeon, Republic of Korea. 8. Directorate for Health, Consumers and Reference Materials, Joint Research Centre (JRC), European Commission, Geel, Belgium. 9. INSERM UMR-S1147, CNRS SNC5014, Equipe labellisée Ligue Nationale contre le cancer, Paris Descartes University, Paris, France. 10. Inorganic Analysis Team, LGC, Teddington, Middlesex, UK. 11. Statistics Team, LGC, Teddington, Middlesex, UK. 12. Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK; alison.devonshire@lgcgroup.com j.huggett@surrey.ac.uk. 13. School of Biosciences and Medicine, Faculty of Health and Medical Science, University of Surrey, Guildford, UK.
Abstract
BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.
BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRASG12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.
Authors: Denise M O'Sullivan; Ronan M Doyle; Sasithon Temisak; Nicholas Redshaw; Alexandra S Whale; Grace Logan; Jiabin Huang; Nicole Fischer; Gregory C A Amos; Mark D Preston; Julian R Marchesi; Josef Wagner; Julian Parkhill; Yair Motro; Hubert Denise; Robert D Finn; Kathryn A Harris; Gemma L Kay; Justin O'Grady; Emma Ransom-Jones; Huihai Wu; Emma Laing; David J Studholme; Ernest Diez Benavente; Jody Phelan; Taane G Clark; Jacob Moran-Gilad; Jim F Huggett Journal: Sci Rep Date: 2021-05-19 Impact factor: 4.379
Authors: Gustav Johansson; Daniel Andersson; Stefan Filges; Junrui Li; Andreas Muth; Tony E Godfrey; Anders Ståhlberg Journal: Biomol Detect Quantif Date: 2019-02-13
Authors: Mojca Milavec; Megan H Cleveland; Young-Kyung Bae; Robert I Wielgosz; Maxim Vonsky; Jim F Huggett Journal: Anal Bioanal Chem Date: 2021-11-04 Impact factor: 4.142
Authors: Ana Fernandez-Gonzalez; Simon Cowen; Juhyun Kim; Carole A Foy; Jose Jimenez; Jim F Huggett; Alexandra S Whale Journal: Anal Chem Date: 2022-03-31 Impact factor: 6.986