title: Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.
abstract: Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.
authors.affiliation: Lung Cancer Group, Division of Molecular Pathology, The Institute of Cancer Research, London, United Kingdom; Department of Medicine, The Royal Marsden NHS Foundation Trust, London, United Kingdom; The Francis Crick Institute, London, United Kingdom.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Pender
authors.affiliation: The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Isaac
authors.initials: I
authors.lastname: Garcia-Murillas
authors.affiliation: Lung Cancer Group, Division of Molecular Pathology, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Sareena
authors.initials: S
authors.lastname: Rana
authors.affiliation: The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Rosalind J
authors.initials: RJ
authors.lastname: Cutts
authors.affiliation: The Francis Crick Institute, London, United Kingdom.
authors.forename: Gavin
authors.initials: G
authors.lastname: Kelly
authors.affiliation: The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Kerry
authors.initials: K
authors.lastname: Fenwick
authors.affiliation: The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Iwanka
authors.initials: I
authors.lastname: Kozarewa
authors.affiliation: The Centre for Molecular Pathology, The Institute of Cancer Research, Sutton, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Gonzalez de Castro
authors.affiliation: Department of Medicine, The Royal Marsden NHS Foundation Trust, London, United Kingdom.
authors.forename: Jaishree
authors.initials: J
authors.lastname: Bhosle
authors.affiliation: Department of Medicine, The Royal Marsden NHS Foundation Trust, London, United Kingdom.
authors.forename: Mary
authors.initials: M
authors.lastname: O'Brien
authors.affiliation: Department of Medicine, The Royal Marsden NHS Foundation Trust, London, United Kingdom; The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, United Kingdom.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Turner
authors.affiliation: Department of Medicine, The Royal Marsden NHS Foundation Trust, London, United Kingdom.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Popat
authors.affiliation: Lung Cancer Group, Division of Molecular Pathology, The Institute of Cancer Research, London, United Kingdom; The Francis Crick Institute, London, United Kingdom.
authors.forename: Julian
authors.initials: J
authors.lastname: Downward
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D045744:Cell Line, Tumor; D002999:Clone Cells; D004273:DNA, Neoplasm / Q000235:genetics; D005557:Formaldehyde; D005787:Gene Frequency / Q000235:genetics; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation / Q000235:genetics*; D016612:Paraffin Embedding; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D013696:Temperature; D016707:Tissue Fixation
chemical_list: D004273:DNA, Neoplasm; C117307:KRAS protein, human; D005557:Formaldehyde; D016283:Proto-Oncogene Proteins p21(ras)