Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.
View/ Open
Date
2015-09-28Author
Pender, A
Garcia-Murillas, I
Rana, S
Cutts, RJ
Kelly, G
Fenwick, K
Kozarewa, I
Gonzalez de Castro, D
Bhosle, J
O'Brien, M
Turner, NC
Popat, S
Downward, J
Type
Journal Article
Metadata
Show full item recordAbstract
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.
Subject
Cell Line, Tumor
Clone Cells
Humans
Carcinoma, Non-Small-Cell Lung
Lung Neoplasms
Formaldehyde
DNA, Neoplasm
Paraffin Embedding
Tissue Fixation
Temperature
Gene Frequency
Mutation
Proto-Oncogene Proteins p21(ras)
High-Throughput Nucleotide Sequencing
Genotyping Techniques
Multiplex Polymerase Chain Reaction
Research team
Molecular Oncology
Thoracic Oncology
Treatment of thoracic tumours
Lung Cancer Group
Language
eng
Date accepted
2015-09-09
License start date
2015-01
Citation
PloS one, 2015, 10 (9), pp. e0139074 - ?
Publisher
PUBLIC LIBRARY SCIENCE