Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

Alexandra Pender, Isaac Garcia-Murillas, Sareena Rana, Rosalind J. Cutts, Gavin Kelly, K. Fenwick, I. Kozarewa, D. Gonzalez de Castro, Jaishree Bhosle, Mary O'Brien, Nicolas C. Turner, Sanjay Popat, Julian Downward

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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.
Original languageEnglish
JournalPLoS ONE
Volume10
Issue number9
DOIs
Publication statusPublished - 28 Sep 2015

Keywords

  • Carcinoma, Non-Small-Cell Lung
  • Cell Line, Tumor
  • Clone Cells
  • DNA, Neoplasm
  • Formaldehyde
  • Gene Frequency
  • Genotyping Techniques
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Lung Neoplasms
  • Multiplex Polymerase Chain Reaction
  • Mutation
  • Paraffin Embedding
  • Proto-Oncogene Proteins p21(ras)
  • Temperature
  • Tissue Fixation

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  • Cite this

    Pender, A., Garcia-Murillas, I., Rana, S., Cutts, R. J., Kelly, G., Fenwick, K., Kozarewa, I., Gonzalez de Castro, D., Bhosle, J., O'Brien, M., Turner, N. C., Popat, S., & Downward, J. (2015). Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach. PLoS ONE, 10(9). https://doi.org/10.1371/journal.pone.0139074