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. 2015 Sep 28;10(9):e0139074.
doi: 10.1371/journal.pone.0139074. eCollection 2015.

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

Alexandra Pender  1 , Isaac Garcia-Murillas  2 , Sareena Rana  3 , Rosalind J Cutts  2 , Gavin Kelly  4 , Kerry Fenwick  2 , Iwanka Kozarewa  2 , David Gonzalez de Castro  5 , Jaishree Bhosle  6 , Mary O'Brien  6 , Nicholas C Turner  7 , Sanjay Popat  6 , Julian Downward  8
Affiliations

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

Alexandra Pender et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The funding provided by Pierre‐Fabre Ltd. does not represent a competing interest for any of the authors, and no other relevant declaration relating to employment, consultancy, patents, products in development or marketed products is required. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. KRAS duplex assays at optimal annealing temperature.
Droplet populations observed for each duplex assay tested with wild-type and relevant mutant cell line gDNA or oligonucleotide at the optimal annealing temperature e.g. G12V panel top left shows droplet populations seen with WT for G12V assay, G12V assay, NCI-H727 gDNA and NCI-H1975 gDNA present. HEX amplitude is up to 6000 on the x axis and FAM amplitude up to 11000 on the y-axis of each panel. Key: Black drops- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets.
Fig 2
Fig 2. Four different KRAS multiplex digital PCR assays combining G12C, G12V and G12D mutant assays and corresponding duplex assays.
KRAS G12C mutant droplet populations are indicated by a red dashed square, KRAS G12D mutant populations by a blue dashed square and KRAS G12V mutant populations by a yellow dashed square. Each multiplex assay, combining all relevant FAM and HEX assays, KRAS WT gDNA and G12V, D and C mutant gDNA, is shown in the top panel. The corresponding duplex assay for each mutation, using the same FAM and HEX assay concentration with KRAS WT DNA and the appropriate KRAS mutant DNA present, is shown in the panels below each multiplex assay. Multiplex 1 (top left panel) is an assay combination of 900 nM primers and 500 nM G12C probe, 562.5 nM primers and 312.5 nM G12D probe and 225 nM primers and 125 nM G12V probe with 450 nM primers and 250 nM WT for G12C probe. Multiplex 2 uses the same concentration of G12V and WT for G12C assay as Multiplex 1 but also contains 450 nM primers and 250 nM G12C probe and 675 nM primers and 375 nM G12D probe. Multiplex 3 uses the same concentration of KRAS mutant assays as in Multiplex 2 but with the WT for G12V probe assay present, used at the same concentration as the WT for G12C assay in Multiplex 1. Multiplex 4 is an assay combination of the G12C assay as in Multiplex 2 with 225 nM primers and 125 nM G12D probe and 675 nM primers and 375 nM G12V probe with the WT for G12D assay at the same concentration as the WT for G12C assay in Multiplex 1. Key: black- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets. The same scale is used for all panels (HEX amplitude up to 7000 and FAM amplitude up to 22000).
Fig 3
Fig 3. KRAS multiplex digital PCR assays A-C and corresponding duplex assays.
Multiplex A (top left panel) is an assay combination of 900 nM primers and 500 nM G13C probe (red dashed square), 450 nM primers and 250 nM G12C probe (blue dashed square) and 225 nM primers and 125 nM G12V probe (yellow dashed square). Multiplex B (top middle panel) is an assay combination of 675 nM primers and 375 nM G12S probe (red dashed square), 450 nM primers and 250 nM G12D probe (blue dashed square) and 225 nM primers and 125 nM G13D probe (yellow dashed square). Multiplex C (top right panel) is an assay combination of 675 nM primers and 375 nM G12R probe (red dashed square), 450 nM primers and 250 nM G12A probe (blue dashed square) and 900 nM primers and 500 nM Q61H probe (yellow dashed square). Multiplex C has 900 nM primers and 500 nM Q61H wild-type probe in addition to a G12C wild-type assay. All other wild-type droplet populations shown, except in the Q61H duplex assay, are 450 nM primers and 250 nM G12C wild-type probe. All panels in the left and centre columns show a FAM amplitude up to 18000 and an HEX amplitude up to 6000. Panels in the right column have a FAM amplitude up to 18000 and a HEX amplitude up to 11000.
Fig 4
Fig 4. Correlation of mutant DNA fraction detected in multiplex and duplex assays using cell line gDNA or oligonucleotides (left) and in FFPE tissue DNA (right).
Fig 5
Fig 5. KRAS mutant FFPE tissue DNA analysis using multiplex and duplex assays to detect KRAS mutant clones.
All samples, except for S011, were analysed with multiplexes A, B and C (upper panels) and the KRAS mutation detected was subsequently confirmed with the appropriate duplex assay (lower panels). Mutant DNA droplet populations are highlighted with a red dashed square. Droplet populations caused by cross-reactivity with a KRAS mutant DNA species not present in the multiplex are indicated by a yellow dashed square.
Fig 6
Fig 6. KRAS mutant biclonal gDNA analysis using multiplex and duplex assays to detect KRAS mutant clones.
All samples were analysed with multiplexes A, B and C (upper panels) and the KRAS mutation detected was subsequently confirmed with the appropriate duplex assay (lower panels). Mutant DNA droplet populations are highlighted with a red dashed square. Droplet populations caused by cross-reactivity with a KRAS mutant DNA species not present in the multiplex are indicated by a yellow dashed square.
Fig 7
Fig 7. Correlation of NGS KRAS mutant allele frequency with digital PCR KRAS mutant allele frequency detected in the appropriate multiplex assay for FFPE tissue DNA and cell line gDNA samples.
See this image and copyright information in PMC

References

    1. Ferlay J, Shin H-R, Bray F, Forman D, Mathers C, Parkin D M (2010) Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 127: 2893–2917. 10.1002/ijc.25516 - DOI - PubMed
    1. Health and Social Care Information Centre (2013) National Lung Cancer Audit Report 2013. [Internet] 26 Nov 2013. Available: http://www.hscic.gov.uk/hospital-care. Accessed 14 Jul 2015.
    1. Pao W, Girard N (2011) New driver mutations in non-small-cell lung cancer. Lancet Oncology 12: 175–180. 10.1016/S1470-2045(10)70087-5 - DOI - PubMed
    1. Leighl NB, Rekhtman N, Biermann WA, Huang J, Mino-Kenudson M, Ramalingam SS, et al. (2014) Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: american society of clinical oncology endorsement of the college of American pathologists/international association for the study of lung cancer/association for molecular pathology guideline. Journal of Clinical Oncology 32: 3673–3679. 10.1200/JCO.2014.57.3055 - DOI - PMC - PubMed
    1. Sequist LV, Yang JC-H, Yamamoto N, O'Byrne K, Hirsh V, Mok T, et al. (2013) Phase III Study of Afatinib or Cisplatin Plus Pemetrexed in Patients With Metastatic Lung Adenocarcinoma With EGFR Mutations. Journal of Clinical Oncology 31: 3327–3334. 10.1200/jco.2012.44.2806 - DOI - PubMed

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