Abstract
In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.
Highlights
Droplet-based digital PCR represents an increasingly applied method for quantification and detection of nucleic acids [1]
epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma H1975 (c.2369C>T, p.T790M and c.2573T>G, p.L858R) and H1650 cell lines, Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutant A427 (c.35G>A, p.G12D) and LS123 (c.34G>A, p.G12S) cell lines, Tumoral Protein p53 (TP53)-mutant HT-29 (c.818G>A, p.R273H) cell line were purchased from ATCC (Manassas, VA 20110, US)
Droplet-based digital PCR allows the detection of mutations in circulating tumor DNA from liquid biopsies [34]
Summary
Droplet-based digital PCR (dPCR) represents an increasingly applied method for quantification and detection of nucleic acids [1]. Within the classic TaqMan1 assay fluorescent probes are specific for the WT or the MUT sequence (both placed in exon 21 around p.L858R mutation): we obtained 4000 mutated molecules and 1000 WT-DNA containing droplets.
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