Multiplex Crystal Digital™ PCR · The Naica System for Crystal Digital PCR unambiguously ™...
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Multiplex Crystal Digital™ PCR Quantifying EGFR mutations
Transcript of Multiplex Crystal Digital™ PCR · The Naica System for Crystal Digital PCR unambiguously ™...
Multiplex Crystal Digital™ PCR
Quantifying EGFR mutations
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Three-dimensional representation of droplet fluorescence intensities from a triplex Crystal Digital PCR experiment using the Naica System.
Multiplex Crystal Digital PCR
In Focus: Detecting activating and resistance mutations in a single assay
When confronted with limited sample availability, as is often the case for biological samples, it is critical for researchers and clinicians to obtain as much information as possible from a single assay.
In this context, Crystal Digital PCR is the ideal approach to detect and quantify multiple biomarkers in a single test without sacrificing the precision and reliability of the results.
The Naica System™ for Crystal Digital PCR unambiguously distinguishes between targets through the use of 3 different fluorescence channels. This fast and easy-to-use solution is compatible with various combinations of fluorophores, greatly expanding possibilities for assay design.
In non-small cell lung cancer, the epidermal growth factor receptor (EGFR) is an important therapeutic target. EGFR activating mutations, such as L858R and L861Q are predictive of disease responsiveness to targeted therapy using tyrosine kinase inhibitors (TKIs). On the contrary, the presence of EGFR T790M mutation is associated with tumor resistance to TKIs.
To monitor patient response to TKIs treatment with a simple assay, a multiplex digital PCR assay targeting EGFR L858R, L861Q and T790M, as well as wild-type EGFR was developed. These mutations were successfully and reliably detected in a background of wild-type DNA at concentrations down to 0.25 copies per microliter (final concentration), representing a mutant allele frequency of 0.05% (Figure A, B and C).
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EGFR L858R
R² = 0,99
EGFR T790M
R² = 0,9813R² = 0,9937
EGFR L861Q
Evaluation of multiplex Crystal Digital PCR performance for the EGFR quadruplex assay. Titration (25-0.25 cp/µl) of EGFR L858R (A), EGFR L861Q (B) and EGFR T790M (C), in a background of 500 cp/µl of WT EGFR. The theoretical 95% confidence intervals are represented as shaded areas.
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EGFR
