Multiplexed Detection of Mutant Circulating Tumor DNA Using Peptide Nucleic Acid Clamping Asymmetric Polymerase Chain
Abstract:
Circulating tumor DNA (ctDNA) in blood has been investigated as a feasible substitute for genetic alterations in tumor tissues to predict and assess drug responses, but current techniques of screening clinical relevant mutations still have great limitations in sensitivity, specificity, or multiplexed detection because of highly fragmented ctDNA and its low concentration in a high background of normal DNA. In this study, we developed PNA-aPCR-Liquidchip (PAPL), a novel method that aims to detect multiple mutant ctDNA. In order to demonstrate its utility, we analyzed three high frequent epidermal growth factor receptor (EGFR) mutations (exon 19 deletion, L858R, and T790M) in non-small cell lung cancer (NSCLC). Multiplexed analyses indicated that this method has high specificity and sensitivity which could detect down to 2∼5 copies of mutant EGFR in a background of 10,000 copies of wild-type genomic DNA, achieving the mutant abundance of 0.02%∼0.05%. Furthermore, PAPL had no significant differences with droplet digital PCR (ddPCR) in plasma cell-free DNA (cfDNA) detection. Thus, PAPL can be used to detect EGFR mutations in NSCLC patients' plasma, indicating that this method has great potential for application in the context of precision medicine based on mutant ctDNA detection.
Insights
A new method, PNA-aPCR-Liquidchip (PAPL), accurately detects multiple circulating tumor DNA (ctDNA) mutations, including EGFR mutations in non-small cell lung cancer (NSCLC). This advance offers potential for precision medicine through sensitive ctDNA analysis.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Circulating tumor DNA (ctDNA) shows promise for monitoring cancer and predicting treatment response.
- Current ctDNA detection methods struggle with sensitivity, specificity, and multiplexing due to low mutant DNA concentrations.
- Accurate detection of clinically relevant mutations in ctDNA is crucial for personalized cancer care.
Purpose of the Study:
- To develop and validate a novel method, PNA-aPCR-Liquidchip (PAPL), for sensitive and specific multiplexed detection of ctDNA mutations.
- To assess the utility of PAPL in detecting common epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC).
- To compare the performance of PAPL with existing droplet digital PCR (ddPCR) methods for plasma cell-free DNA (cfDNA) analysis.
Main Methods:
- Development of the PNA-aPCR-Liquidchip (PAPL) assay for simultaneous detection of multiple ctDNA mutations.
- Analysis of three frequent EGFR mutations (exon 19 deletion, L858R, T790M) in NSCLC patient samples.
- Comparison of PAPL assay sensitivity and specificity against droplet digital PCR (ddPCR) using plasma cfDNA.
Main Results:
- PAPL demonstrated high specificity and sensitivity, detecting as few as 2-5 copies of mutant EGFR in 10,000 copies of wild-type DNA (0.02%-0.05% mutant abundance).
- The method successfully performed multiplexed analysis of multiple EGFR mutations.
- PAPL showed no significant difference in performance compared to ddPCR for plasma cfDNA detection.
Conclusions:
- PNA-aPCR-Liquidchip (PAPL) is a highly sensitive and specific method for detecting multiple ctDNA mutations, including key EGFR mutations in NSCLC.
- PAPL offers a viable alternative to ddPCR for cfDNA analysis in clinical settings.
- This novel assay holds significant potential for advancing precision medicine through improved ctDNA-based diagnostics and monitoring.
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