Patients experience relapse after EGFR-TKIs, second tissue biopsy to confirm resistance mutation is required. Despite of this, non-invasive method such as liquid biopsy can be an alternative method. As tumor-derived cell-free DNA from blood sample is often few, establishment of multiplexed assay is meaningful to save samples, analyzing time and cost. Here, we report our novel assay to detect EGFR mutations using ultra-sensitive digital PCR (dPCR) technology.
The appropriate concentration of plasmid DNA was determined empirically to yield a mixture in which the number of mutant EGFR DNA copies was approximately 0.01-1.00% of the number of wild-type EGFR fragments. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. Feasibility of this assay was tested using clinical samples.
Serial-dilution experiments using plasmids and genomic DNAs revealed a linear performance with an analytical sensitivity of approximately 0.01% for each EGFR mutation. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.98-0.99 for each mutations. The limit of blank, defined by the frequency of positive droplets measured in 400 ng of wild-type DNA control samples, was determined as 10 events per assay from the analyses of false events in each mutantion. In the clinical data set, 33 lung cancer patients were enrolled. Results of multiplexed assay completely matched with duplex assay. Concordance between blood and tissue sample were analyzed, and the specificity of EGFR wild-type was 100%. Among 19 EGFR mutated patients, 10 had discordant results. Interestingly, seven did not have extra-thoracic metastasis, and one had brain only recurrence after surgery. The sensitivity was different in patients with or without distant metastasis (77% vs 22%, respectively).
Using ddPCR, we established multiplexed ultra-sensitive genotyping platform for common EGFR mutations. Results of clinical study indicated that liquid biopsy is especially useful in patients with distant metastasis.
Clinical trial identification
All authors have declared no conflicts of interest.