Abstract
BackgroundApproximately 50%–60% of secondary resistance to primary EGFR- tyrosine kinase inhibitors (TKI) therapy is caused by acquired p.Thr790Met (T790M) mutation; however, highly fragmented, low-quantity circulating tumor DNA is an obstacle for detecting mutations. Therefore, more sensitive mutation detection techniques are required. Here, we report a new mutant enrichment technology, the CRISPR system combined with post-polymerase chain reaction (PCR) cell-free DNA (cfDNA) (CRISPR-CPPC) to detect the T790M mutation using droplet digital PCR (ddPCR) from cfDNA.MethodsThe CRISPR-CPPC process comprises the following three steps: (1) cfDNA PCR, (2) assembly of post-PCR cfDNA and CRISPR/CRISPR associated protein 9 complex, and (3) enrichment of the target DNA template. After CRISPR-CPPC, the target DNA was detected using ddPCR. We optimized and validated CRISPR-CPPC using reference cfDNA standards and cfDNA from patients with non-small cell lung cancer who underwent TKI therapy. We then compared the detection sensitivity of CRISPR-CPPC assay with the results of real-time PCR and those of ddPCR.ResultsCRISPR-CPPC aided detection of T790M with 93.9% sensitivity and 100% specificity. T790M mutant copies were sensitively detected achieving an approximately 13-fold increase in the detected allele frequency. Furthermore, positive rate of detecting a low T790M copy number (< 10 copies/mL) were 93.8% (15/16) and 43.8% (7/16) for CRISPR-CPPC assay and ddPCR, respectively.ConclusionsCRISPR-CPPC is a useful mutant enrichment tool for the sensitive detection of target mutation. When tested in patients with progressive disease, the diagnostic performance of CRISPR-CPPC assay is exceptionally better than that of any other currently available methods.
Highlights
50%–60% of secondary resistance to primary epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitors (TKI) therapy is caused by acquired p.Thr790Met (T790M) mutation; highly fragmented, low-quantity circulat‐ ing tumor DNA is an obstacle for detecting mutations
We evaluated the performance of Clustered regularly interspaced short palindromic repeats (CRISPR)-CPPC assay using the samples containing low copies of T790M mutant alleles from patients with EGFR-mutated non-small cell lung cancer (NSCLC) who had clinically progressed after EGFR-TKI treatment
We evaluated the diagnostic performance of CRISPR-CPPC assay by comparing results of Real-time PCR (qPCR), droplet digital polymerase chain reaction (PCR) (ddPCR), and CRISPR-CPPC assay tested in 60 cell-free DNA (cfDNA) samples of patients with NSCLC
Summary
50%–60% of secondary resistance to primary EGFR- tyrosine kinase inhibitors (TKI) therapy is caused by acquired p.Thr790Met (T790M) mutation; highly fragmented, low-quantity circulat‐ ing tumor DNA is an obstacle for detecting mutations. Many researchers suggest that sensitive detection of cell-free DNA (cfDNA) mutations can be accomplished using droplet digital PCR (ddPCR) and next-generation sequencing (NGS) [12,13,14]; mutations with less than 0.1% allele frequency can be randomly detected using current techniques [15]. Strategies to improve the detection capability of clinically significant mutant alleles with exceptionally low copy numbers among circulating nucleic acids are needed [13]
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