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Application of CRISPR/Cas9-based mutant enrichment technique to improve the clinical sensitivity of plasma EGFR testing in patients with non-small cell lung cancer.
- Source :
-
Cancer Cell International . 2/15/2022, Vol. 22 Issue 1, p1-11. 11p. - Publication Year :
- 2022
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Abstract
- Background: Approximately 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. Methods: The 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. Results: CRISPR-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. Conclusions: CRISPR-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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 14752867
- Volume :
- 22
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Cancer Cell International
- Publication Type :
- Academic Journal
- Accession number :
- 155261125
- Full Text :
- https://doi.org/10.1186/s12935-022-02504-2