Abstract
The detection of EGFR mutations in circulating cell-free DNA can enable personalized therapy for cancer. The current techniques for detecting circulating EGFR mutations are expensive and time-consuming with moderate sensitivity. Emerging CRISPR is revolutionizing medical diagnostics and showing a great promise for nucleic acid detection. This study aims to develop CRISPR-Cas12a as a simple test to sensitively detect circulating EGFR mutations in plasma. Serially diluted samples of DNA containing heterozygous EGFR mutations (L858R and T790M) in wild-type genomic DNA are concurrently tested for the mutations by a CRISPR-Cas12a system and droplet digital PCR (ddPCR). The CRISPR-Cas12a system can detect both L858R and T790M with a limit of detection of 0.005% in less than three hours. ddPCR detects the mutations with a limit of detection of 0.05% for more than five hours. Plasma samples of 28 lung cancer patients and 20 cancer-free individuals are tested for the EGFR mutations by CRISPR-Cas12a system and ddPCR. The CRISPR-Cas12a system could detect L858R in plasma of two lung cancer patients whose tissue biopsies are positive for L858R, and one plasma sample of three lung cancer patients whose tissue biopsies are positive for T790M. ddPCR detects L858R in the same two plasm samples, however, does not detect T790M in any of the plasma samples. This proof of principle study demonstrates that the CRISPR-Cas12a system could rapidly and sensitively detect circulating EGFR mutations, and thus, has potential prognostic or therapeutic implications.
Highlights
Lung cancer has a dismal prognosis with the highest mortality rate among all cancer types.Non–small cell lung cancer (NSCLC) accounts for 85% of all lung cancers
Plasma samples of 28 lung cancer patients and 20 cancer-free individuals are tested for the Epidermal growth factor receptor (EGFR) mutations by Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system and droplet digital Polymerase Chain Reaction (PCR) (ddPCR)
We investigate whether the CRISPR-Cas12a-system could detect the EGFR sensitizing (L858R) and resistance (T790M) mutations in plasma
Summary
Non–small cell lung cancer (NSCLC) accounts for 85% of all lung cancers. Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase [1]. Activation of EGFR induces the proliferation and growth of cancer cells, and provides a therapeutic target in advanced NSCLC [1]. EGFR-tyrosine kinase inhibitors (EGFR-TKI) can significantly prolong the five-year survival of advanced. NSCLC patients harboring EGFR mutations [1]. Osimertinib, a third-generation EGFR-TKI, represents the new standard of care for treatment of EGFR mutated NSCLC patients in 1st line [2]. L858R point mutation of EGFR is a major mutation of the gene, which causes EGFR-TKI sensitivity [1]. T790M point mutation of EGFR is the key source of resistance to first-generation EGFR-TKIs, comprising 70%
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