Multiplex Droplet Digital PCR Assay for Detection of and Genes Amplification in Non-Small Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, remains one of the most common tumors with a high mortality and morbidity rate. Numerous targeted drugs were implemented or are now developed for the treatment of NSCLC. Two genes, and , are among targets for these specific therapeutic agents. Alterations in and could lead to primary or acquired resistance to commonly used anti-EGFR drugs. Using current methods for detecting and amplifications is time and labor-consuming; alternative methods are required for and testing. We developed the first multiplex droplet digital PCR assay for the simultaneous detection of and amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR. The optimal elongation temperature, reference genes for DNA quantification, and amplicon length were selected. The developed ddPCR was validated on control samples with various DNA concentrations and ratios of and genes. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed. Among the tested samples, five specimens (1.15%) showed a higher ratio of and six samples (1.38%) showed a higher ratio of . The reported multiplex ddPCR assay could be used for the routine screening of and amplification in NSCLC samples.