Abstract
Simple SummaryNon-small-cell lung cancer (NSCLC) remains one of the most common tumors with a high mortality and morbidity rate. Alterations in HER2 and MET could be a target for anti-tumor drugs or lead to resistance to anti-EGFR therapeutics. Existing methods for detecting HER2 and MET amplifications are time and labor-consuming, and alternative methods are needed. We report the first multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection of MET and HER2 amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR, and optimal ddPCR conditions were selected. The developed ddPCR was validated on artificial samples with various DNA concentrations and MET and HER2 ratios. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed, and, among them, five specimens (1.15%) were MET-positive and six samples (1.38%) were HER2-positive. The multiplex ddPCR assay could be used for screening MET and HER2 amplification in NSCLC samples.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, HER2 and MET, are among targets for these specific therapeutic agents. Alterations in HER2 and MET could lead to primary or acquired resistance to commonly used anti-EGFR drugs. Using current methods for detecting HER2 and MET amplifications is time and labor-consuming; alternative methods are required for HER2 and MET testing. We developed the first multiplex droplet digital PCR assay for the simultaneous detection of MET and HER2 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 MET and HER2 genes. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed. Among the tested samples, five specimens (1.15%) showed a higher ratio of MET, and six samples (1.38%) showed a higher ratio of HER2. The reported multiplex ddPCR assay could be used for the routine screening of MET and HER2 amplification in NSCLC samples.
Highlights
Lung cancer remains one of the most common malignancies
The observed concentrations of targets allow for the calculation of a ratio between each target and the other three loci, and enable us to make a conclusion about the presence of MET and HER2 amplification in the sample
The increased ratio of MET was in the range of 1.92–3.22, Siberia and the Far East of Russia and blinded before the analysis to hide the personal data and the increased ratio of HER2 was in the range of 1.62–2.76
Summary
Lung cancer remains one of the most common malignancies. In 2020, lung cancer became the second most frequently diagnosed cancer, with 2.2 million estimated new cases (11.4% of all new cancer cases), and remained a leading reason for cancer-associated mortality, with an estimated 1.8 million deaths [1]. Developing new lung cancer diagnostics and treatment approaches is a pressing challenge that can hardly be overestimated. Based on cell morphology and immunohistochemistry, lung cancer is divided into several subtypes: small cell lung cancer (15% of all cases) and non-small-cell lung cancer (NSCLC). NSCLC is further classified into the most common adenocarcinomas Of NSCLC), large-cell carcinomas (7% of NSCLC), and squamous cell carcinomas (18% of NSCLC) [2,3]. Over the past few decades, extensive studies revealed the genetic landscape of NSCLC, which allowed the identification of promising targets for therapeutic agents
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