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The aim of the present study was to develop a procedure for the isolation of circulating tumor cells (CTCs), and to evaluate its application in the detection of epidermal growth factor receptor (EGFR) mutations, and potential heterogeneity in patients with non-small-cell lung cancer (NSCLC). Peripheral blood samples were collected from 91 patients with lung cancer, 10 patients with benign disease and 10 healthy volunteers. CTCs were enriched by positive immunomagnetic separation, detected by immunocytochemistry, and processed for single-cell capture. Pure CTC DNA was amplified, and the EGFR gene was analyzed using the amplification refractory mutation system (ARMS) and digital polymerase chain reaction (dPCR). The CTC capture rate in patients with lung cancer was 61.5% (56/91), whereas no CTCs were detected in patients with benign lung disease or in healthy volunteers. The CTC-positive detection rates were 69.3% (52/75) and 25.0% (4/16) in patients with TNM stage III and IV disease, respectively. Markedly more CTCs were captured from patients with small-cell lung cancer compared with patients with other types of cancer. In patients who were positive for EGFR mutations, the detection rate of these mutations was low (16.67%, 2/12), at the single CTC level. The sensitivity increased as the number of CTCs per sample increased. A total of four patients displayed consistent detection of EGFR mutations at the 10-cell level, and one patient exhibited a clear, inconsistent and rare mutation (G719×) between CTCs. A simplified technique for isolating CTCs from blood was established, though multiple CTCs were required to sensitively detect mutations in these cells. The detection of EGFR mutations in CTCs and tissue specimens was generally homogeneous, and therefore, the CTC-level mutation analysis may potentially contribute to the discovery of heterogeneous mutations.

Isolation of circulating tumor cells and detection of EGFR mutations in patients with non‑small‑cell lung cancer