Detection of tumor-associated gene mutations in cell-free DNA of early-stage non-small cell lung cancer patients

Introduction: Recently, the gene analysis using circulating cell-free DNA (cfDNA) gives us an increasing interest as a tool of a non-invasive biomarker for treatment selection in advanced non-small cell lung cancer (NSCLC). However, few studies have reported in early-stage NSCLC. The aims of this study are to elucidate the sensitivity of tumor-associated gene mutations in cell-free DNA and the correlation between the sensitivity and patients’ characteristics.

Patients and methods: From January 2017 to June 2017, patients with suspected NSCLC who underwent curative resection in our hospital were enrolled in this study. This study was approved by the Institutional Review Board of Kanazawa University Hospital. Written informed consent was obtained from all patients. Tumor DNA was extracted from 25 mg of tumor tissues using the QIAamp DNA Mini Kit (QIAGEN). Blood samples were collected within 14 days of resections, and cfDNA was extracted immediately using QIAamp Circulating Nucleic Acid Kit (QIAGEN). EGFR and KRAS mutation were analyzed by droplet digital PCR (Prime PCR, Bio-RAD). Concentrations of cfDNA were calculated by real-time quantitative PCR targeted ALU gene.

Results: 40 patients were enrolled, and the mutations were detected in 14 (35.0%). All patients with the mutations had adenocarcinoma histology. Deletional mutation (DEL) and L858R in EGFR, and KRAS mutation were detected in 5, 6 and 4 respectively. The sensitivity of mutations for paired tumor DNA and cfDNA was 33% (5/14). All patients with mutations in cfDNA had DEL. The concentrations in patients with cfDNA mutations were significantly higher than that without cfDNA mutations (35.4 ng/mL vs. 13.0 ng/mL).

Conclusions: The study demonstrated that tumor-associated mutations were detected from about a quarter of early-stage NSCLC patients using droplet digital PCR as a highly sensitive assay. Deletional mutation of EGFR and high concentrations of cfDNA were factors for which tumor-associated mutations are likely to be detected in cfDNA.