Comprehensive Evaluation of the Factors Affecting Plasma Circulating Cell-Free DNA Levels and Their Application in Diagnosing Nonsmall Cell Lung Cancer

Aims: Circulating cell-free DNA (ccfDNA) is a valuable biomarker, but the ccfDNA levels are influenced by variations that occur during sample processing. The feasibility of using ccfDNA as a diagnostic biomarker requires further examination. Materials and Methods: We established a real-time PCR assay with an external standard to comprehensively evaluate the factors affecting ccfDNA levels, including the extraction kit used, freeze-thaw stability, and stability of delayed extraction. Then we compared the ccfDNA levels between benign controls (64 cases, including 23 sarcoidosis patients, 19 pneumonia patients, and 22 other lung disease patients) and nonsmall cell lung cancer (NSCLC) patients (74 patients). Results: The different kits showed different recovery rates. Moreover, the ccfDNA present in plasma or stored in extraction buffer was stable after freeze-thawing, and the ccfDNA concentration remained consistent for 24 h at 4 degrees C and for 12 h at room temperature. The patients with NSCLC-III/IV exhibited significantly higher ccfDNA levels than the patients with NSCLC-I/II (293 copies/mu L vs. 190 copies/mu L, p = 0.0339). However, no significant differences in the plasma ccfDNA levels were observed between the benign controls and NSCLC patients (241 copies/mu L vs. 233 copies/mu L, p > 0.05). Conclusions: Variations in sample processing procedures led to variable results. The lack of differences between the NSCLC patients and benign controls indicates that further research is necessary to better characterize ccfDNA as a biomarker for diagnosing NSCLC.