Feasibility Analysis of Cell-Free DNA Derived from Plasma of Lung Cancer Patients for Next-Generation Sequencing

Abstract

Purpose: The quality of specimens directly affects the experimental results. The stability and structural integrity of nucleic acids in samples have a decisive influence on high-throughput sequencing results. Next-generation sequencing (NGS) provides the most comprehensive criteria for evaluating the specimen quality. To test the quality of cell-free DNA (cfDNA) from lung cancer plasma samples stored in our biobank, we conducted a study to evaluate the quality in terms of the genetic level. Methods: A total of 189 peripheral blood samples were collected from patients from patients with EGFR-positive nonsmall cell lung cancer who were seen and treated in Jilin Provincial Cancer Hospital from August 2012 to March 2018. Twelve milliliters of peripheral blood samples were collected and centrifuged at 4°C, 2000 rpm for 15 minutes. Plasma samples were dispensed into cryotubes and stored at -80°C. Plasma cfDNA was extracted by a DNA extraction kit (Qiagen) and the DNA concentration was detected by a Qubit 3.0 fluorometer. Results: The total volume of cfDNA extraction at baseline was 50 μL, the median concentration according to Qubit was 0.633 ng/μL, the range was 0.331-6.09 ng/μL, and the median total DNA was 34.25 ng, ranging from 20.35 to 304.5 ng. The median value of the Qubit concentration in advanced plasma samples was 0.838 ng/μL, ranging from 0.24 to 21.9 ng/μL, and median total DNA was 41.9 ng, ranging from 12.0 to 1095.0 ng. Based on the aforementioned quality assessment factors, 4 of 189 frozen lung cancer baseline plasma samples were not included in further analyses, and for the remaining 185 cases of cfDNA >20 ng, the pass rate was 97.9%. In 143 frozen lung cancer advanced stage plasma samples, 133 cases of cfDNA >20 ng, the pass rate was 93%. Conclusion: Frozen lung cancer plasma samples stored in the biobank for 1-6 years at -80°C under certain conditions still retain a high level of cfDNA, which is suitable for NGS detection.