Abstract:

Objective To establish and evaluate the performance and clinical application of plasma cfDNA determination in colorectal cancer patients based on next generation sequencing（NGS） platform. Methods Plasma circulating free DNA（cfDNA） determination system（cfDNA panel） for colorectal cancer patients had been established. The accuracy,precision,minimum sample amount and input of extraction,sensitivity and specificity of cfDNA panel were evaluated. The results were compared with both the results of Miseq Dx NGS platform and amplification refractory mutation system（ARMS）. Results The accuracy of cfDNA panel for colorectal cancer was 99.97%,and the determination rate of expected mutation was 100%. The minimum amount of plasma extraction was 1 mL,and the minimum amount of cfDNA input was 10 ng.The sensitivity was 0.2%. The 10 000 mg/L hemoglobin,500 mg/L bilirubin or 2% fat emulsion had no effet on the extraction of colorectal cancer cfDNA panel. Compared with the results of Miseq Dx NGS platform and ARMS,the consistency rates were 94.12% and 90.91%,respectively,and the positive and negative predictive rates of KRAS,NRAS,BRAF and PIK3CA were 100% and 99.60%,respectively. Conclusions The performance of cfDNA panel in colorectal cancer meets the needs of clinical application. The results of plasma cfDNA determination are highly consistent with the determination results of matched tumor tissue,which is a useful supplement for tissue biopsy.

Key words: Circulating free DNA, Colorectal cancer, Next generation sequencing, Laboratory developed test