Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer
The aims of the study were:i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures,ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, andiii) to use next generation sequencing (NGS) technology to analyzeKRAS,BRAF, andNRASsomatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated inKRASand one patient with aBRAFmutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.