title: Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer.
abstract: 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, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic 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 in KRAS and one patient with a BRAF mutation. 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.
authors.affiliation: Department of Biochemical Sciences, Charles University, Faculty of Pharmacy in Hradec Králové, Czech Republic. beranek@lfhk.cuni.cz.|Institute of Clinical Biochemistry and Diagnostics, University Hospital Hradec Králové, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic. beranek@lfhk.cuni.cz.
authors.forename: Martin
authors.initials: M
authors.lastname: Beránek
authors.affiliation: Department of Oncology, University Hospital Hradec Králové, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic.
authors.forename: Igor
authors.initials: I
authors.lastname: Sirák
authors.affiliation: Department of Oncology, University Hospital Hradec Králové, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic.
authors.forename: Milan
authors.initials: M
authors.lastname: Vošmik
authors.affiliation: Department of Oncology, University Hospital Hradec Králové, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Petera
authors.affiliation: Institute of Clinical Biochemistry and Diagnostics, University Hospital Hradec Králové, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic.
authors.forename: Monika
authors.initials: M
authors.lastname: Drastíková
authors.affiliation: Institute of Clinical Biochemistry and Diagnostics, University Hospital Hradec Králové, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Palička
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002352:Carrier Proteins / Q000097:blood*; D015179:Colorectal Neoplasms / Q000097:blood / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D009362:Neoplasm Metastasis; D011379:Prognosis; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D002352:Carrier Proteins; D004273:DNA, Neoplasm
keywords: Carrier; Circulating tumor DNA; Extraction; Next generation sequencing; Real-time PCR