Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection
During colorectal cancer (CRC) development tumor-derived cell-free DNA (cfDNA) can be released into the bloodstream. Many different cfDNA isolation methods and specific blood collection tubes preventing the release of genomic DNA and stabilizing cfDNA with preservative reagents became available. These factors may affect greatly on the further liquid biopsy analyses. Our aim was to test different blood collection tubes and cfDNA isolation methods to determine whether these factors influence the cfDNA amount and the promoter methylation of four previously described hypermethylated biomarkers. Three manual isolation methods (High Pure Viral Nucleic Acid Large Volume Kit; Epi proColon 2.0 Kit; Quick-cfDNA™ Serum & Plasma Kit) and automated sample preparation systems (InviGenius and InviGenius PLUS) were examined. Furthermore, K3EDTA Vacuette tubes and Streck Cell-Free DNA BCT® tubes were compared. After cfDNA isolation and bisulfite conversion of samples, the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 were defined with MethyLight assays. We have ascertained that there are differences between the cfDNA amounts depending on the isolation methods. Higher cfDNA yield was observed using InviGenius system than column-based manual isolation method; however, InviGenius PLUS has produced lower cfDNA amounts. No remarkable variance could be found between K3EDTA and Streck tubes; slightly higher cfDNA quantity was detected in 60% of plasma samples using Streck tubes. In point of methylation level and frequency, manual column-based isolation produced more consistent results. Automated cfDNA extraction systems are easy-to-use and high-throughput; however, further improvements in the isolation protocols might lead to the increase of the sensitivity of further methylation analysis.
KeywordsLiquid biopsy Plasma cfDNA isolation Blood collection Colorectal cancer DNA methylation
This study was supported by the ÚNKP 16-3 New National Excellence Program of The Ministry of Human Capacities (ÚNKP-16-4-DSZ-2016-0010), by the National Research, Development and Innovation Office (KMR-12-1-2012-0216 grant) and by the Hungarian Scientific Research Fund (OTKA-K111743 grant).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no competing interests.
- 3.Janz T, Lu K, Povlow MR, Urso B (2016) A review of colorectal cancer detection modalities, stool DNA, and fecal immunochemistry testing in adults over the age of 50. Cureus 8(12):e931Google Scholar
- 8.Kurdyukov S, Bullock M (2016) DNA methylation analysis: choosing the right method. Biology (Basel) 5(1)Google Scholar
- 10.Lam K, Pan K, Linnekamp JF, Medema JP, Kandimalla R (2016) DNA methylation based biomarkers in colorectal cancer: a systematic review. Biochim Biophys Acta 1866(1):106–120Google Scholar
- 15.Malentacchi F, Pizzamiglio S, Verderio P, Pazzagli M, Orlando C, Ciniselli CM, Günther K, Gelmini S (2015) Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas external quality assessment experience. Clin Chem Lab Med 53(12):1935–1942CrossRefGoogle Scholar
- 21.Barták BK, Kalmár A, Péterfia B, Patai ÁV, Galamb O, Valcz G, Spisák S, Wichmann B, Nagy ZB, Tóth K, Tulassay Z, Igaz P, Molnár B (2017) Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1, SFRP2, SDC2, and PRIMA1 in plasma samples. Epigenetics. https://doi.org/10.1080/15592294.2017.1356957
- 24.Romero A, Acosta-Eyzaguirre D, Sanz J, Moreno F, Serrano G, Díaz-Rubio E, Caldés T, Garcia-Saenz JÁ (2015) Identification of E545k mutation in plasma from a PIK3CA wild-type metastatic breast cancer patient by array-based digital polymerase chain reaction: circulating-free DNA a powerful tool for biomarker testing in advance disease. Transl Res 166(6):783–787CrossRefGoogle Scholar
- 32.Toro PV, Erlanger B, Beaver JA, Cochran RL, VanDenBerg DA, Yakim E, Cravero K, Chu D, Zabransky DJ, Wong HY, Croessmann S, Parsons H, Hurley PJ, Lauring J, Park BH (2015) Comparison of cell stabilizing blood collection tubes for circulating plasma tumor DNA. Clin Biochem 48(15):993–998CrossRefGoogle Scholar