Effects of Pre-analytical Variables on Cell-free DNA Extraction for Liquid Biopsy
1
조회
0
다운로드
2
인용
0
참고문헌
키워드
초록
Background: Extraction of cell-free DNA (cfDNA) is a key step for determining the quality of cfDNA-related molecular diagnostics. We evaluated the effect of sample containers and sample storage conditions on cfDNA extraction. Methods: The cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit from five healthy controls and five lung cancer patients was evaluated according to the type of sample container and storage conditions: K2-EDTA container, <1, 6, 24, and 48 hr storage at 4˚C after immediate plasma separation; and Cell-Free DNA BCT container, <1, 3, 7, and 14 days stored at room temperature. Mutation analysis of EGFR exons 18–21 was performed. To assess the effect of a delay in centrifugation, EDTA whole blood samples from five healthy individuals were stored at 4˚C for 6, 12, and 24 hr before plasma separation. Results: There was no significant difference in the amount and nucleic acid size of cfDNA in both controls and patients with cancer when EDTA plasma was stored at 4˚C up to 48 hr. The amount and size of cfDNA in the BCT container were not different up to 7 days; however, the 14-day sample showed an increase in cfDNA concentration due to genomic DNA contamination. EGFR mutations were detected on EDTA containers up to 48 hr and with BCT containers up to 14 days. When EDTA whole blood was stored at 4˚C and plasma separation was delayed, the cfDNA concentration increased from 24 hr. Conclusions: The cfDNA extraction was affected by the sample containers and storage conditions.
Abstract
Background: Extraction of cell-free DNA (cfDNA) is a key step for determining the quality of cfDNA-related molecular diagnostics. We evaluated the effect of sample containers and sample storage conditions on cfDNA extraction. Methods: The cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit from five healthy controls and five lung cancer patients was evaluated according to the type of sample container and storage conditions: K2-EDTA container, <1, 6, 24, and 48 hr storage at 4˚C after immediate plasma separation; and Cell-Free DNA BCT container, <1, 3, 7, and 14 days stored at room temperature. Mutation analysis of EGFR exons 18–21 was performed. To assess the effect of a delay in centrifugation, EDTA whole blood samples from five healthy individuals were stored at 4˚C for 6, 12, and 24 hr before plasma separation. Results: There was no significant difference in the amount and nucleic acid size of cfDNA in both controls and patients with cancer when EDTA plasma was stored at 4˚C up to 48 hr. The amount and size of cfDNA in the BCT container were not different up to 7 days; however, the 14-day sample showed an increase in cfDNA concentration due to genomic DNA contamination. EGFR mutations were detected on EDTA containers up to 48 hr and with BCT containers up to 14 days. When EDTA whole blood was stored at 4˚C and plasma separation was delayed, the cfDNA concentration increased from 24 hr. Conclusions: The cfDNA extraction was affected by the sample containers and storage conditions.
참고문헌 (26)
- Volik S, "Cell-free DNA(cfDNA) : Clinical significance and utility in cancer shaped by emerging technologies", Mol Cancer Res, 14, pp.898~908 (2016)
- Wan JCM, "Liquid biopsies come of age : towards implementation of circulating tumour DNA", Nat Rev Cancer, 17, pp.223~238 (2017)
- Maheswaran S, "Detection of mutations in EGFR in circulating lung-cancer cells", N Engl J Med, 359, pp.366~377 (2008)
- Kwapisz D, "The first liquid biopsy test approved. Is it a new era of mutation testing for non-small cell lung cancer?", Ann Transl Med, 5, pp.46~ (2017)
- Diaz LA Jr, "Liquid biopsies : genotyping circulating tumor DNA", J Clin Oncol, 32, pp.579~586 (2014)
- Ilie M, "Pros: Can tissue biopsy be replaced by liquid biopsy?", Transl Lung Cancer Res, 5, pp.420~423 (2016)
- Krishnamurthy N, "Liquid biopsies for cancer : Coming to a patient near you", J Clin Med, 6, pp.3~ (2017)
- Gerlinger M, "Intratumor heterogeneity and branched evolution revealed by multiregion sequencing", N Engl J Med, 366, pp.883~892 (2012)
- Bedard PL, "Tumour heterogeneity in the clinic", Nature, 501, pp.355~364 (2013)
- Thierry AR, "Origins, structures, and functions of circulating DNA in oncology", Cancer Metastasis Rev, 35, pp.347~376 (2016)
- Yao W, "Evaluation and comparison of in vitro degradation kinetics of DNA in serum, urine and saliva : A qualitative study", Gene, 590, pp.142~148 (2016)
- Bronkhorst AJ, "Cell-free DNA : Preanalytical variables", Clin Chim Acta, 450, pp.243~253 (2015)
- Heitzer E, "Circulating tumor DNA as a liquid biopsy for cancer", Clin Chem, 61, pp.112~123 (2015)
- Chen W, "Strategies of reducing input sample volume for extracting circulating cell-free nuclear DNA and mitochondrial DNA in plasma", Clin Chem Lab Med, 50, pp.261~265 (2011)
- Devonshire AS, "Towards standardisation of cell-free DNA measurement in plasma : controls for extraction efficiency, fragment size bias and quantification", Anal Bioanal Chem, 406, pp.6499~6512 (2014)
- van Dessel LF, "Application of circulating tumor DNA in prospective clinical oncology trials-standardization of preanalytical conditions", Mol Oncol, 11, pp.295~304 (2017)
- Medina Diaz I, "Performance of Streck cfDNA blood collection tubes for liquid biopsy testing", PLoS One, 11, pp.e0166354~ (2016)
- Sherwood JL, "Optimised pre-analytical methods improve KRAS mutation detection in circulating tumour DNA(ctDNA)from patients with non-small cell lung cancer(NSCLC)", PLoS One, 11, pp.e0150197~ (2016)
- Parpart-Li S, "The effect of preservative and temperature on the analysis of circulating tumor DNA", Clin Cancer Res, 23, pp.2471~2477 (2017)
- van Ginkel JH, "Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics", Cancer Med, 6, pp.2297~2307 (2017)
- Warton K, "Evaluation of Streck BCT and PAXgene stabilised blood collection tubes for cell-free circulating DNA studies in plasma", Mol Diagn Ther, 21, pp.563~570 (2017)
- "Cell-Free DNA BCT: INSTRUCTIONS FOR USE" (2014)[URL]
- Alix-Panabieres C, "Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy", Cancer Discov, 6, pp.479~491 (2016)
- Zhang R, "Synthetic circulating cell-free DNA as quality control materials for somatic mutation detection in liquid biopsy for cancer", Clin Chem, 63, pp.1465~1475 (2017)
- Xue X, "Optimizing the yield and utility of circulating cell-free DNA from plasma and serum", Clin Chim Acta, 404, pp.100~104 (2009)
- Leung F, "Circulating tumor DNA as a cancer biomarker : fact or fiction", Clin Chem, 62, pp.1054~1060 (2016)