Abstract
Purpose
Recently, ‘solid tumor biopsies’ have been challenged by the emergence of ‘liquid biopsies’, which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC).
Methods
Blood and tissue samples from 28 CRC patients were included for the detection of KRAS mutations. For the tissue samples, mutation analysis was conducted by high resolution melting (HRM) analysis and sequencing. For the SCC-MAG method, ctDNA was isolated from 200 µl plasma from patients with a mutant KRAS gene. For comparison, ctDNA extraction was carried out using a silica membrane-based method, after which mutations were detected using Intplex allele-specific PCR.
Results
The mean ctDNA integrity index in plasma samples of cancer patients was 1.03, comparable with that of silica membrane-derived ctDNA (1.011). Notably, the limit of detection for the SCC-MAG approach was lower than that of the silica membrane method and measured 2.25 pg/ml ctDNA in plasma. Our analyses showed that while the silica membrane-based approach was capable of collecting ctDNA from two out of six CRC patient samples (average Cq 34.23), the SCC-MAG captured ctDNA from all samples with an average Cq of 29.76.
Conclusions
We present a robust, reproducible, and highly sensitive method for the analysis of mutation statuses in liquid biopsies. The SCC-MAG method can readily be applied to any nucleic acid target for diagnostic purposes upon careful design of the specific capture probes, and can be multiplexed by several probes to identify multiple targets.



References
A.R. Thierry, S. El Messaoudi, P.B. Gahan, P. Anker, M. Stroun, Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev. 35, 347–376 (2016)
H. Fettke, E.M. Kwan, A.A.J.C.O. Azad, Cell-free DNA in cancer: current insights. Cell. Oncol. 42, 13–28 (2019)
M. Lim, C.J. Kim, V. Sunkara, M.H. Kim, Y.K. Cho, Liquid biopsy in lung cancer: Clinical applications of circulating biomarkers (CTCs and ctDNA). Micromachines 9, 100 (2018)
P.L. Bedard, A.R. Hansen, M.J. Ratain, L.L. Siu, Tumour heterogeneity in the clinic. Nature 501, 355–364 (2013)
M.A. Kerachian, A. Poudineh, J.P. Thiery, Cell free circulating tumor nucleic acids, a revolution in personalized cancer medicine. Crit. Rev. Oncol. Hematol. 2, 102827 (2019)
C. Perez-Barrios, I. Nieto-Alcolado, M. Torrente, C. Jimenez-Sanchez, V. Calvo, L. Gutierrez-Sanz, M. Palka, E. Donoso-Navarro, M. Provencio, A. Romero, Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing. Transl. Lung Cancer Res. 5, 665–672 (2016)
W.A. Al-Soud, P. Radstrom, Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39, 485–493 (2001)
C. Bettegowda, M. Sausen, R.J. Leary, I. Kinde, Y. Wang, N. Agrawal, B.R. Bartlett, H. Wang, B. Luber, R.M. Alani, E.S. Antonarakis, N.S. Azad, A. Bardelli, H. Brem, J.L. Cameron, C.C. Lee, L.A. Fecher, G.L. Gallia, P. Gibbs, D. Le, R.L. Giuntoli, M. Goggins, M.D. Hogarty, M. Holdhoff, S.M. Hong, Y. Jiao, H.H. Juhl, J.J. Kim, G. Siravegna, D.A. Laheru, C. Lauricella, M. Lim, E.J. Lipson, S.K. Marie, G.J. Netto, K.S. Oliner, A. Olivi, L. Olsson, G.J. Riggins, A. Sartore-Bianchi, K. Schmidt, M. Shih l, S.M. Oba-Shinjo, S. Siena, D. Theodorescu, J. Tie, T.T. Harkins, S. Veronese, T.L. Wang, J.D. Weingart, C.L. Wolfgang, L.D. Wood, D. Xing, R.H. Hruban, J. Wu, P.J. Allen, C.M. Schmidt, M.A. Choti, V.E. Velculescu, K.W. Kinzler, B. Vogelstein, N. Papadopoulos, L.A. Diaz Jr., Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci. Transl. Med. 6, 224ra224 (2014)
D. Whitney, J. Skoletsky, K. Moore, K. Boynton, L. Kann, R. Brand, S. Syngal, M. Lawson, A. Shuber, Enhanced retrieval of DNA from human fecal samples results in improved performance of colorectal cancer screening test. J. Mol. Diagn. 6, 386–395 (2004)
C. McCarthy, Chromas version 1.45, School of Health science (Griffifth University, Gold Coast Campus, Queensland, Australia, 1996)
J.T. Hill, B. Demarest, M.J. Hill, S. BiocStyle, S. BiocViews Sequencing, Package ‘sangerseqR’, (2014)
A.R. Thierry, F. Mouliere, S. El Messaoudi, C. Mollevi, E. Lopez-Crapez, F. Rolet, B. Gillet, C. Gongora, P. Dechelotte, B. Robert, Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat. Med. 20, 430 (2014)
M. Burns, H. Valdivia, Modelling the limit of detection in real-time quantitative PCR. Eur. Food Res. Technol. 226, 1513–1524 (2008)
H. Wickham, ggplot: An implementation of the Grammar of Graphics in R, R package version 0.4.0. (2006)
V. Kloten, N. Ruchel, N.O. Bruchle, J. Gasthaus, N. Freudenmacher, F. Steib, J. Mijnes, J. Eschenbruch, M. Binnebosel, R. Knuchel, E. Dahl, Liquid biopsy in colon cancer: comparison of different circulating DNA extraction systems following absolute quantification of KRAS mutations using Intplex allele-specific PCR. Oncotarget 8, 86253–86263 (2017)
C.J. Jorgez, D.D. Dang, J.L. Simpson, D.E. Lewis, F.Z. Bischoff, Quantity versus quality: optimal methods for cell-free DNA isolation from plasma of pregnant women. Genet. Med. 8, 615–619 (2006)
M. Elazezy, S.A. Joosse, Techniques of using circulating tumor DNA as a liquid biopsy component in cancer management. Comput. Struct. Biotechnol. J. 16, 370–378 (2018)
L. Sorber, K. Zwaenepoel, V. Deschoolmeester, G. Roeyen, F. Lardon, C. Rolfo, P. Pauwels, A comparison of cell-free DNA isolation kits: Isolation and quantification of cell-free DNA in plasma. J. Mol. Diagn. 19, 162–168 (2017)
F. Nishimura, N. Uno, P.-C. Chiang, N. Kaku, Y. Morinaga, H. Hasegawa, K. Yanagihara, The effect of in vitro hemolysis on measurement of cell-free DNA. J. Appl. Lab. Med. 4, 235–240 (2019)
K. Page, D.S. Guttery, N. Zahra, L. Primrose, S.R. Elshaw, J.H. Pringle, K. Blighe, S.D. Marchese, A. Hills, L. Woodley, J. Stebbing, R.C. Coombes, J.A. Shaw, Influence of plasma processing on recovery and analysis of circulating nucleic acids. PLoS One 8, e77963 (2013)
Acknowledgements
We would like to thank Ehsan Shams Davodly for designing and preparing the figures, and the charity of Reza Radiotherapy and Oncology Center for supporting this study. This work is in memorial of Mrs Robabeh Aminian for her contribution to cancer research studies.
Funding
This study was supported financially by the Reza Radiotherapy and Oncology Center; Mashhad University of Medical Sciences (Grant number: 961467) and the Iran National Science Foundation (INSF) (Grant number: 93048371).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that there is no conflict of interest.
Ethics approval
The current study was approved by the Mashhad University of Medical Sciences (MUMS) ethics committee (approval number: 961467) and all analyses were performed in accordance with the relevant guidelines and regulations of MUMS.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdiction-al claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Kerachian, M., Azghandi, M., Javadmanesh, A. et al. Selective capture of plasma cell-free tumor DNA on magnetic beads: a sensitive and versatile tool for liquid biopsy. Cell Oncol. 43, 949–956 (2020). https://doi.org/10.1007/s13402-020-00536-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13402-020-00536-2