Precision cell-free DNA extraction for liquid biopsy by integrated microfluidics

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Abstract

Cell-free DNA (cfDNA) has been implicated as an important biomarker in cancer management. Thus, efficient techniques for cfDNA extraction are necessary for precision medicine. We developed a centrifugation-free cfDNA extraction microfluidic chip capable of extracting cfDNA from plasma samples through microfluidic circuits within 15 min under vacuum pressure using an immiscible solvent. The microfluidic chip had excellent performance that was comparable to the most widely used commercial product (QIAamp kit) in terms of extraction efficiency, purity, and quality of DNA samples. The microfluidic chip was validated for the continuous monitoring of HER-2 type breast cancer and was able to successfully detect a point mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase ( PIK3CA) during severe liver metastasis. The chip effectively eliminates the repetitive centrifugation processes and dramatically shortened the sample preparation time. The proposed platform could facilitate the development of a sample-to-answer system for use in liquid biopsy of cancers.

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Circulating nucleic acids (CNAs) and cancer--a survey.

M Fleischhacker, B. Schmidt (2006)

It has been known for decades that it is possible to detect small amounts of extracellular nucleic acids in plasma and serum of healthy and diseased human beings. The unequivocal proof that part of these circulating nucleic acids (CNAs) is of tumor origin, initiated a surge of studies which confirmed and extended the original observations. In the past few years many experiments showed that tumor-associated alterations can be detected at the DNA and RNA level. At the DNA level the detection of point mutations, microsatellite alterations, chromosomal alterations, i.e. inversion and deletion, and hypermethylation of promoter sequences were demonstrated. At the RNA level the overexpression of tumor-associated genes was shown. These observations laid the foundation for the development of assays for an early detection of cancer as well as for other clinical means.

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Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing.

Rebecca Leary, Mark Sausen, Isaac Kinde … (2012)

Clinical management of cancer patients could be improved through the development of noninvasive approaches for the detection of incipient, residual, and recurrent tumors. We describe an approach to directly identify tumor-derived chromosomal alterations through analysis of circulating cell-free DNA from cancer patients. Whole-genome analyses of DNA from the plasma of 10 colorectal and breast cancer patients and 10 healthy individuals with massively parallel sequencing identified, in all patients, structural alterations that were not present in plasma DNA from healthy subjects. Detected alterations comprised chromosomal copy number changes and rearrangements, including amplification of cancer driver genes such as ERBB2 and CDK6. The level of circulating tumor DNA in the cancer patients ranged from 1.4 to 47.9%. The sensitivity and specificity of this approach are dependent on the amount of sequence data obtained and are derived from the fact that most cancers harbor multiple chromosomal alterations, each of which is unlikely to be present in normal cells. Given that chromosomal abnormalities are present in nearly all human cancers, this approach represents a useful method for the noninvasive detection of human tumors that is not dependent on the availability of tumor biopsies.

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Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification

Alison Devonshire, Alexandra S. Whale, Alice Gutteridge … (2014)

Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp® circulating nucleic acid (CNA) kit, NucleoSpin® Plasma XS (NS) kit and FitAmp™ plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity. Figure Comparison of single and multiple reference gene normalisation for quantification of plasma cell free DNA Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7835-3) contains supplementary material, which is available to authorized users.

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Author and article information

Contributors

Sehyun Shin:

ORCID: http://orcid.org/0000-0002-2611-5610

lexerdshin@korea.ac.kr

Journal

Journal ID (nlm-ta): NPJ Precis Oncol

Journal ID (iso-abbrev): NPJ Precis Oncol

Title: NPJ Precision Oncology

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2397-768X

Publication date (Electronic): 24 February 2020

Publication date PMC-release: 24 February 2020

Publication date Collection: 2020

Volume: 4

Electronic Location Identifier: 3

Affiliations

[1 ]ISNI 0000 0001 0840 2678, GRID grid.222754.4, School of Mechanical Engineering, , Korea University, ; Seoul, 02841 Republic of Korea

[2 ]ISNI 0000 0001 0840 2678, GRID grid.222754.4, Nano-Biofluigonstic Research Center, , Korea University, ; Seoul, 02841 Republic of Korea

[3 ]ISNI 0000 0001 0840 2678, GRID grid.222754.4, Division of Oncology/Hematology, Department of Internal Medicine, , Korea University College of Medicine, ; Seoul, 02841 Republic of Korea

Author information

Sehyun Shin http://orcid.org/0000-0002-2611-5610

Article

Publisher ID: 107

DOI: 10.1038/s41698-019-0107-0

PMC ID: 7039987

PubMed ID: 32133418

SO-VID: e3be89f3-b6c3-469b-bf65-76856d1de380

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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 12 May 2019

Date accepted : 20 December 2019

Funding

Funded by: FundRef https://doi.org/10.13039/501100003725, National Research Foundation of Korea (NRF);

Award ID: 2016R1A5A1010148

Award ID: 2015M3A9D7031026

Award Recipient : Sehyun Shin

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Keywords: molecular medicine,breast cancer,nanobiotechnology,prognostic markers

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Keywords: molecular medicine, breast cancer, nanobiotechnology, prognostic markers

Precision cell-free DNA extraction for liquid biopsy by integrated microfluidics

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Abstract

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Karger: Oncology

Most cited references 35

Circulating nucleic acids (CNAs) and cancer--a survey.

Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing.

Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification

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