Name: detection of cell-free dna in blood plasma samples of cancer patients
Uploaded: 2020-09-09T13:30:00
Description: Watch this Scientific Journal Video about Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients at JoVE.com

The authors would like to thank the members of the Laboratory of Cancer Genomics and Biocomputing of Complex Diseases for their keen observational inputs and their participation in multiple discussions at different stages of this project. The funding support includes Israel Cancer Association (ICA grant for M.F-M 2017-2019) and Kamin grant of Israel Innovation Authority (for M.F-M.).

Prior to blood collection, informed consent from subjects participating in the research is required and must be obtained. The research described in this manuscript was performed in accordance and compliance with the Rabin Medical Center, Israel ethic committee (ethic code: 0039-17-RMC) and the Faculty of Medicine Der Christian-Albrechts-Universit&#228;t zu Kiel, Germany ethic committee (ethic code: D 405/14).
1. Blood sample collection and storage in cfDNA or cfRNA preservative tubes
<ol>
	<...

The collection of a patient&#8217;s blood in a tube, shipment and storage are crucial initial steps in liquid biopsy. Improper handling can impair the quality of the plasma and, therefore, can interfere with the results of the liquid biopsy47. If a blood sample is collected in an EDTA blood tube, the plasma must be separated within two hours of blood collection to avoid lysis of WBCs and release of its genomic DNA into the plasma48. WBCs can also undergo apoptosis in an EDT...

Technological advances have led to the sequencing of hundreds of cancer genomes and transcriptomes1. This has contributed to understanding landscapes of molecular changes across different cancer types2. Further studies on these landscapes have helped characterize the sequential somatic alterations and gene-gene fusions3 that are involved in cancer or tumor progression, by serially disrupting apoptosis pathways4. Therefore, somatic mutations and gene-gene fusions can provide information about tumors by serving as biomarkers in individual patients for a particular tumor type5, identifying existing primary tumors prognosis6, categorizing secondary tumors based on molecular changes7, and identifying druggable tumor targets8. Such information may facilitate in selecting personalized treatment for cancer patients and in determining positive and negative treatment responses9. However, obtaining tumor material for identifying genomic profiling of tumor tissue is an invasive procedure10. Moreover, a tumor biopsy comprises only a small part of a heterogeneous tumor; and may, therefore, not be representative for the molecular profile of the whole tumor11. Serial monitoring and tumor genotyping require&#160;a repeated collection of tumor tissues, which, usually, is not feasible due to the invasiveness of tumor biopsy procedure and the safety issues that arise&#160;from such procedures12.
The liquid biopsy technique, on the other hand, has gained tremendous attention in precision oncology over the last decade13,14. It&#160;is mainly due to the non-invasiveness of this technique, and the possibility of it being repeated at multiple time points, thereby enabling an easy-to-use and safe monitoring technique for the disease courses15,16. Liquid biopsy is based on a phenomenon that tumor cells multiply rapidly, and simultaneously many of them undergo apoptosis and necrosis. This leads to the release of apoptotic cell debris into the patients&#39; blood, together with the DNA fragments that are cut at precise sizes during apoptosis17. The apoptosis of non-cancerous cells also leads to the release of its cellular debris into the blood, however, the apoptosis rate in these cells is relatively much lower than tumor cells18. The rational of the liquid biopsy technique is to capture tumor-associated molecules such as DNA, RNA, proteins, and tumor cells14,19 which circulate continuously in the blood. Various techniques20 can be used for the analysis of these molecules including Next-Generation Sequencing (NGS), digital droplet polymerase chain reaction (ddPCR), real-time PCR, and enzyme-linked immunosorbent assay (ELISA). Liquid biopsy technique enables identifying biomarkers that are characteristics of tumor cells. These biomarker molecules are not just released from specific parts of a tumor, but rather from all parts of the tumor21. Hence, markers identified in liquid biopsy represents the molecular profiling of an entire heterogeneous tumor, in addition to other tumors in the body, thus, having advantages over the tissue biopsy-based technique22.
The cfDNA has a short half-life time in the circulating blood ranging from a few minutes to 1&#8211;2 hours23. However, the short half-life time of cfDNA facilitates real-time analyses by evaluating treatment response and dynamic tumor assessments. The tumor-derived cfDNA levels indicate prognostication of tumor stage/size evidenced by several studies, which showed a relationship between cfDNA levels and the survival outcomes24. Moreover, studies have proved that the cfDNA has a better prediction capacity than existing tumor markers25. The prognostication of cfDNA is even more pronounced after cancer treatment, higher levels of cfDNA following treatment correlates well with a reduced rate of survival, and resistance to treatment. Whereas, lower levels of cfDNA following therapy generally corresponds with positive treatment response. Additionally, the cfDNA facilitates early detection of treatment response than the traditional detection methods.
The cfDNA increases the possibility of early detection of cancer-associated mutations: during early-stage disease15, the onset of symptoms26 and before cancer diagnosis up to 2 years27. As cfDNA is released from multiple tumor regions or foci, its analysis provides a comprehensive view of the tumor genome it represents28. Therefore, the cfDNA enables to detect somatic mutations that might have been missed in the tissue samples29. As intra-tumor heterogeneity and subclonal mutations can be detected by deep sequencing of genomic regions spanning thousands of bases, hence the analysis of the cfDNA enables to uncover specific molecular subtypes with distinct genomic signatures13. To obtain a similar level of information through tissue sample many solid biopsies would have been needed.
Furthermore, the cfDNA levels in patients with a localized disease such as colon, ovarian, and lung cancer after a surgical treatment and/or chemotherapy, demonstrated to be a powerful prognostic marker for cancer recurrence and treatment outcomes20. Moreover, in patients with colon, breast, and lung cancer, analyses of cfDNA from the blood could successfully detect the tumor-specific changes, which led to the precise prediction of recurrence several months in advance13. Furthermore, the treatment resistance markers, such as KRAS mutations in patients with CRC receiving anti-EGFR therapy30; VAFs for genes such as PIK3CA, MED1 or EGFR in patients with breast cancer after the treatment with various therapies31; and EGFR T790M resistance mutation in lung cancer patients treated with EGFR-targeted TKIs32 can also be identified by cfDNA analysis.
In summary, the cfDNA analysis can be used to identify precise biomarkers in the field of oncology13,33. In this protocol, blood samples of 3 glioma patients and 3 healthy controls were processed to obtain genomic DNA from WBCs and cfDNA from the plasma. In glioma cancer, mutations in IDH, TERT, ATRX, EGFR, and TP53 serves as a diagnostic as well as prognostic markers that may help in the early diagnosis of glioma tumors, classifying different types of glioma tumors, guiding the accurate treatment for the individual patient and understanding the treatment response34,35. Mutational status of these genes can be identified using blood-derived cfDNA. In this manuscript, we present a detailed protocol of plasma-derived cfDNA that has been used for studying mutational changes in glioma cancer12. Such cfDNA-based liquid biopsy protocol explained in this article can be used for studying mutational changes in many other types of cancers. Moreover, a recent study has shown that cfDNA-based liquid biopsy can detect 50 different types of cancers36.
Blood sample collection, storage, and shipment are crucial steps in this protocol, as uncontrolled temperature during these steps causes lysis of WBCs, leading to the release of genomic DNA from the WBC into the plasma and causing contamination of the cfDNA sample, which affects the rest of the procedure37. Hemolysis due to uncontrolled temperature can impair downstream sample preparation processes of cfDNA, such as the PCR steps38. The serum contains a high proportion of germline cfDNA rather than plasma, although it presents a large background noise for tumor-associated cfDNA39. Therefore, for isolating tumor-associated cfDNA, plasma is a&#160;suitable sample39. Blood drawn in an anti-coagulant containing blood collection tube should be centrifuged immediately or within up to two hours, to separate the plasma and to avoid cfDNA contamination. In this protocol, dedicated commercial cfDNA preservation blood collection tubes are used (see Table of Materials), which are an alternative to anticoagulant containing blood collection tubes. These dedicated blood collection tubes preserve cfDNA and cfRNA, and prevents lysis of WBCs for up to 30 days at ambient temperature, and up to 8 days at 37 &#176;C. This facilitates maintaining the appropriate temperature during a blood sample shipment and until the plasma and WBC are separated40.
There are three types of cfDNA extraction methodologies currently available: phase isolation, silicon-membrane based spin column, and magnetic bead-based isolation41. The silicon-membrane based spin column method yielded a high quantity of cfDNA with high integrity compared to other cfDNA extraction methods42.
The quantitative evaluation of DNA is a fundamental requirement in liquid biopsy, there is a need to develop a simple, affordable, and standardized procedure for their easy implementation and wide usage. Three commonly used methods for cfDNA quantification are spectrophotometric, fluorimetric, and qPCR. The fluorimetric method is proved better over the other methods concerning the accuracy, cost, and ease of conducttion43.
The&#160;integrity and purity of the cfDNA can be estimated by either agarose electrophoresis or capillary electrophoresis. Agarose electrophoresis neither shows sensitivity at low concentration of cfDNA nor has high resolution to show precise fragment size of cfDNA. On the other hand, capillary electrophoresis has an advantage over the agarose electrophoresis by overcoming the associated challenges and, therefore, widely used by the researchers for cfDNA fragment size analysis. In this protocol, the fragment size distribution of isolated cfDNA was estimated using an automated capillary electrophoresis instrument (see Table of Materials).

<table>
	<tbody>
		<tr>
			<td>2100 Bioanalyzer Instrument</td>
			<td>Agilent Technologies, Inc.</td>
			<td>G2939BA</td>
			<td>The 2100 Bioanalyzer system is an established automated electrophoresis tool for the sample quality control of biomolecules.</td>
		</tr>
		<tr>
			<td>Adjustable Clip for Priming Station</td>
			<td>Agilent Technologies, Inc.</td>
			<td>5042-1398</td>
			<td>Used in combination with syringe to apply defined pressure for chip priming.</td>
		</tr>
		<tr>
			<td>Agilent High Sensitivity DNA Kit</td>
			<td>Agilent Technologies, Inc.</td>
			<td>5067-4626</td>
			<td>The High Sensitivity DNA assays are often used for sample quality control for next-generation sequencing libraries</td>
		</tr>
		<tr>
			<td>cf-DNA/cf-RNA Preservative Tubes</td>
			<td>Norgen Biotek Corp.</td>
			<td>63950</td>
			<td>Norgen&#39;s cf-DNA/cf-RNA Preservative Tubes are closed, evacuated plastic tubes for the collection and the preservation of cf-DNA, circulating tumor DNA, cf-RNA and circulating tumor cells in human whole blood samples during storage and shipping</td>
		</tr>
		<tr>
			<td>Chip Priming Station</td>
			<td>Agilent Technologies, Inc.</td>
			<td>5065-4401</td>
			<td>Used to load gel matrix into a chip with a syringe provided with each assay kit&#8212; used for RNA, DNA, and protein assays. Includes priming station, stop watch, and 1 syringe clip</td>
		</tr>
		<tr>
			<td>Electrode Cleaner Kit</td>
			<td>Agilent Technologies, Inc.</td>
			<td>5065-9951</td>
			<td>Prevents cross-contamination. Removes bacterial or protein contaminants from electrodes.</td>
		</tr>
		<tr>
			<td>Filters for Gel Matrix</td>
			<td>Agilent Technologies, Inc.</td>
			<td>185-5990</td>
			<td>Used for proper mixing of DNA dye concentrate and DNA gel matrix</td>
		</tr>
		<tr>
			<td>IKA Basic Chip Vortex</td>
			<td>IKA-Werke GmbH &#38; Co. KG</td>
			<td>MS-3-S36</td>
			<td>Used for proper mixing of DNA ladder and DNA sample on Bioanalyzer assay chips</td>
		</tr>
		<tr>
			<td>NucleoSpin Tissue kit</td>
			<td>MACHEREY-NAGEL</td>
			<td>740952.5</td>
			<td>With the NucleoSpin Tissue kit, genomic DNA can be prepared from tissue, cells 
			(e.g., bacteria), and many other sources.</td>
		</tr>
		<tr>
			<td>QIAamp circulating nucleic acid kit</td>
			<td>Qiagen</td>
			<td>55114</td>
			<td>The QIAamp Circulating Nucleic Acid Kit enables efficient purification of these circulating nucleic acids from human plasma or serum and other cell-free body fluids.</td>
		</tr>
		<tr>
			<td>QIAvac 24 Plus vacuum manifold</td>
			<td>Qiagen</td>
			<td>19413</td>
			<td>The QIAvac 24 Plus vacuum manifold is designed for vacuum processing of QIAGEN columns in parallel.</td>
		</tr>
		<tr>
			<td>QIAvac Connecting System</td>
			<td>Qiagen</td>
			<td>19419</td>
			<td>In combination with the QIAvac Connecting System, the QIAvac 24 Plus vacuum manifold can be used as a flow-through system. The sample flow-through, containing possibly infectious material, is collected in a separate waste bottle.</td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td>The Qubit 2.0 Fluorometer is an easy-to-use, analytical instrument designed to work with the Qubit assays for DNA, RNA, and protein quantitation.</td>
		</tr>
		<tr>
			<td>Qubit assay tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32856</td>
			<td>Qubit assay tubes are 500 &#181;L thin-walled polypropylene tubes for use with the Qubit Fluorometer.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32851</td>
			<td>The Qubit dsDNA HS (High Sensitivity) Assay Kit is designed specifically for use with the Qubit Fluorometer. The assay is highly selective for double-stranded DNA (dsDNA) over RNA and is designed to be accurate for initial sample concentrations from 10 pg/&#181;L to 100 ng/&#181;L.</td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td>Qiagen</td>
			<td>84010</td>
			<td>used for vacuum processing of QIAGEN columns</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crushed ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (96&#8211;100%)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block or similar at 56 &#176;C (capable of holding 2 ml collection tubes)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (100%)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes (adjustable)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipette tips (pipette tips with aerosol barriers are recommended to help prevent cross-contamination)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath or heating block capable of holding 50 mL centrifuge tubes at 60 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of cell-free dna in blood plasma samples of cancer patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Plasma Separation 
8.5-9 mL blood collected in cfDNA or cfRNA preservative tubes yields around ~4 mL plasma in volume. The volume of plasma separated from blood collected in EDTA tubes may vary depending on the temperature. Exposure of EDTA tubes containing blood at a temperature higher than 37 &#176;C leads to decreased plasma volume yield44.
Fluorometer Assay Results 
cfDNA concentration in 1 mL plasma of each of glioma pati...

Watch this Scientific Journal Video about Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients at JoVE.com

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Research

JoVE Journal

Cancer Research

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, ...

Diagnosis of Vitreoretinal Lymphoma Using Cell-Free DNA

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor (Video) | JoVE

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...