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.).

The authors declare that they have no competing financial interests.

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 EDTA tube if kept for a longer time, and the resulting cfDNA fragments can contaminate original cfDNA in the plasma48. Exposure of a blood sample to a high temperature (&#62;37 &#176;C) or excessive shaking of the tube before plasma separation causes hemolysis. The hemoglobin that consequently remains as a contaminant in plasma affects the downstream processes of cfDNA such as during library preparation49. High temperature (&#62;37 &#176;C) also affects the yield of plasma in an EDTA blood tube. Therefore, prompt separation of plasma after its collection in an EDTA tube50 is a significant step in the quality control of the method. If blood is collected in commercial cfDNA or cfRNA Preservation Tube, the preservative chemical should be mixed with the blood immediately after the blood collection40. Failure to properly mix the preservative chemical leads to the formation of microclots in the plasma. These clog the silica membranes in a column during the cfDNA extraction process and lowers the yield of cfDNA. Moderate shaking of blood in the tube does not cause hemolysis; although&#160;excessive shaking can cause hemolysis even in these tubes40. Therefore, during the shipment or handling of these tubes, excessive shaking should be avoided. Blood tubes are recommended to be shipped in their upright position.
The accurate quantification and assessment of cfDNA concentration per unit plasma is an important step in liquid biopsy, since plasma cfDNA concentration is directly associated with physiological and pathological processes51. The cfDNA concentration is higher in patients with cancer than in healthy subjects52. Increased cfDNA concentration in human blood is not specific to cancer; pregnant women and patients who receive transplantations also tend to have elevated cfDNA concentrations in their blood21. Associations have also been shown for inflammation, tissue trauma, diabetes, myocardial infarction, and sepsis with an increased level of cfDNA concentration53. The concentration of plasma cfDNA in early-stage cancer patients tends to be lower than in patients with advanced or metastatic tumors. This supports a direct association of cfDNA with a tumor burden54. The cfDNA concentration in the blood varies significantly ranged between 0.5 and &#62;1000 ng/mL in cancer patients and between 0 and 100 ng/mL in healthy subjects18. For the library preparation, a minimum of 0.5 ng of cfDNA required. To achieve the minimum demand quantity of cfDNA for sequencing, one can start with a minimum of 2 mL of a blood sample or 1 mL plasma sample. Moreover, technical errors during the plasma cfDNA extraction also reduce the cfDNA yield, and the resultant concentration, thereby deviating from the actual plasma cfDNA concentration. cfDNA degradation may occur due to anticoagulants in EDTA tubes or due to the process of multiple thawing and freezing of plasma samples. A poor activity of proteinase K during the lysis process also results in a low yield of cfDNA. Proteinase K activity decreases due to its prolonged exposure to high temperatures. The use of low concentration ethanol instead of 96-100% during the cfDNA extraction washing step also causes a low yield of cfDNA. Therefore, avoiding technical errors in the liquid biopsy technique is important for achieving accurate and reliable results.
Qualitative analysis of cfDNA is a central step, during which the actual distribution of DNA fragments present in the extracted cfDNA sample is&#160;estimated. Quantitative results of cfDNA are reliable only after confirming its purity by qualitative analysis. Therefore, an ideal graph of cfDNA generated in the DNA fragment analyzer assay shows enrichment of the major peak of cfDNA fragments near 166 bp, and a smaller peak near 332 bp length. This indicates that the extracted cfDNA sample is not contaminated with genomic DNA from WBCs. If a fragment is enriched from 7,000 bp to the reference ladder (10,380bp) and beyond, this indicates contamination of a cfDNA sample with WBC genomic DNA. If genomic DNA contamination is seen in a cfDNA sample after&#160;DNA fragment size estimation assay&#160;the sample can be run on 2% agarose gel with a 100 bp ladder, and gel can be excised between the range ~150-200 bp, followed by a gel extraction assay. Some amount of cfDNA is lost during the agarose gel size selection process. However, the NGS library preparation step requires a minimum of 0.5 ng and a maximum of 1,000 ng of input DNA material to prepare libraries for sequencing. Therefore, if the total quantity of cfDNA is equal to or more than 0.5 ng, it is enough quantity for library preparation step. Moreover, such samples pass the library preparation process successfully, and the resulting library shows a major peak close to 291 bp and a smaller peak at 457 bp, due to the addition of 125 bp indexes and adapters.
The NGS data analysis is the last step in the NGS-based liquid biopsy technique, and lists of mutations and gene-gene fusions3,55,56,57 are generated in this step. Mutations and gene-gene fusions that are identified can be assessed according to previous studies on cancer mutation landscapes58,59, previously characterized gene mutations60 or gene-gene fusions61,62,63. These may serve as prognostic64 and diagnostic7 biomarkers in cancer patients. For example, the R132H mutation in the isocitrate dehydrogenase 1 (IDH1) gene is a decisive signpost of secondary glioblastoma cancer, and also distinguishes it from primary glioblastoma in which this mutation is normally absent65. Moreover, chronic myelogenous leukemia (CML) is characterized by a BCR/ABL1 gene fusion resulting from a balanced translocation between chromosome 9 and 2261. The BCR/ABL1 gene and its mRNA and fusion protein are unique to CML progenitors and, therefore, constitute a good target for therapy66.
Advanced diagnosis, the correct staging of the tumor and treatment monitoring is a current strategy for the efficient management of cancer. Until recently, tissue biopsy has been the gold standard for the histological evaluation of tumor tissues. Yet, following vast research in this area and the advent of technology, it has&#160;proven that a single biopsy is unable to provide the whole mutational landscape of a tumor and that serial biopsies are usually impractical to be performed due to their invasiveness, particularly in glioblastoma and lymphoma, where biopsies are highly invasive. Additionally, approximately 16% of needle biopsies are associated with procedural complications67. Moreover, high-quality genomic profiling requires a sufficient amount of tissue, a demand that is usually not fulfilled by the needle biopsy68.
The liquid biopsies, from a routine blood sample by analyzing tumor-derived DNA&#160;is found to replace or adjunct the routine needle tissue biopsy procedures12. Analysis of tumor-derived DNA/ cfDNA through plasma by liquid biopsy is a minimally invasive and effective procedure for molecular profiling of tumors. It can essentially allow treatment monitoring by serial sampling over time without the potential risks and complications usually associated with traditional tumor tissue biopsy due to its invasive nature69. Moreover, studies comparing the needle biopsy with cfDNA analysis by liquid biopsy of a single tumor lesion found that cfDNA profiling can offer a full molecular heterogeneity that a tumor can be harbored by multiple distinct clonal populations70.
However, although plasma cfDNA based diagnosis has many advantages over tumor tissue biopsy, there are still some crucial limitations that hamper its applicability in the general clinical practice. Limitations of cfDNA based liquid biopsy diagnosis includes the higher cost of the overall procedure, need for high-quality DNA and a dedicated bioinformatician for the necessity of a vast data analysis71. Due to data management issue associated with cfDNA analysis, NGS also poses problems in its immediate application in the clinic. The challenges of the data management that NGS poses is primarily due to the difficulty in distinguishing between the intrinsic background noise of deep sequencing and the aberrations that associated&#160;with the tumour72. Lastly, due to the unavailability of the clinical validity and utility data for the majority of the liquid biopsy assays, they are currently confined only to the clinical studies and basic researches13.
The future developments in the liquid biopsy technique, are expected through combined efforts by basic research in the field of biology, physiology, molecular biology, assay techniques, statistical analysis for data management, and machine-learn technology73,74. Additionally, numerous clinical trials based on cfDNA biomarker evaluation are ongoing, and their results will help establish the validity of the technique. These combined efforts hopefully will establish the liquid biopsy platform through cfDNA analysis as a powerful prognostic, diagnostic, and treatment monitoring tool in the oncology clinical setting within the next five years70,75.
To conclude, the cfDNA based liquid biopsy offers a safe and non-invasive approach for identifying genomic biomarkers that are useful in cancer disease management. However, to achieve reliable and accurate results, standardized protocols for blood collection, plasma/WBC separation, cfDNA extraction, and cfDNA quantitative and qualitative analysis are particularly required74,75.

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&#039;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&amp;mdash; 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 &amp;amp; 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&lt;br/&gt; (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 &amp;micro;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/&amp;micro;L to 100 ng/&amp;micro;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>&lt;strong&gt;Miscellaneous&lt;/strong&gt;</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&amp;ndash;100%)</td><td></td><td></td><td></td></tr><tr><td>Heating block or similar at 56 &amp;deg;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 &amp;deg;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 patients #1 and #3 and healthy controls #H1, #H2, and #H3 were 137 ng, 12.6 ng, 6.52 ng, 2.26 ng, and 2.48 ng, respectively. However, in glioma patient #2 cfDNA concentration was too low to be detected in a fluorometer. cfDNA concentration in a cancer patient&#8217;s 1 mL plasma ranges from 0.5 to &#62;1000 ng18, with a mean value of 20 ng. In healthy persons, cfDNA concentration ranges from an undetectable concentration to 16 ng, with a mean value of 8 ng45. Genomic DNA isolated from 200 &#181;L buffy coat (PBMCs) yields around 25- 50 &#181;g of DNA.
DNA fragment analyzer assay results 
According to a previous study, cfDNA is enriched in 166-bp long fragments, which account for ~85% of the total cfDNA in a patient&#8217;s plasma. In contrast, 332-bp long fragments account for 10%, and 498 bp long fragments account for 5% of cfDNA46. Two main factors that affect the cfDNA fragment analyzer assay results are genomic DNA contamination from PBMCs and low concentration of cfDNA. Figure 1 shows the expected cfDNA bioanalyzer graph of glioma patient #1, in which cfDNA fragments are enriched at 166 bp and there is no genomic DNA contamination in the sample21. Figure 2 shows the fragment enrichment graph after the NGS library preparation of the cfDNA of glioma patient #1 and fragment enrichment is shifted from 166 bp to 291 bp, due to attachment of 125 bp indexes and adapters to it. Figure 3 shows very slight enrichment of the cfDNA fragments of glioma patient #2. Despite the low cfDNA concentration in glioma patient #2, the addition of NGS adapters to the cfDNA and PCR amplification steps lead&#160;to the visible cfDNA library peak on the fragment enrichment graph Figure 4. Therefore, the library was prepared successfully from this low concentration cfDNA sample, as shown in Figure 4. In Figure 5, the cfDNA fragments of glioma patient #3 are shown to peak near 166 bp but genomic DNA contamination is also apparent near the 10380 bp reference ladder peak.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61449/61449fig01.jpg" /> 
Figure 1: An ideal cfDNA fragment analyzer assay graph of glioma sample #1 cfDNA.&#160;A major peak was observed at 166 bp and a smaller peak was observed at 332 bp. There was no genomic contamination seen in this sample, which occurs close to the 10,380 bp upper marker. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61449/61449fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61449/61449fig02.jpg" /> 
Figure 2: An ideal cfDNA fragment analyzer assay graph of glioma sample #1 cfDNA library.&#160;After ligating 125 bp next-generation sequencing library adapters, 166 bp and 332 bp cfDNA fragments showed a major peak at 291 bp and a smaller peak at 457 bp respectively. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61449/61449fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61449/61449fig03.jpg" /> 
Figure 3: cfDNA fragment analyzer assay graph of glioma sample #2 cfDNA, showing a very small peak at 166 bp.&#160;Due to the low concentration of cfDNA, peak at 166 bp was barely visible. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61449/61449fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61449/61449fig04.jpg" /> 
Figure 4: cfDNA fragment analyzer assay graph of cfDNA library of glioma sample #2.&#160;After ligating 125 bp next-generation sequencing library adapters and performing PCR cycles during library preparation, the barely visible cfDNA peak at 166 bp or completely invisible peaks at 332 bp and 498 bp, were clearly visible. A major peak was present close to 291 bp and a smaller peak at 457 bp, 623 bp, 789 bp and near 2,500 bp were observed. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61449/61449fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61449/61449fig05.jpg" /> 
Figure 5: cfDNA fragment analyzer assay showing genomic DNA contamination in glioma sample #3 cfDNA. A small peak at 166 bp indicated the presence of cfDNA and enrichment near upper marker (10,380 bp) was genomic DNA contamination. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61449/61449fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table S1: Volumes of the lysis buffer and carrier RNA (dissolved in Buffer AVE) required for processing 1 mL of plasma samples. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61449/Supplementary_Table_S1.xlsx" target="_blank">Please click here to download this table.</a>

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

10.9K Views.  Bar-Ilan University. 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.

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

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 cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

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, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

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 (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

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, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

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

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 sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

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

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Isolation of Circulating Cell-free DNA (cfDNA): A Silica-based Membrane Procedure to Extract cfDNA from Blood Plasma of Cancer Patients

Source: Palande, V. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/61449" target="_blank">Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients.</a> J. Vis. Exp. (2020)
This video describes a silica-based membrane procedure to isolate circulating cell-free DNA or cfDNA from human plasma sample. The cfDNA is a promising biomarker for cancer diagnosis and assessment.&#160;

Izolacja krążącego DNA wolnego od komórek (cfDNA): procedura membranowa na bazie krzemionki w celu ekstrakcji cfDNA z osocza krwi pacjentów z rakiem

Source: Palande, V. et al. Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients. J. Vis. Exp. (2020)
This video describes a silica-based ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

Automated Capillary Electrophoresis Based Fragment Size Distribution Analysis of cfDNA: A DNA Microchip-based Method to Study Size Distribution of DNA Fragments Isolated from Biofluids

Source: Palande, V. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/61449" target="_blank">Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients.</a> J. Vis. Exp. (2020)
In this video, we analyze the size distribution of cell-free DNA or cfDNA fragments using automated capillary electrophoresis. This technique helps gain insights into the profile of DNA fragments released by cancer cells during apoptosis.

Zautomatyzowana analiza rozkładu wielkości fragmentów cfDNA oparta na elektroforezie kapilarnej: metoda oparta na mikroprocesorze DNA do badania rozkładu wielkości fragmentów DNA wyizolowanych z płynów biologicznych

Source: Palande, V. et al. Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients. J. Vis. Exp. (2020)
In this video, we analyze the size ...