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
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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 ...

Liquid biopsy using cell-free DNA may identify tumor mutations and fusions to potentially diagnose the tumor type and its drug response. In contrast to tissue biopsy, cell-free DNA based liquid biopsy offers a non-invasive approach that can be performed multiple times to monitor cancer disease progression. To isolate circulating cell-free DNA from a patient plasma sample, add 100 microliters of Proteinase K and 800 microliters of lysis buffer supplemented with one microgram of carrier RNA to one milliliter of patient plasma.Thoroughly pulse vortex the solution for 30 seconds before incubating for 30 minutes at 60 degrees Celsius. At the end of the incubation, add 1.8 milliliters of binding buffer to the tube and thoroughly mix with 15 to 30 seconds of pulse vortexing. After a five minute incubation on ice, insert a silica membrane column into a vacuum apparatus connected to a vacuum pump and firmly insert a 20 milliliter tube extender into the open column to prevent sample leakage.At the end of the incubation, carefully pour the mixture into the tube extender and switch on the vacuum pump. When all of the lysate has completely run through the columns, switch off the vacuum pump, release the pressure to zero millibars and carefully discard the tube extender without contaminating the adjacent columns. Transfer the column into a collection tube for centrifugation to remove any residual lysate.After discarding the flow-through, add 600 microliters of wash buffer 1 to the column for a second centrifugation. After discarding the flow-through, centrifuge the column again with 750 microliters of wash buffer 2. After discarding the flow-through, add 750 microliters of 96 to 100%ethanol to the column for an additional centrifugation.Transfer the column into a new two milliliter collection tube for another centrifugation before placing the membrane column assembly into a new two milliliter collection tube at 56 degrees Celsius for 10 minutes. At the end of the incubation, transfer the column into a new 1.5 milliliter elution tube and add 50 microliters of elution buffer to the column for a three minute incubation at room temperature. Then, centrifuge the recovered solution for one minute at 20, 000 times G to elute the nucleic acids.To analyze the size of the DNA fragments, first secure the base plate to the chip priming station and adjust the clip at the lowest position. Place a new high sensitivity DNA chip onto the chip priming station and add nine microliters of gel dye mix to the bottom of the G chip well. Position the plunger at one milliliter and close the chip priming station.Close the lock of the latch until it clicks, set the timer to 60 seconds, and press down the plunger until it is held by the clip. After exactly 60 seconds, use the clip release mechanism to release the plunger. When the plunger retreats to at least the 300 microliter mark, wait for five seconds before slowly retracting the plunger to the one milliliter position.Open the chip priming station to remove nine microliters of the gel dye mix and transfer the gel to the bottom of the high sensitivity DNA chip G well. To load the DNA marker, dispense five microliters of DNA marker into the ladder well and to the 11 sample wells. To load the ladder and samples, add one microliter of the DNA ladder to the DNA ladder well, one microliter of sample to the used sample wells and one microliter of marker to the unused sample wells.Place the high sensitivity DNA chip horizontally in the adapter for 60 seconds of vortexing at 2, 400 revolutions per minute, taking care that the bulge that fixes the high sensitivity DNA chip is not damaged. After vortexing, confirm that the electrode cartridge is properly inserted and that the chip selector is positioned to double stranded high sensitivity DNA. Carefully mount the high sensitivity DNA chip into the receptacle and confirm that the electrode cartridge is fit exactly into the wells of the chip before closing the lid.The fragment analyzer software screen will display a chip icon to indicate that the chip has been inserted and the lid has been closed. To initiate the analysis, open the assay menu and select the double stranded DNA high sensitivity assay. Enter the sample names into the table and click start to initiate the chip run.At the end of the run, immediately remove and discard the chip according to good laboratory practice. Here are the expected cell-free DNA bioanalyzer graph from a representative glioma patient in which cell-free DNA fragments are enriched at 166 base pairs and there is no genomic DNA contamination in the sample can be observed. Here, the same fragment enrichment graph can be observed after next generation sequencing library preparation of the cell-free DNA.Note the fragment enrichment shift from 166 to 291 base pairs due to the attachment of 125 base pair indexes and adapters. For this second representative patient, a very slight enrichment of the cell-free DNA fragments can be observed. Despite the low cell-free DNA concentration, the addition of next generation sequencing adapters to the cell-free DNA and PCR amplification leads to a visible cell-free DNA library peak on the fragment enrichment graph, indicating that the library was prepared successfully from this low concentration cell-free DNA sample.For this third representative patient, the cell-free DNA fragments peaked near 166 base pairs, but genomic DNA contamination was also apparent near the 10, 380 base pair reference ladder peak. Plasma sample lysis is a crucial step in this protocol, as incomplete lysis will affect the cell-free DNA yield and the entire downstream process.

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

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 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 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 biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

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 reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

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 healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

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 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 ...

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, protein amounts, or other relevant molecules. These methods have shown to be substantial alternatives to PCR or ELISA for clinical and research applications. Moreover, the detection of protein amount (by Western blot or immunohistochemistry) is often insufficient to provide information for cancer diagnosis, whereas the measurement of enzyme activity represents a valuable biomarker. Measurement of enzyme activity also allows for the diagnosis and potential treatment of pathogen-borne diseases. In all eukaryotes, topoisomerases are the key DNA-binding enzymes involved in the control of the DNA topological state during important cellular processes and are among the important biomarkers for cancer prognosis and treatment.
Over the years, topoisomerases have been substantially investigated as a potential target of antiparasitic and anticancer drugs with libraries of natural and synthetic small-molecule compounds that are investigated every year. Here, the rolling circle amplification method, termed rolling circle enhanced enzyme activity detection (REEAD) assay that allows for the quantitative measurement of topoisomerase 1 (TOP1) activity in a simple, fast, and gel-free manner is presented.By cleaving and ligating a specially designed DNA substrate, TOP1 converts a DNA oligonucleotide into a closed circle, which becomes the template for rolling circle amplification, yielding ~103 tandem repeat rolling circle products. Depending on the nucleotide incorporation during the amplification, there is the possibility of different readout methods, from fluorescence to chemiluminescence to colorimetric. As each TOP1-mediated cleavage-ligation generates one closed DNA circle, the assay is highly sensitive and directly quantitative.

A protocol for the sensitive and quantitative detection of topoisomerase 1 activity using the rolling circle enhanced enzyme activity detection assay is described. The method allows detection of topoisomerase 1 activity from purified components or cell/tissue extracts. This protocol has wide-ranging applications in any field involving detection of enzymatic activity.

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

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor 

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

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