We thank Chiara Molinari and Filippo Martignano for support in data analysis.

The protocol consists of the isolation of DNA from serum or plasma samples to perform real-time PCR for copy number analysis. DNA extraction, DNA quantity control (spectrophotometer) and real-time PCR for specific targets were performed. In Figure 1, a summary of the procedures and time line are reported.
The protocol follows the guidelines of IRST Human Research Ethics Committee.
1. Serum Collection and Processing
<ol>
	<li>Collect approximately 5 mL whole blood in a serum tube without anticoagulant to obtain serum. Maintain blood tubes at 4 &#176;C until processing.</li>
	<li>Centrifuge tubes at 1,000 x g for 15 min at 4 &#176;C.</li>
	<li>Carefully transfer serum into 2 mL tubes.</li>
	<li>Discard the collecting tube and immediately freeze the supernatant at -80 &#176;C until use.</li>
</ol>
2. Plasma Collection and Processing
<ol>
	<li>Collect approximately 10 mL whole blood in a plasma tube with EDTA to obtain plasma. Fill the tube completely and then invert it 8 times. Maintain blood tubes at 4 &#176;C until processing.</li>
	<li>Centrifuge the tube at 1,800 x g for 15 min at room temperature with no brake setting on the centrifuge.</li>
	<li>Carefully transfer the upper part of the plasma into 2 mL tubes. Repeat the transfer, leaving at least 1 mL of the supernatant above the white cell layer. 
	NOTE: Transferring the upper part of the supernatant reduces the risk of contamination by cells or cell debris.</li>
	<li>Discard the collecting tube and immediately freeze the supernatant at -80 &#176;C until use.</li>
</ol>
3. DNA Isolation from Serum or Plasma
NOTE: Isolation of DNA from serum or plasma should be performed using the commercial protocol modified in the following steps.
<ol>
	<li>Thaw one aliquot (containing from 0.5 to 2 mL) of serum or plasma at room temperature.</li>
	<li>Vortex and mix the sample and transfer 0.5 mL of serum or plasma into a clear 1.5 mL tube. Freeze the rest of material at -80 &#176;C.</li>
	<li>Add 50 &#181;L of proteinase k (20 mg/mL) directly to the sample.</li>
	<li>Add 0.5 mL of lysis buffer to the sample and mix well by pipetting.</li>
	<li>Close the tubes and incubate samples at 56 &#176;C for 15 min in the heat block.</li>
	<li>Add the indicated amount of 100% ethanol at wash buffer 1 and 2, as indicated by the manufacturer&#39;s instructions.</li>
	<li>Bring the samples back to room temperature and add 0.5 mL of absolute ethanol and mix well by pipetting.</li>
	<li>Add 650 &#181;L of the mixture obtained in step 3.7 to the separation column and centrifuge at 6,000 x g for 1 min.</li>
	<li>Discard the tube containing the flow through and place the column on a new clean collection tube. Repeat steps 3.8 and 3.9 one more time.</li>
	<li>Add 500 &#181;L of wash buffer 1, without wetting the rim of the column and centrifuge at 6,000 x g for 1 min.</li>
	<li>Discard the tube containing the flow through and replace it with a new clean collection tube.</li>
	<li>Add 500 &#181;L of wash buffer 2, without wetting the rim of the column and centrifuge at full speed (20,000 x g) for 3 min.</li>
	<li>Discard the tube containing the flow through and replace it with a new clean collection tube.</li>
	<li>Centrifuge at full speed (20,000 x g) for 3 min to remove any residual washing buffer.</li>
	<li>Place the column into a clean 1.5 mL tube. Add 150 &#181;L of elution buffer and wait 7 min to ensure that buffer wets the column.</li>
	<li>Centrifuge at 8,000 x g for 1 min.</li>
	<li>Pipette the elute from step 3.16 again into the same column and centrifuge at full speed (20,000 x g) for 1 min to ensure maximum recovery of DNA.</li>
</ol>
4. DNA Quantification and Dilution
<ol>
	<li>Perform quantification of DNA using a spectrophotometer. Use 2 &#181;L of sample on a bench-top spectrophotometer per the manufacturer&#39;s instructions. 
	NOTE: If the DNA quantity is not sufficient to proceed with real-time PCR (at least 120 ng), perform a new DNA isolation process.</li>
	<li>Dilute DNA from serum or plasma to 2.5 ng/&#181;L in a final volume sufficient for all real-time PCR (about 50 &#181;L).</li>
</ol>
5. Real-time PCR
<ol>
	<li>Thaw copy number assays, reference gene assay, DNA diluted samples, and calibrators pooled in ice. Equilibrate at room temperature the master mix that is stored at 4 &#176;C.</li>
	<li>Prepare a mix of 1 &#181;L of target assay, 1 &#181;L of reference gene assay, and 10 &#181;L of master mix for 3 replicates of each sample and calibrators pooled. Prepare the mix including a negative control (water) and 2 extra samples.</li>
	<li>In a 96 well plate, aliquot 12 &#181;L of the mix into each well. Do not pipette or spin tubes.</li>
	<li>Add 8 &#181;L (concentration of 2.5 ng/&#181;L) of each DNA sample and calibrators pooled into each well. Do not pipette or spin tubes. Change the tip for each well. 
	NOTE: 30 DNA samples can be processed for each Real-time experiment using the 96-well plate: 30 x 3 samples + 1 x 3 calibrators pooled + negative control = 94.</li>
	<li>Briefly spin down the plate at 1,000 x g.</li>
	<li>Run the plate using the following protocol: hold stage at 95 &#176;C for 10 min, 40 cycles at 95 &#176;C for 15 s, and 60 &#176;C for 1 min.</li>
</ol>
6. Data Analysis and Interpretation
<ol>
	<li>In the section &#34;option&#34;, below the amplification plot results, set the Ct threshold at 0.2 for the first target gene analyzed. Repeat the set for each target or reference genes. To Export the real-time PCR file in .txt extension, press &#34;Export&#34; into toolbar. Flag only &#34;results&#34; in the select data to export &#8594; choose as file type the extension .txt &#8594; press &#34;start export&#34; &#8594; close the export tool.</li>
	<li>Open the analysis software and import the file .txt (toolbar &#8594; import).</li>
	<li>Select the file name to analyze and select the analysis setting by toolbar (play symbol).</li>
	<li>Specify the calibrator sample and the known number of copies of the target (e.g., 1 copy for AR gene). Press &#34;apply.&#34;</li>
	<li>For each sample, omit one well with different delta Ct (&#916;Ct) in comparison with other wells.</li>
	<li>Reanalyze the experiment (press play symbol).</li>
	<li>Evaluate the results by the copy number bar plot or the table. 
	NOTE: The results table contains numerous parameters like confidence, Z-score, and replicates analyzed that could be useful for results interpretation.</li>
</ol>

<ol>
	<li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+as+a+diagnostic+marker+for+cancer:+current+insights.">Cell-free DNA as a diagnostic marker for cancer: current insights.</a> Onco Targets Ther. 9, 6549-6559 (2016).</li><li>Heitzer, E., Ulz, P., Geigl, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+as+a+liquid+biopsy+for+cancer.">Circulating tumor DNA as a liquid biopsy for cancer.</a> Clin. Chem. 61 (1), 112-123 (2015).</li><li>Murtaza, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+analysis+of+acquired+resistance+to+cancer+therapy+by+sequencing+of+plasma+DNA.">Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA.</a> Nature. 497 (7447), 108-112 (2013).</li><li>Heitzer, E., Ulz, P., Geigl, J. B., Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+genome-wide+somatic+copy+number+alterations+by+liquid+biopsies.">Non-invasive detection of genome-wide somatic copy number alterations by liquid biopsies.</a> Mol. Oncol. 10 (3), 494-502 (2016).</li><li>Diaz, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+evolution+of+acquired+resistance+to+targeted+EGFR+blockade+in+colorectal+cancers.">The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.</a> Nature. 486 (7404), 537-540 (2012).</li><li>Dawson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circulating+tumor+DNA+to+monitor+metastatic+breast+cancer.">Analysis of circulating tumor DNA to monitor metastatic breast cancer.</a> N. Engl. J. Med. 368 (13), 1199-1209 (2013).</li><li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+cell-free+AR+and+CYP17A1+copy+number+variations+may+associate+with+outcome+of+metastatic+castration-resistant+prostate+cancer+patients+treated+with+abiraterone.">Circulating cell-free AR and CYP17A1 copy number variations may associate with outcome of metastatic castration-resistant prostate cancer patients treated with abiraterone.</a> Br. J. Cancer. 112 (10), 1717-1724 (2015).</li><li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+AR+copy+number+and+outcome+to+enzalutamide+in+docetaxel-treated+metastatic+castration-resistant+prostate+cancer.">Circulating AR copy number and outcome to enzalutamide in docetaxel-treated metastatic castration-resistant prostate cancer.</a> Oncotarget. 7 (25), 37839-37845 (2016).</li><li>Romanel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+AR+and+abiraterone-resistant+prostate+cancer.">Plasma AR and abiraterone-resistant prostate cancer.</a> Sci. Transl. Med. 7 (312), (2015).</li><li>Conteduca, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgen+receptor+gene+status+in+plasma+DNA+associates+with+worse+outcome+on+enzalutamide+or+abiraterone+for+castration-resistant+prostate+cancer:+a+multi-institution+correlative+biomarker+study.">Androgen receptor gene status in plasma DNA associates with worse outcome on enzalutamide or abiraterone for castration-resistant prostate cancer: a multi-institution correlative biomarker study.</a> Ann Oncol. , (2017).</li><li>Attard, G., Antonarakis, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+cancer:+AR+aberrations+and+resistance+to+abiraterone+or+enzalutamide.">Prostate cancer: AR aberrations and resistance to abiraterone or enzalutamide.</a> Nat. Rev. Urol. 13 (12), 697-698 (2016).</li><li>Lee, T. H., Montalvo, L., Chrebtow, V., Busch, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+genomic+DNA+in+plasma+and+serum+samples:+higher+concentrations+of+genomic+DNA+found+in+serum+than+in+plasma.">Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma.</a> Transfusion. 41 (2), 276-282 (2001).</li><li>Chan, K. C., Yeung, S. W., Lui, W. B., Rainer, T. H., Lo, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+preanalytical+factors+on+the+molecular+size+of+cell-free+DNA+in+blood.">Effects of preanalytical factors on the molecular size of cell-free DNA in blood.</a> Clin. Chem. 51 (4), 781-784 (2005).</li></ol>

The authors declare no competing financial interests.

AR CN analysis in serum and plasma sample represents a new, non-invasive approach for the stratification of castration resistant prostate cancer (CRPC) patients. It has been recently demonstrated that the AR CN is able to predict outcomes in CRPC patients treated with abiraterone and enzalutamide, before and after chemotherapy7,8,9,10.
The main advantage of our approach is that the protocol is very simple to perform, consisting of only a DNA isolation process, one real-time PCR analysis and easy data interpretations. Moreover, the test could be quickly performed in 1 working day, and the procedure is inexpensive ($20 USD for each sample, approximately). We have demonstrated that this method is highly reproducible and the results obtained are in line with those coming from more expensive and accurate methods, including digital PCR7,8. Moreover, we found a similar AR gain frequency detected with targeted NGS approaches on plasma samples treated with abiraterone9,10. Another important advantage is that this approach is flexible and could be applicable to other diseases in which the AR gene has an important role. Furthermore, other target and reference genes could be chosen based on the disease of interest.
CN analysis of the AR gene in serum has also some limitations. Firstly, the DNA spectrophotometric quantification method is often imprecise and could be replaced with other, more accurate fluorometric approaches. A more precise quantification could give more precise CN results. Secondly, some studies have suggested that it could be better to analyze plasma DNA instead of serum DNA. The quantity of circulating cell free DNA is generally higher in serum than in plasma12,13, due to the clotting of white blood cells in serum, suggesting that serum is potentially a worse source for tumor-specific DNA analysis because of the possible presence of wild-type DNA.
CN analysis of the AR gene in serum using real-time PCR approaches has been performed on 112 CRPC patients before they started treatment with either enzalutamide or abiraterone. Our data showed that AR CN on cfDNA is a powerful genetic biomarker of clinical outcome and resistance to abiraterone and enzalutamide, and can be used to identify men who might benefit a priori from anti-AR therapies or chemotherapy using a rapid and low-cost PCR assay. These findings highlight the utility of studying blood as a minimally invasive method for interrogating mechanisms of therapeutic resistance in both early and advanced CRPC. In the future, prospective and larger studies should stratify patients by circulating AR status in view of the subsequent differences in clinical outcomes and treatment response.

Circulating cell free DNA (cfDNA) in blood has been demonstrated to be an optimal source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance1,2. Many studies have shown a good concordance between DNA alterations (mutations, copy number variations, epigenetic modifications) in tissues and those found in corresponding plasma samples1, confirming that circulating tumor DNA (ctDNA) is informative for primary and metastatic tumor tissue alterations3. The possibility of studying ctDNA thus allows for the reconstruction of genomic rearrangements and copy number variations (CNVs) at specific oncogenes4, identifying potentially metastatic clonal and subclonal cells. CtDNA has been shown to be clinically useful especially for cancer treatment monitoring as it harbors specific mutations and CNVs, related to specific targeted therapies5,6. It also overcomes the need for tissue biopsies and allows results to be obtained at different times during a specific cancer treatment in a non-invasive manner.
With regard to prostate cancer, a significant correlation between circulating cell-free androgen receptor (AR) CNVs and treatment response to abiraterone and enzalutamide has been shown, indicating AR gene copy number (CN) in cfDNA may be a promising biomarker capable of predicting treatment resistance7,8,9,10,11. CNVs of specific genes in ctDNA can be evaluated using different approaches with different sensitivity, cost, and rapidity (e.g. real-time, Digital PCR, and Next Generation Sequencing).
Here we describe a simple and fast approach, based on duplex assays in real-time PCR technology, for evaluating AR CN in cfDNA from serum and plasma samples7,8. We considered two different PCR assays designed on two different genomic regions within intron 5 of AR (Xq12) and two other genes, as internal standard reference genes known to have a normal copy number status in prostate cancer (RNaseP, located on 14q11; AGO1, located on 1p34). We selected two reference genes, rather than one, to increase the precision and sensitivity of the results. A DNA amount of 60 ng was amplified for each assay combination (combined assay for AR-assay_1+RNaseP and for AR-assay_2+AGO1). Three serum or plasma DNA samples from healthy males were pooled and used as a calibrator. We considered cutoffs of &#62;1.5 for AR gain and &#60;0.5 for deletion. One of the main advantages of this method is that it is flexible and that other genes can also be evaluated, changing the standard internal reference genes, on the basis of tumor type and characteristics.

<table>
	<tbody>
		<tr>
			<td>BD Vacutainer Serum Tubes</td>
			<td>Becton Dickinson</td>
			<td>367814</td>
			<td>whole blood tube for serum</td>
		</tr>
		<tr>
			<td>BD Vacutainer EDTA Tubes</td>
			<td>Becton Dickinson</td>
			<td>366643</td>
			<td>whole blood tube for plasma</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR</td>
			<td></td>
			<td>the user could use also other companies</td>
		</tr>
		<tr>
			<td>TaqMan Copy Number assay</td>
			<td>Thermo Fisher Scientific</td>
			<td>4400291</td>
			<td>Pre-designed and validated assays with FAM-dye. We used the followig assay: AR_assay1: hs04107225 adn AR_assay2: hs04511283</td>
		</tr>
		<tr>
			<td>TaqMan Copy Number assay (modified)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4467084</td>
			<td>Pre-designed assay modified with VIC-dye: AGO1: Hs02320401_cn</td>
		</tr>
		<tr>
			<td>TaqMan Copy Number Reference Assay, human, RNase P</td>
			<td>Thermo Fisher Scientific</td>
			<td>4403326</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4326708</td>
			<td>Master mix for Real Time PCR</td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit (50)</td>
			<td>Qiagen</td>
			<td>51304</td>
			<td>DNA extraction kit</td>
		</tr>
		<tr>
			<td>MicroAmp 96-Well Plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>N8010560</td>
			<td>plates for realt time PCR</td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>-</td>
			<td>the user could use also other spectrophotometric methods to quantify DNA</td>
		</tr>
		<tr>
			<td>7500 Fast Real-Time PCR System</td>
			<td>Applied Biosystem</td>
			<td>-</td>
			<td>the user could use also other real time instrument</td>
		</tr>
		<tr>
			<td>CopyCaller Software</td>
			<td>Applied Biosystem</td>
			<td>-</td>
			<td>Software for copy number analysis</td>
		</tr>
	</tbody>
</table>

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.

Total cfDNA concentration was quantifiable by spectrophotometry for all samples analyzed, showing a median of 6.12 ng/&#181;L (range: 2.00 - 23.71 ng/&#181;L) for serum samples and a median of 3.21 ng/&#181;L (range: 2.31 - 8.49 ng/&#181;L) for plasma samples. We analyzed a total of 115 samples by real-time PCR experiments.
Test sensitivity was assessed using mixed serum DNA from patients with high or low gain for AR, and DNA from healthy donors, which are also used as calibrator samples for copy number analysis. As represented in Figure 2, AR CN is sufficiently detected in 0.375% of DNA from patients with AR gain mixed with healthy donor DNA to obtain a gene gain detectable (Figure 2). Thus, this approach detected a very low amount of copy number gain. The standard deviation of &#916;Ct in CN assays for each patient were calculated (median &#177; SD= 0.1, range: 0.00 - 0.36). We used a median of the standard deviation values for choosing copy number gain cutoff.
We analyzed the copy number of AR in patients treated with abiraterone (n = 53), finding 16 (30.2%) patients exhibited AR gain7. Similarly, we analyzed the copy number variation for AR gene in patients treated with enzalutamide (n = 59), finding AR gain in 21 (36%) of patients8.
Moreover, we evaluated the association between AR CN and clinical outcomes using the Kaplan-Meier method and log-rank test and found a significant association. In particular, abiraterone treated case series patients with AR gene gain showed a median progression free survival (PFS) of 2.8 vs. 9.5 months of non-gained cases (p&#160;&#60;0.0001). A lower overall survival (OS) was also reported in patients with AR CN gain (p&#160;&#60;0.0001).
Similarly, for enzalutamide case series, we found a median PFS of 2.4 vs. 4.0 months for patients with AR gain vs. patients with no gain (p = 0.0004). Moreover, median OS of patients with AR CN gain was 6.1 vs. 14.1 months for those without gain (p = 0.0003). All AR CN gain results obtained from the real-time PCR approach were confirmed using other methods, including digital PCR (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56502/56502fig1.jpg" /> 
Figure 1. Workflow and timeline. The method workflow is divided into different steps and times. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56502/56502fig1large.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/56502/56502fig2.jpg" /> 
Figure 2. Approach sensitivity for AR CN detection tested using 2 distinct pools (sample 1 and sample 2) of DNA from healthy donors and from CRPC patients, gained for AR gene, mixed at different concentrations. Patient DNA were mixed at percentages of 0.375, 0.75, 1.5, 3, 6, 12, 25, 50, and 100, in respect to total DNA. Red line: cutoff for AR CN gain (1.5). This figure has been modified from Salvi et al.7
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56502/56502fig3.jpg" /> 
Figure 3. AR CN results comparison between real-time PCR (qPCR) and digital PCR (dPCR) for several patients (x-axis). Black line: cutoff for AR CN gain (1.5). This figure has been modified from Salvi et al.7

Watch this Scientific Journal Video about Serum and Plasma Copy Number Detection Using Real-time PCR at JoVE.com

Serum and Plasma Copy Number Detection Using Real-time PCR

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, ...

serum-and-plasma-copy-number-detection-using-real-time-pcr

Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.3K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 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.

Video: Serum and Plasma Copy Number Detection Using Real-time PCR

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as &#34;hot&#34; LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3&#180; transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3&#180; transduction events stemming from a particular hot LINE-1.

This article outlines a simple PCR-based assay to monitor the activity of an active LINE-1 retrotransposon and to map de novo retrotranspositions in a given genome. Using the MCF7 cell line, we demonstrate herein how this method can be applied to detect activity of a LINE-1 located at 22q12.1.

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...