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, 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.
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 >1.5 for AR gain and <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.
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
2. Plasma Collection and Processing
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.
4. DNA Quantification and Dilution
5. Real-time PCR
6. Data Analysis and Interpretation
Total cfDNA concentration was quantifiable by spectrophotometry for all samples analyzed, showing a median of 6.12 ng/µL (range: 2.00 – 23.71 ng/µL) for serum samples and a median of 3.21 ng/µL (range: 2.31 – 8.49 ng/µL) 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 ΔCt in CN assays for each patient were calculated (median ± 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 <0.0001). A lower overall survival (OS) was also reported in patients with AR CN gain (p <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).
Figure 1. Workflow and timeline. The method workflow is divided into different steps and times. Please click here to view a larger version of this figure.
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
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
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.
The authors have nothing to disclose.
We thank Chiara Molinari and Filippo Martignano for support in data analysis.
BD Vacutainer Serum Tubes | Becton Dickinson | 367814 | whole blood tube for serum |
BD Vacutainer EDTA Tubes | Becton Dickinson | 366643 | whole blood tube for plasma |
Ethanol absolute | VWR | the user could use also other companies | |
TaqMan Copy Number assay | Thermo Fisher Scientific | 4400291 | Pre-designed and validated assays with FAM-dye. We used the followig assay: AR_assay1: hs04107225 adn AR_assay2: hs04511283 |
TaqMan Copy Number assay (modified) | Thermo Fisher Scientific | 4467084 | Pre-designed assay modified with VIC-dye: AGO1: Hs02320401_cn |
TaqMan Copy Number Reference Assay, human, RNase P | Thermo Fisher Scientific | 4403326 | |
TaqMan Universal PCR Master Mix | Thermo Fisher Scientific | 4326708 | Master mix for Real Time PCR |
QIAamp DNA Mini Kit (50) | Qiagen | 51304 | DNA extraction kit |
MicroAmp 96-Well Plates | Thermo Fisher Scientific | N8010560 | plates for realt time PCR |
NanoDrop 1000 Spectrophotometer | Thermo Fisher Scientific | – | the user could use also other spectrophotometric methods to quantify DNA |
7500 Fast Real-Time PCR System | Applied Biosystem | – | the user could use also other real time instrument |
CopyCaller Software | Applied Biosystem | – | Software for copy number analysis |