Background
Prostate cancer is the second most commonly diagnosed cancer and a leading cause of cancer-related mortality in men worldwide [
1‐
3]. Despite possessing similar clinicopathological features, some prostate cancer patients are at high risk of developing local and/or distant recurrence and succumbing to their disease, whereas many others will have clinically indolent disease and will not benefit from further intervention. Determining which patients are unlikely to derive a therapeutic benefit from radiation treatment after surgery will prevent overtreatment, removing the burden of unnecessary therapy and side-effects from patients and healthcare systems. Conversely, the ability to accurately identify which patients harbour residual aggressive disease that require more intensive therapy (i.e., dose intensification or early integration of systemic therapies) could improve patient outcomes.
It has been estimated that upwards of 30% of patients will develop biochemical recurrence (BCR) after radical prostatectomy [
4‐
6]. High-risk features for BCR include high Gleason score, high pathological T score, extraprostatic extension, seminal vesicle invasion, positive margins, and rapid prostate-specific antigen (PSA) doubling time [
7‐
9]. Adjuvant radiotherapy can reduce the risk of BCR by approximately 50%, but is associated with higher incidence of acute and late normal tissue toxicity [
10]. Unfortunately, we are currently limited by the existing assays to accurately predict high-risk prostate cancer BCR. Although PSA doubling time is used for monitoring recurrence after radial prostatectomy, it is not able to determine in advance which patients will recur or are at a high risk of recurrence in the future. Furthermore, PSA does not provide information on which patients may benefit from adjuvant or salvage therapy. In the post-radical prostatectomy setting, Decipher [
11,
12] is the one commercially available genomic test to combat the limitations of PSA. It uses surgical tissue to predict disease aggressiveness and the probability of progression after radical prostatectomy. Although this test is very valuable to guide treatment decisions, it cannot be used to monitor treatment response and disease progression over time, as samples are taken from a single timepoint. Furthermore, only the index lesion is macrodissected for Decipher analysis, which does not take into consideration the extensive heterogeneity within a tumour [
11]. Therefore, there is a significant clinical need to find non-invasive biomarkers to identify patients at a high risk of recurrence, monitor their disease progression and treatment response, and optimize their personalized treatment regimens. There is recent evidence that liquid biopsies (biomarkers found in patient biofluids, i.e. blood and urine) are likely to be more representative of the whole tumour’s genomic landscape compared to tumour sampling [
13,
14].
Using blood collected from patients after radical prostatectomy, we profiled the expression of circulating miRNAs (miRNA, miR) and identified a signature of four microRNAs that predict for high-risk prostate cancer: hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-106a-5p (henceforth referred to as miR-17, miR-20a, miR-20b, miR-106a, respectively). Intriguingly, these four miRNAs are all from the same miRNA family, which describes miRNA that contain the same seed sequence and should therefore theoretically target the same downstream pathways. Our research expands the current knowledge of these four members of the oncogenic miR-17 family by describing them as circulating biomarkers that can independently predict for high-risk prostate cancer after radical prostatectomy.
Materials and methods
Patient selection
Patients who had previously received radical prostatectomy and were referred for postoperative radiotherapy to a multidisciplinary genitourinary clinic at the Odette Cancer Centre in Sunnybrook Health Sciences Centre were prospectively recruited and consented. Patients were recruited using an institutional Research Ethics Board-approved protocol. The protocol and methods were approved by the Sunnybrook Research Ethics Board (REB# 035-2015), and the study was carried out in accordance with institutional guidelines. Informed consent was obtained from all patients. Patient characteristics and tumour features were collected and analyzed. A total of 78 male patients who had previously undergone radical prostatectomy were selected.
Risk stratification
Patients were classified into low-risk and high-risk categories based on pathological T stage, Gleason Score, diagnostic PSA level and margin status, which were all obtained from patients’ surgical pathology report (Additional file
1: Table S1). Criteria for high-risk prostate cancer is as follows: pT3a and positive margin; pT3b any margin; Gleason 8 and above; or diagnostic PSA greater than 20 ng/mL. All other patients were considered low-risk.
Sample processing and miRNA isolation
Blood samples were obtained simultaneously for PSA measurement and serum miRNA extraction. Serum was collected within 2 h of blood draw. Mean and median time after radical prostatectomy to sample collection was 31 and 9 months, respectively (range = 3 weeks to 16 years). Circulating miRNAs were collected from serum using the Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit (Norgen Biotek Corp., Canada).
miRNA expression analysis
miRNA expression was performed using the NanoString nCounter technology. NanoString output was normalized using the R package NanoStringNorm (version 1.2.1) with the parameters CodeCount = “geo.mean”, Background = “mean” and SampleContent = “housekeeping.geo.mean”. After normalizing the expression, three samples were removed from downstream analysis due to insufficient RNA quantity and/or quality. Abundant miRNAs were selected by filtering out any miRNAs with zero expression in more than 10% samples. All samples had expression values for at least 90% miRNAs.
Upon normalization, the count matrix where each row is a miRNA and each column is a sample was subjected to negative binomial test for differential expression analysis. This was done using the R package DESeq [
15]. The sizeFactors() function of DESeq was set to zero to avoid over-normalization before estimating dispersion using the function estimateDispersions(). The nbinomTest() function was used to compute the differential expression between two groups of patients—either for high-risk versus low-risk, high Gleason score versus low Gleason score, etc. The fold change cutoff was set to 1.5 fold and
p value cutoff was set to 0.05 across the study if not otherwise specified. The plots were generated using the R package ggplot2.
TCGA data analysis
The miRNA-seq data from TCGA dataset were downloaded from the GDC portal. The clinical data for BCR-free survival analysis were downloaded from the cBioPortal. The survival analysis was performed using the R package Survival and the Kaplan–Meier plots were generated using the R package survminer.
Cell lines and cell culture
PC3 prostate cell line was purchased from the American Type Culture Collection (ATCC, USA). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose (Invitrogen), and supplemented with 10% FBS (Invitrogen) and penicillin (100 U/mL)—streptomycin (100 μg/mL) (Invitrogen). Cells were maintained in a humidified 37 °C incubator with 5% CO2. Cells were passaged upon 70–90% confluency and tested regularly with MycoAlert™ Mycoplasma Detection Kit (Lonza).
Transfection of miRNA mimics
Cells were seeded into 6-well plates at a density of 2 × 10
5 cells per well. The following day, miRNA mimics (5 μM) (Shanghai GenePharma Co. Shanghai, China; Additional file
2: Table S2) were transiently transfected into cells using Lipofectamine 2000 (Invitrogen, Canada) and Opti-MEM I (1X) reduced serum media (Invitrogen), as per manufacturer’s instructions. miRNA overexpression with mimic was verified with qRT-PCR (Additional file
3: Figure S1). Cells were collected at 24 h after transfection for proliferation, soft agar, and clonogenic assays.
Cell proliferation assay
Transfected cells were seeded in triplicate in 6-well plates at a density of 5 × 105 cells/well. Four days later, cells were trypsinized and the total number of viable cells was determined using the Countess automated cell counter (Life Technologies). This experiment was performed in three biological replicates.
Transfected cells were resuspended in DMEM with 0.5% Agar-A (Bio Basic Inc.) and 1000 cells were seeded into 24-well plate with 0.8% Agar-A coated wells, to prevent cells from adhering to plate surface, and placed in a humidified incubator at 37 °C. Colonies were counted approximately 6 weeks later and averaged among three biological replicates.
Clonogenic survival assay after radiation
Control and miRNA mimic–transfected PC3 cells were seeded in triplicate at the following density per well of a 6-well plate: 0 Gy—250 cells, 2 Gy—500 cells, 4 Gy—2000 cells, 6 Gy—4000 cells, and 8 Gy—6000 cells. Cells were irradiated 4 h after seeding and returned to 37 °C incubator. At day 11 after irradiation, cells were stained with crystal violet stain (0.5% crystal violet (Sigma Alderich, Canada), 25% methanol). The number of colonies (> 50 cells) were counted and surviving fraction was determined based on plating efficiency of IR-treated cells relative to mock-radiated cells. Clonogenic survival was represented in dose–response curves by fitting relative surviving fraction to the linear quadratic formula equation S = e−αD−βD2 using GraphPad Prism 5.0 (GraphPad Software Inc., USA), where S is the surviving fraction, α and β are inactivation constants, and D is the radiation dose in Gy.
Statistical analysis
Statistical tests and data representation were performed using GraphPad Prism v5.01 software (GraphPad Software) for in vitro experiments. Data are represented as mean values ± standard error of the mean (SEM) unless otherwise mentioned. Statistical significance defined as p < 0.05.
Discussion
Although patients can possess similar clinicopathological features, a subset of patients are at a high risk of cancer recurring after radical prostatectomy, whereas many others will have clinically insignificant disease. There is a need for non-invasive tests that correlate with histopathological data to better identify these patients who are at risk of recurrence.
To address this gap, we identified four circulating miRNAs that stratify prostate cancer patients after radical prostatectomy. We found that high expression of miR-17, -20a, -20b, and -106a predict for high-risk disease and high pathological T stage (pT3 and above). Furthermore, in combination with previously published works and our new data, we found that these miRNAs confer an aggressive phenotype as demonstrated utilizing in vitro validation studies, providing biological evidence to support their role in high-risk prostate cancer. Our results were consistent with analysis from the TCGA dataset where miR-17, miR-20a, and miR-106a [
20] were associated with shorter time to BCR. This suggests that these miRNAs not only accurately stratify patients into risk categories, but also serve as promising candidates for prognostic biomarkers.
Our results are supported by previous studies. As previously discussed, miR-17 has been described as an oncomiR in prostate cancer [
21]. miR-20a has previously been identified as a plasma biomarker that could accurately distinguish high- versus low-risk disease in treatment naïve prostate cancer patients using the D’Amico risk classification [
22]. miR-20a was significantly overexpressed in patients with high-risk Cancer of the Prostate Risk Assessment (CAPRA) score, and in patients with pathological T stage 3/4 compared to T stage 1/2 [
22]. Another study found that miR-20a was enriched in high/intermediate-grade prostate cancer (Gleason 7–10) compared to low-grade prostate cancer (Gleason 6 and below) [
23]. Our group previously described miR-106a as an oncomiR that is associated with high-grade prostate cancer and confers an aggressive phenotype in vitro and in vivo [
20]. We found that miR-106a is not only overexpressed in prostate cancer tissue compared to normal tissue but is overexpressed in high-grade tumours (Gleason 8–10) compared to low-grade tumours (Gleason 6–7). We also previously found that high miR-106a predicts for shorter time to recurrence after radical prostatectomy, which supports our results in this study. Furthermore, miR-106a has previously been described by Alhasan et al. [
24] as a five-miRNA serum biomarker signature to identify patients with treatment-naïve high-risk prostate cancer.
Although there are various studies identifying prostate cancer circulating biomarkers for prostate cancer diagnosis [
25‐
28] and treatment-naïve disease progression [
24,
29,
30], there are few in the post-radical prostatectomy setting. There are numerous tissue-based miRNAs that have been shown to predict for prostate cancer biochemical recurrence after radical prostatectomy [
31‐
35]. However, these biomarker signatures are hindered by their use of radical prostatectomy tissue, which cannot monitor tumour progression over time like circulating biomarkers.
miRNAs are small, non-coding RNA that regulate the expression of mRNA by inhibiting translation and facilitating target mRNA degradation. miRNAs have been implicated in many pathological states but are especially known for their role in cancer. In prostate cancer in particular, circulating miRNAs have been described to have diagnostic, predictive, and prognostic capabilities [
36,
37]. miR-1290 and miR-375 were identified from blood samples as prognostic markers that correlated with overall survival in castrate-resistant prostate cancer [
38]. miR-1246 has also been identified as a prognostic biomarker for prostate cancer; its expression correlated with pathological grade, positive metastasis, poor prognosis, and tumour aggression in vitro and in vivo [
39].
Circulating miRNAs are uniquely helpful for stratification and they have intriguing qualities that would make them particularly promising biomarkers. Circulating miRNAs are non-invasive, as they can be extracted from a simple blood test at various timepoints. They are highly stable in the blood and during laboratory processing [
40,
41]. Circulating miRNAs, in particular exosomal miRNAs, are thought to be involved in various aspects of tumour maintenance and progression, including local proliferation, invasion and treatment resistance; recruitment and activation of tumour-promoting immune cells; and formation of pre-metastatic niche at distant locations [
42]. Specifically, exosomal miRNAs have been shown to be largely representative of cellular miRNA contents [
43], and exosomes may be selectively secreted from cancer cells [
44]. It was recently found that circulating tumour DNA (ctDNA) is detectable in men with de novo metastatic castrate-sensitive prostate cancer, and ctDNA was significantly reduced after ADT [
45]. Furthermore, somatic mutations in ctDNA were found to be highly consistent (83%) with those in the matched prostate biopsy specimen, suggesting that ctDNA can provide a non-invasive method to identify important tumour alterations.
Although our signature can predict for the presence of high-risk disease, our clinical follow-up is not yet mature enough to establish its impact on predicting BCR (mean and median time to follow-up is 5.3 and 3.7 years after radical prostatectomy, respectively). We expect that high expression of these miRNAs will translate to increased biochemical failure, given that this signature predicts for high-risk disease. Furthermore, using the TCGA dataset as a surrogate, we did find that high expression of miR-17, -20a, and -106a [
20] correlate with shorter time to BCR. However, future studies will need to determine whether this signature provides additional prognostic benefits to current clinicopathological protocols and be validated in larger multi-institutional datasets.
Furthermore, we did not have access to preoperative circulating miRNA levels for these patients. This would provide additional clinical utility by identifying a miRNA profile to distinguish men who could be spared radical prostatectomy and should undergo upfront radiation therapy instead.
A limitation of this study is that due to the small sample size we are unable to accurately assess the predictive power, which is dependent on sample size. Future studies should assess the predictive power of these miRNAs using a larger patient cohort.
Another limitation is that we did not have access to the radical prostatectomy specimens to correlate tissue miRNA expression with blood expression. However, as a surrogate analysis, we mined the TCGA and showed that these miRNAs are enriched in high-grade prostate cancer compared to low-grade prostate cancer to validate our findings.
The final limitation of this study is that the NanoString technology was unable to differentiate between miR-17 and miR-106a, as well as miR-20a and miR-20b. We therefore cannot further establish their individual contributions.
During the course of this study, other groups have developed tests to identify patients at high-risk of recurrence and disease progression after radical prostatectomy. OncotypeDx (San Diego, USA) developed Decipher—a prognostic test to identify patients’ aggressive prostate cancer that is likely to metastasize after radical prostatectomy. Decipher captures a single timepoint of the disease using radical prostatectomy tissue. An advantage of our study is that a liquid biopsy allows for serial monitoring of disease progression. With the ever-changing tumoral landscape, serial monitoring is an important feature of a biomarker. Future studies should evaluate the utility of serial timepoint collections to delineate whether circulating miRNA expression is correlated with disease progression and treatment response.
Despite these limitations, our study has significant novel data showing proof-of-principle that circulating miRNAs are detectable after radical prostatectomy, where the entire prostate has been surgically removed, and can accurately stratify patients into high- and low-risk categories. These results are hypothesis-driving, suggesting that high expression of these four miRNAs in patient blood samples is indicative of high-risk disease that is likely to recur after radical prostatectomy. Future studies will confirm this hypothesis and reveal the clinical utility of these miRNAs as predictive or prognostic biomarkers.
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