Abstract
Background/Aim: Current research of prostate cancer (PCa) offers a promising way of identifying patients with adverse prognosis who do benefit from radical treatment that can affect quality of life as resections are associated with numerous side-effects. The aim of our study was to evaluate the relationship of TMPRSS2-ERG fusion gene status, tumor tissue prostate-specific antigen (PSA), prostate cancer antigen 3 (PCA3), miR-23b, miR-26a and miR-221 expression levels in combination with preoperative serum PSA level to the risk of PCa recurrence after radical prostatectomy. Patients and Methods: The study group consisted of 108 patients who underwent radical prostatectomy. PSA was measured in peripheral blood collected preoperativelly. The expression of TMPRSS2-ERG transcript and the expression of miR-23b, miR-26a and miR-221 in formalin-fixed, paraffin-embedded (FFPE) tumor tissues was analyzed by reverse transcription (RT) real-time polymerase chain reaction (PCR). Results: Significantly shorter time to recurrence was observed in patients with high expression of TMPRSS2-ERG (p=0.0020). High levels of preoperative PSA (>10.0 ng/ml) proved to be marker of shorter time to recurrence (p=0.0153). The most promising marker of the risk of recurrence after radical prostatectomy was a combination of high level of preoperative serum PSA and high expression of TMPRSS2-ERG fusion transcript in tumor tissue (p=0.0001). Conclusion: A combination of high preoperative serum PSA and high expression of TMPRSS2-ERG could be promising in distinguishing those tumors that are aggressive and life-threatening.
Prostate cancer (PCa) belongs to the most commonly diagnosed cancers worldwide having a higher incidence in Western countries, which might be due to environmental and lifestyle factors, as well as the greater scale of prostate-specific antigen (PSA) screening in the developed countries. PCa has been proven to be a very heterogenous disease, with individual cases differing in both the speed of progression and overall prognosis (1). The main issue of PCa management is to distinguish those tumors that are subsequently progressing. This would make it possible to limit the number of patients undergoing radical surgery to cases of life-threatening tumors as radical prostatectomy is associated with numerous side-effects (2, 3). To assure radical treatment in cases of aggressive prostate cancer a set of markers reliably indicating the nature of the individual case needs to be identified.
Current research offers a promising way of identifying patients with adverse prognosis. It is based on the knowledge of the pathogenesis of PCa and on the role of the fusion gene TMPRSS2-ERG described and linked to PCa by Tomlin et al. in 2005 (4). The origin of the TMPRSS2-ERG oncogene can be traced to a recurrent rearrangement (translocation, interstitial deletion) on long arm of chromosome 21 that fuses androgen-regulated prostate-specific gene promoter of the transmembrane protease serine 2 (TMPRSS2), locus 21q22.3, and the gene ERG, locus 21q22.2, a member of the transcription factor erytroblastosis virus E26 transforming sequence family (ETS) (5). Prostate tissue has a strong androgen dependency and so does PCa, with the signal pathways being mediated by androgen receptors (6). As a result of a TMPRSS2-ERG fusion, overexpression of oncogenic transcription factor ERG is driven by androgens through androgen-responsive elements of TMPRSS2 5’-untranslated region. Nevertheless, as the disease evolves into castration-resistant PCa, TMPRSS2-ERG fusion gene overcomes androgen regulation (7). Fusion gene TMPRSS2-ERG appears in early high grade prostatic intraepithelial neoplasia (PIN) and it has been observed that its presence in PCa cells promotes the loss of the tumor supressor gene PTEN and other alterations, which in turn lead to the speed up of progression of the disease (8).
In addition to analysis of TMPRSS2-ERG fusion gene significance, we also focused on selected microRNAs. MicroRNAs are non-coding RNAs consisting of a small number of nucleotides (~22 nt) that can influence gene expression by interfering with translation or by destabilising target mRNAs. In this way, microRNAs can affect cancerogenesis in both directions; slow the progression by inbibiting oncogen expression or fasten it by down-regulating tumor supressors (9). Many studies have described changes in expression of microRNAs in PCa tissue and their involvement in initiation, progression and the effects of treatment (10). Based on our research of published literature, we chose three tumor-suppressive miRNAs (miR-23b, miR-26a and miR-221) whose levels could be of interest in making prediction of tumor aggressiveness.
The expression level of miR-23b has been observed to be down-regulated in PCa and restoration of the expression inhibited cancer cell proliferation, migration and invasion in PCa cell lines (11). In PCa, miR-23b supresses proto-oncogene Src kinase and its up-regulation shows a strong positive correlation to better prognosis (12). Both miR-26a and miR-221 function as cell proliferation inhibitors, miR-26a inhibits prostate cancer progression by repression of Wnt5a (13) and La-related protein 1 (LARP1) (14). Lower miR-221 expression was associated with a higher risk of recurrence after radical prostatectomy (15).
Furthermore, we included analysis of tumor tissue expression of PSA and prostate cancer antigen 3 (PCA3), formerly referred to as differential display code 3 (DD3). PCA3 is a long non-coding RNA (lncRNA) beginning to be used as a diagnostic marker of PCa. Recent meta-analysis concluded that urine PCA3 test had acceptable sensitivity and specificity for the diagnosis of PCa (16). There are studies showing association of high PCA3 expression with pathological features of PCa, being predictive of high Gleason score, high-stage and high-volume disease (17, 18). However, less is known about prognostic significance of PCA3 on the basis of the follow-up of patients.
The aim of our study was to evaluate the relationship of TMPRSS2-ERG fusion gene status, tumor tissue PSA, PCA3, miR-23b, miR-26a and miR-221 expression levels in combination with routinely used preoperative serum PSA level to the risk of PCa recurrence after radical prostatectomy and, in this way, to predict the aggressivity of the tumor.
Patients and Methods
Patients. Our study group consisted of 108 patients who underwent radical prostatectomy between January 2011 and June 2012 at the Department of Urology of the University Hospital in Pilsen, Czech Republic. All the patients exhibited elevated serum PSA levels or abnormal digital rectal examination. Indication for surgery was confirmed by positive biopsy results, namely the detection of cancerous cells. The median age was 62.9 years (range=43.8-73.9). Clinicopathological data, such as age at the time of surgery, preoperative PSA level, stage of the disease according to the International Union against Cancer (IUCC) and Gleason score are listed in Table I. Approval was obtained from the Institutional Ethics Committee and written informed consent from each patient.
Tissue samples and RNA extraction. The FFPE (formalin-fixed, paraffin-embedded) tissue samples were used for RNA extraction as we described previously (19, 20). Briefly, areas selected for analysis highlighted by pathologist (either tumor tissue or adjacent normal prostate tissue) were manually macrodisected from 15-μm thick FFPE tissue sections prepared with microtome (Leica RM 2135; Leica Biosystems, Nussloch, Germany). Total RNA was isolated using the RNeasy FFPE Kit (Qiagen, GmbH, Hilden, Germany) according to the protocol of the manufacturer. The concentration of the isolated RNA was measured with NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA); in case of lower concentration than 15 ng/μl the isolation was repeated.
Quantitative estimation of TMPRSS2-ERG, PSA and PCA3 expression. Reverse transcription (RT) was performed from 250 ng of total RNA with Superscript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) and random hexamers as primers. Quantitative estimation of TMPRSS2-ERG, PSA and PCA3 transcripts was performed by real-time polymerase chain reaction (PCR) method with UPL probes (Universal ProbeLibrary; Roche, Mannheim, Germany) on Stratagene Mx3005P apparatus (Agilent Technologies, Santa Clara, CA, USA). The sequences of primers for TMPRSS2-ERG fusion gene (forward primer 5’-TAGGCGCGAGCTAAGCAG-3’ targeting TMPRSS2 exon 1 and reverse primer 5’-GTCCATAGTCGCTGGAGGAG-3’ targeting ERG exon 4) were previously designed and validated by other researchers (21, 22). We found the appropriate UPL probe (probe #1) for this pair of primers by ProbeFinder Software (Roche, Mannheim, Germany). The sequences of primers and corresponding UPL probes for PSA mRNA (forward primer 5’-GTGCTTGTGG CCTCTCGT-3’, reverse primer 5’-CAGCAAGATCACGCTTTTGT-3’, probe #44) and PCA3 lncRNA (forward primer 5’-TGGGAAGGACCTGATGATACA-3’, reverse primer 5’-TGTGTGGCCTCAGA TGGTAA-3’, probe #66) were generated by ProbeFinder Software. The PCR reactions were carried out in 96-well plates in a volume of 20 μl containing 1.0 μl of RT product, 2.0 μl of each primer and 2.0 μl of UPL probe and FastStart TaqMan Probe Master reaction mix (Roche, Mannheim, Germany). The reaction conditions were initial denaturation at 95°C for 10 min, followed by 50 cycles of 95°C for 10 s and 60°C for 30 s. The expression levels were normalized to total RNA and PSA (PCA3/PSA ratio). All samples were assessed in technical duplicates. If Ct values obtained from technical duplicates were in discrepancy, the sample assessment was repeated.
Quantitative estimation of microRNAs. A quantitative estimation of miR-23b, miR-26a and miR-221 was performed by a RT real-time PCR method using TaqMan® MicroRNA Assays (Applied Biosystems, manufactured by Roche, Branchburg, NJ, USA). We used RNU6B (U6snRNA) as a normalizer. The Ct values were corrected using calibrators to eliminate differences between individual runs of the Stratagene Mx3005P Real-Time PCR apparatus (Agilent Technologies, Santa Clara, CA, USA). The results are presented as normalized values as a ratio of the number of copies of the given gene to that of the reference gene. To obtain gene expression data we used the ΔΔCt approach (2−ΔΔCt algorithm).
Quantitative estimation of blood serum PSA protein. Preoperative blood samples were taken from the cubital vein before any procedures involving the prostate manipulation, collected in VACUETTE® blood collection tubes (Greiner Bio-One, Kremsmünster, Austria). The serum was separated by centrifugation at 1700 × g for 10 minutes and PSA determined using the UniCel DxI 800 chemiluminescent immunoassay system (Beckman Coulter, Brea, CA, USA) as described previously (23).
Statistical analysis. SAS version 9.3 statistical software (SAS Institute Inc., Cary, NC, USA) was used for all statistical calculations. The results with p<0.05 were considered statistically significant. Non-parametric two-sided Wilcoxon signed-rank test was used for comparing the two groups (tumor tissue and normal prostate tissue) and Pearson's Chi-squared test for evaluating categorical data. Evaluation of prognostic significance (the relation of markers to time to recurrence) was performed as analysis of maximum likelihood estimates (Cox regression hazard model); the Kaplan-Meier survival distribution functions were generated for markers significant in Cox model.
Results
We have previously analyzed the TMPRSS2-ERG fusion gene status by the RT-qPCR method with UPL probes quantifying mRNA transcripts, so we were able to classify cases not only into categories (fusion gene present/absent) but according to the expression level as well. We detected presence of the TMPRSS2-ERG fusion in 34 out of 108 samples (31.5%). Nevertheless, in about two thirds of positive cases it was a late amplification with cycle threshold (Ct) above 40. Therefore, we decided to distinguish a category of high level of TMPRSS2-ERG fusion transcript expression (Ct≤40.0) in which 13 out of 108 samples (12.0 % fell within). We did not record statistically significant differences of the presence or high expression of TMPRSS2-ERG fusion gene in relationship to the stage of the disease, but there were differences related to Gleason score (Pearson's Chi-squared test, p=0.0259). Higher Gleason score was associated with higher percentage of TMPRSS2-ERG positivity (Table II).
The TMPRSS2-ERG fusion gene status was also evaluated in relation to time to recurrence (disease-free interval (DFI)). The mere presence of TMPRSS2-ERG had no relation to DFI; however, we found significantly shorter DFI in patients with high expression of TMPRSS2-ERG in the carcinoma tissue (p=0.0020, Cox model, analysis of maximum likelihood estimates); details in Table III. Preoperative PSA level, the currently available biomarker for PCa, was also evaluated by Cox model and its high levels proved to be marker of shorter DFI (p=0.0153). There was no correlation between preoperative PSA level and TMPRSS2-ERG fusion gene status. Kaplan-Meier survival curves were generated for both TMPRSS2-ERG fusion gene status (Figure 1A) and preoperative PSA (Figure 1B). Kaplan-Meier survival curves were also computed for the combinations of TMPRSS2-ERG status and preoperative serum PSA level (Figure 2). The category of patients with high preoperative PSA level splits according to the TMPRSS2-ERG status into groups with different prognosis; those with high expression of TMPRSS2-ERG have shorter DFI.
Furthermore, we evaluted the expression levels of microRNAs and found miR-23b, miR-26a and miR-221 to be down-regulated in tumor tissue compared to normal prostate tissue (p=0.0031, p=0.0022, p<0.0001, respectively). We found no correlation of the expression of these microRNAs to clinical stage, Gleason score, TMPRSS2-ERG status or prognosis.
There was no significant difference in the expression of PSA mRNA in tumor tissue compared to normal prostate tissue. PCA3 was significantly overexpressed in tumor tissue compared to normal prostate tissue for both ways of expression values normalization, to total RNA (p<0.0001) and as a PCA3/PSA ratio (p<0.0001). We found no correlation of the expression of either PSA or PCA3 to clinical stage, Gleason score, TMPRSS2-ERG status or prognosis.
The expression of miR-23b, miR-26a and miR-221 had no relation to TMPRSS2-ERG status and DFI. We recorded significantly lower expression of miR-221 in tumor tissue compared to normal prostate tissue (p=0.0005).
Discussion
It is known that some prostate carcinomas may not cause any trouble or only progress very slowly. Microscopic foci of prostate cancer are frequently randomly found in autopsies of men over the age of 50 who died from other reasons (24). Due to the progress in PCa screening, a rising number of men are diagnosed with PCa; nevertheless, not in all cases immediate radical treatment is necessary. There is an increasing need to distinguish tumors that are rapidly progressing to spare patients with slowly growing tumors the strain of overtreatment. To identify those patients with tumors that tend to progress, we used a panel of genes whose expression is significantly involved in PCa carcinogenesis (TMPRSS2-ERG, PCA3, miR-23b, miR-26a and miR-221) (12, 14, 25). We assessed their expression on RNA level that allowed their quantification using RT real time PCR. We combined these markers with serum PSA, as a routinely used marker of high senzitivity for detection of PCa, but with a limited prognostic value.
As the most promising indicator of worse outcome, we have revealed a combination of high level of preoperative serum PSA and a presence of high level expression of TMPRSS2-ERG fusion gene in tumor tissue. Despite of limitation in specificity, serum PSA is one of the most useful biomarkers in oncology and still has not lost its potential that is evident from currently developing concept of prostate health index (Phi) (23). Therefore, the identification of new biomarkers that can be used alone or in combination with PSA is welcome to improve distinguishing the aggressive from the indolent ones (26).
The TMPRSS2-ERG fusion was identified to be the most common gene rearrangement in PCa; published data report its prevalence approximately 50% in PSA screened localized PCa (27). In our cohort, we detected presence of the TMPRSS2-ERG fusion in 31.5% of PCa samples. The mere presence of the fusion gene in our cohort had no relation to prognosis. The meta-analysis of Pettersson et al. concluded that TMPRSS2-ERG or ERG overexpression did not strongly predict recurrence or mortality among men treated with radical prostatectomy; the cohort of this meta-analysis included patients from Europe, North America and Asia (28). It appears, however, that the levels of TMPRSS2-ERG, not merely its presence, have the dicisive effect on progression (29). Similarly, in our cohort, we found significantly shorter time to biochemical recurence in patients with high expression of TMPRSS2-ERG transcript.
TMPRSS2-ERG fusion gene presence can be detected non-invasivelly from samples of urine. Stephan et al. in their work focused on diagnostics of PCa and concluded that PCA3 and Phi were superior to the other parameters and further stated that the advantage of TMPRSS2-ERG might be seen in subgroups of aggressive PCa (30).
MicroRNAs, whose expression was assessed (miR-23b, miR-26a and miR-221) in our study, were chosen on the base of described role in pathogenesis of PCa (12, 14, 25). Nevertheless, we did not record any prognostic significance. We observed lower expression of miR-221 in PCa tumor tissue. This finding supports tumor suppressor role of miR-221 in PCa, which was described in several previously published studies (15, 31, 32). However, some other studies (33, 34) indicate up-regulation of miR-221 in PCa.
In conclusion, a combination of high preoperative serum PSA and high expression of TMPRSS2-ERG could be promising in distinguishing those tumors that are aggressive and together with patient status make it possible to decide if radical resection is beneficial.
Acknowledgements
This study was supported by the grant of Ministry of Health of the Czech Republic - Conceptual Development of Research Organization (Faculty Hospital in Pilsen - FNPl, 00669806) and the project of Faculty of Medicine in Pilsen SVV-2016-260283.
Footnotes
Conflicts of Interest
The Authors declared they have no conflicts of interest.
- Received July 1, 2016.
- Revision received August 17, 2016.
- Accepted August 18, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved