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Prostate cancer (PCa) is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in men, accounting for 14% of total new cancer cases and 6% of total cancer deaths in males in 2008 . With an increased mean survival rate, PCa is now one of the most significant health problems of developed countries .
Several clinical and pathological variables have been shown to indicate the probability of recurrence after radical prostatectomy (RP) [3–5]. Constructed algorithms and nomograms using preoperative or postoperative variables can help patients and physicians to decide on the best treatment options depending on the probability of recurrence-free survival after RP [6–9]. However, even with the combined use of these prognostic factors, there is still a lack of individualized prognosis. Therefore, there is a strong clinical need to evaluate new cancer biomarkers.
Extensive research efforts, as well as biotechnological advances, have led to a remarkable source of new predictive and prognostic molecular biomarkers of PCa in tissue, serum and urine [10,11]. However, very few of them have found, to date, a clinical application . Moreover, novel biological markers were added to a RP predictive model to improve a preoperative nomogram . There is an increasing need to develop non-invasive tests to predict the behaviour of PCa.
L-3,4-dihydroxyphenylalanine decarboxylase (L-dopa decarboxylase/DDC) is an enzyme that participates in the biosynthesis of both 5-hydroxytryptamine (serotonin, 5-HT) and catecholamines . Intense research has focused on the role of L-dopa decarboxylase in the diagnosis and treatment of a number of neoplasias in recent years [15–19].
The enzyme seems to have a particular biological role in the physiology of the prostate gland as well as in the development and progression of prostate malignancy. In PC-3 and LNCaP PCa cell lines, the overexpression of DDC promotes the increased activity of androgen receptors (ARs), highlighting the dependence of AR action on the levels of DDC expression . Recent studies also support the ability of DDC to activate ARs [21–23]. Furthermore, DDC is a key molecule in the neuroendocrine (NE) differentiation of PCa cells .
To our knowledge, this is the first study to analyse the expression profile of DDC in patients with PCa so as to reveal its correlation with patients' biochemical recurrence and disease prognosis after RP.
PATIENTS AND METHODS
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Between January 2005 and February 2008, 82 prostatic tissue specimens were obtained from patients with PCa at the 1st Department of Urology of ‘Laiko’ University Hospital. The study was approved by the ethical board and informed consent was obtained from all patients. A tissue sample of ≈200 mg was received from the peripheral zone of the gland specimen by radical retropubic prostatectomy, guided by the preoperative features of the biopsy and macroscopic findings of the specimen. The sample was separated into two mirror-image sections, one of which was always evaluated by the same pathologist to confirm the presence of malignancy. The other section was rapidly frozen in liquid nitrogen and stored at −80 °C until its analysis. In all 70 tissue specimens were included; some samples were deemed inadequate for inclusion because of poor preservation and processing. Patients who had received radiotherapy or hormonal therapy before surgery were also excluded from the present study.
In the study of DDC expression as a prognostic marker of PCa, 56/70 patients were included; 10 patients were excluded either because they were not found or because sufficient and clear monitoring data were not available. Four more patients were also excluded because they had received adjuvant therapy before any biochemical recurrence due to the fact that they were at high risk (e.g. there were positive surgical margins).
Time to biochemical recurrence was defined as the interval between surgery and the measurement of two consecutive values of PSA level ≥0.2 ng/mL . Regarding patients who did not show biochemical recurrence, disease-free survival (DFS) was defined as the interval between the RP and the most recent measurement of PSA level.
The total RNA of the prostate tissue was isolated after pulverization of 50–100 mg of specimen, using TRI-reagent (Ambion Inc., Austin, TX, USA). First-strand cDNA was synthesized by the reverse transcription of total RNA using an oligo(dT) as reverse transcription primer and M-MuLV Reverse Transcriptase RNase H- (Finnzymes Oy, Espoo, Finland).
A SYBR Green fluorescence-based quantitative real-time PCR was developed to determine DDC expression levels in the prostate tissue specimens on an ABI Prism 7500 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). A DDC-specific sequence of 90 bp was amplified by the DDC forward 5′-GAACAGACTTAACGGGAGCCTTT-3′ and the reverse 5′-AATGCCGGTAGTCAGTGATAAGC-3′ pair of primers, whereas the use of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward 5′-ATGGGGAAGGTGAAGGTCG-3′ and reverse 5′-GGGTCATTGATGGCAACAATATC-3′ set gave rise to a 107 bp amplicon.
The expression of the DDC target and the GAPDH reference genes was evaluated in separate triplicate reactions for each tested sample. To verify the DDC- and GAPDH-specific amplification from the accumulation of non-specific amplicons or primer-dimers, the melting temperature values of the PCR products were determined by construction of the dissociation curve after amplification.
The evaluation of DDC expression was completed by relative quantification analysis using the 2–ΔΔCT comparative CT method, whereas GAPDH expression was used as the endogenous reference gene for the DDC expression normalization and the LNCaP PCa cell line as a calibrator.
To test the association between DDC expression levels and the clinicopathological characteristics of the patients with PCa, we selected as a threshold the median value (50th percentile) from the population. The association of the DDC expression status with the patients' staging and Gleason score was analysed using Fisher's exact test and the chi-squared test, respectively. The analysis of DDC expression levels, as a continuous variable, and the patients' Gleason score was evaluated using the non-parametric Kruskal–Wallis test.
The assessment of the prognostic value of DDC expression for patients with PCa was done using Cox proportional regression and Kaplan–Meier survival analysis. The P value of the Cox proportional hazard regression analysis was evaluated by the test for trend approach. The construction of the Kaplan–Meier curve was carried out with the graphic representation of the percentage probability of the DFS to the interval time after RP, using as the threshold the expression level of the DDC gene corresponding to the median value (50th percentile) of the patient cohort. The P value was calculated using the log-rank test. The statistical analyses used in the present study are in accordance with current trends regarding the clinical evaluation of novel tissue biomarkers [26,27].
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The age range (mean, median) of the 70 patients with PCa who underwent radical retropubic prostatectomy was 52.0–76.0 (64.9, 65.0) years. In the same patient cohort, the preoperative serum PSA level had a range (mean, median) of 2.56–41.80 (9, 7.65) ng/mL. The follow-up period of the 56 patients with PCa who were successfully monitored had a range (mean, median) of 1.0–62.0 (28.6, 31.5) months. During the follow-up period of the monitored patients with PCa, the serum PSA level varied from 0.00 to 4.00 ng/mL, with a mean of 0.231 ng/mL and a median of 0.075 ng/mL.
There was a statistically significant association (P= 0.003) between the expression level of the DDC gene and pathological (TNM) stage of the disease (Table 1). In particular, the expression of the gene at higher levels than the adopted threshold point went up from 32.5% of patients with localized PCa (stages pT1 and pT2) to 70% of those with locally advanced disease (stages pT3 and pT4). Regarding the grade of differentiation (Gleason score), there was also a statistically significant association (P= 0.039) with DDC expression. More precisely, 33.3% of the patients with Gleason score ≤6 were shown to be DDC(+) (i.e. they had an expression level higher than the median value threshold), compared with 52.8% of patients with Gleason score = 7, and 85.7% of patients with Gleason score >7. The same conclusions were reached concerning the association of DDC expression with Gleason score using the study of gene expression as a continuous variable (Fig. 1).
Table 1. Correlation between DDC expression status and clinicopathological features of patients with PCa
|Variable||No. of patients (%)||Patients, n (%)|| P value|
| DDC(–)*|| DDC(+)*|
|DRE|| || || ||0.44†|
| Positive||30 (42.8)||17 (56.7)||13 (43.3)|
| Negative||31 (44.3)||14 (45.2)||17 (54.8)|
| Suspicious||9 (12.9)|| || |
|Patient stage|| || || ||0.003†|
| pT1/pT2||40 (57.2)||27 (67.5)||13 (32.5)|
| pT3/pT4||30 (42.8)||9 (30.0)||21 (70.0)|
|Gleason score|| || || ||0.039‡|
| ≤6||27 (38.6)||18 (66.7)||9 (33.3)|
| 7||36 (51.4)||17 (47.2)||19 (52.8)|
| >7||7 (10.0)||1 (14.3)||6 (85.7)|
Figure 1. Box plots representing the DDC mRNA expression levels in Gleason score ≤6 (n= 27), 7 (n= 36), >7 (n= 7). The horizontal bold line represents the median value (50th percentile) of the patients' DDC expression. The level of statistical significance (P value) was calculated by the non-parametric Kruskal–Wallis test. c/Kc, DDC mRNA copies/103 GAPDH mRNA copies.
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The study of the prognostic significance of DDC expression levels was carried out using the DFS, as none of the patients with PCa died during follow-up. Consequently, it cannot be related to the overall survival of patients. Patients with PCa who had an expression level lower than the median value, were described as ‘negative’DDC, i.e. DDC(–) (cf. DDC(+) for those with expression levels higher than the median value).
There was a statistically significant correlation (P= 0.021) between the DDC expression and the relative risk of patients with PCa recurrence (Table 2). In particular, DDC(+) patients had an almost threefold higher risk (hazard ratio [HR]= 2.72, 95% CI: 1.16–6.36) of biochemical recurrence than DDC(–) patients (HR = 1.00). Moreover, the prognostic significance of preoperative PSA serum level, age, DRE, Gleason score and pathological stage was also evaluated in the same patient cohort (Table 2). As expected, there was a statistically significant association between patient outcome and preoperative PSA level (HR = 1.10, 95% CI: 1.03–1.17, P= 0.004), Gleason score (HR = 3.80, 95% CI: 1.81–8.00, P < 0.001) and TNM stage (HR = 1.65, 95% CI: 1.23–2.23, P= 0.001).
Table 2. Cox proportional regression analysis for the prediction of DFS in patients with patients'
|Covariant||Univariate analysis||Multivariate analysis|
|HR*||95% CI†|| P value‡||HR*||95% CI†|| P value‡|
| DDC || || || || || || |
| Positive||2.72|| || ||2.76§||0.67–4.32¶||0.26¶|
| || || || ||1.71¶|| || |
|DRE|| || || || || || |
| Positive||1.65|| || ||1.25§|| || |
|Gleason score (orbital)||3.80||1.81–8.00||<0.001||2.47¶||1.11–5.51¶||0.026¶|
In the present study, models of multivariate Cox proportional hazard regression analysis were adjusted for DDC expression, preoperative PSA, age and DRE, as well as DDC expression, Gleason score and pathological stage of the patients who were followed (Table 2). The study of the expression of the DDC gene as a partition variable denotes its independence from preoperative PSA, age and DRE clinical value for the prediction of DFS in patients with PCa (HR = 2.76, 95% CI: 1.06–7.18, P= 0.036). Focusing on the multivariate model adjusted for the expression level of the DDC gene, Gleason score and pathological stage, an independent clinical value for the prediction of patients' DFS was shown only for Gleason score (HR = 2.47, 95% CI: 1.11–5.51, P= 0.026). The DDC expression profile (HR = 1.71, 95% CI: 0.67–4.32, P= 0.26) was not independent of Gleason score or TNM stage.
The predictive value of DDC expression in the prognosis of DFS among patients with PCa was also proved using Kaplan–Meier survival analysis (Fig. 2). On the Kaplan–Meier curve there was a statistically significant difference (P= 0.015) in the DFS between the two groups. More specifically, DDC(+) patients had a less favourable outcome, as depicted by the significantly shorter DFS period than for DDC(–) patients.
Figure 2. Kaplan–Meier curves of the DFS of patients with PCa in relation to DDC gene expression levels. *Threshold = 9.00 c/Kc, equal to the median value.
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This is the first study indicating the potential use of DDC expression as a novel prognostic biomarker in patients with PCa who have undergone RP. Understanding the biology of PCa is essential for the discovery of new therapeutic strategies and for obtaining a prognosis for the disease. This has led to an increased research interest to find novel tumour biomarkers with advanced clinical benefits . Prognostic biomarkers in PCa aim to predict the natural course of the disease and the tumour's outcome .
Several factors have been extensively studied in PCa with regard to their prognostic value, both clinically and biologically. Preoperative PSA level, Gleason score, seminal vesicle invasion and margin status have to be considered to predict biochemical failure after RP [3–5]. Surgical margins and Gleason score are independent prognostic markers of biochemical recurrence [30,31]. Factors such as PSA level, Gleason score, pathological stage and PSA doubling time are the strongest variables in predicting the time course to the development of metastatic disease after initial biochemical recurrence and survival [6–8].
It is important that patients are properly informed by their physicians about the likelihood of treatment success, complications and morbidity. Cancer recurrence is a concern for those patients who decide to undergo RP as a definitive treatment with curative intent for localized PCa. Physicians' clinical judgment has long formed the basis for the prediction of outcome and patient counselling. It's clear, however, that this prediction process is not accurate enough. Another approach for risk estimation is the stratification of groups of patients at risk. This method offers no possibility for individualization . To obtain more accurate predictions of biochemical recurrence and disease progression among patients with PCa who have undergone RP as a definitive treatment, in recent years researchers have developed several predictive and prognostic tools, including nomograms, probability tables, risk groupings, artificial neural networks and classification and regression tree (CART) analyses . Nomograms are currently the most accurate tools for predicting the outcome of patients who undergo definitive treatment for localized PCa [9,33]. It is also important for investigators to be aware of the pitfalls and difficulties of developing statistically valid classifiers that will truly benefit patients by improving the ability to predict a patient's risk of progression .
The major discovery of PSA in the 1980s superseded biomarkers for PCa that had been in use for nearly a century. However, the use of PSA as a cancer-specific test has several well recognized limitations. Many studies have aimed to improve the performance of PSA as well as identify additional biomarkers. Biomarkers for the prognosis of PCa include DNA-based, RNA-based and protein-based markers . Detection of biomarkers in bodily fluids (serum and urine) could, in the near future, provide valuable clinical information while avoiding unnecessary invasive procedures . This is why the focus of molecular diagnostics is rapidly moving from tissues to bodily fluids, particularly to blood and urine . Analysis of all tissue, blood and urine biomarkers in PCa is difficult and goes beyond the scope of this article. New research methods and current advances in molecular techniques have provided new tools facilitating the discovery of new biomarkers for PCa. Discoveries in genetics could represent a new revolution in PCa research . The extensive clinical validation of novel biomarkers remains one of the most significant challenges; however, the process of searching for new biomarkers should be continued because we might discover new pathways of PCa that could lead to a cure .
The interaction of the DDC enzyme with ARs amplifies the function of the latter as a transcriptional factor as well as their ability to be activated by low blood concentrations of androgens . The co-expression of DDC and ARs in a large proportion of cells in the prostate is particularly significant. A study also found significant increases in the expression levels of DDC in NE prostate cells after long-term hormonal therapy in patients with PCa . The increased expression of DDC induces the progression of the disease to a higher stage, both through the activation of AR transcriptional activity, upsetting the balance of the cellular circle, and through the mitotic activity of NE agents, whose population is increased in the disease . A recent study showed that carbidopa, a known DDC inhibitor, can restrain PCa progression in vitro and in vivo, via the blockage of the DDC-dependent co-activation of AR .
In the present study we analysed, for the first time to our knowledge, the mRNA expression levels of the DDC gene in patients with PCa and their association with biochemical recurrence and prognosis after RP. The quantification of DDC expression was accomplished using a highly sensitive reverse transcriptase quantitative real-time PCR methodology. RP specimens allow better pathological assessment than do prostate biopsy specimens. Thus, in the present study, the correlation between DDC and prognosis is probably more accurate than in most studies that have based pathological assessment on biopsy samples . The results of a recent study showed higher gene expression in patients with PCa than in those with BPH, as well as highlighting the significant clinical value of the quantification of DDC expression as a new marker for the differential diagnosis of PCa from BPH . In the present study, with a bigger number of tested PCa samples, higher levels of gene expression were associated with advanced disease stages and a higher degree of malignancy, thus indicating a poorer prognosis and a shorter life expectancy for the patients, as well as highlighting the need to determine the best possible treatment.
Between 27% and 53% of patients undergoing RP will have a detectable rise in serum PSA within 10 years after surgery [39,40]. Two-thirds of biochemical recurrence occurs during the first 2 years after RP . In the present study 25/56 (44.6%) patients had a biochemical recurrence during the monitoring period, the mean value of which was almost 29 months. A large proportion of the patient cohort had PCa of stage ≥ pT3a and Gleason score ≥7.
The DDC(+) patients had an almost threefold higher risk of recurrence than the DDC(–) patients (P= 0.021). This association of the DDC expression levels and the shorter DFS for patients with PCa was independent of preoperative PSA, age and DRE (P= 0.036). However, the multivariate Cox proportional hazard regression model adjusted for DDC expression, Gleason score and TNM stage showed an independent correlation of patients' DFS with Gleason score only (P= 0.026). To emphasize the prognostic value of this specific molecular marker regarding the DFS expectancy of patients with PCa, a Kaplan–Meier survival analysis was carried out. There was a statistically significant difference (P= 0.015) in DFS between DDC(+) and DDC(–) patients. The probability of DFS at 1 year after RP for DDC(–) patients is 97%, while for DDC(+) patients it is 80%;p at 2 years the figures are 93% and 60%, and at 3 years 80% and 55%, respectively. Thus, DDC(+) patients with PCa have a significantly lower expectancy of DFS. All of this reinforces the prognostic value of this specific molecular marker and illustrates the potential benefits of its clinical application for predicting DFS expectancy in patients with PCa.
The present study has several limitations. Specifically, we had to analyse the expression levels in a larger number of samples and use longer follow-up periods. Undoubtedly, the analysis of the mRNA expression of the DDC gene in tissues is not of great benefit in daily clinical practice. This should be carried out before surgery. However, this increase of the mRNA levels in tissues constitutes a first step in researching its clinical value, suggesting a direct analysis of mRNA and/or its protein expression level in preoperative biopsy, blood serum and urine. It would also be valuable if we were able to test DDC biomarker reactivity by immunohistochemistry or fluorescence in situ hybridization.
There was a statistically significant positive association between the mRNA expression levels of the DDC gene and the pathological stage and grade of differentiation of PCa. There was also a statistically significant correlation between the DDC gene expression levels and the relative risk of recurrence as well as the shorter DFS period of patients with PCa treated with RP. While only Gleason score was shown to be an overall independent prognostic marker for the prediction of biochemical recurrence in RP-treated patients, the unfavourable prognostic value of DDC expression levels in relation to the DFS period for patients with PCa was shown to be independent of the preoperative PSA level, age and the DRE.