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Keywords:

  • prostate cancer;
  • epithelial–mesenchymal transition;
  • radical prostatectomy;
  • biochemical recurrence

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

What's known on the subject? and What does the study add?

  • There have been few studies evaluating the prognostic value of epithelial–mesenchymal transition markers in prostate cancer; therefore the significance of these markers in the prognosis of patients with prostate cancer, particularly those with localized disease, remains largely unknown.
  • Consideration of the expression levels of potential epithelial–mesenchymal transition markers, particularly Twist and vimentin, in addition to conventional prognostic parameters would contribute to the prediction of biochemical recurrence after radical prostatectomy for localized prostate cancer.

Objective

  • To analyse the expression patterns of multiple molecular markers implicated in epithelial–mesenchymal transition (EMT) in localized prostate cancer (PC), in order to clarify the significance of these markers in patients undergoing radical prostatectomy (RP).

Patients and Methods

  • Expression levels of 13 EMT markers, namely E-cadherin, N-cadherin, β-catenin, γ-catenin, fibronectin, matrix metalloproteinase (MMP) 2, MMP-9, Slug, Snail, Twist, vimentin, ZEB1 and ZEB2, in RP specimens from 197 consecutive patients with localized PC were evaluated by immunohistochemical staining.

Results

  • Of the 13 markers, expression levels of E-cadherin, Snail, Twist and vimentin were closely associated with several conventional prognostic factors.
  • Univariate analysis identified these four EMT markers as significant predictors for biochemical recurrence (BR), while serum prostate-specific antigen, Gleason score, seminal vesicle invasion (SVI), surgical margin status (SMS) and tumour volume were also significant.
  • Of these significant factors, expression levels of Twist and vimentin, SVI and SMS appeared to be independently related to BR on multivariate analysis.
  • There were significant differences in BR-free survival according to positive numbers of these four independent factors. That is, BR occurred in four of 90 patients who were negative for risk factors (4.4%), 21 of 83 positive for one or two risk factors (25.3%) and 19 of 24 positive for three or four risk factors (79.2%).

Conclusion

  • Measurement of expression levels of potential EMT markers, particularly Twist and vimentin, in RP specimens, in addition to conventional prognostic parameters, would contribute to the accurate prediction of the biochemical outcome in patients with localized PC following RP.

Abbreviations
RP

radical prostatectomy

PC

prostate cancer

BR

biochemical recurrence

SMS

surgical margin status

EMT

epithelial–mesenchymal transition

SVI

seminal vesicle invasion

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

Although radical prostatectomy (RP) has been the mainstay of treatment for men with clinically organ confined prostate cancer (PC), biochemical recurrence (BR), defined as a persistent increase in the serum value of PSA, occurs in approximately 30% of men undergoing RP [1]. To date, a number of studies have been carried out in order to identify parameters closely associated with postoperative prognostic outcomes of RP, which would be of potential utility for planning both additional postoperative therapy and follow-up schedule in an individual patient. Consequently, there are various factors shown to have a significant impact on the development of BR following RP, such as PSA, Gleason score, pathological stage, nodal involvement, surgical margin status (SMS) and tumour volume [2, 3]. Moreover, several recent studies have demonstrated the usefulness of nomograms predicting survival after definitive therapy for localized PC, which were developed by considering the significance of multiple clinicopathological parameters [4, 5]. However, PC has been shown to be characterized by unique biological features as well as heterogeneous genetic backgrounds [6], suggesting the limitations for predicting postoperative outcomes in patients with localized PC using conventional clinicopathological parameters alone.

Epithelial–mesenchymal transition (EMT) is a process in which polarized epithelial cells are converted into motile mesenchymal cells, accompanying alterations during this process in adhesion, morphology, cellular architecture and migration potential [7]. Initially, EMT was investigated focusing on its role in embryonic development; however, this process has also been shown to be implicated in the progression of a wide variety of malignant tumours, including PC [8-12]. For example, Xu et al. [9] reported that human PC cells undergo EMT through cellular interaction with the host microenvironment, resulting in an increased metastatic potential to bone and adrenal gland.

In recent years, the induction of EMT in tumour cells has been demonstrated to require the involvement of multiple complex signalling pathways [13]; accordingly, a wide variety of molecular markers mediating the execution of EMT have been identified, and the usefulness of these markers as prognostic predictors in some types of malignant tumour has recently been reported [7, 8]. However, there have been few studies assessing the impact of EMT markers on the progression of PC [14, 15], and it remains largely unknown whether these markers have significance in the prognosis of patients with PC, particularly those with clinically localized disease. Considering these findings, we evaluated the expression patterns of multiple EMT markers, namely E-cadherin, N-cadherin, β-catenin, γ-catenin, fibronectin, matrix metalloproteinase (MMP) 2, MMP-9, Slug, Snail, Twist, vimentin, ZEB1 and ZEB2, in RP specimens from 197 consecutive patients with localized PC to clarify the prognostic impact of these markers in this category of patients.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

The study included a total of 197 consecutive patients who were diagnosed as having clinically organ confined PC and subsequently underwent RP and bilateral pelvic lymphadenectomy without any neoadjuvant therapies between 2005 and 2008 at our institution. The staging procedures included DRE, transrectal ultrasonography, serum PSA assay, CT, MRI and/or bone scan. In this series, RP was performed based on the procedure described by Walsh [16] in combination with that modified by others [17]. The median duration of postoperative follow-up was 61 months (range 19–107 months). Informed consent for performing immunohistochemical staining of RP specimens was obtained from all these patients, and the study design was approved by the research ethics committee of our institution.

All pathological examinations were generally performed under the guidance of a single pathologist according to the 2002 TNM classification system. The surface of the resected specimen was inked and fixed, and whole-mount step sections were cut transversely at 3 mm intervals from the apex of the prostate to the tips of the seminal vesicles. A positive surgical margin was defined as the presence of cancer cells at the inked margin. As for Gleason grading, a modified system based on the International Society of Urological Pathology consensus conference [18] was used. The total tumour volume was determined by planimetry using a digitizer tablet, as described previously [19]. After RP, patients were followed by periodic measurement of the serum PSA value at least every 3 months for the first 2 years and every 6 months thereafter. In this series, biochemical recurrence was defined as PSA ≥ 0.2 ng/mL on two consecutive measurements. Irrespective of the pathological findings suggesting a poor prognosis, none of the patients was treated with any adjuvant therapies until their serum PSA values reached 0.4 ng/mL or more.

Immunohistochemical staining of RP specimens was performed as previously described [20]. Briefly, sections from formaldehyde-fixed, paraffin-embedded tissue from 197 specimens were deparaffinized by xylene and rehydrated in ethanol. After blocking endogenous peroxidase with hydrogen peroxidase, sections were boiled in 0.01 M citrate buffer for 10 min and incubated with 5% normal blocking serum for 20 min. The sections were then incubated with the following anti-human antibodies: E-cadherin mouse monoclonal antibody, N-cadherin mouse monoclonal antibody (Dako, Carpinteria, CA, USA), β-catenin mouse monoclonal antibody (BD Transduction Laboratories, Franklin Lakes, NJ, USA), γ-catenin mouse monoclonal antibody, fibronectin mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP-2 mouse monoclonal antibody, MMP-9 mouse monoclonal antibody (Daiichi Fine Chemical, Toyama, Japan), Slug rabbit polyclonal antibody, Snail rabbit polyclonal antibody, Twist rabbit polyclonal antibody (Abcam, Cambridge, UK), vimentin mouse monoclonal antibody, ZEB1 rabbit polyclonal antibody (Santa Cruz Biotechnology) and ZEB2 rabbit polyclonal antibody (Assay Biotechnology, Sunnyvale, CA, USA). The sections were then incubated with biotinylated goat anti-mouse or rabbit IgG (Vector Laboratories, Burlingame, CA, USA). After incubation in an avidin–biotin peroxidase complex for 30 min, the samples were exposed to diaminobenzidine tetrahydrochloride solution and counterstained with methyl green.

Staining outcomes were evaluated by two independent observers who were blinded to the clinicopathological data on the 197 patients. According to previous studies [21, 22], the staining intensity was visually scored as 0 (no staining at all), 1 (weak), 2 (medium) or 3 (strong), and the staining extent was also scored as 0 (0%–5%), 1 (5%–25%), 2 (26%–75%) or 3 (75%–100%). The intensity and extent scores were summed to obtain a composite score, and a score ≤3 and >3 was considered as weak and strong expression, respectively.

All statistical analyses were performed using Statview 5.0 software (Abacus Concepts Inc., Berkeley, CA, USA), and P values less than 0.05 were considered significant. A chi-squared test was used to analyse the association between several clinicopathological factors and expression levels of EMT markers. The BR-free survival rates were calculated employing the Kaplan–Meier method, and differences were determined by the log-rank test. The prognostic significance of certain factors was assessed using the Cox proportional hazards regression model.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

Table 1 summarizes the clinicopathological characteristics of the 197 patients included in the study. We initially analysed the association between several conventional clinicopathological parameters and expression levels of 13 EMT markers in RP specimens. As shown in Table 2, expression levels of E-cadherin, Snail, Twist and vimentin were significantly associated with several conventional prognostic factors; however, the remaining nine markers showed no significant correlation with conventional prognostic factors.

Table 1. Patient characteristics.
Median age (years, range)68.5 (51–79)
Median preoperative serum PSA (ng/mL, range)9.1 (3.7–50.6)
Pathological stage (%) 
pT2118 (59.9)
pT3a50 (25.4)
pT3b25 (12.7)
pT44 (2.0)
Gleason score (%) 
6 or less53 (26.9)
7107 (54.3)
8 or greater37 (18.8)
Seminal vesicle invasion (%) 
Negative170 (86.3)
Positive27 (13.7)
Surgical margin status (%) 
Negative142 (72.1)
Positive55 (27.9)
Median tumour volume (mL, range)1.18 (0.24–5.51)
Table 2. Correlation of expression levels of EMT markers in RP specimens with conventional prognostic factors.
Molecular markeraAge (years)PSA (ng/mL)Pathological stage (%)Gleason scoreSeminal vesicle invasionSurgical margin statusTumour volume (mL)
<7070?P<1010?PpT2pT3, pT4P?78?P+P+P<1.01.0?P
  1. a

    Values are expressed as the number of patients with strong expression of each molecule.

E-cadherin29250.7440140.01636180.235040.0124950.2644100.0713420<0.001
N-cadherin61500.3171400.2560510.05789220.6796150.9375360.1142690.68
β-catenin20180.9123150.9923150.9328100.193350.9127110.8815230.96
γ-catenin26260.7732200.8530220.7141110.614480.6837150.8621310.82
Fibronectin24220.9527190.7926200.593790.884060.8832140.6621250.48
MMP-247400.5749380.3053340.7970170.8174130.6562250.8232550.56
MMP-970690.5480590.2184550.81111280.45118210.66103360.3359800.13
Slug70650.9885500.2885500.20108270.52118170.50100350.3657780.18
Snail57570.5664500.01165490.3387270.03993210.02474400.00863084<0.001
Twist45420.993057<0.0013156<0.0015730<0.00166210.00265037<0.0011770<0.001
Vimentin40390.792554<0.0012950<0.0015128<0.0016019<0.0014633<0.0011168<0.001
ZEB164640.5078500.8475530.61103250.71111170.8193350.8149790.75
ZEB267610.8372560.1073550.26103250.71109190.5395330.3655730.13
Overall10295119781187916037170271425577120

During the observation period of the study, BR occurred in 44 of the 197 patients (22.3%), and 1-, 3- and 5-year BR-free survival rates were 95.5%, 85.7% and 78.2%, respectively (Fig. 1). We then evaluated the impact of the expression patterns of EMT markers in addition to conventional prognostic parameters on the prediction of BR-free survival in the 197 patients. Univariate analysis using the Cox proportional hazards regression model showed that expression levels of E-cadherin, Snail, Twist and vimentin were identified as significant factors associated with BR-free survival, while serum PSA, Gleason score, seminal vesicle invasion (SVI), SMS and tumour volume were also significant among several conventional parameters examined (Table 3). Moreover, overexpression of Twist and vimentin and SVI and SMS appeared to be independently related to BR-free survival on multivariate analysis of the nine significant parameters determined by univariate analysis (Table 3). Typical findings of an immunohistochemical study for evaluating Twist and vimentin expression in RP specimens are presented in Fig. 2. BR-free survival curves according to the expression level of Twist and vimentin and SVI and SMS are shown in Fig. 3, and significant differences in BR-free survival with respect to all these four factors were noted.

figure

Figure 1. BR-free survival of 197 patients with clinically localized PC who underwent RP.

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figure

Figure 2. Representative findings on immunohistochemical staining of RP specimens with a Twist or vimentin antibody: (A) PC with weak expression of Twist; (B) PC with strong expression of Twist; (C) PC with weak expression of vimentin; (D) PC with strong expression of vimentin.

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figure

Figure 3. BR-free survival of patients with clinically localized PC who underwent RP according to (A) the expression level of Twist, (B) the expression level of vimentin, (C) SVI and (D) SMS.

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Table 3. Univariate and multivariate analyses of the association between various parameters with recurrence-free survival in 197 patients with PC who underwent RP.
VariableNo. of patients (no. of patients with biochemical recurrence)Univariate analysisMultivariate analysis
Hazard ratioPHazard ratioP
Age (years) (<70 vs 70?)102 (22) vs 95 (22)0.980.89
Serum PSA (ng/mL) (<10 vs 10?)119 (20) vs 78 (24)2.690.0391.670.25
Pathological stage (pT2 vs pT3 or pT4)118 (19) vs 79 (25)2.550.0451.100.44
Gleason score (?7 vs 8?)160 (25) vs 37 (19)2.980.0231.710.19
Seminal vesicle invasion (negative vs positive)170 (29) vs 27 (15)3.830.00852.150.039
Surgical margin status (negative vs positive)142 (21) vs 55 (23)4.33<0.0012.970.0098
Tumour volume (mL) (<1.0 vs 1.0?)77 (11) vs 120 (33)2.800.0351.600.29
E-cadherin (weak vs strong expression)143 (22) vs 54 (22)3.090.019
N-cadherin (weak vs strong expression)86 (18) vs 111 (26)1.690.18
β-catenin (weak vs strong expression)159 (35) vs 38 (9)1.630.21
γ-catenin (weak vs strong expression)145 (32) vs 52 (12)1.600.47
Fibronectin (weak vs strong expression)151 (31) vs 46 (13)1.090.52
MMP-2 (weak vs strong expression)110 (24) vs 87 (20)1.550.29
MMP-9 (weak vs strong expression)58 (14) vs 139 (30)1.680.19
Slug (weak vs strong expression)62 (13) vs 135 (31)3.090.011
Snail (weak vs strong expression)83 (15) vs 114 (29)3.610.00711.900.14
Twist (weak vs strong expression)110 (14) vs 87 (30)4.13<0.0012.330.023
Vimentin (weak vs strong expression)118 (15) vs 79 (29)4.37<0.0013.510.0079
ZEB1 (weak vs strong expression)69 (20) vs 128 (24)1.250.52
ZEB2 (weak vs strong expression)69 (18) vs 128 (26)1.200.59

To more precisely assess the biochemical outcome following RP, we categorized patients according to positive numbers of four independent risk factors for BR identified by multivariate analysis. BR occurred in four of 90 patients who were negative for risk factors (4.4%), 21 of 83 positive for one or two risk factors (25.3%) and 19 of 24 positive for three or four risk factors (79.2%). As shown in Fig. 4, there were significant differences in BR-free survival among these three groups.

figure

Figure 4. BR-free survival of patients with clinically localized PC who underwent RP according to the number of independent risk factors for postoperative BR, including strong expression of Twist, strong expression of vimentin, SVI and SMS.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

It is important to precisely predict the prognostic outcome of patients following surgical resection of malignant tumours using a widely accepted consensus in order to determine the postoperative follow-up schedule as well as additional treatment. In patients with PC as well, a number of studies have been carried out to characterize factors associated with BR following RP [2, 3]. In addition, various types of nomogram for calculating survival after definitive treatment for localized PC have been developed based on multiple clinicopathological factors [4, 5]. Although some of these systems were shown to be useful, several limitations concerning their abilities to precisely predict outcomes following RP using conventional parameters alone have also been suggested [23]. Therefore, the prognostic significance of numerous molecular markers involved in the pathogenesis of PC has been investigated [24, 25]. Recently, EMT has been demonstrated to play crucial roles during the progression of malignant tumours, and several molecules implicated in EMT could be used as useful prognostic markers in patients with some types of malignant tumour [8-12]. However, the significance of EMT markers as prognostic indicators for patients with PC, particularly those with localized disease, remains largely unknown [14, 15]. Considering these findings, we analysed the values of multiple potential EMT markers in addition to conventional clinicopathological prognostic factors for predicting the clinical course in 197 patients undergoing RP for clinically localized PC.

It has been well documented that EMT, consisting of multiple molecular steps, could have a significant influence on diverse events driving the progression of malignant tumours, such as carcinogenesis, cell adhesion, invasion, metastasis and resistance to pro-apoptotic signals; accordingly, the execution of this process has been shown to be achieved by several types of molecule, such as epithelial markers, mesenchymal markers and transcriptional factors [7, 8]. Of such markers underlying EMT, expression levels of 13 potential markers in RP specimens were evaluated in this series. Despite the expression of these EMT markers, even slightly, in the majority of RP specimens, there appeared to be varied expression patterns of the markers with respect to the relation with conventional parameters. That is, expression levels of E-cadherin, Snail, Twist and vimentin, but not those of the remaining nine markers, were significantly associated with major prognostic indicators. Collectively, these findings suggest that the loss of E-cadherin as well as overexpression of Snail, Twist and vimentin may play important roles in the acquisition of a malignant phenotype in localized PC. However, the spatial and/or temporal heterogeneity of EMT in a comparatively early stage of malignant diseases could be observed [13], suggesting the need for careful interpretation of the findings of immunohistochemical staining reported in the present study.

The impacts of established prognostic parameters and potential EMT markers on BR-free survival following RP were then compared. Univariate analysis showed that the preoperative PSA value, Gleason score, SVI, SMS and tumour volume and expression levels of E-cadherin, Snail, Twist and vimentin were significantly associated with BR; however, only SVI, SMS and expression levels of Twist and vimentin were identified as independent predictors of the biochemical outcome on multivariate analysis. SVI and SMS are well accepted prognostic indicators in patients with PC undergoing RP [1-3]. The utility of vimentin expression as a predictor for BR was confirmed in a previous study; i.e. Zhang et al. [14] performed an immunohistochemical study using a tissue microarray, and reported that there was a high risk of BR associated with tumours that displayed high levels of vimentin expression. As for the expression of Twist, a helix–loop–helix transcriptional factor [26], there have been no data demonstrating the impact on the prognosis of patients with PC; however, our present outcome could be theoretically supported by the elucidated function of Twist, which is characterized by the transcriptional repression of E-cadherin expression, resulting in a loss of E-cadherin-mediated cell–cell adhesion and an enhancement of cell motility [26].

It is of interest to develop the system to more precisely predict the biochemical outcome following RP by combining potential prognostic indicators in order to allow better individualization of the postoperative management. Based on the outcomes achieved by multivariate analysis, the 197 patients were classified into the following three groups according to the positive number of independent risk factors for BR: negative for any risk factors, positive for one or two risk factors and positive for three or four risk factors. Significant differences in BR-free survival among these three groups were noted. Furthermore, if classification is performed using only SVI and SMS, there was no significant difference in BR-free survival between patients positive for either factor and those positive for both factors (data not shown). Although it should be strictly analysed whether the improved prediction of BR by additional evaluation of EMT markers justifies the efforts and costs compared with use of the clinicopathological parameters alone, these findings suggest that a simultaneous consideration of the four major risk factors identified in this series (SVI, SMS, Twist and vimentin expression) may contribute to develop a novel predicting system that can be more closely related to BR after RP.

Here, the limitations of the present study should be described. A sample size of 197 men in such a common disease like PC is not large enough to draw definitive conclusions on prognostic issues. In addition, there have been several studies demonstrating significant differences between Japanese and western populations in the characteristics of PC [27]; hence, it would be difficult to apply the present findings to the general population with PC. In fact, although the study included men who were diagnosed as having organ confined PC alone, clinicopathological features were relatively adverse compared with large series performed in western countries in the recent PSA era [1]. Furthermore, it should be addressed whether additional analysis of EMT markers to the established combination of conventional clinicopathological parameters, such as nomogram and look-up table, substantially improves the accuracy for predicting BR in order to define the real prognostic impact of these markers in patients undergoing RP. Finally, the study included a total of 13 EMT markers as potential predictors for BR following RP; however, these 13 markers had been selected based on subjective rather than scientifically objective criteria. Therefore, there might be some other molecules more closely associated with biochemical outcome than these 13 markers. Even in the field of EMT, the involvement of molecules relevant to other molecular events, such as cancer microRNA and stem cells [28, 29], has been clarified, suggesting the need for additional assessment, considering such a novel concept.

In the present study, we analysed the value of several conventional prognostic parameters and multiple molecular markers implicated in the process of EMT as predictors of BR following RP for clinically organ confined PC, and expression levels of Twist and vimentin in addition to SVI and SMS were identified as independent factors associated with the biochemical outcome after RP. Furthermore, the usefulness of the combined assessment of these four parameters for further refinement of this predicting system was suggested. However, a prospective study including additional candidate markers underlying the malignant progression of localized PC is warranted before a definitive conclusion can be drawn.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References
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