Scandinavian Journal of Urology. 2015; 49: 51–57
ORIGINAL ARTICLE
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TWIST overexpression predicts biochemical recurrence-free survival in prostate cancer patients treated with radical prostatectomy
SAMI RAATIKAINEN1,2, SIRPA AALTOMAA1, JORMA J. PALVIMO3, VESA KÄRJÄ4 & YLERMI SOINI5 Departments of 1Surgery and, 4Clinical Pathology, Kuopio University Hospital, Kuopio, Finland, 2Department of Surgery, Institute of Clinical Medicine, School of Medicine, and, 3Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland, and 5Department of Pathology and Forensic Medicine, Institute of Clinical Medicine, School of Medicine, University of Eastern Finland, Cancer Center of Eastern Finland, Kuopio, Finland
Abstract Objective. The aim of this study was to determine whether TWIST and androgen receptor (AR) expression can predict the outcome in radical prostatectomy (RP) patients. Material and methods. Samples from different tumour areas of 181 prostate cancer patients were analysed for TWIST and AR expression, and the results were correlated with known clinicopathological data and biochemical recurrence-free survival (BFS). Results. TWIST overexpression in the margin area of the tumour (M-TWIST) was related to positive surgical margin (p = 0.047), capsule invasion (p = 0.006) and biochemical recurrence (BCR) (p = 0.004). AR expression in the margin area of the tumour (M-AR) was associated with high Gleason score (p = 0.004), positive surgical margin (p = 0.004) and BCR (p = 0.05). M-TWIST overexpression was clearly associated with M-AR expression (p < 0.0001). Four parameters, i.e. M-TWIST overexpression (p < 0.0001), positive surgical margin (p = 0.003), high Gleason score (p < 0.0001) and M-AR expression (p = 0.008), predicted BFS. In the multivariate analysis, M-TWIST overexpression (p = 0.011) and Gleason score (p = 0.002) were the only independent predictors of BFS. Conclusions. M-TWIST overexpression is associated with clinicopathological prognosis factors and M-AR overexpression and is a powerful independent predictor of BFS in conjunction with the Gleason score in prostate cancer patients treated with RP.
Key Words: androgen receptor, prognosis, prostate cancer, radical prostatectomy, TWIST
Introduction Prostate cancer (PC) is the most common neoplasm in men living in Western countries. Nowadays, as a result of prostate-specific antigen (PSA) testing, it is diagnosed more often at an early stage without clinical symptoms and in young men with a long life expectancy [1,2]. The curative treatments, radical prostatectomy (RP) and radiation therapy, may decrease quality of life since they are associated with undesirable side-effects [3]. Alternatively, when PC is not aggressive and the lesion of the PC is small, the recommended option is active surveillance [4]. The treatment decision is traditionally
based on recognized clinicopathological prognostic factors, such as T class, PSA value and Gleason score; of these, the Gleason score is considered the most powerful prognosticator [5,6]. Many genetic and molecular factors have been examined in efforts to achieve biochemical recurrence-free survival (BFS) in localized PC that would be better than the clinicopathological prognostic factors. There are several studies demonstrating that expression of growth signal factors and factors associated with apoptosis, angiogenesis and tissue invasion [Ki-67, androgen receptor (AR), P53, BCL2, vascular endothelial growth factor, transforming growth factor-b] are linked with poor outcome in PC patients [7].
Correspondence: S. Raatikainen, Kuopio University Hospital, PO Box 100, FI-70029 Kuopio, Finland. Tel: +358 44 7175712. Fax: +358 17 173749. E-mail: sami.raatikainen@kuh.fi
(Received 13 December 2013; revised 16 February 2014; accepted 24 March 2014) ISSN 2168-1805 print/ISSN 2168-1813 online 2015 Informa Healthcare DOI: 10.3109/21681805.2014.909529
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However, although several of these biomolecular markers have been claimed to be promising, none of them is actually used in clinical practice. TWIST is a basic helix-loop transcription factor involved in the regulation of several pathways that control cell growth, differentiation and epithelial–mesenchymal transition (EMT). During EMT, epithelial cells lose their cell contacts owing to the downregulation of adhesion molecules such as E-cadherin, and they exhibit a remodelling of the cytoskeleton via increased expression of a-smooth muscle actin and vimentin. In addition to its physiological functions, EMT is involved in tumour progression processes where early-stage tumours are converted into invasive malignancies. TWIST is an inducer of EMT and therefore its overexpression triggers EMT, allowing tumour cells to become invasive and metastatic [8–10]. Research conducted in the past decade has revealed that TWIST and the EMT process can be linked with progression of many malignancies, such as head and neck cancer, squamous cell carcinoma, rhabdomyosarcoma and breast cancer [10–13]. In PC cell lines, TWIST is known to act as a trigger of the EMT process, which could be envisaged to promote the progression of PC [14–16]. Kwok et al. examined human prostate samples and found that there was greater TWIST expression in malignant than in benign tissue. TWIST overexpression has also been associated with a high Gleason score [17]. Behnsawy et al. explored the outcome of PC patients and noted that TWIST overexpression was associated with many clinicopathological factors and also with BFS [18]. It has been found that induction of TWIST leads to AR expression in PC cells. Consequently, AR expression has been linked to castration resistance of PC, which is a condition with a poor prognosis [16]. The prognostic value of AR is controversial [7], although in several studies AR expression was shown to predict aggressive behaviour, biochemical recurrence (BCR) and clinical recurrence of PC [19–23]. This study investigated the prognostic value of TWIST and AR expression compared with clinicopathological prognostic factors and BFS in 181 PC patients treated with RP. Material and methods This study was approved by the research ethics committee of Kuopio University Hospital.
1998 and 2009. Clinical data, including PSA value, and clinical follow-up data were extracted from the laboratory database and patient records. All of the patients had a clinically localized tumour according to digital rectal examination and transrectal ultrasonography. Lymphadenectomy was performed on some of the patients, but none of these patients had positive lymph nodes. Bone scans were used to exclude distant metastases when there was a need for confirmation of clinical status. The follow-up was conducted after 2, 6 and 12 months, and then according to clinical practice. None of the patients received hormone therapy before RP. In 38 cases (21%), the immeasurable PSA was not reached. An elevation of the PSA value of 0.2 ng/ml or more was considered as a biochemical recurrence [24]. Histopathological analyses All the samples had been fixed in neutral-buffered formalin and embedded in paraffin. They were re-evaluated for pT class, Gleason score, capsule invasion and surgical marginal status by two pathologists (YS and VK) who were blinded to the clinical data and a consensus assessment was made in each case. The clinical tumour, node, metastasis (TNM) classification was conducted according to Union for International Cancer Control (UICC) guidelines [25]. The samples for the immunohistochemical analyses were selected from the PC tissues from four representative tumour regions (high Gleason, low Gleason, central and margin). The margin area or invasive front was defined as the boundary area between histological tumour mass and adjacent benign prostate tissue as visualized in haematoxylin–eosin-stained slides. The high and low Gleason areas represented the highest and lowest Gleason pattern found in the tumour. Samples were also collected from the corresponding benign prostate tissue, and all samples were placed into multitissue microarray blocks with a Beecher Instruments Manual Tissue Arrayer (Beecher Instruments, Silver Spring, MD, USA). The microarray sample diameter was 1300 mm. Adequate immunostaining of AR expression was available in 165 cases (91.2%). TWIST expression in different tumour areas was obtained in the samples as follows: high Gleason (n = 170, 93.9%), low Gleason (n = 169, 93.4%), central (n = 169, 93.4%) and margin (n = 167, 92.2%). These cases were included in the analyses. Immunohistochemistry
Study population In total, 181 PC patients were treated with RP in Kuopio University Hospital, Finland, between
Immunostaining was performed as follows. Tissue sections (4 mm thick) were cut from the paraffinembedded blocks. After deparaffinization and
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TWIST expression in prostate cancer rehydration, the sections were heated in a microwave oven for 2 5 min in Tris–EDTA buffer (pH 9.0), incubated in Tris–EDTA buffer for 20 min and washed twice for 5 min in phosphate-buffered saline (PBS). Hydrogen peroxide (5%, 5 min) was used to block endogenous peroxidase. Non-specific binding was blocked with 1.5% normal serum in PBS for 35 min at room temperature. The sections were incubated overnight at 4 C with mouse monoclonal anti-TWIST and anti-AR antibodies (dilutions 1:500 and 1:500, respectively). The TWIST antibody was purchased from Abcam (ab50887; Abcam, Cambridge, UK). The AR antibody has been described in an earlier publication [26]. The slides were then incubated with a biotinylated secondary antibody and avidin–biotin–peroxidase complex (ABC Vectastain Elite Kit; Vector Laboratories, Burlingame, CA, USA). Careful rinses were performed with PBS in each step of the immunostaining procedure. The colour was developed with diaminobenzidine tetrahydrochloride (DAP) (Sigma, St Louis, MO, USA). The slides were counterstained with Mayer’s haematoxylin, washed, dehydrated, cleared and mounted with Depex (BDH, Poole, UK). Ovarian tumour tissue with known positive TWIST expression was used as a positive control. In negative controls, the primary antibody was omitted. The immunoreactivity for TWIST and AR was first analysed in tumour cells as follows: 0–5% = negative, 6–50% = weak positive and 51–100% = strong positive. The data were then divided into two groups: negative = 0–5% and positive = 5–100%. The evaluation was performed by two pathologists (YS and VK) who were blinded to the clinical data and a consensus assessment was made in each case. In the samples, TWIST expression was nuclear and cytoplasmic whereas AR expression was nuclear. Statistical analysis The SPSS 19.0 program package was used for the statistical analyses. Differences between groups were analysed with the t test. The chi-squared test was used to assess differences in AR and TWIST expression compared with the clinical prognostic factors. BFS was analysed by the Kaplan–Meyer method. Multivariate analyses were conducted according to Cox’s method. A p value less than 0.05 was considered statistically significant.
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Table I. Demographic data of the patients. Characteristic Age (years)
62.3 ± 5.3
Follow-up (years)
7.6 (2.3–14.1)
PSA at diagnosis (ng/ml)
7.8 ± 6.7
PSA (ng/ml) 20
10 (5.5)
pT category 2
130 (71.8)
3
51 (28.2)
Gleason score 2–6
111 (61.4)
7
58 (32.0)
8–10
12 (6.6)
Capsule invasion No
131 (72.4)
Yes
50 (27.6)
Surgical marginal status Negative
115 (63.5)
Positive
66 (36.5)
BCR Yes
67 (37.0)
No
114 (63.0)
Data are shown as median ± SD, median (range) or n (%). PSA = prostate-specific antigen; BCR = biochemical recurrence.
tumour according to the histopathological analyses and belonged to pT categories 2 and 3. During the follow-up period, 67 patients (37.0%) experienced a BCR. There was no TWIST expression in benign tissue, in contrast to the situation in PC tissue, where TWIST was expressed in all samples taken from different tumour areas (high Gleason 8.2%, low Gleason 3.0%, central 4.7% and margin 11.4%) (Figure 1). Differences in TWIST expression in the malign parts of tumour were observed between the following tumour areas: high Gleason and low
A Twist
B BPH
Results The demographic data of the patients are presented in Table I. The median follow-up time was 7.6 years (range 2.3–14.1 years). All the patients had a localized
Figure 1. (A) In this TWIST-positive case, positive nuclear expression can be seen. The sample is taken from the margin part of the tumour. (B) In benign prostatic hyperplasia (BPH), no TWIST expression was detected.
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Table II. TWIST expression in benign samples and different parts of the tumour.
Benign
Positive expression
Negative expression
Benign vs malign
High Gleason vs other parts of tumour
Low Gleason vs central and margin
Central vs margin
n (%)
n (%)
p
p
p
p
0 (0)
169 (100)
High Gleason
14 (8.2)
156 (91.8)
< 0.0001
Low Gleason
5 (3.0)
164 (97.0)
0.025
0.029
Central
8 (4.7)
161 (95.3)
0.004
NS
NS
Margin
19 (11.4)
148 (88.6)
< 0.0001
NS
0.002
0.016
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NS = not significant.
Gleason (p = 0.029), low Gleason and margin (p = 0.002), and central and margin (p = 0.016) (Table II). No association was found between clinical prognostic factors and TWIST overexpression in the samples taken from high Gleason, low Gleason and central parts of the tumour. Furthermore, the average score of TWIST overexpression as a group in four malign areas exhibited no association with clinical outcome. In contrast, TWIST overexpression in the invasive, margin front of the tumour (M-TWIST) was associated with a positive surgical marginal status (p = 0.047), capsule invasion (p = 0.006) and BCR (p = 0.004). The analysis was continued with the
samples from the margin area, because of the significant findings found there for M-TWIST. AR expression in the margin area of the tumour (M-AR) was associated with higher Gleason score (p = 0.004), positive surgical marginal status (p = 0.004) and BCR (p = 0.05) (Table III). To test the observation that TWIST was regulating AR expression, the association between M-TWIST and M-AR expression was explored, and it was found to be highly significant (p < 0.0001). M-TWIST overexpression was clearly associated with shortened BFS (p < 0.0001) (Figure 2). An association of BFS was also found with a positive
Table III. Association between clinical prognosis factors and TWIST and androgen receptor (AR) expression in the margin areas of prostate cancer samples. TWIST expression (n = 167)
AR expression (n = 165)
Negative
Positive
p
Negative
Positive
p
pT2
105 (62.9)
15 (9.0)
NS
76 (46.1)
42 (25.5)
NS
pT3
43 (25.7)
4 (2.4)
35 (21.2)
12 (7.3)
20
N/A
8 (4.8)
0 (0)
2–6
90 (53.9)
10 (6.0)
77 (46.7)
23 (13.9)
7
50 (29.9)
6 (3.6)
29 (17.6)
25 (15.2)
8 (4.8)
3 (1.8)
5 (3.0)
6 (3.6)
Positive
51 (30.5)
11 (6.6)
32 (19.4)
28 (17.0)
Negative
97 (58.1)
8 (4.8)
79 (47.9)
26 (15.8)
Yes
34 (20.4)
10 (6.0)
No
114 (68.3)
9 (5.4)
Yes
51 (30.5)
13 (7.8)
No
97 (58.1)
6 (3.6)
NS
Gleason score
8–10
NS
0.004
Margin 0.047
0.004
Capsule invasion 0.006
28 (17.0)
16 (9.7)
83 (50.3)
38 (23.0)
NS
Biochemical recurrence 0.004
Data are shown as n (%). PSA = prostate-specific antigen; NS = not significant; N/A = not applicable.
36 (21.8)
26 (15.8)
75 (45.5)
28 (17.0)
0.050
TWIST expression in prostate cancer
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M-TWIST expression
1.0
Negative Positive Negative-censored Positive-censored
0.8
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BFS
0.6
0.4
Log-rank p < 0.0001 0.2
0.0 .00
5.00 10.00 Time to recurrence, years
15.00
Figure 2. Kaplan–Maier estimates of biochemical recurrence-free survival (BFS) according to margin (M)-TWIST expression.
surgical margin status (p = 0.003), Gleason score (p < 0.0001) and M-AR expression (p = 0.008). In the multivariate analysis, M-TWIST overexpression [hazard ratio (HR) = 2.516, 95% confidence interval (CI) 1.235–5.125, p = 0.011) and Gleason score (HR = 1.483, 95% CI 1.155–1.904, p = 0.002) were the only independent predictors of BFS, when the factors (positive surgical marginal status, Gleason score and M-AR expression) were included in the analysis. Discussion The decision to undertake radical treatment or active surveillance, or secondary treatments after radical procedures, is based exclusively on the clinicopathological factors, i.e. PSA, T class, tumour volume and Gleason score [6]. It is appreciated that these factors are still rather inaccurate for clinical use and that even many high-risk radically treated organ-confined PC patients never have BCR and primarily need multimodal treatments. Although it had been hoped that biochemical markers would confer benefits for clinicians in deciding optimal treatment protocols, the value of the biochemical markers is still virtually zero [7]. In earlier studies examining PC and other malignancies, such as rhabdomyosarcoma, breast cancer and lung cancer, TWIST expression was found to be elevated in malignant tissue in comparison to benign tissue [11,13,17,27]. Similar observations were made
in the present clinical PC material. In particular, more abundant TWIST expression was found in the margin and in high Gleason areas than in central and low Gleason parts of the tumour, evidence of the more aggressive nature of the cancer cells. TWIST overexpression was associated with clinicopathological prognostic factors in the samples taken from the margins of malignant tissue. In general, in cancer biology the most active area is the invasive front of the tumour, where one observes the highest proliferation and clearest signs of the EMT process. In accordance with this hypothesis, a significant association has been detected in the tumour invasive front, where TWIST acts as an activator of the EMT process [15]. In the recently published article by Behnsawy et al., the study design and the immunohistochemical method seem to be identical to those in the current study, with regard to clinical data, TWIST antibody, staining and categorical expression of the markers. In agreement with the present results, the association between TWIST overexpression and conventional prognostic factors, such as invasion and surgical margin status, was emphasized [18]. In contrast to the present study, Behnsawy et al. found an association between TWIST overexpression and pT class, Gleason score and PSA, even though the two study populations seemed similar. However, they did not specify the location from which the samples were taken. In addition, PC incidence and mortality differ between European and Asian populations, possibly accounting for discrepant results [18,28].
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TWIST has been shown to up-regulate AR. The most likely mechanism involved in this regulation is the ability of TWIST to act as an AR gene promoter [16]. In support of this observation, a clear association was also seen in the present work between M-TWIST and M-AR expression. Previous reports indicate that AR expression is associated with unfavourable prognostic factors and shortened BFS in PC, as also found here [19–23]. However, in this study M-AR did not remain as an independent predictor in the multivariate analyses, probably owing to the more powerful effect of M-TWIST. The observation of EMT process-related molecular markers, such as TWIST, is linked with the progression of many malignancies. For example, in squamous cell carcinoma and breast cancer, TWIST overexpression is indicative of disease progression and poorer survival of the patients [29–31]. In earlier PC cell line studies, TWIST overexpression was related to aggressive cell behaviour [15,16]. In their study of patients with localized PC, Behnsawy et al. found that TWIST overexpression was linked with shortened BFS [18]. Similarly, the present investigation found that TWIST overexpression had independent value to predict BFS. Emphasizing the coherence of the findings, BCR was defined as a rise in PSA value by 0.2 ng/ml in both studies. Since BCR is the first sign of recurrence of PC and rising PSA levels precede clinical progression, it is important to note that TWIST overexpression also has prognostic value in organ-confined PC [32]. However, a prospective study design will be needed to evaluate the usefulness of TWIST in clinical work as a prognostic factor in PC. In addition, it may be reasonable to analyse TWIST expression in prostate biopsy samples compared with clinical outcome, to assess the pretreatment prognostic value of TWIST. In summary, this study found that TWIST overexpression in the margins of PC samples is linked with unfavourable conventional clinicopathological prognostic factors and poor outcome in patients with localized PC after RP. Therefore, TWIST may represent a valuable prognostic marker since it associates with aggressive behaviour of PC. Acknowledgements The study was supported by the Finnish Cancer Society, the Finnish Anti-Tuberculosis Association and the strategic funding of the University of Eastern Finland. Declaration of interest: The authors declare that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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[26] Karvonen U, Kallio PJ, Janne OA, Palvimo JJ. Interaction of androgen receptors with androgen response element in intact cells. Roles of amino- and carboxyl-terminal regions and the ligand. J Biol Chem 1997;272:15973–9. [27] Merikallio H, Paakko P, Salmenkivi K, Kinnula V, Harju T, Soini Y. Expression of snail, twist, and Zeb1 in malignant mesothelioma. APMIS 2013;121:1–10. [28] Center MM, Jemal A, Lortet-Tieulent J, Ward E, Ferlay J, Brawley O, et al. International variation in prostate cancer incidence and mortality rates. Eur Urol 2012;61:1079–92. [29] Yang MH, Hsu DS, Wang HW, Wang HJ, Lan HY, Yang WH, et al. Bmi1 is essential in Twist1-induced epithelial–mesenchymal transition. Nat Cell Biol 2010;12: 982–92. [30] Jouppila-Matto A, Narkio-Makela M, Soini Y, Pukkila M, Sironen R, Tuhkanen H, et al. Twist and snai1 expression in pharyngeal squamous cell carcinoma stroma is related to cancer progression. BMC Cancer 2011;11:350. [31] Riaz M, Sieuwerts AM, Look MP, Timmermans MA, Smid M, Foekens JA, et al. High TWIST1 mRNA expression is associated with poor prognosis in lymph node-negative and estrogen receptor-positive human breast cancer and is coexpressed with stromal as well as ECM related genes. Breast Cancer Res 2012;14:R123. [32] Stephenson AJ, Kattan MW, Eastham JA, Dotan ZA, Bianco FJ Jr, Lilja H, et al. Defining biochemical recurrence of prostate cancer after radical prostatectomy: a proposal for a standardized definition. J Clin Oncol 2006;24:3973–8.
ORIGINAL ARTICLE
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TWIST overexpression predicts biochemical recurrence-free survival in prostate cancer patients treated with radical prostatectomy
SAMI RAATIKAINEN1,2, SIRPA AALTOMAA1, JORMA J. PALVIMO3, VESA KÄRJÄ4 & YLERMI SOINI5 Departments of 1Surgery and, 4Clinical Pathology, Kuopio University Hospital, Kuopio, Finland, 2Department of Surgery, Institute of Clinical Medicine, School of Medicine, and, 3Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland, and 5Department of Pathology and Forensic Medicine, Institute of Clinical Medicine, School of Medicine, University of Eastern Finland, Cancer Center of Eastern Finland, Kuopio, Finland
Abstract Objective. The aim of this study was to determine whether TWIST and androgen receptor (AR) expression can predict the outcome in radical prostatectomy (RP) patients. Material and methods. Samples from different tumour areas of 181 prostate cancer patients were analysed for TWIST and AR expression, and the results were correlated with known clinicopathological data and biochemical recurrence-free survival (BFS). Results. TWIST overexpression in the margin area of the tumour (M-TWIST) was related to positive surgical margin (p = 0.047), capsule invasion (p = 0.006) and biochemical recurrence (BCR) (p = 0.004). AR expression in the margin area of the tumour (M-AR) was associated with high Gleason score (p = 0.004), positive surgical margin (p = 0.004) and BCR (p = 0.05). M-TWIST overexpression was clearly associated with M-AR expression (p < 0.0001). Four parameters, i.e. M-TWIST overexpression (p < 0.0001), positive surgical margin (p = 0.003), high Gleason score (p < 0.0001) and M-AR expression (p = 0.008), predicted BFS. In the multivariate analysis, M-TWIST overexpression (p = 0.011) and Gleason score (p = 0.002) were the only independent predictors of BFS. Conclusions. M-TWIST overexpression is associated with clinicopathological prognosis factors and M-AR overexpression and is a powerful independent predictor of BFS in conjunction with the Gleason score in prostate cancer patients treated with RP.
Key Words: androgen receptor, prognosis, prostate cancer, radical prostatectomy, TWIST
Introduction Prostate cancer (PC) is the most common neoplasm in men living in Western countries. Nowadays, as a result of prostate-specific antigen (PSA) testing, it is diagnosed more often at an early stage without clinical symptoms and in young men with a long life expectancy [1,2]. The curative treatments, radical prostatectomy (RP) and radiation therapy, may decrease quality of life since they are associated with undesirable side-effects [3]. Alternatively, when PC is not aggressive and the lesion of the PC is small, the recommended option is active surveillance [4]. The treatment decision is traditionally
based on recognized clinicopathological prognostic factors, such as T class, PSA value and Gleason score; of these, the Gleason score is considered the most powerful prognosticator [5,6]. Many genetic and molecular factors have been examined in efforts to achieve biochemical recurrence-free survival (BFS) in localized PC that would be better than the clinicopathological prognostic factors. There are several studies demonstrating that expression of growth signal factors and factors associated with apoptosis, angiogenesis and tissue invasion [Ki-67, androgen receptor (AR), P53, BCL2, vascular endothelial growth factor, transforming growth factor-b] are linked with poor outcome in PC patients [7].
Correspondence: S. Raatikainen, Kuopio University Hospital, PO Box 100, FI-70029 Kuopio, Finland. Tel: +358 44 7175712. Fax: +358 17 173749. E-mail: sami.raatikainen@kuh.fi
(Received 13 December 2013; revised 16 February 2014; accepted 24 March 2014) ISSN 2168-1805 print/ISSN 2168-1813 online 2015 Informa Healthcare DOI: 10.3109/21681805.2014.909529
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However, although several of these biomolecular markers have been claimed to be promising, none of them is actually used in clinical practice. TWIST is a basic helix-loop transcription factor involved in the regulation of several pathways that control cell growth, differentiation and epithelial–mesenchymal transition (EMT). During EMT, epithelial cells lose their cell contacts owing to the downregulation of adhesion molecules such as E-cadherin, and they exhibit a remodelling of the cytoskeleton via increased expression of a-smooth muscle actin and vimentin. In addition to its physiological functions, EMT is involved in tumour progression processes where early-stage tumours are converted into invasive malignancies. TWIST is an inducer of EMT and therefore its overexpression triggers EMT, allowing tumour cells to become invasive and metastatic [8–10]. Research conducted in the past decade has revealed that TWIST and the EMT process can be linked with progression of many malignancies, such as head and neck cancer, squamous cell carcinoma, rhabdomyosarcoma and breast cancer [10–13]. In PC cell lines, TWIST is known to act as a trigger of the EMT process, which could be envisaged to promote the progression of PC [14–16]. Kwok et al. examined human prostate samples and found that there was greater TWIST expression in malignant than in benign tissue. TWIST overexpression has also been associated with a high Gleason score [17]. Behnsawy et al. explored the outcome of PC patients and noted that TWIST overexpression was associated with many clinicopathological factors and also with BFS [18]. It has been found that induction of TWIST leads to AR expression in PC cells. Consequently, AR expression has been linked to castration resistance of PC, which is a condition with a poor prognosis [16]. The prognostic value of AR is controversial [7], although in several studies AR expression was shown to predict aggressive behaviour, biochemical recurrence (BCR) and clinical recurrence of PC [19–23]. This study investigated the prognostic value of TWIST and AR expression compared with clinicopathological prognostic factors and BFS in 181 PC patients treated with RP. Material and methods This study was approved by the research ethics committee of Kuopio University Hospital.
1998 and 2009. Clinical data, including PSA value, and clinical follow-up data were extracted from the laboratory database and patient records. All of the patients had a clinically localized tumour according to digital rectal examination and transrectal ultrasonography. Lymphadenectomy was performed on some of the patients, but none of these patients had positive lymph nodes. Bone scans were used to exclude distant metastases when there was a need for confirmation of clinical status. The follow-up was conducted after 2, 6 and 12 months, and then according to clinical practice. None of the patients received hormone therapy before RP. In 38 cases (21%), the immeasurable PSA was not reached. An elevation of the PSA value of 0.2 ng/ml or more was considered as a biochemical recurrence [24]. Histopathological analyses All the samples had been fixed in neutral-buffered formalin and embedded in paraffin. They were re-evaluated for pT class, Gleason score, capsule invasion and surgical marginal status by two pathologists (YS and VK) who were blinded to the clinical data and a consensus assessment was made in each case. The clinical tumour, node, metastasis (TNM) classification was conducted according to Union for International Cancer Control (UICC) guidelines [25]. The samples for the immunohistochemical analyses were selected from the PC tissues from four representative tumour regions (high Gleason, low Gleason, central and margin). The margin area or invasive front was defined as the boundary area between histological tumour mass and adjacent benign prostate tissue as visualized in haematoxylin–eosin-stained slides. The high and low Gleason areas represented the highest and lowest Gleason pattern found in the tumour. Samples were also collected from the corresponding benign prostate tissue, and all samples were placed into multitissue microarray blocks with a Beecher Instruments Manual Tissue Arrayer (Beecher Instruments, Silver Spring, MD, USA). The microarray sample diameter was 1300 mm. Adequate immunostaining of AR expression was available in 165 cases (91.2%). TWIST expression in different tumour areas was obtained in the samples as follows: high Gleason (n = 170, 93.9%), low Gleason (n = 169, 93.4%), central (n = 169, 93.4%) and margin (n = 167, 92.2%). These cases were included in the analyses. Immunohistochemistry
Study population In total, 181 PC patients were treated with RP in Kuopio University Hospital, Finland, between
Immunostaining was performed as follows. Tissue sections (4 mm thick) were cut from the paraffinembedded blocks. After deparaffinization and
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TWIST expression in prostate cancer rehydration, the sections were heated in a microwave oven for 2 5 min in Tris–EDTA buffer (pH 9.0), incubated in Tris–EDTA buffer for 20 min and washed twice for 5 min in phosphate-buffered saline (PBS). Hydrogen peroxide (5%, 5 min) was used to block endogenous peroxidase. Non-specific binding was blocked with 1.5% normal serum in PBS for 35 min at room temperature. The sections were incubated overnight at 4 C with mouse monoclonal anti-TWIST and anti-AR antibodies (dilutions 1:500 and 1:500, respectively). The TWIST antibody was purchased from Abcam (ab50887; Abcam, Cambridge, UK). The AR antibody has been described in an earlier publication [26]. The slides were then incubated with a biotinylated secondary antibody and avidin–biotin–peroxidase complex (ABC Vectastain Elite Kit; Vector Laboratories, Burlingame, CA, USA). Careful rinses were performed with PBS in each step of the immunostaining procedure. The colour was developed with diaminobenzidine tetrahydrochloride (DAP) (Sigma, St Louis, MO, USA). The slides were counterstained with Mayer’s haematoxylin, washed, dehydrated, cleared and mounted with Depex (BDH, Poole, UK). Ovarian tumour tissue with known positive TWIST expression was used as a positive control. In negative controls, the primary antibody was omitted. The immunoreactivity for TWIST and AR was first analysed in tumour cells as follows: 0–5% = negative, 6–50% = weak positive and 51–100% = strong positive. The data were then divided into two groups: negative = 0–5% and positive = 5–100%. The evaluation was performed by two pathologists (YS and VK) who were blinded to the clinical data and a consensus assessment was made in each case. In the samples, TWIST expression was nuclear and cytoplasmic whereas AR expression was nuclear. Statistical analysis The SPSS 19.0 program package was used for the statistical analyses. Differences between groups were analysed with the t test. The chi-squared test was used to assess differences in AR and TWIST expression compared with the clinical prognostic factors. BFS was analysed by the Kaplan–Meyer method. Multivariate analyses were conducted according to Cox’s method. A p value less than 0.05 was considered statistically significant.
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Table I. Demographic data of the patients. Characteristic Age (years)
62.3 ± 5.3
Follow-up (years)
7.6 (2.3–14.1)
PSA at diagnosis (ng/ml)
7.8 ± 6.7
PSA (ng/ml) 20
10 (5.5)
pT category 2
130 (71.8)
3
51 (28.2)
Gleason score 2–6
111 (61.4)
7
58 (32.0)
8–10
12 (6.6)
Capsule invasion No
131 (72.4)
Yes
50 (27.6)
Surgical marginal status Negative
115 (63.5)
Positive
66 (36.5)
BCR Yes
67 (37.0)
No
114 (63.0)
Data are shown as median ± SD, median (range) or n (%). PSA = prostate-specific antigen; BCR = biochemical recurrence.
tumour according to the histopathological analyses and belonged to pT categories 2 and 3. During the follow-up period, 67 patients (37.0%) experienced a BCR. There was no TWIST expression in benign tissue, in contrast to the situation in PC tissue, where TWIST was expressed in all samples taken from different tumour areas (high Gleason 8.2%, low Gleason 3.0%, central 4.7% and margin 11.4%) (Figure 1). Differences in TWIST expression in the malign parts of tumour were observed between the following tumour areas: high Gleason and low
A Twist
B BPH
Results The demographic data of the patients are presented in Table I. The median follow-up time was 7.6 years (range 2.3–14.1 years). All the patients had a localized
Figure 1. (A) In this TWIST-positive case, positive nuclear expression can be seen. The sample is taken from the margin part of the tumour. (B) In benign prostatic hyperplasia (BPH), no TWIST expression was detected.
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Table II. TWIST expression in benign samples and different parts of the tumour.
Benign
Positive expression
Negative expression
Benign vs malign
High Gleason vs other parts of tumour
Low Gleason vs central and margin
Central vs margin
n (%)
n (%)
p
p
p
p
0 (0)
169 (100)
High Gleason
14 (8.2)
156 (91.8)
< 0.0001
Low Gleason
5 (3.0)
164 (97.0)
0.025
0.029
Central
8 (4.7)
161 (95.3)
0.004
NS
NS
Margin
19 (11.4)
148 (88.6)
< 0.0001
NS
0.002
0.016
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NS = not significant.
Gleason (p = 0.029), low Gleason and margin (p = 0.002), and central and margin (p = 0.016) (Table II). No association was found between clinical prognostic factors and TWIST overexpression in the samples taken from high Gleason, low Gleason and central parts of the tumour. Furthermore, the average score of TWIST overexpression as a group in four malign areas exhibited no association with clinical outcome. In contrast, TWIST overexpression in the invasive, margin front of the tumour (M-TWIST) was associated with a positive surgical marginal status (p = 0.047), capsule invasion (p = 0.006) and BCR (p = 0.004). The analysis was continued with the
samples from the margin area, because of the significant findings found there for M-TWIST. AR expression in the margin area of the tumour (M-AR) was associated with higher Gleason score (p = 0.004), positive surgical marginal status (p = 0.004) and BCR (p = 0.05) (Table III). To test the observation that TWIST was regulating AR expression, the association between M-TWIST and M-AR expression was explored, and it was found to be highly significant (p < 0.0001). M-TWIST overexpression was clearly associated with shortened BFS (p < 0.0001) (Figure 2). An association of BFS was also found with a positive
Table III. Association between clinical prognosis factors and TWIST and androgen receptor (AR) expression in the margin areas of prostate cancer samples. TWIST expression (n = 167)
AR expression (n = 165)
Negative
Positive
p
Negative
Positive
p
pT2
105 (62.9)
15 (9.0)
NS
76 (46.1)
42 (25.5)
NS
pT3
43 (25.7)
4 (2.4)
35 (21.2)
12 (7.3)
20
N/A
8 (4.8)
0 (0)
2–6
90 (53.9)
10 (6.0)
77 (46.7)
23 (13.9)
7
50 (29.9)
6 (3.6)
29 (17.6)
25 (15.2)
8 (4.8)
3 (1.8)
5 (3.0)
6 (3.6)
Positive
51 (30.5)
11 (6.6)
32 (19.4)
28 (17.0)
Negative
97 (58.1)
8 (4.8)
79 (47.9)
26 (15.8)
Yes
34 (20.4)
10 (6.0)
No
114 (68.3)
9 (5.4)
Yes
51 (30.5)
13 (7.8)
No
97 (58.1)
6 (3.6)
NS
Gleason score
8–10
NS
0.004
Margin 0.047
0.004
Capsule invasion 0.006
28 (17.0)
16 (9.7)
83 (50.3)
38 (23.0)
NS
Biochemical recurrence 0.004
Data are shown as n (%). PSA = prostate-specific antigen; NS = not significant; N/A = not applicable.
36 (21.8)
26 (15.8)
75 (45.5)
28 (17.0)
0.050
TWIST expression in prostate cancer
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M-TWIST expression
1.0
Negative Positive Negative-censored Positive-censored
0.8
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BFS
0.6
0.4
Log-rank p < 0.0001 0.2
0.0 .00
5.00 10.00 Time to recurrence, years
15.00
Figure 2. Kaplan–Maier estimates of biochemical recurrence-free survival (BFS) according to margin (M)-TWIST expression.
surgical margin status (p = 0.003), Gleason score (p < 0.0001) and M-AR expression (p = 0.008). In the multivariate analysis, M-TWIST overexpression [hazard ratio (HR) = 2.516, 95% confidence interval (CI) 1.235–5.125, p = 0.011) and Gleason score (HR = 1.483, 95% CI 1.155–1.904, p = 0.002) were the only independent predictors of BFS, when the factors (positive surgical marginal status, Gleason score and M-AR expression) were included in the analysis. Discussion The decision to undertake radical treatment or active surveillance, or secondary treatments after radical procedures, is based exclusively on the clinicopathological factors, i.e. PSA, T class, tumour volume and Gleason score [6]. It is appreciated that these factors are still rather inaccurate for clinical use and that even many high-risk radically treated organ-confined PC patients never have BCR and primarily need multimodal treatments. Although it had been hoped that biochemical markers would confer benefits for clinicians in deciding optimal treatment protocols, the value of the biochemical markers is still virtually zero [7]. In earlier studies examining PC and other malignancies, such as rhabdomyosarcoma, breast cancer and lung cancer, TWIST expression was found to be elevated in malignant tissue in comparison to benign tissue [11,13,17,27]. Similar observations were made
in the present clinical PC material. In particular, more abundant TWIST expression was found in the margin and in high Gleason areas than in central and low Gleason parts of the tumour, evidence of the more aggressive nature of the cancer cells. TWIST overexpression was associated with clinicopathological prognostic factors in the samples taken from the margins of malignant tissue. In general, in cancer biology the most active area is the invasive front of the tumour, where one observes the highest proliferation and clearest signs of the EMT process. In accordance with this hypothesis, a significant association has been detected in the tumour invasive front, where TWIST acts as an activator of the EMT process [15]. In the recently published article by Behnsawy et al., the study design and the immunohistochemical method seem to be identical to those in the current study, with regard to clinical data, TWIST antibody, staining and categorical expression of the markers. In agreement with the present results, the association between TWIST overexpression and conventional prognostic factors, such as invasion and surgical margin status, was emphasized [18]. In contrast to the present study, Behnsawy et al. found an association between TWIST overexpression and pT class, Gleason score and PSA, even though the two study populations seemed similar. However, they did not specify the location from which the samples were taken. In addition, PC incidence and mortality differ between European and Asian populations, possibly accounting for discrepant results [18,28].
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TWIST has been shown to up-regulate AR. The most likely mechanism involved in this regulation is the ability of TWIST to act as an AR gene promoter [16]. In support of this observation, a clear association was also seen in the present work between M-TWIST and M-AR expression. Previous reports indicate that AR expression is associated with unfavourable prognostic factors and shortened BFS in PC, as also found here [19–23]. However, in this study M-AR did not remain as an independent predictor in the multivariate analyses, probably owing to the more powerful effect of M-TWIST. The observation of EMT process-related molecular markers, such as TWIST, is linked with the progression of many malignancies. For example, in squamous cell carcinoma and breast cancer, TWIST overexpression is indicative of disease progression and poorer survival of the patients [29–31]. In earlier PC cell line studies, TWIST overexpression was related to aggressive cell behaviour [15,16]. In their study of patients with localized PC, Behnsawy et al. found that TWIST overexpression was linked with shortened BFS [18]. Similarly, the present investigation found that TWIST overexpression had independent value to predict BFS. Emphasizing the coherence of the findings, BCR was defined as a rise in PSA value by 0.2 ng/ml in both studies. Since BCR is the first sign of recurrence of PC and rising PSA levels precede clinical progression, it is important to note that TWIST overexpression also has prognostic value in organ-confined PC [32]. However, a prospective study design will be needed to evaluate the usefulness of TWIST in clinical work as a prognostic factor in PC. In addition, it may be reasonable to analyse TWIST expression in prostate biopsy samples compared with clinical outcome, to assess the pretreatment prognostic value of TWIST. In summary, this study found that TWIST overexpression in the margins of PC samples is linked with unfavourable conventional clinicopathological prognostic factors and poor outcome in patients with localized PC after RP. Therefore, TWIST may represent a valuable prognostic marker since it associates with aggressive behaviour of PC. Acknowledgements The study was supported by the Finnish Cancer Society, the Finnish Anti-Tuberculosis Association and the strategic funding of the University of Eastern Finland. Declaration of interest: The authors declare that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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