Prostate Cancer and Prostatic Disease (2014) 17, 310–314 © 2014 Macmillan Publishers Limited All rights reserved 1365-7852/14 www.nature.com/pcan
ORIGINAL ARTICLE
Prognostic significance of YWHAZ expression in localized prostate cancer K Rüenauver1,6, R Menon1,6, MA Svensson2,3,4,5, J Carlsson3,4,5, W Vogel1, O Andrén3,4,5, M Nowak1,6 and S Perner1,6 BACKGROUND: Prostate cancer (PCa) patients are often over-treated because of the lack of biomarkers needed to distinguish the lethal from the indolent form of PCa. YWHAZ was recently identified as a potential therapeutic target in castration-resistant PCa (CRPC). Therefore, this study focused on determining the prognostic significance of YWHAZ in localized PCa. METHODS: YWHAZ expression was assessed by immunohistochemistry on formalin-fixed paraffin-embedded tissue from 213 men who underwent radical prostatectomy. Kaplan–Meier analysis and Cox proportional-hazards models were used to assess the prognostic value of YWHAZ intensity. RESULTS: High YWHAZ expression was strongly associated with high Gleason score at the time of diagnosis (P o 0.001) and PSA relapse (P = 0.001). Importantly, patients with high expression of YWHAZ had a higher risk of CRPC development (P = 0.002) and reduced survival time (P = 0.002). CONCLUSIONS: Our findings indicate that YWHAZ could serve as a promising prognostic biomarker in localized PCa to predict poor prognosis and to identify a subgroup of tumors, which might benefit from earlier adjuvant or YWHAZ-targeted therapy. Prostate Cancer and Prostatic Disease (2014) 17, 310–314; doi:10.1038/pcan.2014.32; published online 26 August 2014
INTRODUCTION Prostate cancer (PCa) is the second most common cause of cancerrelated death among men in the western world.1 Owing to widespread screening efforts, the majority of PCa is detected at an early stage. Although early detection of PCa is important, it is currently challenging to distinguish between indolent and aggressive subtypes of the disease. This has lead to the debate regarding the ideal treatment option for patients. The ongoing challenge is to identify patients who require further treatment, like radical prostatectomy, and patients who would benefit from active surveillance.2 In cases of indolent PCa, early detection of the disease often leads to overtreatment with potentially severe side effects, such as urinary incontinence and erectile dysfunction.3 On the other hand, patients harboring the aggressive subtype of the disease could be undertreated, eventually developing a lethal phenotype.4 This difficult situation emphasizes the urgent need to identify novel prognostic biomarkers of PCa progression and outcome. YWHAZ, also known as 14-3-3ζ, belongs to the family of 14-3-3 proteins, which are highly conserved expressed in all eukaryotic organisms.5 There are seven different isoforms of 14-3-3 proteins, which have a flattened horseshoe structure and form homodimers as well as heterodimers.6 Although they are not enzymes they are able to bind to phosphorylated serine/threonine motifs on their target proteins and can thereby influence them. They are able to facilitate protein complex formation, to influence protein activity and structure and can change the intracellular localization of the protein.5,7 By binding to their numerous target proteins they regulate a wide array of cellular events such as signal transduction, apoptosis, cell cycle progression, metabolic processes, cell growth, and cell migration.5 Of the seven 14-3-3 isoforms, several studies 1
demonstrated that elevated expression of YWHAZ has been linked to tumor aggressiveness, chemotherapy resistance, poor prognosis and reduced disease-free survival in various types of cancer.8–14 Recently, our group identified YWHAZ as a potential therapeutic target in castration-resistant PCa (CRPC).15 However, until now, the potential prognostic significance of YWHAZ in localized PCa has not been investigated. This study focused on an in-depth immunohistochemical analysis of YWHAZ expression in a large, clinically well-characterized prostatectomy cohort to determine the relationship between YWHAZ expression and clinico-pathological parameters in localized PCa. MATERIALS AND METHODS Patient cohort This study was approved by the Regional Ethics Review Board in Uppsala/ Örebro, Sweden (2009/016), and the Institutional Review Board of the University Hospital of Bonn, Germany (264/11). The cohort consists of formalin-fixed paraffin-embedded primary prostate tissue of 289 tumor foci from 213 patients, who had undergone radical prostatectomy at the University Hospital of Örebro, Sweden, between 1989 and 2005, as recently described.16 For 61 patients, more than one tumor foci was investigated. A tumor focus was defined as an independent concentration of tumor cells within the prostate. Tumor stage was defined according to the criteria of the UICC. Clinico-pathological data included in this study are summarized in Table 1. The histological staging included 117 T1 tumors, 84 pT2 tumors and 12 pT3 tumors. The Gleason score was determined in a centralized pathology review by SP using the 2005 modified Gleason scoring system. In all, 59 tumors had a Gleason score of 6 and lower, 127 tumors had a Gleason score of 7 and 27 tumors had a Gleason score of 8 and higher. Follow-up data until January 2011 were available in 94% of patients for
Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Bonn, Germany; 2Department of Laboratory Medicine, University Hospital of Örebro, Örebro, Sweden; 3Department of Urology, University Hospital of Örebro, Örebro, Sweden; 4Institute of Health and Medical Science, University of Örebro, Örebro, Sweden and 5 Member of the Transdisciplinary Prostate Cancer Partnership (ToPCaP). Correspondence: Professor S Perner, Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany. E-mail: [email protected] 6 These authors contributed equally to this work. Received 20 January 2014; revised 30 May 2014; accepted 27 June 2014; published online 26 August 2014
YWHAZ as a potential prognostic marker in prostate cancer K Rüenauver et al
311 CRPC, in 89% for PSA relapse and in 94% for PCa-specific death, with a median follow-up period of 122 months (range: 14–273 months). The tissue microarray was constructed as previously described.17 In brief, nontumor and tumor sections were identified by a pathologist (SP) and triplicates of 0.6 mm cores from each tumor focus were taken and reembedded into a recipient paraffin block.
Table 1. Clinico-pathological characteristics of patients included in the study Patients Parameters
Immunohistochemistry (IHC) IHC for YWHAZ was performed using the Ventana Discovery XT automated staining system (Ventana Medical Systems,Tucson, AZ, USA). In brief, 4-μm thick sections were obtained from the tissue microarray blocks. The sections were deparaffinized, and NaCi, pH 6.0 was used to demask the antigens. The rabbit antibody 14-3-3ζ (C-16) (Santa Cruz Biotechnology, Dallas, TX, USA) was used at a dilution of 1:300. Secondary staining was performed with a biotinylated Ig cocktail (universal secondary antibody; Ventana). Counter staining was performed using Hematoxyline II and Blueing Reagent (Ventana). The stained tissue microarrays were then digitized using the Zeiss MIRAX DESK scanner (Carl Zeiss, Oberkochen, Germany). Staining intensity of YWHAZ was determined by a semiautomated quantitative image analysis software (Definiens Tissue Studio 2.1, Definiens, Munich, Germany) as previously described.18 The average nuclear and cytoplasmic IHC marker intensity (average IHC marker intensity of the cell) was used to correlate YWHAZ expression with clinico-pathological data. Mean expression values were specifically calculated for each tumor focus of a patient. The mean staining intensity derived using all foci of a patient were then used to calculate the overall YWHAZ expression level of this patient.
Statistics All statistical analyses were performed using the SPSS 21.0 statistical software package (SPSS, Chicago, IL, USA). Bivariate correlation using the Spearman rank correlation test and χ2 test were calculated to test for correlation and association. By using Student’s independent sample t-tests, the significance of differences between mean values was determined. These calculations were based on the intensities of YWHAZ expression. For further data analysis, the average YWHAZ marker intensity was divided into quartiles and subsequently grouped into low YWHAZ intensity (quartiles 1–3) and high YWHAZ intensity (quartile 4). The quartile-based analysis was performed in order to figure out if there was a difference in the prognosis between high and low YWHAZexpressing tumors. Kaplan–Meier analysis was used to perform survival analyses, and the log rank test was used to detect differences between the survival curves. Univariate and multivariate Cox proportional-hazards models were fitted to assess the prognostic and predictive value of YWHAZ intensity. Statistical significance was defined as Po0.05.
RESULTS Correlation of YWHAZ and clinico-pathologic variables In the majority of cases, benign prostate tissue showed a very low YWHAZ expression. Representative images of YWHAZ expression in prostate tissue are shown in Figure 1. The average YWHAZ staining intensity of the tumor foci was significantly correlated to the Gleason score (r2 = 0.296; P o 0.001) and was significantly higher in tumor foci with a high Gleason score. For further analysis, the Gleason score was divided into four groups (⩽GS 6, GS 3+4, GS 4+3 and ⩾ GS 8). YWHAZ protein expression also significantly correlated with these Gleason groups (r2 = 0.260; P o0.001). A positive association was also found for YWHAZ expression and development of CRPC (r2 = 0.207; P = 0.003), where YWHAZ expression was significantly higher in tumors of patients who developed CRPC than of those of who did not (P = 0.048). Furthermore, YWHAZ expression significantly associated with PCa-specific death (r2 = 0.164, P = 0.020). Prognostic significance of YWHAZ expression In order to determine the prognostic value of YWHAZ, we compared survival rates of patients who were stratified by quartiles based on YWHAZ staining intensity (Table 2). Cumulative survival curves were calculated according to the Kaplan–Meier method. The log-rank test was used to assess differences in © 2014 Macmillan Publishers Limited
n Age median (range), years Follow-up time median (range) PSA at diagnosis median (range) Benign tissue
Foci
Total
%
Total
%
213 64 (45–74)
100.0
289 64 (45–74)
100.0
122 (14–273)
128 (14–273)
8.9 (3.0–52.0)
9.2 (3.0–52.0)
84
39.4
88
30.4
Gleason score 5 6 7 8 9 10
1 58 127 19 8 0
0.5 27.2 59.6 8.9 3.8 0.0
1 86 168 25 9 0
0.3 29.8 58.1 8.7 3.1 0.0
T stage T1 pT2 pT3 pT4
117 84 12 0
54.9 39.4 5.6 0.0
157 115 17 0
54.3 39.8 5.9 0.0
106 50 57
49.8 23.5 26.8
138 66 85
47.8 22.8 29.4
CRPC Yes No Not available
16 184 13
7.5 86.4 6.1
20 254 15
6.9 87.9 5.2
PSA relapse Yes No Not available
59 130 24
27.7 61.0 11.3
78 182 29
27.0 63.0 10.0
16 122 (14–273)
7.5
20 128 (14–273)
6.9
185 12
86.9 5.6
255 14
88.2 4.8
Surgical margin status R0 R1 Rx (distance of tumor to margin o0.5 mm)
PCa-specific death Yes Median survival time (months) (range) No Not available
Abbreviations: CRPC, castration-resistant PCa; PCa, prostate cancer. PSA failure was defined as two consecutive rises in PSA above 0.4 ng ml −1. PCa mortality was defined as PCa-specific death or death due to castrationresistant state of PCa.
survival times. Kaplan–Meier analysis indicated that patients with tumors belonging to the highest quartile had a significantly greater rate of PCa-specific mortality than patients with a lower YWHAZ expression (P = 0.002; Figure 2). The mean survival of these patients was reduced by 59.6 months. In addition, patients with high YWHAZ expression had a significantly higher risk of PSA relapse (P = 0.001) and showed an increased risk of developing the CRPC subtype (P = 0.002). Comparing the two groups low YWHAZ expression (quartiles 1–3) and high YWHAZ expression (quartile 4) showed an association between high YWHAZ expression and younger patient age (P = 0.001; Table 2). Cox regression analysis was performed to Prostate Cancer and Prostatic Disease (2014), 310 – 314
YWHAZ as a potential prognostic marker in prostate cancer K Rüenauver et al
312
a
50 µm 100 µm
b
50 µm 100 µm
Figure 1. Representative images of YWHAZ immunohistochemistry staining of prostate cancer (PCa) tissue. Two representative cores with low (a) and high (b) YWHAZ expression are shown. YWHAZ expression was observed in the nucleus as well as in the cytoplasm of PCa cells.
further analyze the prognostic value of YWHAZ using quartiles 1–3 of YWHAZ expression as the reference. Univariate analysis showed that patients with high YWHAZ expression (quartile 4) had a 4.28 times higher risk of PCa-related death, in comparison to patients with low YWHAZ expression (95% confidence interval (CI): 1.589–11.525, P = 0.004). Furthermore, these patients also had a 4.13 times increased risk of developing CRPC (95% CI: 1.544–11.050, P = 0.005). The risk of a PSA relapse was 2.37 times higher for quartile 4 patients (95% CI: 1.389–4.043, P = 0.002). Moreover, YWHAZ expression remained a significant predictor for PCa-specific death (P = 0.015) with a hazard ratio of 3.516 (95% CI: 1.275–9.699) in multivariate analysis including Gleason score and T-stage. Similar results were observed for YWHAZ expression and PSA relapse in a multivariate analysis including Gleason score and T-stage (hazard ratio: 1.916, 95% CI: 1.115–3.293, P = 0.019). However, when adjusting for PSA at diagnosis, age at diagnosis and surgical margin status the association disappeared. Whereas YWHAZ expression remained an independent prognostic marker for CRPC in a multivariate model adjusted for Gleason score, T-stage, PSA at diagnosis and age at diagnosis (hazard ratio: 5.072, 95% CI: 1.529–16.827, P = 0.008). DISCUSSION The rate of PCa diagnosis has dramatically increased in the recent years because of an aging population and increased screening efforts.19 These efforts have lead to the detection of locally defined, low-grade, microscopic PCa with unpredictable growth potential. Therefore, there is an urgent need for the identification of new molecular markers that can effectively distinguish low-risk indolent PCa from the clinically aggressive subtype of the disease. Prostate Cancer and Prostatic Disease (2014), 310 – 314
YWHAZ belongs to the family of 14-3-3 proteins involved in several cellular functions. Former studies indicated a direct involvement of YWHAZ in cellular transformation and proliferation and proposed its oncogenic character.5,15,20 In this study, we showed that YWHAZ overexpression is significantly associated with PCa-specific death and development of CRPC. Consistent with our findings, YWHAZ overexpression was also found to decrease disease-free survival of patients with head-and-neck/oral squamous cell carcinomas.10 Furthermore, we found a significant positive correlation between YWHAZ expression and Gleason score. In addition, Kaplan–Meier and Cox regression analyses revealed a significant decrease in survival and an increase in PSA relapse and CRPC in patients with high YWHAZ-expressing tumors. Similar results were derived from YWHAZ studies on non-small-cell lung cancer and breast cancer. Here, YWHAZ was significantly associated with reduced survival and disease recurrence. In nonsmall-cell lung cancer patients, YWHAZ was even the only independent predictor of disease recurrence and reduced overall survival.11,13 Recently, we were able to show that YWHAZ amplification is associated with YWHAZ overexpression in PCa, indicating that gene amplification might be a common mechanism leading to YWHAZ overexpression.15 Furthermore, this hypothesis is supported by the significantly higher YWHAZ expression in the patients that later developed CRPC. Although, in 2012, Murata et al. analyzed the protein expression of YWHAZ in PCa tissue of 90 patients and found a significant higher YWHAZ expression in cases with lymph node metastasis, they did not determine the significance of YWHAZ as a prognostic marker in PCa. 21 We identified YWHAZ to be a prognostic factor for PCa-specific death, development of CRPC and PSA relapse. Furthermore, Murata et al. © 2014 Macmillan Publishers Limited
YWHAZ as a potential prognostic marker in prostate cancer K Rüenauver et al
313 Table 2. Clinico-pathological features of the cohort based on YWHAZ expression
1.0
YWHAZ protein expression 0.8
Parameters
Total
n 159 Age median 65 (57–73) (range), years Follow-up time 115 (29–239) median (range) PSA at diagnosis 8.1 (3.2–52.0) median (range)
Quartile 4 %
Total
%
100.0
54 63 (45–74)
100.0 0.001
127 (14–273)
0.072
9.2 (3.0–49.0)
0.862 0.001
Gleason score 5 6 7 8 9 10
1 54 89 10 5 0
0.6 34.0 56.0 6.3 3.1 0.0
0 4 38 9 3 0
0.0 7.4 70.4 16.7 5.6 0.0
T stage T1 pT2 pT3 pT4
95 54 10 0
59.7 34.0 6.3 0.0
22 30 2 0
40.7 55.6 3.7 0.0
Surgical margin status R0 R1 Rx (distance of tumor to margin o0.5 mm)
P Survival rate
Quartiles 1–3
0.6
0.4
0.2 Quartiles 1-3 Quartile 4
0.0
0.067
0
50
100
150
200
250
300
Survival time (months)
Figure 2. Kaplan–Meier curve of PCa patients divided into two groups (quartiles 1–3 and quartile 4) according to the average YWHAZ marker intensity of the tumors. 0.275
82 33 44
51.6 20.8 27.7
24 17 13
44.4 31.5 24.1
CRPC Yes No Not available
8 146 5
5.0 91.8 3.1
8 38 8
14.8 70.4 14.8
PSA relapse Yes No Not available
38 108 13
23.9 67.9 8.2
21 22 11
38.9 40.7 20.4
PCa specific death Yes No Not available
8 147 4
5.0 92.5 2.5
8 38 8
14.8 70.4 14.8
0.013
0.008
cohort size and patient history available to this study. Another obstacle is the relation of YWHAZ expression in tumors and patient age. Further experiments are needed to stratify PCa cohorts into patients of ages of disease onset to clarify this point. Furthermore, efforts are needed to analyze the YWHAZ expression in a cohort of low-risk patients with localized PCa to delineate the prognostic value of YWHAZ in patients with yet undefined disease progression and outcome. Moreover, validation in biopsies is needed, as biopsies are one of the first steps in the diagnosis of PCa. As our results in prostatectomy samples indicate, high YWHAZ expression is associated with poor patient outcome. Validation of these results in PCa biopsies would further support the prognostic potential of YWHAZ expression in PCa and might help to determine the patient's further treatment at the earliest possible time.
0.012
Abbreviation: CRPC, castration-resistant PCa; PCa, prostate cancer. YWHAZ expression was divided into two groups (quartiles 1–3 and quartile 4) for Kaplan–Meier and Cox regression analysis.
demonstrated that YWHAZ is an androgen-responsive gene.21 As younger men have higher androgen levels than older men, this might be an explanation for the association between younger patient age and high YWHAZ expression.22 The subgroup of tumors showing a high YWHAZ expression seems to be more aggressive than tumors with low YWHAZ expression. Therefore, this subgroup might benefit from an early adjuvant therapy or YWHAZ-targeted therapy. Our findings show that YWHAZ expression in localized PCa could be used to predict the prognosis of PCa patients that in turn might help in the decision on treatment regimes for patients with early diagnosed locally defined PCa. LIMITATIONS Prognostic tools to distinguish low-risk indolent PCa from clinically aggressive disease are still elusive. Clearly, the use of YWHAZ expression levels as a prognostic tool is still hampered by the © 2014 Macmillan Publishers Limited
CONCLUSION In this study, we demonstrated that overexpression of YWHAZ significantly correlates with adverse clinico-pathological parameters. These results suggest that increased YWHAZ expression, determined by IHC in localized PCa, might be a novel prognostic marker for PCa progression and disease-specific death. Therefore, we propose to conduct further experiments to delineate the value of YWHAZ expression as a tool for identifying ideal treatment options. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by grants from the Wilhelm Sander Foundation (2011.077.1), the Rudolf-Becker-Foundation and the German Research Foundation (Deutsche Forschungsgemeinschaft (DFG), Emmy-Noether-Program, PE1179/2-1) to SP. Furthermore, the project was supported by the medical student research grant of the Medical Faculty of the University of Bonn (BONFOR) to KR.
REFERENCES 1 Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013; 63: 11–30.
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314 2 Turkbey B, Mena E, Aras O, Garvey B, Grant K, Choyke PL. Functional and molecular imaging: applications for diagnosis and staging of localised prostate cancer. Clin Oncol 2013; 25: 451–460. 3 Sanda MG, Dunn RL, Michalski J, Sandler HM, Northouse L, Hembroff L et al. Quality of life and satisfaction with outcome among prostate-cancer survivors. N Engl J Med 2008; 358: 1250–1261. 4 Schwartz KL. Alibhai, Shabbir MH, Tomlinson G, Naglie G, Krahn MD. Continued undertreatment of older men with localized prostate cancer. Urology 2003; 62: 860–865. 5 Tzivion G, Gupta VS, Kaplun L, Balan V. 14-3-3 proteins as potential oncogenes. Semin Cancer Biol 2006; 16: 203–213. 6 Gardino AK, Smerdon SJ, Yaffe MB. Structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the X-ray crystal structures of all human 14-3-3 isoforms. Semin Cancer Biol 2006; 16: 173–182. 7 Matta A, Siu K W, Ralhan R. 14-3-3 zeta as novel molecular target for cancer therapy. Expert Opin Ther Targets 2012; 16: 515–523. 8 Bajpai U, Sharma R, Kausar T, Dattagupta S, Chattopadhayay TK, Ralhan R. Clinical significance of 14-3-3 zeta in human esophageal cancer. Int J Biol Markers 2008; 23: 231–237. 9 Matta A, Bahadur S, Duggal R, Gupta SD, Ralhan R. Over-expression of 14-3-3zeta is an early event in oral cancer. BMC Cancer 2007; 7: 169. 10 Matta A, DeSouza LV, Shukla NK, Gupta SD, Ralhan R, Siu KW. Prognostic significance of head-and-neck cancer biomarkers previously discovered and identified using iTRAQ-labeling and multidimensional liquid chromatographytandem mass spectrometry. J Proteome Res 2008; 7: 2078–2087. 11 Fan T, Li R, Todd NW, Qiu Q, Fang H, Wang H et al. Up-regulation of 14-3-3zeta in lung cancer and its implication as prognostic and therapeutic target. Cancer Res 2007; 67: 7901–7906. 12 Yang X, Cao W, Zhou J, Zhang W, Zhang X, Lin W et al. 14-3-3zeta positive expression is associated with a poor prognosis in patients with glioblastoma. Neurosurgery 2011; 68: 932–938.
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13 Neal CL, Yao J, Yang W, Zhou X, Nguyen NT, Lu J et al. 14-3-3zeta overexpression defines high risk for breast cancer recurrence and promotes cancer cell survival. Cancer Res 2009; 69: 3425–3432. 14 Neal CL, Yu D. 14-3-3zeta as a prognostic marker and therapeutic target for cancer. Expert Opin Ther Targets 2010; 14: 1343–1354. 15 Menon R, Deng M, Ruenauver K, Queisser A, Offermann A, Boehm D et al. Somatic copy number alterations by whole exome sequencing implicates YWHAZ and PTK2 in castration-resistant prostate cancer. J Pathol 2013; 231: 505–516. 16 Nowak M, Svensson MA, Carlsson J, Vogel W, Kebschull M, Wernert N et al. Prognostic significance of phospho-histone H3 in prostate carcinoma. World J Urol 2013; 32: 703–707. 17 Scheble VJ, Braun M, Beroukhim R, Mermel CH, Ruiz C, Wilbertz T et al. ERG rearrangement is specific to prostate cancer and does not occur in any other common tumor. Mod Pathol 2010; 23: 1061–1067. 18 Braun M, Kirsten R, Rupp NJ, Moch H, Fend F, Wernert Net al. Quantification of protein expression in cells and cellular subcompartments on immunohistochemical sections using a computer supported image analysis system. Histol Histopathol 2013; 28: 605–610. 19 Bangma CH, Roemeling S, Schroder FH. Overdiagnosis and overtreatment of early detected prostate cancer. World J Urol 2007; 25: 3–9. 20 Niemantsverdriet M, Wagner K, Visser M, Backendorf C. Cellular functions of 14-33 zeta in apoptosis and cell adhesion emphasize its oncogenic character. Oncogene 2008; 27: 1315–1319. 21 Murata T, Takayama K, Urano T, Fujimura T, Ashikari D, Obinata D et al. 14-3-3zeta, a novel androgen-responsive gene, is upregulated in prostate cancer and promotes prostate cancer cell proliferation and survival. Clin Cancer Res 2012; 18: 5617–5627. 22 Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR. Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab 2001; 86: 724–731.
© 2014 Macmillan Publishers Limited
ORIGINAL ARTICLE
Prognostic significance of YWHAZ expression in localized prostate cancer K Rüenauver1,6, R Menon1,6, MA Svensson2,3,4,5, J Carlsson3,4,5, W Vogel1, O Andrén3,4,5, M Nowak1,6 and S Perner1,6 BACKGROUND: Prostate cancer (PCa) patients are often over-treated because of the lack of biomarkers needed to distinguish the lethal from the indolent form of PCa. YWHAZ was recently identified as a potential therapeutic target in castration-resistant PCa (CRPC). Therefore, this study focused on determining the prognostic significance of YWHAZ in localized PCa. METHODS: YWHAZ expression was assessed by immunohistochemistry on formalin-fixed paraffin-embedded tissue from 213 men who underwent radical prostatectomy. Kaplan–Meier analysis and Cox proportional-hazards models were used to assess the prognostic value of YWHAZ intensity. RESULTS: High YWHAZ expression was strongly associated with high Gleason score at the time of diagnosis (P o 0.001) and PSA relapse (P = 0.001). Importantly, patients with high expression of YWHAZ had a higher risk of CRPC development (P = 0.002) and reduced survival time (P = 0.002). CONCLUSIONS: Our findings indicate that YWHAZ could serve as a promising prognostic biomarker in localized PCa to predict poor prognosis and to identify a subgroup of tumors, which might benefit from earlier adjuvant or YWHAZ-targeted therapy. Prostate Cancer and Prostatic Disease (2014) 17, 310–314; doi:10.1038/pcan.2014.32; published online 26 August 2014
INTRODUCTION Prostate cancer (PCa) is the second most common cause of cancerrelated death among men in the western world.1 Owing to widespread screening efforts, the majority of PCa is detected at an early stage. Although early detection of PCa is important, it is currently challenging to distinguish between indolent and aggressive subtypes of the disease. This has lead to the debate regarding the ideal treatment option for patients. The ongoing challenge is to identify patients who require further treatment, like radical prostatectomy, and patients who would benefit from active surveillance.2 In cases of indolent PCa, early detection of the disease often leads to overtreatment with potentially severe side effects, such as urinary incontinence and erectile dysfunction.3 On the other hand, patients harboring the aggressive subtype of the disease could be undertreated, eventually developing a lethal phenotype.4 This difficult situation emphasizes the urgent need to identify novel prognostic biomarkers of PCa progression and outcome. YWHAZ, also known as 14-3-3ζ, belongs to the family of 14-3-3 proteins, which are highly conserved expressed in all eukaryotic organisms.5 There are seven different isoforms of 14-3-3 proteins, which have a flattened horseshoe structure and form homodimers as well as heterodimers.6 Although they are not enzymes they are able to bind to phosphorylated serine/threonine motifs on their target proteins and can thereby influence them. They are able to facilitate protein complex formation, to influence protein activity and structure and can change the intracellular localization of the protein.5,7 By binding to their numerous target proteins they regulate a wide array of cellular events such as signal transduction, apoptosis, cell cycle progression, metabolic processes, cell growth, and cell migration.5 Of the seven 14-3-3 isoforms, several studies 1
demonstrated that elevated expression of YWHAZ has been linked to tumor aggressiveness, chemotherapy resistance, poor prognosis and reduced disease-free survival in various types of cancer.8–14 Recently, our group identified YWHAZ as a potential therapeutic target in castration-resistant PCa (CRPC).15 However, until now, the potential prognostic significance of YWHAZ in localized PCa has not been investigated. This study focused on an in-depth immunohistochemical analysis of YWHAZ expression in a large, clinically well-characterized prostatectomy cohort to determine the relationship between YWHAZ expression and clinico-pathological parameters in localized PCa. MATERIALS AND METHODS Patient cohort This study was approved by the Regional Ethics Review Board in Uppsala/ Örebro, Sweden (2009/016), and the Institutional Review Board of the University Hospital of Bonn, Germany (264/11). The cohort consists of formalin-fixed paraffin-embedded primary prostate tissue of 289 tumor foci from 213 patients, who had undergone radical prostatectomy at the University Hospital of Örebro, Sweden, between 1989 and 2005, as recently described.16 For 61 patients, more than one tumor foci was investigated. A tumor focus was defined as an independent concentration of tumor cells within the prostate. Tumor stage was defined according to the criteria of the UICC. Clinico-pathological data included in this study are summarized in Table 1. The histological staging included 117 T1 tumors, 84 pT2 tumors and 12 pT3 tumors. The Gleason score was determined in a centralized pathology review by SP using the 2005 modified Gleason scoring system. In all, 59 tumors had a Gleason score of 6 and lower, 127 tumors had a Gleason score of 7 and 27 tumors had a Gleason score of 8 and higher. Follow-up data until January 2011 were available in 94% of patients for
Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Bonn, Germany; 2Department of Laboratory Medicine, University Hospital of Örebro, Örebro, Sweden; 3Department of Urology, University Hospital of Örebro, Örebro, Sweden; 4Institute of Health and Medical Science, University of Örebro, Örebro, Sweden and 5 Member of the Transdisciplinary Prostate Cancer Partnership (ToPCaP). Correspondence: Professor S Perner, Department of Prostate Cancer Research, Institute of Pathology, University Hospital of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany. E-mail: [email protected] 6 These authors contributed equally to this work. Received 20 January 2014; revised 30 May 2014; accepted 27 June 2014; published online 26 August 2014
YWHAZ as a potential prognostic marker in prostate cancer K Rüenauver et al
311 CRPC, in 89% for PSA relapse and in 94% for PCa-specific death, with a median follow-up period of 122 months (range: 14–273 months). The tissue microarray was constructed as previously described.17 In brief, nontumor and tumor sections were identified by a pathologist (SP) and triplicates of 0.6 mm cores from each tumor focus were taken and reembedded into a recipient paraffin block.
Table 1. Clinico-pathological characteristics of patients included in the study Patients Parameters
Immunohistochemistry (IHC) IHC for YWHAZ was performed using the Ventana Discovery XT automated staining system (Ventana Medical Systems,Tucson, AZ, USA). In brief, 4-μm thick sections were obtained from the tissue microarray blocks. The sections were deparaffinized, and NaCi, pH 6.0 was used to demask the antigens. The rabbit antibody 14-3-3ζ (C-16) (Santa Cruz Biotechnology, Dallas, TX, USA) was used at a dilution of 1:300. Secondary staining was performed with a biotinylated Ig cocktail (universal secondary antibody; Ventana). Counter staining was performed using Hematoxyline II and Blueing Reagent (Ventana). The stained tissue microarrays were then digitized using the Zeiss MIRAX DESK scanner (Carl Zeiss, Oberkochen, Germany). Staining intensity of YWHAZ was determined by a semiautomated quantitative image analysis software (Definiens Tissue Studio 2.1, Definiens, Munich, Germany) as previously described.18 The average nuclear and cytoplasmic IHC marker intensity (average IHC marker intensity of the cell) was used to correlate YWHAZ expression with clinico-pathological data. Mean expression values were specifically calculated for each tumor focus of a patient. The mean staining intensity derived using all foci of a patient were then used to calculate the overall YWHAZ expression level of this patient.
Statistics All statistical analyses were performed using the SPSS 21.0 statistical software package (SPSS, Chicago, IL, USA). Bivariate correlation using the Spearman rank correlation test and χ2 test were calculated to test for correlation and association. By using Student’s independent sample t-tests, the significance of differences between mean values was determined. These calculations were based on the intensities of YWHAZ expression. For further data analysis, the average YWHAZ marker intensity was divided into quartiles and subsequently grouped into low YWHAZ intensity (quartiles 1–3) and high YWHAZ intensity (quartile 4). The quartile-based analysis was performed in order to figure out if there was a difference in the prognosis between high and low YWHAZexpressing tumors. Kaplan–Meier analysis was used to perform survival analyses, and the log rank test was used to detect differences between the survival curves. Univariate and multivariate Cox proportional-hazards models were fitted to assess the prognostic and predictive value of YWHAZ intensity. Statistical significance was defined as Po0.05.
RESULTS Correlation of YWHAZ and clinico-pathologic variables In the majority of cases, benign prostate tissue showed a very low YWHAZ expression. Representative images of YWHAZ expression in prostate tissue are shown in Figure 1. The average YWHAZ staining intensity of the tumor foci was significantly correlated to the Gleason score (r2 = 0.296; P o 0.001) and was significantly higher in tumor foci with a high Gleason score. For further analysis, the Gleason score was divided into four groups (⩽GS 6, GS 3+4, GS 4+3 and ⩾ GS 8). YWHAZ protein expression also significantly correlated with these Gleason groups (r2 = 0.260; P o0.001). A positive association was also found for YWHAZ expression and development of CRPC (r2 = 0.207; P = 0.003), where YWHAZ expression was significantly higher in tumors of patients who developed CRPC than of those of who did not (P = 0.048). Furthermore, YWHAZ expression significantly associated with PCa-specific death (r2 = 0.164, P = 0.020). Prognostic significance of YWHAZ expression In order to determine the prognostic value of YWHAZ, we compared survival rates of patients who were stratified by quartiles based on YWHAZ staining intensity (Table 2). Cumulative survival curves were calculated according to the Kaplan–Meier method. The log-rank test was used to assess differences in © 2014 Macmillan Publishers Limited
n Age median (range), years Follow-up time median (range) PSA at diagnosis median (range) Benign tissue
Foci
Total
%
Total
%
213 64 (45–74)
100.0
289 64 (45–74)
100.0
122 (14–273)
128 (14–273)
8.9 (3.0–52.0)
9.2 (3.0–52.0)
84
39.4
88
30.4
Gleason score 5 6 7 8 9 10
1 58 127 19 8 0
0.5 27.2 59.6 8.9 3.8 0.0
1 86 168 25 9 0
0.3 29.8 58.1 8.7 3.1 0.0
T stage T1 pT2 pT3 pT4
117 84 12 0
54.9 39.4 5.6 0.0
157 115 17 0
54.3 39.8 5.9 0.0
106 50 57
49.8 23.5 26.8
138 66 85
47.8 22.8 29.4
CRPC Yes No Not available
16 184 13
7.5 86.4 6.1
20 254 15
6.9 87.9 5.2
PSA relapse Yes No Not available
59 130 24
27.7 61.0 11.3
78 182 29
27.0 63.0 10.0
16 122 (14–273)
7.5
20 128 (14–273)
6.9
185 12
86.9 5.6
255 14
88.2 4.8
Surgical margin status R0 R1 Rx (distance of tumor to margin o0.5 mm)
PCa-specific death Yes Median survival time (months) (range) No Not available
Abbreviations: CRPC, castration-resistant PCa; PCa, prostate cancer. PSA failure was defined as two consecutive rises in PSA above 0.4 ng ml −1. PCa mortality was defined as PCa-specific death or death due to castrationresistant state of PCa.
survival times. Kaplan–Meier analysis indicated that patients with tumors belonging to the highest quartile had a significantly greater rate of PCa-specific mortality than patients with a lower YWHAZ expression (P = 0.002; Figure 2). The mean survival of these patients was reduced by 59.6 months. In addition, patients with high YWHAZ expression had a significantly higher risk of PSA relapse (P = 0.001) and showed an increased risk of developing the CRPC subtype (P = 0.002). Comparing the two groups low YWHAZ expression (quartiles 1–3) and high YWHAZ expression (quartile 4) showed an association between high YWHAZ expression and younger patient age (P = 0.001; Table 2). Cox regression analysis was performed to Prostate Cancer and Prostatic Disease (2014), 310 – 314
YWHAZ as a potential prognostic marker in prostate cancer K Rüenauver et al
312
a
50 µm 100 µm
b
50 µm 100 µm
Figure 1. Representative images of YWHAZ immunohistochemistry staining of prostate cancer (PCa) tissue. Two representative cores with low (a) and high (b) YWHAZ expression are shown. YWHAZ expression was observed in the nucleus as well as in the cytoplasm of PCa cells.
further analyze the prognostic value of YWHAZ using quartiles 1–3 of YWHAZ expression as the reference. Univariate analysis showed that patients with high YWHAZ expression (quartile 4) had a 4.28 times higher risk of PCa-related death, in comparison to patients with low YWHAZ expression (95% confidence interval (CI): 1.589–11.525, P = 0.004). Furthermore, these patients also had a 4.13 times increased risk of developing CRPC (95% CI: 1.544–11.050, P = 0.005). The risk of a PSA relapse was 2.37 times higher for quartile 4 patients (95% CI: 1.389–4.043, P = 0.002). Moreover, YWHAZ expression remained a significant predictor for PCa-specific death (P = 0.015) with a hazard ratio of 3.516 (95% CI: 1.275–9.699) in multivariate analysis including Gleason score and T-stage. Similar results were observed for YWHAZ expression and PSA relapse in a multivariate analysis including Gleason score and T-stage (hazard ratio: 1.916, 95% CI: 1.115–3.293, P = 0.019). However, when adjusting for PSA at diagnosis, age at diagnosis and surgical margin status the association disappeared. Whereas YWHAZ expression remained an independent prognostic marker for CRPC in a multivariate model adjusted for Gleason score, T-stage, PSA at diagnosis and age at diagnosis (hazard ratio: 5.072, 95% CI: 1.529–16.827, P = 0.008). DISCUSSION The rate of PCa diagnosis has dramatically increased in the recent years because of an aging population and increased screening efforts.19 These efforts have lead to the detection of locally defined, low-grade, microscopic PCa with unpredictable growth potential. Therefore, there is an urgent need for the identification of new molecular markers that can effectively distinguish low-risk indolent PCa from the clinically aggressive subtype of the disease. Prostate Cancer and Prostatic Disease (2014), 310 – 314
YWHAZ belongs to the family of 14-3-3 proteins involved in several cellular functions. Former studies indicated a direct involvement of YWHAZ in cellular transformation and proliferation and proposed its oncogenic character.5,15,20 In this study, we showed that YWHAZ overexpression is significantly associated with PCa-specific death and development of CRPC. Consistent with our findings, YWHAZ overexpression was also found to decrease disease-free survival of patients with head-and-neck/oral squamous cell carcinomas.10 Furthermore, we found a significant positive correlation between YWHAZ expression and Gleason score. In addition, Kaplan–Meier and Cox regression analyses revealed a significant decrease in survival and an increase in PSA relapse and CRPC in patients with high YWHAZ-expressing tumors. Similar results were derived from YWHAZ studies on non-small-cell lung cancer and breast cancer. Here, YWHAZ was significantly associated with reduced survival and disease recurrence. In nonsmall-cell lung cancer patients, YWHAZ was even the only independent predictor of disease recurrence and reduced overall survival.11,13 Recently, we were able to show that YWHAZ amplification is associated with YWHAZ overexpression in PCa, indicating that gene amplification might be a common mechanism leading to YWHAZ overexpression.15 Furthermore, this hypothesis is supported by the significantly higher YWHAZ expression in the patients that later developed CRPC. Although, in 2012, Murata et al. analyzed the protein expression of YWHAZ in PCa tissue of 90 patients and found a significant higher YWHAZ expression in cases with lymph node metastasis, they did not determine the significance of YWHAZ as a prognostic marker in PCa. 21 We identified YWHAZ to be a prognostic factor for PCa-specific death, development of CRPC and PSA relapse. Furthermore, Murata et al. © 2014 Macmillan Publishers Limited
YWHAZ as a potential prognostic marker in prostate cancer K Rüenauver et al
313 Table 2. Clinico-pathological features of the cohort based on YWHAZ expression
1.0
YWHAZ protein expression 0.8
Parameters
Total
n 159 Age median 65 (57–73) (range), years Follow-up time 115 (29–239) median (range) PSA at diagnosis 8.1 (3.2–52.0) median (range)
Quartile 4 %
Total
%
100.0
54 63 (45–74)
100.0 0.001
127 (14–273)
0.072
9.2 (3.0–49.0)
0.862 0.001
Gleason score 5 6 7 8 9 10
1 54 89 10 5 0
0.6 34.0 56.0 6.3 3.1 0.0
0 4 38 9 3 0
0.0 7.4 70.4 16.7 5.6 0.0
T stage T1 pT2 pT3 pT4
95 54 10 0
59.7 34.0 6.3 0.0
22 30 2 0
40.7 55.6 3.7 0.0
Surgical margin status R0 R1 Rx (distance of tumor to margin o0.5 mm)
P Survival rate
Quartiles 1–3
0.6
0.4
0.2 Quartiles 1-3 Quartile 4
0.0
0.067
0
50
100
150
200
250
300
Survival time (months)
Figure 2. Kaplan–Meier curve of PCa patients divided into two groups (quartiles 1–3 and quartile 4) according to the average YWHAZ marker intensity of the tumors. 0.275
82 33 44
51.6 20.8 27.7
24 17 13
44.4 31.5 24.1
CRPC Yes No Not available
8 146 5
5.0 91.8 3.1
8 38 8
14.8 70.4 14.8
PSA relapse Yes No Not available
38 108 13
23.9 67.9 8.2
21 22 11
38.9 40.7 20.4
PCa specific death Yes No Not available
8 147 4
5.0 92.5 2.5
8 38 8
14.8 70.4 14.8
0.013
0.008
cohort size and patient history available to this study. Another obstacle is the relation of YWHAZ expression in tumors and patient age. Further experiments are needed to stratify PCa cohorts into patients of ages of disease onset to clarify this point. Furthermore, efforts are needed to analyze the YWHAZ expression in a cohort of low-risk patients with localized PCa to delineate the prognostic value of YWHAZ in patients with yet undefined disease progression and outcome. Moreover, validation in biopsies is needed, as biopsies are one of the first steps in the diagnosis of PCa. As our results in prostatectomy samples indicate, high YWHAZ expression is associated with poor patient outcome. Validation of these results in PCa biopsies would further support the prognostic potential of YWHAZ expression in PCa and might help to determine the patient's further treatment at the earliest possible time.
0.012
Abbreviation: CRPC, castration-resistant PCa; PCa, prostate cancer. YWHAZ expression was divided into two groups (quartiles 1–3 and quartile 4) for Kaplan–Meier and Cox regression analysis.
demonstrated that YWHAZ is an androgen-responsive gene.21 As younger men have higher androgen levels than older men, this might be an explanation for the association between younger patient age and high YWHAZ expression.22 The subgroup of tumors showing a high YWHAZ expression seems to be more aggressive than tumors with low YWHAZ expression. Therefore, this subgroup might benefit from an early adjuvant therapy or YWHAZ-targeted therapy. Our findings show that YWHAZ expression in localized PCa could be used to predict the prognosis of PCa patients that in turn might help in the decision on treatment regimes for patients with early diagnosed locally defined PCa. LIMITATIONS Prognostic tools to distinguish low-risk indolent PCa from clinically aggressive disease are still elusive. Clearly, the use of YWHAZ expression levels as a prognostic tool is still hampered by the © 2014 Macmillan Publishers Limited
CONCLUSION In this study, we demonstrated that overexpression of YWHAZ significantly correlates with adverse clinico-pathological parameters. These results suggest that increased YWHAZ expression, determined by IHC in localized PCa, might be a novel prognostic marker for PCa progression and disease-specific death. Therefore, we propose to conduct further experiments to delineate the value of YWHAZ expression as a tool for identifying ideal treatment options. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by grants from the Wilhelm Sander Foundation (2011.077.1), the Rudolf-Becker-Foundation and the German Research Foundation (Deutsche Forschungsgemeinschaft (DFG), Emmy-Noether-Program, PE1179/2-1) to SP. Furthermore, the project was supported by the medical student research grant of the Medical Faculty of the University of Bonn (BONFOR) to KR.
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