Expression of Estrogen Receptor Beta Predicts Oncologic Outcome of pT3 Upper Urinary Tract Urothelial Carcinoma Better Than Aggressive Pathological Features

Upper urinary tract urothelial carcinoma (UT-UC) is rare and treatment options or prognostic markers are limited. There is increasing evidence indicating that urothelial carcinoma may be an endocrine-related cancer. The aim of this study was to analyze the prognostic effect of estrogen receptor beta (ERβ) on the outcome of UT-UC. From 2005 to 2012, this study included 105 patients with pT3 UT-UC. Perioperative factors, pathological features, and ERβ immunostaining were reviewed and prognostic effects were examined by multivariate analysis. This study divided patients into either the ERβ-high (n = 52) or ERβ-low (n = 53) group and analyzed their oncologic outcomes. All pathological features except infiltrating tumor architecture (significantly higher incidence in ERβ-low group, p = 0.004) are symmetric in both groups. Low ERβ expression was significantly correlated with local recurrence and distant metastasis in univariate analysis (p = 0.035 and 0.004, respectively) and multivariate analysis (p = 0.05 and 0.008, respectively). Cell line study also proved that knock down of ERβ cause less UTUC proliferation and migration. In addition, ERβ agonist also enhanced the cytotoxic and migration inhibition effect of cisplatin and ERβ antagonist cause the UTUC cell more resistant to cisplatin. This result may help identify patients in need of adjuvant therapy or develop potential targeted therapy.

Pathological features and clinical outcome assessment. Perioperative data were obtained from patient charts. Data on commonly known prognostic pathological features, including lymphovascular invasion, carcinoma in situ, squamous differentiation, and tumor necrosis were recorded 10 . The follow-up protocol at our institution is postoperative fiber-cystoscopy and renal echo every 3 months during the first 2 years, every 6 months during the third to fifth year, and every year thereafter during the follow-up period. Abdominal computed tomography was performed annually to assess local or regional recurrence of the tumor and lymph node status. Local recurrence is defined as disease occurrence in the ipsilateral retroperitoneal space, and distant metastasis is defined as disease occurrence outside the residual urinary tract system and ipsilateral retroperitoneal space, such as in other organs or in the contralateral retroperitoneal space. Immunohistochemistry and patient grouping. We have tested the AR and ER-alpha in thirty randomized fifty T3 UTUC specimens initially. However, there is little AR and ER alpha expression in our series and this result might suggest that AR and ER-alpha may not occupy an important role in prediction of clinical prognosis in our series (Supplement file, Fig. 1). Therefore we choose ERβ as out study target. Immunostaining for estrogen receptor beta (ERβ ) was performed on a fully automated Bond-Max system (Leica Microsystems). Slides carrying tissue sections cut from formalin-fixed, paraffin-embedded tissue microarray blocks were dried for 1 hour at 60 °C. These slides were then covered by Bond Universal Covertiles and placed into the Bond-Max instrument. All subsequent steps were performed automatically by the instrument according to the manufacturer's instructions (Leica Microsystems). The following procedure was used: (1) deparaffinization of tissue on slides by rinsing with Bond Dewax Solution at 72 °C; (2) heat-induced epitope retrieval (antigen unmasking) with Bond Epitope Retrieval Solution 2 for 20 minutes at 100 °C; (3) peroxide block placement on the slides for 5 minutes at room temperature; (4) incubation with a mouse monoclonal anti-ERβ antibody at a dilution of 1:100 for 30 minutes at room temperature; (5) incubation with Post Primary reagent for 8 minutes at room temperature; (6) Bond Polymer placement on the slides for 8 minutes at room temperature; (7) color development  Representative conventional tumor sections were selected to examine the pattern of ERβ presentation instead of tissue microarray (Fig. 1). Pathologist M.T.S., who was blinded to the oncologic outcome, scored the percentage of ERβ expression in UTUC representative slide and we choose 10% as the cut-off value. The percentage of ERβ expression more than 10% was thought to be high ERβ expression.
Cell line study. We use BFTC-909 cell line (a cell line from renal pelvis patient), which is an ERβ expression cell line, for cancer behavioral analysis (Supplement file, Fig. 2). This cell line was cultured in Dulbecco's modified Eagle's medium, containing 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in an atmosphere of 5% CO2. ERβ was knock down adequately by Origene ER-beta shRNA transfection (Supplement file, Fig. 3). Wound healing assay was performed to test the proliferation ability of cell line. Cell monolayers were then wounded by sterile pipette tips (1 ml) that generated a gap. Wounded monolayers were then washed three times with PBS to remove cell debris and incubated in 10% CD FBS medium for 24-hour observation. Migration trans-well assay was performed to assess the aggressiveness of cell line. In the upper layer of a cell permeable membrane and a solution containing the test agent is placed below the cell permeable membrane. Following an incubation period (37 °C, 5% CO2, 24 hours), the cells that have migrated through the membrane are stained and counted.

Results
This retrospective study included samples collected between 2005 and 2012 from 105 patients with locally advanced UT-UC (pT3) and adequate quality of tissue for immunohistochemical stain, divided patients into ERβ -high (n = 52) or ERβ -low (n = 53) groups and then analyzed their oncologic outcomes. Table 1 reveals patient characteristics and all pathological features except infiltrating tumor architecture, which had a significantly higher incidence in the ERβ -low group (p = 0.004), were identical in both groups. Upper urinary tract urothelial carcinoma with low ERβ expression had worse local recurrence and distant metastasis-free survival compared with high ERβ expression (p = 0.05 and 0.02 respectively, Fig. 2). The results of multivariate analysis are listed in Table 2. All commonly known prognostic pathological factors were included for examination. Low ERβ expression was significantly correlated with local recurrence and distant metastasis in univariate analysis (p = 0.035 and 0.004 respectively) and multivariate analysis (p = 0.05 and 0.008 respectively). The result of cell line validation showed that knock down of ERβ cause aggressive UTUC cancer cell proliferation behavior by wound healing assay (Fig. 3). The migration assay also revealed more aggressive UTUC cancer cell migration if ERβ was knocked down (Fig. 4). The cytotoxic and migration-inhibiting effect of ERβ on UTUC cell line was observed and compared by cisplatin treatment. ERβ agonist enhanced the cisplatin effect while ERβ antagonist cause UTUC cell more resistant to cisplatin treatment (Fig. 5).

Discussion
UT-UC is rare and the treatment options are limited. The TNM staging for UT-UC is relatively simple and there are few markers for sub-classification of advanced stage UT-UC 11 . The current standard treatment for UT-UC is nephroureterectomy with bladder cuff excision. Much effort has been focused on prognostic pathological features or sub-classification of the current TNM staging system [3][4][5] . The subclassification of locally advanced UT-UC is a clinically important issue. Unlike localized UT-UC (pT0-2), patients with locally advanced stage pT3 UT-UC are thought to experience much higher disease recurrence, even after radical surgery. However, variant prognoses are still noted in pT3 UT-UC in clinical practice. Further sub-classification of such locally advanced stage disease will be helpful to identify patients with need of early adjuvant therapy. Therefore, patients with pT3 UT-UC were selected to identify markers predictive for oncologic outcome in this study.
The most common challenge of advanced UT-UC is the high prevalence of renal insufficiency. Ineligibility for cisplatin-based chemotherapy due to impaired renal function, especially after nephroureterectomy, leads to poor prognosis in such patients 12 . Several molecular markers have been proposed to be associated with oncologic outcomes and may be potential targets for treatment of UT-UC [13][14][15][16] . However, little has been reported about the independent prognostic role of markers in comparison with other aggressive pathological features. In this study, we selected common prognostic pathological features from a systemic review 10 in order to identify if the expression of ERβ had an independent and predictive prognostic effect that might initiate further translational investigations.
Increasing evidence indicates that urothelial carcinoma is a potential endocrine-related malignancy 6,-8 . The most well known examples of biomarkers in endocrine-related cancer are estrogen receptors in breast cancer, where individualized hormonal therapy has improved overall prognosis 17 . However, research on hormone receptors has focused mainly on UB-UC 6,-8 . Though UB-UC and UT-UC have similar cellular origins, their cancer behaviors differ 18 . ERβ has been reported to be a potential target associated with urothelial carcinoma behavior 8,9 . To our knowledge, this is the first study focusing on the prognostic effect of ERβ on UT-UC.
The most common method for prognostic marker screening is tissue array. However, asymmetric distribution of ERβ was noted in our specimens. We think that a tissue array might lead to detection bias in the subsequent outcome analysis. In this study, formalin-fixed representative conventional tumor blocks were used to thoroughly examine ERβ expression. The urologist assessing the clinical oncological outcome was blinded to ERβ expression and the pathologist examining ERβ expression was blinded to the oncologic outcome. Our result revealed that ERβ independently predicts the oncologic outcome for pT3 UT-UC. Its prognostic role has been reported in several malignancies 8,[19][20][21] . ERβ suppresses the cadherin switch and correlates with better oncologic outcome. It is thought that ERβ expression associates with better survival 8 . For non-organ-confined UT-UC, the cancer behavior is reasonably an important mechanism for local recurrence and even distant metastasis. In our cell line study, knock down of ERβ does cause aggressive UTUC cancer cell behavior. In addition, agonist activation of ERβ has been shown to be associated with cell sensitization to cisplatin cytotoxicity, which is compatible with our cell line study 21 . These reports further support our finding that positive ERβ expression is correlated with better prognosis.
The major limitation of this study is its retrospective design and we could only observe the natural course of the UT-UC behavior without any adjuvant or targeted treatment. A prospective study with ERβ and the response to adjuvant or targeted interventions may provide further useful clinical information. The advantages of this study were a careful examination of the prognostic role of ERβ compared with established prognostic factors by multivariate analysis and a focus on the oncologic outcome of advanced UT-UC (pT3). Multivariate analysis can eliminate the potential crossed bias from other significant pathological factors. In addition, a focused analysis on T3 UT-UC avoids the curative effect of radical surgery on organ-confined disease (pT0-2). In this study, ERβ still showed a significant association with oncologic prognosis. Further studies on adjuvant or targeted therapy on ERβ for UT-UC are deemed worthwhile.

Conclusion
This study proposes that positive presentation of ERβ is correlated with better prognosis of pT3 UT-UC compared with commonly known aggressive pathological features. This result may help to identify patients requiring early adjuvant therapy or develop potential targeted therapies.