STAG2 Coupled With p53 Alteration Has a Significant Impact on Bladder Cancer Recurrence and Progression

Background:There is an unmet need for additional biomarkers for stratifying tumors based on their risk of recurrence and progression. Stromal antigen 2 (STAG2) and p53 are the most common mutations in bladder cancer and their association with the prognosis of bladder cancer remains unclear.Methods:Patients were divided into two cohorts according to stage and surgical procedure. The value of combined STAG2 expression (+/-) and P53 mutation (+/-) was evaluated for prediction of recurrence in 334 patients diagnosed by transurethral resection of bladder tumor (TURBT) and for prediction of survival in 144 patients who underwent radical cystectomy and pelvic lymphadenectomy( RCPLT).Results:We found that,in the 334 TURBT-treated patients, recurrence rate was significantly greater for STAG2(+) tumors than STAG2(-) tumors (73.5% vs. 55.0%, P=0.001). Of 144 RCPLT-treated patients, 127 (88.2%) were STAG2(+), of which 71 (55.9%) survived 5 years, compared to none of the 17 STAG2(-) patients. Patients with combined STAG2(+)/P53 mutation(+) tumors had the highest recurrence rate (83.5%) while patients with STAG2(-)/P53(-) tumors had the lowest recurrence rate (50%). Patients with STAG2(+)/P53(-) tumors achieved the highest 5-year survival rate (69.7%). Systemic chemotherapy did not improve prognosis of RCPLT-treated patients, but appeared to benefit STAG2(-) patients.Conclusions: Our results suggest that combined STAG2 expression and P53 status is a valuable biomarker for BCa prognosis, accurately predicting recurrence in TURBT-treated patients. STAG2(+)/P53(-) predicted a higher five-year survival and STAG2(-) predicted low five-year survival in RCPLT-treated patients. Chemotherapy may interfere with this predictive efficacy, especially in STAG2(-) patients. previous an important gene for homologous recombination repair. 34 STAG2 overexpression may increase chemotherapy resistance by enhancing tumor cell DNA damage repair capacity. We found that both STAG2 mutation [STAG2(-)] and P53 mutation [P53(+)] promoted progression in MIBC patients who underwent RCPLT. Indeed, no STAG2(-)/P53(+) patient in our study survived for 5 years, while 5-year survival was relatively high in STAG2(+)/P53(-) patients (69.7%). This suggests that like P53, STAG2 may be a suppressor of tumor progression. But why do STAG2(+) BCa patients relapse more easily after TURBT? Poor sensitivity to chemotherapy in patients with STAG2 (+) may be one of the causes of its recurrence. interaction between the tumour suppressor STAG2 and its paralog STAG1.


Abstract
Background:There is an unmet need for additional biomarkers for stratifying tumors based on their risk of recurrence and progression. Stromal antigen 2 (STAG2) and p53 are the most common mutations in bladder cancer and their association with the prognosis of bladder cancer remains unclear.Methods:Patients were divided into two cohorts according to stage and surgical procedure.
The value of combined STAG2 expression (+/-) and P53 mutation (+/-) was evaluated for prediction of recurrence in 334 patients diagnosed by transurethral resection of bladder tumor (TURBT) and for prediction of survival in 144 patients who underwent radical cystectomy and pelvic lymphadenectomy( RCPLT).Results:We found that,in the 334 TURBT-treated patients, recurrence rate was significantly greater for STAG2(+) tumors than STAG2(-) tumors (73.5% vs.

Background
Bladder cancer (BCa) or urothelial cell carcinoma is the fourth most common neoplasm in Western males, and most are early-stage tumors known as papillary non-muscle-invasive bladder cancers (NMIBCs). 1,2 After resection, up to 70% of NMIBCs recur locally, and up to 20% of these recurrences progress to muscle invasion [3][4][5] and lethal metastatic bladder cancer, with only 5% of patients surviving 5 years. 6 Additional biomarkers are required for stratifying tumors based on risk of recurrence and progression.
Established BCa risk factors include exposure to industrial aromatic amines, cigarette smoke, and various other DNA-damaging agents 7-10 that can cause DNA mutation and functional protein loss. 11,12 A recent study found that STAG2 is one of the most commonly mutated genes in BCa. 13 The STAG2 protein is a component of the cohesin complex, which functions in diverse genomic processes such as chromosome segregation, regulation of chromatin structure, gene expression, and DNA repair. [14][15][16] Loss of STAG2 expression or STAG2 mutation is associated with BC prognosis, 17  Inactivating mutations of STAG2 have recently been identified in human cancer and were demonstrated to cause chromosome segregation defects and aneuploidy. 20,21 As the STAG2 gene is on the X chromosome, complete genetic inactivation requires only a single mutational event in males, 22 leading to a significantly higher prevalence of BCa in males than females. 23 Further, male mice develop and die from N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder cancer than frequently female mice. 24 This sex difference in BBN-induced BCa is unlikely due to differences in exposure and initial response to the carcinogen because the BBN-induced DNA mutation rates did not differ between sexes. 25 A more recent study reported that the X chromosome protects against BCa via upregulation of the canonical P53 pathway. 26 We and others found that p53 mutation has a significant impact on BCa outcome, 27-29 suggesting that both STAG2 and P53 mutations are associated with BCa recurrence and progression. Previous studies on the relationship between STAG2 and BCa prognosis have yielded discrepant results, possibly because they did not account for P53 gene mutations. To test this hypothesis, we conducted immunohistochemical analysis of STAG2 and P53 expression in 478 bladder tumors of different stages and grades. We also investigated the relationships among STAG2 expression, P53 expression, BCa recurrence, and BCa progression.

Patients and Tissue Specimens
The entire study population consisted of two cohorts divided according to the source of tumor tissue.
Once patients were evaluated against exclusion criteria, the final cohort was composed of 478 patients. Exclusions included patients: (i) whose cut tumor block displayed poor integrity on the slide mount, (ii) whose cut slides had no identifiable tumor, (iii) whose tumor was carcinoma insitu (CIS), (iv) whose tumor was adenocarcinoma,(v) whose clinical information in the cancer registry was sufficiently ambiguous that the presence or absence of recurrence could not be determined,(vi) who received preoperative chemotherapy or radiotherapy,(vii) who underwent total resection received chemotherapy, but the chemotherapeutic drugs were not platinum and (or) gemcitabine.All of these exclusions were performed prior to data analysis. Patients were followed-up using hospital records and by telephone interviews with patients or families. Recurrence was defined as a new tumor in the bladder after primary treatment regardless of their pathological type: bladder ultrasonography was performed monthly and cystoscopy was performed once every three months within twelve months. Bladder ultrasonography was performed every three months after a year, and cystoscopy was performed every six months. Patients who underwent RCPLT were examined by ultrasonography two months after operation and platinum and (or) gemcitabine chemotherapy was started if tumor metastasis was found. Only BCa-related deaths were considered for survival analysis. The study was conducted according to Chinese law and ethics board approval was obtained for gathering all study material and patient data. All patients provided informed consent before participation in the study. The study was approved by the Ethics Committee of Chongqing Medical University.

Immunohistochemistry
We used STAG2 monoclonal antibody (1:100, Santa Cruz Biotechnology) and mouse monoclonal antihuman p53 (DO-1) antibody (1:200, Santa Cruz Biotechnology). Immunostaining was performed in an automated immunostainer (Leica Bond-Max) following 30-min of heat-induced antigen retrieval at high pH using epitope retrieval buffer (Bond-Max). Primary antibody was applied for 30 min, and Bond-Max polymer was applied for 15 min. Diaminobenzidine was used as the chromogen, and samples were counterstained with hematoxylin. Levels of STAG2 and P53 staining were scored in a blinded fashion by two independent observers at two different institutions. Results were scored for transitional cell carcinoma by estimating the proportion of tumor cells with characteristic nuclear staining. An arbitrarily defined 15% cutoff was taken to classify the TCC data into categorical groups(positive versus negative)

Statistical Analysis
All statistical analyses were conducted using SPSS® 13.0. Statistical power analysis was conducted to estimate the required numbers in each cohort. Continuous variables are presented as mean (standard deviation) and categorical variables as count (percentage). Kaplan-Meier curves were constructed for survival analysis and results compared by the log rank test and Cox proportional hazards models.
Statistical significance was set at P = 0.05 (two-tailed) for all tests.

Association between STAG2 expression and bladder cancer recurrence
The relationship between STAG2 expression and recurrence of bladder cancer was analyzed in 334 patients who underwent TURBT (Table 1) (Table 3, log rank test P = 0.000). Kaplan-Meier analysis also demonstrated higher recurrence rate in STAG2(+) patients than STAG2(-) patients ( Fig. 2A, P = 0.001).

Association of combined STAG2 expression and P53 mutation with bladder cancer progression
The relationship between combined STAG2(+)/P53 mutation status and 5-year survival rate of bladder cancer was analyzed in the second cohort. Of 127 STAG2(+) patients, 5-year survival was significantly lower in those with p53 mutation (+) compared to those with normal P53 expression (41% vs. 69.7%, P<0.05 chi square test) ( Table 1). Thus, P53 mutation may promotes bladder cancer progression. We also performed a univariate analysis to evaluate whether combined STAG2(+)/P53(+) status was predictive of survival (Table 5). Indeed, the Kaplan-Meier curve showed that combined status predicted survival more accurately than individual STAG2 or P53 status (Fig. 3B, P = 0.00). In STAG2(+) patients, P53 mutation reduces survival rate compared to P53(-) patients (Fig. 3D, P = 0.01). In STAG2(-) patients, the effect of P53 mutation on prognosis was even stronger (Fig. 3F, P = 0.001). To further study the factors affecting bladder cancer survival, multivariate analysis of the Cox proportional hazard model was conducted and revealed that STAG2 and P53 expression had significant clinical predictive value for cancer-specific survival (Table 6)

Affects of chemotherapy on bladder cancer progression
Chemotherapy is one of the main treatments for bladder cancer. After TURBT, all patients were treated with intravesical chemotherapy, so the chemical treatment effect was difficult to assess among STAG2/P53 expression subgroups. We thus studied the effect of chemotherapy on the prognosis of patients with invasive bladder cancer in the second cohort of 144 patients. Systemic chemotherapy did not improve prognosis, as 5-year survival rate did not differ significantly between treatment subgroups (Table 6). Since the five-year survival rate of STAG2(-) patients was 0%, we compared the 3-year survival rate and found greater survival among the chemotherapy subgroup than the no chemotherapy subgroup ( we performed a univariate analysis to evaluate whether chemotherapy was significantly associated with survival. Chemotherapy did not reduce overall mortality (Fig. 4A, P = 0.292) or significantly improve the prognosis of STAG2(+) patients ( Fig. 4C and Fig. 4D, P>0.05), but may have modest effects for STAG2(-).

Discussion
Bladder cancer is a heterogeneous epithelial malignancy that presents most commonly as an exophytic tumor confined to the mucosa or lamina propria. 30 The recurrence and invasiveness of bladder cancer are associated with poor prognosis. Thus, additional biomarkers are required assessment of recurrence risk, progression, and treatment response.
Recent studies indicate that STAG2 mutation is strongly associated with prognosis. [17][18][19] However, some studies have reached the opposite conclusion. In the current study, high expression of STAG2 was closely related to recurrence and poor prognosis in NMIBC patients. In MIBC patients, however, low STAG2 expression promoted bladder cancer progression. In our study, we also found similar conclusions. The biological basis for the different effects of STAG2 expression on the clinical outcomes of non-muscle-invasive papillary carcinomas versus muscle-invasive carcinomas is currently unknown.
To further examine the relationship between STAG2 and bladder cancer prognosis, we analyzed other prognostic factors, including expression of the tumor suppressor p53. Indeed, P53 gene mutation(the mutant P53 gene was highly expressed after immunohistochemical staining)significantly affected prognosis of STAG2(+) patients. Recurrences was higher in STAG2(+)/P53(+) patients than STAG2(+)/P53(-) patients (84.5% vs. 58%), indicating that P53 mutation is a major cause of BCa recurrence. 31,32 All NMIBC patients were treated with intravesical chemotherapy after operation, and P53 mutation may reduce chemosensitivity. 33 Similarly, previous studies have shown that STAG2 is an important gene for homologous recombination repair. 34  Chemotherapy may improves tumor prognosis and many chemotherapeutic drugs induce tumor cell apoptosis by damaging DNA. 35,36 The sensitivity of tumor cells to chemotherapeutic drugs influences tumor prognosis. 37,38 In this study, we found that systemic chemotherapy did not improve the 5-year survival rate of MIBC patients. Alternatively, STAG2(-) patients receiving chemotherapy demonstrated higher 3-year survival than those not receiving chemotherapy (45.5% vs. 16.7%). Alternatively, chemotherapy provided no survival benefit to STAG2(+) patients. Furthermore, univariate analysis suggested that STAG2(-) patients were more sensitive to chemotherapy than STAG2(+) patients, but it was difficult to draw conclusions because of the insufficient sample size. From these results, we speculate that chemotherapy (intravesical instillation chemotherapy) reduces relapse of STAG2(-) NMIBC due to a superior effect of intravesical instillation chemotherapy versus systemic chemotherapy for bladder cancer. 39 Cancer prognosis is strongly related to the genotype of tumor cells and specifically how different genotypes and expression phenotypes affect growth, progression, and treatment response. 40,41 In this study, we also compared the prognosis of MIBC patients with different expression phenotypes after chemotherapy. STAG2(+)/P53(-) patients still had better prognosis than STAG2(-)/P53(+) patients even after chemotherapy, possibly due to sustaining progression of mutant tumor cells and (or) inefficacy of chemotherapy. In bladder cancer, mutations in the P53 gene and STAG2 gene lead to tumor progression, and systemic chemotherapy cannot effectively inhibit this process, resulting in poor prognosis in STAG2(-)/P53(+) patients.
Our previous colleagues found that total resection of early bladder cancer significantly reduced recurrence and mortality. In clinical treatment, patients are willing to choose total cystectomy and bladder substitution after knowing that the diagnosis of bladder cancer is bladder cancer, which leads to partial bladder surgery for early stage tumors. We will follow up these patients for a long time to see if their long-term survival rate has improved. Preoperative chemotherapy may cause changes in STAG2 or P53 expression in surgical specimens. We excluded patients who received preoperative chemotherapy or radiotherapy. After total cystectomy, some patients received cisplatin combined with gemcitabine systemic chemotherapy (other drug chemotherapy patients were excluded), and some patients did not undergo systemic chemotherapy because of the low stage of the tumor during the operation.
However, this does not affect our purpose. We focus on identifying the factors that influence the relationship between STAG2 and the prognosis of bladder cancer. STAG2 is a functional gene on X chromosome. Recently, it has been found that X chromosome can regulate P53 pathway. Therefore, we first speculate that STAG2 may affect the prognosis of bladder cancer through its association with P53. In addition, the most important way to affect the prognosis of bladder cancer is clinical treatment and chemotherapy. Therefore, the focus of our study is to analyze whether these factors are related to STAG2.
We tested different thresholds used to define positivity or loss of expression of STAG2. We found that defined 15% as the cutoff frequency for STAG2 and P53 to classify transitional cell carcinoma data into categorical groups is the most reasonable result.

Conclusions
we confirm the association between combined STAG2/P53 status and bladder cancer prognosis. Ethics approval and consent to participate All pa¬tients provided informed consent before participation in the study. The study was approved by the Ethics Committee of Chongqing Medical University.

Availability of data and materials
The basic patient information and IHC staining results have been shown inTable 1, Table 2 and Table   5.

Consent for publication
Not applicable

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
Jiangchuan Chen, Zhigang Xu and Xiao-Dong Meng for acquisition of data, analysis and interpretation of data,statistical analysis and drafting of the manuscript. Yin Chen and Jie Li for technical and material support. Jie Li for study concept and design ,analysis and interpretation of data, drafting of the manuscript, obtained funding and study supervision. All authors read and approved the final manuscript.        Three-year cancer-specific survival of RCPLT-treated STAG2(-) patients with and without chemotherapy. P values correspond to results from multivariable analysis.