Co-expression of cancer-testis antigens of MAGE-A6 and MAGE-A11 is associated with tumor aggressiveness in patients with bladder cancer

Melanoma antigen gene (MAGE)-A6 and MAGE-A11 are two of the most cancer-testis antigens overexpressed in various types of cancers. However, the clinical and prognosis value of MAGE-A6 and MAGE-A11 co-expression in the pathophysiology of the bladder is unknown. Three studies were selected from GEO databases in order to introduce the common genes that are involved in bladder cancer. Then immunohistochemical analysis for staining pattern and clinicopathological significance of suggested markers, MAGE-A6 and MAGE-A11, were performed in 199 and 213 paraffin-embedded bladder cancer with long adjacent normal tissues, respectively. A significant and positive correlation was found between both nuclear and cytoplasmic expressions of MAGE-A6 as well as expression of cytoplasmic MAGE-A11 with histological grade, PT stage, lamina propria invasion, and LP/ muscularis (L/M) involvement (all of the p-values in terms of H-score were < 0.0001). Additionally, significant differences were found between both nuclear and cytoplasmic MAGE-A6/MAGE-A11 phenotypes with tumor size (P = 0.007, P = 0.043, respectively), different histological grades, PT stage, LP involvement, and L/M involvement (all of the p-values for both phenotypes were < 0.0001). The current study added the value of these novel markers to the bladder cancer clinical settlement that might be considered as an admirable target for immunotherapy.


Results
Bioinformatics approach. Three studies GSE27448 [30][31][32] , GSE100926 33 , and GSE61615 34 that each included the information of BC tissue and control tissues were explored. GSE27448 included: 1) GSE89figure dataset (GDS183), comprised of 40 BC samples; 2) GSE3167 dataset (GDS1479), comprised of 60 samples (9 controls and 51 BC samples); 3) GSE7476 dataset, composed of 12 samples (3 controls and 9 bladder cancer samples) and 4) GSE12630 dataset, comprised of 19 BC samples. In total, their pooled microarray analysis was composed of 17 control samples and 129 BC samples. GSE100926 consisted of three controls and three BC samples and GSE61615 consisted of two controls and two BC samples. The results were analyzed and the statistically significant differential expression of genes in tumor tissues in comparison to control tissues was obtained from selected previous studies (P < 0.05, supplementary Table 1). Venn diagram analysis was performed to find common significant differential in this GSE BC mentioned (Fig. 1A).
Our list was the LINGO2 (Leucine-Rich Repeat and Ig Domain Containing 2), SLC17A9 (Solute Carrier Family 17 Member 9), KCNS1 (Potassium Voltage-Gated Channel Modifier Subfamily S Member 1), MAGEA6, and MAGEA11. LINGO2 encodes a transmembrane protein in addition to its role in Parkinson 's disease 35 , identified as a cancer stem cell-associated protein in gastric cancer initiation and progression by altering cell motility, stamens, and tumorigenicity both in vitro and in patient-derived tissues 36 . SLC17A9 can be used as a new molecular marker to predict the poor prognosis of patients with hepatocellular carcinoma 37 . It is also may play a role in the progression of colorectal cancer 38 and may potentially be used as an independent biomarker for gastric carcinoma prognostic evaluation as well 39,40 . KCNS1 was reported as a bone metastasis signature using a supervised classification approach in a large series of breast cancer patients 41 and variations in this potassium channel genes were associated with the occurrence of preoperative breast pain 42 . Accumulating data proved that MAGE-A11 contributes to the genetic susceptibility and prognosis for renal cell carcinoma as a biomarker for occurrence and prognosis 43 . Besides, studies underscore that MAGE-A11 expression has a negative predictive Scientific Reports | (2022) 12:599 | https://doi.org/10.1038/s41598-021-04510-2 www.nature.com/scientificreports/ role in head and neck cancer 44,45 and valuable diagnostic or prognostic marker as well as a potential molecular therapeutic target 46 . This gene not only portrays DNA hypermethylation but also is important in histone deacetylation for the mechanism underlying gene silencing in breast cancer patients 25,47,48 . Additionally, more research was conducted in identifying new approaches for developing related to the clustered MAGE-A expression analysis cancer-specific therapeutics in esophageal squamous cell carcinoma (ESCC) 49,50 and prostate cancer as well 22,51 .
Overall we intend to focus on MAGE family members in BC for the first time and provide their involvement in the development of new cancer treatment strategies. Common genes with the highest degree of connectivity of PPI network analysis (highest confidence > 0.9) were identified (Fig. 1B). Gene ontology (GO) analysis of these common genes were included on Enrichr and their related GO and Reactome pathways were shown in Supplementary Table 1. In our evaluation, potassium channel complex among cellular components, purine/adenosin nucleotide transmembrane transporter activity,  66 represents the overlaps of differential protein expressions between three subtypes of BC. Five common differential expressions genes (LINGO2, SLC17A9, KCNS1, MAGEA6, and MAGEA11) were obtained from the GEO database, including GSE61615, GSE27448, and GSE100926. (B) PPI via STRING (https:// string-db. org/) 69 of common differential protein expressions was obtained by the string with the highest degree of connectivity (highest confidence > 0.9) in three GEO databases of BC. (C) The Box plot expression results of the BC Match TCGA normal and GTEx data showed that increased expression of MAGE-A6 protein on GEPIA database (p < 0.05, |Log2FC| Cutoff: 2) (http:// gepia. cance rpku. cn/ index. html) 73 . (D) The Box plot expression results of the BC Match TCGA normal and GTEx data showed that increased expression of MAGE-A11 protein on GEPIA database (p < 0.05, |Log2FC| Cutoff: 2) . (E) The Co-expression results of genes predicted by the GEPIA database online analysis showed that there is a statistically significant spearman correlation between MAGE-A6 and MAGE-A11 protein in BC and normal based on TCGA samples (Log-rank test; P = 4e-23). (F) Box plot analysis of the relative expression levels of MAGE-A6 in normal tissues and BC patient's individual cancer stage (I-IV) tissues by UALCAN database (http:// ualcan. path. uab. edu/) 72 . Based on the standard definitions, each box-plot shows the median (bold line) and interquartile lines (box). The result of Mann-Whitney U test showed that there is an association for the median of expression between Normal-vs-Stage2 (p = 2.541000E-03), Normal-vs-Stage3 (p = 4.42790000000359E-05), Normal-vs-Stage4 (p = 1.189250E-04), Stage2-vs-Stage3 (p = 1.790020E-01), Stage2-vs-Stage4 (p = 2.900000E-01), Stage3-vs-Stage4 (p = 7.813600E-01), and there were no statistically significant differences in the median level of MAGE-A6 mRNA expression between Stage1-vs-Normal and the other stages. (G) Box plot analysis of the relative expression levels of MAGE-A11 in normal tissues and BC patient's individual cancer stage (I-IV) tissues by UALCAN database. Based on the standard definitions, each box-plot shows the median (bold line) and interquartile lines (box). The result of Mann-Whitney U test showed that there is an association for the median of expression between Normal-vs-Stage1 (p = 4.896000E-01), Normal-vs-Stage2 (p = 2.821200E-03), Normal-vs-Stage3 (p = 1.76258999999845E-05), Normal-vs-Stage4 (p = 4.155100E-04), Stage1-vs-Stage2 (p = 7.867800E-01), Stage1-vs-Stage3 (p = 6.622000E-01), Stage1-vs-Stage4 (p = 7.758200E-01), Stage2-vs-Stage3 (p = 2.979400E-01), Stage2-vs-Stage4 (p = 5.875000E-01), Stage3-vs-Stage4 (p = 7.793800E-02). BC: bladder cancer, GEPIA: gene expression profiling interactive analysis web server, GTEx: genotype-tissue expression project PPI: protein protein interaction, TCGA: the cancer genome atlas. www.nature.com/scientificreports/ ATP/ADP transmembrane transporter activity, potassium channel regulator activity between molecular function were spotlighted. In addition to the above-mentioned activities, regulation of autophagy and cellular catabolic process throughout the biological process were considered significantly highlighted as well. It was shown that potassium channels regulate membrane potential, ion homeostasis, and electric signaling 52,53 . Furthermore, the presence and also activity of ion pumps/ channels has correlation with the cancer development via its proliferation, differentiation, apoptosis, and migration 54 . In cBioportal, our genes among copy number alterations (CNA) and mutations of BC (12 studies, 2410 samples) were checked. In addition, a confirmatory analysis was conducted using the gene expression profiling interactive analysis (GEPIA) database to acquire more reliable analytic results related to tumor/control differential expression (Fig. 1C,D), and correlation analysis (Fig. 1E). MAGEA6 and MAGEA11 through UALCAN database based on cancer genome atlas (TCGA) were reported (Fig. 1F,G). Next, our genes were shown in BC ( Supplementary Fig. 1). Finally, these two genes were selected for evaluation of expression using the IHC method in bladder tissues. Additionally, it was cleared that these two genes are expressed high, medium and low in the BC/ bladder tumor lines ( Supplementary Fig. 2).
Characteristics of study population. To evaluate the MAGE-A6 and MAGE-A11 expressions and their clinical relevance, the expression of these markers was determined in 199 and 213 BC tissues, respectively. Overall, the median age of the study population both MAGE-A6 and MAGE-A11 expression was 67 years (range 20-95). The study population consisted of 156 (78.4%) male and 43 (21.6%), female patients, with a male/female ratio of 3.6 for MAGE-A6 expression. For MAGE-A11 expression was 170 (80%) male and 43 (20%) female patients, with a male/female ratio of 3.9. This ratio is consistent with the prevalence of BC in men to women, estimated at 2: 1 to 4: 1 55 . Tumor size (at the largest diameter) ranged from 1 to 13 cm. Based on mean tumor size  Table 3). The mean of nuclear MAGE-A6 H-score was 157 for cancerous tissue vs 11 for normal tissues and a strong significant was found between normal and cancerous tissues (P < 0.0001). Of 213 bladder cases stained for nuclear MAGE-A11, negative, weak, intermediate, and strong intensities were observed in 141(66.2%), 30 (14.1%), 24 (11.3%), and 18 (5.8%) of cases, respectively. For cytoplasmic expression, negative, weak, intermediate, and strong intensities were observed in 1(0.5%), 52(24.4%), 68 (31.9%), and 92 (43.2%) of cases, respectively (Table 3).
Significant differences were found between nuclear MAGE-A6 expression with age (P = 0.038), different histological grades (P < 0.0001), PT stage (P < 0.0001), LP involvement (P < 0.0001), and lamina propria / muscularis (L/M) involvement (P < 0.0001) in terms of intensity of staining. In this regard, significant differences were observed between nuclear MAGE-A6 expression with age (P = 0.016), tumor size (P = 0.044), different histological grade (P < 0.0001), PT stage (P < 0.0001), LP involvement (P < 0.0001), and L/M involvement (P < 0.0001) in terms of H-score (Table 1). Pearson's χ2 analysis showed that there was a direct and positive relationship between the mentioned parameters with nuclear MAGE-A6 expression. As age, tumor size, histological grade, tumor invasion to LP, and L/M increased, nuclear MAGE-A6 expression increased. Significant differences were also found between cytoplasmic MAGE-A6 expression with different histological grades (P < 0.0001), PT stage (P < 0.0001), LP involvement (P < 0.0001) both in terms of intensity of staining and H-score. P values of intensity of staining and H-score for L/M involvement were 0.0001 and < 0.0001, respectively (Table 1). Pearson's χ 2 analysis showed that there was a direct and positive relationship between the mentioned parameters with cytoplasmic MAGE-A6 expression. As a histological grade, PT stage, tumor invasion to LP, and L/M increased, cytoplasmic MAGE-A6 expression increased. These results indicate an increase in both nuclear and cytoplasmic MAGE-A6 expression in advanced stages of BC.
No significant differences were found between nuclear MAGE-A11 expressions with clinicopathological parameters ( Table 2). Significant differences were found between cytoplasmic MAGE-A11 expression with different histological grades (P < 0.0001), PT stage (P < 0.0001), LP involvement (P < 0.0001) both in terms of intensity of staining and H-score. P values of intensity of staining and H-score for L/M involvement were 0.0001 and < 0.0001, respectively ( Table 2). Pearson's χ 2 analysis showed that there was a direct and positive relationship between the mentioned parameters with cytoplasmic MAGE-A11 expression. As a histological grade, PT stage, tumor invasion to LP and L/M increased, MAGE-A11 expression increased. These results indicate increased expression of cytoplasmic MAGE-A11 in advanced stages of BC.
Further analysis based on the Mann-Whitney U test showed a significant difference between both nuclear and cytoplasmic MAGE-A6 expressions with histological grade (P < 0.0001), such that in high grades, an increased www.nature.com/scientificreports/ expression was observed compared to low grades (Fig. 3A). In addition, there was a significant difference between the cytoplasmic MAGE-A11 expression and histological grade (P < 0.0001, Fig. 3B).
Kaplan-Meier analysis (with log-rank test) was used to investigate the association between MAGE-A6 and MAGE-A11 expressions with disease-specific survival (DSS) and progression-free survival (PFS). Based on the H-score described in the method section, nuclear and cytoplasmic expressions of MAGE-A6 and MAGE-A11 were divided into low, moderate or intermediate, and high expressions. Table 1. Association between MAGE-A6 expressions (staining intensity and H-score) and clinic-pathological parameters of BC cases (P-value, Pearson's chi-square test). Bold numbers represent significant p-values.

Patients and tumor characteristics Total samples N (%)
Nuclear expression of MAGE-A6   The Pearson's chi-square analysis was used to examine the correlation between expression of MAGE-A6/ MAGE-A11 phenotypes and clinicopathological parameters. Along with significant correlation of MAGE-A6 and MAGE-A11 with some clinicopathological parameters described above, significant differences were also found between both nuclear and cytoplasmic MAGE-A6/MAGE-A11 phenotypes with tumor size (P = 0.007, P = 0.043, respectively), different histological grades, PT stage, LP involvement, L/M involvement (all of the p-values for both phenotypes was P < 0.0001) ( Tables 4 and 5). Pearson's χ2 analysis showed that there was a direct and positive relationship between the mentioned parameters with MAGE-A6/MAGE-A11 phenotypes. As tumor size, histological grade, PT stage, tumor invasion to Laminia propia and L/M increased, simultaneous expression of MAGE-A6/MAGE-A11 increased.
To know which phenotypes caused significant differences, one-way ANOVA and Tukey s post hoc analysis were used to examine the correlation between expressions of MAGE-A6/MAGE-A11 phenotypes and clinicopathological parameters. As shown in Table 6, there was a significant correlation mainly between MAGE-A6 h /

Discussion
Despite recent progress in BC prognostic, we faced a wide range of failures in bladder patients' treatment 56 . Thus, novel and practical clinical prognostic markers are needed to be introduced for future BC decision-making in a clinical settlement. In this way, the bioinformatics analysis would be conducive to defining novel molecular markers. In the current study, we tend to identify the biomarkers that were potentially involved in the BC. Regarding this, we focused on GEO microarray analysis and some bioinformatics online software investigations of important genes. Besides, enrichment analysis determined the involved pathways and their molecular function. Among them, we selected the MAGE-A gene family that is expressed at a high frequency in various tumors. The expression of MAGE-A11 and MAGE-A6 genes were examined only in two studies that were performed by Duan et al. 57 and Laiseca et al. 58 based on coremine data (https:// www. corem ine. com/) to clarify MAGE proteins collaboration in tumorigenesis and the potential importance of their detection to prognosis purposes. Therefore, for the first time, to validate the MAGE-A11 and MAGE-A6 proteins as a prognostic marker for BC was investigated in a well-characterized series of BC tissues specimens. www.nature.com/scientificreports/  (20) pT3  www.nature.com/scientificreports/ In this study, we evaluated immunohistochemical analysis for the expression pattern of MAGE-A6 and MAGE-A11 in 199 and 213 BC samples, respectively. The expression patterns were analyzed with the clinicopathological data of the patients with BC including age, gender, tumor size, histological grade, PT stage, LP involvement, muscle invasion, L/M involvement, recurrence, and distance metastasis. Survival analysis was also evaluated to find the vitality of the MAGE-A6 and MAGE-A11 as potential prognostic factors.

Lamina propria involvement
MAGE-A11 contributes to the AR signaling pathway in prostate cancer cells 23 . It binds directly to the AR, promoting transcriptional through direct binding to the AR FXXLF motif region 23 . Previous studies have demonstrated that AR activation generally associates with the promotion of the growth and development of BC [59][60][61] . Such that AR deletion in AR-positive bladder cell lines using siRNA led to a significantly decreased cell proliferation, cyclin-D1, Bcl-x(L) as well as migration, metastasis-related matrix metallopeptidase (MMP)-9 compared to control 62 . Moreover, it has been shown that androgen-mediated AR signals are correlated with the tumor development and progression of cancer, which may obviously justify some sex-specific differences in BC 59 . Database search confirmed that MAGE-A6 and MAGE-A11 are co-expressed in samples of human prostate cancer. The interaction between these two markers in cancer progression is clearly elucidated in an experimental study 27 .
In the current study, MAGE-A6 and MAGE-A11 were detected in cancer cells either in the cytoplasm or the combined pattern of staining as the nucleus & cytoplasm pattern. Our staining pattern was consistent with findings of Jia Sh et al. for MAGE-A11 expression in head and neck Squamous cell carcinoma 24 . Endo et al. also evaluate MAGE-A6 in gastric cancer by immunohistochemistry, but there is no explanation for the pattern of MAGE-A6 expression in this study 28 . Our immunohistochemical staining showed different expression patterns, from negative to strong staining, so that there was a differentiation between low grades and high grades of BC for both nuclear and cytoplasmic MAGE-A6 expressions as well as cytoplasmic MAGE-A11 expression. Simultaneous nuclear and cytoplasmic expression of other members of the MAGE-A family including MAGE-A2 and MAGE-A3 in patients with prostate cancer revealed a significant correlation with clinic-pathological outcomes and recurrence-free survival 13,63 . Similarly, the expression pattern of MAGE-A6 and MAGE-A11 in BC and www.nature.com/scientificreports/ their correlation with clinic-pathological findings indicate the importance of expression of some members of the MAGE-A family in the diagnosis and prognosis of cancer patients. It was suggested that the pattern of subcellular expression of these antigens may indicate a difference in their biological activity. A high level of MAGE-A11 protein was found in castration-recurrent prostate cancer. Increased MAGE-A11 levels participate in AR transactivation and the growth and progression of prostate cancer 23 . On the side, Endo et al. revealed that a high level of MAGE-A6 is associated with a worse prognosis in patients with gastric cancer. Its expression level in primary lesions predicted the possibility of disease recurrence. MAGE-A6 mRNA levels were higher in gastric cancer tissues in comparison with normal tissues. A positive correlation was also found between the mRNA of MAGE-A6 and matrix metallopeptidase 9 mRNA 28 . In a systematic literature search, MAGE-A6 was also significant in thymoma, esophageal adenocarcinoma, and kidney renal papillary cell carcinoma, while MAGE-A11 was in pheochromocytoma and paraganglioma 64 . Increased MAGE-A11 was also an independent prognostic factor for the overall survival in patients with head and neck squamous cell carcinomas 24 .
In this study, a strong significant direct correlation was observed between the expression of nuclear and cytoplasmic MAGE-A6 as well as cytoplasmic MAGE-A11 with histological grade, PT stage, LP involvement, and L/M involvement, so that with increasing grade, stage, and tumor invasion into LP and L/M, the expression of these two markers increased. Consistently, when the pathological grade of patients was analyzed using Mann-Whitney U test, a direct correlation was also found between both nuclear and cytoplasmic expressions of MAGE-A6 and cytoplasmic expression of MAGE-A11. These findings indicate the importance of the high expression of these markers in the progression of BC. Moreover, the significance of cytoplasmic expression of the MAGE-A11 was valuable with clinicopathological features that could indicate the activity of this marker in the cytoplasm of cancer cells, while both nuclear and cytoplasmic expression of MAGE-A6 was valuable with clinicopathological features. Although the exact role of MAGE-A6 is unknown, these findings indicate the importance of the expression of this marker as well as its possible function in the nucleus and cytoplasm of BC cells.
In our study, survival analysis was performed and the association of MAGE-A6 and MAGE-A11 expressions with DSS and PFS was determined. Univariate analysis indicated that there was no association for both MAGE-A6 and MAGE-A11 expressions with DSS and PFS. Although the PFS was longer in patients with cytoplasmic poor expression of the MAGE-A11; however, no significant association was observed for their expression. This may be due to the higher number of patients in the censored group than the death group, such that the number of cancer-related deaths was low during the follow-up period; if the follow-up period extended, the number of deaths may increase relative to patient survival.
It has been shown that the MHD domain of MAGE-A6 enhances AR activation through MAGE-A11 and is critical for MAGE-A11 interaction and AR regulation. When MAGE-A6 is co-expressed, a lower degree of MAGE-A11 was ubiquitinylation suggesting that it could protect MAGE-A11 from proteasome-dependent degradation proteins 27 . However, no effect on the dynamics of AR translocation to the nucleus was observed upon MAGE-A6 expression 27 . Therefore, due to the interaction of these two markers in the activation of AR and the spread of malignant progression, we attempted to identify different phenotypes with regard to MAGE-A6 and MAGE-A11 expressions in BC tissues. We compared the significance of MAGE-A6 and MAGE-A11 co-expression in clinical samples in BC. For cytoplasmic expression, the highest percentage among bladder samples was allocated to the MAGE-A6 h /MAGE-A11 h phenotype with 47.5% cases. In addition, statistical analysis showed that there is a bivariate correlation between cytoplasmic MAGE-A6 and MAGE-A11 expressions in bladder samples. It is suggested that these markers affect the expression of each other in the cytoplasm of cancer cells. According to the analyzes described for each marker separately above, combined analyzes also showed a significant association of various phenotypes of the MAGE-A6/MAGE-A11 with clinicopathological parameters including tumor size, histological grade, PT stage, LP involvement, and L/M involvement for both nuclear and cytoplasmic expression. The major phenotype that caused significant differences in clinicopathological parameters was the MAGE-A6 h /MAGE-A11 h . In other words, when the expression of both markers increases simultaneously, the rate of disease progression based on clinicopathological parameters was significant that indicate the importance of high expression of these two markers in tissue samples of patients with BC. These immunopathological data were in line with the previous in vitro study that showed the MAGE-A6 and MAGE-A11 form a protein complex resulting in the stabilization of MAGE-A11 and consequently the enhancement of AR activity 27 .
On the other hand, given that the importance of the complex formation of MAGE-A6/MAGE-A11 in activating AR, however, our immunohistochemical study failed to reveal significant sex-related differences for both MAGE-A6 and MAGE-A11 expressions in male versus female samples. This finding is consistent with previous findings that showed no significant sex-related differences in AR expression in male versus female tissues in patients 59 . However, in order to determine the exact mechanism of action of the MAGE-A6 and MAGE-A11 in BC cells, future studies are needed to answer the question of whether these two markers really function in the progression of BC through ARs?
On the other hand, in recent years, immunotherapy has played a major role in the treatment of cancer patients. To establish immunotherapy or vaccination against tumor immunogenic antigens and eventually prolonged survival of patients, expression of these antigens should first be examined in a well-characterized series of cancer tissue specimens, which this experiment currently carried out for MAGE-A6 and MAGE-11 expressions in BC. Poor presentation of MAGE-As in normal tissues, increased expression of theses antigens in malignant tissues, and their high immunogenicity has directed experiments into utilizing them as targets for cancer immunotherapies 19 . We found that MAGE-A6 and MAGE-A11 are highly expressed in BC tissues with high-grade malignancy, therefor it is promising interest to establish the BC immunotherapy for restricting tumor cells through activating specific CD8 + T cells (cytotoxic T lymphocyte; CTL) against tumor cells expressing MAGE-A6 or MAGE-A11.
Taken together, the strength of the association between clinicopathological parameters and immunoreactive MAGE-A6 and MAGE-A11 scoring as well as MAGE-A6/MAGE-A11 co-expression can promote the potentials of these markers for diagnosis and progression of BC. It is suggested that the increased MAGE-A6 expression

Material and methods
Bioinformatics approach. GEO 65 . Then, the Venn diagram analysis was performed to find a common significant differential in the output by Venny (https:// bioin fogp. cnb. csic. es/ tools/ venny/ index2. 0.2. html) 66 . Common genes of these three types of research were selected for subsequent analysis on Enrichr (amp.pharm.mssm.edu/Enrichr/) based on GO (http:// geneo ntolo gy. org/) 67 . GO analysis consist of cellular component (CC), biological process (BP), and molecular function (MF). Besides, pathways including Reactome (https:// react ome. org/) were applied 68 . Additionally, we tried to use a PPI network with more related genes connections 69 . Next, we applied the cBioPortal (https:// www. cbiop ortal. org/) which is a tool for collecting next-generation sequencing data from the TCGA and the international cancer genome consortium (ICGC) that is a repository of user-friendly cancer genomics datasets 70,71 . Furthermore, the online database GEPIA, for analyzing the RNA sequencing expression data and prognostic value were used. All samples in the GEPIA database were derived from the genotype-tissue expression (GTEx) and TCGA projects (http:// gepia. cance rpku. cn/ index. html) 72 . Also, the protein expression level of these selected genes was considered in BC on the UALCAN database (http:// ualcan. path. uab. edu/) which provides protein expression analysis options using data from TCGA 73 . Additionally, in the Cytoscape (https:// cytos cape. org/) our genes were investigated in BC samples 74 . Additionally, we applied MAGE-A6 and MAGEA-11 in the EBI's Expression Atlas website (https:// www. ebi. ac. uk/ gxa/ home) in order to confirm these invaluable targets expression in bladder carcinoma/ bladder tumor cell lines 75 . Patients' characteristics and tissue collection. A total of 250 formalin-fixed, paraffin-embedded (FFPE) specimens were collected from BC patients after transurethral resection of bladder tumor (TURB) with no preoperative chemotherapy or radiotherapy in the Hasheminejad Urology-Nephrology Hospital, Iran, between 2008 to 2011. Of this collection, 51 specimens during MAGE-A6 staining and 37 specimens during MAGE-A11 staining were excluded from the study due to technical problems in tissue processing, leaving a total of 199 and 213 cases for the final evaluation, respectively. Medical documents and hematoxylin and eosin (H&E) stained slides were reviewed to collect the following pathological and clinical characteristics: age, gender, tumor size (maximum tumor diameter), histological grade, pT stage, lamina propria 60 involvement, muscularis invasion, L/M involvement, distant metastasis, and tumor recurrence. Furthermore, 11 adjacent normal tissue samples were used to evaluate the expression of MAGE-6 or MAGE-11 compared to cancerous tissues. The cutoff size and the pT stage of cancers were defined based on the American joint committee on cancer/international union against cancer (AJCC/UICC) and pTNM classification, respectively [76][77][78] . In addition, the patient's survival data, including DSS and PFS, was recorded. DSS was calculated from the time of surgery to the time of death related to the patient's tumor and PFS was considered as an interval between the primary surgery and the last follow-up visit if the case exhibited no sign of disease, recurrence, or distant metastasis.
Tissue microarray construction. BC tissue microarrays were constructed as described previously 79 .
Briefly, H&E slides of all specimens were evaluated by an experienced pathologist (M.A) to select and mark out three suitable regions of cancer in each block. Then, selected spots of each primary block were punched out with a diameter of 0.6 mm and transferred into the TMA recipient paraffin blocks through a precision arraying instrument (ALPHELYS, Plaisir, France). In the present research, due to the issue of tumor heterogeneity, three cores were constructed for each specimen and, then, scored separately to obtain better results 80 . The mean of the three cores was considered as the final score for each tissue specimen. Immunohistochemistry staining. The expression of MAGE-6 and MAGE-11 was immunohistochemically assessed through our laboratory protocol 81 . Briefly, all TMA sections were deparaffinized in xylene, and then rehydrated through immersion in reducing grades of ethanol. Subsequently, methanol containing 0.3% hydrogen peroxide (H2O2) was used to suppress the activity of endogenous peroxidase. After washing the tissue sections three times in Tris Buffered Saline (TBS), the slides were autoclaved for 10 min in sodium citrate buffer (pH 6.0) to retrieve the antigens. The slides were treated overnight at 4 °C with the following antibody dilutions as the optimal dilution for subsequent use: anti-MAGE-6 antibody (Sigma Aldrich, USA) using a 1:100 dilution, and anti-MAGE-11 antibody (Abcam, Inc., Cambridge, MA) using a 1:150 dilution. The next day, TMA slides were washed with TBS and, then, incubated with the secondary antibody, EnVision™ + /HRP, DualLink Rabbit/Mouse (Dako, Denmark) for 1 h at room temperature. Subsequently, visualization of the antigen was achieved through 3, 3′-diaminobenzidine (DAB) chromogen substrate for 10 min at room temperature followed by counterstaining with hematoxylin (Dako). Finally, sections were dehydrated with graded alcohol, cleared in xylene (Dako), and mounted for evaluation. Moreover, a non-reactive primary antibody was used instead of the primary MAGE-A6 or MAGE-A11 antibody as the negative controls. Liver and prostate tissues were used as a positive control for MAGE-A6 and MAGE-A11, respectively.
Immunostaining evaluation and scoring system. The immunohistochemical staining of tissue slides was scored through a semi-quantitative scoring system by a professional pathologist (M.A) who was blinded to pathological and clinical data associated with each sample.

Statistical analysis.
Statistical analyses were carried out through the SPSS statistical software package version 25 (SPSS, Chicago, IL, USA). The association and correlation between MAGE-A6 or MAGE-A11 expression and clinicopathological characteristics were analyzed using Pearson's χ2, R tests, and One-way ANOVA. The pairwise comparisons across the groups were performed through Mann-Whitney U test. Survival analysis was estimated through the Kaplan-Meier method and the estimated curves across the groups were compared using the log-rank test. A p-value of < 0.05 was regarded as statistically significant. Charts were drawn through Prism version 8.3.0 software (Graph Pad Inc., San Diego, CA, USA) and SPSS graphs.
Ethical approval. This