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Abstract
Context: Despite the follow-up protocols developed in non–muscle-invasive bladder cancer patients, progression and recurrence could not be prevented. Aims: We aimed to investigate whether proteins such as OCT-4, CD47, p53, Ki-67, and Survivin, which increase in bladder cancer cells, can be used as prognostic markers for patients with non–muscle-invasive bladder cancer. Settings and Design: The study included a total of 89 patients with newly diagnosed non–muscle-invasive bladder cancer between January 2015 and December 2020. Materials and Methods: Levels of OCT-4, CD47, p53, Kİ-67, and Survivin proteins in cancer cells were determined with a semi-quantitative immunohistochemical experiment. Pathological data and survival rates were compared according to the staining rates. Statistical Analysis Used: Data obtained in the study were analyzed statistically with SPSS 22.0 (SPSS, Chicago, IL, USA). Results: The mean age of the patients was 64.25 ± 9.91 years, and the median follow-up period was 55 months. Recurrence rate was determined to be 36% (n = 32), and the rate of progression at 40.4% (n = 36). The staining rates were stronger for each marker in the progression group and advanced-stage tumors (p < 0.001). The findings of the multivariate analysis carried out as part of the study showed that older age and higher tumor stage were independent risk factors for recurrence-free survival (HR = 1.048 and 7.074, respectively; P = 0.02). Also, higher tumor stages, diameters, and grades were associated with reduced progression-free survival (HR = 0.105, 0.395, 0.225, respectively; P < 0.05). Conclusions: Although immunohistochemical staining rates are promising, it is more appropriate to use tumor characteristics when assessing survival rate in patients with non–muscle-invasive bladder cancer.
Keywords: Bladder cancer, CD47, Kİ-67, OCT-4, p53, Survivin
How to cite this article: Introduction 
Bladder cancer is the tenth most frequently seen type among all cancers. The worldwide mortality rate associated with bladder cancer is approximately 2 per 100,000.[1] According to the 2017 Tumour, Node, Metastasis (TNM) classification, tumours at pTa, pT1, and carcinoma in situ stages are classified as non–muscle-invasive bladder tumours (NMIBT). NMIBT constitute 75% of newly diagnosed bladder cancers; 60-70% of patients in this group are at risk of recurrence, and 10-20% can potentially become muscle-invasive cases.[2]
The stage and grade of the tumor have been defined as two important risk factors to determine the prognosis in bladder tumor patients. In addition, various scoring tables have been developed to determine the risk of progression and recurrence, and patients have been separated into risk groups according to these tables. However, there are heterogeneous groups within bladder tumors, and the existing algorithms cannot reflect all patient groups. Tumours within the diverticulum, even if low grade, require radical treatment, and patients with lymphovascular invasion, carcinoma in situ in the prostatic urethra, and variant histology have worse prognosis than other patients at the same grade. Therefore, determination of other parameters and the implementation of those parameters in clinical practice are important.[3],[4]
Cystoscopy and urine cytology are two essential tools in the follow-up of bladder cancer patients. However, patients are often reluctant to attend follow-up appointments due to the relative invasiveness of these methods. However, even though patients can be monitored closely with these methods, the methods still fall short of providing adequate predictions in relation to cancer progression and recurrence. With increased knowledge of bladder cancer histopathology, promising biomarkers have been developed. Research shows evidence for the increased production of proteins such as CD47, OCT4, p53, Ki67, and Survivin in solid tumors, and this increase has been shown to be closely related to tumor aggressiveness. It has also been shown that these proteins are produced in urothelial cancer cells.[5] However, thus far, no consensus has emerged on the routine use of these markers in bladder cancer.[2] Therefore, the present study aims to determine the levels of the expression of biomarkers CD 47, OCT-4, p53, Ki-67, and Survivin according to the pathological parameters and investigate their prognostic value in patients newly diagnosed with NMIBT.
Materials and Methods Approval for this retrospective study was granted by the Ethics Committee of Aksaray University (Decision no: 2020/01-02). The study included 89 patients diagnosed for the first time with NIMBT and followed up in the Urology Clinic between January 2015 and December 2020. Patients were excluded from the study if they had metastatic disease at the time of diagnosis, had a diagnosis other than transitional cell bladder cancer, had a history of any malignancy other than bladder cancer, left follow-up, or did not regularly attend follow-up appointments.
For each patient included in the study, a record was made of age, gender, body mass index (BMI), chronic diseases, Charlson Comorbidity Index (CCI), and smoking status. The progression risk of patients was classified according to the risk classification defined in the European Association of Urology (EAU) NIMBC guidelines as low, moderate, high, and very high categories of risk. In determining the risk of recurrence, the 2006 European Organisation for Research and Treatment of Cancer (EORTC) scoring model was used, in which a score of 0 marks low risk; 1–4, moderate risk; and >4, high risk.[3],[4]
According to the EAU guidelines, low-risk patients were applied with TUR-BT (Transurethral Resection of Bladder Tumor) followed by early single-dose intravesical treatment, and if no tumor was determined with cystoscopy at the 3-month follow-up, a further follow-up was made with cystoscopy at 9 months, and then, annually, for 5 years. High-risk patients and very high-risk patients who did not accept radical cystectomy were subjected to thoraco-abdominal computed tomography (CT) each year as well as with full-dose Bacillus Calmette-Guerin (BCG) treatment for up to one year. If tumor was not determined in the 3-month cystoscopy and following urinary cytology, a further follow-up was made that included cystoscopy and urinary cytology once every 3 months for 2 years, once every 6 months for up to 5 years subsequently, and annually thereafter. Moderate-risk patients were followed up on an individual basis between these two patient groups.[2] During follow-up, the determination of the same pathological grade of tumor was evaluated as recurrence, and a more advanced grade as progression. The total follow-up period of the patients was recorded in months and the time from first diagnosis to progression and/or recurrence if it occurred.
Histomorphological analysis and ımmunohistochemistry
Using the transurethral resection method and/or punch biopsy during cystoscopy, tissue samples were taken from the tumor. All the tumor samples were graded for histological differentiation according to the 2016 WHO classification, and the clinical stage of the cancer was assessed based on UIIC's (UICC—Union Internationale Contre le Cancer, 8th edition) Tumor-Node-Metastasis (TNM) classification criteria. An experienced uropathologist who was blinded to the clinical history of the patients performed the assessments.
For immunohistochemistry, tissue samples were first fixed in formalin and then embedded in paraffin blocks. Sections 3 μm in thickness were cut from the blocks with a microtome and mounted on poly L-lysine-coated microscopy slides. Immunohistochemistry was performed in a Leica BOND MAX Immunostainer (Leica, Wetzlar, Germany) using the avidin-biotin-peroxidase method with diaminobenzidine as the chromogen, in accordance with the manufacturer's instructions. In each sample, the extent of staining (Weak ≤25%; Strong >25%) was examined under an Olympus BX43 microscope, and a semi-quantitative evaluation of the immunohistochemistry results was carried out which used a scoring system [Figure 1].
Figure 1: Magnification 5x, weak; ≤25% staining, strong: >25% staining; CD47 staining rates (a=weak, b=strong); OCT-4 staining rates (c=weak, d=strong); p53 staining rates (e=weak, f=strong); Survivin staining rates (g=weak, h=strong); KI-67 staining rates (j=weak, k=strong)The primary antibodies were as follows: CD47 rabbit polyclonal EPR21794; Abcam, Cambridge, UK, 1/100 dilution, p53 mouse monoclonal D0–7; Leica, USA, 1/500 dilution, Ki-67: mouse monoclonal, Sp 6; Cell marque, USA, 1/150 dilution, Survivin: rabbit monoclonal Ep119; Abcam Epitomics, Cambridge, UK, 1/100 dilution, OCT-4: mouse monoclonal MRQ-10; Cell marque, USA, 1/100 dilution.
Statistical analysis
Data obtained in the study were analyzed statistically using the SPSS software (SPSS Version 22.0, Chicago, IL, USA). Continuous quantitative variables were stated as mean ± standard deviation (SD) values, discrete quantitative data as median (interquartile range [IQR]) values, and categorical variables as number (n) and percentage (%). Conformity of the data to normal distribution was assessed with the Shapiro–Wilk test. In the comparison of more than two independent groups, the one-way ANOVA of data was carried out that showed normal distribution and the nonparametric Kruskal–Wallis test was carried out when parametric assumptions were not met. The χ2 test was used for comparisons between categorical variables. To evaluate the variables' effect on progression and recurrence, univariate and multivariate Cox Regression analyses were performed. Kaplan–Meier analysis was used to determine both recurrence and progression-free survival. A value of P < 0.05 was accepted as statistically significant.
Results The demographic and clinical data of all patients are presented in [Table 1]. In terms of gender distribution in the study sample, 66.3% (n = 59) were male and their mean age was 64.25 ± 9.91 years. The duration of median follow-up was 55 (n = 34) months. Low-grade tumors were determined in 47 (52.8%) patients and high-grade tumors in 42 (47.2%). Progression was observed in 40.4% (n = 36) of patients and recurrence in 36% (n = 32).
OCT-4 staining and clinicopathological variables
Strong staining with OCT-4 was observed in 32 (34.8%) patients. In tumors of diameter <3 cm, and in isolated tumors, staining with OCT-4 was seen to be weak (p = 0.03 and P = 0.04, respectively). A strong staining and thus high-grade tumors were determined in the T1 stage (p < 0.001). Strong staining with OCT-4 indicated both a high risk of progression and recurrence of tumor, with a majority of patients out of the said 32 whose samples with strong stains presenting clear evidence of tumor progression (p < 0.05) [Table 2]a.
Survivin staining and clinicopathological variables
Strong staining with Survivin was observed in 27 (30.3%) patients. T1 stage tumors, and those including high-grade cancer cells, were evident in these patients whose samples were strongly stained (p < 0.001). Staining with Survivin was weak in the patient group at low-moderate risk of progression and recurrence, and progression was at a higher rate in the group whose samples were strongly stained (p < 0.001, P = 0.007, P < 0.001, respectively) [Table 2]a.
CD-47 staining and clinicopathological variables
Strong staining with CD-47 was observed in 27 (30.3%) patients. Isolated tumors and tumors that were not carcinoma in situ were weakly stained (p = 0.001 and P < 0.001, respectively). A strong staining with CD-47 was evident of T1 stage and high-grade tumors (p < 0.001). Staining with CD-47 was weak in the patient group with low-moderate risk of recurrence, strong in the group with a high risk of progression, and progression was seen at a higher rate in the group with strong staining (p = 0.002, P < 0.001, and P = 0.001, respectively) [Table 2]b.
p53 Staining and clinicopathological variables
Strong staining with p53 was observed in 36 (40.4%) patients. Samples strongly stained with p53 indicated multiple, high-grade, T1-stage tumors, and carcinoma in situ (p = 0.017, P < 0.001, P < 0.001, and P = 0.001, respectively). Staining with p53 was strong in the patient group at high risk of recurrence and progression, and progression was seen at a higher rate in this group (p < 0.001) [Table 2]b.
Ki-67 Staining and clinicopathological variables
Strong staining with Ki-67 was observed in 18 (20.2%) patients. In the weakly stained sample group, more low-grade and T1 stage tumors were seen (p < 0.001). The risk of progression in this patient group was low, and the actual progression observed was low as well (p < 0.001) [Table 2]c.
Kaplan–Meier analysis of progression- and recurrence-free survival
The effects of staining rates on progression-free survival were examined separately. The increase in staining rates was determined to shorten the time to progression; especially in patients with strong staining rates, a statistically significant decrease could be observed in the duration of progression-free survival (p < 0.001). When the effects of staining rates on recurrence-free survival were examined separately, no significant difference was found (p > 0.05) [Figure 2] and [Figure 3].
Figure 2: Kaplan–Meier analysis of progression-free survival, based on staining rates
Figure 3: Kaplan–Meier analysis of recurrence-free survival, based on staining ratesUnivariate and multivariate analyses of the risk factors
In the univariate analysis, tumor diameter, grade, stage, concomitant carcinoma in situ, and strong staining in any of the stains were seen to be significantly associated with progression-free survival, and age and tumor grade had a significant association with recurrence-free survival (p < 0.05). In the multivariate analysis, tumor diameter, grade and stage were each found to be independent risk factors for progression-free survival, and patient age and tumor grade for recurrence-free survival (p < 0.05) [Table 3] and [Table 4].
Table 3: Univariable Cox regression analysis of clinical and pathological parameters
Table 4: Multivariable Cox regression analysis of clinical and pathological parameters Discussion To increase survival in patients with NMIBT, risk groups have been formed and follow-up protocols have been developed according to the criteria used for classifying the patient groups in the present study. However, the fact that there is heterogeneity within these patients themselves, and that progression and recurrence are still determined despite the recommended treatments and follow-up, has driven urologists to research whether or not other markers can be used in the prediction of survival. The roles of angiogenesis, which may play a role in the development of tumor cells, and molecular markers, which may be responsible in apoptosis, have been investigated in bladder tissues, but so far no clear consensus has been reached on the subject of their clinical use.[6]
One of the most evaluated markers of the relationship with urothelial cancers is p53, which plays a role in the response to tumor suppressor gene stress, the regulation of the cell cycle, and in apoptosis. It has been determined that mutations that could develop in the p53 gene could cause an increase in the level of immunohistochemical p53 nuclear staining in bladder cancer cells.[7] Although the rate of staining with p53 was higher in patients at T1 G3 grade, no relationship between the strong staining and the tumor could be seen in prognosis.[8] In a review that examined 117 studies, it was found that an increase in p53 corresponded to a 1.6-fold increase in the risk of recurrence (95% CI, 1.2-2.1), a 3.1-fold increase in the risk of progression (95% CI, 1.9-4.9), and a 1.4-fold increase in mortality (95% CI, 1.2-1.7); however, as the rate of muscle-invasive tumors was greater in the studies on this subject, these risks could have been found to be higher. It was also reported that the results were insufficient to comment on non–muscle-invasive tumors.[7] In the present study, in respect of both tumor grade and stage, the tumors were perhaps more aggressive in patient samples with strong p53 staining, and progression-free survival was also lower in this group. However, the results of the multivariate analysis showed that this effect was not clinically significant.
CD-47 protein, which is a receptor on the cell membrane, ensures that the cell they are on does not undergo phagocytosis as an immunomodulator, and this is achieved by binding to the signal regulatory protein alpha (SIRPα) in macrophages.[9] The CD-47 level was seen to reflect an increase in bladder cancer cells, in addition to many solid tumours. Cystoscopy for diagnosis of bladder cancer showed that CD-47 antibodies marked with fluorescence could be useful both in identifying variant histologies which are difficult to determine and in spotting residual tumor. Numerous immunotherapies targeting this molecule are already under various stages of developments in the field.[10] Some studies have reported differences in CD-47 staining rates according to pathological grades and staining rate in patients with pT1 grade bladder tumor being possibly higher than that of patients with pTa grade (p = 0.034).[11] However, our literature review showed just a few studies that examined the effect of an increase in CD-47 expression on bladder tumor prognosis. In the present study, the presence of combination of low-grade tumor with carcinoma in situ was found to be low in the group with weak CD-47 staining; although such an effect could prove advantageous for progression-free survival, it was not determined in the multivariate analysis. These results from the present study can be considered important because they are new for literature, and to be able to compare these results, there is a need for further studies showing the importance of CD-47 in the determination of risk groups and prognosis in addition to diagnosis and treatment.
OCT-4 protein is encoded by the POU5F1 gene in humans. It functions in the regeneration and differentiation of embryonic stem cells. Stem cell markers in cancer cells which have greater OCT-4 expression in vivo have been shown to be at a higher level compared to a control group, suggesting that OCT-4 could play a role in the development of cancer stem cells, which are held responsible for the development of cancer cells and lack of response to treatment.[12] Although there are studies showing an increase in the level of OCT-4 in bladder tumor cells and that this increase is correlated with tumor grade, there are also studies showing no relationship between tumor grade and staining level.[13],[14] However, there are very few studies in literature which have examined the relationship between prognosis and OCT-4, which is a very new marker for the examination of the effect on bladder cancer. A feature distinguishing this study from previous research was that the effect of OCT-4 was examined on prognosis in patients with NMIBT. Although strong staining with OCT-4 was observed in high-risk groups and had a significant effect on progression-free survival, when evaluated together with other factors such as tumor diameter, grade, and stage, this effect was not seen to be significant.
As Survivin protein is not found in normal cells and has an inhibiting role in apoptosis in addition to regulating mitosis, it is used as a target protein in cancer diagnosis and treatment. An increase in Survivin protein is greater in high-grade tumors, and this has been determined to have a negative effect on progression-free and recurrence-free survival. A previous study has shown an association between tumor grade and Survivin synthesis. A review that examined 2,165 patients reported that sufficient data could not be established for Survivin in the determination of prognosis in bladder cancer patients.[15],[16],[17],[18] Ki-67 protein, which was also examined in this study, functions in cell proliferation, and it has been reported that the level increases with the grade of non–muscle-invasive bladder disease and there is a close relationship with prognosis. However, it has been emphasized in literature that it should be evaluated together with existing parameters in the risk tables. The fact that there are studies that have not determined a relationship between this marker and recurrence-free survival and progression-free survival is the reason that this marker has not been proven for clinical use.[19] Although strong staining with Survivin and Ki-67 was seen in the specimens of patients at high-risk of progression, these two markers were seen to be insufficient if used alone in the determination of prognosis.
The limitations of this study were the low number of patients, its retrospective design, and the inability to evaluate a model of clinical, pathological, and some combination of biomarker expressions that could identify higher-risk groups.
[TAG:2]Conclusions[/TAG:2]
Despite the formation of risk groups and the development of follow-up protocols for non–muscle-invasive bladder tumors, progression and recurrence have not been overcome. Therefore, it is necessary to establish new risk assessment protocols, including easily detectable and adaptable markers that can be used with traditional clinical follow-up tools. Although immunohistochemical staining rates have been determined to be high in tumor specimens in aggressive tumors and in the presence of progression, tumor grade and stage can be considered to be two important determining factors in the prediction of survival of patients with non–muscle-invasive bladder tumor.
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Conflicts of interest
There are no conflicts of interest.
References 
Correspondence Address:
Aykut Demirci
Nevşehir Street No: 117, 68200 Aksaray
Turkey
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_1236_21
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