Invasive bladder cancer biomarkers, and methods and a kit for the detection thereof

Abstract

The invention provides biomarkers, methods involving their use, and a diagnostic kit that enables the distinction between muscle-invasive bladder cancer (MIBC) and non-muscle- invasive bladder cancer (NMIBC) which represents a critical step in the diagnosis and treatment of bladder cancer. This kit, based on widely used and cost-effective immunohistochemistry (IHC) techniques in pathology practice, comprises an effective biomarker panel for distinguishing between MIBC and NMIBC. This invention is suitable for routine use in clinical pathology practice, enabling better guidance of patient treatment processes and more accurate determination of disease progression.

Description

[0001] INVASIVE BLADDER CANCER BIOMARKERS, AND METHODS AND A KIT FOR THE DETECTION THEREOF

[0002] Technical Field

[0003] This invention relates to a kit that enables the detection of protein biomarkers SLC9A1, ALDH4A1, ID3, TMC4, and TYSND1 in combination with the p53 protein biomarker to distinguish between cases of muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC).

[0004] The state of the Art

[0005] Bladder cancer (BC) is one of the most common types of cancer worldwide and arises from the uncontrolled growth of cells in the bladder mucosal epithelium [1], BC imposes a significant burden in terms of treatment costs and health-related quality of life (HRQoL). Its prevalence is expected to increase substantially (by 3-4 fold) by 2040 [2,3],

[0006] One of the main factors in the treatment and prognosis of bladder cancer is the determination of the pathological tumor (pT) stage of the tumor. Pathological staging is obtained through conventional histopathological examinations performed by surgical pathologists. However, the clinical behavior of the disease does not always align with this staging, which may require reevaluation of the pathological diagnosis [2], Pathological staging covers approximately 10 stages of BC, ranging from Ta to T4b. The transition from T1 to T2 is a critical point, as treatment strategies at stage T2 become more aggressive, potentially requiring radical surgical interventions such as cystectomy and lymph node dissection [1,4,5],

[0007] Currently, the first step in diagnosing bladder cancer involves the removal of the tumor mass from the bladder through Transurethral Resection of Bladder Tumor (TURBT), which serves both therapeutic and diagnostic purposes [2,4-6], In approximately 70% of BC cases, the disease is observed as pTa, pTis, or pTl, that is, non-muscle-invasive bladder cancer (NMIBC) [2,4,7], Cancers at stage pT2 and beyond are classified as muscle-invasive bladder cancers (MIBC). Approximately 10^40% of NMIBC cases eventually progress to the MIBC class over time [2,4],

[0008] At the critical T1-T2 transition stage, it is important to determine whether the tumor has reached the Muscularis Propria (MP) layer of the bladder [8-10], If it has not, the tumor is classified as an NMIBC. The pathological criterion for this is the detection of the MP layer containing invasive cancer cells in Hematoxylin & Eosin (H&E ) sections of TURBT material [9,10], The presence of MP can be confirmed by a positive result for muscle biomarkers such as desmin. However, because TURBT material is delivered to the pathology laboratory in a fragmented form and with thermal artifacts in some parts due to cautery during TUR, evaluating MP invasion can be challenging, which may lead to a false-negative result for MIBC [8,10,11], Conversely, the Muscularis Mucosae (MM) layer, located near the epithelium within the MP, may be mistaken for the MP due to hyperproliferation and desmin positivity; this can cause a T1 tumor in the MM layer to be misclassified as T2, resulting in a false-positive outcome [9],

[0009] The first and most important component of clinical staging, TURBT, enables the removal of the tumor mass detected in the bladder by cystoscopy in small fragments through the urethra, ensuring that the lesion is excised from the bladder mucosa and / or wall [2,4-6], However, piecemeal tumor resection is not compatible with the principles of oncological surgery [2,11,12], En-bloc tumor resection is possible but still debated as an alternative surgical method; its superiority over TURBT has not yet been clarified, and it is not widely used [11,12], Small resection fragments obtained from single or multiple lesions are collected from the same recipient and fixed with formaldehyde. Tumor sections are routinely examined in the pathology laboratory, and the observations are reported under the main headings of grading (low- and high-grade), typing (urothelial variant histology and others), and pT staging. In the evaluation of pT staging, both understaging and overstaging are major concerns of the currently used histopathological examination methods.

[0010] The main reasons why the currently used pT staging methodology is insufficient to guide patient treatment are as follows: 1. During conventional cystoscopic examination and TURBT, complete resection of neoplastic tissue in muscle-invasive cases may not be possible. The likelihood of obtaining inadequate samples that demonstrate the relationship between the tumor tissue and the bladder wall muscle layer may reduce diagnostic accuracy [2,11,12],

[0011] 2. A tumor mass that is initially intact in the bladder is obtained after TURBT as small tumor fragments divided into smaller pieces. Because the anatomical integrity of the tumor mass is disrupted and it often contains thermal artifacts caused by cautery, difficulties and errors may arise in detecting bladder muscle invasion [2,5,8,12],

[0012] Various genomic and immunohistochemical biomarkers have been defined for the diagnosis and prognosis of bladder cancer. Genomic biomarkers such as TERT and FGFR3 and immunohistochemical biomarkers such as Ki67, VEGF, FGFR3, and p53 have been reported as diagnostic and prognostic tools. However, their predictive success in terms of prognosis remains controversial, and they cannot be used as staging markers.

[0013] The TERT and FGFR3 biomarkers are generally genomic in nature and are more oriented toward urine-based diagnostic approaches, with no established role in routine clinical practice [2,13],

[0014] p53 is an important tumor suppressor gene involved in genomic stability, apoptosis, and cell cycle regulation. p53 is commonly inactivated in cancers either by inhibitors such as MDM2 or gene mutations (mostly missense, but also nonsense mutations), frequently leading to its accumulation in tumor cells.

[0015] p53 IHC positivity has long been investigated for its potential in prognosis and treatment, but its role in diagnosis or prognosis remains debatable. In earlier years, positivity was generally accepted only when the staining rate exceeded 10-40% [14,15], Stadler et al. and Ziaran et al., who considered >10% nuclear staining as positive, reported unsuccessful results in predicting treatment response [15-18], However, this approach is biologically flawed because certain mutations and alternative pathway mechanisms can lead either to p53 protein accumulation or, less commonly, to complete loss of expression due to nonsense mutations [19,20], None of the biomarkers mentioned above are used to differentiate between MIBC and NMIBC, and their predictive power in this area remains unknown [10,13,18], Therefore, there is a need for an accuracy test for new biomarkers that can be used to distinguish between NMIBC and MIBC bladder cancer

[0013] ,

[0016] In a prior study, patent KR2632423B1 described the use of SKA3, MKI67, and TTK genes as biomarkers for the prognosis of NMIBC, along with a kit for detecting these biomarkers. The biomarkers and kit mentioned in this document are used to predict patient survival in non-muscle-invasive bladder cancer. The document refers to biomarkers intended for NMIBC prognosis, but they are not used for the differential diagnosis between MIBC and NMIBC.

[0017] In a prior technique, patent application WO2023230617A2 disclosed a kit in which ANG, Al AT, APOE, CA9, IL8, MMP9, MMP10, PAI-1, SDC1, and VEGFA genes were used as biomarkers, and the mRNA levels of these genes were measured to calculate the survival probability of patients with bladder cancer. To predict the long-term survival probability of a bladder cancer patient using this application, the following steps must be performed:

[0018] (a) Obtaining tumor tissue or other biological samples from patients at different stages.

[0019] (b) Isolation of mRNA from the biological sample.

[0020] (c) Determining the mRNA levels of approximately 10 genes.

[0021] (d) Normalizing these mRNA values with reference mRNA measurements, followed by analyzing various parameters with specialized computer software to achieve the goal of predicting patient survival.

[0022] Thus, the aim of this patent application is survival prediction (prognosis determination), not the differential diagnosis between MIBC and NMIBC. Furthermore, because the method involves multiple steps, it is laborious, expensive, and prone to errors. The application also describes methods for measuring these biomarkers as diagnostic markers for upper urinary tract urothelial cancers. However, this cancer type differs from bladder cancer and represents a rare form of urinary tract cancer.

[0023] In the prior art, patent KR2066712B1 presents a composition for predicting bladder cancer prognosis by measuring the expression of ARHGAP9 protein or the nucleotide sequences encoding it. This method provides the information necessary for prognosis prediction and includes obtaining a biological sample from a subject diagnosed with bladder cancer, measuring the expression level of the ARHGAP9 gene or protein from the sample, comparing the measured expression level with a reference value, and predicting the bladder cancer prognosis based on the results of the comparative analysis. The reference value was determined from the obtained data, which included the expression levels of ARHGAP9 protein or ARHGAP9 mRNA, along with prognostic information from multiple proteins. The purpose of this patent application is to predict patient survival through ARHGAP9 analysis of biological material already diagnosed with bladder cancer. In other words, it is not a method aimed at predicting whether a newly diagnosed bladder tumor is muscle invasive.

[0024] Considering the limitations and shortcomings of current diagnostic methods, there is a need for kits that can reliably distinguish between NMIBC and MIBC cases, are cost-effective, easy to use in clinical practice, and can perform effective differential diagnosis with a small number of biomarkers possessing high sensitivity and specificity, especially in light of the rising prevalence of the disease. In this regard, the development of alternative biomarkers is critical to improve diagnostic processes and enhance the efficiency of healthcare services.

[0025] Brief Description and Objectives of the Invention

[0026] This invention relates to a kit that enables the detection of protein biomarkers SLC9A1, ALDH4A1, ID3, TMC4, and TYSND1 in combination with the p53 protein biomarker, in order to distinguish between muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC) at the initial diagnostic stage based on TURBT material analysis. The kit of the present invention was developed to ensure accurate staging and prognosis of the disease. In particular, the positive correlation between p53 and MIBC and the inverse correlation between ALDH4A1 and SLC9A1 with MIBC were the key factors that enhanced the diagnostic accuracy of this kit.

[0027] Additionally, ID3 and TMC4 showed decreased expression in MIBC compared with the NMIBC class, similar to ALDH4A1 and SLC9A1, while TYSND1 showed increased expression in MIBC compared with NMIBC, similar to p53 (Figure 1). The ROC AUC graphs and AUC values (shown at the bottom right of the graphs) obtained from pairing each protein with p53 to distinguish between the NMIBC and MIBC groups are presented in Figure 4.

[0028] Most interestingly, these tumor markers are tumor-intrinsic factors independent of non-tumor cells, such as muscular cells; therefore, their predictivity solely depends on their expression pattern in BC cells.

[0029] The purpose of this invention is to distinguish MIBC from NMIBC cases using samples resected from patients for the first diagnostic examination. For this purpose, the invention employs the biomarkers SLC9A1, ALDH4A1, ID3, TMC4, and TYSND1, whose association with bladder cancer has been identified. This invention also combines the data generated with these biomarkers with data from a known tumor marker, p53.

[0030] Another purpose of this invention is to make predictions regarding the prognosis of bladder cancer. Analyses performed with the biomarkers of the invention were also correlated with survival rates.

[0031] Thus, predictions about disease progression can be made by measuring the biomarkers described in this invention.

[0032] Another objective of the invention is to develop a diagnostic assay kit that is easy to apply and inexpensive. This kit offers a molecular biomarker panel that can be tested using immunohistochemistry (IHC) methods to distinguish between MIBC and NMIBC in clinical pathology practice. Therefore, the kit of the present invention is widely applicable in clinical settings.

[0033] Description of Figures

[0034] • Figure 1: Examples of staining results for (a) H&E sections and (b) SLC9A1, (c) ALDH4A1, (d) ID3, (e) TMC4, and (f) TYSND1 proteins, which showed significant differences between the NMIBC and MIBC patient groups across stages pTa, pTl, and pT2.

[0035] • Figure 2: Examples of staining results for the p53 protein in the NMIBC and MIBC patient groups at stages (a) pTa, (b) pTl, and (c) pT2. • Figure 3: Examples of staining results for the AXL protein in the NMIBC and MIBC patient groups at stages (a) pTa, (b) pTl, and (c) pT2.

[0036] • Figure 4: Column charts showing the relationships between novel biomarkers and p53 (left) and ROC AUC values for the differential diagnosis of NMIMC and MIBC (right): (a) AEDH4A1, (b) SLC9A1, (c) ID3, (d) TMC4, and (e).

[0037] • Figure 5: In the NMIBC and MIBC groups: (a) ROC AUC analysis results of all independent variables with and without p53; (b) ROC AUC analysis results of SLC9A1 and p53 versus AEDH4A1 and p53 biomarkers; (c, d) column charts of analysis results showing the performance of SLC9A1 and p53 versus AEDH4A1 and p53 biomarkers in distinguishing the two groups.

[0038] • Figure 6: ROC AUC analysis results of the SLC9Al&p53 and AEDH4Al&p53 biomarker panels combined with selected clinical and histopathological parameters in the NMIBC and MIBC groups.

[0039] • Figure 7: Cox regression graphs related to survival analyses of (a) SLC9A1, (b) AEDH4A1, (c) ID3, (d) TMC4, (e) TYSND1, and (f) p53 biomarkers in NMIBC and MIBC groups.

[0040] Detailed Description of the Invention

[0041] The invention relates to biomarkers capable of distinguishing between muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC), and to an immunohistochemical assay kit in which these biomarkers are used to differentiate such cases and to diagnose the course of the disease. In this invention, the expression of ALDH4A1, SLC9A1, ID3, TMC4, and TYSND1 proteins, together with the p53 protein, was detected in tumor tissue, and based on these data, MIBC and NMIBC cases were distinguished. This differentiation was primarily achieved by analyzing the direct correlation of p53 with the inverse correlation of ALDH4A1 or SLC9A1.

[0042] In addition, ID3 and TMC4 showed decreased expression in MIBC compared to the NMIBC group, similar to AEDH4A1 and SLC9A1, whereas TYSND1 showed increased expression in MIBC compared to NMIBC, similar to p53 (Figure 1). The ROC AUC graphs and AUC values (shown at the bottom right corner of the graphs) obtained from pairing each protein with p53 to distinguish between the NMIBC and MIBC groups are presented in Figure 4.

[0043] There was a significant relationship between SLC9A1, ALDH4A1, ID3, TMC4, p53, and overall survival (OS) (p-values: < 0.0001, < 0.0001, < 0.0001, 0.002, and <0.0001, respectively) (Figure 7, Table 6). In addition, there was a significant relationship between SLC9A1, ALDH4A1, ID3, TMC4, TYSND1, and p53 and disease-free survival (DFS) (p-values: <0.0001, <0.0001, <0.0001, 0.004, 0.015, <0.0001) (Table 6).

[0044] In the present invention, it was determined that the measurements of SLC9A1 with SEQ ID NO:1, ALDH4A1 with SEQ ID NO:2, ID3 with SEQ ID NO:3, TMC4 with SEQ ID NO:4, TYSND1 with SEQ ID NO:5, and p53 with SEQ ID NO:6 exhibited a significant relationship in distinguishing MIBC and NMIBC cases in bladder cancer. The contents of the kit used to differentiate these cases by detecting biomarkers in a biological sample are described below.

[0045] A kit for distinguishing NMIBC from bladder cancer cases by detecting the biomarkers of the invention in a biological sample.

[0046] • Sample placement compartment.

[0047] • Detection compartment.

[0048] • A substance capable of detecting at least one of the proteins SLC9A1, ALDH4A1, ID3, TMC4, or TYSND1 in combination with the p53 protein

[0049] In one embodiment of the invention, the substance used for protein detection in the kit is selected as a material that can bind one or more biomarkers or specifically bind to these biomarkers.

[0050] In another embodiment of the invention, the substance used for protein detection in the kit is a biospecific capture agent selected from the group consisting of antibodies or their antigenbinding fragments, interactive fusion proteins, aptamers, and affibodies.

[0051] In one embodiment of the invention, the biological sample used in the kit is a bladder cancer tissue section on a pathological slide. The method of the kit of the present invention comprises the following steps:

[0052] i. Placing the biological sample obtained from the patient into the sample compartment.

[0053] ii. Selecting biomarkers to be measured from among SLC9A1, ALDH4A1, ID3, TMC4, or TYSND1 proteins in combination with p53 protein

[0054] iii. Placing the samples obtained from the biological material into the detection compartments containing substances that detect the selected biomarkers.

[0055] iv. The selected biomarkers were detected using medical pathology methods.

[0056] In one embodiment of the invention, the medical pathology methods described in step (iv) of the kit method are selected from the group consisting of immunohistochemistry (IHC), immunocytochemistry, immunofluorescence, matrix-assisted laser desorption / ionization imaging mass spectrometry (MALDI-IMS), mass spectrometry-immunohistochemistry (MSIHC), scanning mass cytometry (SMC), and multiplexed ion beam imaging (MIBI). A comprehensive study was conducted to evaluate the effectiveness of the biomarkers. This study focused on detecting the presence of biomarkers using immunohistochemical (IHC) methods, which are routinely applied in pathology centers, for the classification of bladder cancer into nonmuscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC).

[0057] In the first stage, extensive bioinformatics analyses were performed on genes expressed in NMIBC and MIBC tumor tissue. These analyses included thousands of gene transcripts and hundreds of tissue samples, from which 30 gene transcripts that best distinguished the NMIBC and MIBC groups were identified (Table 1). From these 30 genes, the best candidates were prioritized based on the literature, and ultimately, 11 genes were selected: ALDH4, SLC9A1, ID3, TMC4, TYSND1, UNC5B, CDC25B, SPHK1, ST3GAL5, GFPT2, and FAP

[0058] Simultaneously, a broad tumor panel consisting of NMIBC (pTa and pTl stages) and MIBC (pT2 stage) was used to prepare tumor tissue microarrays. The tumor panel was subjected to immunohistochemical analysis using the selected antibodies. In the same tissue material, the expression of p53 and AXL was examined using IHC. Using all obtained IHC results via the Immune Reactivate Scoring (IRS) method, various statistical analyses were performed, the best discriminating biomarkers were identified, and were analyzed both individually and in groups. From this, the most suitable biomarker combinations for IHC-based differential diagnosis were identified.

[0059] The following steps were carried out to detect the biomarkers of the invention.

[0060] Table 1. Thirty genes were identified by bioinformatics analysis as the best distinguishing factors between MIBC and NMIBC cases.

[0061]

[0062] The staining results showed significant differences between the NMIBC and MIBC groups. In particular, statistically significant differences (P<0.05) were observed in the expression levels of ALDH4, SLC9A1, ID3, TMC4, and TYSND1. The statistical differences are presented in Table 2 and Figure 1. In contrast, no significant differences were observed in the expression of ST3GAL5, UNC5B, CDC25B, and SPHK1 proteins (P>0.05).

[0063] Table 2. Clinical and histopathological parameters and candidate biomarkers for which statistically significant differences were identified between NMIBC and MIBC.

[0064] <

[0065] <

[0066] <

[0067] <

[0068]

[0069]

[0070] As shown in Table 2, statistical analyses indicated that with a p-value of 0.0365, TYSND1 was a weak diagnostic parameter, whereas the low expression levels of the other proteins produced significant results. Increased p53 expression has been documented in the literature to play a critical role in the assessment of bladder cancer, and similar findings have been reported for the AXL protein. Therefore, p53 and AXL were included as candidate biomarkers with increased expression in advanced bladder cancer for evaluation in this cohort. The staining results for p53 are shown in Figure 2, visually supporting its significant role at high expression levels (considering both prevalence and staining intensity) in the more advanced stages of bladder cancer.

[0071] To evaluate the diagnostic effectiveness of the biomarkers, Receiver Operating Characteristic (ROC) and Area Under the Curve (AUC) analyses were performed. Notably, combinations of SLC9A1 and p53 were used together achieved the highest AUC values. Similarly, the combined evaluation of ALDH4 and p53 also showed very high AUC values. In subsequent evaluations, only the tumor grading data (low and high grade) were included as histopathological parameters alongside the biomarker group that distinguished NMIBC from MIBC. The findings are presented in Table 3 and visually illustrated in Figures 5 and 6. These analyses statistically demonstrate the success of the biomarkers of the invention in distinguishing between the MIBC and NMIBC groups.

[0072] Table 3. AUC values of diagnostic biomarkers

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[0079]

[0080] The diagnosis of low- and high-grade carcinoma is based on the characterization of the cellular and structural features of bladder cancer [21,22], The criteria for the grading assessment are presented in Table 8. The sensitivity and specificity values of the defined kits are presented in Tables 4 and 5, respectively. These values demonstrate the diagnostic accuracy and reliability of these biomarkers. As can also be seen from the data in Tables 4 and 5, the combination of SLC9A1, ALDH4A1, and p53 biomarkers and their joint analysis with the tumor grading score showed superior performance in correctly distinguishing MIBC and NMIBC cases.

[0081] Table 4. Sensitivity (true positive likelihood ratio) and specificity (false negative likelihood ratio) of the defined kits.

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[0085]

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[0087]

[0088] Table 5. Statistical data on the sensitivity and specificity (false-negative likelihood ratio) of the defined kits.

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[0090]

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[0094]

[0095] The associations of biomarkers with overall survival (OS) and disease-free survival (DFS) were investigated using the Kaplan-Meier statistical method. ALDH4, SLC9A1, ID3, TMC4, ST3GAL5, SPHK1, and p53 were significantly associated with OS and DFS (P<0.05). As shown in Table 6 and Figure 7, TYSND1 showed a significant association only with DFS (P>0.05), whereas no significant association was detected for UNC5B and CDC25B.

[0096] Table 6. Case numbers based on staining scores with candidate biomarkers in Ta+Tl+T2 bladder carcinoma and their associations with Overall Survival (OS) and Disease-Free Survival (DFS) durations / Kaplan-Meier (Mantel-Cox).

[0097] < <

[0098] < <

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[0100]

[0101] < <

[0102]

[0103] Statistical analyses revealed that SLC9A1, ALDH4, and p53 were independent biomarkers. Additionally, a negative correlation was identified between the positivity of ALDH4, SLC9A1, and p53 and poor prognosis indicators such as vascular invasion, prostate invasion, lymph node metastasis, and distant metastasis, which was found to be statistically significant. These correlations demonstrate that detecting the biomarkers of the invention provides safe and consistent predictions of disease progression. The numerical data regarding these associations are presented in Table 7. Table 7. Relationship between effective biomarkers and other prognostic parameters.

[0104] <

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[0107]

[0108] The results showed that ALDH4, SLC9A1, ID3, TMC4, and TYSND1 proteins showed significant differences between the NMIBC and MIBC tumor groups, as detailed in Table 2. These statistical findings demonstrate that ALDH4, SLC9A1, ID3, TMC4, and TYSND1 proteins can be used as biomarkers to differentiate between the NMIBC and MIBC tumor groups.

[0109] Among these biomarkers, ALDH4, SLC9A1, ID3, and TYSND1 were identified as independent biomarkers across the two tumor groups. In particular, ALDH4 and SLC9A1 maintained their importance as independent biomarkers, showing decreased expression in MIBC when analyzed together with the p53 biomarker, as shown in Figures 3 and 4. AUC analyses revealed that the combination of ALDH4, SLC9A1, and p53 biomarkers reached an AUC value of 84.2%, indicating high diagnostic accuracy in bladder cancer classification; these results are presented in Table 3. Furthermore, in analyses where biomarker results were combined with the tumor grading score, this combination reached the highest AUC value of 88.1%, further enhancing diagnostic accuracy; these findings are also detailed in Table 3.

[0110] The capacities of ALDH4 and SLC9A1 biomarkers to predict overall survival (OS) and disease-free survival (DFS) durations were found to be high, and a negative correlation was observed between the positivity of these biomarkers and poor-prognosis indicators, such as vascular invasion, prostate invasion, lymph node metastasis, and distant metastasis. This correlation was statistically significant, as shown in Tables 6 and 7. The tumor grading parameters are presented in Table 8.

[0111] Table 8. Histological and cytological features of low- and high-grade bladder carcinoma.

[0112]

[0113]

[0114] In conclusion, the conducted studies demonstrate that the biomarkers subject to the invention can serve as powerful tools for distinguishing between MIBC and NMIBC, as well as for the survival diagnosis of bladder cancer. The integration of these biomarkers and the kits used for their detection into clinical practice enables more accurate and reliable diagnostic outcomes in bladder cancer.

[0115] References

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Claims

1. CLAIMS1. A bladder cancer biomarker, characterized by the following:

1. SLC9A1 having the amino acid sequence of SEQ ID NO: 1,4.ii. ALDH4A1 having the amino acid sequence of SEQ ID NO: 2,5.iii. ID3 having the amino acid sequence of SEQ ID NO: 3,6.iv. TMC4 having the amino acid sequence of SEQ ID NO: 4,7.v. TYSND1 having the amino acid sequence of SEQ ID NO: 5,8.in combination with one or more of the above five proteins, together with the p53 protein having the amino acid sequence of SEQ ID NO: 6, or mutant variants thereof.

2. The bladder cancer biomarker according to Claim 1, characterized in that said biomarker distinguishes between non-muscle-invasive bladder cancer (Ta / Tl stage / NMIBC) and muscle-invasive bladder cancer (T2 stage / MIBC)3. The bladder cancer biomarker according to Claim 1 or 2, characterized in that the biomarker comprises SLC9A1 protein having the amino acid sequence of SEQ ID NO: 1 and / or ALDH4A1 protein having the amino acid sequence of SEQ ID NO: 2 in combination with p53 protein having the amino acid sequence of SEQ ID NO: 6.

4. The bladder cancer biomarker according to any one of Claims 1-3, characterized in that the biomarker is used in conjunction with tumor grading parameters.

5. A kit for distinguishing non-muscle-invasive bladder cancer (NMIBC) cases from muscle-invasive bladder cancer (MIBC) with biological samples, characterized by the following:13.i. a sample placement compartment,14.ii. one or more detection compartments,15.iii. a substance in the detection compartment capable of detecting one or more biomarkers according to any of claims 1-4.

6. The kit according to Claim 4, wherein the substance capable of detecting biomarkers in the kit is a substance that can bind to one or more biomarkers or specifically bind to such biomarkers.

7. The kit according to Claim 4 or 5, wherein the substance capable of binding to or specifically binding to biomarkers is a biospecific capture agent selected from the group consisting of antibodies or antigen-binding fragments thereof, interactive fusion proteins, aptamers, and affibodies.

8. The kit according to any one of Claims 4-6, wherein the biological sample used in the kit is a bladder cancer tissue slide section.

9. A method related to a kit according to any one of Claims 4-7, characterized by comprising the following steps:19.i. Placing a biological sample obtained from a patient into the sample compartment,20.ii. Selecting at least one of SLC9A1, ALDH4A1, ID3, TMC4, TYSND1 proteins in combination with p53 protein as biomarkers to be measured,21.iii. Placing the samples obtained from the biological specimen into the detection compartments containing substances capable of detecting the selected biomarkers,22.iv. The selected biomarkers were detected using medical pathology methods.

10. The method according to Claim 8, wherein the medical pathology methods are selected from the group consisting of immunohistochemistry, immunocytochemistry, immunofluorescence, matrix-assisted laser desorption / ionization imaging mass spectrometry (MALDI IMS), mass spectrometry-immunohistochemistry (MSIHC), scanning mass cytometry (SMC), and multiplexed ion beam imaging (MIBI).

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