Expression of CREPT as marker for discovery and auxiliary diagnosis of bladder cancer in urine exfoliated epithelial cells and application of CREPT
By detecting the expression of CREPT in urine, combined with traditional urine exfoliative cytology, and utilizing immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), the problem of insufficient sensitivity and specificity in bladder cancer diagnosis has been solved, enabling early diagnosis and prediction of treatment outcomes.
Patent Information
- Application Number
- CN202511663225.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-13
- Publication Date
- 2026-02-06
AI Technical Summary
Existing auxiliary diagnostic methods for bladder cancer lack sufficient sensitivity and specificity, especially in the detection of low-grade tumors, and there is a lack of effective biomarkers for predicting treatment outcomes and monitoring recurrence.
Immunohistochemical detection of CREPT protein or mRNA was used as a marker of exfoliated cells in urine. Combined with traditional urine exfoliative cytology, the expression level of CREPT in urine was detected by immunostaining. The presence or expression level of CREPT was detected by methods such as enzyme-linked immunosorbent assay (ELISA) and electrochemical immunoassay (ECIA).
It improves the specificity and sensitivity of bladder cancer diagnosis, especially showing high specificity in early diagnosis and recurrence monitoring, and can predict treatment outcomes.
Smart Images
Figure CN121476599A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically relating to the expression of CREPT as a marker for the detection and auxiliary diagnosis of bladder cancer in urinary exfoliated epithelial cells, as well as a marker for bladder cancer recurrence and its applications. Background Technology
[0002] CREPT (Cell Cycle-Related and Expression-Elevated Protein in Tumor) is an oncogene discovered in recent years that is highly expressed in various tumors and is closely related to tumor occurrence and development. Studies have shown that CREPT is highly expressed in various malignant tumors, including gastric cancer, liver cancer, pancreatic cancer, and non-small cell lung cancer, and is associated with tumor invasion, metastasis, and poor prognosis. Although there are currently few direct studies on CREPT in bladder cancer, based on its expression patterns in other urinary system tumors (such as kidney cancer and prostate cancer), it can be inferred that CREPT may also show a high expression trend in bladder cancer.
[0003] Currently, the main method used for the auxiliary diagnosis of bladder cancer is urine exfoliative cytology. Its specificity and sensitivity are limited by tumor grade, diagnostic subjectivity, and interference from dynamic monitoring. The sensitivity for high-grade urothelial carcinoma is 60%-80%, while the sensitivity for low-grade tumors drops sharply to 30%-50%.
[0004] Commercially available test kits each have their advantages and disadvantages. The Twist1 gene methylation detection kit, based on fluorescent PCR, has shown in clinical studies to have a sensitivity of 88.2% and a specificity of 86.8% for bladder cancer detection. However, it requires specialized laboratory equipment and technicians, limiting its application in primary healthcare institutions. Compared to traditional cytology testing, it is more expensive. The NMP22 (nuclear matrix protein 22) detection kit is currently a widely used non-invasive urinary tumor marker detection tool for bladder cancer, based on ELISA. However, benign conditions such as urinary tract infections, stones, and hematuria can also cause elevated NMP22 levels. Its sensitivity is approximately 50-70%, and its specificity is approximately 60-80%.
[0005] However, current technologies lack applications for biomarkers that have better sensitivity and specificity for detecting bladder cancer, and also lack experimental detection methods for predicting the effectiveness of bladder cancer treatment and / or assessing prognosis. Summary of the Invention
[0006] In view of the shortcomings of the prior art, the first aspect of this application is to provide the application of a reagent for detecting the expression or expression level of CREPT in the preparation of a kit for diagnosing bladder cancer.
[0007] CREPT, as a biomarker for immunohistochemical staining of exfoliated cells in urine, is simple to perform, low in cost, highly specific and sensitive, produces clear staining patterns suitable for standardized interpretation, has minimal background interference, and offers high reliability. Combining IHC with traditional urine exfoliative cytology further enhances the specificity and sensitivity of the diagnosis.
[0008] This patent explores the diagnostic value of CREPT for bladder cancer through immunostaining: by detecting the expression level of CREPT in cells in urine, it helps to screen for bladder cancer in its early stages and assist in diagnosis and treatment through a simple method.
[0009] As some embodiments of this application, the diagnosis includes early diagnosis and / or auxiliary diagnosis.
[0010] As some embodiments of this application, the expression or expression level of CREPT is the expression or expression level of CREPT in exfoliated urinary epithelial cells.
[0011] As some embodiments of this application, the reagents for detecting the expression or expression level of CREPT include reagents for detecting CREPT protein and CREPT mRNA.
[0012] As some embodiments of this application, the reagent for detecting CREPT expression is a reagent capable of detecting the presence of CREPT protein or its expression level.
[0013] As some embodiments of this application, bladder cancer includes bladder cancer that has recurred after bladder cancer treatment.
[0014] As some embodiments of this application, the reagent capable of detecting CREPT expression is used to determine the presence or expression level of CREPT protein by in vitro detection.
[0015] As some embodiments of this application, the in vitro detection method is selected from immunological detection, mass spectrometry, and spectroscopy.
[0016] As some embodiments of this application, the immunological detection is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescence immunoassay (FIA), and radioimmunoassay (RIA).
[0017] As some embodiments of this application, the reagent capable of detecting CREPT expression comprises an antibody capable of specifically binding to CREPT.
[0018] As some embodiments of this application, the reagent capable of detecting CREPT expression comprises an antibody capable of specifically binding to CREPT and a detectable label linked to the antibody.
[0019] As some embodiments of this application, the reagent capable of detecting CREPT expression comprises an antibody capable of specifically binding to CREPT, and a second antibody specifically recognizing the antibody; optionally, the second antibody further comprises a detectable label.
[0020] As some embodiments of this application, the detectable marker is selected from enzymes, chemiluminescent reagents, electrochemiluminescent reagents, electroactive substances, fluorescent dyes, radionuclides, and biotin.
[0021] As some embodiments of this application, the enzyme includes horseradish peroxidase or alkaline phosphatase, the chemiluminescent reagent includes picolinic esters, the electrochemiluminescent reagent includes ruthenium tripyridine, the fluorescent dye includes FITC, TRITC, PE, Texas Red and cyanine dye derivatives, the radionuclide includes radioactive sulfonium, and the cyanine dye derivatives include Cy3, Cy5, Cy7 and Alexa 750.
[0022] As some embodiments of this application, the reagent for detecting CREPT expression is a reagent capable of detecting the presence or level of CREPT mRNA.
[0023] As some embodiments of this application, the reagent capable of detecting CREPT expression is used to determine the presence or level of CREPT mRNA by in vitro detection.
[0024] As some embodiments of this application, the in vitro detection method is selected from qPCR, nucleic acid sequencing or hybridization, mass spectrometry, and chromatography.
[0025] As some embodiments of this application, the reagent capable of detecting CREPT expression comprises primers and / or probes that specifically bind to CREPT mRNA.
[0026] In some embodiments of this application, the expression level of CREPT is determined by the Allred Score.
[0027] A third aspect of this application is to provide a kit for diagnosing bladder cancer, the kit comprising a reagent for detecting CREPT expression or expression level, wherein the reagent for detecting CREPT expression or expression level is used to detect whether CREPT is expressed or at what level in exfoliated urinary epithelial cells, and if CREPT is expressed, the patient has bladder cancer.
[0028] As some embodiments of this application, the reagent for detecting the expression or expression level of CREPT is a reagent for detecting the presence or expression level of CREPT protein, or the reagent for detecting the expression or expression level of CREPT is a reagent capable of detecting the presence or expression level of CREPT mRNA.
[0029] The fourth aspect of this application is to provide a reagent for detecting the expression or expression level of CREPT in the screening of bladder cancer treatment drugs. After administration of the bladder cancer treatment drug, the level of CREPT expression predicts the therapeutic effect of the bladder cancer treatment drug. A high level of CREPT expression predicts a good therapeutic effect of the bladder cancer treatment drug, while a low level of CREPT expression predicts a poor therapeutic effect of the bladder cancer treatment drug.
[0030] As some embodiments of this application, the expression or expression level of CREPT is the expression or expression level of CREPT in exfoliated urinary epithelial cells.
[0031] As described above, the expression of CREPT in this application serves as a biomarker for the detection and auxiliary diagnosis of bladder cancer in exfoliated urinary epithelial cells, offering the following beneficial effects: 1. This application shows that CREPT is highly expressed in bladder cancer tissues, with a positive rate of up to 100%. At the same time, CREPT is negatively expressed in exfoliated epithelial cells of urine from patients with benign lesions of the urinary system and non-urothelial carcinoma.
[0032] 2. This application demonstrates that the combination of CREPT's IHC with traditional urinary exfoliative epithelial cytology further enhances the specificity and sensitivity of the diagnosis.
[0033] 3. This application also shows that the detection of CREPT can detect weak positive, positive and strong positive expression, demonstrating that the detection of CREPT can be used for the early diagnosis of bladder cancer, for patients in the early stage of clinical diagnosis of bladder cancer.
[0034] 4. The CREPT test in this application can predict the effectiveness of bladder cancer treatment.
[0035] 5. The CREPT detection method of this application can be used to monitor the recurrence of bladder cancer and has high specificity. Attached Figure Description
[0036] Figure 1 The study showed that CREPT was highly expressed in the bladder tissue of bladder cancer patients. Figure 1 Immunohistochemical staining in A showed the expression pattern of CREPT in tumor tissue. Figure 1 B shows the statistical analysis of the immunohistochemical All Red Score in 96 bladder cancer samples; Figure 2 The results of immunohistochemical staining of exfoliated urinary epithelial cells by CREPT in different urinary tract diseases are shown. Figure 2 A shows the expression of CREPT in exfoliated cells of urine from healthy individuals, patients with benign urinary tract lesions, non-urothelial carcinoma, and bladder cancer patients, as detected by immunohistochemical staining. Figure 2 B shows the statistical chart of CREPT IHC scores for each group of samples. Healthy individuals (n=9), benign urinary tract lesions (n=10), non-urothelial carcinoma (n=10), bladder cancer (n=10); Figure 2 C shows the ROC curves plotted based on the percentage of positive cells in the control and bladder cancer groups using CREPT; Figure 3 Immunohistochemical staining results of CREPT in exfoliated epithelial cells of urine from recurrent bladder cancer are shown (urine specimens from clinical patients are pending). Detailed Implementation
[0037] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0038] Experimental materials Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0039] Example 1. CREPT is highly expressed in bladder cancer patient tissues. CREPT is highly expressed in various malignant tumors, including gastric cancer, liver cancer, pancreatic cancer, and non-small cell lung cancer, and is associated with tumor invasion, metastasis, and poor prognosis. Currently, direct studies on CREPT in bladder cancer are limited. Based on the expression patterns of CREPT in other urinary system tumors (such as kidney cancer and prostate cancer), this example explores the high expression trend of CREPT in bladder cancer. Furthermore, this example also investigates the close correlation between CREPT expression levels and clinical indicators of the tumor (such as pathological grade, lymph node metastasis, recurrence, and survival).
[0040] In this embodiment, the expression of CREPT in bladder cancer was detected by immunohistochemistry. The expression of CREPT in bladder cancer was quantitatively analyzed according to the All Red Score. Specifically, it was evaluated based on two aspects: staining intensity and the proportion of positive cells. Table 1 shows the scoring from 0 to 5 based on the proportion of positively stained cells. Table 1. Positive cell proportion score
[0041] Table 2 shows the scoring based on staining intensity: Table 2 Staining Intensity Scoring
[0042] The final Allred total score is the sum of the percentage of positively stained cells and the staining intensity, ranging from 0 to 8. CREPT is highly expressed in bladder cancer, and its expression pattern is as follows: Figure 1 As shown in A, we define a score of 8 as +++; a score of 7 as ++; and a score of 6 or less as +. Representative images are shown below. Figure 1 As shown in Figure A.
[0043] Furthermore, an All Red Score statistical analysis was performed on the immunohistochemical scores of 96 bladder cancer samples. The results showed that the highest proportion of strong positive (+++) samples was approximately 68%, followed by positive (++) and weak positive (+) samples at 12% and 20%, respectively. These results indicate that CREPT is highly expressed in bladder cancer tissues, with a positive rate as high as 100%. Figure 1 B shows the statistical results for each rating level of the All Red Score for these samples.
[0044] Example 2. CREPT is specifically highly expressed in exfoliated urinary epithelial cells of bladder cancer patients. Urine samples were collected from healthy individuals, patients with benign urinary tract lesions, patients with non-urothelial carcinoma (including renal cell carcinoma and prostate cancer), and patients with bladder cancer (BLCA). Exfoliated epithelial cells were isolated and subjected to CREPT immunohistochemical staining. Results showed that, statistically, exfoliated cells from the urine of bladder cancer patients were CREPT-positive, indicating high expression of CREPT in bladder cancer exfoliated cells, while expression was almost negative in healthy individuals, patients with benign urinary tract lesions, and patients with non-urothelial carcinoma. Figure 2 A).
[0045] Furthermore, the percentage of positive CREPT-positive cells was analyzed for each group. The results showed that CREPT expression was significantly elevated only in the bladder cancer group, while the expression levels in the other three groups (healthy, benign lesions, and non-urothelial carcinoma) were low and showed no significant difference. ROC curve analysis revealed its potential as a biomarker, with an AUC value reaching 0.9 (…). Figure 2 C). The above results suggest that CREPT may serve as a potential biomarker to differentiate bladder cancer from other non-malignant or non-epithelial diseases.
[0046] Example 3. CREPT is specifically highly expressed in exfoliated urinary epithelial cells of patients with recurrent bladder cancer. Examples 1 and 2 validated high expression of CREPT in tissue samples from bladder cancer patients and demonstrated the specificity and sensitivity of CREPT expression in urinary epithelial cells from bladder cancer patients. This example investigates the potential of CREPT as a prognostic biomarker in urinary exfoliated epithelial cells in cases of recurrent bladder cancer.
[0047] The experiment collected urine samples from 9 patients. Following the same processing method described above, immunohistochemical experiments demonstrated that CREPT can also serve as a biomarker for the detection of recurrent bladder cancer in the urine, exhibiting the same specificity and sensitivity. The positive detection rate of CREPT in the urine of the 9 patients was 100% (i.e., high specificity, see...). Figure 3 ).
[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.
Claims
1. Application of reagents for detecting CREPT expression or expression levels in the preparation of kits for diagnosing bladder cancer.
2. The application according to claim 1, characterized in that, The diagnosis includes early diagnosis and / or auxiliary diagnosis; Alternatively, the expression or expression level of CREPT may be the expression or expression level of CREPT in exfoliated urinary epithelial cells.
3. The application according to claim 1, characterized in that, Bladder cancer includes bladder cancer that has recurred after treatment.
4. The application according to any one of claims 1 to 3, characterized in that, The reagents for detecting CREPT expression or expression levels include reagents for detecting CREPT protein and CREPT mRNA.
5. The application according to claim 4, characterized in that, The reagent used to detect CREPT expression is one that can detect the presence of CREPT protein or its expression level. Preferably, the reagent capable of detecting CREPT expression is used to determine the presence of CREPT protein or its expression level by in vitro detection; Preferably, the in vitro detection method is selected from immunological detection, mass spectrometry, and spectroscopy. Preferably, the immunological assay is selected from enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay (ECIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescence immunoassay (FIA), and radioimmunoassay (RIA). Preferably, the reagent capable of detecting CREPT expression comprises an antibody capable of specifically binding to CREPT; Preferably, the reagent capable of detecting CREPT expression comprises an antibody capable of specifically binding to CREPT and a detectable label linked to the antibody; Preferably, the reagent capable of detecting CREPT expression comprises an antibody capable of specifically binding to CREPT, and a second antibody specifically recognizing the antibody; optionally, the second antibody further comprises a detectable label; Preferably, the detectable label is selected from enzymes, chemiluminescent reagents, electrochemiluminescent reagents, electroactive substances, fluorescent dyes, radionuclides, and biotin; More preferably, the enzyme includes horseradish peroxidase or alkaline phosphatase, the chemiluminescent reagent includes pyridine esters, the electrochemiluminescent reagent includes ruthenium tripyridine, the fluorescent dye includes FITC, TRITC, PE, Texas Red and cyanine dye derivatives, the radionuclide includes radioactive sulfonium, and the cyanine dye derivatives include Cy3, Cy5, Cy7 and Alexa 750.
6. The application according to claim 4, characterized in that, The reagent used to detect CREPT expression is one that can detect the presence or level of CREPT mRNA. Preferably, the reagent capable of detecting CREPT expression is used to determine the presence or level of CREPT mRNA by in vitro detection; Preferably, the in vitro detection method is selected from qPCR, nucleic acid sequencing or hybridization, mass spectrometry, and chromatography; Preferably, the reagent capable of detecting CREPT expression comprises primers and / or probes that specifically bind to CREPT mRNA.
7. The application according to any one of claims 1 to 3, characterized in that, The level of CREPT expression was determined using the Allred Score.
8. A reagent kit for diagnosing bladder cancer, characterized in that, The kit includes reagents for detecting CREPT expression or expression level. The reagents for detecting CREPT expression or expression level are used to detect whether CREPT is expressed or at what level in exfoliated urinary epithelial cells. If CREPT is expressed, the patient has bladder cancer. Preferably, the reagent for detecting the expression or expression level of CREPT is a reagent for detecting the presence or expression level of CREPT protein, or the reagent for detecting the expression or expression level of CREPT is a reagent capable of detecting the presence or expression level of CREPT mRNA.
9. The application of a reagent for detecting the expression or expression level of CREPT in screening drugs for bladder cancer treatment, characterized in that... After administration of the bladder cancer treatment drug, the expression level of CREPT predicted the therapeutic effect of the bladder cancer treatment drug. A high expression level of CREPT predicted a good therapeutic effect of the bladder cancer treatment drug, while a low expression level of CREPT predicted a poor therapeutic effect of the bladder cancer treatment drug.
10. The application according to claim 9, characterized in that, The expression or expression level of CREPT refers to the expression or expression level of CREPT in exfoliated epithelial cells of urine.