Urinary tract epithelial cell marker and application thereof in diagnosis of urinary tract epithelial cell carcinoma
By using cytoplasmic thyroid hormone-binding protein CRYM as a molecular marker for urothelial carcinoma cells, the problem of distinguishing urothelial carcinoma cells from normal cells in urine has been solved, enabling efficient diagnosis and non-invasive screening of urothelial carcinoma.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-01
- Publication Date
- 2026-03-10
AI Technical Summary
Existing technologies are insufficient to efficiently distinguish reactive hyperplastic urothelial cells from urothelial carcinoma cells in urine, and commonly used markers lack specificity, leading to difficulties in identification.
Cytoplasmic thyroid hormone-binding protein CRYM was used as a molecular marker for urothelial carcinoma cells, and immunohistochemical detection was used to distinguish urothelial carcinoma cells in urothelial tissue and urine samples.
This technology enables the effective differentiation between normal urothelial cells and urothelial carcinoma cells in the histopathological diagnosis of urothelial carcinoma, and develops non-invasive urine screening technology, solving the problem of identifying exfoliated cells in urine.
Smart Images
Figure CN121633480A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular biomedical technology, specifically relating to a urothelial cell marker and its application in the diagnosis of urothelial cell carcinoma. Background Technology
[0002] The cellular composition of urine directly reflects the health of the urinary system (including the kidneys, ureters, bladder, and urethra). The main cellular components in urine include urothelial cells, non-epithelial blood cells, and tumor cells shed from malignant tumors of the urinary system. A significant increase in the number of a certain type of cell in the urine, or the presence of abnormal cells such as red blood cells or tumor cells, usually indicates lesions such as inflammation, injury, stones, or tumors in the urinary system.
[0003] The urothelial tract is a specialized, expandable and contractile lining tissue that covers the interior of the urinary system. When urothelial carcinoma occurs, cancer cells detach and enter the urine, becoming targets for urine testing. However, differentiating reactive, proliferating urothelial cells detached from urine from urothelial carcinoma cells remains a significant challenge in clinical cytological diagnosis.
[0004] Cancer cells and reactive urothelial cells exhibit significant morphological overlap, making accurate differentiation difficult to achieve solely through morphological observation. Immunohistochemical testing can provide crucial auxiliary diagnostic value. Currently, commonly used urothelial lineage markers include GATA3, CK20, Ki-67, and P53, but all suffer from insufficient marker specificity. For example, the expression patterns of CK20 in reactive and malignant cells partially overlap: in reactive hyperplastic urothelial cells, CK20 is typically expressed in the cytoplasm of surface umbrella cells; while in urothelial carcinoma in situ, CK20 is usually positively expressed in the full-thickness cytoplasm, showing characteristic differences but also easily leading to confusion. Ki-67 expression is increased in reactive urothelial cells, but its expression in urothelial carcinoma in situ and reactive dysplasia significantly overlaps, limiting its diagnostic value. GATA3 is the preferred marker for determining urinary tract differentiation and is positively expressed in 80% of high-grade urothelial carcinomas. However, it is not a completely specific marker. Studies have shown that GATA3 is highly expressed in breast cancer, invasive ductal carcinoma, and lobular carcinoma, and is also expressed to varying degrees in diseases such as basal cell carcinoma, squamous cell carcinoma, and paraganglioma of the skin. In particular, it is significantly expressed in normal urothelial cells.
[0005] Currently, no single biomarker can simultaneously achieve 100% sensitivity and specificity to distinguish between normal urothelial cells and urothelial carcinoma cells. Effectively differentiating reactive proliferating urothelial cells from urothelial carcinoma cells in urine remains a critical bottleneck that urgently needs to be overcome in clinical practice.
[0006] The discovery of novel molecular markers has important clinical significance in distinguishing malignant urothelial carcinoma cells from reactive urothelial cells. Summary of the Invention
[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical means: This invention first provides the application of cytoplasmic thyroid hormone-binding protein CRYM as a molecular marker to distinguish between urothelial carcinoma cells and non-cancerous urothelial cells.
[0008] Preferably, by detecting the expression differences of cytoplasmic thyroid hormone-binding protein CRYM, urothelial carcinoma cells and non-cancerous urothelial cells in urothelial tissue samples, or urothelial carcinoma cells and non-cancerous urothelial cells in urine samples, can be distinguished.
[0009] Preferably, the non-cancerous urothelial cells include normal urothelial cells and reactively proliferating urothelial cells.
[0010] Preferably, when the staining results show that the expression of cytoplasmic thyroid hormone-binding protein CRYM is absent or the expression level is significantly downregulated, it is judged to be urothelial carcinoma.
[0011] The present invention further provides the application of cytoplasmic thyroid hormone-binding protein CRYM in the preparation of products for screening, diagnosis or auxiliary diagnosis of urothelial carcinoma.
[0012] Preferably, the product includes reagents, test strips, and kits.
[0013] The present invention also provides a product for screening, diagnosing or assisting in the diagnosis of urothelial carcinoma, the product comprising reagents for detecting the content and / or expression of cytoplasmic thyroid hormone-binding protein CRYM.
[0014] Preferably, the product is an antibody or its antigen-binding fragment that specifically binds to the cytoplasmic thyroid hormone-binding protein CRYM.
[0015] Preferably, the object of detection is a urothelial tissue sample or a urine sample.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) This application screened out 8 characteristic genes in the gene expression matrix through machine learning, and further verified at the protein level by immunochemical staining that the urothelial cell marker CRYM has the potential to be used as a single immunomarker to distinguish reactive hyperplastic urothelial cells from urothelial carcinoma cells, thereby playing an important role in the histopathological diagnosis of urothelial carcinoma.
[0017] 2) Utilizing CRYM as an effective marker for reactive hyperplasia of normal urothelial cells, it is possible to distinguish between reactive hyperplasia of normal urothelial cells and urothelial carcinoma cells, thus enabling the development of non-invasive urine screening technology. This solves the challenge of differentiating between reactive hyperplasia of urothelial cells and urothelial carcinoma cells shed in urine during clinical cytological diagnosis.
[0018] 3) CRYM, the cytoplasmic thyroid hormone-binding protein involved in this application, actually belongs to the aldehyde-ketone reductase (AKR) superfamily and is an NADPH-dependent enzyme. Its unique "enzyme-hormone coupling" mechanism involves the irreversible binding of thyroid hormone T3 to its active site while consuming NADPH cofactors, thereby achieving the "isolation" and "storage" of T3 in the cytoplasm. Current functional reports on CRYM are limited to regulating the bioavailability of intracellular thyroid hormones, participating in the transport and localization of thyroid hormones, and indirectly regulating cell proliferation and differentiation. The application of the cytoplasmic thyroid hormone-binding protein CRYM as a marker for urothelial cells in the diagnosis of urothelial carcinoma and the differentiation of benign and malignant urine is a first in the industry. Attached Figure Description
[0019] Figure 1 The receiver operating characteristic (ROC) curve analysis results of the eight candidate genes screened in the test set to distinguish between normal urothelial cells and urothelial carcinoma cells.
[0020] Figure 2 The receiver operating characteristic (ROC) curve analysis results for 8 candidate genes in the validation set to distinguish between normal urothelial cells and urothelial carcinoma cells.
[0021] Figure 3 The expression of CRYM in urothelial tissue. Figure 3 In Figure A, the immunohistochemical results of CRYM staining on normal urothelial cells are shown, while in Figure B, the immunohistochemical results of CRYM staining on urothelial carcinoma are shown.
[0022] Figure 4 For statistical analysis of CRYM expression in tissues, Figure 4 Figure A shows the statistical analysis of the positive area ratio, and Figure B shows the statistical analysis results of the average optical density.
[0023] Figure 5 The results of immunocytochemical (ICC) staining of the characteristic gene CRYM in urine exfoliated cell smears are shown in Figure A, which shows the staining results of CRYM in the main type of normal exfoliated urothelial cells, and Figure B shows the staining results of urothelial carcinoma cells and reactive hyperplastic urothelial cells. Detailed Implementation
[0024] The technical solution of this application will be described in more detail below with reference to experiments and accompanying drawings.
[0025] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art.
[0026] Example 1
[0027] Machine learning to screen for characteristic molecular biomarkers
[0028] Two machine learning methods were employed, including the Least Absolute Shrinkage and Selection Operator (LASSO) model and the Weighted Gene Co-expression Network (WGCNA) analysis, to screen for characteristic genes that can effectively distinguish between urothelial cells and urothelial carcinoma cells based on gene expression profiles.
[0029] The specific operational procedure is as follows: Lasso logistic regression is implemented using the R package "glmnet," and 10-fold cross-validation is used to select the optimal penalty parameter. A correlation matrix is constructed using the R package WGCNA, and the optimal soft threshold is selected to transform the correlation matrix into an adjacency matrix. A topological overlap matrix (TOM) is then generated from the adjacency matrix. Using a TOM-based phase dissimilarity measure, average linkage hierarchical clustering is employed to divide genes with similar expression patterns into gene modules. Gene modules with significant characteristic associations are selected as key modules for subsequent analysis. Finally, the intersection of differentially expressed genes and genes in the key modules is taken to obtain the target gene. Receiver operating characteristic (ROC) curve analysis is used to calculate the sensitivity and specificity of the characteristic gene in distinguishing between urothelial cells and urothelial carcinoma cells.
[0030] Results: Potential diagnostic biomarkers that can distinguish between normal urothelial cells and urothelial carcinoma cells were screened in the test set using WGCNA and LASSO regression machine learning methods. 2064 genes were identified by WGCNA and 13 genes were identified by LASSO. The intersection of these genes yielded 8 characteristic genes, namely: PRAC1, ESM1, CRYM, ABI3BP, VIPR2, TMEM100, SCARA5, and PI16, which were selected as potential diagnostic biomarkers.
[0031] like Figure 1 The ROC analysis results for the eight genes are shown. The AUC values for PRAC1, ESM1, CRYM, ABI3BP, VIPR2, TMEM100, SCARA5, and PI16 are 0.935, 0.994, 0.937, 0.989, 0.976, 0.992, 0.979, and 0.995, respectively. The AUC values for all eight characteristic genes exceed 0.93, indicating good sensitivity and specificity.
[0032] Simultaneously, the eight selected genes were independently validated in the TCGA-BLCA (Cancer Genome Atlas Database - Bladder Urothelial Carcinoma) validation set. The results showed expression patterns consistent with the experimental group: PRAC1, CRYM, VIPR2, ABI3BP, SCARA5, PI16, and TMEM100 were expressed at lower levels in cancer cells than in normal cells, while ESM1 was expressed at higher levels in cancer cells than in normal cells. See also Figure 2 ROC analysis showed that the AUC values of all eight characteristic genes exceeded 0.88, indicating that the eight selected genes have good diagnostic potential and are potential leading candidate biomarkers.
[0033] Example 2
[0034] Immunohistochemical staining to verify the specific expression of CRYM in urothelial tissue
[0035] Immunohistochemical (IHC) data were collected from the Human Protein Atlas (HPA) database to screen for characteristic molecular markers that can effectively distinguish urothelial cells and urothelial carcinoma cells at the protein expression level.
[0036] To validate the eight candidate genes selected, 36 paraffin-embedded sections were collected from pathologically diagnosed urothelial tissue, reactive urothelial hyperplasia, urothelial carcinoma, or adjacent non-cancerous tissue. Of these, 14 cases were diagnosed as normal urothelial tissue or epithelial hyperplasia, and 22 cases as urothelial carcinoma. Immunohistochemical experiments were used to validate the characteristic molecular markers at the protein expression level.
[0037] The standard procedure for immunohistochemical staining is as follows: Paraffin-embedded tissue sections are dewaxed and dehydrated, then antigen retrieval is performed using 1% sodium citrate solution. After the container cools to room temperature, it is incubated in 3% hydrogen peroxide solution for 10 minutes, washed three times with PBS aqueous solution and once with PBST aqueous solution, each time for 4 minutes. Then, it is blocked at room temperature with 10% goat serum solution (AR1009 Boster) for 45 minutes. The tissue is then incubated overnight at 4 °C with a 1:300 dilution of the first antibody. The next day, the humidified chamber is removed, and the tissue is allowed to return to room temperature. It is then washed three times with PBS aqueous solution and once with PBST aqueous solution, followed by incubation with the second antibody (horseradish peroxidase polymer anti-mouse / rabbit IHC kit, KIT 5020; Maxim) for 25 minutes, followed by three washes with PBS aqueous solution and once with PBST aqueous solution. Finally, it is stained with diaminobenzidine staining solution (DAB-0031; Maxim). After 2 minutes, place the slide in pure water to stop staining, immerse it in hematoxylin staining solution for 5 seconds, and immediately rinse it under running water. Then immerse it in 95% ethanol and anhydrous ethanol for 5 minutes each. After drying, mount the slide and observe the expression of diagnostic markers in benign and malignant tissues under a microscope.
[0038] Image acquisition for immunohistochemical staining sections followed a standardized procedure to ensure objective and reproducible data, as follows: First, a low-power microscope (10× eyepiece, 10× objective, total magnification 100×) was used for whole-section scanning to clearly define the location of the target tissue region, tissue integrity, and overall staining distribution trend, excluding non-target areas such as necrotic areas and edge contamination areas. Then, a high-power microscope (10× eyepiece, 40× objective, total magnification 400×) was used as the core acquisition magnification to clearly present the expression details of target proteins and cellular structural characteristics. All quantitative analyses were based on 40× magnification images, ensuring that five high-power images were randomly acquired for each tissue sample from each section targeting the target tissue region. The acquired high-power images were analyzed using Fiji (imageJ) software. After segmenting the positive regions, the positive region area ratio (PAR) and mean optical density (Mean OD) were calculated, and the results were statistically analyzed and visualized using GraphPad Prism 10.1.2 software.
[0039] Results: Immunohistochemical (IHC) data from the Human Protein Atlas (HPA) database showed that CRYM, PRAC1, ABI3BP, VIPR2, and SCARA5, which were predicted to be highly expressed in normal urothelial tissue but downregulated in cancerous tissue, showed varying degrees of staining in normal urothelial tissue. Only CRYM and PRAC1 were negatively expressed in almost all urothelial carcinoma samples, as shown in Table 1.
[0040] Table 1. Expression of 8 candidate genes
[0041] Furthermore, immunohistochemical analysis was performed on the collected tissue samples, among which... Figure 3 The expression of CRYM in urothelial tissue. Figure 3 Figures A and B in the figure represent the immunohistochemical results of CRYM staining of normal urothelial cells and the immunohistochemical results of CRYM staining of urothelial carcinoma, respectively. Figure 4 For statistical analysis of CRYM expression in tissues, Figure 4 Figures A and B in the figure represent the statistical analysis of the positive area ratio and the statistical analysis of the average optical density, respectively. CRYM is significantly positive in normal urothelial tissue or epithelial hyperplasia, but almost undetectable in urothelial carcinoma tissue. Quantitative evaluation of the immunohistochemical results based on the positive area ratio and staining intensity shows that CRYM can significantly distinguish between normal urothelial cells and urothelial carcinoma cells. These experiments demonstrate that CRYM has the potential to serve as a diagnostic marker for differentiating between normal urothelial tissue and urothelial carcinoma.
[0042] Example 3
[0043] Immunocytochemical experiments have verified that CRYM staining can be used for non-invasive urine screening.
[0044] To evaluate the efficiency of CRYM as a marker of exfoliated urothelial cells in urine, immunocytochemical (ICC) analysis was performed on 43 urine cytology samples. Samples were obtained from patients with a history of urinary tract diseases, including bladder cancer, renal pelvis cancer, ureteral cancer, urinary tract infection, and urinary tract stones. Thin-layer smears of exfoliated cells from the urine were prepared for ICC staining.
[0045] The aforementioned immunohistochemical (IHC) results showed that CRYM was negatively expressed in urothelial carcinoma but positively expressed in normal urothelial cells. It is expected that various types of normal urothelial cells and cancer cells can be effectively distinguished in urine through immunostaining.
[0046] The standard procedure for immunocytochemical staining is as follows: Use urine samples collected clinically. Let the samples stand at room temperature for 30 minutes, discard the supernatant, mix the precipitate, and transfer 50 mL to a 50 mL centrifuge tube. Centrifuge at 3000 rpm for 5 minutes using a Beckman Avanti J-15R centrifuge. Discard the supernatant, aspirate the cell pellet onto a glass slide, and use the spreading method to evenly distribute the cell pellet on the slide. After drying at room temperature, immerse the slide in 95% ethanol solution for 7 minutes. After fixation, gently rinse the slide twice with water, then wash three times with PBS solution. Next, perform antigen retrieval with 1% sodium citrate solution. Once the container has cooled to room temperature, incubate in 3% hydrogen peroxide solution for 10 minutes. Wash three times with PBS solution and once with PBST solution for 4 minutes each time. Finally, block with 10% goat serum solution (AR1009Boster) at room temperature for 45 minutes. The tissue was then incubated overnight at 4 °C with the primary antibody at a 1:300 dilution. The next day, the humidified chamber was removed, and the tissue was allowed to return to room temperature. It was washed three times with PBS aqueous solution and once with PBST aqueous solution, followed by incubation with the secondary antibody (horseradish peroxidase polymer anti-mouse / rabbit IHC kit, KIT 5020; Maxim) for 25 minutes, followed by three washes with PBS aqueous solution and once with PBST aqueous solution. Subsequently, the tissue was stained with diaminobenzidine staining solution (DAB-0031; Maxim). After 2 minutes, staining was stopped by placing the tissue in pure water, followed by immersion in hematoxylin staining solution for 5 seconds, and immediately rinsed under running water. The tissue was then immersed in 95% ethanol and anhydrous ethanol for 5 minutes each. After drying, the tissue was mounted, and the expression of diagnostic markers in exfoliated urine cells was observed under a microscope.
[0047] See Figure 5 ICC results showed that CRYM stained moderately to strongly positively in major exfoliated urothelial cells (including superficial and basal transitional epithelial cells); and showed almost no staining in squamous epithelial cells. Figure 5 (A) For urothelial carcinoma cells, the characteristics are enlarged nuclei, increased nuclear-cytoplasmic ratio, and significantly irregular nuclear membrane, showing CRYM negative staining (in the figure, (+) indicates positive expression, (-) indicates negative expression; arrows indicate target cells) Figure 5 (B)
[0048] Clinical observations have revealed that while reactive hyperplasia cells fall within the normal range, they can undergo morphological changes resembling cancer cells in benign urinary tract diseases. This makes differentiating reactive hyperplasia urothelial cells shed in urine from urothelial carcinoma cells a significant challenge in clinical cytological diagnosis. The results of this application show that these reactive hyperplasia urothelial cells still maintain strong CRYM staining (…). Figure 5(Middle B). Therefore, using ICC staining of CRYM or other alternative techniques to detect its expression level, such as quantitative PCR, ELISA, and in situ hybridization, can serve as a valuable tool for distinguishing morphologically abnormal reactive urothelial cells from cancer cells in urine cytology.
[0049] In summary, CRYM staining can effectively label various types of exfoliated urothelial cells in urine, including morphologically abnormal reactive proliferative cells. Therefore, CRYM staining is helpful in differentiating proliferative cells from cancer cells in urine smears.
[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. Use of cytoplasmic thyroid hormone binding protein CRYM as a molecular marker for distinguishing urothelial cancer cells from non-cancerous urothelial cells.
2. Use according to claim 1, wherein Urothelial cancer cells and non-cancerous urothelial cells in urothelial tissue samples, or urothelial cancer cells and non-cancerous urothelial cells in urine samples are distinguished by detecting the expression difference of cytoplasmic thyroid hormone binding protein CRYM.
3. Use according to claim 1 or 2, characterized in that, The non-cancerous urothelial cells include normal urothelial cells and urothelial cells with reactive hyperplasia.
4. The use according to claim 2, wherein the compound is ###0002### When the staining result shows that cytoplasmic thyroid hormone binding protein CRYM is absent or has a significantly down-regulated expression level, it is judged as urothelial cancer cells.
5. Use of cytoplasmic thyroid hormone binding protein CRYM in the preparation of a product for screening, diagnosing or aiding in the diagnosis of urothelial cancer.
6. The use according to claim 5, wherein the compound is ###0002### The product includes reagents, test strips and kits.
7. A product for screening, diagnosing or aiding in the diagnosis of urothelial carcinoma, characterized in that, The product contains a reagent for detecting the content and / or expression of cytoplasmic thyroid hormone binding protein CRYM.
8. The product of claim 7, wherein, The product is an antibody or antigen-binding fragment thereof that specifically binds to cytoplasmic thyroid hormone binding protein CRYM.
9. The product of claim 7, wherein, The detection object is a urothelial tissue sample or a urine sample.