A protein marker and its application in preparing a product for immunohistochemical typing detection and / or prognosis evaluation of cholangiocarcinoma

Abstract

The present invention provides an application of a protein marker in the preparation of a product for immunohistochemical typing and / or prognosis evaluation of cholangiocarcinoma, wherein the protein marker includes POSTN, and cholangiocarcinoma includes fiber-driven cholangiocarcinoma; and / or; the protein marker includes CLDN6, and cholangiocarcinoma includes proliferative cholangiocarcinoma; and / or; the protein marker includes ADH1A, and cholangiocarcinoma includes metabolic cholangiocarcinoma. The protein marker provided by the present invention provides a new idea for molecular typing and diagnosis, prognosis evaluation and treatment of cholangiocarcinoma, and has a very broad application prospect in the medical field.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor diagnosis, and specifically, relates to a protein marker and its application in preparing a product for immunohistochemical typing detection and / or prognosis evaluation of cholangiocarcinoma. Background Art

[0002] Cholangiocarcinoma (CCA) originates from the bile duct epithelial cells of the liver or bile duct. It is a highly heterogeneous and malignant digestive system tumor. In recent years, many large-sample studies have conducted multi-omics studies and molecular typing of cholangiocarcinoma on the transcriptome, genome, and metabolome. The Cancer Research Center of the National Institutes of Health (NIH) in the United States and the University of Barcelona in Spain have conducted DNA, RNA, and metabolite tests on more than 100 cholangiocarcinoma samples, identified high-risk genes associated with tumor recurrence and poor prognosis, and divided cholangiocarcinoma into multiple subtypes with significant differences in clinical characteristics and prognosis. These studies explored the heterogeneity of cholangiocarcinoma based on large samples, indicating that cholangiocarcinoma is a group of diseases with high heterogeneity in clinical pathological characteristics, biological behavior, and gene expression, and has different molecular subtypes.

[0003] In the clinical treatment of cholangiocarcinoma, there is a lack of effective targeted drugs due to the large individual differences among patients and tumor heterogeneity. Therefore, the development of personalized precision treatment strategies based on the heterogeneity of cholangiocarcinoma is a new direction for the clinical treatment of cholangiocarcinoma. However, there is currently a lack of personalized treatment strategy formulation based on the heterogeneity of cholangiocarcinoma. Although the proteomic molecular typing of cholangiocarcinoma based on high-precision mass spectrometry can reveal its heterogeneous tumor biological characteristics and potential treatment strategies, high-precision mass spectrometry has problems such as high cost, complex process, and large batch effect, which greatly limits its clinical application. Therefore, we urgently need to find an alternative method that can easily, efficiently and accurately identify the molecular subtypes of cholangiocarcinoma in clinical practice, and provide a scientific basis for the formulation of personalized treatment strategies.

[0004] In most cases, proteins are the direct executors of cell physiological functions. However, the existing technology has not yet screened out characteristic proteins or protein markers that can be used for immunohistochemical typing of cholangiocarcinoma. Therefore, there is an urgent need for a protein marker that can be used to diagnose and evaluate the prognosis of cholangiocarcinoma of different proteomic subtypes through immunohistochemical typing, thereby providing specific therapeutic targets for cholangiocarcinoma and achieving accurate diagnosis and treatment of cholangiocarcinoma. Summary of the invention

[0005] The purpose of the present invention is to provide a protein marker and its use in the preparation of products for immunohistochemical typing detection and / or prognosis evaluation of cholangiocarcinoma. The protein marker can diagnose and evaluate the prognosis of cholangiocarcinoma of different proteomic subtypes using immunohistochemical methods, and has a very broad application prospect.

[0006] According to one aspect of the present invention, there is provided a use of a protein marker in the preparation of a product for immunohistochemical typing detection and / or prognosis assessment of cholangiocarcinoma, wherein the protein marker comprises POSTN (POSTN protein is an extracellular matrix protein secreted by fibroblasts), and cholangiocarcinoma includes fiber-driven cholangiocarcinoma; and / or; the protein marker comprises CLDN6 (CLDN6 protein is a skeletal protein of tight junctions between epithelial cells), and cholangiocarcinoma includes proliferative cholangiocarcinoma; and / or; the protein marker comprises ADH1A (ADH1A protein is a member of the alcohol dehydrogenase family, involved in the metabolism and detoxification of alcohols in the body), and cholangiocarcinoma includes metabolic cholangiocarcinoma.

[0007] This application divides cholangiocarcinoma into three subtypes based on cluster analysis of protein expression profiles: 1) fibrosis-driven; 2) proliferative; 3) metabolic. 1) Fiber-driven cholangiocarcinoma has a high degree of fibrosis and is rich in fibroblasts. Its biological characteristics are extracellular matrix remodeling and high expression of POSTN. It promotes lymph node metastasis of cholangiocarcinoma and poor prognosis by binding to integrin receptors of cholangiocarcinoma cells and lymphatic endothelial cells; 2) Proliferative cholangiocarcinoma enhances biological processes such as DNA replication and cell division. Its biological characteristics are cell division regulation, and CLDN6 is its characteristic protein; 3) Metabolic cholangiocarcinoma has alcohol-aldehyde metabolism as its biological characteristics, and ADH1A is its characteristic protein.

[0008] The protein markers provided by the present invention have the functions of diagnosing cholangiocarcinoma and evaluating the prognosis of cholangiocarcinoma. Among them, POSTN can diagnose and evaluate the prognosis of fiber-driven cholangiocarcinoma, CLDN6 can diagnose and evaluate the prognosis of proliferative cholangiocarcinoma, and ADH1A can diagnose and evaluate the prognosis of metabolic cholangiocarcinoma. Therefore, the molecular marker provides a new idea for the molecular typing and diagnosis, prognosis evaluation and treatment of cholangiocarcinoma, and has a very broad application prospect in the medical field.

[0009] This application screens out proteins for immunohistochemical typing of cholangiocarcinoma, which are highly consistent with the results of proteomics molecular typing based on high-precision mass spectrometry. This immunohistochemical typing has important implications for the diagnosis of cholangiocarcinoma and the prognosis of patients. The present invention makes up for the shortcomings of molecular typing of cholangiocarcinoma by high-precision mass spectrometry, such as complex technology, high cost, long detection cycle, and difficulty in clinical application, and provides a basis for the accurate typing of cholangiocarcinoma and the formulation of personalized treatment strategies.

[0010] Preferably, the protein markers include POSTN, CLDN6 and ADH1A, and cholangiocarcinoma includes fibrous-driven cholangiocarcinoma, proliferative cholangiocarcinoma and metabolic cholangiocarcinoma.

[0011] Preferably, the product comprises a kit and / or a chip.

[0012] According to another aspect of the present invention, there is provided the use of a reagent for specifically detecting protein markers in the preparation of a product for immunohistochemical typing detection and / or prognosis assessment of cholangiocarcinoma, wherein the protein markers include POSTN, and cholangiocarcinoma includes fiber-driven cholangiocarcinoma; and / or; the protein markers include CLDN6, and cholangiocarcinoma includes proliferative cholangiocarcinoma; and / or; the protein markers include ADH1A, and cholangiocarcinoma includes metabolic cholangiocarcinoma.

[0013] Preferably, the protein markers include POSTN, CLDN6 and ADH1A, and cholangiocarcinoma includes fibrous-driven cholangiocarcinoma, proliferative cholangiocarcinoma and metabolic cholangiocarcinoma.

[0014] Preferably, the reagent includes a reagent for specifically detecting the expression amount of a protein marker.

[0015] Preferably, the reagent for specifically detecting the expression amount of the protein marker includes at least one of POSTN antibody, CLDN6 antibody and ADH1A antibody.

[0016] According to another aspect of the present invention, a protein marker for immunohistochemical typing and / or prognostic evaluation of cholangiocarcinoma is provided, including POSTN, CLDN6 and ADH1A. The molecular markers provided by the present invention are not only related to the proteomic characteristics of cholangiocarcinoma, but also help to diagnose and evaluate the prognosis of cholangiocarcinoma of different proteomic subtypes (fiber-driven cholangiocarcinoma, proliferative cholangiocarcinoma, metabolic cholangiocarcinoma) using immunohistochemical methods, so as to achieve accurate diagnosis and treatment of cholangiocarcinoma.

[0017] Specifically, the above-mentioned protein markers for immunohistochemical typing and / or prognostic evaluation of cholangiocarcinoma include protein markers for immunohistochemical typing and / or prognostic evaluation of fibrous-driven cholangiocarcinoma, protein markers for immunohistochemical typing and / or prognostic evaluation of proliferative cholangiocarcinoma, and protein markers for immunohistochemical typing and / or prognostic evaluation of metabolic cholangiocarcinoma; the protein markers for immunohistochemical typing and / or prognostic evaluation of fibrous-driven cholangiocarcinoma include POSTN; the protein markers for immunohistochemical typing and / or prognostic evaluation of proliferative cholangiocarcinoma include CLDN6; the protein markers for immunohistochemical typing and / or prognostic evaluation of metabolic cholangiocarcinoma include ADH1A.

[0018] According to another aspect of the present invention, a product for immunohistochemical typing and / or prognostic evaluation of cholangiocarcinoma is provided, including a product for immunohistochemical typing and / or prognostic evaluation of fiber-driven cholangiocarcinoma, a product for immunohistochemical typing and / or prognostic evaluation of proliferative cholangiocarcinoma, and a product for immunohistochemical typing and / or prognostic evaluation of metabolic cholangiocarcinoma; the product for immunohistochemical typing and / or prognostic evaluation of fiber-driven cholangiocarcinoma includes a reagent for specific detection of POSTN; the product for immunohistochemical typing and / or prognostic evaluation of proliferative cholangiocarcinoma includes a reagent for specific detection of CLDN6; the product for immunohistochemical typing and / or prognostic evaluation of metabolic cholangiocarcinoma includes a reagent for specific detection of ADH1A. The product provided by the present invention can realize the diagnosis and prognostic evaluation of different proteomic subtypes of cholangiocarcinoma (fiber-driven cholangiocarcinoma, proliferative cholangiocarcinoma, metabolic cholangiocarcinoma) by immunohistochemical typing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A is a heat map of cholangiocarcinoma samples and clinical information in Example 1 of the present invention.

[0020] Figure 1 B is the protein expression profile of the cholangiocarcinoma sample in Example 1 of the present invention.

[0021] Figure 2 A is a consensus clustering matrix obtained by performing unsupervised consensus clustering on cholangiocarcinoma samples in Example 1 of the present invention when the number of clusters is 2 to 6.

[0022] Figure 2 B is a cumulative distribution function (CDF) diagram of the consensus clustering matrix when the number of clusters is 2 to 6 in Example 1 of the present invention and a relative change diagram of the area under the CDF curve.

[0023] Figure 3 A is a comparison of the differences in clinical characteristics among fiber-driven cholangiocarcinoma, proliferative cholangiocarcinoma, and metabolic cholangiocarcinoma in Example 1 of the present invention.

[0024] Figure 3 B is a comparison of the prognostic differences among fiber-driven cholangiocarcinoma, proliferative cholangiocarcinoma, and metabolic cholangiocarcinoma in Example 1 of the present invention.

[0025] Figure 4 A is a volcano plot of differential proteins among fiber-driven cholangiocarcinoma, proliferative cholangiocarcinoma, and metabolic cholangiocarcinoma in Example 1 of the present invention.

[0026] Figure 4 B is the functional enrichment analysis result of differential proteins of fiber-driven cholangiocarcinoma, proliferative cholangiocarcinoma, and metabolic cholangiocarcinoma in Example 1 of the present invention.

[0027] Figure 5 A is the specific expression of POSTN protein, CLDN6 protein and ADH1A protein in different proteomic subtypes of cholangiocarcinoma verified in Example 1 of the present invention.

[0028] Figure 5 B is Example 1 of the present invention, verifying the ability of POSTN protein, CLDN6 protein, and ADH1A protein to distinguish different proteomic subtypes of cholangiocarcinoma.

[0029] Figure 6 A is the immunohistochemical staining result of POSTN protein, CLDN6 protein and ADH1A protein in the sample sections of fiber-driven, proliferative and metabolic cholangiocarcinoma in Example 2 of the present invention.

[0030] Figure 6 B is the immunohistochemical scores of POSTN protein, CLDN6 protein, and ADH1A protein in the sections of fiber-driven, proliferative, and metabolic cholangiocarcinoma samples in Example 2 of the present invention.

[0031] Figure 7 A is a receiver operating characteristic curve (ROC curve) for verifying the ability of immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein to distinguish different proteomic subtypes of cholangiocarcinoma in Example 2 of the present invention.

[0032] Figure 7 B is the consistency of immunohistochemical typing of POSTN protein and fibrosis-driven cholangiocarcinoma subtype, immunohistochemical typing of CLDN6 protein and proliferative cholangiocarcinoma subtype, and immunohistochemical typing of ADH1A protein and metabolic cholangiocarcinoma subtype evaluated in Example 2 of the present invention.

[0033] Figure 8 A is Example 2 of the present invention verifying the correlation between the immunohistochemical scores of POSTN protein, CLDN6 protein, and ADH1A protein and the clinical characteristics of lymph node metastasis.

[0034] Figure 8 B is a survival curve of Example 2 of the present invention verifying the correlation between the immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein and the patient's prognosis.

[0035] Fig. 9 A is the immunohistochemical staining result of α-smooth muscle actin (αSMA) in the sample sections of fiber-driven, proliferative, and metabolic cholangiocarcinoma in Example 2 of the present invention.

[0036] Fig. 9 B is the αSMA immunohistochemical score in the fiber-driven, proliferative, and metabolic cholangiocarcinoma sample sections of Example 2 of the present invention.

[0037] Fig. 9 C is Example 2 of the present invention verifying the correlation between the αSMA immunohistochemical score and the POSTN protein immunohistochemical score.

[0038] Fig.10 A is the immunohistochemical staining result of tumor proliferation antigen (Ki67) in the sample sections of fiber-driven, proliferative, and metabolic cholangiocarcinomas in Example 2 of the present invention.

[0039] Fig.10 B is the Ki67 immunohistochemical score in the fiber-driven, proliferative, and metabolic cholangiocarcinoma sample sections of Example 2 of the present invention.

[0040] Fig.10 C is the correlation between Ki67 immunohistochemical score and CLDN6 protein immunohistochemical score verified in Example 2 of the present invention.

[0041] Fig.11 A is the correlation between the immunohistochemical scores of POSTN protein, CLDN6 protein, and ADH1A protein and the clinical characteristics of lymph node metastasis, which is externally verified in Example 2 of the present invention.

[0042] Fig.11 B is a survival curve for externally validating the correlation between the immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein and the patient's prognosis in Example 2 of the present invention. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0044] Example 1 Screening and verification of molecular markers for different proteomic subtypes of cholangiocarcinoma

[0045] 1. Collect cholangiocarcinoma samples and perform proteomic testing: Tumor samples from 113 cholangiocarcinoma patients who underwent radical resection at Sun Yat-sen Memorial Hospital of Sun Yat-sen University were collected. The heat map of cholangiocarcinoma samples and clinical information (primary tumor, lymph node metastasis, age, carbohydrate antigen CA199 content, distant metastasis) is shown in the figure below. Figure 1 As shown in A; high-precision mass spectrometry was performed on cholangiocarcinoma samples to obtain their protein expression profiles. Figure 1 As shown in B.

[0046] Depend on Figure 1A shows that among the 113 patients with cholangiocarcinoma, there were 64 cases of hilar cholangiocarcinoma (hCCA) and 49 cases of intrahepatic cholangiocarcinoma (iCCA); Figure 1 B shows that the number of proteins in each cholangiocarcinoma sample is stable between 7000 and 9000, which meets the quality control requirements of proteomics.

[0047] 2. Proteomic classification of cholangiocarcinoma based on protein expression profile: Unsupervised consensus clustering was performed based on the protein expression profile of cholangiocarcinoma samples. The consensus clustering matrix (consensus matrix) results when the number of clusters was 2 to 6 were as follows: Figure 2 As shown in Figure 2A; the consensus cumulative distribution function (consensus CDF) of the consensus clustering matrix when the number of clusters is 2 to 6 and the relative change of the area under the CDF curve are shown in Figure 2B.

[0048] Depend on Figure 2 A and 2B show that the optimal clustering number k for cholangiocarcinoma samples is 3. This shows that it is best to divide cholangiocarcinoma into three proteomic subtypes: fibrosis-driven, proliferative, and metabolic.

[0049] 3. Compare the clinical characteristics and prognosis of different proteomic subtypes of cholangiocarcinoma (fibroblast-driven, proliferative, and metabolic subtypes). Figure 3 A, 3B.

[0050] Depend on Figure 3 A shows that different proteomic subtypes of cholangiocarcinoma (fibroblast-driven, proliferative, and metabolic) are associated with clinical features such as lymph node metastasis, distant metastasis, age, and biliary stones. Figure 3 B shows that there are significant differences in the prognosis of different proteomic subtypes of cholangiocarcinoma (fibroblast-driven, proliferative, and metabolic). Among them, patients with fibroblast-driven cholangiocarcinoma have the worst prognosis, patients with proliferative cholangiocarcinoma have a poor prognosis, and patients with metabolic cholangiocarcinoma have the best prognosis.

[0051] 4. Screening of differentially expressed proteins in different proteomic subtypes of cholangiocarcinoma (fibroblast-driven, proliferative, and metabolic subtypes):

[0052] (1) The R language limma package was used to perform differential protein analysis. The differential proteins of different proteomic subtypes of cholangiocarcinoma (fibroblast-driven, proliferative, and metabolic) were screened using log2 (Fold change) ≥ 2 (log2FC ≥ 2) and adjusted P value < 0.05 as the criteria. The results are as follows: Figure 4 As shown in A. Figure 4 A shows that the most significant differential proteins in fibrous-driven cholangiocarcinoma, proliferative cholangiocarcinoma, and metabolic cholangiocarcinoma are POSTN protein (log2FC=3.42, adjusted.P=2.34E-18), CLDN6 protein (log2FC=2.84, adjusted.P=5.13E-17), and ADH1A protein (log2FC=2.77, adjusted.P=3.18E-8), respectively.

[0053] (2) Functional enrichment analysis was performed on the above differentially expressed proteins (functional enrichment analysis of differentially expressed proteins can reveal characteristic signaling pathways of different proteomic subtypes of cholangiocarcinoma). The results are as follows: Figure 4 As shown in B. Figure 4 B shows that the biological characteristics of fiber-driven cholangiocarcinoma, proliferative cholangiocarcinoma, and metabolic cholangiocarcinoma are extracellular matrix remodeling, cell division regulation, and alcohol-aldehyde metabolism, respectively.

[0054] 5. Verify the specific expression of POSTN protein, CLDN6 protein, and ADH1A protein in different proteomic subtypes of cholangiocarcinoma. The results are as follows Figure 5 As shown in A.

[0055] Depend on Figure 5 A shows that the expression level of POSTN protein in fiber-driven cholangiocarcinoma is significantly higher than that in other subtypes, the expression level of CLDN6 protein in proliferative cholangiocarcinoma is significantly higher than that in other subtypes, and the expression level of ADH1A protein in metabolic cholangiocarcinoma is significantly higher than that in other subtypes.

[0056] 6. Verify the ability of POSTN protein, CLDN6 protein, and ADH1A protein to distinguish different proteomic subtypes of cholangiocarcinoma: The receiver operating characteristic (ROC) and area under the curve (AUC) were used to evaluate the accuracy of POSTN protein expression in distinguishing fiber-driven cholangiocarcinoma, CLDN6 protein expression in distinguishing proliferative cholangiocarcinoma, and ADH1A protein expression in distinguishing metabolic cholangiocarcinoma. The results are as follows: Figure 5 As shown in B.

[0057] Depend on Figure 5B shows that the area under the ROC curve (AUC) of fibroblast-driven cholangiocarcinoma is 0.905 (0.871-0.946), the area under the ROC curve (AUC) of proliferative cholangiocarcinoma is 0.881 (0.844-0.912), and the area under the ROC curve (AUC) of metabolic cholangiocarcinoma is 0.816 (0.776-0.853). This shows that POSTN protein, as a protein marker, can accurately distinguish fibroblast-driven cholangiocarcinoma, CLDN6 protein, as a protein marker, can accurately distinguish proliferative cholangiocarcinoma, and ADH1A protein, as a protein marker, can accurately distinguish metabolic cholangiocarcinoma subtypes.

[0058] Example 2 Verification of the role of immunohistochemical scoring of POSTN, CLDN6, and ADH1A in the detection and prognosis assessment of fibrosis-driven, proliferative, and metabolic cholangiocarcinoma

[0059] 1. Verify the expression of POSTN, CLDN6, and ADH1A in different proteomic subtypes of cholangiocarcinoma by immunohistochemistry: Paraffin-embedded tissues of 113 cholangiocarcinoma samples in Example 1 were sliced, and immunohistochemistry was performed to evaluate the expression levels of POSTN protein, CLDN6 protein, and ADH1A protein.

[0060] The immunohistochemical antibodies used in the immunohistochemical process are as follows:

[0061] protein company Part Number Dilution multiple POSTN Abcam ab215199 1:1000 CLDN6 Abcam ab314134 1:100 ADH1A Abcam ab108203 1:200

[0062] Immunohistochemical staining results Figure 6 As shown in A, immunohistochemical scores were Figure 6 As shown in B. Among them, POSTN protein is an extracellular matrix protein, and its immunohistochemical score is the percentage of the distribution area of ​​POSTN protein to the total area of ​​the section; CLDN6 protein is a cell membrane protein, and its immunohistochemical score is the percentage of cholangiocarcinoma cells expressing CLDN6 protein to all cholangiocarcinoma cells; ADH1A protein is a widely distributed cytoplasmic protein, and its immunohistochemical score is the percentage of cells expressing ADH1A protein to all cells.

[0063] Depend on Figure 6 A and 6B show that the immunohistochemical score of POSTN protein is significantly higher in fibroblast-driven cholangiocarcinoma samples than in other subtypes, the immunohistochemical score of CLDN6 protein is significantly higher in proliferative cholangiocarcinoma samples than in other subtypes, and the immunohistochemical score of ADH1A protein is significantly higher in metabolic cholangiocarcinoma samples than in other subtypes. This shows that POSTN protein, as a protein marker, can be used to diagnose fibroblast-driven cholangiocarcinoma, CLDN6 protein, as a protein marker, can be used to diagnose proliferative cholangiocarcinoma, and ADH1A protein, as a protein marker, can be used to diagnose metabolic cholangiocarcinoma subtypes.

[0064] 2. According to the immunohistochemical scores of POSTN protein, CLDN6 protein and ADH1A protein, immunohistochemical typing of POSTN protein, CLDN6 protein and ADH1A protein was performed:

[0065] POSTN high expression group: immunohistochemical score>40%; POSTN low expression group: immunohistochemical score≤40%.

[0066] CLDN6 high expression group: immunohistochemical score>30%; CLDN6 low expression group: immunohistochemical score≤30%.

[0067] ADH1A high expression group: immunohistochemical score>20%; ADH1A low expression group: immunohistochemical score≤20%.

[0068] 3. Verify the ability of immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein to distinguish different proteomic subtypes of cholangiocarcinoma: ROC curve was used to evaluate the ability of immunohistochemical scoring of POSTN protein, CLDN6 protein, and ADH1A protein to distinguish different proteomic subtypes of cholangiocarcinoma. The results are as follows: Figure 7 As shown in A.

[0069] Depend on Figure 7 A shows that the AUC for distinguishing fibrous-driven cholangiocarcinoma using the POSTN high expression group (immunohistochemical score>40%) is 0.874 (0.843-0.911), the AUC for distinguishing proliferative cholangiocarcinoma using the CLDN6 high expression group (immunohistochemical score>30%) is 0.865 (0.828-0.906), and the ADH1A protein high expression group (immunohistochemical score>20%) is 0.841 (0.809-0.877). This shows that POSTN protein, CLDN6 protein, and ADH1A protein as protein markers can accurately distinguish different proteomic subtypes of cholangiocarcinoma.

[0070] 4. Evaluate the consistency of immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein with the corresponding cholangiocarcinoma proteomic subtypes. Figure 7 As shown in B.

[0071] Depend on Figure 7B shows that the consistency of immunohistochemical typing of POSTN protein for fibrosis-driven cholangiocarcinoma, immunohistochemical typing of CLDN6 protein for proliferative cholangiocarcinoma, and immunohistochemical typing of ADH1A protein for metabolic cholangiocarcinoma is 90.3% (102 / 113), 87.6% (99 / 113), and 94.7% (107 / 113), respectively. This shows that the immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein is highly consistent with the corresponding cholangiocarcinoma proteomic subtypes.

[0072] 5. Verify the correlation between the immunohistochemical scores of POSTN protein, CLDN6 protein, and ADH1A protein and clinical characteristics of lymph node metastasis. The results are as follows Figure 8 As shown in A.

[0073] Depend on Figure 8 A shows that the immunohistochemical score of POSTN protein is significantly correlated with lymph node metastasis LNM(+). This shows that the immunohistochemical score of POSTN protein is related to clinical characteristics and is consistent with the clinical characteristics of fibrous-driven cholangiocarcinoma (see Figure 3 A).

[0074] 6. Verify the relationship between immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein and prognosis, and draw the survival curve of immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein, such as Figure 8 As shown in B.

[0075] Depend on Figure 8 B shows that the prognosis of the POSTN high expression group and the CLDN6 high expression group is poor, and the prognosis of the POSTN low expression group and the CLDN6 low expression group is good; the prognosis of the ADH1A high expression group is good, and the prognosis of the ADH1A low expression group is poor. This shows that the immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein is significantly correlated with the prognosis of patients.

[0076] 7. Verify the correlation between POSTN protein and the biological characteristics of fibrosis-driven cholangiocarcinoma, and the correlation between CLDN6 protein and abnormal tumor proliferation, the biological characteristics of proliferative cholangiocarcinoma: Immunohistochemical staining of α-smooth muscle actin (αSMA) was performed on fibrous-driven, proliferative, and metabolic cholangiocarcinomas (characterizing the degree of tumor fibrosis). The results are as follows Fig. 9 As shown in A to C. Immunohistochemical staining of tumor proliferation antigen (Ki67) (characterizing the degree of tumor proliferation) was performed on fibrous-driven, proliferative, and metabolic cholangiocarcinomas. The results are shown in Fig.10 As shown in A~C.

[0077] Depend on Fig. 9From A to C, we can see that POSTN protein is significantly positively correlated with the degree of tumor fibrosis (Pearson correlation coefficient is r=0.8479, P<0.0001). Fig.10 As shown in A to C, CLDN6 protein was significantly positively correlated with the degree of tumor proliferation (Pearson correlation coefficient was r=0.7854, P<0.0001).

[0078] The above results suggest that fibrous-driven cholangiocarcinoma with POSTN as a protein marker (characteristic protein) may benefit from treatments that inhibit tumor fibrosis and block POSTN and integrin receptor signals; proliferative cholangiocarcinoma with CLDN6 as a protein marker (characteristic protein) may be more sensitive to treatments that can inhibit tumor proliferation and target CLDN6.

[0079] 8. External validation of the correlation between the immunohistochemical scores of POSTN protein, CLDN6 protein, and ADH1A protein and the clinical characteristics and lymph node metastasis of fibroblast-driven, proliferative, and metabolic cholangiocarcinoma: Cholangiocarcinoma samples (N=52) were obtained from an independent medical center, and immunohistochemical evaluation of the correlation between the expression levels of POSTN protein, CLDN6 protein, and ADH1A protein and the clinical characteristics and lymph node metastasis of fibroblast-driven, proliferative, and metabolic cholangiocarcinoma was performed. The results are as follows Fig.11 As shown in A.

[0080] Depend on Fig.11 A shows that the immunohistochemical score of POSTN protein is significantly correlated with lymph node metastasis LNM(+).

[0081] 9. Externally verify the relationship between the immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein and the prognosis of patients with fibrosis-driven, proliferative, and metabolic cholangiocarcinoma, and draw the survival curve of the immunohistochemical typing of POSTN protein, CLDN6 protein, and ADH1A protein, such as Fig.11 As shown in B.

[0082] Depend on Fig.11 B shows that the immunohistochemical scores of POSTN protein, CLDN6 protein and ADH1A protein are related to the prognosis of patients with cholangiocarcinoma. Among them, the prognosis of the POSTN high expression group (immunohistochemical score>40%) and the CLDN6 high expression group (immunohistochemical score>30%) is poor, and the prognosis of the POSTN low expression group (immunohistochemical score≤40%) and the CLDN6 low expression group (immunohistochemical score≤30%) is good; the prognosis of the ADH1A high expression group (immunohistochemical score>20%) is good, and the prognosis of the ADH1A low expression group (immunohistochemical score≤20%) is poor.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. Use of a protein marker in the preparation of a product for the prognosis assessment of cholangiocarcinoma, characterized in that: The protein markers include POSTN, CLDN6 and ADH1A.

2. The use according to claim 1, characterized in that: The product includes a kit and / or a chip.

3. Use of a reagent for specifically detecting a protein marker in the preparation of a product for the prognosis assessment of cholangiocarcinoma, characterized in that: The protein markers include POSTN, CLDN6 and ADH1A.

4. The use according to claim 3, characterized in that: The reagents include reagents for specifically detecting the expression amount of the protein marker.

5. The use according to claim 4, characterized in that: The reagents for specifically detecting the expression amount of the protein marker include POSTN antibody, CLDN6 antibody, and ADH1A antibody.

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